MiSynPat.org

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Uzun Gunes A.

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N'Gbo N'Gbo Ikazabo Rosy, Mostosi Christian, Jissendi Patrice, Labaisse Marie-Anne, Vandernoot Isabelle.

We report a case of an adult patient suffering from leukoencephalopathy with brainstem and spinal cord involvement and elevated white matter lactate (LBSL) caused by a DARS2 polymorphism. DARS2 mutation was identified by combining MRI and genetic analysis. Our patient was affected by compound heterozygosity for a pathogenic mutation and a common variant, but with reduced aspartyl-tRNA synthetase activity. Brain and spinal cord magnetic resonance imaging revealed extensive white matter abnormalities; spectroscopy revealed no lactate elevation. A new compound heterozygous DARS2 variant combined with a polymorphism in the other allele in an adult patient with LBSL was identified, resulting in reduced DARS2 activity. This combination is rare and has consequences on how we should consider benign variant polymorphisms in the future.

Jin Xiaofen, Zhang Zengming, Nie Zhipeng, Wang Chenghui, Meng Feilong, Yi Qiuzi, Chen Mengquan, Sun Jiji, Zou Jian, Jiang Pingping, Guan Min-Xin.

Mitochondria maintain a distinct pool of ribosomal machinery, including tRNAs and tRNAs activating enzymes, such as mitochondrial tyrosyl-tRNA synthetase (YARS2). Mutations in YARS2, which typically lead to the impairment of mitochondrial protein synthesis, have been linked to an array of human diseases including optic neuropathy. However, the lack of YARS2 mutation animal model makes us difficult to elucidate the pathophysiology underlying YARS2 deficiency. To explore this system, we generated YARS2 knockout (KO) HeLa cells and zebrafish using CRISPR/Cas9 technology. We observed the aberrant tRNATyr aminoacylation overall and reductions in the levels in mitochondrion- and nucleus-encoding subunits of oxidative phosphorylation system (OXPHOS), which were especially pronounced effects in the subunits of complex I and complex IV. These deficiencies manifested the decreased levels of intact supercomplexes overall. Immunoprecipitation assays showed that YARS2 bound to specific subunits of complex I and complex IV, suggesting the posttranslational stabilization of OXPHOS. Furthermore, YARS2 ablation caused defects in the stability and activities of OXPHOS complexes. These biochemical defects could be rescued by the overexpression of YARS2 cDNA in the YARS2KO cells. In zebrafish, the yars2KO larva conferred deficient COX activities in the retina, abnormal mitochondrial morphology, and numbers in the photoreceptor and retinal ganglion cells. The zebrafish further exhibited the retinal defects affecting both rods and cones. Vision defects in yars2KO zebrafish recapitulated the clinical phenotypes in the optic neuropathy patients carrying the YARS2 mutations. Our findings highlighted the critical role of YARS2 in the stability and activity of OXPHOS and its pathological consequence in vision impairments.

Ngo Julie, Prokop Jeremy W, Umfleet Jason, Seaver Laurie H.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a progressive disorder associated with deficiency of mitochondrial aspartyl-tRNA synthetase, a homodimer encoded by the gene DARS2. There is a wide range in age of onset of symptoms, typically from childhood to adulthood, with very few cases of infantile onset disease reported. We report a child at age 10 years with perinatal onset of symptoms evidenced by congenital microcephaly with progression to severe but non-lethal epileptic encephalopathy and spastic quadriplegia. A comprehensive epilepsy focused gene panel performed as a trio with parents detected a novel homozygous DARS2 variant. This variant is located at the dimer interface in a critical catalytic domain and is expected to result in markedly reduced enzyme activity which likely explains the severe and early onset symptoms in this case.

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Bostanova Fatima M, Tsygankova Polina G, Larshina Elena A, Nagornov Ilya O, Evseeva Yulia V, Krutikhina Irina L, Dzhentemirova Marina E, Kashlakova Marina N, Petukhova Marina S, Sharkova Inna V, Zakharova Ekaterina Y.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation is an inherited disease caused by pathogenic biallelic variants in the gene DARS2, which encodes mitochondrial aspartyl-tRNA synthetase. This disease is characterized by slowly progressive spastic gait, cerebellar symptoms, and leukoencephalopathy with brainstem and spinal cord involvement.

Pickup Elizabeth, Moore Steven A, Suwannarat Pim, Grant Christina, Ah Mew Nicholas, Gropman Andrea, Sen Kuntal.

Exome sequencing (ES) is a useful tool in diagnosing suspected mitochondrial disease but can miss pathogenic variants for several reasons. Additional testing, such as muscle biopsy or biochemical testing, can be helpful in exome-negative cases.

Huang Wei-Lin, Steenari Maija R, Barrick Rebekah, Simon Mariella T, Chang Richard, Eftekharian Shaya S, Stover Alexander, Schwartz Philip H, Latini Alexandra, Abdenur Jose E.

LBSL is a mitochondrial disorder caused by mutations in the mitochondrial aspartyl-tRNA synthetase gene DARS2, resulting in a distinctive pattern on brain magnetic resonance imaging (MRI) and spectroscopy. Clinical presentation varies from severe infantile to chronic, slowly progressive neuronal deterioration in adolescents or adults. Most individuals with LBSL are compound heterozygous for one splicing defect in an intron 2 mutational hotspot and a second defect that could be a missense, non-sense, or splice site mutation or deletion resulting in decreased expression of the full-length protein.

Licchetta Laura, Di Giorgi Lucia, Santucci Margherita, Taruffi Lisa, Stipa Carlotta, Minardi Raffaella, Carelli Valerio, Bisulli Francesca.

Biallelic pathogenic variants in the mitochondrial prolyl-tRNA synthetase 2 gene (PARS2, OMIM * 612036) have been associated with Developmental and Epileptic Encephalopathy-75 (DEE-75, MIM #618437). This condition is typically characterized by early-onset refractory infantile spasms with hypsarrhythmia, intellectual disability, microcephaly, cerebral atrophy with hypomyelination, lactic acidemia, and cardiomyopathy. Most affected individuals do not survive beyond the age of 10 years.

Khodaeian Mehrnoosh, Bitarafan Fatemeh, Garrousi Fatemeh, Sardehie Elham Amjadi, Pak Neda, Hosseinpour Sareh, Shakiba Marjan, Falah Masoumeh, Garshasbi Masoud, Tavasoli Ali Reza.

NARS2 encodes mitochondrial Asparaginyl-tRNA Synthetase 2, which catalyzes the aminoacylation of tRNA-Asn in the mitochondria. To date, 24 variants have been reported in NARS2 gene in 35 patients. The phenotypic variability of NARS2-associated disorder is broad, ranging from neurodevelopmental disorders to hearing loss. In this study, we report some novel imaging findings in an Iranian patient suffering from epileptic encephalopathy, caused by a previously reported variant, c.500A > G; p.(His167Arg), in NARS2.

Hu Xuyun, Guo Ruolan, Hao Chanjuan, Hao Lijuan.

Biallelic variants in mitochondrial prolyl-tRNA synthetase 2 (PARS2) are associated with developmental and epileptic encephalopathy-75 (DEE75), which is characterized by global developmental delay, seizures and brain imaging anomalies. To date, fewer than 20 patients with PARS2 mutation have been reported in previous literature, and only ten of them had detailed phenotype information.

Podmanicky Oliver, Gao Fei, Munro Benjamin, Jennings Matthew J, Boczonadi Veronika, Hathazi Denisa, Mueller Juliane S, Horvath Rita.

Mitochondrial aminoacyl-tRNA synthetase (mt-ARS) mutations cause severe, progressive, and often lethal diseases with highly heterogeneous and tissue-specific clinical manifestations. This study investigates the molecular mechanisms triggered by three different mt-ARS defects caused by biallelic mutations in AARS2, EARS2, and RARS2, using an in vitro model of human neuronal cells. We report distinct molecular mechanisms of mitochondrial dysfunction among the mt-ARS defects studied. Our findings highlight the ability of proliferating neuronal progenitor cells (iNPCs) to compensate for mitochondrial translation defects and maintain balanced levels of oxidative phosphorylation (OXPHOS) components, which becomes more challenging in mature neurons. Mutant iNPCs exhibit unique compensatory mechanisms, involving specific branches of the integrated stress response, which may be gene-specific or related to the severity of the mitochondrial translation defect. RNA sequencing revealed distinct transcriptomic profiles showing dysregulation of neuronal differentiation and protein translation. This study provides valuable insights into the tissue-specific compensatory mechanisms potentially underlying the phenotypes of patients with mt-ARS defects. Our novel in vitro model may more accurately represent the neurological presentation of patients and offer an improved platform for future investigations and therapeutic development.

Ahmed Iftikhar, Muzammal Muhammad, Khan Muzammil Ahmad, Ullah Hafiz, Farid Arshad, Yasin Muhammad, Khan Jabbar, Alam Khurshid, Mir Asif.

Intellectual disability, a genetically and clinically varied disorder and is a significant health problem, particularly in less developed countries due to larger family size and high ratio of consanguineous marriages. In the current genetic study, we investigate and find the novel disease causative factors in the four Pakistani families with severe type of non-syndromic intellectual disability. For genetic analysis whole-exome sequencing (WES) and Sanger sequencing was performed. I-TASSER and Cluspro tools were used for Protein modeling and Protein-protein docking. Sanger sequencing confirms the segregation of novel homozygous variants in all the families i.e., c.245 T > C; p.Leu82Pro in SLC50A1 gene in family 1, missense variant c.1037G > A; p.Arg346His in TARS2 gene in family 2, in family 3 and 4, nonsense mutation c.234G > A; p.Trp78Term and missense mutation c.2200G > A; p.Asp734Asn in TBC1D3 and ANAPC2 gene, respectively. In silico functional studies have found the drastic effect of these mutations on protein structure and its interaction properties. Substituted amino acids were highly conserved and present on highly conserved region throughout the species. The discovery of pathogenic variants in SLC50A1, TARS2, TBC1D1 and ANAPC2 shows that the specific pathways connected with these genes may be important in cognitive impairment. The decisive role of pathogenic variants in these genes cannot be determined with certainty due to lack of functional data. However, exome sequencing and segregation analysis of all filtered variants revealed that the currently reported variants were the only variations from the respective families that segregated with the phenotype in the family.

Zhao Shichao, Lian Ruofei, Jin Liang, Li Mengchun, Jia Tianming, Xu Falin, Du Kaixian, Wang Lijun, Guo Qiliang, Dong Yan.

Defects in RARS2 cause cerebellopontine hypoplasia type 6 (pontocerebellar hypoplasia type 6, PCH6, OMIM: #611523), a rare autosomal recessive inherited mitochondrial disease. Here, we report two male patients and their respective family histories.

Hu Jui-Lin, Hsu Chih-Chien, Hsiao Yu-Jer, Lin Yi-Ying, Lai Wei-Yi, Liu Yu-Hao, Wang Chia-Lin, Ko Yu-Ling, Tsai Ming-Long, Tseng Huan-Chin, Chien Yueh, Yang Yi-Ping.

A maternal inheritance disorder called Leber's hereditary optic neuropathy (LHON) is the most common primary mitochondrial DNA disorder. In most studies, there are more male patients than female patients, which contradicts the usual pattern in mitochondrial hereditary diseases. This suggests that nuclear DNA (nDNA) may influence the degeneration of retinal ganglion cells (RGCs) in LHON. The primary cause of this is dysfunction in complex I of the electron transport chain, leading to ineffective ATP production. In addition to MT-ND4 or MT-ND1 mutations, genes such as PRICKLE3, YARS2, and DNAJC30, which come from nuclear DNA, also play a role in LHON. These three genes affect the electron chain transport differently. PRICKLE3 interacts with ATP synthase (complex V) at Xp11.23, while YARS2 is a tyrosyl-tRNA synthetase 2 involved in mitochondria. DNAJC30 mutations result in autosomal recessive LHON (arLHON). Understanding how genes impact the disease is crucial for developing new treatments. Idebenone has been approved for treating LHON and has shown safety and efficacy in clinical trials. Mesenchymal stem cell-based therapy has also emerged as a potential treatment for LHON by transferring mitochondria into target cells. Gene therapy research focuses on specific gene mutations, and the wild-type ND4 gene target in the AAV vector has shown promise in clinical trials as a potential treatment for LHON.

Tyynismaa Henna.

Mitochondrial aminoacyl-tRNA synthetases (mtARS) are enzymes critical for the first step of mitochondrial protein synthesis by charging mitochondrial tRNAs with their cognate amino acids. Pathogenic variants in all 19 nuclear mtARS genes are now recognized as causing recessive mitochondrial diseases. Most mtARS disorders affect the nervous system, but the phenotypes range from multisystem diseases to tissue-specific manifestations. However, the mechanisms behind the tissue specificities are poorly understood, and challenges remain in obtaining accurate disease models for developing and testing treatments. Here, some of the currently existing disease models that have increased our understanding of mtARS defects are discussed.

Li Yingjie, Xu Jiaming, Xu Yan, Li Chuanzhou, Wu Yan, Liu Zhijun.

Leukodystrophies are a diverse group of rare inherited disorders that affect the white matter of the central nervous system with a wide phenotypic spectrum. We aimed to characterize the clinical and genetic features of leukodystrophies in a central-southern Chinese cohort.

Francesco Saettini, Fabiola Guerra, Grazia Fazio, Cristina Bugarin, J McMillan Hugh, Akira Ohtake, Anna Ardissone, Masayuki Itoh, Sabrina Giglio, Gerarda Cappuccio, Giuliana Giardino, Roberta Romano, Manuel Quadri, Serena Gasperini, Daniele Moratto, Marco Chiarini, Akira Ishiguro, Yasuyuki Fukuhara, Itaru Hayakawa, Yasushi Okazaki, Mario Mauri, Rocco Piazza, Gianni Cazzaniga, Andrea Biondi.

Biallelic KARS1 mutations cause KARS-related diseases, a rare syndromic condition encompassing central and peripheral nervous system impairment, heart and liver disease, and deafness. KARS1 encodes the t-RNA synthase of lysine, an aminoacyl-tRNA synthetase, involved in different physiological mechanisms (such as angiogenesis, post-translational modifications, translation initiation, autophagy and mitochondrial function). Although patients with immune-hematological abnormalities have been individually described, results have not been collectively discussed and functional studies investigating how KARS1 mutations affect B cells have not been performed. Here, we describe one patient with severe developmental delay, sensoneurinal deafness, acute disseminated encephalomyelitis, hypogammaglobulinemia and recurrent infections. Pathogenic biallelic KARS1 variants (Phe291Val/ Pro499Leu) were associated with impaired B cell metabolism (decreased mitochondrial numbers and activity). All published cases of KARS-related diseases were identified. The corresponding authors and researchers involved in the diagnosis of inborn errors of immunity or genetic syndromes were contacted to obtain up-to-date clinical and immunological information. Seventeen patients with KARS-related diseases were identified. Recurrent/severe infections (9/17) and B cell abnormalities (either B cell lymphopenia [3/9], hypogammaglobulinemia [either IgG, IgA or IgM; 6/15] or impaired vaccine responses [4/7]) were frequently reported. Immunoglobulin replacement therapy was given in five patients. Full immunological assessment is warranted in these patients, who may require detailed investigation and specific supportive treatment.

Guang S, O'Brien B M, Fine A S, Ying M, Fatemi A, Nemeth C L.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare neurological disorder caused by the mutations in the DARS2 gene, which encodes the mitochondrial aspartyl-tRNA synthetase. The objective of this study was to understand the impact of DARS2 mutations on cell processes through evaluation of LBSL patient stem cell derived cerebral organoids and neurons. We generated human cerebral organoids (hCOs) from induced pluripotent stem cells (iPSCs) of seven LBSL patients and three healthy controls using an unguided protocol. Single cells from 70-day-old hCOs were subjected to SMART-seq2 sequencing and bioinformatic analysis to acquire high-resolution gene and transcript expression datasets. Global gene expression analysis demonstrated dysregulation of a number of genes involved in mRNA metabolism and splicing processes within LBSL hCOs. Importantly, there were distinct and divergent gene expression profiles based on the nature of the DARS2 mutation. At the transcript level, pervasive differential transcript usage and differential spliced exon events that are involved in protein translation and metabolism were identified in LBSL hCOs. Single-cell analysis of DARS2 (exon 3) showed that some LBSL cells exclusively express transcripts lacking exon 3, indicating that not all LBSL cells can benefit from the "leaky" nature common to splice site mutations. At the gene- and transcript-level, we uncovered that dysregulated RNA splicing, protein translation and metabolism may underlie at least some of the pathophysiological mechanisms in LBSL. To confirm hCO findings, iPSC-derived neurons (iNs) were generated by overexpressing Neurogenin 2 using lentiviral vector to study neuronal growth, splicing of DARS2 exon 3 and DARS2 protein expression. Live cell imaging revealed neuronal growth defects of LBSL iNs, which was consistent with the finding of downregulated expression of genes related to neuronal differentiation in LBSL hCOs. DARS2 protein was downregulated in iNs compared to iPSCs, caused by increased exclusion of exon 3. The scope and complexity of our data imply that DARS2 is potentially involved in transcription regulation beyond its canonical role of aminoacylation. Nevertheless, our work highlights transcript-level dysregulation as a critical, and relatively unexplored, mechanism linking genetic data with neurodegenerative disorders.

Mihaylova Violeta, Herenger Yvan, Bethge Tobias, Bohlhalter Stephan.

Accogli Andrea, Lin Sheng-Jia, Severino Mariasavina, Kim Sung-Hoon, Huang Kevin, Rocca Clarissa, Landsverk Megan, Zaki Maha, Al-Maawali Almundher, Srinivasan Varunvenkat M, Al-Thihli Khalid, Schaefer G Bradly, Davis Monica, Tonduti Davide, Doneda Chiara, Marten Lara M, Mühlhausen Chris, Gomez Maria, Lamantea Eleonora, Mena Rafael, Nizon Mathilde, Procaccio Vincent, Begtrup Amber, Telegrafi Aida, Cui Hong, Schulz Heidi L, Mohr Julia, Biskup Saskia, Loos Mariana Amina, Aráoz Hilda Verónica, Salpietro Vincenzo, Keppen Laura Davis, Chitre Manali, Petree Cassidy, Raymond Lucy, Vogt Julie, Swayer Lindsey B, Basinger Alice A, Pedersen Signe Vandal, Pearson Toni S, Grange Dorothy K, Lingapp Lokesh, McDunnah Paige, Horvath Rita, Cogne Benjamin, Isidor Bertrand, Hahn Andreas, Gripp Karen, Jafarnejad Seyed Mehdi, Ostergaard Elsebet, Prada Carlos E, Ghezzi Daniele, Gowda Vykuntaraju K, Taylor Robert W, Sonenberg Nahum, Houlden Henry, Sissler Marie, Varshney Gaurav K, Maroofian Reza.

Biallelic variants in TARS2, encoding the mitochondrial threonyl-tRNA-synthetase, have been reported in a small group of individuals displaying a neurodevelopmental phenotype, but with limited neuroradiological data and insufficient evidence for causality of the variants.

Chen Wenqian, Rehsi Preeya, Thompson Kyle, Yeo Mildrid, Stals Karen, He Langping, Schimmel Paul, Chrzanowska-Lightowlers Zofia M A, Wakeling Emma, Taylor Robert W, Kuhle Bernhard.

FARS2 encodes the mitochondrial phenylalanyl-tRNA synthetase (mtPheRS), which is essential for charging mitochondrial (mt-) tRNAPhe with phenylalanine for use in intramitochondrial translation. Many biallelic, pathogenic FARS2 variants have been described previously, which are mostly associated with two distinct clinical phenotypes; an early onset epileptic mitochondrial encephalomyopathy or a later onset spastic paraplegia. In this study, we report on a patient who presented at 3 weeks of age with tachypnoea and poor feeding, which progressed to severe metabolic decompensation with lactic acidosis and seizure activity followed by death at 9 weeks of age. Rapid trio whole exome sequencing identified compound heterozygous FARS2 variants including a pathogenic exon 2 deletion on one allele and a rare missense variant (c.593G > T, p.(Arg198Leu)) on the other allele, necessitating further work to aid variant classification. Assessment of patient fibroblasts demonstrated severely decreased steady-state levels of mtPheRS, but no obvious defect in any components of the oxidative phosphorylation system. To investigate the potential pathogenicity of the missense variant, we determined its high-resolution crystal structure, demonstrating a local structural destabilization in the catalytic domain. Moreover, the R198L mutation reduced the thermal stability and impaired the enzymatic activity of mtPheRS due to a lower binding affinity for tRNAPhe and a slower turnover rate. Together these data confirm the pathogenicity of this FARS2 variant in causing early-onset mitochondrial epilepsy.

Nicolle Romain, Altin Nami, Siquier-Pernet Karine, Salignac Sherlina, Blanc Pierre, Munnich Arnold, Bole-Feysot Christine, Malan Valérie, Caron Barthélémy, Nitschké Patrick, Desguerre Isabelle, Boddaert Nathalie, Rio Marlène, Rausell Antonio, Cantagrel Vincent.

Bi-allelic variants in the mitochondrial arginyl-transfer RNA synthetase (RARS2) gene have been involved in early-onset encephalopathies classified as pontocerebellar hypoplasia (PCH) type 6 and in epileptic encephalopathy. A variant (NM_020320.3:c.-2A > G) in the promoter and 5'UTR of the RARS2 gene has been previously identified in a family with PCH. Only a mild impact of this variant on the mRNA level has been detected. As RARS2 is non-dosage-sensitive, this observation is not conclusive in regard of the pathogenicity of the variant.We report and describe here a new patient with the same variant in the RARS2 gene, at the homozygous state. This patient presents with a clinical phenotype consistent with PCH6 although in the absence of lactic acidosis. In agreement with the previous study, we measured RARS2 mRNA levels in patient's fibroblasts and detected a partially preserved gene expression compared to control. Importantly, this variant is located in the Kozak sequence that controls translation initiation. Therefore, we investigated the impact on protein translation using a bioinformatic approach and western blotting. We show here that this variant, additionally to its effect on the transcription, also disrupts the consensus Kozak sequence, and has a major impact on RARS2 protein translation. Through the identification of this additional case and the characterization of the molecular consequences, we clarified the involvement of this Kozak variant in PCH and on protein synthesis. This work also points to the current limitation in the pathogenicity prediction of variants located in the translation initiation region.

Boutaud Lucile, Ruzzenente Benedetta, Tessier Aude, Anselem Olivia, Pannier Emmanuelle, Grotto Sarah, Talhi Naïma, Amram Daniel, Willems Marjolaine, Wells Constance, Blanchet Patricia, Musizzano Yuri, Jauny Clémence, Nitschke Patrick, Bole-Feysot Christine, Bessières Bettina, Salhi Houria, Achaiaa Amale, Metodiev Metodi D, Razavi Ferechte, Rötig Agnès, Loeuilllet Laurence, Attié-Bitach Tania.

Corpus callosum defects are frequent congenital cerebral disorders caused by mutations in more than 300 genes. These include genes implicated in corpus callosum development or function, as well as genes essential for mitochondrial physiology. However, in utero corpus callosum anomalies rarely raise a suspicion of mitochondrial disease and are characterized by a very large clinical heterogeneity. Here, we report a detailed pathological and neuro-histopathological investigation of nine foetuses from four unrelated families with prenatal onset of corpus callosum anomalies, sometimes associated with other cerebral or extra-cerebral defects. Next generation sequencing allowed the identification of novel pathogenic variants in three different nuclear genes previously reported in mitochondrial diseases: TIMMDC1, encoding a Complex I assembly factor never involved before in corpus callosum defect; MRPS22, a protein of the small mitoribosomal subunit; and EARS2, the mitochondrial tRNA-glutamyl synthetase. The present report describes the antenatal histopathological findings in mitochondrial diseases and expands the genetic spectrum of antenatal corpus callosum anomalies establishing OXPHOS function as an important factor for corpus callosum biogenesis. We propose that, when observed, antenatal corpus callosum anomalies should raise suspicion of mitochondrial disease and prenatal genetic counselling should be considered.

Poquérusse Jessie, Nolan Melinda, Thorburn David R, Van Hove Johan L K, Friederich Marisa W, Love Donald R, Taylor Juliet, Powell Christopher A, Minczuk Michal, Snell Russell G, Lehnert Klaus, Glamuzina Emma, Jacobsen Jessie C.

Disorders of mitochondrial function are a collectively common group of genetic diseases in which deficits in core mitochondrial translation machinery, including aminoacyl tRNA synthetases, are key players. Biallelic variants in the CARS2 gene (NM_024537.4), which encodes the mitochondrial aminoacyl-tRNA synthetase for cysteine (CARS2, mt-aaRScys; MIM*612800), result in childhood onset epileptic encephalopathy and complex movement disorder with combined oxidative phosphorylation deficiency (MIM#616672). Prior to this report, eight unique pathogenic variants in the CARS2 gene had been reported in seven individuals. Here, we describe a male who presented in the third week of life with apnoea. He rapidly deteriorated with paroxysmal dystonic crises and apnoea resulting in death at 16 weeks. He had no evidence of seizure activity or multisystem disease and had normal brain imaging. Skeletal muscle biopsy revealed a combined disorder of oxidative phosphorylation. Whole-exome sequencing identified biallelic variants in the CARS2 gene: one novel (c.1478T>C, p.Phe493Ser), and one previously reported (c.655G>A, p.Ala219Thr; rs727505361). Northern blot analysis of RNA isolated from the patient's fibroblasts confirmed a clear defect in aminoacylation of the mitochondrial tRNA for cysteine (mt-tRNACys). To our knowledge, this is the earliest reported case of CARS2 deficiency with severe, early onset dystonia and apnoea, without epilepsy.

Wu Teng-Hui, Peng Jing, Yang Li, Chen Yan-Hui, Lu Xiu-Lan, Huang Jiao-Tian, You Jie-Yu, Ou-Yang Wen-Xian, Sun Yue-Yu, Xue Yi-Nan, Mao Xiao, Yan Hui-Ming, Ren Rong-Na, Xie Jing, Chen Zhi-Heng, Zhang Victor-Wei, Lyu Gui-Zhen, He Fang.

Mitochondrial diseases (MDs) were a large group multisystem disorders, attributable in part to the dual genomic control. The advent of massively sequencing has improved diagnostic rates and speed, and was increasingly being used as a first-line diagnostic test. Paediatric patients (aged < 18 years) who underwent dual genomic sequencing were enrolled in this retrospective multicentre study. We evaluated the mitochondrial disease criteria (MDC) and molecular diagnostic yield of dual genomic sequencing. Causative variants were identified in 177 out of 503 (35.2%) patients using dual genomic sequencing. Forty-six patients (9.1%) had mitochondria-related variants, including 25 patients with nuclear DNA (nDNA) variants, 15 with mitochondrial DNA (mtDNA) variants, and six with dual genomic variants (MT-ND6 and POLG; MT-ND5 and RARS2; MT-TL1 and NARS2; MT-CO2 and NDUFS1; MT-CYB and SMARCA2; and CHRNA4 and MT-CO3). Based on the MDC, 15.2% of the patients with mitochondria-related variants were classified as "unlikely to have mitochondrial disorder". Moreover, 4.5% of the patients with non-mitochondria-related variants and 1.43% with negative genetic tests, were classified as "probably having mitochondrial disorder". Dual genomic sequencing in suspected MDs provided a more comprehensive and accurate diagnosis for pediatric patients, especially for patients with dual genomic variants.

De Michele Giovanna, Maione Luigi, Cocozza Sirio, Tranfa Mario, Pane Chiara, Galatolo Daniele, De Rosa Anna, De Michele Giuseppe, Sacc� Francesco, Filla Alessandro.

The association of hypogonadism and cerebellar ataxia was first recognized in 1908 by Gordon Holmes. Since the seminal description, several heterogeneous phenotypes have been reported, differing for age at onset, associated features, and gonadotropins levels. In the last decade, the genetic bases of these disorders are being progressively uncovered. Here, we review the diseases associating ataxia and hypogonadism and the corresponding causative genes. In the first part of this study, we focus on clinical syndromes and genes (RNF216, STUB1, PNPLA6, AARS2, SIL1, SETX) predominantly associated with ataxia and hypogonadism as cardinal features. In the second part, we mention clinical syndromes and genes (POLR3A, CLPP, ERAL1, HARS, HSD17B4, LARS2, TWNK, POLG, ATM, WFS1, PMM2, FMR1) linked to complex phenotypes that include, among other features, ataxia and hypogonadism. We propose a diagnostic algorithm for patients with ataxia and hypogonadism, and we discuss the possible common etiopathogenetic mechanisms.

Pauly Martje G, Korenke G Christoph, Diaw Sokhna Haissatou, Grözinger Anne, Cazurro-Gutiérrez Ana, Pérez-Dueñas Belén, González Victoria, Macaya Alfons, Serrano Antón Ana Teresa, Peterlin Borut, Božović Ivana Babić, Maver Aleš, Münchau Alexander, Lohmann Katja.

Biallelic variants in the mitochondrial form of the tryptophanyl-tRNA synthetases (WARS2) can cause a neurodevelopmental disorder with movement disorders including early-onset tremor-parkinsonism syndrome. Here, we describe four new patients, who all presented at a young age with a tremor-parkinsonism syndrome and responded well to levodopa. All patients carry the same recurrent, hypomorphic missense variant (NM_015836.4: c.37T>G; p.Trp13Gly) either together with a previously described truncating variant (NM_015836.4: c.797Cdel; p.Pro266ArgfsTer10), a novel truncating variant (NM_015836.4: c.346C>T; p.Gln116Ter), a novel canonical splice site variant (NM_015836.4: c.349-1G>A), or a novel missense variant (NM_015836.4: c.475A>C, p.Thr159Pro). We investigated the mitochondrial function in patients and found increased levels of mitochondrially encoded cytochrome C Oxidase II as part of the mitochondrial respiratory chain as well as decreased mitochondrial integrity and branching. Finally, we conducted a literature review and here summarize the broad phenotypical spectrum of reported WARS2-related disorders. In conclusion, WARS2-related disorders are diagnostically challenging diseases due to the broad phenotypic spectrum and the disease relevance of a relatively common missense change that is often filtered out in a diagnostic setting since it occurs in ~0.5% of the general European population.

Guang Shiqi, O'Brien Brett, Fine Amena Smith, Ying Mingyao, Fatemi Ali, Nemeth Christina.

Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) is a rare neurological disorder caused by the mutations in the DARS2 gene, which encodes the mitochondrial aspartyl-tRNA synthetase. The objective of this study was to understand the impact of DARS2 mutations on cell processes through evaluation of LBSL patient stem cell derived cerebral organoids and neurons. We generated human cerebral organoids (hCOs) from induced pluripotent stem cells (iPSCs) of seven LBSL patients and three healthy controls using an unguided protocol. Single cells from 70-day-old hCOs underwent SMART-seq2 sequencing and multiple bioinformatic analysis tools were applied to high-resolution gene and transcript expression analyses. To confirm hCO findings, iPSC-derived neurons (iNs) were generated by overexpressing Neurogenin 2 using lentiviral vector to study neuronal growth, splicing of DARS2 exon 3 and DARS2 protein expression. Global gene expression analysis demonstrated dysregulation of a number of genes involved in mRNA metabolism and splicing processes within LBSL hCOs. Importantly, there were distinct and divergent gene expression profiles based on the nature of the DARS2 mutation. At the transcript level, pervasive differential transcript usage and differential spliced exon events that are involved in protein translation and metabolism were identified in LBSL hCOs. Single-cell analysis of DARS2 (exon 3) showed that some LBSL cells exclusively express transcripts lacking exon 3, indicating that not all LBSL cells can benefit from the "leaky" nature common to splice site mutations. Live cell imaging revealed neuronal growth defects of LBSL iNs, which was consistent with the finding of downregulated expression of genes related to neuronal differentiation in LBSL hCOs. DARS2 protein was downregulated in iNs compared to iPSCs, caused by increased exclusion of exon 3. At the gene- and transcript-level, we uncovered that dysregulated RNA splicing, protein translation and metabolism may underlie at least some of the pathophysiological mechanisms in LBSL. The scope and complexity of our data imply that DARS2 is potentially involved in transcription regulation beyond its canonical role of aminoacylation. Nevertheless, our work highlights transcript-level dysregulation as a critical, and relatively unexplored, mechanism linking genetic data with neurodegenerative disorders.

Oswald Susanne, Steinbruecker Katja, Achleitner Melanie, Goeschl Elisabeth, Bittner Reginald, Schmidt Wolfgang, Tiefenthaler Elke, Hammerl Emma, Eisl Anna, Mayr Doris, Mayr Johannes Adalbert, Wortmann Saskia B.

By loading transfer RNAs with their cognate amino acids, aminoacyl-tRNA synthetases (ARS) are essential for protein translation. Both cytosolic ARS1-deficiencies as well as mitochondrial ARS2-deficiencies can cause severe diseases. Amino acid supplementation has shown to positively influence the clinical course of four individuals with cytosolic ARS1-deficiencies. We hypothesize that this intervention could also benefit individuals with mitochondrial ARS2-deficiencies.

Chen Jia-Rong, Chen Chao, Chen Jie, Ji Yanchun, Lian Yanna, Zhang Juanjuan, Yu Jialing, Li Xiang-Yao, Qu Jia, Guan Min-Xin.

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted eye disease due to the degeneration of retinal ganglion cells (RGC). Mitochondrial 11778G > A mutation is the most common LHON-associated mitochondrial DNA (mtDNA) mutation. Our recent studies demonstrated some LHON families manifested by synergic interaction between m.11778G > A mutation and YARS2 allele (c.572G > T, p.Gly191Val) encoding mitochondrial tyrosyl-tRNA synthetase. However, the RGC-specific effects of LHON-associated mtDNA mutations remains elusive and there is no highly effective therapy for LHON. Here, we generated patients-derived induced pluripotent stem cells (iPSCs) from fibroblasts derived from a Chinese LHON family (both m.11778G > A and c.572G > T mutations, only m.11778G > A mutation, and control subject). The c.572G > T mutation in iPSC lines from a syndromic individual was corrected by CRISPR/Cas9. Those iPSCs were differentiated into neural progenitor cells (NPCs) and subsequently induced RGC-like cells using a stepwise differentiation procedure. Those RGC-like cells derived from symptomatic individual harboring both m.11778G > A and c.572G > T mutations exhibited greater defects in neuronal differentiation, morphology including reduced area of soma, numbers of neurites, and shortened length of axons, electrophysiological properties than those in cells bearing only m.11778G > A mutation. Furthermore, these RGC-like cells revealed more drastic reductions in oxygen consumption rates, levels of mitochondrial ATP and increasing productions of reactive oxygen species than those in other cell models. These mitochondrial dysfunctions promoted the apoptotic process for RGC degenerations. Correction of YARS2 c.572G > T mutation rescued deficiencies of patient-derived RGC-like cells. These findings provide new insights into pathophysiology of LHON arising from RGC-specific mitochondrial dysfunctions and step toward therapeutic intervention for this disease.

Kistol Denis, Tsygankova Polina, Krylova Tatiana, Bychkov Igor, Itkis Yulia, Nikolaeva Ekaterina, Mikhailova Svetlana, Sumina Maria, Pechatnikova Natalia, Kurbatov Sergey, Bostanova Fatima, Migiaev Ochir, Zakharova Ekaterina.

Leigh syndrome (LS), also known as infantile subacute necrotizing encephalopathy, is the most frequent mitochondrial disorder in children. Recently, more than 80 genes have been associated with LS, which greatly complicates the diagnosis. In this article, we present clinical and molecular findings of 219 patients with LS and give the detailed description of three cases with rare findings in nuclear genes MORC2, NARS2 and VPS13D, demonstrating wide genetic heterogeneity of this mitochondrial disease. The most common cause of LS in Russian patients are pathogenic variants in the SURF1 gene (44.3% of patients). The most frequent pathogenic variant is c.845_846delCT (66.0% of mutant alleles; 128/192), which is also widespread in Eastern Europe. Five main LS genes, SURF1, SCO2, MT-ATP6, MT-ND5 and PDHA1, account for 70% of all LS cases in the Russian Federation. Using next generation sequencing (NGS) technique, we were able to detect pathogenic variants in other nuclear genes: NDUFV1, NDUFS2, NDUFS8, NDUFAF5, NDUFAF6, NDUFA10, SUCLG1, GFM2, COX10, PMPCB, NARS2, PDHB and SLC19A3, including two genes previously associated with Leigh-like phenotypes-MORC2 and VPS13D. We found 49 previously undescribed nucleotide variants, including two deep intronic variants which affect splicing.

Panzeri Elena, Citterio Andrea, Martinuzzi Andrea, Ancona Vera, Martini Eleonora, Bassi Maria Teresa.

Defects in FARS2 are associated with either epileptic phenotypes or a spastic paraplegia subtype known as SPG77. Here, we describe an 8-year-old patient with severe and complicated spastic paraplegia, carrying a missense variant (p.Pro361Leu) and a novel intragenic deletion in FARS2. Of note, the disease is unexpectedly progressing rapidly and in a biphasic way differently from the previously reported cases. Our study provides the first detailed molecular characterization of a FARS2 deletion and its underlying molecular mechanism, and demonstrates the need for combining different tools to improve the diagnostic rate.

Abdulkareem Angham Abdulrhman, Zaman Qaiser, Khan Hamza, Khan Sabar, Rehman Gauhar, Tariq Nabeel, Ahmad Mashal, Owais Muhammad, Najumuddin , Muthaffar Osama Yousef, Bibi Fehmida, Khang Rin, Ryu Seung Woo, Naseer Muhammad Imran, Jelani Musharraf.

Introduction: Epilepsy is a group of neurological disorders characterized by recurring seizures and fits. The Epilepsy genes can be classified into four distinct groups, based on involvement of these genes in different pathways leading to Epilepsy as a phenotype. Genetically the disease has been associated with various pathways, leading to pure epilepsy-related disorders caused by CNTN2 variations, or involving physical or systemic issues along with epilepsy caused by CARS2 and ARSA, or developed by genes that are putatively involved in epilepsy lead by CLCN4 variations. Methods: In this study, five families of Pakistani origin (EP-01, EP-02, EP-04, EP-09, and EP-11) were included for molecular diagnosis. Results: Clinical presentations of these patients included neurological symptoms such as delayed development, seizures, regression, myoclonic epilepsy, progressive spastic tetraparesis, vision and hearing impairment, speech problems, muscle fibrillation, tremors, and cognitive decline. Whole exome sequencing in index patients and Sanger sequencing in all available individuals in each family identified four novel homozygous variants in genes CARS2: c.655G>A p.Ala219Thr (EP-01), ARSA: c.338T>C: p.Leu113Pro (EP-02), c.938G>T p.Arg313Leu (EP-11), CNTN2: c.1699G>T p.Glu567Ter (EP-04), and one novel hemizygous variant in gene CLCN4: c.2167C>T p.Arg723Trp (EP-09). Conclusion: To the best of our knowledge these variants were novel and had not been reported in familial epilepsy. These variants were absent in 200 ethnically matched healthy control chromosomes. Three dimensional protein analyses revealed drastic changes in the normal functions of the variant proteins. Furthermore, these variants were designated as "pathogenic" as per guidelines of American College of Medical Genetics 2015. Due to overlapping phenotypes, among the patients, clinical subtyping was not possible. However, whole exome sequencing successfully pinpointed the molecular diagnosis which could be helpful for better management of these patients. Therefore, we recommend that exome sequencing be performed as a first-line molecular diagnostic test in familial cases.

Gedikbasi Asuman, Toksoy Guven, Karaca Meryem, Gulec Cagri, Balci Mehmet Cihan, Gunes Dilek, Gunes Seda, Aslanger Ayca Dilruba, Unverengil Gokcen, Karaman Birsen, Basaran Seher, Demirkol Mubeccel, Gokcay Gulden Fatma, Uyguner Zehra Oya.

Background: Mitochondrial diseases are the most common group of inherited metabolic disorders, causing difficulties in definite diagnosis due to clinical and genetic heterogeneity. Clinical components are predominantly associated with pathogenic variants shown in nuclear or mitochondrial genomes that affect vital respiratory chain function. The development of high-throughput sequencing technologies has accelerated the elucidation of the genetic etiology of many genetic diseases that previously remained undiagnosed. Methods: Thirty affected patients from 24 unrelated families with clinical, radiological, biochemical, and histopathological evaluations considered for mitochondrial diseases were investigated. DNA isolated from the peripheral blood samples of probands was sequenced for nuclear exome and mitochondrial DNA (mtDNA) analyses. MtDNA sequencing was also performed from the muscle biopsy material in one patient. For segregation, Sanger sequencing is performed for pathogenic alterations in five other affected family members and healthy parents. Results: Exome sequencing revealed 14 different pathogenic variants in nine genes encoding mitochondrial function peptides (AARS2, EARS2, ECHS1, FBXL4, MICOS13, NDUFAF6, OXCT1, POLG, and TK2) in 12 patients from nine families and four variants in genes encoding important for muscle structure (CAPN3, DYSF, and TCAP) in six patients from four families. Three probands carried pathogenic mtDNA variations in two genes (MT-ATP6 and MT-TL1). Nine variants in five genes are reported for the first time with disease association: (AARS2: c.277C>T/p.(R93*), c.845C>G/p.(S282C); EARS2: c.319C>T/p.(R107C), c.1283delC/p.(P428Lfs*); ECHS1: c.161G>A/p.(R54His); c.202G>A/p.(E68Lys); NDUFAF6: c.479delA/p.(N162Ifs*27); and OXCT1: c.1370C>T/p.(T457I), c.1173-139G>T/p.(?). Conclusion: Bi-genomic DNA sequencing clarified genetic etiology in 67% (16/24) of the families. Diagnostic utility by mtDNA sequencing in 13% (3/24) and exome sequencing in 54% (13/24) of the families prioritized searching for nuclear genome pathologies for the first-tier test. Weakness and muscle wasting observed in 17% (4/24) of the families underlined that limb-girdle muscular dystrophy, similar to mitochondrial myopathy, is an essential point for differential diagnosis. The correct diagnosis is crucial for comprehensive genetic counseling of families. Also, it contributes to making treatment-helpful referrals, such as ensuring early access to medication for patients with mutations in the TK2 gene.

Kazakova Ekaterina, Téllez-Martínez José Alberto, Flores-Lagunes Leonardo, Sosa-Ortiz Ana Luisa, Carillo-Sánchez Karol, Molina-Garay Carolina, González-Domínguez Carlos Alberto, Jimenez-Olivares Marco, Fernandez-Valverde Francisca, Vargas-Cañas Edwin Steven, Vázquez-Memije Martha Elisa, Garcia-Latorre Ethel Awilda, Martínez-Duncker Iván, Alaez-Verson Carmen.

To report the first Mexican case with two novel AARS2 mutations causing primary ovarian failure, uterus infantilis, and early-onset dementia secondary to leukoencephalopathy.

Ait-El-Mkadem Saadi Samira, Kaphan Elsa, Morales Jaurrieta Amaya, Fragaki Konstantina, Chaussenot Annabelle, Bannwarth Sylvie, Maues De Paula André, Paquis-Flucklinger Véronique, Rouzier Cécile.

Biallelic rare variants in NARS2 that encode the mitochondrial asparaginyl-tRNA synthetase are associated with a wide spectrum of clinical phenotypes ranging from severe neurodegenerative disorders to isolated mitochondrial myopathy or deafness. To date, only a small number of patients with NARS2 variants have been reported, and possible genotype-phenotype correlations are still lacking. Here, we present three siblings who had an early-onset hearing loss, while one developed severe symptoms in adulthood associated with early intellectual impairment, refractory seizures, moderate axonal sensorimotor neuropathy, and atypical psychiatric symptoms. Biochemical analysis revealed impairment of the activity and assembly of the respiratory chain complexes in this patient's muscle and fibroblasts. Whole Exome Sequencing allowed identification of a heterozygous variant NM_024678.5(NARS2):c.822G > C (p.Gln274His) that is known to be pathogenic and to affect splicing of the NARS2 gene, but was unable to detect a second variant in this gene. Coverage analysis and Sanger sequencing led to identification of a novel intronic deletion NM_024678.5(NARS2):c.922-21_922-19del in the three siblings in trans with the c.822G > C. Functional analysis by RT-PCR showed that this deletion was causing aberrant splicing and led to exon 9 skipping in NARS2 mRNA in patient fibroblasts. Our work expands the phenotype and genotype spectrum of NARS2-related disorders. We provide evidence of the pathogenic effect of a novel intronic deletion in the NARS2 gene and report on additional adult patients with a large intrafamilial variability associated with splice variants in this gene. More specifically, we detail the phenotype of the oldest living patient to date with NARS2 variants and, for the first time, we report the psychiatric symptoms associated with this gene. Our work confirms the complexity of genotype-phenotype correlation in patients with pathogenic NARS2 variants.

Neyroud Anne Sophie, Rudinger-Thirion Joëlle, Frugier Magali, Riley Lisa G, Bidet Maud, Akloul Linda, Simpson Andrea, Gilot David, Christodoulou John, Ravel Célia, Sinclair Andrew H, Belaud-Rotureau Marc-Antoine, Tucker Elena J, Jaillard Sylvie.

Premature ovarian insufficiency (POI) affects 1 in 100 women and is a leading cause of female infertility. There are over 80 genes in which variants can cause POI, with these explaining only a minority of cases. Whole exome sequencing (WES) can be a useful tool for POI patient management, allowing clinical care to be personalized to underlying cause. We performed WES to investigate two French sisters, whose only clinical complaint was POI. Surprisingly, they shared one known and one novel likely pathogenic variant in the Perrault syndrome gene, LARS2. Using amino-acylation studies, we established that the novel missense variant significantly impairs LARS2 function. Perrault syndrome is characterized by sensorineural hearing loss in addition to POI. This molecular diagnosis alerted the sisters to the significance of their difficulty in following conversation. Subsequent audiology assessment revealed a mild bilateral hearing loss. We describe the first cases presenting with perceived isolated POI and causative variants in a Perrault syndrome gene. Our study expands the phenotypic spectrum associated with LARS2 variants and highlights the clinical benefit of having a genetic diagnosis, with prediction of potential co-morbidity and prompt and appropriate medical care, in this case by an audiologist for early detection of hearing loss.

Del Greco Christina, Antonellis Anthony.

Aminoacyl-tRNA synthetases (ARSs) are highly conserved essential enzymes that charge tRNA with cognate amino acids-the first step of protein synthesis. Of the 37 nuclear-encoded human ARS genes, 17 encode enzymes are exclusively targeted to the mitochondria (mt-ARSs). Mutations in nuclear mt-ARS genes are associated with rare, recessive human diseases with a broad range of clinical phenotypes. While the hypothesized disease mechanism is a loss-of-function effect, there is significant clinical heterogeneity among patients that have mutations in different mt-ARS genes and also among patients that have mutations in the same mt-ARS gene. This observation suggests that additional factors are involved in disease etiology. In this review, we present our current understanding of diseases caused by mutations in the genes encoding mt-ARSs and propose explanations for the observed clinical heterogeneity.

Jauss Robin-Tobias, Schließke Sophia, Abou Jamra Rami.

Routine diagnostics is biased towards genes and variants with satisfactory evidence, but rare disorders with only little confirmation of their pathogenicity might be missed. Many of these genes can, however, be considered relevant, although they may have less evidence because they lack OMIM entries or comprise only a small number of publicly available variants from one or a few studies. Here, we present 89 individuals harbouring variants in 77 genes for which only a small amount of public evidence on their clinical significance is available but which we still found to be relevant enough to be reported in routine diagnostics. For 21 genes, we present case reports that confirm the lack or provisionality of OMIM associations (ATP6V0A1, CNTN2, GABRD, NCKAP1, RHEB, TCF7L2), broaden the phenotypic spectrum (CC2D1A, KCTD17, YAP1) or substantially strengthen the confirmation of genes with limited evidence in the medical literature (ADARB1, AP2M1, BCKDK, BCORL1, CARS2, FBXO38, GABRB1, KAT8, PRKD1, RAB11B, RUSC2, ZNF142). Routine diagnostics can provide valuable information on disease associations and support for genes without requiring tremendous research efforts. Thus, our results validate and delineate gene-disorder associations with the aim of motivating clinicians and scientists in diagnostic departments to provide additional evidence via publicly available databases or by publishing short case reports.

Cokyaman Turgay, Cetin Huriye, Dogan Durmus, Silan Fatma.

NARS2 mutations are known to cause various clinical phenotypes such as nonsyndromic hearing loss, Leigh/Alpers syndrome, refractory epilepsy, developmental delay, intellectual disability and myopathy. We presented the first Turkish variant of NASR2 and added type 1 diabetes mellitus (DM), which was not previously described in the phenotype spectrum of this disease. A 4.5-month-old girl presented with hearing loss, hypotonia, refractory myoclonic epilepsy, severe developmental delay and large subdural hemorrhage. In the first year of the follow-up, type 1 DM developed. A homozygous missense mutation, [c.500 A>G, p.H167R] in the NARS2 gene was detected in the trio-based whole-exome sequencing (WES). In this disease, in addition to multi-organ involvement, type 1 DM may also develop, as in our case. Since it is a mitochondrial disease, the decision to treat with valproic acid should be reconsidered. The long diagnostic process can be shortened with WES.

Yu Tingting, Zhang Yi, Zheng Wen-Qiang, Wu Siqi, Li Guoqiang, Zhang Yong, Li Niu, Yao Ruen, Fang Pengfei, Wang Jian, Zhou Xiao-Long.

Mitochondrial translation is of high significance for cellular energy homeostasis. Aminoacyl-tRNA synthetases (aaRSs) are crucial translational components. Mitochondrial aaRS variants cause various human diseases. However, the pathogenesis of the vast majority of these diseases remains unknown. Here, we identified two novel SARS2 (encoding mitochondrial seryl-tRNA synthetase) variants that cause a multisystem disorder. c.654-14T > A mutation induced mRNA mis-splicing, generating a peptide insertion in the active site; c.1519dupC swapped a critical tRNA-binding motif in the C-terminus due to stop codon readthrough. Both mutants exhibited severely diminished tRNA binding and aminoacylation capacities. A marked reduction in mitochondrial tRNASer(AGY) was observed due to RNA degradation in patient-derived induced pluripotent stem cells (iPSCs), causing impaired translation and comprehensive mitochondrial function deficiencies. These impairments were efficiently rescued by wild-type SARS2 overexpression. Either mutation caused early embryonic fatality in mice. Heterozygous mice displayed reduced muscle tissue-specific levels of tRNASers. Our findings elucidated the biochemical and cellular consequences of impaired translation mediated by SARS2, suggesting that reduced abundance of tRNASer(AGY) is a key determinant for development of SARS2-related diseases.

Al-Sharif Fawzia, Alsadeq Hussain, Rozan Aahid, Halabi Molham B, Badwilan Hamzah, Mohammed Adel A, Rahman Moshiur, Balgith Turki.

Mitochondrial diseases disrupt the process of energy generation by the mitochondria, leading to manifestations that can affect almost any organ in the body. Although various possible clinical phenotypes can result, neurological and neuromuscular affection is most frequently encountered. NARS2 encodes an enzyme responsible for the conjugation of asparagine to its cognate mitochondrial transfer ribonucleic acid (tRNA) molecule, representing an essential step necessary for effective mitochondrial protein synthesis. As such, mutations in this gene can lead to poor mitochondrial gene expression and, consequently, poor energy output resulting in disease. Pathogenic variants in NARS2 have been known to cause neurodegenerative and myopathic syndromes in combined oxidative phosphorylation deficiency 24 (COXPD24). However, nonsyndromic autosomal recessive deafness 94 (DFNB94), with which only one family is known to be affected, has also been reported concerning NARS2. Our report demonstrates the association of a new pathogenic variant in mitochondrial asparaginyl-tRNA synthetase (NARS2) with nonsyndromic sensorineural hearing loss, thus confirming biallelic mutations in NARS2 as a cause of nonsyndromic deafness.

He Peiqing, Wang Qingming, Hong Xiaochun, Yuan Haiming.

Biallelic pathogenic variants in the TARS2 gene cause combined oxidative phosphorylation deficiency, subtype 21 (COXPD21, MIM #615918), which is a rare mitochondrial encephalomyopathy (ME) characterized by early-onset severe axial hypotonia, limb hypertonia, delayed psychomotor development, epilepsy, and brain anomalies. Currently, eight COXPD21 patients have been reported in the literature, and 11 pathogenic variants in TARS2 have been identified. Here, we report a 2-year-6-month-old Chinese female who presented with severe dystonia, developmental regression, absent speech, and intractable epilepsy. Laboratory examination showed persistently increased serum lactate. Brain MRI showed that the head of the caudate and partial lenticular nucleus were bilateral symmetrical T2-weighted imaging (T2WI) hyperintense and the corpus callosum was very thin. The clinical characteristics pointed to a ME. Trio-based whole-exome sequencing (WES) was employed to detect the causative variants. WES revealed novel compound heterozygous variants, c.470G>C (p.Thr157Arg) and c.2051C>T (p.Arg684Gln), in TARS2 in our patient that were inherited from the mother and father, respectively. Next, we systematically reviewed the available clinical features of COXPD21 patients and noticed that the reduced fetal movement observed in our patient may be a novel phenotype of COXPD21. These findings expand the mutation spectrum of TARS2 and provide insights into the genotype-phenotype relationship in COXPD21 as well as a foundation for its genetic counseling, diagnosis and treatment.

Stutterd C A, Vanderver A, Lockhart P J, Helman G, Pope K, Uebergang E, Love C, Delatycki M B, Thorburn D, Mackay M T, Peters H, Kornberg A J, Patel C, Rodriguez-Casero V, Waak M, Silberstein J, Sinclair A, Nolan M, Field M, Davis M R, Fahey M, Scheffer I E, Freeman J L, Wolf N I, Taft R J, van der Knaap M S, Simons C, Leventer R J.

Next generation sequencing studies have revealed an ever-increasing number of causes for genetic disorders of central nervous system white matter. A substantial number of disorders are identifiable from their specific pattern of biochemical and/or imaging findings for which single gene testing may be indicated. Beyond this group, the causes of genetic white matter disorders are unclear and a broader approach to genomic testing is recommended.

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Hereditary spastic paraplegia type 4 is extremely variable in age at onset; the same variant can cause onset at birth or in the eighth decade. We recently discovered that missense variants in SPAST, which influences microtubule dynamics, are associated with earlier onset and more severe disease than truncating variants, but even within the early and late-onset groups there remained significant differences in onset. Given the rarity of the condition, we adapted an extreme phenotype approach to identify genetic modifiers of onset.

Gao Xin, Xin Guoyan, Tu Ya, Liang Xiaoping, Yang Huimin, Meng Hong, Wang Yumin.

To explore the clinical characteristics and genetic characteristics of the combined oxidative phosphorylation defect type 21 (COXPD21) caused by the TARS2 compound heterozygous varians, and to improve clinicians' awareness of the disease.

Li Liangshan, Ma Jianhua, Wang Jingli, Dong Liping, Liu Shiguo.

As a rare mitochondrial disease, combined oxidative phosphorylation deficiency 14 (COXPD14) is caused by biallelic variants in the phenylalanyl-tRNA synthetase 2, mitochondrial gene (FARS2) with clinical features of developmental delay, an elevated lactate level, early-onset encephalopathy, liver failure, and hypotonia. The objectives of this study were to analyze the clinical and molecular features of two Chinese siblings affected with COXPD14, and to review relevant literature.

Wu Xiao-Hui, Lin Shuang-Zhu, Zhou Yan-Qiu, Wang Wan-Qi, Li Jia-Yi, Chen Qian-Dui.

The mitochondrial respiratory chain defects have become the most common cause of neurometabolic disorders in children and adults, which can occur at any time in life, often associated with neurological dysfunction, and lead to chronic disability and premature death. Approximately one-third of patients with mitochondrial disease have biochemical defects involving multiple respiratory chain complexes, suggesting defects in protein synthesis within the mitochondria. We here report a child with VARS2 gene mutations causing mitochondrial disease.

Chen Xihui, Liu Fangfang, Li Bowen, Wang Yufeng, Yuan Lijuan, Yin Anan, Chen Qi, Hu Weihong, Yao Yan, Zhang Mengjie, Wu YuanMing, Chen Kun.

Neurodegenerative diseases encompass an extensive and heterogeneous group of nervous system disorders which are characterized by progressive degeneration and death of neurons. Many lines of evidence suggest the participation of mitochondria dysfunction in these diseases. Mitochondrial phenylalanyl-tRNA synthetase, encoded by FARS2, catalyzes the transfer of phenylalanine to its cognate tRNA for protein synthesis. As a member of mt-aaRSs genes, FARS2 missense homozygous mutation c.424G > T (p.D142Y) found in a Chinese consanguineous family first built the relationship between pure hereditary spastic paraplegia (HSP) and FARS2 gene. More FARS2 variations were subsequently found to cause heterogeneous group of neurologic disorders presenting three main phenotypic manifestations: infantile-onset epileptic mitochondrial encephalopathy, later-onset spastic paraplegia and juvenile onset refractory epilepsy. Studies showed that aminoacylation activity is frequently disrupt in cases with FARS2 mutations, indicating a loss-of-function mechanism. However, the underlying pathogenesis of neuropathy-associated Fars2 deficiency is still largely unknown.

Fan Yan, Han Jinming, Yang Yanyan, Chen Tuanzhi.

Missense mutations in the mitochondrial alanyl-tRNA synthetase 2 (AARS2) gene are clinically associated with infantile mitochondrial cardiomyopathy or adult-onset leukoencephalopathy with early ovarian failure. To date, approximately 40 cases have been reported related to AARS2 mutations, while its genetic and phenotypic spectrum remains to be defined.

Ferrer Isidro.

Primary microglial leukodystrophy or leukoencephalopathy are disorders in which a genetic defect linked to microglia causes cerebral white matter damage. Pigmented orthochromatic leukodystrophy, adult-onset orthochromatic leukodystrophy associated with pigmented macrophages, hereditary diffuse leukoencephalopathy with (axonal) spheroids, and adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) are different terms apparently used to designate the same disease. However, ALSP linked to dominantly inherited mutations in CSF1R (colony stimulating factor receptor 1) cause CSF-1R-related leukoencephalopathy (CRP). Yet, recessive ALSP with ovarian failure linked to AARS2 (alanyl-transfer (t)RNA synthase 2) mutations (LKENP) is a mitochondrial disease and not a primary microglial leukoencephalopathy. Polycystic membranous lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL; Nasu-Hakola disease: NHD) is a systemic disease affecting bones, cerebral white matter, selected grey nuclei, and adipose tissue The disease is caused by mutations of one of the two genes TYROBP or TREM2, identified as PLOSL1 and PLOSL2, respectively. TYROBP associates with receptors expressed in NK cells, B and T lymphocytes, dendritic cells, monocytes, macrophages, and microglia. TREM2 encodes the protein TREM2 (triggering receptor expressed on myeloid cells 2), which forms a receptor signalling complex with TYROBP in macrophages and dendritic cells. Rather than pure microglial leukoencephalopathy, NHD can be considered a multisystemic "immunological" disease.

Kayvanpour Elham, Wisdom Michael, Lackner Maximilian K, Sedaghat-Hamedani Farbod, Boeckel Jes-Niels, Müller Marion, Eghbalian Rose, Dudek Jan, Doroudgar Shirin, Maack Christoph, Frey Norbert, Meder Benjamin.

Mutations in mitochondrial aminoacyl-tRNA synthetases (mtARSs) have been reported in patients with mitochondriopathies: most commonly encephalopathy, but also cardiomyopathy. Through a GWAS, we showed possible associations between mitochondrial valyl-tRNA synthetase (VARS2) dysregulations and non-ischemic cardiomyopathy. We aimed to investigate the possible consequences of VARS2 depletion in zebrafish and cultured HEK293A cells. Transient VARS2 loss-of-function was induced in zebrafish embryos using Morpholinos. The enzymatic activity of VARS2 was measured in VARS2-depleted cells via northern blot. Heterozygous VARS2 knockout was established in HEK293A cells using CRISPR/Cas9 technology. BN-PAGE and SDS-PAGE were used to investigate electron transport chain (ETC) complexes, and the oxygen consumption rate and extracellular acidification rate were measured using a Seahorse XFe96 Analyzer. The activation of the integrated stress response (ISR) and possible disruptions in mitochondrial fatty acid oxidation (FAO) were explored using RT-qPCR and western blot. Zebrafish embryos with transient VARS2 loss-of-function showed features of heart failure as well as indications of CNS and skeletal muscle involvements. The enzymatic activity of VARS2 was significantly reduced in VARS2-depleted cells. Heterozygous VARS2-knockout cells showed a rearrangement of ETC complexes in favor of complexes III2, III2 + IV, and supercomplexes without significant respiratory chain deficiencies. These cells also showed the enhanced activation of the ISR, as indicated by increased eIF-2α phosphorylation and a significant increase in the transcript levels of ATF4, ATF5, and DDIT3 (CHOP), as well as disruptions in FAO. The activation of the ISR and disruptions in mitochondrial FAO may underlie the adaptive changes in VARS2-depleted cells.

Tanaka Ryosuke, Takeguchi Ryo, Kuroda Mami, Suzuki Nao, Makita Yoshio, Yanagi Kumiko, Kaname Tadashi, Takahashi Satoru.

Leigh syndrome is the most genetically heterogenous phenotype of mitochondrial disease. We describe a patient with Leigh syndrome whose diagnosis had not been confirmed because of normal metabolic screening results at the initial presentation. Whole-exome sequencing identified pathogenic variants in NARS2, the gene encoding a mitochondrial asparaginyl-tRNA synthetase. One of the biallelic variants was novel. This highlights the essential role of genetic testing for a definite diagnosis of Leigh syndrome.

Tragni Vincenzo, Primiano Guido, Tummolo Albina, Cafferati Beltrame Lucas, La Piana Gianluigi, Sgobba Maria Noemi, Cavalluzzi Maria Maddalena, Paterno Giulia, Gorgoglione Ruggiero, Volpicella Mariateresa, Guerra Lorenzo, Marzulli Domenico, Servidei Serenella, De Grassi Anna, Petrosillo Giuseppe, Lentini Giovanni, Pierri Ciro Leonardo.

Mitochondrial diseases (MDs) may result from mutations affecting nuclear or mitochondrial genes, encoding mitochondrial proteins, or non-protein-coding mitochondrial RNA. Despite the great variability of affected genes, in the most severe cases, a neuromuscular and neurodegenerative phenotype is observed, and no specific therapy exists for a complete recovery from the disease. The most used treatments are symptomatic and based on the administration of antioxidant cocktails combined with antiepileptic/antipsychotic drugs and supportive therapy for multiorgan involvement. Nevertheless, the real utility of antioxidant cocktail treatments for patients affected by MDs still needs to be scientifically demonstrated. Unfortunately, clinical trials for antioxidant therapies using α-tocopherol, ascorbate, glutathione, riboflavin, niacin, acetyl-carnitine and coenzyme Q have met a limited success. Indeed, it would be expected that the employed antioxidants can only be effective if they are able to target the specific mechanism, i.e., involving the central and peripheral nervous system, responsible for the clinical manifestations of the disease. Noteworthily, very often the phenotypes characterizing MD patients are associated with mutations in proteins whose function does not depend on specific cofactors. Conversely, the administration of the antioxidant cocktails might determine the suppression of endogenous oxidants resulting in deleterious effects on cell viability and/or toxicity for patients. In order to avoid toxicity effects and before administering the antioxidant therapy, it might be useful to ascertain the blood serum levels of antioxidants and cofactors to be administered in MD patients. It would be also worthwhile to check the localization of mutations affecting proteins whose function should depend (less or more directly) on the cofactors to be administered, for estimating the real need and predicting the success of the proposed cofactor/antioxidant-based therapy.

Sevinç Selin, İnci Aslı, Ezgü Fatih S, Eminoğlu Fatma T.

Pontocerebellar hypoplasia (PCH) is a heterogeneous neurodevelopmental disorder that is characterized by decreased brainstem and cerebellum volume. Pontocerebellar hypoplasia type 6 (PCH6) is a mitochondrial disease associated with autosomal recessive inheritance that results from mutations in the RARS2 gene. In this case report, we describe a new clinical presentation with a novel RARS2 pathogenic variant. We report here on 2 siblings who presented with neonatal lactic acidosis, microcephaly, growth retardation, persistent seizures, and cholestasis with a previously undefined RARS2 pathogenic variant. In our literature review, we evaluated the clinical features and pathogenic variants of 34 patients reported in 16 publications since the initial identification of RARS2 pathogenic variants in PCH6 in 2007. Both siblings were detected with c.1564G>A (p.Val522Ile), a novel homozygous pathogenic variant of the RARS2 gene. Imaging revealed advanced cerebral atrophy and cerebellar hypoplasia, while the basal ganglia and pons were preserved. At follow-up, the elevations in liver function test results and cholestasis had regressed while the LDH and GGT elevations persisted. Both siblings showed microcephaly on follow-up and started to suffer seizures. Severe developmental delay and nutritional problems were observed, and both died in infancy. RARS2 pathogenic variant is a mitochondrial disease that causes severe mental, motor, and developmental retardation, as well as short life expectancy. Our patients are the first cases with liver involvement in PCH6 and a novel homozygous RARS2 pathogenic variant to be reported in the literature. This additional phenotype can be considered as making a valid contribution to the literature.

Zhang Yi, Zhao Xiangyue, Xu Yufei, Chen Lina, Li Niu, Yao Ruen, Wang Xiumin, Wang Jian, Yu Tingting.

NARS2 catalyzes the attachment of asparagine amino acids to mitochondrial tRNAAsn and is critical for efficient mitochondrial protein synthesis. Biallelic variants in NARS2 are associated with combined oxidative phosphorylation deficiency 24 (COXPD24) and autosomal recessive deafness-94.

Upadia Jariya, Li Yuwen, Walano Nicolette, Deputy Stephen, Gajewski Kelly, Andersson Hans C.

Isoleucyl-tRNA synthetase 2 (IARS2) encodes mitochondrial isoleucine-tRNA synthetase. Pathogenic variants in the IARS2 gene are associated with mitochondrial disease. We report a female with IARS2 compound heterozygous variants, p.Val499Glyfs*14 and p.Arg784Trp who presented with infantile spasms, Leigh disease and Wolff-Parkinson White (WPW) pattern. This report expands the phenotypic spectrum of IARS2-related disease.

Zhang Xiao, Li Jie, Zhang Yanyan, Gao Meina, Peng Tao, Tian Tian.

Mutations in the alanyl-transfer RNA synthase 2 (AARS2) represent a heterogenous group of autosomal recessive leukodystrophy characterized by cognitive decline, ataxia, spasticity, and Parkinsonism. AARS2-related leukodystrophy (AARS2-L) is extremely rare. To date, only 45 genetically confirmed cases, explaining the frequent diagnostic delay. Here, we report a 21-year-old male presented with unsteady gait and weakness in the bilateral lower extremities. Examination revealed dysarthria, cerebellar ataxia, paraparesis, and Parkinsonism with generalized hyperreflexia. MRI findings showed extensive white matter lesions in bilateral frontoparietal lobes, immediate periventricular regions, and corpus callosum. Focused exome sequencing revealed compound heterozygous mutations in the AARS2 gene confirming the diagnosis of AARS2-L; two heterogeneous missense mutations (c.452 T > C, p. M151T; c. 2557C > T, p. R853W) appeared together for the first time. We also reviewed phenotypic spectra of AARS2-related leukodystrophies from a total of 45 reported cases.

Buonfiglio Paula I, Bruque Carlos D, Lotersztein Vanesa, Luce Leonela, Giliberto Florencia, Menazzi Sebastián, Francipane Liliana, Paoli Bibiana, Goldschmidt Ernesto, Elgoyhen Ana Belén, Dalamón Viviana.

Hearing loss is a heterogeneous disorder. Identification of causative mutations is demanding due to genetic heterogeneity. In this study, we investigated the genetic cause of sensorineural hearing loss in patients with severe/profound deafness. After the exclusion of GJB2-GJB6 mutations, we performed whole exome sequencing in 32 unrelated Argentinean families. Mutations were detected in 16 known deafness genes in 20 patients: ACTG1, ADGRV1 (GPR98), CDH23, COL4A3, COL4A5, DFNA5 (GSDDE), EYA4, LARS2, LOXHD1, MITF, MYO6, MYO7A, TECTA, TMPRSS3, USH2A and WSF1. Notably, 11 variants affecting 9 different non-GJB2 genes resulted novel: c.12829C > T, p.(Arg4277*) in ADGRV1; c.337del, p.(Asp109*) and c.3352del, p.(Gly1118Alafs*7) in CDH23; c.3500G > A, p.(Gly1167Glu) in COL4A3; c.1183C > T, p.(Pro395Ser) and c.1759C > T, p.(Pro587Ser) in COL4A5; c.580 + 2 T > C in EYA4; c.1481dup, p.(Leu495Profs*31) in LARS2; c.1939 T > C, p.(Phe647Leu), in MYO6; c.733C > T, p.(Gln245*) in MYO7A and c.242C > G, p.(Ser81*) in TMPRSS3 genes. To predict the effect of these variants, novel protein modeling and protein stability analysis were employed. These results highlight the value of whole exome sequencing to identify candidate variants, as well as bioinformatic strategies to infer their pathogenicity.

Ilinca Andreea, Kafantari Efthymia, Puschmann Andreas.

Finsterer J.

Rumyantseva Anastasia, Popovic Milica, Trifunovic Aleksandra.

Mitochondria are essential organelles found in every eukaryotic cell, required to convert food into usable energy. Therefore, it is not surprising that mutations in either mtDNA or nuclear DNA-encoded genes of mitochondrial proteins cause diseases affecting the oxidative phosphorylation system, which are heterogeneous from a clinical, genetic, biochemical and molecular perspective and can affect patients at any age. Despite all this, it is surprising that our understanding of the mechanisms governing mitochondrial gene expression and its associated pathologies remain superficial and therapeutic interventions largely unexplored. We recently showed that loss of the mitochondrial matrix protease caseinolytic protease proteolytic subunit (CLPP) ameliorates phenotypes in cells characterized by defects in oxidative phosphorylation maintenance. Here, we build upon this finding by showing that CLPP depletion is indeed beneficial in vivo for various types of neuronal populations, including Purkinje cells in the cerebellum and cortical and hippocampal neurons in the forebrain, as it strongly improves distinct phenotypes of mitochondria encephalopathy, driven by the deficiency of the mitochondrial aspartyl tRNA synthase DARS2. In the absence of CLPP, neurodegeneration of DARS2-deficient neurons is delayed as they present milder oxidative phosphorylation dysfunction. This in turn leads to a decreased neuroinflammatory response and significantly improved motor functions in both double-deficient models (Purkinje cell-specific or forebrain neuron-specific Dars2/Clpp double knockout mice). We propose that diminished turnover of respiratory complex I caused by the loss of CLPP is behind the improved phenotype in Dars2/Clpp double knockout animals, even though this intervention might not restore respiratory complex I activity but rather improve mitochondrial cristae morphology or help maintain the NAD+/NADH ratio inside mitochondria. These results also open the possibility of targeting CLPP activity in many other mitochondrial encephalopathies characterized by respiratory complex I instability.

Liu Min, Liang Yue, Huang Bixue, Sun Jincangjian, Chen Kaitian.

Many hearing-impaired patients carry mutations in rare or novel genes undetected in regular genetic hot regions/genes screening.

Yalcouyé Abdoulaye, Esoh Kevin, Guida Landouré, Wonkam Ambroise.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy characterised by a high clinical and genetic heterogeneity. While most cases were described in populations with Caucasian ancestry, genetic research on CMT in Africa is scant. Only a few cases of CMT have been reported, mainly from North Africa. The current study aimed to summarise available data on CMT in Africa, with emphasis on the epidemiological, clinical, and genetic features.

Zhang Yi, Yu Yafen, Zhao Xiangyue, Xu Yufei, Chen Lina, Li Niu, Yao Ruen, Wang Jian, Yu Tingting.

Pontocerebellar hypoplasia type 6 (PCH6) is an early-onset encephalopathy with/without mitochondrial respiratory complex defects caused by recessive mutations in mitochondrial arginyl-tRNA synthetase (RARS2). Highly heterogeneous clinical phenotypes and numerous missense variations of uncertain significance make diagnosis difficult. Pathogenesis of PCH6 remains unclear.

Rudaks Laura I, Watson Eloise, Oboudiyat Carly, Kumar Kishore R, Sullivan Patricia, Cowley Mark J, Davis Ryan L, Sue Carolyn M.

Myopathy, lactic acidosis, and sideroblastic anemia 2 (MLASA2) is an autosomal recessive mitochondrial disorder caused by pathogenic variants in YARS2. YARS2 variants confer heterogeneous phenotypes ranging from the full MLASA syndrome to a clinically unaffected state. Symptom onset is most common in the first decade of life but can occur in adulthood and has been reported following intercurrent illness. Early death can result from respiratory muscle weakness and cardiomyopathy. We report a case of MLASA2 with compound heterozygous YARS2 pathogenic variants; a known pathogenic nonsense variant [NM_001040436.3:c.98C>A (p.Ser33Ter)] and a likely pathogenic missense variant not previously associated with disease [NM_001040436.3:c.948G>T (p.Arg316Ser)]. The proband initially presented with a relatively mild phenotype of myopathy and lactic acidosis. During pregnancy, anemia emerged as an additional feature and in the postpartum period she experienced severe decompensation of cardiorespiratory function. This is the first reported case of pregnancy-related complications in a patient with YARS2-related mitochondrial disease. This case highlights the need for caution and careful counseling when considering pregnancy in mitochondrial disease, due to the risk of disease exacerbation and pregnancy complications.

Yagasaki Hideaki, Sano Fumikazu, Narusawa Hiromune, Watanabe Daisuke, Kaga Yoshimi, Kobayashi Koji, Asano Yoshihiro, Nagata Miho, Yonei Ayumi, Inukai Takeshi.

Neonatal diabetes mellitus (NDM) with developmental delay and epilepsy is classified as developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome. The majority of DEND syndrome are due to severely damaging variants of K-ATP channels, and few mitochondria-related genes have been reported. We report here two Japanese siblings who were clinically diagnosed with DEND syndrome in whom NARS2 compound heterozygous variants were detected. Patient 1 was a 3-year-old girl and presented with diabetes ketoacidosis at 3 months old. Patient 2 was a 1-year-old boy who presented with severe hyperglycemia and started insulin therapy at 3 days old. After the first episodes, they both presented with severe developmental delay, hearing loss and treatment-resistant epilepsy accompanied by progressive brain atrophy. Whole-exome sequencing revealed compound heterozygous NARS2 p.R159C and p.L217V variants, and the GATA4 p.P407Q variant in both patients. They were treated by mitochondrial supportive therapy of vitamin B1, L-carnitine, and coenzyme Q10. Patient 2 was withdrawn from insulin therapy at 6 months old. This is the first report of NDM in which variants of the NARS2 gene coding mitochondrial protein were detected. Genetic analysis including mitochondrial genes should be considered in patients with neonatal onset diabetes associated with neurogenic symptoms.

Wongkittichote Parith, Magistrati Martina, Shimony Joshua S, Smyser Christopher D, Fatemi Seyed Ali, Fine Amena S, Bellacchio Emanuele, Dallabona Cristina, Shinawi Marwan.

Biallelic pathogenic variants in the nuclear gene DARS2 (MIM# 610956), encoding the mitochondrial enzyme aspartyl-tRNA synthetase (MT-ASPRS) cause leukoencephalopathy with Brain Stem and Spinal Cord Involvement and Lactate Elevation (LBSL) (MIM# 611105), a neurometabolic disorder characterized by progressive ataxia, spasticity, developmental arrest or regression and characteristic brain MRI findings. Most patients exhibit a slowly progressive disease course with motor deterirartion that begins in childhood or adolescence, but can also occasionaly occur in adulthood. More severe LBSL presentations with atypical brain MRI findings have been recently described. Baker's yeast orthologue of DARS2, MSD1, is required for growth on oxidative carbon sources. A yeast with MSD1 knockout (msd1Δ) demonstrated a complete lack of oxidative growth which could be rescued by wild-type MSD1 but not MSD1 with pathogenic variants. Here we reported two siblings who exhibited developmental regression and ataxia with different age of onset and phenotypic severity. Exome sequencing revealed 2 compound heterozygous missense variants in DARS2: c.473A>T (p.Glu158Val) and c.829G>A (p.Glu277Lys); this variant combination has not been previously reported. The msd1Δ yeast transformed with plasmids expressing p.Glu259Lys, equivalent to human p.Glu277Lys, showed complete loss of oxidative growth and oxygen consumption, while the strain carrying p.Gln137Val, equivalent to human p.Glu158Val, showed a significant reduction of oxidative growth, but a residual ability to grow was retained. Structural analysis indicated that p.Glu158Val may interfere with protein binding of tRNAAsp, while p.Glu277Lys may impact both homodimerization and catalysis of MT-ASPRS. Our data illustrate the utility of yeast model and in silico analysis to determine pathogenicity of DARS2 variants, expand the genotypic spectrum and suggest intrafamilial variability in LBSL.

Zhang Anli, Lu Jieping, Xiao Fang.

Leucoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) is commonly induced by DARS2 abnormalities and accompanied by slowly progressing pyramidal and cerebellar dysfunction, as well as concomitant dorsal column dysfunction. In this study, an LBSL induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells of a female patient carrying biallelic mutations in DARS2. Pluripotency, differentiation potential, and karyotypic normality of this cell line were confirmed. This iPSC line offers a useful cellular model to investigate LBSL phenotypes, mechanisms, and therapy.

Feng Shujun, Wan Shan, Liu Shuangying, Wang Wei, Tang Minyue, Bai Long, Zhu Yimin.

Several studies have indicated that mutations of LARS2 are associated with premature ovarian insufficiency (POI). However, the pathogenic mechanism of LARS2 in POI has not been reported yet. In the present study, the expression levels of LARS2 and E2F1 in granulosa cells (GCs) of POI patients were examined. CCK-8 and Edu assay were performed to determine the effect of LARS2 on cell proliferation. Apoptosis rate, mitochondrial membrane potential, reactive oxygen species (ROS), and cytoplasm Ca2+ levels were analyzed by flow cytometry. Western blot was conducted to evaluate the expression level of genes affected by LARS2. Transmission electron microscopy (TEM) was used to observe mitochondrial structure in GCs. Chromatin immunoprecipitation (ChIP) was used to evaluate the regulatory effect of E2F1 on Mfn-2 expression. Our results showed that LARS2 expression was downregulated in GCs of POI patients. Silencing of LARS2 inhibited cell proliferation and promoted the apoptosis of GCs. Meanwhile, LARS2 knockdown could induce mitochondrial dysfunction and accumulation of ROS levels. Moreover, ROS was found to be involved in the antiproliferation, proapoptotic, and endoplasmic reticulum (ER) stress effects of LARS2 knockdown. Furthermore, we also found that the expression level of E2F1 was positively correlated with LARS2. In addition, E2F1 could bind at the -61/-46 region of Mfn-2 promoter and regulated MFN-2 transcription. These findings demonstrated that LARS2 could promote the expression of E2F1. E2F1 mediated the effect of LARS2 on Mfn-2 expression via targeting the promoter region of Mfn-2, in which subsequently regulated cell proliferation and apoptosis, which resulted in the etiology of POI. This study will provide useful information for further investigations on the LARS2 in the occurrence of POI.

Chakraborty Arka Prava, Mukherjee Adreesh, Bhattacharyya Aishee, Bhattacharyya Dwaipayan, Ray Biman Kanti, Biswas Atanu.

A 55-year-old male presented with apraxia of gait with exaggerated upper limb movement with relative preservation of cognition and mild spasticity of limbs. His investigations reveal posterior-predominant leukodystrophy in brain magnetic resonance imaging (MRI) and compound heterozygous mutations in mitochondrial alanyl-transfer RNA synthetase 2 (AARS2) by next generation sequencing. His asymptomatic brother also has MRI changes with subtle mild pyramidal signs. AARS2 mutation is a rare cause of mitochondrial encephalopathy which may give rise to leukodystrophy with premature ovarian failure, infantile cardiomyopathy, lung hypoplasia and myopathy. Gait apraxia as primary presenting feature of this rare variant of mitochondrial encephalomyopathy is hitherto un-reported.

Sun Lianhua, Lin Zhengyu, Zhang Jifang, Shen Jiali, Wang Xiaowen, Yang Jun.

Auditory neuropathy spectrum disease (ANSD) is caused by both environmental and genetic causes and is defined by a failure in peripheral auditory neural transmission but normal outer hair cells function. To date, 13 genes identified as potentially causing ANSD have been documented. To study the etiology of ANSD, we collected 9 probands with ANSD diagnosed in the clinic and performed targeted next-generation sequencing.

Hu Wenjing, Fang Hongjun, Peng Yu, Li Li, Guo Danni, Tang Jingwen, Yi Jurong, Liu Qingqing, Qin Wei, Wu Liwen, Ning Zeshu.

Biallelic NARS2 mutations can cause various neurodegenerative diseases, leading to growth retardation, intractable epilepsy, and hearing loss in early infancy and further progressing to spastic paraplegia, neurodegeneration, and even death. NARS2 mutations are associated with mitochondrial dysfunction and cause combined oxidative phosphorylation deficiency 24 (COXPD24). Relatively few cases have been reported worldwide; therefore, the pathogenesis of COXPD24 is poorly understood. We studied two unrelated patients with COXPD24 with similar phenotypes who presented with intractable refractory epilepsia partialis continua, hearing loss, and growth retardation. One patient died from epilepsy. Three novel NARS2 variants (case 1: c.185T > C and c.251 + 2T > G; case 2: c.185T > C and c.509T > G) were detected with whole-exome sequencing. c.251 + 2T > G is located at the donor splicing site in the non-coding sequence of the gene. The minigene experiment further verified that c.251 + 2T > G caused variable splicing abnormalities and produced truncated proteins. Molecular dynamics studies showed that c.185T > C and c.509T > G reduced the binding free energy of the NARS2 protein dimer. The literature review revealed fewer than 30 NARS2 variants. These findings improved our understanding of the disease phenotype and the variation spectrum and revealed the potential pathogenic mechanism of non-coding sequence mutations in COXPD24.

Yang Fan, Wang Dan, Zhang Xuehua, Fan Haoqin, Zheng Yu, Xiao Zhenghui, Chen Zhi, Xiao Yunbin, Liu Qiming.

Hyperuricemia, pulmonary hypertension, and renal failure in infancy and alkalosis syndrome (HUPRA syndrome) is an ultrarare mitochondrial disease that is characterized by hyperuricemia, pulmonary hypertension, renal failure, and alkalosis. Seryl-tRNA synthetase 2 (SARS2) gene variants are believed to cause HUPRA syndrome, and these variants result in the loss of function of seryl-tRNA synthetase. Eventually, mutated seryl-tRNA synthetase is unable to catalyze tRNA synthesis and leads to the inhibition of the biosynthesis of mitochondrial proteins. This causes oxidative phosphorylation (OXPHOS) system impairments. To date, five mutation sites in the SARS2 gene have been identified. We used whole-exome sequencing and Sanger sequencing to find and validate a novel compound heterozygous variants of SARS2 [c.1205G>A (p.Arg402His) and c.680G>A (p.Arg227Gln)], and in silico analysis to analyze the structural change of the variants. We found that both variants were not sufficient to cause obvious structural damage but changed the intermolecular bond of the protein, which could be the cause of HUPRA syndrome in this case. We also performed the literature review and found this patient had significant pulmonary hypertension and minor renal dysfunction compared with other reported cases. This study inspired us to recognize HUPRA syndrome and broaden our knowledge of gene variation in PH.

Gong Yan, Lan Xiao Ping, Guo Sheng.

IARS2 (EC6.1.5) is a mitochondrial isoleucine-tRNA synthetase. Despite the fact that only fewer than 30 patients have been reported in the literature, mitochondrial disorders caused by pathogenic variants in the IARS2 gene (OMIM: 616007) have a very broad and variable clinical phenotype spectrum. We present a child who has sideroblastic anemia and hypoparathyroidism as a result of a previously unreported mutation in the IARS2 gene.

Colin Elia, Courtois Geneviève, Brouzes Chantal, Pulman Juliette, Rabant Marion, Rötig Agnès, Taffin Hélène, Lion-Lambert Mathilde, Fabrega Sylvie, Da Costa Lydie, De Montalembert Mariane, Salomon Rémi, Hermine Olivier, Couronné Lucile.

Skorvanek Matej, Rektorova Irena, Mandemakers Wim, Wagner Matias, Steinfeld Robert, Orec Laura, Han Vladimir, Pavelekova Petra, Lackova Alexandra, Kulcsarova Kristina, Ostrozovicova Miriam, Gdovinova Zuzana, Plecko Barbara, Brunet Theresa, Berutti Riccardo, Kuipers Demy J S, Boumeester Valerie, Havrankova Petra, Tijssen M A J, Kaiyrzhanov Rauan, Rizig Mie, Houlden Henry, Winkelmann Juliane, Bonifati Vincenzo, Zech Michael, Jech Robert.

Sixteen subjects with biallelic WARS2 variants encoding the tryptophanyl mitochondrial aminoacyl-tRNA synthetase, presenting with a neonatal- or infantile-onset mitochondrial disease, have been reported to date. Here we present six novel cases with WARS2-related diseases and expand the spectrum to later onset phenotypes including dopa-responsive early-onset parkinsonism and progressive myoclonus-ataxia.

Xu Manting, Duan Xin, Ren Xiaotun, Liu Zhimei, Chen Shuhua, Fang Fang.

Mutations in CARS2 gene, encoding for the mitochondrial cysteinyl-tRNA synthetase, has been reported to be associated with early-onset epileptic encephalopathy (EOEE). Here, we generated an induced pluripotent stem cell (iPSC) line from the human dermal fibroblasts (HDFs) of an one-year-old boy with EOEE carrying homozygous c.1426G > A mutation in CARS2 gene. These iPSCs exhibited stable amplification, expressed pluripotent markers, and differentiated spontaneously into three germ layers in vitro.

Štěrbová K, Vlčková M, Hansíková H, Sebroňová V, Sedláčková L, Pavlíček P, Laššuthová P.

Lipponen Joonas, Helisalmi Seppo, Raivo Joose, Siitonen Ari, Doi Hiroshi, Rusanen Harri, Lehtilahti Maria, Ryytty Mervi, Laakso Markku, Tanaka Fumiaki, Majamaa Kari, Kytövuori Laura.

The genetics of cerebellar ataxia is complex. Hundreds of causative genes have been identified, but only a few cause more than single cases. The spectrum of ataxia-causing genes differs considerably between populations. The aim of the study was to investigate the molecular epidemiology of ataxia in the Finnish population.

Li Jeng-Lin, Lee Ni-Chung, Chen Pin-Shiuan, Lee Gin Hoong, Wu Ruey-Meei.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is characterized by slowly progressive spastic gait, cerebellar symptoms, and posterior cord dysfunction. DARS2, which encodes mitochondrial aspartyl tRNA synthase, is associated with the rare disease.

de Valles-Ibáñez Guillem, Hildebrand Michael S, Bahlo Melanie, King Chontelle, Coleman Matthew, Green Timothy E, Goldsmith John, Davis Suzanne, Gill Deepak, Mandelstam Simone, Scheffer Ingrid E, Sadleir Lynette G.

Recessive variants in RARS2, a nuclear gene encoding a mitochondrial protein, were initially reported in pontocerebellar hypoplasia. Subsequently, a recessive RARS2 early-infantile (<12 weeks) developmental and epileptic encephalopathy was described with hypoglycaemia and lactic acidosis. Here, we describe two unrelated patients with a novel RARS2 phenotype and reanalyse the published RARS2 epilepsy phenotypes and variants. Our novel cases had infantile-onset myoclonic developmental and epileptic encephalopathy, presenting with a progressive movement disorder from 9 months on a background of normal development. Development plateaued and regressed thereafter, with mild to profound impairment. Multiple drug-resistant generalized and focal seizures occurred with episodes of non-convulsive status epilepticus. Seizure types included absence, atonic, myoclonic, and focal seizures. Electroencephalograms showed diffuse slowing, multifocal, and generalised spike-wave activity, activated by sleep. Both patients had compound heterozygous RARS2 variants with likely impact on splicing and transcription. Remarkably, of the now 52 RARS2 variants reported in 54 patients, our reanalysis found that 44 (85%) have been shown to or are predicted to affect splicing or gene expression leading to protein truncation or nonsense-mediated decay. We expand the RARS2 phenotypic spectrum to include infantile encephalopathy and suggest this gene is enriched for pathogenic variants that disrupt splicing.

Axelsen Tobias Melton, Vammen Tzvetelina Lubenova, Bak Mads, Pourhadi Nelsan, Stenør Christian Midtgaard, Grønborg Sabine.

Mitochondrial alanyl-tRNA synthetase 2 gene (AARS2) related disease is a rare genetic disorder affecting mitochondrial metabolism, leading to severe cardiac disease in infants or progressive leukodystrophy in young adults. The disease is considered ultra-rare with only 39 cases of AARS2-leukodystrophy previously reported.

Zou Yulin, Yang Yanyan, Fu Xiuxiu, He Xiangqin, Liu Meixin, Zong Tingyu, Li Xiaolu, Htet Aung Lynn, Wang Zhibin, Yu Tao.

Aminoacyl-tRNA synthetases (ARSs) are widely found in organisms, which can activate amino acids and make them bind to tRNA through ester bond to form the corresponding aminoyl-tRNA. The classic function of ARS is to provide raw materials for protein biosynthesis. Recently, emerging evidence demonstrates that ARSs play critical roles in controlling inflammation, immune responses, and tumorigenesis as well as other important physiological and pathological processes. With the recent development of genome and exon sequencing technology, as well as the discovery of new clinical cases, ARSs have been reported to be closely associated with a variety of cardiovascular diseases (CVDs), particularly angiogenesis and cardiomyopathy. Intriguingly, aminoacylation was newly identified and reported to modify substrate proteins, thereby regulating protein activity and functions. Sensing the availability of intracellular amino acids is closely related to the regulation of a variety of cell physiology. In this review, we summarize the research progress on the mechanism of CVDs caused by abnormal ARS function and introduce the clinical phenotypes and characteristics of CVDs related to ARS dysfunction. We also highlight the potential roles of aminoacylation in CVDs. Finally, we discuss some of the limitations and challenges of present research. The current findings suggest the significant roles of ARSs involved in the progress of CVDs, which present the potential clinical values as novel diagnostic and therapeutic targets in CVD treatment.

Zheng Wen-Qiang, Pedersen Signe Vandal, Thompson Kyle, Bellacchio Emanuele, French Courtney E, Munro Benjamin, Pearson Toni S, Vogt Julie, Diodato Daria, Diemer Tue, Ernst Anja, Horvath Rita, Chitre Manali, Ek Jakob, Wibrand Flemming, Grange Dorothy K, Raymond Lucy, Zhou Xiao-Long, Taylor Robert W, Ostergaard Elsebet.

TARS2 encodes human mitochondrial threonyl tRNA-synthetase that is responsible for generating mitochondrial Thr-tRNAThr and clearing mischarged Ser-tRNAThr during mitochondrial translation. Pathogenic variants in TARS2 have hitherto been reported in a pair of siblings and an unrelated patient with an early onset mitochondrial encephalomyopathy and a combined respiratory chain enzyme deficiency in muscle. We here report five additional unrelated patients with TARS2-related mitochondrial diseases, expanding the clinical phenotype to also include epilepsy, dystonia, hyperhidrosis and severe hearing impairment. Additionally, we document seven novel TARS2 variants-one nonsense variant and six missense variants-that we demonstrate are pathogenic and causal of the disease presentation based on population frequency, homology modelling and functional studies that show the effects of the pathogenic variants on TARS2 stability and/or function.

Okamoto Nobuhiko, Miya Fuyuki, Tsunoda Tatsuhiko, Kanemura Yonehiro, Saitoh Shinji, Kato Mitsuhiro, Yanagi Kumiko, Kaname Tadashi, Kosaki Kenjiro.

Aminoacyl tRNA synthetases (ARSs) are highly conserved enzymes that link amino acids to their cognate tRNAs. Thirty-seven ARSs are known and their deficiencies cause various genetic disorders. Variants in some ARSs are associated with the autosomal dominant inherited form of axonal neuropathy, including Charcot-Marie-Tooth (CMT) disease. Variants of genes encoding ARSs often cause disorders in an autosomal recessive fashion. The clinical features of cytosolic ARS deficiencies are more variable, including systemic features. Deficiencies of ARSs localized in the mitochondria are often associated with neurological disorders including Leigh and early-onset epileptic syndromes. Whole exome sequencing (WES) is an efficient way to identify the genes causing various symptoms in patients. We identified 4 pedigrees with novel compound heterozygous variants in ARS genes (WARS1, MARS1, AARS2, and PARS2) by WES. Some unique manifestations were noted. The number of patients with ARSs has been increasing since the application of WES. Our findings broaden the known genetic and clinical spectrum associated with ARS variants.

Sawada Daisuke, Naito Sachiko, Aoyama Hiromi, Shiohama Tadashi, Ichikawa Tomohiko, Imagawa Eri, Miyake Noriko, Matsumoto Naomichi, Fujii Katsunori.

Leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) is a hereditary disorder caused by biallelic variants in the EARS2 gene. Patients exhibit developmental delay, hypotonia, and hyperreflexia. Brain magnetic resonance imaging (MRI) reveals T2-hyperintensities in the deep white matter, thalamus, and brainstem, which generally stabilize over time. Herein, we report a case of LTBL, showing remitting and exacerbating white matter lesions.

Faridi Rabia, Rea Alessandro, Fenollar-Ferrer Cristina, O'Keefe Raymond T, Gu Shoujun, Munir Zunaira, Khan Asma Ali, Riazuddin Sheikh, Hoa Michael, Naz Sadaf, Newman William G, Friedman Thomas B.

Hearing loss and impaired fertility are common human disorders each with multiple genetic causes. Sometimes deafness and impaired fertility, which are the hallmarks of Perrault syndrome, co-occur in a person. Perrault syndrome is inherited as an autosomal recessive disorder characterized by bilateral mild to severe childhood sensorineural hearing loss with variable age of onset in both sexes and ovarian dysfunction in females who have a 46, XX karyotype. Since the initial clinical description of Perrault syndrome 70 years ago, the phenotype of some subjects may additionally involve developmental delay, intellectual deficit and other neurological disabilities, which can vary in severity in part dependent upon the genetic variants and the gene involved. Here, we review the molecular genetics and clinical phenotype of Perrault syndrome and focus on supporting evidence for the eight genes (CLPP, ERAL1, GGPS1, HARS2, HSD17B4, LARS2, RMND1, TWNK) associated with Perrault syndrome. Variants of these eight genes only account for approximately half of the individuals with clinical features of Perrault syndrome where the molecular genetic base remains under investigation. Additional environmental etiologies and novel Perrault disease-associated genes remain to be identified to account for unresolved cases. We also report a new genetic variant of CLPP, computational structural insight about CLPP and single cell RNAseq data for eight reported Perrault syndrome genes suggesting a common cellular pathophysiology for this disorder. Some unanswered questions are raised to kindle future research about Perrault syndrome.

Vafaee-Shahi Mohammad, Farhadi Mohammad, Razmara Ehsan, Morovvati Saeid, Ghasemi Saeide, Abedini Seyedeh Sedigheh, Bagher Zohreh, Alizadeh Rafieh, Falah Masoumeh.

Mutations in NARS2 (MIM: 612803) are associated with combined oxidative phosphorylation deficiency 24 (COXPD24; MIM: 616239) that is a rare mitochondrial and a multisystem autosomal recessive disorder.

Souissi Amal, Ben Said Mariem, Frikha Fakher, Elloumi Ines, Masmoudi Saber, Megarbane Andre.

Background: Variants in the HARS2 gene have been reported to be associated with nonsyndromic hearing loss (HL) and Perrault syndrome (PS), a rare recessive disorder marked by bilateral sensorineural HL and ovarian dysgenesis. Given the low number of pathogenic variants described in the HARS2 gene, no genotype/phenotype correlations have been established between variants in this gene and the clinical data. Materials and Methods: Whole blood was collected from four members of a Lebanese family with PS. An affected woman was evaluated for HL by clinical examination and audiological tests. Primary ovarian failure was analyzed according to age of primary or secondary amenorrhea, follicle stimulating hormone levels, and pelvic ultrasound. The existence of neurological symptoms and other associated conditions was checked. To identify the causative variant, we used a custom HaloPlexHS panel for next-generation sequencing of the coding sequences of six genes implicated in this syndrome. Results: We identified a novel homozygous HARS2 missense variant (c.260G>A; p.Arg87His), which is only the second homozygous variant in the HARS2 gene identified to date worldwide. This variant is predicted to be deleterious by multiple in silico analysis tools, moreover the Arg87 amino acid nearly is invariant among eight species. Based on molecular modeling analysis, this variation is predicted to disturb the proper folding of HARS2, which may reduce its aminoacylation efficiency. Clinical data are compared with the other cases recorded in the literature to help gain further knowledge with regard to the phenotype. Conclusion: Our results provide strong evidence corroborating the etiological association of this mutation with the HARS2-PS phenotype. HARS2 variants need to be searched for in patients with early-onset bilateral sensorineural HL and ovarian dysfunction in women so as to guarantee accurate endocrinological surveillance and management to minimize secondary complications.

Pauly Martje G, Hellenbroich Yorck, Grundmann-Hauser Kathrin, Hinrichs Frauke, Lohmann Katja, Brüggemann Norbert.

Murofushi Yuka, Hayakawa Itaru, Abe Yuichi, Ohto Tatsuyuki, Murayama Kei, Suzuki Hisato, Takenouchi Toshiki, Kosaki Kenjiro, Kubota Masaya.

KARS encodes lysyl-tRNA synthetase, which is essential for protein translation. KARS mutations sometimes cause impairment of cytoplasmic and mitochondrial protein synthesis, and sometimes lead to progressive leukodystrophies with mitochondrial signature and psychomotor regression, and follow a rapid regressive course to premature death. There has been no disease-modifying therapy beyond supportive treatment. We present a 5-year-old male patient with an asymmetrical leukodystrophy who showed overt evidence of mitochondrial dysfunction, including elevation of lactate on brain MR spectroscopy and low oxygen consumption rate in fibroblasts. We diagnosed this patient's condition as KARS-related leukodystrophy with cerebral calcification, congenital deafness, and evidence of mitochondrial dysfunction. We employed a ketogenic diet as well as multiple vitamin supplementation with the intention to alleviate mitochondrial dysfunction. The patient showed alleviation of his psychomotor regression and even partial restoration of his abilities within 4 months. This is an early report of a potential disease-modifying therapy for KARS-related progressive leukodystrophy without appreciable adverse effects.

Carreño-Gago Lidia, Juárez-Flores Diana Luz, Grau Josep Maria, Ramón Javier, Lozano Ester, Vila-Juli� Ferran, Martí Ramon, Garrabou Glòria, Garcia-Arumí Elena.

Pathogenic variants in the mitochondrial tyrosyl-tRNA synthetase gene (YARS2) were associated with myopathy, lactic acidosis, and sideroblastic anemia (MLASA). However, patients can present mitochondrial myopathy, with exercise intolerance and muscle weakness, leading from mild to lethal phenotypes. Genes implicated in mtDNA replication were studied by Next Generation Sequencing (NGS) and whole exome sequence with the TruSeq Rapid Exome kit (Illumina, San Diego, CA, USA). Mitochondrial protein translation was studied following the Sasarman and Shoubridge protocol and oxygen consumption rates with Agilent Seahorse XF24 Analyzer Mitostress Test, (Agilent, Santa Clara, CA, USA). We report two siblings with two novel compound heterozygous pathogenic variants in YARS2 gene: a single nucleotide deletion in exon 1, c.314delG (p.(Gly105Alafs*4)), which creates a premature stop codon in the amino acid 109, and a single nucleotide change in exon 5 c.1391T>C (p.(Ile464Thr)), that cause a missense variant in amino acid 464. We demonstrate the pathogenicity of these new variants associated with reduced YARS2 mRNA transcript, reduced mitochondrial protein translation and dysfunctional organelle function. These pathogenic variants are responsible for late onset MLASA, herein accompanied by pancreatic insufficiency, observed in both brothers, clinically considered as Pearson's syndrome. Molecular study of YARS2 gene should be considered in patients presenting Pearson's syndrome characteristics and MLASA related phenotypes.

Diamond Tamir, DiVito Donna, Savoca Melanie, Mascarenhas Maria, Goldstein Amy.

Primary mitochondrial disorders (PMDs) comprise a group of hundreds of individual genetic diseases affecting mitochondrial function, including oxidative phosphorylation and energy production. The estimated prevalence of these disorders ranges from 2.9 to 20 cases per 100,000. PMDs are commonly associated with malnutrition and growth failure. There is a paucity of literature regarding nutrition assessment and long-term data in the PMD population. We present three patients with various PMDs who presented complications related to malnutrition: (1) a 16-year-old male with Kearns-Sayre syndrome developed type 2 insulin-requiring diabetes mellitus after the initiation of high-calorie nutrition rehabilitation via gastrostomy tube (G-tube); (2) an 11-year-old female with myoclonic epilepsy associated with ragged red fibers developed diarrhea with metabolic decompensation and profound neurological and respiratory deterioration during nutrition rehabilitation after surgical G-tube placement; and (3) a 19-year-old male with a WARS2-associated PMD manifesting with developmental delay and severe parkinsonism presented complications related to poor wound healing after gastrojejunostomy tube placement. The last patient required prolonged hospitalization in the intensive care unit. Clinicians should be vigilant in monitoring these possible complications, as no standards of care exist for the initiation of enteral nutrition for this unique population.

Cappuccio Gerarda, Ceccatelli Berti Camilla, Baruffini Enrico, Sullivan Jennifer, Shashi Vandana, Jewett Tamison, Stamper Tara, Maitz Silvia, Canonico Francesco, Revah-Politi Anya, Kupchik Gabriel S, Anyane-Yeboa Kwame, Aggarwal Vimla, Benneche Andreas, Bratland Eirik, Berland Siren, D'Arco Felice, Alves Cesar A, Vanderver Adeline, Longo Daniela, Bertini Enrico, Torella Annalaura, Nigro Vincenzo, , D'Amico Alessandra, van der Knaap Marjo S, Goffrini Paola, Brunetti-Pierri Nicola.

KARS1 encodes a lysyl-transfer RNA synthetase (LysRS) that links lysine to its cognate transfer RNA. Two different KARS1 isoforms exert functional effects in cytosol and mitochondria. Bi-allelic pathogenic variants in KARS1 have been associated to sensorineural hearing and visual loss, neuropathy, seizures, and leukodystrophy. We report the clinical, biochemical, and neuroradiological features of nine individuals with KARS1-related disorder carrying 12 different variants with nine of them being novel. The consequences of these variants on the cytosol and/or mitochondrial LysRS were functionally validated in yeast mutants. Most cases presented with severe neurological features including congenital and progressive microcephaly, seizures, developmental delay/intellectual disability, and cerebral atrophy. Oculo-motor dysfunction and immuno-hematological problems were present in six and three cases, respectively. A yeast growth defect of variable severity was detected for most variants on both cytosolic and mitochondrial isoforms. The detrimental effects of two variants on yeast growth were partially rescued by lysine supplementation. Congenital progressive microcephaly, oculo-motor dysfunction, and immuno-hematological problems are emerging phenotypes in KARS1-related disorder. The data in yeast emphasize the role of both mitochondrial and cytosolic isoforms in the pathogenesis of KARS1-related disorder and supports the therapeutic potential of lysine supplementation at least in a subset of patients.

Roux Charles-Joris, Barcia Giulia, Schiff Manuel, Sissler Marie, Levy Raphaël, Dangouloff-Ros Volodia, Desguerre Isabelle, Edvardson Shimon, Elpeleg Orli, Rötig Agnès, Munnich Arnold, Boddaert Nathalie.

Mitochondrial aminoacyl-tRNA synthetases-encoded by ARS2 genes-are evolutionarily conserved enzymes that catalyse the attachment of amino acids to their cognate tRNAs, ensuring the accuracy of the mitochondrial translation process. ARS2 gene mutations are associated with a wide range of clinical presentations affecting the CNS.

Jin Xiaofen, Zhang Juanjuan, Yi Qiuzi, Meng Feilong, Yu Jialing, Ji Yanchun, Mo Jun Q, Tong Yi, Jiang Pingping, Guan Min-Xin.

To investigate the mechanism underlying the synergic interaction between Leber's hereditary optic neuropathy (LHON)-associated ND1 and mitochondrial tyrosyl-tRNA synthetase (YARS2) mutations.

van Helden Ruben W J, Birket Matthew J, Freund Christian, Arendzen Christaan H, Mikkers Harald M, Orlova Valeria, de Coo René I, Mummery Christine L, Bellin Milena.

Combined Oxidative Phosphorylation Deficiency 8 (COXPD8) is an autosomal recessive disorder causing lethal childhood-onset hypertrophic cardiomyopathy. Homozygous or compound heterozygous mutations in the nuclear-encoded mitochondrial alanyl-tRNA synthetase 2 (AARS2) gene underly the pathology. We generated induced pluripotent stem cells (hiPSCs) from two patients carrying the heterozygous compound c.1774 C>T, c.2188 G>A and c.2872 C>T AARS2 mutations, as well as a related healthy control carrying the c.2872 C>T AARS2 mutation. All hiPSC-lines expressed pluripotency markers, maintained a normal karyotype, and differentiated towards the three germ layer derivatives in vitro. These lines can be used to model COXPD8 or mitochondrial dysfunction.

Kose Melis, Isik Esra, Aykut Ayça, Durmaz Asude, Kose Engin, Ersoy Melike, Diniz Gulden, Adebali Ogun, Ünalp Aycan, Yilmaz Ünsal, Karaoğlu Pakize, Edizer Selvinaz, Tekin Hande Gazeteci, Özdemir Taha Reşid, Atik Tahir, Onay Hüseyin, Özkınay Ferda.

Diagnostic process of mitochondrial disorders (MD) is challenging because of the clinical variability and genetic heterogeneity of these conditions. Next-Generation Sequencing (NGS) technology offers a high-throughput platform for nuclear MD.

Figuccia Sonia, Degiorgi Andrea, Ceccatelli Berti Camilla, Baruffini Enrico, Dallabona Cristina, Goffrini Paola.

In most eukaryotes, mitochondrial protein synthesis is essential for oxidative phosphorylation (OXPHOS) as some subunits of the respiratory chain complexes are encoded by the mitochondrial DNA (mtDNA). Mutations affecting the mitochondrial translation apparatus have been identified as a major cause of mitochondrial diseases. These mutations include either heteroplasmic mtDNA mutations in genes encoding for the mitochondrial rRNA (mtrRNA) and tRNAs (mttRNAs) or mutations in nuclear genes encoding ribosomal proteins, initiation, elongation and termination factors, tRNA-modifying enzymes, and aminoacyl-tRNA synthetases (mtARSs). Aminoacyl-tRNA synthetases (ARSs) catalyze the attachment of specific amino acids to their cognate tRNAs. Differently from most mttRNAs, which are encoded by mitochondrial genome, mtARSs are encoded by nuclear genes and then imported into the mitochondria after translation in the cytosol. Due to the extensive use of next-generation sequencing (NGS), an increasing number of mtARSs variants associated with large clinical heterogeneity have been identified in recent years. Being most of these variants private or sporadic, it is crucial to assess their causative role in the disease by functional analysis in model systems. This review will focus on the contributions of the yeast Saccharomyces cerevisiae in the functional validation of mutations found in mtARSs genes associated with human disorders.

Stellingwerff Menno D, Figuccia Sonia, Bellacchio Emanuele, Alvarez Karin, Castiglioni Claudia, Topaloglu Pinar, Stutterd Chloe A, Erasmus Corrie E, Sanchez-Valle Amarilis, Lebon Sebastien, Hughes Sarah, Schmitt-Mechelke Thomas, Vasco Gessica, Chow Gabriel, Rahikkala Elisa, Dallabona Cristina, Okuma Cecilia, Aiello Chiara, Goffrini Paola, Abbink Truus E M, Bertini Enrico S, Van der Knaap Marjo S.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is regarded a relatively mild leukodystrophy, diagnosed by characteristic long tract abnormalities on MRI and biallelic variants in DARS2, encoding mitochondrial aspartyl-tRNA synthetase (mtAspRS). DARS2 variants in LBSL are almost invariably compound heterozygous; in 95% of cases, 1 is a leaky splice site variant in intron 2. A few severely affected patients, still fulfilling the MRI criteria, have been described. We noticed highly unusual MRI presentations in 15 cases diagnosed by WES. We examined these cases to determine whether they represent consistent novel LBSL phenotypes.

Sofou Kalliopi, Kollberg Gittan, Hedberg-Oldfors Carola, Oldfors Anders.

The phenotypic variability of NARS2 associated disease is vast, yet not thoroughly explored. We present the phenotypic and genetic features of 2 siblings with early-onset mitochondrial encephalopathy due to pathogenic variant in NARS2, along with the results from a systematic literature review.

Zehravi Mehwish, Wahid Mohsin, Ashraf Junaid, Fatima Tehseen.

Background: Muscular dystrophies are a heterogeneous group of inherited disorders that cannot be diagnosed clinically due to overlapping clinical phenotypes. Whole-exome sequencing is considered as the diagnostic strategy of choice in these cases. In this study we aimed to determine the mutational spectrum of multiplex ligation-dependent probe amplification (MLPA)-negative muscular dystrophy patients in Pakistan using whole-exome sequencing. Subsequently the mutations identified via WES were used to screen additional dystrophinopathy patients by Sanger sequencing. Materials and Methods: DNA extracted from the peripheral blood of three MLPA-negative muscular dystrophy patients was sent for whole-exome sequencing. The identified variants in these 3 patients were then checked in 18 dystrophinopathy patients using Sanger sequencing. Results: Four missense variants and one nonsense variant in the Duchenne muscular dystrophy (DMD) gene were detected. WES diagnosed a DMD patient carrying a nonsense variant c.4375C>T (rs398123953) who can benefit from Ataluren therapy. The other two patients carried missense variant (c.572G>T) in the YARS2 gene (rs11539445) labeling them as patients of MLASA (myopathy, lactic acidosis, and sideroblastic anemia). The identified missense and nonsense variants in the DMD gene were detected in 18 clinically diagnosed dystrophinopathy patients using Sanger sequencing. Three missense variants were detected in our cohort of 18 dystrophinopathy patients. One missense variant c.3406A>T (rs3827462) and a nonsense variant c.4375C>T (rs398123953) were not detected in our cohort of 18 dystrophinopathy patients. Conclusions: Whole-exome sequencing identified a nonsense variant in Pakistani muscular dystrophy patients, which is amenable to treatment by Ataluren and a missense variant in YARS2 gene responsible for causing MLASA.

Nuovo Sara, Micalizzi Alessia, Romaniello Romina, Arrigoni Filippo, Ginevrino Monia, Casella Antonella, Serpieri Valentina, D'Arrigo Stefano, Briguglio Marilena, Salerno Grazia Gabriella, Rossato Sara, Sartori Stefano, Leuzzi Vincenzo, Battini Roberta, Ben-Zeev Bruria, Graziano Claudio, Mirabelli Badenier Marisol, Brankovic Vesna, Nardocci Nardo, Spiegel Ronen, Petković Ramadža Danijela, Vento Giovanni, Marti Itxaso, Simonati Alessandro, Dipresa Savina, Freri Elena, Mazza Tommaso, Bassi Maria Teresa, Bosco Luca, Travaglini Lorena, Zanni Ginevra, Bertini Enrico Silvio, Vanacore Nicola, Borgatti Renato, Valente Enza Maria.

Pontocerebellar hypoplasias (PCH) comprise a group of genetically heterogeneous disorders characterised by concurrent hypoplasia of the pons and the cerebellum and variable clinical and imaging features. The current classification includes 13 subtypes, with ~20 known causative genes. Attempts have been made to delineate the phenotypic spectrum associated to specific PCH genes, yet clinical and neuroradiological features are not consistent across studies, making it difficult to define gene-specific outcomes.

De Cocker Laurens J L, Castillo Mauricio.

Genetic leukoencephalopathies are inherited disorders characterized by progressive white matter involvement. Although most are paediatric conditions, late-onset adult leukoencephalopathies are being increasingly recognized. Adult leukoencephalopathies may present as neurodegenerative diseases with cognitive decline and motor symptoms. Similar to their paediatric counterparts, different adult leukoencephalopathies often have distinctive MRI appearances. In particular, DWI has been recently shown to demonstrate specific patterns of persistent diffusion restriction in several adult-onset leukoencephalopathies. As such, DWI may provide important clues to the diagnosis of adult-onset leukoencephalopathy. The purpose of this review is to discuss characteristic DWI features in some late-onset leukoencephalopathies.

Peluso Francesca, Palazzo Viviana, Indolfi Giuseppe, Mari Francesco, Pasqualetti Roberta, Procopio Elena, Nesti Claudia, Guerrini Renzo, Santorelli Filippo, Giglio Sabrina.

Mutations in lysyl-tRNA synthetase (KARS1), an enzyme that charges tRNA with the amino acid lysine in both the cytoplasm and mitochondria, have been associated thus far with autosomal recessive Charcot-Marie-Tooth type CMTRIB, hearing loss type DFNB89, and mitochondrial encephalohepatopathy (MEH) featuring neurodevelopmental disorders with microcephaly, white matter changes, and cardiac and hepatic failure in less than 30 patients.

Zhou Yi, Zhong Cheng, Yang Qin, Zhang Gaofu, Yang Haiping, Li Qiu, Wang Mo.

Hyperuricemia, pulmonary hypertension, renal failure, and alkaline intoxication syndrome (HUPRA syndrome) is a rare autosomal recessive mitochondrial disease. SARS2 gene encoding seryl-tRNA synthetase is the only pathogenic gene of HUPRA syndrome. All the previously reported cases with HUPRA syndrome were detected for homozygous mutation.

Wu Siqi, Hei Zhoufei, Zheng Li, Zhou Jintong, Liu Zaizhou, Wang Jing, Fang Pengfei.

Aminoacyl-tRNA synthetases (AARSs) catalyze the ligation of amino acids to their cognate tRNAs and therefore play an essential role in protein biosynthesis in all living cells. The KARS gene in human encodes both cytosolic and mitochondrial lysyl-tRNA synthetase (LysRS). A recent study identified a missense mutation in KARS gene (c.517T > C) that caused autosomal recessive nonsyndromic hearing loss. This mutation led to a tyrosine to histidine (YH) substitution in both cytosolic and mitochondrial LysRS proteins, and decreased their aminoacylation activity to different levels. Here, we report the crystal structure of LysRS YH mutant at a resolution of 2.5 Å. We found that the mutation did not interfere with the active center, nor did it cause any significant conformational changes in the protein. The loops involved in tetramer interface and tRNA anticodon binding site showed relatively bigger variations between the mutant and wild type proteins. Considering the differences between the cytosolic and mitochondrial tRNAlyss, we suggest that the mutation triggered subtle changes in the tRNA anticodon binding region, and the interferences were further amplified by the different D and T loops in mitochondrial tRNAlys, and led to a complete loss of the aminoacylation of mitochondrial tRNAlys.

Cotta Ana, Carvalho Elmano, da-Cunha-Junior AntonioLopes, Navarro Mônica Machado, Paim Julia Filardi, Valicek Jaquelin, Baptista-Junior Sidney, da Silveira Eni Braga, Lima Maria Isabel, Carellos Ericka Viana Machado, de-La-Rocque-Ferreira Alessandra, Takata Reinaldo Issao, Horvath Rita.

Reversible infantile respiratory chain deficiency is a severe neonatal mitochondrial myopathy that resolves spontaneously. It is caused by the homoplasmic m.14674T>C mtDNA mutation and additional nuclear variants in genes interacting with mt-tRNAGlu have been detected in some patients. We present detailed clinical, imaging, and muscle biopsy findings in a boy and a girl with neonatal hypotonia, feeding difficulties, lactic acidosis, and ragged red fibers. Both patients show fat replacement on muscle imaging, which was mild in the boy, but severe in the girl, affecting mostly the posterior leg muscles. In addition to the homoplasmic m.14674T>C, both patients carried heterozygous variants in QRSL1 (c. 686T>G; p.Val299Gly) and EARS2 (c.358C>T; p.Arg120Trp), respectively. It is very important to recognize the clinical and morphological signs of reversible infantile respiratory chain deficiency as patients should receive intensive supportive care in the first 6 months of life. Understanding the mechanism of the spontaneous recovery may lead to novel therapeutic perspectives in other mitochondrial diseases.

Ni Min, Black Lauren F, Pan Chunxiao, Vu Hieu, Pei Jimin, Ko Bookyung, Cai Ling, Solmonson Ashley, Yang Chendong, Nugent Kimberly M, Grishin Nick V, Xing Chao, Roeder Elizabeth, DeBerardinis Ralph J.

Glutamyl-tRNA synthetase 2 (encoded by EARS2) is a mitochondrial aminoacyl-tRNA synthetase required to translate the 13 subunits of the electron transport chain encoded by the mitochondrial DNA. Pathogenic EARS2 variants cause combined oxidative phosphorylation deficiency, subtype 12 (COXPD12), an autosomal recessive disorder involving lactic acidosis, intellectual disability, and other features of mitochondrial compromise. Patients with EARS2 deficiency present with variable phenotypes ranging from neonatal lethality to a mitigated disease with clinical improvement in early childhood. Here, we report a neonate homozygous for a rare pathogenic variant in EARS2 (c.949G>T; p.G317C). Metabolomics in primary fibroblasts from this patient revealed expected abnormalities in TCA cycle metabolites, as well as numerous changes in purine, pyrimidine, and fatty acid metabolism. To examine genotype-phenotype correlations in COXPD12, we compared the metabolic impact of reconstituting these fibroblasts with wild-type EARS2 versus four additional EARS2 variants from COXPD12 patients with varying clinical severity. Metabolomics identified a group of signature metabolites, mostly from the TCA cycle and amino acid metabolism, that discriminate between EARS2 variants causing relatively mild and severe COXPD12. Taken together, these findings indicate that metabolomics in patient-derived fibroblasts may help establish genotype-phenotype correlations in EARS2 deficiency and likely other mitochondrial disorders.

Štěrbová K, Vlčková M, Hansíková H, Sebroňová V, Sedláčková L, Pavlíček P, Laššuthová Petra.

Biallelic variants in the NARS2 gene are the cause of a continuous spectrum of neurodegenerative disorders presenting with various severity-from spastic paraplegia, progressive neurodegeneration to Leigh and Alpers syndrome. Common clinical signs result from a mitochondrial dysfunction based on OXPHOS deficiency. Here, we present a patient with infantile-onset severe epilepsy leading to fatal refractory status epilepticus. Whole exome sequencing with Exomiser analysis based on HPO terms detected two novel NARS2 variants in a compound heterozygous state. To date, 18 different NARS2 disease-causing mutations have been described. Our study adds to the understanding of this mitochondrial disorder.

Parra Sahyli Perez, Heckers Stephan H, Wilcox William R, Mcknight Colin David, Jinnah H A.

The AARS2 gene encodes a mitochondrial alanyl-transfer RNA synthetase. Defects in this gene have been linked with autosomal recessive inheritance of a variety of different clinical phenotypes.

Fan Wenlu, Jin Xiaoye, Xu Man, Xi Yongmei, Lu Weiguo, Yang Xiaohang, Guan Min-Xin, Ge Wanzhong.

Mutations in genes encoding mitochondrial aminoacyl-tRNA synthetases are linked to diverse diseases. However, the precise mechanisms by which these mutations affect mitochondrial function and disease development are not fully understood. Here, we develop a Drosophila model to study the function of dFARS2, the Drosophila homologue of the mitochondrial phenylalanyl-tRNA synthetase, and further characterize human disease-associated FARS2 variants. Inactivation of dFARS2 in Drosophila leads to developmental delay and seizure. Biochemical studies reveal that dFARS2 is required for mitochondrial tRNA aminoacylation, mitochondrial protein stability, and assembly and enzyme activities of OXPHOS complexes. Interestingly, by modeling FARS2 mutations associated with human disease in Drosophila, we provide evidence that expression of two human FARS2 variants, p.G309S and p.D142Y, induces seizure behaviors and locomotion defects, respectively. Together, our results not only show the relationship between dysfunction of mitochondrial aminoacylation system and pathologies, but also illustrate the application of Drosophila model for functional analysis of human disease-causing variants.

Finsterer Josef.

Gong Pan, Xue Jiao, Jiao Xianru, Zhang Yuehua, Yang Zhixian.

Recently, the electroencephalogram pattern of electrical status epilepticus during sleep (ESES) had been reported in some genetic disorders, and most of them were noted with developmental and epileptic encephalopathy (DEE) or epileptic encephalopathy (EE). This study aimed to determine the genetic etiologies and clinical characteristics of ESES in DEE/EE.

Kušíková Katarína, Feichtinger René Günther, Csillag Bernhard, Kalev Ognian Kostadinov, Weis Serge, Duba Hans-Christoph, Mayr Johannes Adalbert, Weis Denisa.

Mitochondriopathies represent a wide spectrum of miscellaneous disorders with multisystem involvement, which are caused by various genetic changes. The establishment of the diagnosis of mitochondriopathy is often challenging. Recently, several mutations of the VARS2 gene encoding the mitochondrial valyl-tRNA synthetase were associated with early onset encephalomyopathies or encephalocardiomyopathies with major clinical features such as hypotonia, developmental delay, brain MRI changes, epilepsy, hypertrophic cardiomyopathy, and plasma lactate elevation. However, the correlation between genotype and phenotype still remains unclear. In this paper we present a male Caucasian patient with a recurrent c.1168G>A (p.Ala390Thr) and a new missense biallelic variant c.2758T>C (p.Tyr920His) in the VARS2 gene which were detected by whole exome sequencing (WES). VARS2 protein was reduced in the patient's muscle. A resulting defect of oxidative phosphorylation (OXPHOS) was proven by enzymatic assay, western blotting and immunohistochemistry from a homogenate of skeletal muscle tissue. Clinical signs of our patient included hyperlactatemia, hypertrophic cardiomyopathy (HCM) and pulmonary hypertension, which led to early death at the age of 47 days without any other known accompanying signs. The finding of novel variants in the VARS2 gene expands the spectrum of known mutations and phenotype presentation. Based on our findings we recommend to consider possible mitochondriopathy and to include the analysis of the VARS2 gene in the genetic diagnostic algorithm in cases with early manifesting and rapidly progressing HCM with hyperlactatemia.

Wang Fei, Zhang Deyu, Zhang Dejiu, Li Peifeng, Gao Yanyan.

Mitochondria are one of the most important organelles in cells. Mitochondria are semi-autonomous organelles with their own genetic system, and can independently replicate, transcribe, and translate mitochondrial DNA. Translation initiation, elongation, termination, and recycling of the ribosome are four stages in the process of mitochondrial protein translation. In this process, mitochondrial protein translation factors and translation activators, mitochondrial RNA, and other regulatory factors regulate mitochondrial protein translation. Mitochondrial protein translation abnormalities are associated with a variety of diseases, including cancer, cardiovascular diseases, and nervous system diseases. Mutation or deletion of various mitochondrial protein translation factors and translation activators leads to abnormal mitochondrial protein translation. Mitochondrial tRNAs and mitochondrial ribosomal proteins are essential players during translation and mutations in genes encoding them represent a large fraction of mitochondrial diseases. Moreover, there is crosstalk between mitochondrial protein translation and cytoplasmic translation, and the imbalance between mitochondrial protein translation and cytoplasmic translation can affect some physiological and pathological processes. This review summarizes the regulation of mitochondrial protein translation factors, mitochondrial ribosomal proteins, mitochondrial tRNAs, and mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) in the mitochondrial protein translation process and its relationship with diseases. The regulation of mitochondrial protein translation and cytoplasmic translation in multiple diseases is also summarized.

Li Bowen, Chen Kun, Liu Fangfang, Zhang Juan, Chen Xihui, Chen Tangdong, Chen Qi, Yao Yan, Hu Weihong, Wang Li, Wu Yuanming.

Background: Mitochondrial aminoacyl-tRNA synthetases (mtARSs) catalyze the binding of specific amino acids to their cognate tRNAs and play an essential role in the synthesis of proteins encoded by mitochondrial DNA. Defects in mtARSs have been linked to human diseases, but their tissue-specific pathophysiology remains elusive. Here we examined the role of mitochondrial phenylalanyl-tRNA synthetase (FARS2) in developmental angiogenesis and its potential contribution to the pathogenesis of cardiovascular disease. Methods: Morpholinos were injected into fertilized zebrafish ova to establish an in vivo fars2 knock-down model. A visualization of the vasculature was achieved by using Tg (fli1: EGFP)y1 transgenic zebrafish. In addition, small interference RNAs (siRNAs) were transferred into human umbilical vein endothelial cells (HUVECs) to establish an in vitro FARS2 knock-down model. Cell motility, proliferation, and tubulogenesis were determined using scratch-wound CCK8, transwell-based migration, and tube formation assays. In addition, mitochondria- and non-mitochondria-related respiration were evaluated using a Seahorse XF24 analyzer and flow cytometry assays. Analyses of the expression levels of transcripts and proteins were performed using qRT-PCR and western blotting, respectively. Results: The knock-down of fars2 hampered the embryonic development in zebrafish and delayed the formation of the vasculature in Tg (fli1: EGFP)y1 transgenic zebrafish. In addition, the siRNA-mediated knock-down of FARS2 impaired angiogenesis in HUVECs as indicated by decreased cell motility and tube formation capacity. The knock-down of FARS2 also produced variable decreases in mitochondrial- and non-mitochondrial respiration in HUVECs and disrupted the regulatory pathways of angiogenesis in both HUVECs and zebrafish. Conclusion: Our current work offers novel insights into angiogenesis defects and cardiovascular diseases induced by FARS2 deficiency.

Li Hua, Wang Wei, Han Xiaodi, Zhang Yujia, Dai Lifang, Xu Manting, Deng Jie, Ding Changhong, Wang Xiaohui, Chen Chunhong, Yang Xiaofeng, Fang Fang.

Alpers' syndrome is an early inceptive neurodegenerative disorder with a poor prognosis, characterized by developmental regression, intractable epilepsy, and hepatic dysfunction. Candidate genes, such as POLG, PARS2, CARS2, FARS2, NARS2, and GABRB2 are distinguished and registered following research on large cohorts that portray the clinical phenotype in such patients using expanded access to whole-exome sequencing (WES). In this study, we aimed to better understand the electroencephalogram (EEG) characteristics and clinical phenotype of different genotypes of the Alpers' syndrome, which are currently insufficiently studied. We conducted a study on seven patients with Alpers' syndrome who received treatment in Beijing Children's Hospital and had a detailed clinical EEG. Furthermore, a substantial literature search of the Chinese Biomedical Literature Database, PubMed, and Cochrane Central Register of Controlled Trials EMBASE was also conducted, which revealed a total of 22 reported cases between January 2008 to January 2021. We analyzed 29 cases of Alpers' syndrome caused by different gene variants, of which 22 cases were related to POLG gene mutation and 7 cases were related to PARS2, CARS2, FARS2, NARS2, and GABRB2 gene mutation, and found that patients with distinctive pathogenic variants exhibited comparable phenotypes and similar EEG patterns. And we defined EEG characteristics found specifically in Alpers' syndrome. Rhythmic high-amplitude delta with superimposed (poly) spikes (RHADS) is a characteristic EEG finding in the early stages of Alpers' syndrome and is a kind of epileptic phenomenon, which can provide clues for the early diagnosis of the disease.

Kapoor Dipti, Majethia Purvi, Anand Aakanksha, Shukla Anju, Sharma Suvasini.

Biallelic variants in CARS2 (Cysteinyl-tRNA synthetase 2; MIM*612800), are known to cause combined oxidative phosphorylation deficiency 27 (MIM#616672), characterized by severe myoclonic epilepsy, neuroregression and complex movement disorders. To date, six individuals from five families have been reported with variants in CARS2. Herein, we present an 11-year-old boy who presented with neuroregression, dysfluent speech, aggressive behavior and tremors for 2 years. An electroencephalogram (EEG) revealed a highly abnormal background with generalized spike-and-wave discharges suggestive of Electrical Status Epilepticus during Sleep (ESES). A known homozygous c.655G > A(p.Ala219Thr) pathogenic variant in exon 6 of the CARS2(NM_024537.4) was identified on exome sequencing. Our report expands the electro-clinical spectrum of the phenotype with presence of severe behavioral abnormalities, continuous tremors and ESES pattern on EEG, not previously reported.

Hines Timothy J, Lutz Cathleen, Murray Stephen A, Burgess Robert W.

As sequencing technology improves, the identification of new disease-associated genes and new alleles of known genes is rapidly increasing our understanding of the genetic underpinnings of rare diseases, including neuromuscular diseases. However, precisely because these disorders are rare and often heterogeneous, they are difficult to study in patient populations. In parallel, our ability to engineer the genomes of model organisms, such as mice or rats, has gotten increasingly efficient through techniques such as CRISPR/Cas9 genome editing, allowing the creation of precision human disease models. Such in vivo model systems provide an efficient means for exploring disease mechanisms and identifying therapeutic strategies. Furthermore, animal models provide a platform for preclinical studies to test the efficacy of those strategies. Determining whether the same mechanisms are involved in the human disease and confirming relevant parameters for treatment ideally involves a human experimental system. One system currently being used is induced pluripotent stem cells (iPSCs), which can then be differentiated into the relevant cell type(s) for in vitro confirmation of disease mechanisms and variables such as target engagement. Here we provide a demonstration of these approaches using the example of tRNA-synthetase-associated inherited peripheral neuropathies, rare forms of Charcot-Marie-Tooth disease (CMT). Mouse models have led to a better understanding of both the genetic and cellular mechanisms underlying the disease. To determine if the mechanisms are similar in human cells, we will use genetically engineered iPSC-based models. This will allow comparisons of different CMT-associated GARS alleles in the same genetic background, reducing the variability found between patient samples and simplifying the availability of cell-based models for a rare disease. The necessity of integrating mouse and human models, strategies for accomplishing this integration, and the challenges of doing it at scale are discussed using recently published work detailing the cellular mechanisms underlying GARS-associated CMT as a framework.

Guerrero Carlos Mario, Bhatia Sonal.

Epilepsy of infancy with migrating focal seizures (EIMFS) is a rare devastating infantile epileptic encephalopathy that is characterized by a unique electroencephalopgraphy (EEG) signature of multifocal simultaneous seizures. Although no definite etiology is understood for EIMFS, mutations in certain ion channels, are implicated. Similarly, phenylalanyl-tRNA synthetase 2 (FARS2) deficiency is a rare, autosomal recessive disorder of dysfunctional mitochondrial translation causing refractory seizures, lactic acidosis, and developmental regression with a variety of EEG findings. However, an EIMFS-like pattern on EEG in FARS2 deficiency has only recently been reported once. Herein, we describe a seven-week-old male with seizures where whole exome sequencing (WES) revealed pathogenic FARS2 variants and an EIMFS pattern on EEG. This case provides an insight on a novel genetic mechanism for EIMFS. We encourage early consideration of WES when EIMFS is detected to evaluate for FARS2 deficiency, especially in the setting of profound lactic acidosis.

Barcia Giulia, Pandithan Dinusha, Ruzzenente Benedetta, Assouline Zahra, Pennisi Alessandra, Ormieres Clothilde, Besmond Claude, Roux Charles-Joris, Boddaert Nathalie, Desguerre Isabelle, Thorburn David R, Bratkovic Drago, Munnich Arnold, Bonnefont Jean-Paul, Rötig Agnès, Steffann Julie.

Not available.

Ribó Pau, Guo Yanru, Aranda Juan, Ainsua-Enrich Erola, Navinés-Ferrer Arnau, Guerrero Mario, Pascal Mariona, Cruz Cinthia de la, Orozco Modesto, Muñoz-Cano Rosa, Martin Margarita.

Anaphylaxis is a severe allergic reaction that can be lethal if not treated adequately. The underlying molecular mechanisms responsible for the severity are mostly unknown.

Minardi Raffaella, Licchetta Laura, Baroni Maria Chiara, Pippucci Tommaso, Stipa Carlotta, Mostacci Barbara, Severi Giulia, Toni Francesco, Bergonzini Luca, Carelli Valerio, Seri Marco, Tinuper Paolo, Bisulli Francesca.

Developmental and epileptic encephalopathies (DEE) encompass rare, sporadic neurodevelopmental disorders and usually with pediatric onset. As these conditions are characterized by marked clinical and genetic heterogeneity, whole-exome sequencing (WES) represents the strategy of choice for the molecular diagnosis. While its usefulness is well established in pediatric DEE cohorts, our study is aimed at assessing the WES feasibility in adult DEE patients who experienced a diagnostic odyssey prior to the advent of this technique. We analyzed exomes from 71 unrelated adult DEE patients, consecutively recruited from an Italian cohort for the EPI25 Project. All patients underwent accurate clinical and electrophysiological characterization. An overwhelming percentage (90.1%) had already undergone negative genetic testing. Variants were classified according to the American College of Medical Genetics and Genomics guidelines. WES disclosed 24 (likely) pathogenic variants among 18 patients in epilepsy-related genes with either autosomal dominant, recessive or X-linked inheritance. Ten of these were novel. We obtained a diagnostic yield of 25.3%, higher among patients with brain malformations, early-onset epilepsy and dysmorphisms. Despite a median diagnostic delay of 38.7 years, WES analysis provided the long-awaited diagnosis for 18 adult patients, which also had an impact on the clinical management of 50% of them.

Bedoyan Jirair K, Hage Rosemary, Shin Ha Kyung, Linard Sharon, Ferren Edwin, Ducich Nicole, Wilson Kirkland, Lehman April, Schillaci Lori-Anne, Manickam Kandamurugu, Mori Mari, Bartholomew Dennis, DeBrosse Suzanne, Cohen Bruce, Parikh Sumit, Kerr Douglas.

Pyruvate dehydrogenase complex deficiencies (PDCDs) and other mitochondrial disorders (MtDs) can (a) result in congenital lactic acidosis with elevations of blood alanine (Ala) and proline (Pro), (b) lead to decreased ATP production, and (c) result in high morbidity and mortality. With ~140,000 live births annually in Ohio and ~1 in 9,000 overall prevalence of MtDs, we estimate 2 to 3 newborns will have PDCD and 13 to 14 others likely will have another MtD annually. We compared the sensitivities of plasma amino acids (AA) Alanine (Ala), Alanine:Leucine (Ala:Leu), Alanine:Lysine and the combination of Ala:Leu and Proline:Leucine (Pro:Leu), in subjects with known primary-specific PDCD due to PDHA1 and PDHB mutations vs controls. Furthermore, in collaboration with the Ohio newborn screening (NBS) laboratory, we determined Ala and Pro concentrations in dried blood spot (DBS) specimens using existing NBS analytic approaches and evaluated Ala:Leu and Pro:Leu ratios from DBS specimens of 123,414 Ohio newborns in a 12-month period. We used the combined Ala:Leu ≥4.0 and Pro:Leu ≥3.0 ratio criterion from both DBS and plasma specimens as a screening tool in our retrospective review of newborn data. The screening tool applied on DBS and/or plasma (or serum) AA specimens successfully identified three unrelated females with novel de novoPDHA1 mutations, one male with a novel de novo X-linked HSD17B10 mutation, and a female with VARS2 mutations. This work lays the first step for piloting an NBS protocol in Ohio for identifying newborns at high risk for primary-specific PDCD and other MtDs who might benefit from neonatal diagnosis and early institution of known therapy and/or potential novel therapies for such disorders.

Yu Jing, Jiang Wei, Cao Li, Na Xiaoxue, Yang Jiyun.

Mutations in HARS2 are one of the genetic causes of Perrault syndrome, characterized by sensorineural hearing loss (SNHL) and ovarian dysfunction. Here, we identified two novel putative pathogenic variants of HARS2 in a Chinese family with sensorineural hearing loss including two affected male siblings, c.349G > A (p.Asp117Asn) and c.908 T > C (p.Leu303Pro), through targeted next-generation sequencing methods. The two affected siblings (13 and 11 years old) presented with early-onset, rapidly progressive SNHL. The affected siblings did not have any inner ear malformations or delays in gross motor development. Combined with preexisting clinical reports, Perrault syndrome may be latent in some families with non-syndromic deafness associated with HARS2 mutations. The definitive diagnosis of Perrault syndrome based on clinical features alone is a challenge in sporadic males, and preadolescent females with no signs of POI. Our findings further expanded the existing spectrum of HARS2 variants and Perrault syndrome phenotypes, which will assist in molecular diagnosis and genetic counselling of patients with HARS2 mutations.

Vargas Adriana, Rojas Jorge, Aivasovsky Ivan, Vergara Sergio, Castellanos Marianna, Prieto Carolina, Celis Luis.

The KARS gene encodes the aminoacyl-tRNA synthetase (aaRS), which activates and joins the lysin with its corresponding transfer RNA (tRNA) through the ATP-dependent aminoacylation of the amino acid. KARS gene mutations have been linked to diverse neurologic phenotypes, such as neurosensorial hearing loss, leukodystrophy, microcephaly, developmental delay or regression, peripheral neuropathy, cardiomyopathy, the impairment of the mitochondrial respiratory chain, and hyperlactatemia, among others. This article presents the case of a Colombian pediatric patient with two pathological missense variants in a compound heterozygous state in the KARS gene and, in addition to the case report, the paper reviews the literature for other cases of KARS1-associated leukodystrophy.

Monfrini Edoardo, Ronchi Dario, Franco Giulia, Garbellini Manuela, Straniero Letizia, Scola Elisa, Arienti Federica, Duga Stefano, Comi Giacomo Pietro, Bresolin Nereo, Di Fonzo Alessio.

Tucker Elena J, Rius Rocio, Jaillard Sylvie, Bell Katrina, Lamont Phillipa J, Travessa André, Dupont Juliette, Sampaio Lurdes, Dulon Jérôme, Vuillaumier-Barrot Sandrine, Whalen Sandra, Isapof Arnaud, Stojkovic Tanya, Quijano-Roy Susana, Robevska Gorjana, van den Bergen Jocelyn, Hanna Chloe, Simpson Andrea, Ayers Katie, Thorburn David R, Christodoulou John, Touraine Philippe, Sinclair Andrew H.

Perrault syndrome is a rare heterogeneous condition characterised by sensorineural hearing loss and premature ovarian insufficiency. Additional neuromuscular pathology is observed in some patients. There are six genes in which variants are known to cause Perrault syndrome; however, these explain only a minority of cases. We investigated the genetic cause of Perrault syndrome in seven affected individuals from five different families, successfully identifying the cause in four patients. This included previously reported and novel causative variants in known Perrault syndrome genes, CLPP and LARS2, involved in mitochondrial proteolysis and mitochondrial translation, respectively. For the first time, we show that pathogenic variants in PEX6 can present clinically as Perrault syndrome. PEX6 encodes a peroxisomal biogenesis factor, and we demonstrate evidence of peroxisomal dysfunction in patient serum. This study consolidates the clinical overlap between Perrault syndrome and peroxisomal disorders, and highlights the need to consider ovarian function in individuals with atypical/mild peroxisomal disorders. The remaining patients had variants in candidate genes such as TFAM, involved in mtDNA transcription, replication, and packaging, and GGPS1 involved in mevalonate/coenzyme Q10 biosynthesis and whose enzymatic product is required for mouse folliculogenesis. This genomic study highlights the diverse molecular landscape of this poorly understood syndrome.

Nielsen Søren K, Hansen Frederikke, Schrøder Henrik Daa, Wibrand Flemming, Gustafsson Finn, Mogensen Jens.

Xu Yan, Wu Bing-Bing, Wang Hui-Jun, Zhou Shui-Zhen, Cheng Guo-Qiang, Zhou Yuan-Feng.

The RARS2 gene encodes mitochondrial arginine-tRNA synthetase. Patients with variants of the RARS2 gene have pontocerebellar hypoplasia type 6 (PCH6), which is characterized by early onset seizures, progressive microcephaly, and developmental delay. PCH6 is a rare mitochondrial encephalopathy. To the best of our knowledge, the onset seizure type which the ictal video-electroencephalogram (VEEG) was compatible with early myoclonic encephalopathy (EME) has not been reported. Here we reported a term female neonate with EME caused by heterozygous variants of the RARS2 gene [NM_020320: exon10: c.773G>A (p. R258H) Maternal, NM_020320: exon4: c.282_285delAGAG Paternal]. Groan was the first symptom manifested, followed by metabolic disorders, and early marked cerebral atrophy. Metabolic disorders were corrected after feeding with extensively hydrolyzed protein formula. Seizures started at the 19th day of life. Interictal VEEG showed a suppression-burst (SB) pattern and ictal VEEG revealed myoclonic seizures that were compatible with early myoclonic encephalopathy (EME). She had frequent myoclonic seizures resistant to multi-antiepileptic drugs including phenobarbital, levetiracetam and oxcarbazepine, and soon developed into convulsive status epilepticus. At 7 months of age, she had severe developmental delay, and developed infantile spasms. Our case report expands the phenotypic spectrum of the PCH6, meanwhile, RARS2 should be considered be a causative gene in patients with EME.

A Almuqbil Mohammed, Vernon Hilary J, Ferguson Marcia, Kline Antonie D.

Biallelic pathogenic variants in mitochondrial aminoacyl-tRNA synthetase (mt-aaRS) PARS2 are associated with mitochondrial cytopathy. Here, we report the tenth case of an individual with biallelic PARS2 pathogenic variants, detected by exome sequencing (ES), and a literature review of ten cases of PARS2 mutations. Our patient displayed symptoms and clinical and laboratory findings similar to those reported previously with normal lactate levels. These symptoms included seizure disorder (which was managed with antiepileptics), developmental delay, and progressive cardiomyopathy which manifested at 19 years of age. The patient received a vitamin regimen including antioxidants as part of his treatment regimen. While further studies are required to conclusively establish the beneficial role of vitamin and cofactor administration on the mitochondria in PARS2-associated mitochondrial disease, these factors may have delayed the onset of cardiomyopathy.

Barbosa-Gouveia Sofia, González-Vioque Emiliano, Hermida Álvaro, Suarez María Unceta, Martínez-González María Jesús, Borges Filipa, Wintjes Liesbeth, Kappen Antonia, Rodenburg Richard, Couce María-Luz.

The EARS2 nuclear gene encodes mitochondrial glutamyl-tRNA synthetase, a member of the class I family of aminoacyl-tRNA synthetases (aaRSs) that plays a crucial role in mitochondrial protein biosynthesis by catalyzing the charging of glutamate to mitochondrial tRNA(Glu). Pathogenic EARS2 variants have been associated with a rare mitochondrial disorder known as leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL). The targeted sequencing of 150 nuclear genes encoding respiratory chain complex subunits and proteins implicated in the oxidative phosphorylation (OXPHOS) function was performed. The oxygen consumption rate (OCR), and the extracellular acidification rate (ECAR), were measured. The enzymatic activities of Complexes I-V were analyzed spectrophotometrically. We describe a patient carrying two heterozygous EARS2 variants, c.376C>T (p.Gln126*) and c.670G>A (p.Gly224Ser), with infantile-onset disease and a severe clinical presentation. We demonstrate a clear defect in mitochondrial function in the patient's fibroblasts, suggesting the molecular mechanism underlying the pathogenicity of these EARS2 variants. Experimental validation using patient-derived fibroblasts allowed an accurate characterization of the disease-causing variants, and by comparing our patient's clinical presentation with that of previously reported cases, new clinical and radiological features of LTBL were identified, expanding the clinical spectrum of this disease.

Yazici Gencdal Isil, Dincer Alp, Obuz Oguzhan, Yapici Zuhal.

Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) is caused by a recessive mutation in the DARS2 gene and can be recognized by specific magnetic resonance imaging patterns.

Zafar Saba, Shahzad Mohsin, Ishaq Rafaqat, Yousaf Ayesha, Shaikh Rehan S, Akram Javed, Ahmed Zubair M, Riazuddin Saima.

We report the underlying genetic causes of prelingual hearing loss (HL) segregating in eight large consanguineous families, ascertained from the Punjab province of Pakistan. Exome sequencing followed by segregation analysis revealed seven potentially pathogenic variants, including four novel alleles c.257G>A, c.6083A>C, c.89A>G, and c.1249A>G of CLPP, CDH23, COL4A5, and LARS2, respectively. We also identified three previously reported HL-causing variants (c.4528C>T, c.35delG, and c.1219T>C) of MYO15A, GJB2, and TMPRSS3 segregating in four families. All identified variants were either absent or had very low frequencies in the control databases. Our in silico analyses and 3-dimensional (3D) molecular modeling support the deleterious impact of these variants on the encoded proteins. Variants identified in MYO15A, GJB2, TMPRSS3, and CDH23 were classified as "pathogenic" or "likely pathogenic", while the variants in CLPP and LARS2 fall in the category of "uncertain significance" based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) variant pathogenicity guidelines. This paper highlights the genetic diversity of hearing disorders in the Pakistani population and reports the identification of four novel mutations in four HL families.

De Michele Giovanna, Galatolo Daniele, Lieto Maria, Maione Luigi, Cocozza Sirio, Santorelli Filippo Maria, Filla Alessandro.

Riley Lisa G, Rudinger-Thirion Joëlle, Frugier Magali, Wilson Meredith, Luig Melissa, Alahakoon Thushari Indika, Nixon Cheng Yee, Kirk Edwin P, Roscioli Tony, Lunke Sebastian, Stark Zornitza, Wierenga Klaas J, Palle Sirish, Walsh Maie, Higgs Emily, Arbuckle Susan, Thirukeswaran Shalini, Compton Alison G, Thorburn David R, Christodoulou John.

LARS2 variants are associated with Perrault syndrome, characterized by premature ovarian failure and hearing loss, and with an infantile lethal multisystem disorder: Hydrops, lactic acidosis, sideroblastic anemia (HLASA) in one individual. Recently we reported LARS2 deafness with (ovario) leukodystrophy. Here we describe five patients with a range of phenotypes, in whom we identified biallelic LARS2 variants: three patients with a HLASA-like phenotype, an individual with Perrault syndrome whose affected siblings also had leukodystrophy, and an individual with a reversible mitochondrial myopathy, lactic acidosis, and developmental delay. Three HLASA cases from two unrelated families were identified. All were males with genital anomalies. Two survived multisystem disease in the neonatal period; both have developmental delay and hearing loss. A 55-year old male with deafness has not displayed neurological symptoms while his female siblings with Perrault syndrome developed leukodystrophy and died in their 30s. Analysis of muscle from a child with a reversible myopathy showed reduced LARS2 and mitochondrial complex I levels, and an unusual form of degeneration. Analysis of recombinant LARS2 variant proteins showed they had reduced aminoacylation efficiency, with HLASA-associated variants having the most severe effect. A broad phenotypic spectrum should be considered in association with LARS2 variants.

Hu Chaoping, Li Xihua, Zhao Lei, Shi Yiyun, Zhou Shuizhen, Wu Bingbing, Wang Yi.

Mitochondrial disorders (MDs) are genetic ailments affecting all age groups. Epidemiological data and frequencies of gene mutations in pediatric patients in China are scarce. This retrospective study assessed 101 patients with suspected MDs treated at the Neurology Department of Children's Hospital, Fudan University, in 2011-2017. Mitochondrial (mtDNA) and nuclear (nDNA) samples were assessed by long-range polymerase chain reaction (PCR)-based whole mtDNA sequencing and whole exome sequencing (WES) for identifying pathogenic mutations. Muscle samples underwent various staining protocols and immunofluorescence for detecting selected proteins. Seventeen mutations in the MT-TL1, MT-COX2, MT-ND4, MT, tRNA TRNE, MT-TN, MT-TK, MT-ATP6, MT-ND6, MT-ND3 and MT-CO3 genes were identified in 39 patients, of which m.3243A > G, m.3303C > T, m.8993T > C/G, m.9176T > C, and m.10191T > C were most common. Mitochondrial myopathy and MELAS were most common for m.3243A > G mutation. Four novel mutations were detected, including m.9478insT, m.5666T > C, m.8265T > C, and m.8380-13600 deletion mutations related to Leigh syndrome, mitochondrial myopathy and KSS, respectively. Thirty-three mutations in the TK2, POLG, IBA57, HADHB, FBXL4, ALDH5A1, FOXRED1, TPK1, NDUFAF5, NDUFAF7, NDUFV1, CARS2, PDHA1, and HIBCH genes were identified in 19 patients, including 23 currently unknown. Higher rates of TK2, POLG, IBA57, and HADHB mutations were found in nDNA-mutated MD compared with the remaining individuals. Besides, IBA57 c.286T > C (p.Y96H), TK2 c.497A > T (p.D166V) founder mutations critically contributed to MDs. Comprehensive genomic analysis plays a critical role in pediatric MD diagnosis. These data summarize the relative frequencies of different gene mutations in a large Chinese population, and identified 23 novel MD-associated nDNA and 4 novel mtDNA mutations.

Wang Yong, Zhou Jing-Bo, Zeng Qi-Yu, Wu Siqi, Xue Mei-Qin, Fang Pengfei, Wang En-Duo, Zhou Xiao-Long.

Aminoacyl-tRNA synthetases (aaRSs) are ubiquitously expressed, essential enzymes, synthesizing aminoacyl-tRNAs for protein synthesis. Functional defects of aaRSs frequently cause various human disorders. Human KARS encodes both cytosolic and mitochondrial lysyl-tRNA synthetases (LysRSs). Previously, two mutations (c.1129G>A and c.517T>C) were identified that led to hearing impairment; however, the underlying biochemical mechanism is unclear. In the present study, we found that the two mutations have no impact on the incorporation of LysRS into the multiple-synthetase complex in the cytosol, but affect the cytosolic LysRS level, its tertiary structure, and cytosolic tRNA aminoacylation in vitro. As for mitochondrial translation, the two mutations have little effect on the steady-state level, mitochondrial targeting, and tRNA binding affinity of mitochondrial LysRS. However, they exhibit striking differences in charging mitochondrial tRNALys, with the c.517T>C mutant being completely deficient in vitro and in vivo. We constructed two yeast genetic models, which are powerful tools to test the in vivo aminoacylation activity of KARS mutations at both the cytosolic and mitochondrial levels. Overall, our data provided biochemical insights into the potentially molecular pathological mechanism of KARS c.1129G>A and c.517T>C mutations and provided yeast genetic bases to investigate other KARS mutations in the future.

Ville Dorothée, Lesca Gaetan, Labalme Audrey, Portes Vincent des, Arzimanoglou Alexis, de Bellescize Julitta.

Epilepsy of infancy with migrating focal seizures (EIMFS) is now a well-recognized early-onset syndrome included in the ILAE classification of the epilepsies. KCNT1 gain-of-function variants are identified in about half of patients. In the remaining cases, the underlying genetic component is far more heterogeneous with sporadic mutations occasionally reported in SCN1A, SCN2A, SLC12A5, TBC1D24, PLCB1, SLC25A22, and KCNQ2. Here, we report, for the first time, a homozygous deleterious variant in the FARS2 gene, identified using a 115-gene panel for monogenic epilepsies, in a patient with EIMFS. This boy was the second child born to healthy consanguineous parents. The first seizures occurred at six weeks of age. The patient rapidly developed severe epilepsy with focal discharges on EEG, migrating from one brain region to another, highly suggestive of EIMFS. At five months of age, he had daily multifocal clonic seizures and erratic myoclonic fits, which were not consistently related to spikes or spike-and-wave discharges. Neurological status was severely abnormal from onset and the patient died at 10 months of age from respiratory distress. Using the gene panel, a homozygous missense variant of FARS2 was identified, at Chr6 (GRCh37):g.5404829C>T, c.667C>T (NM_001318872.1), inherited from both parents, leading to an arginine-to-cysteine substitution, p.(Arg223Cys). FARS2 is a member of the mitochondrial aminoacyl tRNA transferase (ARS) enzymes. ARS variants are increasingly recognized causes of early-onset epileptic and neurodevelopmental encephalopathies, however, the associated epileptic phenotype is not completely described. This case shows that FARS2-related seizures can mimic EIMFS in the early stage of the disease. Furthermore, in the setting of migrating focal seizures of infancy, FARS2 should be considered as a further candidate gene, and increased lactate level and occurrence of refractory myoclonic seizures are possible key features to suspect FARS deficiency.

Felhi Rahma, Charif Majida, Sfaihi Lamia, Mkaouar-Rebai Emna, Desquiret-Dumas Valerie, Kallel Rim, Bris Céline, Goudenège David, Guichet Agnès, Bonneau Dominique, Procaccio Vincent, Reynier Pascal, Amati-Bonneau Patrizia, Hachicha Mongia, Fakhfakh Faiza, Lenaers Guy.

Mitochondrial diseases are a clinically heterogeneous group of multisystemic disorders that arise as a result of various mitochondrial dysfunctions. Autosomal recessive aARS deficiencies represent a rapidly growing group of severe rare inherited mitochondrial diseases, involving multiple organs, and currently without curative option. They might be related to defects of mitochondrial aminoacyl t-RNA synthetases (mtARS) that are ubiquitous enzymes involved in mitochondrial aminoacylation and the translation process. Here, using NGS analysis of 281 nuclear genes encoding mitochondrial proteins, we identified 4 variants in different mtARS in three patients from unrelated Tunisian families, with clinical features of mitochondrial disorders. Two homozygous variants were found in KARS (c.683C>T) and AARS2 (c.1150-4C>G), respectively in two patients, while two heterozygous variants in EARS2 (c.486-7C>G) and DARS2 (c.1456C>T) were concomitantly found in the third patient. Bio-informatics investigations predicted their pathogenicity and deleterious effects on pre-mRNA splicing and on protein stability. Thus, our results suggest that mtARS mutations are common in Tunisian patients with mitochondrial diseases.

Finsterer Josef.

The aim of this review is to summarize and discuss recent findings and new insights in the etiology and phenotype of metabolic myopathies. The review relies on a systematic literature review of recent publications. Metabolic myopathies are a heterogeneous group of disorders characterized by mostly inherited defects of enzymatic pathways involved in muscle cell metabolism. Metabolic myopathies present with either permanent (fixed) or episodic abnormalities, such as weakness, wasting, exercise-intolerance, myalgia, or an increase of muscle breakdown products (creatine-kinase, myoglobin) during exercise. Though limb and respiratory muscles are most frequently affected, facial, extra-ocular, and axial muscles may be occasionally also involved. Age at onset and prognosis vary considerably. There are multiple disease mechanisms and the pathophysiology is complex. Genes most recently related to metabolic myopathy include PGM1, GYG1, RBCK1, VMA21, MTO1, KARS, and ISCA2. The number of metabolic myopathies is steadily increasing. There is limited evidence from the literature that could guide diagnosis and treatment of metabolic myopathies. Treatment is limited to mainly non-invasive or invasive symptomatic measures. In conclusion, the field of metabolic myopathies is evolving with the more widespread availability and application of next generation sequencing technologies worldwide. This will broaden the knowledge about pathophysiology and putative therapeutic strategies for this group of neuromuscular disorders.

Demain Leigh A M, Gerkes Erica H, Smith Richard J H, Molina-Ramirez Leslie P, O'Keefe Raymond T, Newman William G.

HARS2 encodes mitochondrial histidyl-tRNA synthetase (HARS2), which links histidine to its cognate tRNA in the mitochondrial matrix. Biallelic variants in HARS2 are associated with Perrault syndrome, a rare recessive condition characterized by sensorineural hearing loss in both sexes and primary ovarian insufficiency in 46,XX females. Some individuals with Perrault syndrome have a broader phenotypic spectrum with neurological features, including ataxia and peripheral neuropathy. Here, we report a recurrent variant in HARS2 in association with sensorineural hearing loss. In affected individuals from three unrelated families, the variant HARS2 c.1439G>A p.(Arg480His) is present as a heterozygous variant in trans to a putative pathogenic variant. The low prevalence of the allele HARS2 c.1439G>A p.(Arg480His) in the general population and its presence in three families with hearing loss, confirm the pathogenicity of this variant and illustrate the presentation of Perrault syndrome as nonsyndromic hearing loss in males and prepubertal females.

Karstensen Helena Gásdal, Rendtorff Nanna Dahl, Hindbæk Lone Sandbjerg, Colombo Roberto, Stein Amelie, Birkebæk Niels Holtum, Hartmann-Petersen Rasmus, Lindorff-Larsen Kresten, Højland Allan Thomas, Petersen Michael Bjørn, Tranebjærg Lisbeth.

Biallelic variants in HARS2 have been associated with Perrault syndrome, characterized by sensorineural hearing impairment and premature ovarian insufficiency. Here we report three novel families, compound heterozygous for missense variants in HARS2 identified by next-generation sequencing, namely c.172A > G (p.Lys58Glu) and c.448C > T (p.Arg150Cys) identified in two sisters aged 13 and 16 years and their older brother, c.448C > T (p.Arg150Cys) and c.980G > A (p.Arg327Gln) identified in a seven year old girl, and finally c.137T > A (p.Leu46Gln) and c.259C > T (p.Arg87Cys) identified in a 32 year old woman. Clinically, all five individuals presented with early onset, rapidly progressive hearing impairment. Whereas the oldest female fulfilled the criteria of Perrault syndrome, the three younger females, aged 7, 13 and 16, all had apparently normal ovarian function, apart from irregular menstrual periods in the oldest female at age 16. The present report expands the list of HARS2 variants and helps gain further knowledge to the phenotype.

Hübers Annemarie, Huppertz Hans-Jürgen, Wortmann Saskia B, Kassubek Jan.

Al Balushi Aaisha, Matviychuk Diana, Jobling Rebekah, Salomons Gajja S, Blaser Susan, Mercimek-Andrews Saadet.

Mitochondrial aminoacyl-tRNA synthetases play a major role in protein translation, synthesis, and oxidative phosphorylation. We reviewed all patients diagnosed with mitochondrial aminoacyl-tRNA synthetase deficiencies diagnosed in a single neurometabolic clinic. We report five patients with mitochondrial aminoacyl-tRNA synthetase deficiencies including DARS2, EARS2, PARS2, and RARS2 deficiencies. Siblings with DARS2 deficiency presented with global developmental delay within the first year of life. DARS2, EARS2, PARS2, and RARS2 deficiencies were identified by whole exome sequencing. We report coagulation factor abnormalities in PARS2 deficiency for the first time. We also report symmetric increased signal intensity in globus pallidi in FLAIR images in brain MRI in EARS2 deficiency for the first time. One patient with RARS2 deficiency had compound heterozygous variants in RARS2. One of those variants was an intronic variant. We confirmed the pathogenicity by mRNA studies. Mitochondrial aminoacyl-tRNA synthetase deficiencies are diagnosed by molecular genetic investigations. Clinically available non-invasive biochemical investigations are non-specific for the diagnosis of mitochondrial aminoacyl-tRNA synthetase deficiencies. A combination of brain MRI features and molecular genetic investigations should be undertaken to confirm the diagnosis of mitochondrial aminoacyl-tRNA synthetase deficiencies.

Zou Songfeng, Mei Xueshuang, Yang Weiqiang, Zhu Rvfei, Yang Tao, Hu Hongyi.

Genetic causes of hearing loss are highly heterogeneous and often ethnically specific. In recent years, a variety of next-generation sequencing (NGS) panels have been developed to target deafness-causative genes. Whole-exome sequencing (WES), on the other hand, was rarely used for genetic testing for deafness. In this study, we performed WES in 38 sporadic Chinese Han deaf patients who have been pre-excluded for mutations in common deafness genes GJB2, SLC26A4 and MT-RNR1. Non-synonymous variants have been filtered based on their minor allele frequencies in public databases and ethnically matched controls. Bi-allelic pathogenic mutations in eight deafness genes, OTOF, TRIOBP, ESPN, HARS2, CDH23, MYO7A, USH1C and TJP2, were identified in 10 patients, with 17 mutations identified in this study not being associated with deafness previously. For the rest 28 patients, possibly bi-allelic rare non-synonymous variants in an averaged 4.7 genes per patient were identified as candidate pathogenic causes for future analysis. Our study showed that WES may provide a unified platform for genetic testing of deafness and enables retro-analyzing when new causative genes are revealed.

Tarnopolsky Mark, Kozenko Mariya, Jones Kevin.

Martinelli Simone, Cordeddu Viviana, Galosi Serena, Lanzo Ambra, Palma Eleonora, Pannone Luca, Ciolfi Andrea, Di Nottia Michela, Rizza Teresa, Bocchinfuso Gianfranco, Traversa Alice, Caputo Viviana, Farrotti Andrea, Carducci Claudia, Bernardini Laura, Cogo Susanna, Paglione Maria, Venditti Martina, Bentivoglio Annarita, Ng Joanne, Kurian Manju A, Civiero Laura, Greggio Elisa, Stella Lorenzo, Trettel Flavia, Sciaccaluga Miriam, Roseti Cristina, Carrozzo Rosalba, Fucile Sergio, Limatola Cristina, Di Schiavi Elia, Tartaglia Marco, Leuzzi Vincenzo.

To investigate the molecular cause(s) underlying a severe form of infantile-onset parkinsonism and characterize functionally the identified variants.

Meszarosova Anna Uhrova, Seeman Pavel, Jencik Jan, Drabova Jana, Cibochova Renata, Stellmachova Julia, Safka Brozkova Dana.

Hereditary spastic paraplegia (HSP or SPG) is a group of rare upper motor neuron diseases. As some ethnically-specific, disease-causing homozygous variants were described in the Czech Roma population, we hypotesised that some prevalent HSP-causing variant could exist in this population. Eight Czech Roma patients were found in a large group of Czech patients with suspected HSP and were tested using gene panel massively parallel sequencing (MPS). Two of the eight were diagnosed with SPG11 and SPG77, respectively. The SPG77 patient manifests a pure HSP phenotype, which is unusual for this SPG type. Both patients are compound heterozygotes for two different variants in the SPG11 (c.1603-1G>A and del ex. 16-18) and FARS2 (c.1082C>T and del ex.1-2) genes respectively; the three variants are novel. In order to find a potential ethnically-specific, disease-causing variant for HSP, we tested the heterozygote frequency of these variants among 130 anonymised DNA samples of Czech Roma individuals without clinical signs of HSP (HPS-negative). A novel deletion of ex.16-18 in the SPG11 gene was found in a heterozygous state in one individual in the HSP-negative group. Haplotype analysis showed that this individual and the patient with SPG11 shared the same haplotype. This supports the assumption that the identified SPG11 deletion could be a founder mutation in the Czech Roma population. In some Roma patients the disease may also be caused by two different biallelic pathogenic mutations.

Wu Di, Huang Weiyuan, Xu Zhenhang, Li Shuo, Zhang Jie, Chen Xiaohua, Tang Yan, Qiu Jinhong, Wang Zhixia, Duan Xuchu, Zhang Luping.

Nonsyndromic hearing loss is clinically and genetically heterogeneous. In this study, we characterized the clinical features of 12 Chinese Han deaf families in which mutations in common deafness genes GJB2, SLC26A4, and MT-RNR1 were excluded.

Lee Jin Sook, Yoo Taekyeong, Lee Moses, Lee Youngha, Jeon Eunyoung, Kim Soo Yeon, Lim Byung Chan, Kim Ki Joong, Choi Murim, Chae Jong-Hee.

Leigh syndrome (LS), the most common childhood mitochondrial disorder, has characteristic clinical and neuroradiologic features. Mutations in more than 75 genes have been identified in both the mitochondrial and nuclear genome, implicating a high degree of genetic heterogeneity in LS. To profile these genetic signatures and understand the pathophysiology of LS, we recruited 64 patients from 62 families who were clinically diagnosed with LS at Seoul National University Children's Hospital. Mitochondrial genetic analysis followed by whole-exome sequencing was performed on 61 patients. Pathogenic variants in mitochondrial DNA were identified in 18 families and nuclear DNA mutations in 22. The following 17 genes analyzed in 40 families were found to have genetic complexity: MTATP6, MTND1, MTND3, MTND5, MTND6, MTTK, NDUFS1, NDUFV1, NDUFAF6, SURF1, SLC19A3, ECHS1, PNPT1, IARS2, NARS2, VPS13D, and NAXE. Two treatable cases had biotin-thiamine responsive basal ganglia disease, and another three were identified as having defects in the newly recognized genes (VPS13D or NAXE). Variants in the nuclear genes that encoded mitochondrial aminoacyl tRNA synthetases were present in 27.3% of cases. Our findings expand the genetic and clinical spectrum of LS, showing genetic heterogeneity and highlighting treatable cases and those with novel genetic causes.

Carminho-Rodrigues Maria Teresa, Klee Phillipe, Laurent Sacha, Guipponi Michel, Abramowicz Marc, Cao-van Hélène, Guinand Nils, Paoloni-Giacobino Ariane.

Perrault syndrome is a rare recessive and genetically heterogeneous disorder characterized by sensorineural hearing loss in males and females and gonadal dysgenesis in females. Mutations in seven different genes have been identified: HARS2, HSD17B4, CLLP, C10orf, ERAL1, TWNK and LARS2. To date, 19 variants have been reported in 18 individuals with LARS2-Perrault syndrome.

Pan Zhaoyu, Xu Hongen, Tian Yongan, Liu Danhua, Liu Huanfei, Li Ruijun, Dou Qian, Zuo Bin, Zhai Rongqun, Tang Wenxue, Lu Wei.

Perrault syndrome (PRLTS4; OMIM# 615300) is a rare autosomal recessive disorder and only a few cases have been reported worldwide. We report a Chinese female characterized by sensorineural hearing loss and premature ovarian insufficiency.

Li Xiaojing, Peng Bingwei, Hou Chi, Li Jinliang, Zeng Yiru, Wu Wenxiao, Liao Yinting, Tian Yang, Chen Wen-Xiong.

Mitochondrial encephalomyopathy caused by bi-allelic deleterious variants in TARS2 is rare. To date, only two pedigrees were reported in the literature and the connection between the gene and disease needs further study.

Hotait Mostafa, Nasreddine Wassim, El-Khoury Riyad, Dirani Maya, Nawfal Omar, Beydoun Ahmad.

FARS2, a nuclear gene, encodes the mitochondrial phenylalanyl-tRNA synthetase (mtPheRS). Previous reports have described two distinct phenotypes linked to FARS2 gene mutation: an early onset epileptic encephalopathy and spastic paraplegia. This report describes a distinctive phenotype of FARS2-linked, juvenile onset refractory epilepsy, caused by a hemizygous mutation in a compound heterozygous state (p.V197M and exon 2 microdeletion). A 17-year- old woman with normal development presented with a super refractory focal motor status epilepticus. Only an emergency life-saving surgery aborted her status after all therapeutic interventions, including anesthesia, failed to control her seizures. Pathological and biochemical activities on muscle biopsy showed mitochondrial proliferation with enhanced isolated activities of complexes II and IV, suggestive of a compensatory mechanism for the bioenergetic deficiency. Postoperatively, the patient started experiencing focal aware motor seizures originating from the contralateral hemisphere after being seizure free for a few months. This report suggests a third phenotypic manifestation of FARS2 gene mutation.

Webb Bryn D, Diaz George A, Prasun Pankaj.

In eukaryotic cells, mitochondria perform the essential function of producing cellular energy in the form of ATP via the oxidative phosphorylation system. This system is composed of 5 multimeric protein complexes of which 13 protein subunits are encoded by the mitochondrial genome: Complex I (7 subunits), Complex III (1 subunit),Complex IV (3 subunits), and Complex (2 subunits). Effective mitochondrial translation is necessary to produce the protein subunits encoded by the mitochondrial genome (mtDNA). Defects in mitochondrial translation are known to cause a wide variety of clinical disease in humans with high-energy consuming organs generally most prominently affected. Here, we review several classes of disease resulting from defective mitochondrial translation including disorders with mitochondrial tRNA mutations, mitochondrial aminoacyl-tRNA synthetase disorders, mitochondrial rRNA mutations, and mitochondrial ribosomal protein disorders.

Muthiah Annapoorani, Housley Gary D, Klugmann Matthias, Fröhlich Dominik.

Aminoacyl-tRNA synthetases (ARSs) accurately charge tRNAs with their respective amino acids. As such, they are vital for the initiation of cytosolic and mitochondrial protein translation. These enzymes have become increasingly scrutinized in recent years for their role in neurodegenerative disorders caused by the mutations of ARS-encoding genes. This review focuses on two such genes-DARS1 and DARS2-which encode cytosolic and mitochondrial aspartyl-tRNA synthetases, and the clinical conditions associated with mutations of these genes. We also describe attempts made at modeling these conditions in mice, which have both yielded important mechanistic insights. Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a disease caused by a range of mutations in the DARS2 gene, initially identified in 2003. Ten years later, hypomyelination with brainstem and spinal cord involvement and leg spasticity (HBSL), caused by mutations of cytosolic DARS1, was discovered. Multiple parallels have been drawn between the two conditions. The Magnetic Resonance Imaging (MRI) patterns are strikingly similar, but still set these two conditions apart from other leukodystrophies. Clinically, both conditions are characterized by lower limb spasticity, often associated with other pyramidal signs. However, perhaps due to earlier detection, a wider range of symptoms, including peripheral neuropathy, as well as visual and hearing changes have been described in LBSL patients. Both HBSL and LBSL are spectrum disorders lacking genotype to phenotype correlation. While the fatal phenotype of Dars1 or Dars2 single gene deletion mouse mutants revealed that the two enzymes lack functional redundancy, further pursuit of disease modeling are required to shed light onto the underlying disease mechanism, and enable examination of experimental treatments, including gene therapies.

Al-Jaroudi Dania, Enabi Saed, AlThagafi Malak Sameer.

Perrault syndrome is a rare autosomal recessive disorder that affects both males and females. The syndrome causes deafness in males, however females display gonadal dysgenesis along with sensorineural hearing loss. Herein, we present a 27-year-old female patient who is deaf and mute along with primary amenorrhea. Hormonal assays revealed hypergonadotropic hypogonadism and the karyotype was 46 XX. Pelvic ultrasound described a hypoplastic uterus and streak ovaries. MRI of the spine showed degenerative discs and Tarlov cysts. Whole exome sequencing identified a LARS2 mutation and the patient was diagnosed with Perrault syndrome type four (PRLTS4).

Shakya Sunil, Kumari Renu, Suroliya Varun, Tyagi Nishu, Joshi Aditi, Garg Ajay, Singh Inder, Kalikavil Puthanveedu Divya, Cherian Ajith, Mukerji Mitali, Srivastava Achal K, Faruq Mohammed.

Over 100 genetically distinct causal known loci for hereditary ataxia phenotype poses a challenge for diagnostic work-up for ataxia patients in a clinically relevant time and precision. In the present study using next-generation sequencing, we have investigated pathogenic variants in early-onset cerebellar ataxia cases using whole exome sequencing in singleton/family-designed and targeted gene-panel sequencing. A total of 98 index patients were clinically and genetically (whole exome sequencing (WES) in 16 patients and targeted gene panel of 41 ataxia causing genes in 82 patients) evaluated. Four families underwent WES in family based design. Overall, we have identified 24 variants comprising 20 pathogenic and four likely-pathogenic both rare/novel, variations in 21 early onset cerebellar ataxia patients. Among the identified variations, SACS (n = 7) and SETX (n = 6) were frequent, while ATM (n = 2), TTPA (n = 2) and other rare loci were observed. We have prioritized novel pathogenic variants in RARS2 and FA2H loci through family based design in two out of four families.

Fan Wenlu, Zheng Jing, Kong Wanzhong, Cui Limei, Aishanjiang Maerhaba, Yi Qiuzi, Wang Min, Cang Xiaohui, Tang Xiaowen, Chen Ye, Mo Jun Qin, Sondheimer Neal, Ge Wanzhong, Guan Min-Xin.

Nuclear modifier genes have been proposed to modify the phenotypic expression of mitochondrial DNA mutations. Using a targeted exome-sequencing approach, here we found that the p.191Gly>Val mutation in mitochondrial tyrosyl-tRNA synthetase 2 (YARS2) interacts with the tRNASer(UCN) 7511A>G mutation in causing deafness. Strikingly, members of a Chinese family bearing both the YARS2 p.191Gly>Val and m.7511A>G mutations displayed much higher penetrance of deafness than those pedigrees carrying only the m.7511A>G mutation. The m.7511A>G mutation changed the A4:U69 base-pairing to G4:U69 pairing at the aminoacyl acceptor stem of tRNASer(UCN) and perturbed tRNASer(UCN) structure and function, including an increased melting temperature, altered conformation, instability, and aberrant aminoacylation of mutant tRNA. Using lymphoblastoid cell lines derived from symptomatic and asymptomatic members of these Chinese families and control subjects, we show that cell lines harboring only the m.7511A>G or p.191Gly>Val mutation revealed relatively mild defects in tRNASer(UCN) or tRNATyr metabolism, respectively. However, cell lines harboring both m.7511A>G and p.191Gly>Val mutations displayed more severe defective aminoacylations and lower tRNASer(UCN) and tRNATyr levels, aberrant aminoacylation, and lower levels of other tRNAs, including tRNAThr, tRNALys, tRNALeu(UUR), and tRNASer(AGY), than those in the cell lines carrying only the m.7511A>G or p.191Gly>Val mutation. Furthermore, mutant cell lines harboring both m.7511A>G and p.191Gly>Val mutations exhibited greater decreases in the levels of mitochondrial translation, respiration, and mitochondrial ATP and membrane potentials, along with increased production of reactive oxygen species. Our findings provide molecular-level insights into the pathophysiology of maternally transmitted deafness arising from the synergy between tRNASer(UCN) and mitochondrial YARS mutations.

Fine Amena Smith, Nemeth Christina L, Kaufman Miriam L, Fatemi Ali.

The mitochondrial aminoacyl-tRNA synthetase proteins (mt-aaRSs) are a group of nuclear-encoded enzymes that facilitate conjugation of each of the 20 amino acids to its cognate tRNA molecule. Mitochondrial diseases are a large, clinically heterogeneous group of disorders with diverse etiologies, ages of onset, and involved organ systems. Diseases related to mt-aaRS mutations are associated with specific syndromes that affect the central nervous system and produce highly characteristic MRI patterns, prototypically the DARS2, EARS, and AARS2 leukodystrophies, which are caused by mutations in mitochondrial aspartyl-tRNA synthetase, mitochondria glutamate tRNA synthetase, and mitochondrial alanyl-tRNA synthetase, respectively. BODY: The disease patterns emerging for these leukodystrophies are distinct in terms of the age of onset, nature of disease progression, and predominance of involved white matter tracts. In DARS2 and EARS2 disorders, earlier disease onset is typically correlated with more significant brain abnormalities, rapid neurological decline, and greater disability. In AARS2 leukodystrophy cases reported thus far, there is nearly invariable progression to severe disability and atrophy of involved brain regions, often within a decade. Although most mutations are compound heterozygous inherited in an autosomal recessive fashion, homozygous variants are found in each disorder and demonstrate high phenotypic variability. Affected siblings manifest disease on a wide spectrum.

Nemeth Christina L, Tomlinson Sophia N, Rosen Melissa, O'Brien Brett M, Larraza Oscar, Jain Mahim, Murray Connor F, Marx Joel S, Delannoy Michael, Fine Amena S, Wu Dan, Trifunovic Aleksandra, Fatemi Ali.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare, slowly progressive white matter disease caused by mutations in the mitochondrial aspartyl-tRNA synthetase (mt-AspRS, or DARS2). While patients show characteristic MRI T2 signal abnormalities throughout the cerebral white matter, brainstem, and spinal cord, the phenotypic spectrum is broad and a multitude of gene variants have been associated with the disease. Here, Dars2 disruption in CamKIIα-expressing cortical and hippocampal neurons results in slowly progressive increases in behavioral activity at five months, and culminating by nine months as severe brain atrophy, behavioral dysfunction, reduced corpus callosum thickness, and microglial morphology indicative of neuroinflammation. Interestingly, RNAseq based gene expression studies performed prior to the presentation of this severe phenotype reveal the upregulation of several pathways involved in immune activation, cytokine production and signaling, and defense response regulation. RNA transcript analysis demonstrates that activation of immune and cell stress pathways are initiated in advance of a behavioral phenotype and cerebral deficits. An understanding of these pathways and their contribution to significant neuronal loss in CamKII-Dars2 deficient mice may aid in deciphering mechanisms of LBSL pathology.

Chin Hui-Lin, Goh Denise Li-Meng, Wang Furene Sijia, Tay Stacey Kiat Hong, Heng Chew Kiat, Donnini Claudia, Baruffini Enrico, Pines Ophry.

The VARS2 gene encodes a mitochondrial valyl-transfer RNA synthetase which is used in mitochondrial translation. To date, several patients with VARS2 pathogenic variants have been described in the literature. These patients have features of lactic acidosis with encephalomyopathy. We present a case of an infant with lactic acidosis, failure to thrive, and severe primary pulmonary hypertension who was found to be a compound heterozygote for two novel VARS2 variants (c.1940C>T, p.(Thr647Met) and c.2318G>A, p.(Arg773Gln)). The patient was treated with vitamin supplements and a carbohydrate-restricted diet. The lactic acidosis and failure to thrive resolved, and he showed good growth and development. Functional studies and molecular analysis employed a yeast model system and the VAS1 gene (yeast homolog of VARS2). VAS1 genes harboring either one of two mutations corresponding to the two novel variants in the VARS2 gene, exhibited partially reduced function in haploid yeast strains. A combination of both VAS1 variant alleles in a diploid yeast cell exhibited a more significant decrease in oxidative metabolism-dependent growth and in the oxygen consumption rate (reminiscent of the patient who carries two mutant VARS2 alleles). Our results demonstrate the pathogenicity of the biallellic novel VARS2 variants. KEY MESSAGES: • A case of an infant who is a compound heterozygote for two novel VARS2 variants. • This infant displayed lactic acidosis, failure to thrive, and pulmonary hypertension. • Treatment of the patient with a carbohydrate-restricted diet resulted in good growth and development. • Studies with the homologous yeast VAS1 gene showed reduced function of corresponding single mutant in haploid yeast strains. • A combination of both VAS1 variant alleles in diploid yeast exhibited a more significant decrease in function, thereby confirming the pathogenicity of the biallellic novel VARS2 variants.

Prasun Pankaj, Mintz Cassie, Cork Emalyn, Naidich Thomas P, Webb Bryn D.

Seo Go Hun, Oh Arum, Kim Eun Na, Lee Yeonmi, Park Jumi, Kim Taeho, Lim Young-Min, Kim Gu-Hwan, Kim Chong Jai, Yoo Han-Wook, Kang Eunju, Lee Beom Hee.

Whole exome sequencing (WES) is an effective tool for the genetic diagnosis of mitochondrial disorders due to various nuclear genetic defects. In this study, three patients affected by extremely rare mitochondrial disorders caused by nuclear genetic defects are described. The medical records of each patient were reviewed to obtain clinical symptoms, results of biochemical and imaging studies, and muscle biopsies. WES and massive parallel sequencing of whole mtDNA were performed for each patient. The oxygen consumption rate (OCR) and complex activity I and IV was measured. Patients 1 and 2 had exhibited global developmental delay and seizure since early infancy. Blood lactate, the lactate-to-pyruvate ratio, and urinary excretion of Krebs cycle intermediates were markedly elevated. Patient 1 also was noted for ophthalmoplegia. Patient 2 had left ventricular hypertrophy and ataxia. Patient 3 developed dysarthria, gait disturbance, and right-side weakness at age 29. Brain magnetic resonance imaging demonstrated abnormal signal intensity involving the bilateral thalami, midbrain, or pons. Based on WES, patient 1 had p.Glu415Gly and p.Arg484Trp variants in MTO1. In patient 2, p.Gln111ThrfsTer5 and RNA mis-splicing were identified in TSFM. Patient 3 carried p.Met151Thr and p.Met246Lys variants in AARS2. Skin fibroblasts of three patients exhibited decreased OCRs and complex 1 activity, and mitochondrial DNA was normal. These results demonstrate the utility of WES for identifying the genetic cause of extremely rare mitochondrial disorders, which has implications for genetic counseling.

Chen Zhongyun, Zhang Yan.

FARS2 encodes mitochondrial phenylalanyl transfer ribonucleic acid (RNA) synthetase and is implicated in autosomal recessive combined oxidative phosphorylation deficiency 14. The clinical manifestation can be divided into early onset epileptic phenotype and spastic paraplegia phenotype. The purpose of this study was to report a case of juvenile manifesting refractory epilepsy caused by two novel compound heterozygous mutations in the FARS2 gene. Microscopic and histochemical examination as well as next-generation sequencing and reconstruction of the three-dimensional structure of FARS2 protein were performed. A 17-year-old man with no developmental delays suffered from generalized tonic-clonic convulsion since 12 years of age and developed refractory status epilepticus 5 years later. No specific etiology was found following brain imaging, muscle biopsy and metabolic studies. DNA sequencing identified two novel compound heterozygous mutations in FARS2, (p.V197M and p.F402S), derived from each parents, respectively. These mutations affected the structure or thermodynamic stability of the protein. This is a case report of juvenile-onset refractory epilepsy caused by two novel compound heterozygous mutations in the FARS2 gene. This case confirms and expands the clinicalphenotype and the genotypic spectrum of the FARS2 gene.

Ruzman Lucija, Kolic Ivana, Radic Nisevic Jelena, Ruzic Barsic Antonija, Skarpa Prpic Ingrid, Prpic Igor.

Background: Mitochondrial disorders are heterogeneous clinical syndromes caused by defective activity in the mitochondrial respiratory chain, resulting in a faulty oxidative phosphorylation system. These inherited disorders are individually rare, and furthermore they are phenotypic variables. The genetically characterized mitochondrial disorders are rarely associated with epileptic encephalopathies.Case presentation: We present the clinical phenotype, biochemical analysis, and electrographic and neuro-radiological features of a 5-month-old girl with epileptic encephalopathy, microcephaly, severe psychomotor delay, hypertrophic cardiomyopathy, and abnormal MRI scan. Using whole-genome sequencing technique, compound heterozygous mutations of the VARS2 gene were revealed, with one previously unreported frameshift mutation.Conclusion: Our report extends the phenotypic spectrum of VARS2-related disorders with an initial presentation of epileptic encephalopathy and early death due to malignant arrhythmia.

Pergande Matthias, Motameny Susanne, Özdemir Özkan, Kreutzer Mona, Wang Haicui, Daimagüler Hülya-Sevcan, Becker Kerstin, Karakaya Mert, Ehrhardt Harald, Elcioglu Nursel, Ostojic Slavica, Chao Cho-Ming, Kawalia Amit, Duman Özgür, Koy Anne, Hahn Andreas, Reimann Jens, Schoner Katharina, Schänzer Anne, Westhoff Jens H, Schwaibold Eva Maria Christina, Cossee Mireille, Imbert-Bouteille Marion, von Pein Harald, Haliloglu Göknur, Topaloglu Haluk, Altmüller Janine, Nürnberg Peter, Thiele Holger, Heller Raoul, Cirak Sebahattin.

Fetal akinesia has multiple clinical subtypes with over 160 gene associations, but the genetic etiology is not yet completely understood.

Kuo Molly E, Antonellis Anthony, Shakkottai Vikram G.

Mutations in the mitochondrial alanyl-tRNA synthetase gene, AARS2, have been reported to cause leukoencephalopathy associated with early ovarian failure, a clinical presentation described as "ovarioleukodystrophy." We present a sibling pair: one with cerebellar ataxia and one with vision loss and cognitive impairment in addition to ataxia. Neither shows evidence of leukoencephalopathy on MRI imaging. Exome sequencing revealed that both siblings are compound heterozygous for AARS2 variants (p.Phe131del and p.Ile328Met). Yeast complementation assays indicate that p.Phe131del AARS2 dramatically impairs gene function and that p.Ile328Met AARS2 is a hypomorphic allele. This work expands the phenotypic spectrum of AARS2-associated disease to include ataxia without leukoencephalopathy.

Song Chengyuan, Peng Linliu, Wang Shengjun, Liu Yiming.

Leukodystrophies are genetic disorders leading to progressive white matter degeneration in the central nervous system. Mitochondrial aminoacyl tRNA synthase protein is encoded by the nuclear gene AARS2. An autosomal recessive mutation in this gene has been linked to AARS2 mutation-related adult-onset leukodystrophy (AARS2-L) or infantile mitochondrial cardiomyopathy. To date, only 16 AARS2-L cases have been reported in English literature. Thus, the clinical and genetic characteristics of this disease remain to be defined. Through whole-exome sequencing, we identified a Chinese patient with leukodystrophy related to two novel compounds heterozygous mutation in AARS2 (c.965 G > A, p.R322H; c.334 G > C, p.G112R). These two compounds heterozygous variants in AARS2 gene co-segregated with disease in his family. And pyramidal tracts in the spinal cord were involved. Our findings have important implications on genetic counseling for any case with leukodystrophy and extend the mutational spectrum in AARS2 gene.

Abu Diab Alaa, AlTalbishi Ala'a, Rosin Boris, Kanaan Moien, Kamal Lara, Swaroop Anand, Chowers Itay, Banin Eyal, Sharon Dror, Khateb Samer.

To identify the accurate clinical diagnosis of rare syndromic inherited retinal diseases (IRDs) based on the combination of clinical and genetic analyses.

Forman Eva B, Gorman Kathleen M, Ennis Sean, King Mary D.

Herein we present two siblings with hereditary spastic paraplegia caused by novel compound heterozygous variant and deletion in FARS2 and expansion of the disease spectrum to include dysphonia.

Nevanlinna Viivi, Konovalova Svetlana, Ceulemans Berten, Muona Mikko, Laari Anni, Hilander Taru, Gorski Katarin, Valanne Leena, Anttonen Anna-Kaisa, Tyynismaa Henna, Courage Carolina, Lehesjoki Anna-Elina.

Pontocerebellar hypoplasia type 6 (PCH6) is a rare infantile-onset progressive encephalopathy caused by biallelic mutations in RARS2 that encodes the mitochondrial arginine-tRNA synthetase enzyme (mtArgRS). The clinical presentation overlaps that of PEHO syndrome (Progressive Encephalopathy with edema, Hypsarrhythmia and Optic atrophy). The proband presented with severe intellectual disability, epilepsy with varying seizure types, optic atrophy, axial hypotonia, acquired microcephaly, dysmorphic features and progressive cerebral and cerebellar atrophy and delayed myelination on MRI. The presentation had resemblance to PEHO syndrome but sequencing of ZNHIT3 did not identify pathogenic variants. Subsequent whole genome sequencing revealed novel compound heterozygous variants in RARS2, a missense variant affecting a highly conserved amino acid and a frameshift variant with consequent degradation of the transcript resulting in decreased mtArgRS protein level confirming the diagnosis of PCH6. Features distinguishing the proband's phenotype from PEHO syndrome were later appearance of hypotonia and elevated lactate levels in blood and cerebrospinal fluid. On MRI the proband presented with more severe supratentorial atrophy and lesser degree of abnormal myelination than PEHO syndrome patients. The study highlights the challenges in clinical diagnosis of patients with neonatal and early infantile encephalopathies with overlapping clinical features and brain MRI findings.

Zhou Yiran, Chen Beili, Li Lin, Pan Hong, Liu Beihong, Li Tengyan, Wang Ruyi, Ma Xu, Wang Binbin, Cao Yunxia.

To explore the candidate pathogenic gene in a premature ovarian insufficiency (POI) proband from a consanguineous marriage and detect the potential effects of mutation on cellular energy metabolism.

Maffezzini Camilla, Laine Isabelle, Dallabona Cristina, Clemente Paula, Calvo-Garrido Javier, Wibom Rolf, Naess Karin, Barbaro Michela, Falk Anna, Donnini Claudia, Freyer Christoph, Wredenberg Anna, Wedell Anna.

Mutations in mitochondrial aminoacyl tRNA synthetases form a subgroup of mitochondrial disorders often only perturbing brain function by affecting mitochondrial translation. Here we report two siblings with mitochondrial disease, due to compound heterozygous mutations in the mitochondrial tryptophanyl-tRNA synthetase (WARS2) gene, presenting with severe neurological symptoms but normal mitochondrial function in skeletal muscle biopsies and cultured skin fibroblasts.

Chakraborty Shruti, Ibba Michael, Banerjee Rajat.

Mutations in nucleus-encoded mitochondrial aminoacyl-tRNA synthetases (mitaaRSs) lead to defects in mitochondrial translation affecting the expression and function of 13 subunits of the respiratory chain complex leading to diverse pathological conditions. Mutations in the FARS2 gene encoding human mitochondrial phenylalanyl-tRNA synthetase (HsmitPheRS) have been found to be associated with two different clinical representations, infantile Alpers encephalopathy and spastic paraplegia. Here we have studied three pathogenic mutants (Tyr144Cys, Ile329Thr, and Asp391Val) associated with Alpers encephalopathy to understand how these variants affect the biophysical properties of the enzyme. These mutants have already been reported to have reduced aminoacylation activity. Our study established that the mutants are significantly more thermolabile compared to the wild-type enzyme with reduced solubility in vitro. The presence of aggregation-prone insoluble HsmitPheRS variants could have a detrimental impact on organellar translation, and potentially impact normal mitochondrial function.

Begliuomini Chiara, Magli Giorgio, Di Rocco Maja, Santorelli Filippo M, Cassandrini Denise, Nesti Claudia, Deodato Federica, Diodato Daria, Casellato Susanna, Simula Delia M, Dessì Veronica, Eusebi Anna, Carta Alessandra, Sotgiu Stefano.

Mitochondrial respiratory chain consists of five complexes encoded by nuclear and mitochondrial genomes. Mitochondrial aminoacyl-tRNA synthetases are key enzymes in the synthesis of such complexes. Bi-allelic variants of VARS2, a nuclear gene encoding for valyl-tRNA (Val-tRNA) synthetase, are associated to several forms of mitochondrial encephalopathies or cardiomyoencephalopathies. Among these, the rare homozygous c.1100C > T (p.Thr367Ile) mutation variably presents with progressive developmental delay, axial hypotonia, limbs spasticity, drug-resistant epilepsy leading, in some cases, to premature death. Yet only six cases, of which three are siblings, harbouring this homozygous mutation have been described worldwide.

Srivastava Siddharth, Butala Ankur, Mahida Sonal, Richter John, Mu Weiyi, Poretti Andrea, Vernon Hilary, VanGerpen Jay, Atwal Paldeep S, Middlebrooks Erik H, Zee David S, Naidu SakkuBai.

Biallelic pathogenic variants in AARS2, a gene encoding the mitochondrial alanyl-tRNA synthetase, result in a spectrum of findings ranging from infantile cardiomyopathy to adult-onset progressive leukoencephalopathy. In this article, we present three unrelated individuals with novel compound heterozygous pathogenic AARS2 variants underlying diverse clinical presentations. Patient 1 is a 51-year-old man with adult-onset progressive cognitive, psychiatric, and motor decline and leukodystrophy. Patient 2 is a 34-year-old man with childhood-onset progressive tremor followed by the development of polyneuropathy, ataxia, and mild cognitive and psychiatric decline without leukodystrophy on imaging. Patient 3 is a 57-year-old woman with childhood-onset tremor and nystagmus which preceded dystonia, chorea, ataxia, depression, and cognitive decline marked by cerebellar atrophy and white matter disease. These cases expand the clinical heterogeneity of AARS2-related disorders, given that the first and third case represent some of the oldest known survivors of this disease, the second is adult-onset AARS2-related neurological decline without leukodystrophy, and the third is biallelic AARS2-related disorder involving a partial gene deletion.

Wang Jian-Yong, Chen Song-Fang, Zhang Hong-Qiu, Wang Meng-Yan, Zhu Jian-Hong, Zhang Xiong.

Leukodystrophy is a group of hereditary leukoencephalopathies predominantly affecting the white matter. Multiple genes and mutations have been reported to be associated with this disorder. Identification of pathogenic genes can facilitate diagnosis of leukodystrophy and development of therapeutic strategies.

Scheidecker Sophie, Bär Séverine, Stoetzel Corinne, Geoffroy Véronique, Lannes Béatrice, Rinaldi Bruno, Fischer Frédéric, Becker Hubert D, Pelletier Valérie, Pagan Cécile, Acquaviva-Bourdain Cécile, Kremer Stéphane, Mirande Marc, Tranchant Christine, Muller Jean, Friant Sylvie, Dollfus Hélène.

Mutations in genes encoding aminoacyl-tRNA synthetases have been reported in several neurological disorders. KARS is a dual localized lysyl-tRNA synthetase and its cytosolic isoform belongs to the multiple aminoacyl-tRNA synthetase complex (MSC). Biallelic mutations in the KARS gene were described in a wide phenotypic spectrum ranging from nonsyndromic deafness to complex impairments. Here, we report on a patient with severe neurological and neurosensory disease investigated by whole-exome sequencing and found to carry biallelic mutations c.683C>T (p.Pro228Leu) and c.871T>G (p.Phe291Val), the second one being novel, in the KARS gene. The patient presented with an atypical clinical presentation with an optic neuropathy not previously reported. At the cellular level, we show that cytoplasmic KARS was expressed at a lower level in patient cells and displayed decreased interaction with MSC. In vitro, these two KARS variants have a decreased aminoacylation activity compared with wild-type KARS, the p.Pro228Leu being the most affected. Our data suggest that dysfunction of cytoplasmic KARS resulted in a decreased level of translation of the nuclear-encoded lysine-rich proteins belonging to the respiratory chain complex, thus impairing mitochondria functions.

Sun Chong, Song Jie, Jiang Yanjun, Zhao Chongbo, Lu Jiahong, Li Yuxin, Wang Yin, Gao Mingshi, Xi Jianying, Luo Sushan, Li Meixia, Donaldson Kevin, Oprescu Stephanie N, Slavin Thomas P, Lee Sansan, Magoulas Pilar L, Lewis Andrea M, Emrick Lisa, Lalani Seema R, Niu Zhiyv, Landsverk Megan L, Walkiewicz Magdalena, Person Richard E, Mei Hui, Rosenfeld Jill A, Yang Yaping, Antonellis Anthony, Hou Ya-Ming, Lin Jie, Zhang Victor W.

To expand the clinical spectrum of lysyl-tRNA synthetase (KARS) gene-related diseases, which so far includes Charcot-Marie-Tooth disease, congenital visual impairment and microcephaly, and nonsyndromic hearing impairment.

Uittenbogaard Martine, Wang Hao, Zhang Victor Wei, Wong Lee-Jun, Brantner Christine A, Gropman Andrea, Chiaramello Anne.

In this study, we report the metabolic consequences of the m.1630 A > G variant in fibroblasts from the symptomatic proband affected with the mitochondrial encephalomyopathy lactic acidosis and stroke-like episode Syndrome and her asymptomatic mother. By long-range PCR followed by massively parallel sequencing of the mitochondrial genome, we accurately measured heteroplasmy in fibroblasts from the proband (89.6%) and her mother (94.8%). Using complementary experimental approaches, we show a functional correlation between manifestation of clinical symptoms and bioenergetic potential. Our mitochondrial morphometric analysis reveals a link between defects of mitochondrial cristae ultrastructure and symptomatic status. Despite near-homoplasmic level of the m.1630A > G variant, the mother's fibroblasts have a normal OXPHOS metabolism, which stands in contrast to the severely impaired OXPHOS response of the proband's fibroblasts. The proband's fibroblasts also exhibit glycolysis at near constitutive levels resulting in a stunted compensatory glycolytic response to offset the severe OXPHOS defect. Whole exome sequencing reveals the presence of a heterozygous nonsense VARS2 variant (p.R334X) exclusively in the proband, which removes two thirds of the VARS2 protein containing key domains interacting with the mt-tRNAval and may play a role in modulating the penetrance of the m.1630A > G variant despite similar near homoplasmic levels. Our transmission electron microscopy study also shows unexpected ultrastructural changes of chromatin suggestive of differential epigenomic regulation between the proband and her mother that may explain the differential OXPHOS response between the proband and her mother. Future study will decipher by which molecular mechanisms the nuclear background influences the penetrance of the m.1630 A > G variant causing MELAS.

Sirkis Daniel W, Geier Ethan G, Bonham Luke W, Karch Celeste M, Yokoyama Jennifer S.

In this review we highlight recent advances in the human genetics of frontotemporal dementia (FTD). In addition to providing a broad survey of genes implicated in FTD in the last several years, we also discuss variation in genes implicated in both hereditary leukodystrophies and risk for FTD (e.g., TREM2, TMEM106B, CSF1R, AARS2, NOTCH3).

van der Knaap Marjo S, Bugiani Marianna, Mendes Marisa I, Riley Lisa G, Smith Desiree E C, Rudinger-Thirion Joëlle, Frugier Magali, Breur Marjolein, Crawford Joanna, van Gaalen Judith, Schouten Meyke, Willems Marjolaine, Waisfisz Quinten, Mau-Them Frederic Tran, Rodenburg Richard J, Taft Ryan J, Keren Boris, Christodoulou John, Depienne Christel, Simons Cas, Salomons Gajja S, Mochel Fanny.

To describe the leukodystrophy caused by pathogenic variants in LARS2 and KARS, encoding mitochondrial leucyl transfer RNA (tRNA) synthase and mitochondrial and cytoplasmic lysyl tRNA synthase, respectively.

Musier-Forsyth Karin.

Aminoacyl-tRNA synthetases (ARSs) catalyze the attachment of specific amino acids to cognate tRNAs for use in protein synthesis. This historical function of ARSs and tRNAs is fairly well understood. However, ARSs and tRNAs also perform non-canonical functions that are continuing to be unveiled at a rapid pace. The expanded functions of these essential molecules of life range from roles in retroviral replication to stimulation of mTOR activity; DNA repair, splicing, transcriptional and translational regulation; and other aspects of cellular homeostasis. Furthermore, mutations in tRNAs and synthetases are known to drive human maladies, such as the neurodegenerative disorder Charcot-Marie-Tooth (CMT) disease along with other central nervous system dysfunctions, and cancer. This series of reviews focuses on the diseases that result from natural variations in human cytoplasmic tRNAs, as well as from mutations in mitochondrial tRNAs and ARSs. Ultimately, the exciting work in this rapidly emerging area may lead to new therapies for microbial and parasitic infections, cancer, and neurodegenerative diseases.

Finsterer Josef.

Finsterer Josef, Zarrouk-Mahjoub Sinda.

Takezawa Yusuke, Fujie Hiromi, Kikuchi Atsuo, Kure Shigeo.

Yelam Anudeep, Nagarajan Elanagan, Chuquilin Miguel, Govindarajan Raghav.

Leucoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a very rare autosomal recessive, slowly progressive neurological disorder characterised by distinctive clinical findings including cerebellar, pyramidal and dorsal column dysfunction. This is caused by a mutation in the DARS2 gene, which encodes mitochondrial aspartyl-tRNA synthetase. MRI shows distinctive abnormalities in the cerebral white matter and specific brain stem and spinal cord tracts. Here, we present a case of LBSL, with a novel c.1192-2A>G mutation.

Imai-Okazaki Atsuko, Kishita Yoshihito, Kohda Masakazu, Mizuno Yosuke, Fushimi Takuya, Matsunaga Ayako, Yatsuka Yukiko, Hirata Tomoko, Harashima Hiroko, Takeda Atsuhito, Nakaya Akihiro, Sakata Yasushi, Kogaki Shigetoyo, Ohtake Akira, Murayama Kei, Okazaki Yasushi.

Cardiomyopathy is a reported indicator of poor prognosis in children with mitochondrial disease. However, the association between prognosis and the genetic background of cardiomyopathy in children with mitochondrial disease has yet to be fully elucidated.

González-Serrano Ligia Elena, Chihade Joseph W, Sissler Marie.

Mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are essential components of the mitochondrial translation machinery. The correlation of mitochondrial disorders with mutations in these enzymes has raised the interest of the scientific community over the past several years. Most surprising has been the wide-ranging presentation of clinical manifestations in patients with mt-aaRS mutations, despite the enzymes' common biochemical role. Even among cases where a common physiological system is affected, phenotypes, severity, and age of onset varies depending on which mt-aaRS is mutated. Here we review work done thus far and propose a categorization of diseases based on tissue specificity that highlights emerging patterns. We further discuss multiple in vitro and in cellulo efforts to characterize the behavior of wildtype and mutant mt-aaRSs that have shaped hypotheses about the molecular causes of these pathologies. Much remains to do in order to complete our understanding of these proteins. We expect that futher work is likely to result in the discovery of new roles for the mt-aaRSs in addition to their fundamental function in mitochondrial translation, informing the development of treatment strategies and diagnoses.

Itoh Masayuki, Dai Hongmei, Horike Shin-Ichi, Gonzalez John, Kitami Yoshikazu, Meguro-Horike Makiko, Kuki Ichiro, Shimakawa Shuichi, Yoshinaga Harumi, Ota Yoko, Okazaki Tetsuya, Maegaki Yoshihiro, Nabatame Shin, Okazaki Shin, Kawawaki Hisashi, Ueno Naoto, Goto Yu-Ichi, Kato Yoichi.

The leukodystrophies cause severe neurodevelopmental defects from birth and follow an incurable and progressive course that often leads to premature death. It has recently been reported that abnormalities in aminoacyl t-RNA synthetase (ARS) genes are linked to various unique leukodystrophies and leukoencephalopathies. Aminoacyl t-RNA synthetase proteins are fundamentally known as the first enzymes of translation, catalysing the conjugation of amino acids to cognate tRNAs for protein synthesis. It is known that certain aminoacyl t-RNA synthetase have multiple non-canonical roles in both transcription and translation, and their disruption results in varied and complicated phenotypes. We clinically and genetically studied seven patients (six male and one female; aged 2 to 12 years) from five unrelated families who all showed the same phenotypes of severe developmental delay or arrest (7/7), hypotonia (6/7), deafness (7/7) and inability to speak (6/7). The subjects further developed intractable epilepsy (7/7) and nystagmus (6/6) with increasing age. They demonstrated characteristic laboratory data, including increased lactate and/or pyruvate levels (7/7), and imaging findings (7/7), including calcification and abnormal signals in the white matter and pathological involvement (2/2) of the corticospinal tracts. Through whole-exome sequencing, we discovered genetic abnormalities in lysyl-tRNA synthetase (KARS). All patients harboured the variant [c.1786C>T, p.Leu596Phe] KARS isoform 1 ([c.1702C>T, p.Leu568Phe] of KARS isoform 2) either in the homozygous state or compound heterozygous state with the following KARS variants, [c.879+1G>A; c.1786C>T, p.Glu252_Glu293del; p.Leu596Phe] ([c.795+1G>A; c.1702C>T, p.Glu224_Glu255del; p.Leu568Phe]) and [c.650G>A; c.1786C>T, p.Gly217Asp; p.Leu596Phe] ([c.566G>A; c.1702C>T, p.Gly189Asp; p.Leu568Phe]). Moreover, similarly disrupted lysyl-tRNA synthetase (LysRS) proteins showed reduced enzymatic activities and abnormal CNSs in Xenopus embryos. Additionally, LysRS acts as a non-canonical inducer of the immune response and has transcriptional activity. We speculated that the complex functions of the abnormal LysRS proteins led to the severe phenotypes in our patients. These KARS pathological variants are novel, including the variant [c.1786C>T; p.Leu596Phe] (c.1702C>T; p.Leu568Phe) shared by all patients in the homozygous or compound-heterozygous state. This common position may play an important role in the development of severe progressive leukodystrophy. Further research is warranted to further elucidate this relationship and to investigate how specific mutated LysRS proteins function to understand the broad spectrum of KARS-related diseases.

Tang Yi, Qin Qi, Xing Yi, Guo Dongmei, Di Li, Jia Jianping.

Mutations in the mitochondrial alanyl-transfer (t)RNA synthetase 2 (AARS2,OMIM:612035) have been linked to leukoencephalopathy recently. Till now, there have been 19 cases reported so far. However, the clinical and genetic characteristics of this disease are not fully understood. We reported an adult-onset male leukoencephalopathy patient related to novel AARS2 gene mutations and reviewed all previous cases regarding the clinical and genetic features of AARS2 leukoencephalopathy.

Kiraly-Borri Catherine, Jevon Gareth, Ji Weizhen, Jeffries Lauren, Ricciardi Jamie-Lee, Konstantino Monica, Ackerman Kate G, Lakhani Saquib A.

Variants in the mitochondrial alanyl-tRNA synthetase 2 gene AARS2 (OMIM 612035) are associated with infantile mitochondrial cardiomyopathy or later-onset leukoencephalopathy with premature ovarian insufficiency. Here, we report two newborn siblings who died soon after birth with primary pulmonary hypoplasia without evidence of cardiomyopathy. Whole-exome sequencing detected the same compound heterozygous AARS2 variants in both siblings (c.1774C>T, p.Arg592Trp and c.647dup, p.Cys218Leufs*6) that have previously been associated with infantile mitochondrial cardiomyopathy. Segregation analysis in the family confirmed carrier status of the parents and an unaffected sibling. To our knowledge, this is the first report of primary pulmonary hypoplasia in the absence of cardiomyopathy associated with recessive AARS2 variants and further defines the phenotypic spectrum associated with this gene.

Virdee Manveen, Swarnalingam Eroshini, Kozenko Mariya, Tarnopolsky Mark, Jones Kevin.

WARS2 encodes a tryptophanyl tRNA synthetase, which is involved in mitochondrial protein synthesis. Biallelic mutations in WARS2 are rare and have been associated with a spectrum of clinical presentations, including neurodevelopmental disorder with abnormal movements, lactic acidosis with or without seizures (NEMMLAS).

Tiosano Dov, Mears Jason A, Buchner David A.

Primary ovarian insufficiency (POI) is defined by the loss or dysfunction of ovarian follicles associated with amenorrhea before the age of 40. Symptoms include hot flashes, sleep disturbances, and depression, as well as reduced fertility and increased long-term risk of cardiovascular disease. POI occurs in ∼1% to 2% of women, although the etiology of most cases remains unexplained. Approximately 10% to 20% of POI cases are due to mutations in a single gene or a chromosomal abnormality, which has provided considerable molecular insight into the biological underpinnings of POI. Many of the genes for which mutations have been associated with POI, either isolated or syndromic cases, function within mitochondria, including MRPS22, POLG, TWNK, LARS2, HARS2, AARS2, CLPP, and LRPPRC. Collectively, these genes play roles in mitochondrial DNA replication, gene expression, and protein synthesis and degradation. Although mutations in these genes clearly implicate mitochondrial dysfunction in rare cases of POI, data are scant as to whether these genes in particular, and mitochondrial dysfunction in general, contribute to most POI cases that lack a known etiology. Further studies are needed to better elucidate the contribution of mitochondria to POI and determine whether there is a common molecular defect in mitochondrial function that distinguishes mitochondria-related genes that when mutated cause POI vs those that do not. Nonetheless, the clear implication of mitochondrial dysfunction in POI suggests that manipulation of mitochondrial function represents an important therapeutic target for the treatment or prevention of POI.

Lin Tsu-Kung, Chang Yung-Yee, Lin Hung-Yu, Liou Chia-Wei, Wang Pei-Wen, Chuang Jiin-Haur, Chen Shang-Der, Chuang Yao-Chung, Huang Sheng-Teng, Hsu Te-Yao, Peng Cheng-Huei, Lan Min-Yu.

Several inherited human diseases have been linked to mitochondrial aminoacyl-tRNA synthetases (mtARSs). Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a leukodystrophy caused by mutations in the DARS2 gene which encodes mitochondrial aspartyl-tRNA synthetase. As mitochondrial ARSs are key components of the mitochondrial translation apparatus, we investigated the effects of DARS2 mutations on mitochondrial functions and mitochondrial morphology in an LBSL patient. In fibroblasts from the patient with LBSL, biosynthesis of respiratory chain complex proteins encoded by mitochondrial DNA was decreased, while those encoded by nuclear DNA were not. Cellular oxygen consumption rates and respiratory control ratio were decreased in the LBSL patient; in addition, fragmentation of mitochondria was increased, while their tubular elongation and interconnectivity were decreased. Taken together, these findings suggest that DARS2 mutations impair translations of mitochondrial DNA-encoded respiratory chain complex proteins, consequently causing dysfunction of cellular respiration and impediment of mitochondrial dynamics, which highlights the role of mtARSs in the maintenance of normal mitochondrial bioenergetics and dynamics.

Wang Xingao, Wang Qun, Tang Hefei, Chen Bin, Dong Xiang, Niu Songtao, Li Shaowu, Shi Yuzhi, Shan Wei, Zhang Zaiqiang.

The white matter disease spectrum is associated with many genetic diseases, including AARS2, CADASIL, ALD, and others. In this study, to determine the novel alanyl-tRNA synthetase 2 mutation implicated in white matter disease, several families with an autosomal recessive inheritance pattern of white matter disease were analyzed by whole-exome sequencing. Variants were prioritized according to their rarity and pathogenic variants in genes already known to be associated with leukodystrophies and were confirmed by Sanger sequencing using standard protocols. We identified 5 rare variants (c.452T>C chr6:44279256 p.M151T, c.1871G>A chr6:44272054 p.W624X, c.802A>G chr6:44278128 p.M268V, c.1703-1704del chr6:-44272430-44272431 p.Q568fs, and c.179C>A chr6-44280882 p.P60H) with varying expression in 4 independent Chinese families with leukodystrophy. These single nucleotide variants (SNVs), or deletion mutations, each induced a frameshift, causing a missense mutation in alanyl-tRNA synthetase 2. These findings suggested that all mutations might contribute to the development of leukodystrophy in the examined family members. Combined with previous findings, our data confirmed that the novel mutations are located in leukodystrophy-related risk genes. We also summarized all the alanyl-tRNA synthetase 2 mutations related to the onset of leukodystrophies in adults.

Kim Eun-Joo, Kim Young-Eun, Jang Ja-Hyun, Cho Eun-Hae, Na Duk L, Seo Sang Won, Jung Na-Yeon, Jeong Jee H, Kwon Jay C, Park Kee Hyung, Park Kyung Won, Lee Jae-Hong, Roh Jee Hoon, Kim Hee-Jin, Yoon Soo Jin, Choi Seong Hye, Jang Jae-Won, Ki Chang-Seok, Kim Seung Hyun.

To identify pathogenic variants in 107 Korean patients with sporadic frontotemporal dementia (FTD), 46 genes related to FTD, amyotrophic lateral sclerosis, and other dementias were screened by next-generation sequencing. Hexanucleotide repeats in C9orf72 gene were also tested by repeat-primed polymerase chain reaction. Next-generation sequencing revealed one known pathogenic variant (c.708+1G>A) in the GRN gene in a patient with behavioral variant FTD (bvFTD). In addition, a novel in-frame deletion (c.2675_2683del) in the CSF1R gene was identified in a patient with bvFTD who had severe bifrontal atrophy with frontal subcortical white matter changes. Novel compound heterozygous variants in the AARS2 gene, c.1040+1G>A and c.636G>A (p.Met212Ile), were found in a patient with bvFTD. Forty-six variants of uncertain significance were detected in other patients. None of the patients had expanded hexanucleotide repeats in C9orf72. These results show that pathogenic variants of known FTD genes are rare in Korean FTD patients but the CSF1R and AARS2 genes should be screened for a genetic diagnosis of FTD or other dementias.

Ruzzenente Benedetta, Assouline Zahra, Barcia Giulia, Rio Marlène, Boddaert Nathalie, Munnich Arnold, Rötig Agnès, Metodiev Metodi D.

Aminoacyl-tRNA synthetases are ubiquitous enzymes, which universally charge tRNAs with their cognate amino acids for use in cytosolic or organellar translation. In humans, mutations in mitochondrial tRNA synthetases have been linked to different tissue-specific pathologies. Mutations in the KARS gene, which encodes both the cytosolic and mitochondrial isoform of lysyl-tRNA synthetase, cause predominantly neurological diseases that often involve deafness, but have also been linked to cardiomyopathy, developmental delay, and lactic acidosis. Using whole exome sequencing, we identified two compound heterozygous mutations, NM_001130089.1:c.683C>T p.(Pro228Leu) and NM_001130089.1:c.1438del p.(Leu480TrpfsX3), in a patient presenting with sensorineural deafness, developmental delay, hypotonia, and lactic acidosis. Nonsense-mediated mRNA decay eliminated the truncated mRNA transcript, rendering the patient hemizygous for the missense mutation. The c.683C>T mutation was previously described, but its pathogenicity remained unexamined. Molecular characterization of patient fibroblasts revealed a multiple oxidative phosphorylation deficiency due to impaired mitochondrial translation, but no evidence of inhibition of cytosolic translation. Reintroduction of wild-type mitochondrial KARS, but not the cytosolic isoform, rescued this phenotype confirming the disease-causing nature of p.(Pro228Leu) exchange and demonstrating the mitochondrial etiology of the disease. We propose that mitochondrial translation deficiency is the probable disease culprit in this and possibly other patients with mutations in KARS.

Finsterer Josef, Scorza Fulvio A, Scorza Carla A.

Agnew Thomas, Goldsworthy Michelle, Aguilar Carlos, Morgan Anna, Simon Michelle, Hilton Helen, Esapa Chris, Wu Yixing, Cater Heather, Bentley Liz, Scudamore Cheryl, Poulton Joanna, Morten Karl J, Thompson Kyle, He Langping, Brown Steve D M, Taylor Robert W, Bowl Michael R, Cox Roger D.

Mutations in genes essential for mitochondrial function have pleiotropic effects. The mechanisms underlying these traits yield insights into metabolic homeostasis and potential therapies. Here we report the characterization of a mouse model harboring a mutation in the tryptophanyl-tRNA synthetase 2 (Wars2) gene, encoding the mitochondrial-localized WARS2 protein. This hypomorphic allele causes progressive tissue-specific pathologies, including hearing loss, reduced adiposity, adipose tissue dysfunction, and hypertrophic cardiomyopathy. We demonstrate the tissue heterogeneity arises as a result of variable activation of the integrated stress response (ISR) pathway and the ability of certain tissues to respond to impaired mitochondrial translation. Many of the systemic metabolic effects are likely mediated through elevated fibroblast growth factor 21 (FGF21) following activation of the ISR in certain tissues. These findings demonstrate the potential pleiotropy associated with Wars2 mutations in patients.

Samanta Debopam.

Almannai Mohammed, Wang Julia, Dai Hongzheng, El-Hattab Ayman W, Faqeih Eissa A, Saleh Mohammed A, Al Asmari Ali, Alwadei Ali H, Aljadhai Yaser I, AlHashem Amal, Tabarki Brahim, Lines Matthew A, Grange Dorothy K, Benini Ruba, Alsaman Abdulaziz S, Mahmoud Adel, Katsonis Panagiotis, Lichtarge Olivier, Wong Lee-Jun C.

An increasing number of mitochondrial diseases are found to be caused by pathogenic variants in nuclear encoded mitochondrial aminoacyl-tRNA synthetases. FARS2 encodes mitochondrial phenylalanyl-tRNA synthetase (mtPheRS) which transfers phenylalanine to its cognate tRNA in mitochondria. Since the first case was reported in 2012, a total of 21 subjects with FARS2 deficiency have been reported to date with a spectrum of disease severity that falls between two phenotypes; early onset epileptic encephalopathy and a less severe phenotype characterized by spastic paraplegia. In this report, we present an additional 15 individuals from 12 families who are mostly Arabs homozygous for the pathogenic variant Y144C, which is associated with the more severe early onset phenotype. The total number of unique pathogenic FARS2 variants known to date is 21 including three different partial gene deletions reported in four individuals. Except for the large deletions, all variants but two (one in-frame deletion of one amino acid and one splice-site variant) are missense. All large deletions and the single splice-site variant are in trans with a missense variant. This suggests that complete loss of function may be incompatible with life. In this report, we also review structural, functional, and evolutionary significance of select FARS2 pathogenic variants reported here.

Vasilescu Catalina, Ojala Tiina H, Brilhante Virginia, Ojanen Simo, Hinterding Helena M, Palin Eino, Alastalo Tero-Pekka, Koskenvuo Juha, Hiippala Anita, Jokinen Eero, Jahnukainen Timo, Lohi Jouko, Pihkala Jaana, Tyni Tiina A, Carroll Christopher J, Suomalainen Anu.

Childhood cardiomyopathies are progressive and often lethal disorders, forming the most common cause of heart failure in children. Despite severe outcomes, their genetic background is still poorly characterized.

Nan Haitian, Takaki Ryusuke, Hata Takanori, Ichinose Yuta, Tsuchiya Mai, Koh Kishin, Takiyama Yoshihisa.

We report the first family with a glycyl-tRNA synthetase (GARS) mutation with autosomal dominant intermediate Charcot-Marie-Tooth disease (DI-CMT). The proband and the proband's father presented with gait disturbance and hand weakness. Both patients displayed moderately decreased conduction velocities (MNCV) (ranging from 29.2 m/s to 37.8 m/s). A sural nerve biopsy of the father revealed evidence of both axonal loss and demyelination. On exome sequencing, in both the proband and his father we identified a novel missense mutation (c.643G>C, p.Asp215His) in the GARS gene in a heterozygous state, which is considered to be pathogenic for this DI-CMT family. The present study broadens current knowledge about intermediate CMT and the phenotypic spectrum of defects associated with GARS. This article is protected by copyright. All rights reserved.

Vona Barbara, Maroofian Reza, Bellacchio Emanuele, Najafi Maryam, Thompson Kyle, Alahmad Ahmad, He Langping, Ahangari Najmeh, Rad Abolfazl, Shahrokhzadeh Sima, Bahena Paulina, Mittag Falk, Traub Frank, Movaffagh Jebrail, Amiri Nafise, Doosti Mohammad, Boostani Reza, Shirzadeh Ebrahim, Haaf Thomas, Diodato Daria, Schmidts Miriam, Taylor Robert W, Karimiani Ehsan Ghayoor.

IARS2 encodes a mitochondrial isoleucyl-tRNA synthetase, a highly conserved nuclear-encoded enzyme required for the charging of tRNAs with their cognate amino acid for translation. Recently, pathogenic IARS2 variants have been identified in a number of patients presenting broad clinical phenotypes with autosomal recessive inheritance. These phenotypes range from Leigh and West syndrome to a new syndrome abbreviated CAGSSS that is characterised by cataracts, growth hormone deficiency, sensory neuropathy, sensorineural hearing loss, and skeletal dysplasia, as well as cataract with no additional anomalies.

Ma Keze, Xie Mingyu, He Xiaoguang, Liu Guojun, Lu Xiaomei, Peng Qi, Zhong Baimao, Li Ning.

Genetic defects in the mitochondrial aminoacyl-tRNA synthetase are important causes of mitochondrial disorders. VARS2 is one of the genes encoding aminoacyl-tRNA synthetases. Recently, an increasing number of pathogenic variants of VARS2 have been reported.

Lynch David S, Wade Charles, Paiva Anderson Rodrigues Brandão de, John Nevin, Kinsella Justin A, Merwick Áine, Ahmed Rebekah M, Warren Jason D, Mummery Catherine J, Schott Jonathan M, Fox Nick C, Houlden Henry, Adams Matthew E, Davagnanam Indran, Murphy Elaine, Chataway Jeremy.

Adult-onset leukodystrophies and genetic leukoencephalopathies comprise a diverse group of neurodegenerative disorders of white matter with a wide age of onset and phenotypic spectrum. Patients with white matter abnormalities detected on MRI often present a diagnostic challenge to both general and specialist neurologists. Patients typically present with a progressive syndrome including various combinations of cognitive impairment, movement disorders, ataxia and upper motor neuron signs. There are a number of important and treatable acquired causes for this imaging and clinical presentation. There are also a very large number of genetic causes which due to their relative rarity and sometimes variable and overlapping presentations can be difficult to diagnose. In this review, we provide a structured approach to the diagnosis of inherited disorders of white matter in adults. We describe clinical and radiological clues to aid diagnosis, and we present an overview of both common and rare genetic white matter disorders. We provide advice on testing for acquired causes, on excluding small vessel disease mimics, and detailed advice on metabolic and genetic testing available to the practising neurologist. Common genetic leukoencephalopathies discussed in detail include CSF1R, AARS2, cerebral arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), and mitochondrial and metabolic disorders.

Finsterer Josef.

Seaver Laurie H, DeRoos Steven, Betz Brad, Rajasekaran Surender.

Samanta Debopam, Gokden Murat, Willis Erin.

We describe a 13-year-old girl with a past medical history of epilepsy, intellectual impairment, dysphagia with gastric tube dependence, and autism spectrum disorder who presented with focal status epilepticus.

Sommerville Ewen W, Zhou Xiao-Long, Oláhová Monika, Jenkins Janda, Euro Liliya, Konovalova Svetlana, Hilander Taru, Pyle Angela, He Langping, Habeebu Sultan, Saunders Carol, Kelsey Anna, Morris Andrew A M, McFarland Robert, Suomalainen Anu, Gorman Gráinne S, Wang En-Duo, Thiffault Isabelle, Tyynismaa Henna, Taylor Robert W.

Recessively-inherited variants in AARS2 (NM_020745.2) encoding mitochondrial alanyl-tRNA synthetase (mt-AlaRS) were first described in patients presenting with fatal infantile cardiomyopathy and multiple oxidative phosphorylation defects. To date, all described patients with AARS2-related fatal infantile cardiomyopathy are united by either a homozygous or compound heterozygous c.1774C>T (p.Arg592Trp) missense founder mutation that is absent in patients with other AARS2-related phenotypes. We describe the clinical, biochemical and molecular investigations of two unrelated boys presenting with fatal infantile cardiomyopathy, lactic acidosis and respiratory failure. Oxidative histochemistry showed cytochrome c oxidase (COX)-deficient fibres in skeletal and cardiac muscle. Biochemical studies showed markedly decreased activities of mitochondrial respiratory chain complexes I and IV with a mild decrease of complex III activity in skeletal and cardiac muscle. Using next-generation sequencing, we identified a c.1738C>T (p.Arg580Trp) AARS2 variant shared by both patients that was in trans with a loss-of-function heterozygous AARS2 variant; a c.1008dupT (p.Asp337*) nonsense variant or an intragenic deletion encompassing AARS2 exons 5-7. Interestingly, our patients did not harbour the p.Arg592Trp AARS2 founder mutation. In silico modelling of the p.Arg580Trp substitution suggested a deleterious impact on protein stability and folding. We confirmed markedly decreased mt-AlaRS protein levels in patient fibroblasts, skeletal and cardiac muscle, although mitochondrial protein synthesis defects were confined to skeletal and cardiac muscle. In vitro data showed that the p.Arg580Trp variant had a minimal effect on activation, aminoacylation or misaminoacylation activities relative to wild-type mt-AlaRS, demonstrating that instability of mt-AlaRS is the biological mechanism underlying the fatal cardiomyopathy phenotype in our patients.

Yahia Ashraf, Elsayed Liena, Babai Arwa, Salih Mustafa A, El-Sadig Sarah Misbah, Amin Mutaz, Koko Mahmoud, Abubakr Rayan, Idris Razaz, Taha Shaimaa Omer M A, Elmalik Salah A, Brice Alexis, Ahmed Ammar Eltahir, Stevanin Giovanni.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL, OMIM #611105) is a genetic disease of the central nervous system characterized by lower limb spasticity, cerebellar ataxia and involvement of the dorsal column. The disease is caused by mutations in the DARS2 gene but has never been reported in sub-Saharan Africa so far.

Maddirevula Sateesh, Alzahrani Fatema, Al-Owain Mohammed, Al Muhaizea Mohammad A, Kayyali Husam R, AlHashem Amal, Rahbeeni Zuhair, Al-Otaibi Maha, Alzaidan Hamad I, Balobaid Ameera, El Khashab Heba Y, Bubshait Dalal K, Faden Maha, Yamani Suad Al, Dabbagh Omar, Al-Mureikhi Mariam, Jasser Abdulla Al, Alsaif Hessa S, Alluhaydan Iram, Seidahmed Mohammed Zain, Alabbasi Bashair Hamza, Almogarri Ibrahim, Kurdi Wesam, Akleh Hana, Qari Alya, Al Tala Saeed M, Alhomaidi Suzan, Kentab Amal Y, Salih Mustafa A, Chedrawi Aziza, Alameer Seham, Tabarki Brahim, Shamseldin Hanan E, Patel Nisha, Ibrahim Niema, Abdulwahab Firdous, Samira Menasria, Goljan Ewa, Abouelhoda Mohamed, Meyer Brian F, Hashem Mais, Shaheen Ranad, AlShahwan Saad, Alfadhel Majid, Ben-Omran Tawfeg, Al-Qattan Mohammad M, Monies Dorota, Alkuraya Fowzan S.

Establishing links between Mendelian phenotypes and genes enables the proper interpretation of variants therein. Autozygome, a rich source of homozygous variants, has been successfully utilized for the high throughput identification of novel autosomal recessive disease genes. Here, we highlight the utility of the autozygome for the high throughput confirmation of previously published tentative links to diseases.

Sahai Supreet K, Steiner Rebecca E, Au Margaret G, Graham John M, Salamon Noriko, Ibba Michael, Pierson Tyler M.

Mutations in FARS2, the gene encoding the mitochondrial phenylalanine-tRNA synthetase (mtPheRS), have been linked to a range of phenotypes including epileptic encephalopathy, developmental delay, and motor dysfunction. We report a 9-year-old boy with novel compound heterozygous variants of FARS2, presenting with a pure spastic paraplegia syndrome associated with bilateral signal abnormalities in the dentate nuclei. Exome sequencing identified a paternal nonsense variant (Q216X) lacking the catalytic core and anticodon-binding regions, and a maternal missense variant (P136H) possessing partial enzymatic activity. This case confirms and expands the phenotype related to FARS2 mutations with regards to clinical presentation and neuroimaging findings.

González-Serrano Ligia Elena, Karim Loukmane, Pierre Florian, Schwenzer Hagen, Rötig Agnès, Munnich Arnold, Sissler Marie.

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are key enzymes in the mitochondrial protein translation system and catalyze the charging of amino acids on their cognate tRNAs. Mutations in their nuclear genes are associated with pathologies having a broad spectrum of clinical phenotypes, but with no clear molecular mechanism(s). For example, mutations in the nuclear genes encoding mt-AspRS and mt-ArgRS are correlated with the moderate neurodegenerative disorder leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) and with the severe neurodevelopmental disorder pontocerebellar hypoplasia type 6 (PCH6), respectively. Previous studies have shown no or only minor impacts of these mutations on the canonical properties of these enzymes, indicating that the role of the mt-aaRSs in protein synthesis is mostly not affected by these mutations, but their effects on the mitochondrial localizations of aaRSs remain unclear. Here, we demonstrate that three human aaRSs, mt-AspRS, mt-ArgRS, and LysRS, each have a specific sub-mitochondrial distribution, with mt-ArgRS being exclusively localized in the membrane, LysRS exclusively in the soluble fraction, and mt-AspRS being present in both. Chemical treatments revealed that mt-AspRs is anchored in the mitochondrial membrane through electrostatic interactions, whereas mt-ArgRS uses hydrophobic interactions. We also report that novel mutations in mt-AspRS and mt-ArgRS genes from individuals with LBSL and PCH6, respectively, had no significant impact on the mitochondrial localizations of mt-AspRS and mt-ArgRS. The variable sub-mitochondrial locations for these three mt-aaRSs strongly suggest the existence of additional enzyme properties, requiring further investigation to unravel the mechanisms underlying the two neurodegenerative disorders.

Seaver Laurie H, DeRoos Steven, Andersen Nicholas J, Betz Brad, Prokop Jeremy, Lannen Nick, Jordan Renee, Rajasekaran Surender.

Infantile epileptic encephalopathy is a heterogeneous condition that has been associated with variants in more than 200 genes. The variability in findings and prognosis creates challenges to making the correct diagnosis and initiating the appropriate therapy. Biallelic variants in NARS2, a mitochondrial aminoacyl-tRNA synthetase gene, were recently associated with neurodegenerative disorders that include epilepsy.

van der Knaap Marjo S, Abbink Truus E M.

Riley Lisa G, Heeney Matthew M, Rudinger-Thirion Joëlle, Frugier Magali, Campagna Dean R, Zhou Ronghao, Hale Gregory A, Hilliard Lee M, Kaplan Joel A, Kwiatkowski Janet L, Sieff Colin A, Steensma David P, Rennings Alexander J, Simons Annet, Schaap Nicolaas, Roodenburg Richard J, Kleefstra Tjitske, Arenillas Leonor, Fita-Torró Josep, Ahmed Rasha, Abboud Miguel, Bechara Elie, Farah Roula, Tamminga Rienk Yj, Bottomley Sylvia S, Sanchez Mayka, Swinkels Dorine W, Christodoulou John, Fleming Mark D.

YARS2 variants have previously been described in patients with myopathy, lactic acidosis and sideroblastic anemia 2 (MLASA2). YARS2 encodes the mitochondrial tyrosyl-tRNA synthetase, which is responsible for conjugating tyrosine to its cognate mt-tRNA for mitochondrial protein synthesis. Here we describe 14 individuals from 11 families presenting with sideroblastic anemia and with YARS2 variants that we identified using a sideroblastic anemia gene panel or exome sequencing. The phenotype of these patients ranged from MLASA to isolated congenital sideroblastic anemia. As in previous cases, inter- and intra-familial phenotypic variability was observed, however this report includes the first cases with isolated sideroblastic anemia and patients with biallelic YARS2 variants that have no clinically ascertainable phenotype. We identified ten novel YARS2 variants and three previously reported variants. In vitro amino-acylation assays of three five novel missense variants showed they that three had less effect on the catalytic activity of YARS2 than the most commonly reported variant, p.(Phe52Leu), associated with MLASA2, which may explain the milder phenotypes in patients with these variants. However, the other two missense variants had a more severe effect on YARS2 catalytic efficiency. Several patients carried the common YARS2 c.572 G>T, p.(Gly191Val) variant (minor allele frequency = 0.1259) in trans with a rare deleterious YARS2 variant. We have previously shown that the p.(Gly191Val) variant reduces YARS2 catalytic activity. Consequently, we suggest that biallelic YARS2 variants, including severe loss-of-function alleles in trans of the common p.(Gly191Val) variant, should be considered as a cause of isolated congenital sideroblastic anemia, as well as the MLASA syndromic phenotype.

Takezawa Yusuke, Fujie Hiromi, Kikuchi Atsuo, Niihori Tetsuya, Funayama Ryo, Shirota Matsuyuki, Nakayama Keiko, Aoki Yoko, Sasaki Masayuki, Kure Shigeo.

IARS2 encodes isoleucine-tRNA synthetase, which is aclass-1 amino acyl-tRNA synthetase. IARS2 mutations are reported to cause Leigh syndrome or cataracts, growth hormone deficiency, sensory neuropathy, sensorineural hearing loss, and skeletal dysphasia syndrome (CAGSSS). To our knowledge, IARS2 mutations and diseases related to it have only been reported in three families. Here we report a case of two Japanese siblings with Leigh syndrome, some features of CAGSSS, and West syndrome that are found to have compound heterozygous novel IARS2 mutations.

Obeid Rawad, Sogawa Yoshimi, Naik Monica, Goldstein Amy, Gropman Andrea, Asato Miya.

The etiology of hyperlactatemia in newborns could be a challenging diagnosis. In this article we are discussing a diagnostic paradigm using the clinical history, laboratory results, and brain imaging that could be helpful in directing the work up.

Mathew Thomas, Avati Amrutha, D'Souza Delon, Therambil Manjusha.

Pontocerebellar hypoplasia type 6 (PCH6) is an autosomal recessive mitochondrial disease, typically characterized by pontine atrophy, vermian hypoplasia, infantile encephalopathy, generalized hypotonia, and intractable seizures. The purpose of this study is to describe the seizures and other neurological manifestations of RARS2 gene mutations and to compare the clinical features with other causes of progressive myoclonic epilepsy. Detailed history, physical examination, and clinical and genetic work-up were performed in 2 siblings who presented with progressive myoclonic epilepsy. One sibling, a 20-year-old woman, and the other a 24-year-old man, had a homozygous missense variant (c.848T>A; p.Leu283Gln) in exon 10 of the RARS2 gene. The female patient had action and audiogenic myoclonic jerks, postural tremors, spastic dysarthria, and bradykinesia, and her male sibling had similar features with oculomotor apraxia. The RARS2 gene mutation can present with myoclonic epilepsy, mental retardation, and pyramidal and extrapyramidal features, and is an important differential for causes of progressive myoclonic epilepsy.

Yin Xiaomeng, Tang Beisha, Mao Xiao, Peng Jinxin, Zeng Sheng, Wang Yaqin, Jiang Hong, Li Nan.

Mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are a family of enzymes that play critical roles in protein biosynthesis. Mutations in mt-aaRSs are associated with various diseases. As a member of the mt-aaRS family, PARS2 encoding prolyl-tRNA synthetase 2 was recently shown to be associated with Alpers syndrome and certain infantile-onset neurodegenerative disorders in four patients. Here, we present two patients in a pedigree with early developmental delay, epileptic spasms, delayed myelination combined with cerebellar white matter abnormalities, and progressive cortical atrophy. Whole-exome sequencing revealed pathogenic compound heterozygous variants [c.283 G > A (p.95 V > I)] and [c.604 G > C (p.202 R > G)] in PARS2. Nearly all patients had epileptic spasms with early response to treatment, early developmental delay and/or regression followed by generalized hypotonia, postnatal microcephaly, elevated lactate levels, and progressive cerebral atrophy. Our study provides further evidence for validating the role of PARS2 in the pathology of related infantile-onset encephalopathy, contributing to the phenotypic features of this condition, and providing clinical and molecular insight for the diagnosis of this disease entity.

Li Jingyan, Leng Yunji, Han Shirui, Yan Lulu, Lu Chaoxia, Luo Yang, Zhang Xue, Cao Lihua.

Pediatric cataract is a clinically and genetically heterogeneous disease which is a significant cause of lifelong visual impairment and treatable blindness. Our study aims to investigate the genotype spectrum in a group of Chinese patients with pediatric cataract.

Taglia I, Di Donato I, Bianchi S, Cerase A, Monti L, Marconi R, Orrico A, Rufa A, Federico A, Dotti M T.

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), previously known as hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) or pigmentary orthochromatic leukodystrophy (POLD), is the most frequent non-vascular adult-onset leukoencephalopathy. It is caused by autosomal dominant mutations in CSF1R gene. Recently, also autosomal recessive mutations in AARS2 gene were found to be the cause of an adult-onset leukodystrophy with axonal spheroids. Our aim was to achieve a genetic diagnosis in a cohort of CSF1R-negative patients, performing a sequence analysis of AARS2 gene.

Vantroys Elise, Smet Joél, Vanlander Arnaud V, Vergult Sarah, De Bruyne Ruth, Roels Frank, Stepman Hedwig, Roeyers Herbert, Menten Björn, Van Coster Rudy.

The first subjects with deficiency of mitochondrial tryptophanyl-tRNA synthetase (WARS2) were reported in 2017. Their clinical characteristics can be subdivided into three phenotypes (neonatal phenotype, severe infantile onset phenotype, Parkinson-like phenotype).

Ardissone Anna, Tonduti Davide, Legati Andrea, Lamantea Eleonora, Barone Rita, Dorboz Imen, Boespflug-Tanguy Odile, Nebbia Gabriella, Maggioni Marco, Garavaglia Barbara, Moroni Isabella, Farina Laura, Pichiecchio Anna, Orcesi Simona, Chiapparini Luisa, Ghezzi Daniele.

KARS encodes lysyl- transfer ribonucleic acid (tRNA) synthetase, which catalyzes the aminoacylation of tRNA-Lys in the cytoplasm and mitochondria. Eleven families/sporadic patients and 16 different mutations in KARS have been reported to date. The associated clinical phenotype is heterogeneous ranging from early onset encephalopathy to isolated peripheral neuropathy or nonsyndromic hearing impairment. Recently additional presentations including leukoencephalopathy as predominant cerebral involvement or cardiomyopathy, isolated or associated with muscular and cerebral involvement, have been reported. A progressive Leukoencephalopathy with brainstem and spinal cord calcifications was previously described in a singleton patient and in two siblings, without the identification of the genetic cause. We reported here about a new severe phenotype associated with biallelic KARS mutations and sharing some common points with the other already reported phenotypes, but with a distinct clinical and neuroimaging picture. Review of KARS mutant patients published to date will be also discussed.

Dong Qing, Long Ling, Chang Yan-Yu, Lin Yan-Jun, Liu Mei, Lu Zheng-Qi.

Mutations in the mitochondrial alanyl-transfer (t)RNA synthetase 2 (AARS2; OMIM 612035) have been linked to leukoencephalopathy recently. Until now, there have been only 13 cases reported in the literature. Hence, the clinical and genetic characteristics of this disease are not fully understood. Here, we reported an adolescence-onset male leukoencephalopathy patient characterized by progressive limb tremor at the age of 17 years. He had no signs of a cardiomyopathy. Magnetic resonance imaging scanning demonstrated severe cerebellar atrophy and white matter abnormalities involving descending tracts. Focused exome sequencing revealed he had novel compound heterozygous mutations in AARS2 gene (c.2265dupA; p.Arg756fs and c.650C>T; p.Pro217Leu). The patient was diagnosed with AARS2 mutation-related leukodystrophy (AARS2-L). We report a case with novel AARS2 gene mutations with developed striking cerebellar atrophy and leukoencephalopathy, which helps to further understand the clinical and genetic heterogeneity of AARS2-L.

Paley Elena L, Perry George.

Transgenic mice used for Alzheimer's disease (AD) preclinical experiments do not recapitulate the human disease. In our models, the dietary tryptophan metabolite tryptamine produced by human gut microbiome induces tryptophanyl-tRNA synthetase (TrpRS) deficiency with consequent neurodegeneration in cells and mice. Dietary supplements, antibiotics and certain drugs increase tryptamine content in vivo. TrpRS catalyzes tryptophan attachment to tRNAtrp at initial step of protein biosynthesis. Tryptamine that easily crosses the blood-brain barrier induces vasculopathies, neurodegeneration and cell death via TrpRS competitive inhibition. TrpRS inhibitor tryptophanol produced by gut microbiome also induces neurodegeneration. TrpRS inhibition by tryptamine and its metabolites preventing tryptophan incorporation into proteins lead to protein biosynthesis impairment. Tryptophan, a least amino acid in food and proteins that cannot be synthesized by humans competes with frequent amino acids for the transport from blood to brain. Tryptophan is a vulnerable amino acid, which can be easily lost to protein biosynthesis. Some proteins marking neurodegenerative pathology, such as tau lack tryptophan. TrpRS exists in cytoplasmic (WARS) and mitochondrial (WARS2) forms. Pathogenic gene variants of both forms cause TrpRS deficiency with consequent intellectual and motor disabilities in humans. The diminished tryptophan-dependent protein biosynthesis in AD patients is a proof of our model-based disease concept.

Ciara Elżbieta, Rokicki Dariusz, Lazniewski Michal, Mierzewska Hanna, Jurkiewicz Elżbieta, Bekiesińska-Figatowska Monika, Piekutowska-Abramczuk Dorota, Iwanicka-Pronicka Katarzyna, Szymańska Edyta, Stawiński Piotr, Kosińska Joanna, Pollak Agnieszka, Pronicki Maciej, Plewczyński Dariusz, Płoski Rafał, Pronicka Ewa.

Most of the 19 mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) involved in mitochondrial protein synthesis are already linked to specific entities, one of the exceptions being PARS2 mutations for which pathogenic significance is not finally validated. The aim of the study was to characterize the PARS2- related phenotype.Three siblings with biallelic PARS2 mutations presented from birth with infantile spasms, secondary microcephaly, and similar facial dysmorphy. Mental development was deeply impaired with speech absence and no eye contact. A dilated cardiomyopathy and multiorgan failure developed in childhood at the terminal stage, together with mitochondrial dysfunction triggered by valproate administration.Brain MRI showed progressive volume loss of the frontal lobes, both cortical and subcortical, with widening of the cortical sulci and frontal horns of the lateral ventricles. Hypoplasia of the corpus callosum and progressive demyelination were additional findings. Similar brain features were seen in three already reported PARS2 patients and seemed specific for this defect when compared with other mt-aaRSs defects (DARS2, EARS2, IARS2, and RARS2).Striking resemblance of the phenotype and Alpers-like brain MRI changes with predominance of frontal cerebral volume loss (FCVL-AS) in six patients from three families of different ethnicity with PARS2 mutations, justifies to distinguish the condition as a new disease entity.

Kamps Rick, Szklarczyk Radek, Theunissen Tom E, Hellebrekers Debby M E I, Sallevelt Suzanne C E H, Boesten Iris B, de Koning Bart, van den Bosch Bianca J, Salomons Gajja S, Simas-Mendes Marisa, Verdijk Rob, Schoonderwoerd Kees, de Coo Irenaeus F M, Vanoevelen Jo M, Smeets Hubert J M.

This study aims to identify gene defects in pediatric cardiomyopathy and early-onset brain disease with oxidative phosphorylation (OXPHOS) deficiencies. We applied whole-exome sequencing in three patients with pediatric cardiomyopathy and early-onset brain disease with OXPHOS deficiencies. The brain pathology was studied by MRI analysis. In consanguineous patient 1, we identified a homozygous intronic variant (c.850-3A > G) in the QRSL1 gene, which was predicted to cause abnormal splicing. The variant segregated with the disease and affected the protein function, which was confirmed by complementation studies, restoring OXPHOS function only with wild-type QRSL1. Patient 2 was compound heterozygous for two novel affected and disease-causing variants (c.[253G > A];[938G > A]) in the MTO1 gene. In patient 3, we detected one unknown affected and disease-causing variants (c.2872C > T) and one known disease-causing variant (c.1774C > T) in the AARS2 gene. The c.1774C > T variant was present in the paternal copy of the AARS2 gene, the c.2872C > T in the maternal copy. All genes were involved in translation of mtDNA-encoded proteins. Defects in mtDNA-encoded protein translation lead to severe pediatric cardiomyopathy and brain disease with OXPHOS abnormalities. This suggests that the heart and brain are particularly sensitive to defects in mitochondrial protein synthesis during late embryonic or early postnatal development, probably due to the massive mitochondrial biogenesis occurring at that stage. If both the heart and brain are involved, the prognosis is poor with a likely fatal outcome at young age.

Pereira Sandra, Adrião Mariana, Sampaio Mafalda, Basto Margarida Ayres, Rodrigues Esmeralda, Vilarinho Laura, Teles Elisa Leão, Alonso Isabel, Leão Miguel.

Combined oxidative phosphorylation deficiency 20 (COXPD20) is a mitochondrial respiratory chain complex (RC) disorder, caused by disease-causing variants in the VARS2 gene, which encodes a mitochondrial aminoacyl-tRNA synthetase. Here we describe a patient with fatal mitochondrial encephalopathy caused by a homozygous VARS2 gene missense variant.

Çavuşoğlu Dilek, Olgaç-Dündar Nihal, Öztekin Özgür, Özdemir Taha Reşid, Arıcan Pınar, Gençpınar Pınar.

Çavuşoğlu D, Olgaç-Dündar N, Öztekin Ö, Özdemir TR, Arıcan P, Gençpınar P. The first pediatric case of leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) from Turkey. Turk J Pediatr 2018; 60: 216-220. Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is defined as an autosomal recessive inheritance disorder characterized by slowly progressive cerebellar, pyramidal and dorsal column dysfunction. The diagnosis is based on specific magnetic resonance imaging abnormalities (MRI) in the cerebral and cerebellar white matter and selective involvement of white matter tracts in the brain stem and spinal cord. LBSL is caused by mutations in the DARS2 gene which encodes the mitochondrial aspartyl-tRNA synthetase. Herein, we report the first pediatric case from Turkey with a typical MRI course of LBSL associated with a compound heterozygous mutation in DARS2 gene.

Bruni Francesco, Di Meo Ivano, Bellacchio Emanuele, Webb Bryn D, McFarland Robert, Chrzanowska-Lightowlers Zofia M A, He Langping, Skorupa Ewa, Moroni Isabella, Ardissone Anna, Walczak Anna, Tyynismaa Henna, Isohanni Pirjo, Mandel Hanna, Prokisch Holger, Haack Tobias, Bonnen Penelope E, Enrico Bertini, Pronicka Ewa, Ghezzi Daniele, Taylor Robert W, Diodato Daria.

In recent years, an increasing number of mitochondrial disorders have been associated with mutations in mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs), which are key enzymes of mitochondrial protein synthesis. Bi-allelic functional variants in VARS2, encoding the mitochondrial valyl tRNA-synthetase, were first reported in a patient with psychomotor delay and epilepsia partialis continua associated with an oxidative phosphorylation (OXPHOS) Complex I defect, before being described in a patient with a neonatal form of encephalocardiomyopathy. Here we provide a detailed genetic, clinical, and biochemical description of 13 patients, from nine unrelated families, harboring VARS2 mutations. All patients except one, who manifested with a less severe disease course, presented at birth exhibiting severe encephalomyopathy and cardiomyopathy. Features included hypotonia, psychomotor delay, seizures, feeding difficulty, abnormal cranial MRI, and elevated lactate. The biochemical phenotype comprised a combined Complex I and Complex IV OXPHOS defect in muscle, with patient fibroblasts displaying normal OXPHOS activity. Homology modeling supported the pathogenicity of VARS2 missense variants. The detailed description of this cohort further delineates our understanding of the clinical presentation associated with pathogenic VARS2 variants and we recommend that this gene should be considered in early-onset mitochondrial encephalomyopathies or encephalocardiomyopathies.

Zhang Jie, Zhang Zhongbin, Zhang Yao, Wu Ye.

Pontocerebellar hypoplasia type 6 (PCH6) is a rare autosomal recessive disease that occurs due to mutations in the mitochondrial arginyl-tRNA synthetase 2 (RARS2) gene. To the best of our knowledge, 23 cases with relatively complete clinical data have been reported thus far. In the present study, a case with PCH6 caused by novel RARS2 mutations is described, in which distinct magnetic resonance imaging (MRI) features were identified. In addition, 23 PCH6 cases found in the literature were reviewed. Early onset hypotonia (43.48%), epileptic seizures (34.78%), encephalopathy (26.08%) and feeding difficulties (17.39%) were common initial symptoms of PCH6. During disease progression, the patients presented refractory epileptic seizures (94.12%), feeding problems (60.87%), severe developmental delay (100%), microcephaly (88.89%) and hyperlactacidemia (76.47%). The clinical features of the present patient were suggestive of PCH6, with early onset epilepsy, feeding difficulties, severe developmental delay, microcephaly, hearing loss and hyperlactacidemia. According to available MRI data from 20 reported cases with PCH6, the characteristic finding in MRI was pontocerebellar dysplasia or progressive cerebral/pontocerebellar atrophy in 16 cases, while 4 cases did not present pontocerebellar hypoplasia, and no basal ganglia involvement was observed in any of the cases. Distinctive MRI features were also identified in the present case, including pontocerebellar preservation after 1 year of age, as well as a high diffusion-weighted imaging signal suggesting intracellular edema in the cerebellar hemispheres, basal ganglia, thalamus and corpus callosum. Progressive loss of cerebral white matter and cortical volume were common features shared by all patients. In conclusion, in the present study, two novel heterozygous mutations were identified in RARS2, namely c.1718C>T(p.Thr573Ile) and c.991A>G (p.Ile331Val). Thus, the present case enriched the phenotypic and genotypic spectrum of the RARS2 mutations.

Wang Danqing, Yu Meng, Zhang Wei, Wang Zhaoxia, Yuan Yun.

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), usually referred to as hereditary diffuse leukoencephalopathy with spheroids or pigmentary orthochromatic leukodystrophy, is genetically caused by CSF1R mutations. AARS2 was recently confirmed to be another causative gene in a series of CSF1R-negative ALSP cases. We report a case of adult-onset leukoencephalopathy with ALSP with AARS2 variants. A 34-year-old woman presented with 2 years of motor and cognitive deterioration with severely impaired cortical functions and rigid spasticity. Brain magnetic resonance imaging showed a confluent, patchy, and predominantly frontoparietal, periventricular pattern of white matter lesions, with relatively preserved subcortical U-fibers. Brain biopsy revealed axonal spheroids, severe demyelination and pigmented macrophages. Genetic analyses revealed compound heterozygous c.1691T>C and c.179C>A variants in the AARS2 gene. CSF1R mutation testing was negative. Our findings proved adult-onset leukoencephalopathy with spheroids and pigmented glia to be a genetically heterogeneous disease entity. The selective brain involvement without ovarian failure might be a new subtype in AARS2 mutations related to ALSP.

Smith Frances, Hopton Sila, Dallabona Cristina, Gilberti Micol, Falkous Gavin, Norwood Fiona, Donnini Claudia, Gorman Gráinne S, Clark Barnaby, Taylor Robert W, Kulasekararaj Austin G.

Kosaki Rika, Horikawa Reiko, Fujii Eriko, Kosaki Kenjiro.

Perrault syndrome represents a genetically heterogeneous disorder characterized by sensorineural hearing loss in males and females and ovarian dysfunction in females. Causative genes include HARS2, HSD17B4, CLPP, C10orf2, and LARS2. Some patients with Perrault syndrome exhibit neurologic features including learning disability, cerebellar ataxia, and peripheral neuropathy and are classified as type 2 and are clinically separate from those without neurological symptoms other than a hearing loss (type 1). To date, all reported patients with LARS2 mutations (15 patients in 8 families) have been classified as type 1. Here, we report female siblings with biallelic mutations in LARS2, p.Glu294Lys, and p.Thr519Met, who were classified as type 2. The proposita developed progressive sensorineural hearing loss at 18 months and pervasive developmental disorder at 8 years, with repetitive behavior, insistence on sameness, attention deficit, tic, irritability, and an ataxic gait. At age 15 years, she was diagnosed as having primary amenorrhea with elevated FSH and LH and a decreased estradiol; ultrasound and magnetic resonance imaging examinations revealed a small uterus and no detectable ovaries. The proposita's younger sister presented with neonatal sensorineural hearing loss and a mild delay in motor and speech development. She was diagnosed as having primary amenorrhea with endocrinologic and radiographic findings that were comparable to those of her sister. She had difficulty with reading comprehension, and had trouble with open-ended test questions at 12 years of age. We concluded that Perrault syndrome patients with LARS2 mutations are at risk for neurologic problems, despite previous notions otherwise.

Boczonadi Veronika, Jennings Matthew J, Horvath Rita.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes responsible for charging tRNAs with their cognate amino acids, therefore essential for the first step in protein synthesis. Although the majority of protein synthesis happens in the cytosol, an additional translation apparatus is required to translate the 13 mitochondrial DNA-encoded proteins important for oxidative phosphorylation. Most ARS genes in these cellular compartments are distinct, but two genes are common, encoding aminoacyl-tRNA synthetases of glycine (GARS) and lysine (KARS) in both mitochondria and the cytosol. Mutations in the majority of the 37 nuclear-encoded human ARS genes have been linked to a variety of recessive and dominant tissue-specific disorders. Current data indicate that impaired enzyme function could explain the pathogenicity, however not all pathogenic ARSs mutations result in deficient catalytic function; thus, the consequences of mutations may arise from other molecular mechanisms. The peripheral nerves are frequently affected, as illustrated by the high number of mutations in cytosolic and bifunctional tRNA synthetases causing Charcot-Marie-Tooth disease (CMT). Here we provide insights on the pathomechanisms of CMT-causing tRNA synthetases with specific focus on the two bifunctional tRNA synthetases (GARS, KARS).

Burke E A, Frucht S J, Thompson K, Wolfe L A, Yokoyama T, Bertoni M, Huang Y, Sincan M, Adams D R, Taylor R W, Gahl W A, Toro C, Malicdan M C V.

Mitochondrial aminoacyl-tRNA synthetases (mtARSs) are essential, ubiquitously expressed enzymes that covalently attach amino acids to their corresponding tRNA molecules during translation of mitochondrial genes. Deleterious variants in the mtARS genes cause a diverse array of phenotypes, many of which involve the nervous system. Moreover, distinct mutations in mtARSs often cause different clinical manifestations. Recently, the gene encoding mitochondrial tryptophanyl tRNA synthetase (WARS2) was reported to cause two different neurological phenotypes, a form of autosomal recessive intellectual disability and a syndrome of severe infantile-onset leukoencephalopathy. Here, we present the case of a 17 year-old boy with compound heterozygous mutations in WARS2 (p.Trp13Gly, p.Ser228Trp) who presented with infantile-onset, Levodopa responsive parkinsonism at the age of 2 years. Analysis of patient-derived dermal fibroblasts revealed decreased steady state WARS2 protein and normal OXPHOS content. Muscle mitochondrial studies suggested mitochondrial proliferation without obvious respiratory chain deficiencies at age 9 years. This case expands the phenotypic spectrum of WARS2 deficiency and emphasizes the importance of mitochondrial protein synthesis in the pathogenesis of parkinsonism.

Vantroys Elise, Larson Austin, Friederich Marisa, Knight Kaz, Swanson Michael A, Powell Christopher A, Smet Joél, Vergult Sarah, De Paepe Boel, Seneca Sara, Roeyers Herbert, Menten Björn, Minczuk Michal, Vanlander Arnaud, Van Hove Johan, Van Coster Rudy.

Mutations in FARS2 are known to cause dysfunction of mitochondrial translation due to deficient aminoacylation of the mitochondrial phenylalanine tRNA. Here, we report three novel mutations in FARS2 found in two patients in a compound heterozygous state. The missense mutation c.1082C>T (p.Pro361Leu) was detected in both patients. The mutations c.461C>T (p.Ala154Val) and c.521_523delTGG (p.Val174del) were each detected in one patient. We report abnormal in vitro aminoacylation assays as a functional validation of the molecular genetic findings. Based on the phenotypic data of previously reported subjects and the two subjects reported here, we conclude that FARS2 deficiency can be associated with two phenotypes: (i) an epileptic phenotype, and (ii) a spastic paraplegia phenotype.

Zhou Xiao-Long, He Long-Xia, Yu Li-Jia, Wang Yong, Wang Xi-Jin, Wang En-Duo, Yang Tao.

Leukoencephalopathies are a broad class of common neurologic deterioration for which the etiology remain unsolved in many cases. In a Chinese Han family segregated with sensorineural hearing loss and leukoencephalopathy, candidate pathogenic variants were identified by targeted next-generation sequencing of 144 genes associated with deafness and 108 genes with leukoencephalopathy. Novel compound heterozygous mutations p.R477H and p.P505S were identified in KARS, which encodes lysyl-tRNA synthetase (LysRS), as the only candidate causative variants. These two mutations were functionally characterized by enzymatic assays, immunofluorescence, circular dichroism analysis and gel filtration chromatography. Despite no alteration in the dimer-tetramer oligomerization and cellular distribution by either mutation, the protein structure was notably influenced by the R477H mutation, which subsequently released the protein from the multiple-synthetase complex (MSC). Mutant LysRSs with the R477H and P505S mutations had decreased tRNA(Lys) aminoacylation and displayed a cumulative effect when introduced simultaneously. Our studies showed that mutations in KARS lead to a newly defined subtype of leukoencephalopathy associated with sensorineural hearing impairment. The combined effect of reduced aminoacylation and release of LysRS from the MSC likely underlies the pathogenesis of the KARS mutations identified in this study. This article is protected by copyright. All rights reserved.

Wortmann Saskia B, Timal Sharita, Venselaar Hanka, Wintjes Liesbeth T, Kopajtich Robert, Feichtinger René G, Onnekink Carla, Mühlmeister Mareike, Brandt Ulrich, Smeitink Jan A, Veltman Joris A, Sperl Wolfgang, Lefeber Dirk, Pruijn Ger, Stojanovic Vesna, Freisinger Peter, V Spronsen Francjan, Derks Terry Gj, Veenstra-Knol Hermine E, Mayr Johannes A, Rötig Agnes, Tarnopolsky Mark, Prokisch Holger, Rodenburg Richard J.

Mitochondrial protein synthesis involves an intricate interplay between mitochondrial DNA encoded RNAs and nuclear DNA encoded proteins, such as ribosomal proteins and aminoacyl-tRNA synthases. Eukaryotic cells contain 17 mitochondria-specific aminoacyl-tRNA synthases. WARS2 encodes mitochondrial tryptophanyl-tRNA synthase (mtTrpRS), a homodimeric class Ic enzyme (mitochondrial tryptophan-tRNA ligase; EC 6.1.1.2). Here, we report six individuals from five families presenting with either severe neonatal onset lactic acidosis, encephalomyopathy and early death or a later onset, more attenuated course of disease with predominating intellectual disability. Respiratory chain enzymes were usually normal in muscle and fibroblasts, while a severe combined respiratory chain deficiency was found in the liver of a severely affected individual. Exome sequencing revealed rare biallelic variants in WARS2 in all affected individuals. An increase of uncharged mitochondrial tRNA(Trp) and a decrease of mtTrpRS protein content were found in fibroblasts of affected individuals. We hereby define the clinical, neuroradiological and metabolic phenotype of WARS2 defects. This confidently implicates that mutations in WARS2 cause mitochondrial disease with a broad spectrum of clinical presentation. This article is protected by copyright. All rights reserved.

Bruwer Zandrè, Al Riyami Nihal, Al Dughaishi Tamima, Al Murshedi Fathiya, Al Sayegh Abeer, Al Kindy Adila, Meftah Douja, Al Kharusi Khalsa, Al Foori Amel, Al Yarubi Naeema, Scott Patrick, Al-Thihli Khalid.

The purpose of this study was to determine the frequency of non-immune hydrops fetalis (NIHF) among all pregnancies referred for prenatal care at Sultan Qaboos University Hospital (SQUH) during the study period and to evaluate the underlying etiologies of NIH.

Peragallo Jason H, Keller Stephanie, van der Knaap Marjo S, Soares Bruno P, Shankar Suma P.

Optic atrophy may be the sequela of optic nerve injury due to any insult, including isolated and syndromic genetic diseases. Alanyl-tRNA synthetase 2 (AARS2) pathogenic variants have been reported to cause leukodystrophy with ovarian failure, and cardiomyopathy (#615889) as well as combined oxidative phosphorylation deficiency-8 (#614096). We report a young child who presented with decreased vision due to optic atrophy and was found to harbor missense variants in the AARS2 gene expanding the phenotypic expression of the AARS2 gene.

Zerkaoui Maria, Demain Leigh A M, Cherkaoui Jaouad Imane, Ratbi Ilham, Amjoud Karima, Urquhart Jill E, O'Sullivan James, Newman William G, Sefiani Abdelaziz.

The objective of this study was to report the clinical and biological characteristics of two Perrault syndrome cases in a Moroccan family with homozygous variant c.1565C>A in the LARS2 gene and to establish genotype-phenotype correlation of patients with the same mutation by review of the literature. Whole-exome sequencing was performed. Data analysis was carried out and confirmed by Sanger sequencing and segregation. The affected siblings were diagnosed as having Perrault syndrome with sensorineural hearing loss at low frequencies; the female proband had primary amenorrhea and ovarian dysgenesis. Both affected individuals had a marfanoid habitus and no neurological features. Both patients carried the homozygous variant c.1565C>A; p.Thr522Asn in exon 13 of the LARS2 gene. This variant has already been reported as a homozygous variant in three other Perrault syndrome families. Both affected siblings of a Moroccan consanguineous family with LARS2 variants had low-frequency sensorineural hearing loss, marfanoid habitus, and primary ovarian insufficiency in the affected girl. According to the literature, this variant, c.1565C>A; p.Thr522Asn, can be correlated with low-frequency hearing loss. However, marfanoid habitus was been considered a nonspecific feature in Perrault syndrome, but we believe that it may be more specific than considered previously. This diagnosis allowed us to provide appropriate management to the patients and to provide more accurate genetic counseling to this family.

Aradjanski Marijana, Dogan Sukru Anil, Lotter Stephan, Wang Shuaiyu, Hermans Steffen, Wibom Rolf, Rugarli Elena, Trifunovic Aleksandra.

Although mitochondria are ubiquitous, each mitochondrial disease has surprisingly distinctly different pattern of tissue and organ involvement. Congruently, mutations in genes encoding for different mitochondrial tRNA synthetases result in the development of a very flamboyant group of diseases. Mutations in some of these genes, including aspartyl-tRNA synthetase (DARS2), lead to the onset of a white matter disease-leukoencephalopathy with brainstem and spinal cord involvement, and lactate elevation (LBSL) characterized by progressive spastic ataxia and characteristic leukoencephalopathy signature with multiple long-tract involvements. Puzzled by the white matter disease phenotypes caused by DARS2 deficiency when numerous other mutations in the genes encoding proteins involved in mitochondrial translation have a detrimental effect predominantly on neurons, we generated transgenic mice in which DARS2 was specifically depleted in forebrain-hippocampal neurons or myelin-producing cells. Our results now provide the first evidence that loss of DARS2 in adult neurons leads to strong mitochondrial dysfunction and progressive loss of cells. In contrast, myelin-producing cells seem to be resistant to cell death induced by DARS2 depletion despite robust respiratory chain deficiency arguing that LBSL might originate from the primary neuronal and axonal defect. Remarkably, our results also suggest a role for early neuroinflammation in the disease progression, highlighting the possibility for therapeutic interventions of this process.

Oprescu Stephanie N, Chepa-Lotrea Xenia, Takase Ryuichi, Golas Gretchen, Markello Thomas C, Adams David R, Toro Camilo, Gropman Andrea L, Hou Ya-Ming, Malicdan May Christine V, Gahl William A, Tifft Cynthia J, Antonellis Anthony.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes that ligate amino acids onto tRNA molecules. Genes encoding ARSs have been implicated in myriad dominant and recessive disease phenotypes. Glycyl-tRNA synthetase (GARS) is a bi-functional ARS that charges tRNA(Gly) in the cytoplasm and mitochondria. GARS variants have been associated with dominant Charcot-Marie-Tooth disease but have not been convincingly implicated in recessive phenotypes. Here we describe a patient from the NIH Undiagnosed Diseases Program with a multi-system, developmental phenotype. Whole-exome sequence analysis revealed that the patient is compound heterozygous for one frameshift (p.Glu83Ilefs*6) and one missense (p.Arg310Gln) GARS variant. Using in vitro and in vivo functional studies, we show that both GARS variants cause a loss-of-function effect: the frameshift variant results in depleted protein levels and the missense variant reduces GARS tRNA charging activity. In support of GARS variant pathogenicity, our patient shows striking phenotypic overlap with other patients having ARS-related recessive diseases, including features associated with variants in both cytoplasmic and mitochondrial ARSs; this observation is consistent with the essential function of GARS in both cellular locations. In summary, our clinical, genetic, and functional analyses expand the phenotypic spectrum associated with GARS variants. This article is protected by copyright. All rights reserved.

Sissler Marie, González-Serrano Ligia Elena, Westhof Eric.

Dysfunctions in mitochondria - the powerhouses of the cell - lead to several human pathologies. Because mitochondria integrate nuclear and mitochondrial genetic systems, they are richly intertwined with cellular activities. The nucleus-encoded mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are key components of the mitochondrial translation apparatus. Mutations in these enzymes predominantly affect the central nervous system (CNS) but also target other organs. Comparable mutations in mt-aaRSs can lead to vastly diverse diseases, occurring at different stages in life, and within different tissues; this represents a confounding issue. With newer information available, we propose that the pleiotropy and tissue-specificity of mt-aaRS-associated diseases result from the molecular integration of mitochondrial translation events within the cell; namely, through specific crosstalk between the cellular program and the energy demands of the cell. We place particular focus on neuronal cells.

Murray Clark R, Abel Samantha N, McClure Matthew B, Foster Joseph, Walke Maria I, Jayakar Parul, Bademci Guney, Tekin Mustafa.

Patients with unclear patterns of developmental and cognitive delay may go years without a definitive diagnosis despite extensive testing due to overlapping phenotypes of many genetic disorders. In this study, we identified causative variants in DYRK1A, KARS, or KAT6A in four individuals with global developmental delay and various findings including microcephaly and sensorineural hearing loss using whole exome sequencing. We present the cognitive, neurologic, and physical findings of four individuals to expand the clinical knowledge of possible features of the phenotypes of three rare genetic disorders. Through this process, we provide support for the use of whole exome sequencing in the setting of severe, intellectual disability or in those in whom a genetic disorder is suspected despite initial negative testing.

Theisen Benjamin E, Rumyantseva Anastasia, Cohen Julie S, Alcaraz Wendy A, Shinde Deepali N, Tang Sha, Srivastava Siddarth, Pevsner Jonathan, Trifunovic Aleksandra, Fatemi Ali.

Pathogenic variants in the mitochondrial aminoacyl tRNA synthetases lead to deficiencies in mitochondrial protein synthesis and are associated with a broad range of clinical presentations usually with early onset and inherited in an autosomal recessive manner. Of the 19 mitochondrial aminoacyl tRNA synthetases, WARS2, encoding mitochondrial tryptophanyl tRNA synthetase, was as of late the only one that had not been associated with disease in humans. A case of a family with pathogenic variants in WARS2 that caused mainly intellectual disability, speech impairment, aggressiveness, and athetosis was recently reported. Here we substantially extend and consolidate the symptomatology of WARS2 by presenting a patient with severe infantile-onset leukoencephalopathy, profound intellectual disability, spastic quadriplegia, epilepsy, microcephaly, short stature, failure to thrive, cerebral atrophy, and periventricular white matter abnormalities. He was found by whole-exome sequencing to have compound heterozygous variants in WARS2, c.938A>T (p.K313M) and c.298_300delCTT (p.L100del). De novo synthesis of proteins inside mitochondria was reduced in the patient's fibroblasts, leading to significantly lower steady-state levels of respiratory chain subunits compared to control and resulting in lower oxygen consumption rates.

Finsterer Josef, Zarrouk-Mahjoub Sinda.

Ognjenović Jana, Simonović Miljan.

Aminoacyl-tRNA synthetases (AaRSs) are ubiquitously expressed enzymes that ensure accurate translation of the genetic information into functional proteins. These enzymes also execute a variety of non-canonical functions that are significant for regulation of diverse cellular processes and that reside outside the realm of protein synthesis. Associations between faults in AaRS-mediated processes and human diseases have been long recognized. Most recent research findings strongly argue that 10 cytosolic and 14 mitochondrial AaRSs are implicated in some form of pathology of the human nervous system. The advent of modern whole-exome sequencing makes it all but certain that similar associations between the remaining 15 ARS genes and neurological illnesses will be defined in future. It is not surprising that an intense scientific debate about the role of translational machinery, in general, and AaRSs, in particular, in the development and maintenance of the healthy human neural cell types and the brain is sparked. Herein, we summarize the current knowledge about causative links between mutations in human AaRSs and diseases of the nervous system and briefly discuss future directions.

Sellars Elizabeth A, Balmakund Tonya, Bosanko Katherine, Nichols Brandi L, Kahler Stephen G, Zarate Yuri A.

Leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) is a recently described autosomal recessive mitochondrial disease characterized by early onset of neurological symptoms, a biphasic clinical course, and distinctive neuroimaging. Pathogenic variants in the EARS2 gene that encode for mitochondrial glutamyl-tRNA synthetase are responsible for LTBL. Here, we describe the clinical course of an infant diagnosed with an acute crisis of LTBL and severe liver disease. This article illustrates the utility of blood lactate quantification in addition to basic metabolic testing and brain imaging in a child with low tone and poor growth. In addition, this case demonstrates the utility of current genetic diagnostic testing, in lieu of more invasive procedures, in obtaining rapid answers in this very complicated group of disorders.

Lakshmanan Rahul, Adams Matthew E, Lynch David S, Kinsella Justin A, Phadke Rahul, Schott Jonathan M, Murphy Elaine, Rohrer Jonathan D, Chataway Jeremy, Houlden Henry, Fox Nick C, Davagnanam Indran.

To provide an overview of the phenotype of 2 clinically, radiologically, and pathologically similar leukodystrophies, adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) and alanyl-transfer RNA synthetase 2 mutation-related leukodystrophy (AARS2-L), and highlight key differentiating features.

Moosa Shahida, Haagerup Annette, Gregersen Pernille Axel, Petersen Karin Kastberg, Altmüller Janine, Thiele Holger, Nürnberg Peter, Cho Tae-Joon, Kim Ok-Hwa, Nishimura Gen, Wollnik Bernd, Vogel Ida.

Since the original description of the IARS2-related cataracts, growth hormone deficiency, sensory neuropathy, sensorineural hearing loss, skeletal dysplasia syndrome (CAGSSS; OMIM 616007) in an extended consanguineous family of French-Canadian descent, no further patients have been reported. IARS2 (OMIM 612801) encodes the mitochondrial isoleucine-tRNA synthetase which belongs to the class-I aminoacyl-tRNA synthetase family, and has been implicated in CAGSSS and a form of Leigh syndrome. Here, we report on a female Danish patient with a novel homozygous IARS2 mutation, p.Gly874Arg, who presented at birth with bilateral hip dislocation and short stature. At 3 months, additional dysmorphic features were noted and at 18 months her radiographic skeletal abnormalities were suggestive of an underlying spondyloepimetaphyseal dysplasia (SEMD). Retrospective analysis of the neonatal radiographs confirmed that the skeletal changes were present at birth. It was only with time that several of the other manifestations of the CAGSSS emerged, namely, cataracts, peripheral neuropathy, and hearing loss. Growth hormone deficiency has not (yet) manifested. We present her clinical features and particularly highlight her skeletal findings, which confirm the presence of a primary SEMD skeletal dysplasia in a growing list of mitochondrial-related disorders including CAGSSS, CODAS, EVEN-PLUS, and X-linked SEMD-MR syndromes.

Lee J-M, Yang H-J, Kwon J-H, Kim W-J, Kim S-Y, Lee E-M, Park J-Y, Weon Y C, Park S H, Gwon B-J, Ryu J-C, Lee S-T, Kim H-J, Jeon B.

Sommerville Ewen W, Ng Yi Shiau, Alston Charlotte L, Dallabona Cristina, Gilberti Micol, He Langping, Knowles Charlotte, Chin Sophie L, Schaefer Andrew M, Falkous Gavin, Murdoch David, Longman Cheryl, de Visser Marianne, Bindoff Laurence A, Rawles John M, Dean John C S, Petty Richard K, Farrugia Maria E, Haack Tobias B, Prokisch Holger, McFarland Robert, Turnbull Douglass M, Donnini Claudia, Taylor Robert W, Gorman Gráinne S.

YARS2 mutations have been associated with a clinical triad of myopathy, lactic acidosis, and sideroblastic anemia in predominantly Middle Eastern populations. However, the identification of new patients expands the clinical and molecular spectrum of mitochondrial disorders.

Kartvelishvili Ekaterine, Tworowski Dmitry, Vernon Hilary, Moor Nina, Wang Jing, Wong Lee-Jun, Chrzanowska-Lightowlers Zofia, Safro Mark.

Mutations in the mitochondrial aminoacyl-tRNA synthetases (mtaaRSs) can cause profound clinical presentations, and have manifested as diseases with very selective tissue specificity. To date most of the mtaaRS mutations could be phenotypically recognized, such that clinicians could identify the affected mtaaRS from the symptoms alone. Among the recently reported pathogenic variants are point mutations in FARS2 gene, encoding the human mitochondrial PheRS. Patient symptoms range from spastic paraplegia to fatal infantile Alpers encephalopathy. How clinical manifestations of these mutations relate to the changes in three-dimensional structures and kinetic characteristics remains unclear, although impaired aminoacylation has been proposed as possible etiology of diseases. Here, we report four crystal structures of HsmtPheRS mutants, and extensive MD simulations for wild-type and nine mutants to reveal the structural changes on dynamic trajectories of HsmtPheRS. Using steady-state kinetic measurements of phenylalanine activation and tRNA(Phe) aminoacylation, we gained insight into the structural and kinetic effects of mitochondrial disease-related mutations in FARS2 gene.

Sun Bo, Chen Zhaohui, Ling Li, Yang Fei, Huang Xusheng.

Charcot-Marie-Tooth disease (CMT) is a common hereditary motor and sensory neuropathy. Epidemiological data for Chinese CMT patients are few. This study aimed to analyze the electrophysiological and genetic characteristics of Chinese Han patients. A total of 106 unrelated patients with the clinical diagnosis of CMT were included. Clinical examination, nerve conduction studies (NCS), next-generation sequencing (NGS), and bioinformatic analyses were performed. Genetic testing was performed for 82 patients; 27 (33%) patients carried known CMT-associated gene mutations. PMP22 duplication was detected in 10 (12%) patients and GJB1 mutations in 9 (11%) patients. The mutation rate was higher in patients with a positive family history than in the sporadic cases (50% vs. 27%, p < 0.05). Six novel CMT-associated gene mutations including BSCL2 (c.461C>T), LITAF (c.32C>G), MFN2 (c.497C>T), GARS (c.794C>T), NEFL (c.280C>T), and MPZ (c.440T>C) were discovered. All except the LITAF (c.32C>G) mutation were identified as "disease causing" via bioinformatic analyses. In this Chinese Han population, the frequency of PMP22 gene duplication in those with CMT1 was slightly (50% vs. 70%-80%) less than in Western/Caucasian populations. The novel CMT-associated gene mutations broaden the mutation diversity of CMT1. NGS should be considered for genetic analyses in CMT patients.

Nishio Shin-Ya, Takumi Yutaka, Usami Shin-Ichi.

Cochlear implantation (CI), which directly stimulates the cochlear nerves, is the most effective and widely used medical intervention for patients with severe to profound sensorineural hearing loss. The etiology of the hearing loss is speculated to have a major influence of CI outcomes, particularly in cases resulting from mutations in genes preferentially expressed in the spiral ganglion region. To elucidate precise gene expression levels in each part of the cochlea, we performed laser-capture micro dissection in combination with next generation-sequencing analysis and determined the expression levels of all known deafness-associated genes in the organ of Corti, spiral ganglion, lateral wall, and spiral limbs. The results were generally consistent with previous reports based on immunocytochemistry or in situ hybridization. As a notable result, the genes associated with many kinds of syndromic hearing loss (such as Clpp, Hars2, Hsd17b4, Lars2 for Perrault syndrome, Polr1c and Polr1d for Treacher Collins syndrome, Ndp for Norrie Disease, Kal for Kallmann syndrome, Edn3 and Snai2 for Waardenburg Syndrome, Col4a3 for Alport syndrome, Sema3e for CHARGE syndrome, Col9a1 for Sticker syndrome, Cdh23, Cib2, Clrn1, Pcdh15, Ush1c, Ush2a, and Whrn for Usher syndrome and Wfs1 for wolfram syndrome) showed higher levels of expression in the spiral ganglion than in other parts of the cochlea. This dataset will provide a base for more detailed analysis in order to clarify gene functions in the cochlea as well as predict CI outcomes based on gene expression data.

Lynch David S, Rodrigues Brandão de Paiva Anderson, Zhang Wei Jia, Bugiardini Enrico, Freua Fernando, Tavares Lucato Leandro, Macedo-Souza Lucia Inês, Lakshmanan Rahul, Kinsella Justin A, Merwick Aine, Rossor Alexander M, Bajaj Nin, Herron Brian, McMonagle Paul, Morrison Patrick J, Hughes Deborah, Pittman Alan, Laur� Matilde, Reilly Mary M, Warren Jason D, Mummery Catherine J, Schott Jonathan M, Adams Matthew, Fox Nick C, Murphy Elaine, Davagnanam Indran, Kok Fernando, Chataway Jeremy, Houlden Henry.

Leukodystrophies and genetic leukoencephalopathies are a rare group of disorders leading to progressive degeneration of cerebral white matter. They are associated with a spectrum of clinical phenotypes dominated by dementia, psychiatric changes, movement disorders and upper motor neuron signs. Mutations in at least 60 genes can lead to leukoencephalopathy with often overlapping clinical and radiological presentations. For these reasons, patients with genetic leukoencephalopathies often endure a long diagnostic odyssey before receiving a definitive diagnosis or may receive no diagnosis at all. In this study, we used focused and whole exome sequencing to evaluate a cohort of undiagnosed adult patients referred to a specialist leukoencephalopathy service. In total, 100 patients were evaluated using focused exome sequencing of 6100 genes. We detected pathogenic or likely pathogenic variants in 26 cases. The most frequently mutated genes were NOTCH3, EIF2B5, AARS2 and CSF1R. We then carried out whole exome sequencing on the remaining negative cases including four family trios, but could not identify any further potentially disease-causing mutations, confirming the equivalence of focused and whole exome sequencing in the diagnosis of genetic leukoencephalopathies. Here we provide an overview of the clinical and genetic features of these disorders in adults.

Mizuguchi Takeshi, Nakashima Mitsuko, Kato Mitsuhiro, Yamada Keitaro, Okanishi Tohru, Ekhilevitch Nina, Mandel Hanna, Eran Ayelet, Toyono Miyuki, Sawaishi Yukio, Motoi Hirotaka, Shiina Masaaki, Ogata Kazuhiro, Miyatake Satoko, Miyake Noriko, Saitsu Hirotomo, Matsumoto Naomichi.

Musante Luciana, Püttmann Lucia, Kahrizi Kimia, Garshasbi Masoud, Hu Hao, Stehr Henning, Lipkowitz Bettina, Otto Sabine, Jensen Lars R, Tzschach Andreas, Jamali Payman, Wienker Thomas, Najmabadi Hossein, Ropers Hans Hilger, Kuss Andreas W.

Intellectual disability (ID) is the hallmark of an extremely heterogeneous group of disorders that comprises a wide variety of syndromic and non-syndromic phenotypes. Here we report on mutations in two aminoacyl-tRNA synthetases that are associated with ID in two unrelated Iranian families. In the first family, we identified a homozygous missense mutation (c.514G>A, p.Asp172Asn) in the cytoplasmic seryl-tRNA synthetase (SARS) gene. The mutation affects the enzymatic core domain of the protein and impairs its enzymatic activity, probably leading to reduced cytoplasmic tRNA(Ser) concentrations. The mutant protein was predicted to be unstable, which could be substantiated by investigating ectopic mutant SARS in transfected HEK293T cells. In the second family, we found a compound heterozygous genotype of the mitochondrial tryptophanyl-tRNA synthetase (WARS2) gene, comprising a nonsense mutation (c.325delA, p.Ser109Alafs*15), which very likely entails nonsense-mediated mRNA decay, and a missense mutation (c.37T>G, p.Trp13Gly). The latter affects the mitochondrial localization signal of WARS2, causing protein mislocalization. Including AIMP1, which we have recently implicated in the aetiology of ID, three genes with a role in tRNA-aminoacylation are now associated with this condition. We therefore suggest that the functional integrity of tRNAs in general is an important factor in the development and maintenance of human cognitive functions. This article is protected by copyright. All rights reserved.

Lan Min-Yu, Chang Yung-Yee, Yeh Tu-Hseuh, Lin Tsu-Kung, Lu Chin-Song.

Mizuguchi Takeshi, Nakashima Mitsuko, Kato Mitsuhiro, Yamada Keitaro, Okanishi Tohru, Ekhilevitch Nina, Mandel Hanna, Eran Ayelet, Toyono Miyuki, Sawaishi Yukio, Motoi Hirotaka, Shiina Masaaki, Ogata Kazuhiro, Miyatake Satoko, Miyake Noriko, Saitsu Hirotomo, Matsumoto Naomichi.

Here we present four unrelated families with six individuals that have infantile-onset developmental delay/regression and epilepsy. Whole-exome sequencing revealed compound heterozygous mutations, c.[283G>A];[607G>A] in a gene encoding prolyl-tRNA synthetase (PARS2) in one family. Two pairs of compound heterozygous mutations, c.[151C>T];[1184T>G] and c.[707T>G];[594+1G>A], and a homozygous mutation, c.[500A>G];[500A>G], in a gene encoding asparaginyl-tRNA synthetase (NARS2) were also identified in the other three families. Mutations in genes encoding aminoacyl-tRNA synthetases cause gene-specific mitochondrial disorders. Biallelic PARS2 or NARS2 mutations are reported to cause Alpers' syndrome, which is an autosomal recessive neurodegenerative disorder characterized by psychomotor regression and epilepsy with variable degree of liver involvement. Moreover, it is known that NARS2 mutations cause various clinical phenotypes, including non-syndromic hearing loss, Leigh syndrome, intellectual disability with epilepsy and severe myopathy. The individuals with PARS2 and NARS2 mutations, we have reported here demonstrate similar neurological features as those previously reported, with diversity in clinical presentation such as hearing loss and seizure type. Our data broaden the clinical and mutational spectrum of PARS2- and NARS2-related disorders.Journal of Human Genetics advance online publication, 12 January 2017; doi:10.1038/jhg.2016.163.

Nafisinia Michael, Riley Lisa G, Gold Wendy A, Bhattacharya Kaustuv, Broderick Carolyn R, Thorburn David R, Simons Cas, Christodoulou John.

Glycyl-tRNA synthetase (GARS; OMIM 600287) is one of thirty-seven tRNA-synthetase genes that catalyses the synthesis of glycyl-tRNA, which is required to insert glycine into proteins within the cytosol and mitochondria. To date, eighteen mutations in GARS have been reported in patients with autosomal-dominant Charcot-Marie-Tooth disease type 2D (CMT2D; OMIM 601472), and/or distal spinal muscular atrophy type V (dSMA-V; OMIM 600794). In this study, we report a patient with clinical and biochemical features suggestive of a mitochondrial respiratory chain (MRC) disorder including mild left ventricular posterior wall hypertrophy, exercise intolerance, and lactic acidosis. Using whole exome sequencing we identified compound heterozygous novel variants, c.803C>T; p.(Thr268Ile) and c.1234C>T; p.(Arg412Cys), in GARS in the proband. Spectrophotometric evaluation of the MRC complexes showed reduced activity of Complex I, III and IV in patient skeletal muscle and reduced Complex I and IV activity in the patient liver, with Complex IV being the most severely affected in both tissues. Immunoblot analysis of GARS protein and subunits of the MRC enzyme complexes in patient fibroblast extracts showed significant reduction in GARS protein levels and Complex IV. Together these studies provide evidence that the identified compound heterozygous GARS variants may be the cause of the mitochondrial dysfunction in our patient.

Shimojima Keiko, Higashiguchi Takafumi, Kishimoto Kanako, Miyatake Satoko, Miyake Noriko, Takanashi Jun-Ichi, Matsumoto Naomichi, Yamamoto Toshiyuki.

The mitochondrial aspartyl-tRNA synthetase 2 gene (DARS2) is responsible for leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL). A Japanese patient with LBSL showed compound heterozygous DARS2 mutations c.358_359delinsTC (p.Gly120Ser) and c.228-15C>G (splicing error). This provides further evidence that most patients with LBSL show compound heterozygous mutations in DARS2 in association with a common splicing mutation in the splicing acceptor site of intron 2.

Cho Jae So, Kim Seung Hyo, Kim Ha Young, Chung Taesu, Kim Dongsup, Jang Sesong, Lee Seung Bok, Yoo Seung Keun, Shin Jongyeon, Kim Jong-Il, Kim Hunmin, Hwang Hee, Chae Jong-Hee, Choi Jieun, Kim Ki Joong, Lim Byung Chan.

Early-onset epileptic encephalopathy (EOEE) consists of a heterogeneous group of epilepsy phenotypes. Recent technological advances in molecular biology have also rapidly expanded the genotype of EOEE. Genes involved in diverse molecular pathways, including ion channels, synaptic structure, transcription regulation, and cellular growth, have been implicated in EOEE. Mitochondrial aminoacyl tRNA synthetase, which plays a key role in mitochondrial protein synthesis by attaching 20 different amino acids to the tRNA tail, has been recently linked with the epilepsy phenotype. Here, we report a novel homozygous c.925G>A (G309S) missense mutation in the gene that encodes the human mitochondrial phenylalanyl-tRNA synthetase (FARS2) in four patients from two nonconsanguineous Korean families. All four patients suffered from intractable seizures that started at the age of 3 and 4 months. Seizure types were variable, including infantile spasms and myoclonic seizures, and often prolonged. Although their initial development seemed to be normal, relentless regression after seizure onset occurred in all patients. An etiologic investigation, including brain imaging and metabolic studies, did not reveal a specific etiology. We reviewed the epilepsy phenotypes of six additional FARS2 mutation-positive patients and suggest that FARS2 can be considered one of the genetic causes of EOEE.

Mazurova Stella, Magner Martin, Kucerova-Vidrova Vendula, Vondrackova Alzbeta, Stranecky Viktor, Pristoupilova Anna, Zamecnik Josef, Hansikova Hana, Zeman Jiri, Tesarova Marketa, Honzik Tomas.

Cardiomyopathy is a common manifestation in neonates and infants with mitochondrial disorders. In this study, we report two cases manifesting with fatal mitochondrial hypertrophic cardiomyopathy, which include the third known patient with thymidine kinase 2 deficiency and the ninth patient with alanyl-tRNA synthetase 2 deficiency. The girl with thymidine kinase 2 deficiency had hypertrophic cardiomyopathy together with regression of gross motor development at the age of 13 months. Neurological symptoms and cardiac involvement progressed into severe myopathy, psychomotor arrest, and cardiorespiratory failure at the age of 22 months. The imaging methods and autoptic studies proved that she suffered from unique findings of leucoencephalopathy, severe, mainly cerebellar neuronal degeneration, and hepatic steatosis. The girl with alanyl-tRNA synthetase 2 deficiency presented with cardiac failure and underlying hypertrophic cardiomyopathy within 12 hours of life and subsequently died at 9 weeks of age. Muscle biopsy analyses demonstrated respiratory chain complex I and IV deficiencies, and histological evaluation revealed massive mitochondrial accumulation and cytochrome c oxidase-negative fibres in both cases. Exome sequencing in the first case revealed compound heterozygozity for one novel c.209T>C and one previously published c.416C>T mutation in the TK2 gene, whereas in the second case homozygozity for the previously described mutation c.1774C>T in the AARS2 gene was determined. The thymidine kinase 2 mutations resulted in severe mitochondrial DNA depletion (to 12% of controls) in the muscle. We present, for the first time, severe leucoencephalopathy and hepatic steatosis in a patient with thymidine kinase 2 deficiency and the finding of a ragged red fibre-like image in the muscle biopsy in a patient with alanyl-tRNA synthetase 2 deficiency.

Verrigni Daniela, Diodato Daria, Di Nottia Michela, Torraco Alessandra, Bellacchio Emanuele, Rizza Teresa, Tozzi Giulia, Verardo Margherita, Piemonte Fiorella, Tasca Giorgio, D'Amico Adele, Bertini Enrico, Carrozzo Rosalba.

Mutations in KARS, which encodes for both mitochondrial and cytoplasmic lysyl-tRNA synthethase, have been so far associated with three different phenotypes: the recessive form of Charcot Mary-Tooth polyneuropathy, the autosomal recessive non-syndromic hearing loss and the last recently described condition related to congenital visual impairment and progressive microcephaly. Here we report the case of a 14-years-old-girl with severe cardiomyopathy associated to mild psychomotor delay and mild myopathy; moreover, a diffuse reduction of cytochrome C oxidase (COX, complex IV) and a combined enzymatic defect of complex I (CI) and IV (CIV) was evident in muscle biopsy. Using the TruSight One sequencing panel we identified two novel mutations in KARS. Both mutations, never reported previously, occur in a highly conserved region of the catalytic domain and had a dramatic effect on KARS stability. Structural analysis confirmed the pathogenic role of the identified variants. Our findings confirm and emphasize that mt-ARSs enzymes are related to a broad clinical spectrum due to their multiple and still unknown functions.

Hamatani Mio, Jingami Naoto, Tsurusaki Yoshinori, Shimada Shino, Shimojima Keiko, Asada-Utsugi Megumi, Yoshinaga Kenji, Uemura Norihito, Yamashita Hirofumi, Uemura Kengo, Takahashi Ryosuke, Matsumoto Naomichi, Yamamoto Toshiyuki.

Even now, only a portion of leukodystrophy patients are correctly diagnosed, though various causative genes have been identified. In the present report, we describe a case of adult-onset leukodystrophy in a woman with ovarian failure. By whole-exome sequencing, a compound heterozygous mutation consisting of NM_020745.3 (AARS2_v001):c.1145C>A and NM_020745.3 (AARS2_v001):c.2255+1G>A was identified. Neither of the mutations has been previously reported, and this is the first report of alanyl-transfer RNA synthetase 2 mutation in Asia. We anticipate that further studies of the molecular basis of leukodystrophy will provide insight into its pathogenesis and hopefully lead to sophisticated diagnostic and treatment strategies.

Szpisjak Laszlo, Zsindely Nora, Engelhardt Jozsef I, Vecsei Laszlo, Kovacs Gabor G, Klivenyi Peter.

AARS2 gene (NM_020745.3) mutations result in two different phenotypic diseases: infantile mitochondrial cardiomyopathy and late-onset leukoencephalopathy. The patient's first symptoms appeared at the age of 18 years with behavioral changes and psychiatric problems. Some years later, extrapyramidal symptoms, cognitive impairment, nystagmus, dysarthria and pyramidal symptoms also developed. The brain magnetic resonance imaging (MRI) indicated extensive white matter abnormalities. The diagnosis of AARS2 gene mutations causing leukodystrophy was confirmed by genetic testing. Segregation analysis confirmed the compound heterozygous state of the patient. Histological examination of the biopsy did not prove specific pathological alterations. The clinical phenotype of our patient was compared with seven previously described patients suffering from leukoencephalopathy caused by AARS2 mutations. We have documented a new, nonsense AARS2 gene mutation (c.578T>G, p.Leu193*) and a known missense mutation (c.595C>T, p.Arg199Cys) associated with leukoencephalopathy in a male patient. Clinical features, imaging characteristics and genetic testing are presented, and histological data from an AARS2-related leukodystrophy patient are described for the first time.Journal of Human Genetics advance online publication, 13 October 2016; doi:10.1038/jhg.2016.126.

Lühl S, Bode H, Schlötzer W, Bartsakoulia M, Horvath R, Abicht A, Stenzel M, Kirschner J, Grünert S C.

Pontocerebellar hypoplasia type 6 (PCH6) is a mitochondrial disease caused by mutations in the RARS2 gene. RARS2 encodes mitochondrial arginyl transfer RNA synthetase, an enzyme involved in mitochondrial protein translation. A total of 27 patients from 14 families have been reported so far. Characteristic clinical features comprise neonatal lactic acidosis, severe encephalopathy, intractable seizures, feeding problems and profound developmental delay. Most patients show typical neuroradiologic abnormalities including cerebellar hypoplasia and progressive pontocerebellar atrophy.

Lynch David S, Zhang Wei Jia, Lakshmanan Rahul, Kinsella Justin A, Uzun Günes Altiokka, Karbay Merih, Tüfekçioglu Zeynep, Hanagasi Hasmet, Burke Georgina, Foulds Nicola, Hammans Simon R, Bhattacharjee Anupam, Wilson Heather, Adams Matthew, Walker Mark, Nicoll James A R, Chataway Jeremy, Fox Nick, Davagnanam Indran, Phadke Rahul, Houlden Henry.

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a frequent cause of adult-onset leukodystrophy known to be caused by autosomal dominant mutations in the CSF1R (colony-stimulating factor 1) gene. The discovery that CSF1R mutations cause ALSP led to more accurate prognosis and genetic counseling for these patients in addition to increased interest in microglia as a target in neurodegeneration. However, it has been known since the discovery of the CSF1R gene that there are patients with typical clinical and radiologic evidence of ALSP who do not carry pathogenic CSF1R mutations. These patients include those in whom the pathognomonic features of axonal spheroids and pigmented microglia have been found. Achieving a genetic diagnosis in these patients is important to our understanding of this disorder.

Lerat Justine, Jonard Laurence, Loundon Natalie, Christin-Maitre Sophie, Lacombe Didier, Goizet Cyril, Rouzier Cécile, Van Maldergem Lionel, Gherbi Souad, Garabedian Eréa-Nöel, Bonnefont Jean-Paul, Touraine Philippe, Mosnier Isabelle, Munnich Arnold, Denoyelle Françoise, Marlin Sandrine.

Perrault syndrome (PS) is a rare autosomal recessive condition characterized by deafness and gonadic dysgenesis. Recently, mutations in five genes have been identified: C10orf2, CLPP, HARS2, HSD17B4, and LARS2. Probands included are presented with sensorineural deafness associated with gonadic dysgenesis. DNA was sequenced using next-generation sequencing (NGS) with a panel of 35 deafness genes including the five Perrault genes. Exonic variations known as pathogenic mutations or detected with <1% frequency in public databases were extracted and subjected to segregation analysis within each family. Both mutations and low coverage regions were analyzed by Sanger sequencing. Fourteen female index patients were included. The screening in four cases has been extended to four family members presenting with PS phenotype. For four unrelated patients (28.6%), causative mutations were identified: three homozygous mutations in C10orf2, CLPP, and HARS2, and one compound heterozygous mutation in LARS2. Three additional heterozygous mutations in LARS2 and HSD17B4 were found in three independent familial cases. All these missense mutations were verified by Sanger sequencing. Familial segregation analyses confirmed the molecular diagnosis in all cases carrying biallelic mutations. Because of NGS, molecular analysis confirmed the clinical diagnosis of PS in 28.6% of our cohort and four novel mutations were found in four Perrault genes. For the unsolved cases, exome sequencing should be performed to search for a sixth unknown PS gene.

van Dijk Tessa, van Ruissen Fred, Jaeger Bregje, Rodenburg Richard J, Tamminga Saskia, van Maarle Merel, Baas Frank, Wolf Nicole I, Poll-The Bwee Tien.

Mutations in the mitochondrial arginyl tRNA synthetase (RARS2) gene are associated with Pontocerebellar Hypoplasia type 6 (PCH6). Here we report two patients, compound heterozygous for RARS2 mutations, presenting with early onset epileptic encephalopathy and (progressive) atrophy of both supra- and infratentorial structures. Early pontocerebellar hypoplasia was virtually absent and respiratory chain (RC) defects could not be detected in muscle biopsies. Both patients carried a novel missense mutation c.1544A>G (p.(Asp515Gly)) in combination with either a splice site (c.297+2T>G) or a frameshift (c.452_454insC) mutation. The splice site mutation induced skipping of exon 4.These two patients expand the phenotypical spectrum associated with RARS2 mutations beyond the first report of PCH6 by Edvardson and colleagues. We propose to classify RARS2-associated phenotypes as an early onset mitochondrial encephalopathy, since this is more in agreement with both clinical presentation and underlying genetic cause.

Baertling Fabian, Alhaddad Bader, Seibt Annette, Budaeus Sonja, Meitinger Thomas, Strom Tim M, Mayatepek Ertan, Schaper Jörg, Prokisch Holger, Haack Tobias B, Distelmaier Felix.

VARS2 encodes a mitochondrial aminoacyl-tRNA-synthetase. Mutations in VARS2 have recently been identified as a cause of mitochondrial encephalomyopathy in three individuals. However, clinical information remained scarce. Exome sequencing lead us to identify compound heterozygous pathogenic VARS2 variants in a boy presenting with severe lactic acidosis, hypertrophic cardiomyopathy, epilepsy, and abnormalities on brain imaging including hypoplasia of corpus callosum and cerebellum as well as a massive lactate peak on MR-spectroscopy. Studies in patient-derived fibroblasts confirmed the functional relevance of the identified VARS2 variants. Our report expands the phenotypic spectrum associated with this rare mitochondrial defect, in that VARS2 deficiency may also cause severe neonatal presentations with cardiac involvement and structural brain abnormalities.

Raviglione Federico, Conte Giorgio, Ghezzi Daniele, Parazzini Cecilia, Righini Andrea, Vergaro Raffaella, Legati Andrea, Spaccini Luigina, Gasperini Serena, Garavaglia Barbara, Mastrangelo Massimo.

The FARS2 gene encodes the mitochondrial phenylalanyl-tRNA synthetase and is implicated in autosomal recessive combined oxidative phosphorylation deficiency 14, a clinical condition characterized by infantile onset epilepsy and encephalopathy. Mutations in FARS2 have been reported in only few patients, but a detailed description of seizures, electroencephalographic patterns, magnetic resonance imaging findings, and long-term follow-up is still needed. We provide a clinical report of a child with FARS2-related disease manifesting drug-resistant infantile spasms associated with focal seizures. By comparative genomic hybridization analysis we identified a heterozygous microdeletion in the short arm of chromosome 6, inherited from the mother, that encompasses the first coding exon of FARS2. By sequencing of the FARS2 gene we identified a variant c.1156C>G; p.(R386G), inherited from the father. By using standard spectrophotometric techniques in skin fibroblasts, we found a combined abnormality of complexes I and IV of the mitochondrial respiratory chain. The main clinical features of the patient included axial hypotonia, mild distal hypertonia, and psychomotor delay. The magnetic resonance imaging showed microcephaly, frontal cerebral atrophy, and signal changes of dentate nuclei. At the age of 3 years and 6 months, the patient was still under treatment with vigabatrin and he has been seizure free for the last 23 months. © 2016 Wiley Periodicals, Inc.

Oliveira Renata, Sommerville Ewen W, Thompson Kyle, Nunes Joana, Pyle Angela, Grazina Manuela, Chinnery Patrick F, Diogo Luísa, Garcia Paula, Taylor Robert W.

Mitochondrial translation defects are important causes of early onset mitochondrial disease. Although the biochemical (combined respiratory chain deficiency) signature and neuroimaging are usually distinctive, they are not diagnostic as the genetic origin of mitochondrial translation defects is heterogeneous. We report a female child, born at term to non-consanguineous parents, who exhibited global hypotonia, failure to thrive, persistent and progressive hyperlactacidaemia with lactic acidosis, liver dysfunction and encephalopathy and died at the age of 5 months. Brain MRI revealed hypogenesis of the corpus callosum, T2 signal abnormalities in the medulla oblongata, pons, midbrain, thalami, cerebellar white matter, and a lactate peak on MRS. Muscle histochemistry showed cytochrome c oxidase (COX)-deficient and ragged-red fibres, while muscle biochemical studies showed decreased activities of mitochondrial respiratory chain complexes I and IV. Whole exome sequencing (WES) identified biallelic EARS2 (NM_001083614) variants, a previously reported start-loss (c.1>G, p.Met1?) variant and a novel missense (c.184A>T, p.Ile62Phe) variant. Patient fibroblasts and muscle homogenate displayed markedly decreased EARS2 protein levels, although decreased steady-state levels of complex I (NDUFB8) and complex IV (MT-CO1 and MT-CO2) subunits were only observed in muscle. Pathogenic variants in EARS2, encoding mitochondrial glutamyl-tRNA synthetase (mtGluR), are associated with Leukoencephalopathy involving the Thalamus and Brainstem with high Lactate (LTBL), a mitochondrial disorder characterised by a distinctive brain MRI pattern and a biphasic clinical course. We further outline the unique phenotypic spectrum of LTBL and review the neuroradiological features reported in all patients documented in the literature.

Şahin Sevim, Cansu Ali, Kalay Ersan, Dinçer Tuba, Kul Sibel, Çakır İsmet Miraç, Kamaşak Tülay, Budak Gülden Yorgancıoğlu.

Leukoencephalopathy with thalamus and brainstem involvement, and high lactate (LTBL) is a recently identified disease related to mutations in the EARS2 gene encoding glutamyl-tRNA synthetase. We report clinical and radiological findings for two siblings with new pathogenic mutations in the EARS2 gene. Both patients showed symptoms of mild-type disease, but there were clinical differences between the two siblings. While the older brother had hypotonia and delayed developmental milestones, the younger brother had seizures and spasticity in the lower extremities. Brain magnetic resonance imaging (MRI) findings were quite similar for the two siblings. MRI findings were specific to LTBL. MRI lesions of the older sibling had regressed over time. Clinical and radiological improvement, as in the previously reported patients with LTBL, may be an important clue for diagnosis.

Linnankivi Tarja, Neupane Nirajan, Richter Uwe, Isohanni Pirjo, Tyynismaa Henna.

Mitochondrial aminoacyl-tRNA synthetases are an important group of disease genes typically underlying either a disorder affecting an isolated tissue or a distinct syndrome. Missense mutations in the mitochondrial seryl-tRNA synthetase gene, SARS2, have been identified in HUPRA syndrome (hyperuricemia, pulmonary hypertension, renal failure in infancy and alkalosis). We report here a homozygous splicing mutation in SARS2 in a patient with progressive spastic paresis. We show that the mutation leads to diminished levels of the synthetase in patient's fibroblasts. This has a destabilizing effect on the tRNASer(AGY) isoacceptor, but to a lesser degree than in HUPRA syndrome patients. tRNASer(UCN) is largely unaffected in both phenotypes. In conclusion, the level of tRNASer(AGY) instability may be a factor in determining tissue manifestation in patients with SARS2 mutations. This finding exemplifies the sensitivity of the nervous system to partially reduced aminoacylation, which is sufficient in other tissues to maintain respiratory chain function. This article is protected by copyright. All rights reserved.

Pronicka Ewa, Piekutowska-Abramczuk Dorota, Ciara Elżbieta, Trubicka Joanna, Rokicki Dariusz, Karkucińska-Więckowska Agnieszka, Pajdowska Magdalena, Jurkiewicz Elżbieta, Halat Paulina, Kosińska Joanna, Pollak Agnieszka, Rydzanicz Małgorzata, Stawinski Piotr, Pronicki Maciej, Krajewska-Walasek Małgorzata, Płoski Rafał.

Whole-exome sequencing (WES) has led to an exponential increase in identification of causative variants in mitochondrial disorders (MD).

Viering Daan H H M, de Baaij Jeroen H F, Walsh Stephen B, Kleta Robert, Bockenhauer Detlef.

Magnesium is essential to the proper functioning of numerous cellular processes. Magnesium ion (Mg(2+)) deficits, as reflected in hypomagnesemia, can cause neuromuscular irritability, seizures and cardiac arrhythmias. With normal Mg(2+) intake, homeostasis is maintained primarily through the regulated reabsorption of Mg(2+) by the thick ascending limb of Henle's loop and distal convoluted tubule of the kidney. Inadequate reabsorption results in renal Mg(2+) wasting, as evidenced by an inappropriately high fractional Mg(2+) excretion. Familial renal Mg(2+) wasting is suggestive of a genetic cause, and subsequent studies in these hypomagnesemic families have revealed over a dozen genes directly or indirectly involved in Mg(2+) transport. Those can be classified into four groups: hypercalciuric hypomagnesemias (encompassing mutations in CLDN16, CLDN19, CASR, CLCNKB), Gitelman-like hypomagnesemias (CLCNKB, SLC12A3, BSND, KCNJ10, FYXD2, HNF1B, PCBD1), mitochondrial hypomagnesemias (SARS2, MT-TI, Kearns-Sayre syndrome) and other hypomagnesemias (TRPM6, CNMM2, EGF, EGFR, KCNA1, FAM111A). Although identification of these genes has not yet changed treatment, which remains Mg(2+) supplementation, it has contributed enormously to our understanding of Mg(2+) transport and renal function. In this review, we discuss general mechanisms and symptoms of genetic causes of hypomagnesemia as well as the specific molecular mechanisms and clinical phenotypes associated with each syndrome.

Tao Kaixiong, Gao Jinbo, Wang Guobin.

Elderly patients with colorectal cancer have different clincopathological characteristics from younger patients. Colorectal cancers tend to localize in the proximal colon, from cecum to the splenic flexure in the elderly patients. Changes in the stools, rectal bleeding or black stool, abdominal pain, fatigue, weight loss and anemia are the common symptoms. Analysis showed that age is one of independent risk factors for lower completion rates of colonoscopy. Therefore, the choice of diagnosis methods in elderly patients should be careful. Achieving a clear diagnosis and avoiding complications should be considered at the same time. Most colorectal cancers in elderly are highly and moderately differentiated adenocarcinomas and locally advanced, and have less lymphatic and blood metastasis. The proportion of poorly differentiated adenocarcinoma increases with the increase of age, which should be concerned. Multiple colorectal cancers and colorectal cancer with extra-colorectal malignancy are not rare in the elderly patients. The common extra-colorectal tumors consist of gastric cancer, lung cancer, biliary carcinoma, pancreas cancer and malignancy from blood system. Molecular events, such as mutations of KARS, BRAF, TP53 and deficiency of DNA mismatch repair, are more frequent in elderly colorectal cancer patients. Many factors have impact on treatment decision in elderly patients with colorectal cancer, including age, comorbidities, physiological functions of organs and willingness of patients and their relatives. Although surgery is still the main treatment, the proportion of radical surgery is lower and emergency surgery is higher as compared to younger patients. With the development of minimally invasive surgical techniques and advances in anesthesia and perioperative management, laparoscopic surgery has become widespread in elderly patients with colorectal cancer. In addition, more attention should be paid to adjuvant therapy. Comprehensive individualized treatment plan should be taken to improve outcomes.

Ngoh Adeline, Bras Jose, Guerreiro Rita, Meyer Esther, McTague Amy, Dawson Eleanor, Mankad Kshitij, Gunny Roxana, Clayton Peter, Mills Philippa B, Thornton Rachel, Lai Ming, Forsyth Robert, Kurian Manju A.

Pontocerebellar hypoplasia is a group of heterogeneous neurodevelopmental disorders characterized by reduced volume of the brainstem and cerebellum. We report two male siblings who presented with early infantile clonic seizures, and then developed infantile spasms associated with prominent isolated cerebellar hypoplasia/atrophy on magnetic resonance imaging (MRI). Using whole exome sequencing techniques, both were found to be compound heterozygotes for one previously reported and one novel mutation in the gene encoding mitochondrial arginyl-tRNA synthetase 2 (RARS2). Mutations in this gene have been classically described in pontocerebellar hypoplasia type six (PCH6), a phenotype characterized by early (often intractable) seizures, profound developmental delay, and progressive pontocerebellar atrophy. The electroclinical spectrum of PCH6 is broad and includes a number of seizure types: myoclonic, generalized tonic-clonic, and focal clonic seizures. Our report expands the characterization of the PCH6 disease spectrum and presents infantile spasms as an associated electroclinical phenotype.

Jabbour Samir, Harissi-Dagher Mona.

To report the ocular findings of a rare case of mutation in the nuclear-encoded mitochondrial aminoacyl-tRNA synthetase IARS2.

Walker Melissa A, Mohler Kyle P, Hopkins Kyle W, Oakley Derek H, Sweetser David A, Ibba Michael, Frosch Matthew P, Thibert Ronald L.

Mutations in mitochondrial aminoacyl-tRNA synthetases are an increasingly recognized cause of human diseases, often arising in individuals with compound heterozygous mutations and presenting with system-specific phenotypes, frequently neurologic.FARS2encodes mitochondrial phenylalanyl transfer ribonucleic acid (RNA) synthetase (mtPheRS), perturbations of which have been reported in 6 cases of an infantile, lethal disease with refractory epilepsy and progressive myoclonus. Here the authors report the case of juvenile onset refractory epilepsy and progressive myoclonus with compound heterozygousFARS2mutations. The authors describe the clinical course over 6 years of care at their institution and diagnostic studies including electroencephalogram (EEG), brain magnetic resonance imaging (MRI), serum and cerebrospinal fluid analyses, skeletal muscle biopsy histology, and autopsy gross and histologic findings, which include features shared with Alpers-Huttenlocher syndrome, Leigh syndrome, and a previously published case ofFARS2mutation associated infantile onset disease. The authors also present structure-guided analysis of the relevant mutations based on published mitochondrial phenylalanyl transfer RNA synthetase and related protein crystal structures as well as biochemical analysis of the corresponding recombinant mutant proteins.

Güngör Olcay, Özkaya Ahmet Kağan, Şahin Yavuz, Güngör Gülay, Dilber Cengiz, Aydın Kürşad.

Mitochondrial glutamyl-tRNA synthetase is a major component of protein biosynthesis that loads tRNAs with cognate amino acids. Mutations in the gene encoding this enzyme have been associated with a variety of disorders related to oxidative phosphorylation. Here, we present a case of leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) presenting a biphasic clinical course characterized by delayed psychomotor development and seizure. High-throughput sequencing revealed a novel compound heterozygous mutation in mitochondrial glutamyl-tRNA synthetase 2 (EARS2), which appears to be causative of disease symptoms.

Alkhateeb Asem M, Aburahma Samah K, Habbab Wesal, Thompson I Richard.

Intellectual disability is a heterogeneous disease with many genes and mutations influencing the phenotype. Consanguineous families constitute a rich resource for the identification of rare variants causing autosomal recessive disease, due to the effects of inbreeding. Here, we examine three consanguineous Arab families, recruited in a quest to identify novel genes/mutations. All the families had multiple offspring with non-specific intellectual disability. We identified homozygosity (autozygosity) intervals in those families through SNP genotyping and whole exome sequencing, with variants filtered using Ingenuity Variant Analysis (IVA) software. The families showed heterogeneity and novel mutations in three different genes known to be associated with intellectual disability. These mutations were not found in 514 ethnically matched control chromosomes. p.G410C in WWOX, p.H530Y in RARS2, and p.I69F in C10orf2 are novel changes that affect protein function and could give new insights into the development and function of the central nervous system.

Mordaunt Dylan A, Savarirayan Ravi.

Demain Leigh A M, Urquhart Jill E, O'Sullivan James, Williams Simon G, Bhaskar Sanjeev S, Jenkinson Emma M, Lourenco Charles M, Heiberg Arvid, Pearce Simon H, Shalev Stavit A, Yue Wyatt W, Mackinnon Sabrina, Munro Kevin J, Newbury-Ecob Ruth, Becker Kristen, Kim Min Jeong, O' Keefe Raymond T, Newman William G.

Perrault syndrome is a rare autosomal recessive disorder characterised by sensorineural hearing loss (SNHL) in both sexes and primary ovarian insufficiency in 46, XX karyotype females. Biallelic variants in five genes are reported to be causative: HSD17B4, HARS2, LARS2, CLPP and C10orf2. Here we present eight families affected by Perrault syndrome. In five families we identified novel or previously reported variants in HSD17B4, LARS2, CLPP and C10orf2. The proband from each family was whole exome sequenced and variants confirmed by Sanger sequencing. A female was compound heterozygous for a known, p.(Gly16Ser) and novel, p.(Val82Phe) variant in D-bifunctional protein (HSD17B4). A family was homozygous for mtLeuRS (LARS2) p.(Thr522Asn), previously associated with Perrault syndrome. A further family was compound heterozygous for mtLeuRS, p.(Thr522Asn) and a novel variant, p.(Met117Ile). Affected individuals with LARS2 variants had low frequency SNHL, a feature previously described in Perrault syndrome. A female with significant neurological disability was compound heterozygous for p.(Arg323Gln) and p.(Asn399Ser) variants in Twinkle (C10orf2). A male was homozygous for a novel variant in CLPP, p.(Cys144Arg). In three families there were no putative pathogenic variants in these genes confirming additional disease-causing genes remain unidentified. We have expanded the spectrum of disease-causing variants associated with Perrault syndrome.

Nishri Daniella, Goldberg-Stern Hadassa, Noyman Iris, Blumkin Lubov, Kivity Sara, Saitsu Hirotomo, Nakashima Mitsuko, Matsumoto Naomichi, Leshinsky-Silver Esther, Lerman-Sagie Tally, Lev Dorit.

Early onset epileptic encephalopathies (EOEEs) are a group of devastating diseases, manifesting in the first year of life with frequent seizures and/or prominent interictal epileptiform discharges on the electroencephalogram, developmental delay or regression and usually a poor prognosis. There are numerous causes for EOEEs making the diagnostic workup time consuming and costly.

Tzoulis Charalampos, Schwarzlmüller Thomas, Biermann Martin, Haugarvoll Kristoffer, Bindoff Laurence A.

Mitochondrial involvement in the pathogenesis of Parkinson's disease has been suggested by multiple studies, but the mechanisms involved remain unresolved. Here, we sought to identify which mitochondrial defects are associated with degeneration of the nigrostriatal system. Nigrostriatal integrity was assessed in vivo by dopamine transporter (DAT) imaging in twenty-one patients with mitochondrial disorders of different molecular etiology including: maternally inherited mitochondrial DNA (mtDNA) point mutations, primary single mtDNA deletions, nuclear-encoded disorders of mtDNA replication and maintenance due to mutations in POLG or C10orf2 (Twinkle), and mutations in other nuclear mitochondrial genes including the mitochondrial aspartyl-tRNA synthetase (DARS2) and ADCK3 genes. Patients with mitochondrial disease were compared with twenty patients with Parkinson's disease and eighteen controls. Nigrostriatal degeneration occurred exclusively in patients with defective mtDNA replication and maintenance. In these patients, nigrostriatal degeneration was progressive and at least as severe as in patients with advanced Parkinson's disease. None of the patients with other mitochondrial defects showed evidence of nigral involvement. Our findings demonstrate that dopaminergic neurons of the substantia nigra are specifically vulnerable to defective mtDNA replication/repair or quality control and not to primary point mutations of mtDNA. These results support the hypothesis that accumulating somatic mtDNA damage plays an important role in neurodegeneration.

Taskin Birce Dilge, Karalok Zeynep Selen, Gurkas Esra, Aydin Kursad, Aydogmus Ummu, Ceylaner Serdar, Karaer Kadri, Yilmaz Cahide, Pearl Phillip Lawrence.

Childhood leukoencephalopathies are a broad class of diseases, which are extremely rare. The treatment and classification of these disorders are both challenging. Nearly half of children presenting with a leukoencephalopathy remain without a specific diagnosis. Leukoencephalopathy with thalamus and brain stem involvement and high lactate (LTBL) is a newly described childhood leukoencephalopathy caused by mutations in the gene encoding a mitochondrial aminoacyl-tRNA synthetase specific for glutamate, EARS2. Magnetic resonance images show a characteristic leukoencephalopathy with thalamic and brain stem involvement. Here, we report a different clinical course of LTBL supported by typical MRI features in a Turkish patient who presented with a history of failure to walk. The EARS2 gene mutation analysis identified a c.322C>T transition, predicting a p.R108W change. This is the first reported early-onset mild type LTBL caused by a homozygous EARS2 mutation case in the literature.

Danhauser Katharina, Haack Tobias B, Alhaddad Bader, Melcher Marlen, Seibt Annette, Strom Tim M, Meitinger Thomas, Klee Dirk, Mayatepek Ertan, Prokisch Holger, Distelmaier Felix.

Mitochondrial aminoacyl tRNA synthetases are essential for organelle protein synthesis. Genetic defects affecting the function of these enzymes may cause pediatric mitochondrial disease. Here, we report on a child with fatal neonatal lactic acidosis and recurrent hypoglycemia caused by mutations in EARS2, encoding mitochondrial glutamyl-tRNA synthetase 2. Brain ultrasound revealed agenesis of corpus callosum. Studies on patient-derived skin fibroblasts showed severely decreased EARS2 protein levels, elevated reactive oxygen species (ROS) production, and altered mitochondrial morphology. Our report further illustrates the clinical spectrum of the severe neonatal-onset form of EARS2 mutations. Moreover, in this case the live-cell parameters appeared to be more sensitive to mitochondrial dysfunction compared to standard diagnostics, which indicates the potential relevance of fibroblast studies in children with mitochondrial diseases.

Wang Yong, Zhou Xiao-Long, Ruan Zhi-Rong, Liu Ru-Juan, Eriani Gilbert, Wang En-Duo.

Mitochondria require all translational components, including aminoacyl-tRNA synthetases (aaRSs), to complete organelle protein synthesis. Some aaRS mutations cause mitochondrial disorders, including human mitochondrial threonyl-tRNA synthetase (hmtThrRS; encoded by TARS2), whose P282L mutation causes mitochondrial encephalomyopathies. However, its catalytic and structural consequences remain unclear. Herein, we cloned TARS2 and purified the wild-type and P282L mutant hmtThrRS. HmtThrRS mis-activates non-cognate Ser and uses post-transfer editing to clear erroneously synthesized products. In vitro and in vivo analyses revealed that the mutation induces decrease of Thr activation, aminoacylation and proofreading activities, and a change in the protein structure and/or stability that might cause reduced catalytic efficiency. We also identified a splicing variant of TARS2 mRNA lacking exons 8 and 9, whose protein product is targeted into mitochondria. In HEK293T cells, the variant does not dimerize and cannot complement the ThrRS-knockout strain in yeast, suggesting that the truncated protein is inactive and might have a non-canonical function, as observed for other aaRSs fragments. The present study describes the aminoacylation and editing properties of hmtThrRS, clarifies the molecular consequences of the P282L mutation and shows that the yeast ThrRS-deletion model is suitable to test pathology-associated point mutations or alternative splicing variants of mammalian aaRS mRNAs.

Henmyr Viktor, Lind-Halldén Christina, Halldén Christer, Säll Torbjörn, Carlberg Daniel, Bachert Claus, Cardell Lars-Olaf.

Genetic studies of chronic rhinosinusitis (CRS) have identified a total of 53 CRS-associated SNPs that were subsequently evaluated for their reproducibility in a recent study. The rs2873551 SNP in linkage disequilibrium with PARS2 showed the strongest association signal. The present study aims to comprehensively screen for rare variants in PARS2 and evaluate for accumulation of such variants in CRS-patients. Sanger sequencing and long-range PCR were used to screen for rare variants in the putative promoter region and coding sequence of 310 CRS-patients and a total of 21 variants were detected. The mutation spectrum was then compared with data from European populations of the 1000Genomes project (EUR) and the Exome Aggregation Consortium (ExAC). The CRS population showed a significant surplus of low-frequency variants compared with ExAC data. Haplotype analysis of the region showed a significant excess of rare haplotypes in the CRS population compared to the EUR population. Two missense mutations were also genotyped in the 310 CRS patients and 372 CRS-negative controls, but no associations with the disease were found. This is the first re-sequencing study in CRS research and also the first study to show an association of rare variants with the disease.

Jiang Pingping, Jin Xiaofen, Peng Yanyan, Wang Meng, Liu Hao, Liu Xiaoling, Zhang Zhenjun, Ji Yanchun, Zhang Juanjuan, Liang Min, Zhao Fuxin, Sun Yan-Hong, Zhang Minglian, Zhou Xiangtian, Chen Ye, Mo Jun Qin, Huang Taosheng, Qu Jia, Guan Min-Xin.

Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disorder. Nuclear modifier genes are proposed to modify the phenotypic expression of LHON-associated mitochondrial DNA (mtDNA) mutations. By using an exome sequencing approach, we identified a LHON susceptibility allele (c.572G>T, p.191Gly>Val) in YARS2 gene encoding mitochondrial tyrosyl-tRNA synthetase, which interacts with m.11778G>A mutation to cause visual failure. We performed functional assays by using lymphoblastoid cell lines derived from members of Chinese families (asymptomatic individuals carrying m.11778G>A mutation, or both m.11778G>A and heterozygous p.191Gly>Val mutations, and symptomatic subjects harboring m.11778G>A and homozygous p.191Gly>Val mutations) and controls lacking these mutations. The 191Gly>Val mutation reduced the YARS2 protein level in the mutant cells. The aminoacylated efficiency and steady-state level of tRNA(Tyr) were markedly decreased in the cell lines derived from patients both carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The failure in tRNA(Tyr) metabolism impaired mitochondrial translation, especially for polypeptides with high content of tyrosine codon such as ND4, ND5, ND6 and COX2 in cells lines carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The YARS2 p.191Gly>Val mutation worsened the respiratory phenotypes associated with m.11778G>A mutation, especially reducing activities of complex I and IV. The respiratory deficiency altered the efficiency of mitochondrial ATP synthesis and increased the production of reactive oxygen species. Thus, mutated YARS2 worsens mitochondrial dysfunctions associated with the m.11778G>A mutation, exceeding the threshold for the expression of blindness phenotype. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between mtDNA mutation and mutated nuclear-modifier YARS2.

Sold� Giulia, Caccia Sonia, Robusto Michela, Chiereghin Chiara, Castorina Pierangela, Ambrosetti Umberto, Duga Stefano, Asselta Rosanna.

Perrault syndrome (MIM #233400) is a rare autosomal recessive disorder characterized by ovarian dysgenesis and primary ovarian insufficiency in females, and progressive hearing loss in both genders. Recently, mutations in five genes (HSD17B4, HARS2, CLPP, LARS2 and C10ORF2) were found to be responsible for Perrault syndrome, although they do not account for all cases of this genetically heterogeneous condition. We used whole-exome sequencing to identify pathogenic variants responsible for Perrault syndrome in an Italian pedigree with two affected siblings. Both patients were compound heterozygous for two novel missense variants within the mitochondrial leucyl-tRNA synthetase (LARS2): NM_015340.3:c.899C>T(p.Thr300Met) and c.1912G>A(p.Glu638Lys). Both variants cosegregated with the phenotype in the family. p.Thr300 and p.Glu638 are evolutionarily conserved residues, and are located, respectively, within the editing domain and immediately before the catalytically important KMSKS motif. Homology modeling using as template the E. coli leucyl-tRNA synthetase provided further insights on the possible pathogenic effects of the identified variants. This represents the first independent replication of the involvement of LARS2 mutations in Perrault syndrome, contributing valuable information for the further understanding of this disease.Journal of Human Genetics advance online publication, 10 December 2015; doi:10.1038/jhg.2015.149.

Sauter Claude, Lorber Bernard, Gaudry Agnès, Karim Loukmane, Schwenzer Hagen, Wien Frank, Roblin Pierre, Florentz Catherine, Sissler Marie.

Mutations in human mitochondrial aminoacyl-tRNA synthetases are associated with a variety of neurodegenerative disorders. The effects of these mutations on the structure and function of the enzymes remain to be established. Here, we investigate six mutants of the aspartyl-tRNA synthetase correlated with leukoencephalopathies. Our integrated strategy, combining an ensemble of biochemical and biophysical approaches, reveals that mutants are diversely affected with respect to their solubility in cellular extracts and stability in solution, but not in architecture. Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele. The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation.

Riley Lisa G, Rudinger-Thirion Joëlle, Schmitz-Abe Klaus, Thorburn David R, Davis Ryan L, Teo Juliana, Arbuckle Susan, Cooper Sandra T, Campagna Dean R, Frugier Magali, Markianos Kyriacos, Sue Carolyn M, Fleming Mark D, Christodoulou John.

Pathogenic variants in mitochondrial aminoacyl-tRNA synthetases result in a broad range of mitochondrial respiratory chain disorders despite their shared role in mitochondrial protein synthesis. LARS2 encodes the mitochondrial leucyl-tRNA synthetase, which attaches leucine to its cognate tRNA. Sequence variants in LARS2 have previously been associated with Perrault syndrome, characterized by premature ovarian failure and hearing loss (OMIM #615300). In this study, we report variants in LARS2 that are associated with a severe multisystem metabolic disorder. The proband was born prematurely with severe lactic acidosis, hydrops, and sideroblastic anemia. She had multisystem complications with hyaline membrane disease, impaired cardiac function, a coagulopathy, pulmonary hypertension, and progressive renal disease and succumbed at 5 days of age. Whole exome sequencing of patient DNA revealed compound heterozygous variants in LARS2 (c.1289C>T; p.Ala430Val and c.1565C>A; p.Thr522Asn). The c.1565C>A (p.Thr522Asn) LARS2 variant has previously been associated with Perrault syndrome and both identified variants are predicted to be damaging (SIFT, PolyPhen). Muscle and liver samples from the proband did not display marked mitochondrial respiratory chain enzyme deficiency. Immunoblotting of patient muscle and liver showed LARS2 levels were reduced in liver and complex I protein levels were reduced in patient muscle and liver. Aminoacylation assays revealed p.Ala430Val LARS2 had an 18-fold loss of catalytic efficiency and p.Thr522Asn a 9-fold loss compared to wild-type LARS2. We suggest that the identified LARS2 variants are responsible for the severe multisystem clinical phenotype seen in this baby and that mutations in LARS2 can result in variable phenotypes.

Yang Ying, Liu Wei, Fang Zhipeng, Shi Juan, Che Fengyu, He Chunxia, Yao Libo, Wang Enduo, Wu Yuanming.

Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous group of neurodegenerative disorders characterized by spasticity of the lower limbs due to pyramidal tract dysfunction. Here we report that a missense homozygous mutation c.424G>T (p.D142Y) in the FARS2 gene, which encodes a mitochondrial phenylalanyl tRNA synthetase (mtPheRS), causes HSP in a Chinese consanguineous family by using combination of homozygous mapping and whole exome sequencing. Immunohistochemical experiments were performed showing that the FARS2 protein was highly expressed in the Purkinje cells of rat cerebellum. The aminoacylation activity of mtPheRS was severely disrupted by the p.D142Y substitution in vitro not only in the first aminoacylation step but also in the last transfer step. Taken together, our results indicate a missense mutation in FARS2 contributes to HSP, which has the clinical significance of the regulation of tRNA synthetases in human neurodegenerative diseases. This article is protected by copyright. All rights reserved.

Kevelam Sietske H, Klouwer Femke C C, Fock Johanna M, Salomons Gajja S, Bugiani Marianna, van der Knaap Marjo S.

Leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) is caused by autosomal recessive EARS2 mutations. Onset is most often in infancy, but in severe cases in the neonatal period. Patients typically have magnetic resonance imaging (MRI) signal abnormalities involving the thalamus, brainstem, and deep cerebral white matter. Most signal abnormalities resolve, but in severe cases at the expense of tissue loss. Here, we report a patient with an encephalopathy of antenatal onset. His early MRI at 8 months of age showed signal abnormalities in the deep cerebral white matter that improved over time. The thalami were absent with the configuration of a developmental anomaly, without evidence of a lesion. We hypothesized that this was a case of LTBL in which the thalamic damage occurred antenatally and was incorporated in the normal brain development. The diagnosis was confirmed by a novel homozygous EARS2 mutation. Our case adds to the phenotypic and genetic spectrum of LTBL.

Sun Aping, Liu Xiangyi, Zheng Mei, Sun Qingli, Huang Yu, Fan Dongsheng.

To explore the clinical features of a novel glycyl-tRNA synthetase (GARS) gene mutation in a family with Charcot-Marie-Tooth disease type 2D (CMT2D).

Köhler Cornelia, Heyer Christoph, Hoffjan Sabine, Stemmler Susanne, Lücke Thomas, Thiels Charlotte, Kohlschütter Alfried, Löbel Ulrike, Horvath Rita, Kleinle Stephanie, Benet-Pages Anna, Abicht Angela.

Mutations in the DARS2 gene are known to cause leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL), a rare autosomal recessive neurological disorder. It was originally described as juvenile-onset slowly progressive ataxia and spasticity, but recent reports suggest a broader clinical spectrum. Most patients were found to carry compound heterozygous DARS2 mutations, and only very few patients with homozygous mutations have been described so far. We present here an 8-month-old boy carrying a homozygous missense mutation in DARS2 who clinically showed severe neurological deterioration after a respiratory tract infection, followed by an almost complete remission of symptoms. This report further extends the knowledge about the clinical and molecular genetic spectrum of LBSL.

Lax Nichola Z, Alston Charlotte L, Schon Katherine, Park Soo-Mi, Krishnakumar Deepa, He Langping, Falkous Gavin, Ogilvy-Stuart Amanda, Lees Christoph, King Rosalind H, Hargreaves Iain P, Brown Garry K, McFarland Robert, Dean Andrew F, Taylor Robert W.

Autosomal recessive mutations in the RARS2 gene encoding the mitochondrial arginyl-transfer RNA synthetase cause infantile-onset myoencephalopathy pontocerebellar hypoplasia type 6 (PCH6). We describe 2 sisters with novel compound heterozygous RARS2 mutations who presented perinatally with neurologic features typical of PCH6 but with additional features including cardiomyopathy, hydrops, and pulmonary hypoplasia and who died at 1 day and 14 days of age. Magnetic resonance imaging findings included marked cerebellar hypoplasia, gyral immaturity, punctate lesions in cerebral white matter, and unfused deep cerebral grey matter. Enzyme histochemistry of postmortem tissues revealed a near-global cytochrome c oxidase-deficiency; assessment of respiratory chain enzyme activities confirmed severe deficiencies involving complexes I, III, and IV. Molecular genetic studies revealed 2 RARS2 gene mutations: a c.1A>G, p.? variant predicted to abolish the initiator methionine, and a deep intronic c.613-3927C>T variant causing skipping of exons 6-8 in the mature RARS2 transcript. Neuropathologic investigation included low brain weights, small brainstem and cerebellum, deep cerebral white matter pathology, pontine nucleus neuron loss (in 1 sibling), and peripheral nerve pathology. Mitochondrial respiratory chain immunohistochemistry in brain tissues confirmed an absence of complexes I and IV immunoreactivity with sparing of mitochondrial numbers. These cases expand the clinical spectrum of RARS2 mutations, including antenatal features and widespread mitochondrial respiratory chain deficiencies in postmortem brain tissues.

McMillan Hugh J, Humphreys Peter, Smith Amanda, Schwartzentruber Jeremy, Chakraborty Pranesh, Bulman Dennis E, Beaulieu Chandree L, , Majewski Jacek, Boycott Kym M, Geraghty Michael T.

Aminoacyl-transfer ribonucleic acid (RNA) synthetases (ARSs) are a group of enzymes required for the first step of protein translation. Each aminoacyl-transfer RNA synthetase links a specific amino acid to its corresponding transfer RNA component within the cytoplasm, mitochondria, or both. Mutations in ARSs have been linked to a growing number of diseases. Lysyl-transfer RNA synthetase (KARS) links the amino acid lysine to its cognate transfer RNA. We report 2 siblings with severe infantile visual loss, progressive microcephaly, developmental delay, seizures, and abnormal subcortical white matter. Exome sequencing identified mutations within the KARS gene (NM_005548.2):c.1312C>T; p.Arg438Trp and c.1573G>A; p.Glu525Lys occurring within a highly conserved region of the catalytic domain. Our patients' phenotype is remarkably similar to a phenotype recently reported in glutaminyl-transfer RNA synthetase (QARS), another bifunctional ARS gene. This finding expands the phenotypic spectrum associated with mutations in KARS and draws attention to aminoacyl-transfer RNA synthetase as a group of enzymes that are increasingly being implicated in human disease.

Vernon Hilary J, McClellan Rebecca, Batista Denise As, Naidu Sakkubai.

Recently, mutations in FARS2, which encodes for mitochondrial phenylalanyl-tRNA synthetase, have been implicated in autosomal recessive combined oxidative phosphorylation deficiency 14. Associated clinical features in three previously reported patients with confirmed FARS2 mutations include infantile onset epilepsy, and a fatal Alpers-like encephalopathy. Herein, we report on two siblings with global developmental delay, dysarthria and tremor and compound heterozygous FARS2 abnormalities. They have a heterozygous missense mutation, c.1255C>T which predicts p.Arg419Cys in exon 7 of FARS2, inherited from their father and uncovered on exome sequencing, and an interstitial deletion of chromosome 6p25.1 inherited from their mother and uncovered on SNP array. This interstitial deletion includes all of exon 6 and parts of introns 5 and 6 of FARS2. Biochemical studies were also consistent with a mitochondrial disorder. While these siblings had considerable developmental difficulties, they are making consistent developmental progress and appear to be considerably less severely affected than the other patients reported in the literature with FARS2 associated mitochondrial disease. Thus, this study expands the phenotypic spectrum of FARS2 related disease and emphasizes intragenic deletion in the list of causative mutations. © 2015 Wiley Periodicals, Inc.

Li Zejuan, Schonberg Rhonda, Guidugli Lucia, Johnson Amy Knight, Arnovitz Stephen, Yang Sandra, Scafidi Joseph, Summar Marshall L, Vezina Gilbert, Das Soma, Chapman Kimberly, Del Gaudio Daniela.

Pontocerebellar hypoplasia (PCH) is characterized by hypoplasia and atrophy of the cerebellum, variable pontine atrophy, microcephaly, severe mental and motor impairments and seizures. Mutations in 11 genes have been reported in 8 out of 10 forms of PCH. Recessive mutations in the mitochondrial arginyl-transfer RNA synthetase gene (RARS2) have been recently associated with PCH type 6, which is characterized by early-onset encephalopathy with signs of oxidative phosphorylation defect. Here we describe the clinical presentation, neuroimaging findings and molecular characterizations of two siblings with a clinical diagnosis of PCH who displayed a novel variant (c.-2A>G) in the 5'-UTR of the RARS2 gene in the homozygous state. This variant was identified through next-generation sequencing testing of a panel of nine genes known to be involved in PCH. Gene expression and functional studies demonstrated that the c.-2A>G sequence change directly leads to a reduced RARS2 messenger RNA expression in the patients by decreasing RARS2 promoter activity, thus providing evidence that mutations in the RARS2 promoter are likely to represent a new causal mechanism of PCH6.Journal of Human Genetics advance online publication, 26 March 2015; doi:10.1038/jhg.2015.31.

Coughlin Curtis R, Scharer Gunter H, Friederich Marisa W, Yu Hung-Chun, Geiger Elizabeth A, Creadon-Swindell Geralyn, Collins Abigail E, Vanlander Arnaud V, Coster Rudy Van, Powell Christopher A, Swanson Michael A, Minczuk Michal, Van Hove Johan L K, Shaikh Tamim H.

Mitochondrial disease is often suspected in cases of severe epileptic encephalopathy especially when a complex movement disorder, liver involvement and progressive developmental regression are present. Although mutations in either mitochondrial DNA or POLG are often present, other nuclear defects in mitochondrial DNA replication and protein translation have been associated with a severe epileptic encephalopathy.

Samuels Mark E, Alos Nathalie, Deal Cheri L.

Mordaunt Dylan A, Savaririyan Ravi.

Webb Bryn D, Wheeler Patricia G, Hagen Jacob J, Cohen Ninette, Linderman Michael D, Diaz George A, Naidich Thomas P, Rodenburg Richard J, Houten Sander M, Schadt Eric E.

Novel, single-nucleotide mutations were identified in the mitochondrial methionyl amino-acyl tRNA synthetase gene (MARS2) via whole exome sequencing in two affected siblings with developmental delay, poor growth, and sensorineural hearing loss.We show that compound heterozygous mutations c.550C>T:p.Gln 184* and c.424C>T:p.Arg142Trp in MARS2 lead to decreased MARS2 protein levels in patient lymphoblasts. Analysis of respiratory complex enzyme activities in patient fibroblasts revealed decreased complex I and IV activities. Immunoblotting of patient fibroblast and lymphoblast samples revealed reduced protein levels of NDUFB8 and COXII, representing complex I and IV, respectively. Additionally, overexpression of wild-type MARS2 in patient fibroblasts increased NDUFB8 and COXII protein levels. These findings suggest that recessive single-nucleotide mutations in MARS2 are causative for a new mitochondrial translation deficiency disorder with a primary phenotype including developmental delay and hypotonia. Identification of additional patients with single-nucleotide mutations in MARS2 is necessary to determine if pectus carinatum is also a consistent feature of this syndrome.

Simon Mariella, Richard Elodie M, Wang Xinjian, Shahzad Mohsin, Huang Vincent H, Qaiser Tanveer A, Potluri Prasanth, Mahl Sarah E, Davila Antonio, Nazli Sabiha, Hancock Saege, Yu Margret, Gargus Jay, Chang Richard, Al-Sheqaih Nada, Newman William G, Abdenur Jose, Starr Arnold, Hegde Rashmi, Dorn Thomas, Busch Anke, Park Eddie, Wu Jie, Schwenzer Hagen, Flierl Adrian, Florentz Catherine, Sissler Marie, Khan Shaheen N, Li Ronghua, Guan Min-Xin, Friedman Thomas B, Wu Doris K, Procaccio Vincent, Riazuddin Sheikh, Wallace Douglas C, Ahmed Zubair M, Huang Taosheng, Riazuddin Saima.

Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.

Vanlander Arnaud V, Menten Björn, Smet Joél, De Meirleir Linda, Sante Tom, De Paepe Boel, Seneca Sara, Pearce Sarah F, Powell Christopher A, Vergult Sarah, Michotte Alex, De Latter Elien, Vantomme Lies, Minczuk Michal, Van Coster Rudy.

A homozygous missense mutation (c.822G>C) was found in the gene encoding the mitochondrial asparaginyl-tRNA synthetase (NARS2) in two siblings born to consanguineous parents. These siblings presented with different phenotypes: one had mild intellectual disability and epilepsy in childhood, whereas the other had severe myopathy. Biochemical analysis of the oxidative phosphorylation (OXPHOS) complexes in both siblings revealed a combined complex I and IV deficiency in skeletal muscle. In-gel activity staining after blue native-polyacrylamide gel electrophoresis confirmed the decreased activity of complex I and IV, and, in addition, showed the presence of complex V subcomplexes. Considering the consanguineous descent, homozygosity mapping and whole-exome sequencing were combined revealing the presence of one single missense mutation in the shared homozygous region. The c.822G>C variant affects the 3' splice site of exon 7, leading to skipping of the whole exon 7 and a part of exon 8 in the NARS2 mRNA. In EBV-transformed lymphoblasts, a specific decrease in the amount of charged mt-tRNA(Asn) was demonstrated as compared with controls. This confirmed the pathogenic nature of the variant. To conclude, the reported variant in NARS2 results in a combined OXPHOS complex deficiency involving complex I and IV, making NARS2 a new member of disease-associated aaRS2.

Sofou Kalliopi, Kollberg Gittan, Holmström Maria, Dávila Marcela, Darin Niklas, Gustafsson Claes M, Holme Elisabeth, Oldfors Anders, Tulinius Már, Asin-Cayuela Jorge.

Alpers syndrome is a progressive neurodegenerative disorder that presents in infancy or early childhood and is characterized by diffuse degeneration of cerebral gray matter. While mutations in POLG1, the gene encoding the gamma subunit of the mitochondrial DNA polymerase, have been associated with Alpers syndrome with liver failure (Alpers-Huttenlocher syndrome), the genetic cause of Alpers syndrome in most patients remains unidentified. With whole exome sequencing we have identified mutations in NARS2 and PARS2, the genes encoding the mitochondrial asparaginyl-and prolyl-tRNA synthetases, in two patients with Alpers syndrome. One of the patients was homozygous for a missense mutation (c.641C>T, p.P214L) in NARS2. The affected residue is predicted to be located in the stem of a loop that participates in dimer interaction. The other patient was compound heterozygous for a one base insertion (c.1130dupC, p.K378 fs*1) that creates a premature stop codon and a missense mutation (c.836C>T, p.S279L) located in a conserved motif of unknown function in PARS2. This report links for the first time mutations in these genes to human disease in general and to Alpers syndrome in particular.

Biancheri Roberta, Lamantea Eleonora, Severino Mariasavina, Diodato Daria, Pedemonte Marina, Cassandrini Denise, Ploederl Alexandra, Trucco Federica, Fiorillo Chiara, Minetti Carlo, Santorelli Filippo M, Zeviani Massimo, Bruno Claudio.

Leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) is a novel mitochondrial disease caused by mutations in EARS2, which encodes the mitochondrial glutamyl-tRNA synthetase (mtGluRS). A distinctive brain MRI pattern is the hallmark of the disease.A 6-year-old boy presented at 3 months with feeding difficulties and muscle hypotonia. Brain MRI, at 8 months, showed hyperintensity of the deep cerebral and cerebellar white matter, thalamus, basal ganglia, brainstem, and thin corpus callosum. From the second year of life onward, the child reported global clinical improvement, parallel to partial resolution of brain MRI pattern. However, the last neuroimaging assessment revealed novel lesions within the left caudate and pallidum nuclei. DNA genomic sequencing analysis identified a novel EARS2 mutation.This case expands the clinical and neuroradiological phenotype of LTBL presenting intermediate clinical manifestations between the severe and milder forms of the disease and previously unreported brain MRI features.

Euro Liliya, Konovalova Svetlana, Asin-Cayuela Jorge, Tulinius Már, Griffin Helen, Horvath Rita, Taylor Robert W, Chinnery Patrick F, Schara Ulrike, Thorburn David R, Suomalainen Anu, Chihade Joseph, Tyynismaa Henna.

The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs) and the mitochondrial DNA-encoded tRNAs. The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19) is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS) have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations. The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes.

Ardissone Anna, Lamantea Eleonora, Quartararo Jade, Dallabona Cristina, Carrara Franco, Moroni Isabella, Donnini Claudia, Garavaglia Barbara, Zeviani Massimo, Uziel Graziella.

YARS2 encodes the mitochondrial tyrosyl-tRNA synthetase that catalyzes the covalent binding of tyrosine to its cognate mt-tRNA. Mutations in YARS2 have been identified in patients with myopathy, lactic acidosis, and sideroblastic anemia type 2 (MLASA2). We report here on two siblings with a novel mutation and a review of literature. The older patient presented at 2 months with marked anemia and lactic acidemia. He required periodic blood transfusions until 14 months of age. Cognitive and motor development was normal. His younger sister was diagnosed at birth, presenting with anemia and lactic acidosis at 1 month of age requiring periodical transfusions. She is now 14 months old and doing well. For both our patients, there was no clinical evidence of muscle involvement. We found a new homozygous mutation in YARS2, located in the α-anticodon-binding (αACB) domain, involved in the interaction with the anticodon of the cognate mt-tRNA(Tyr).Our study confirms that MLASA must be considered in patients with congenital sideroblastic anemia and underlines the importance of early diagnosis and supportive therapy in order to prevent severe complications. Clinical severity is variable among YARS2-reported patients: our review of the literature suggests a possible phenotype-genotype correlation, although this should be confirmed in a larger population.

Liao Yi-Chu, Liu Yo-Tsen, Tsai Pei-Chien, Chang Chia-Ching, Huang Yen-Hua, Soong Bing-Wen, Lee Yi-Chung.

Mutations in the GARS gene have been identified in a small number of patients with Charcot-Marie-Tooth disease (CMT) type 2D or distal spinal muscular atrophy type V, for whom disease onset typically occurs during adolescence or young adulthood, initially manifesting as weakness and atrophy of the hand muscles. The role of GARS mutations in patients with inherited neuropathies in Taiwan remains elusive.

Hallmann Kerstin, Zsurka Gábor, Moskau-Hartmann Susanna, Kirschner Janbernd, Korinthenberg Rudolf, Ruppert Ann-Kathrin, Ozdemir Ozkan, Weber Yvonne, Becker Felicitas, Lerche Holger, Elger Christian E, Thiele Holger, Nürnberg Peter, Sander Thomas, Kunz Wolfram S.

We report a consanguineous family with 2 affected individuals whose clinical symptoms closely resembled MERRF (myoclonus epilepsy with ragged red fibers) syndrome including severe myoclonic epilepsy, progressive spastic tetraparesis, progressive impairment of vision and hearing, as well as progressive cognitive decline.

Liu Lei, Zhang Ruxu.

Charcot-Marie-Tooth (CMT) disease is a common neurogenetic disorder and its heterogeneity is a challenge for genetic diagnostics. The genetic diagnostic procedures for a CMT patient can be explored according to the electrophysiological criteria: very slow motor nerve conduction velocity (MNCV) (<15 m/s), slow MNCV (15-25 m/s), intermediate MNCV (25-45 m/s), and normal MNCV (>45 m/s). Based on the inheritance pattern, intermediate CMT can be divided into dominant (DI-CMT) and recessive types (RI-CMT). GJB1 is currently considered to be associated with X-linked DI-CMT, and MPZ, INF2, DNM2, YARS, GNB4, NEFL, and MFN2 are associated with autosomal DI-CMT. Moreover, GDAP1, KARS, and PLEKHG5 are associated with RI-CMT. Identification of these genes is not only important for patients and families but also provides new information about pathogenesis. It is hoped that this review will lead to a better understanding of intermediate CMT and provide a detailed diagnostic procedure for intermediate CMT.

Datt Manish, Sharma Amit.

Mutation(s) in proteins are a natural byproduct of evolution but can also cause serious diseases. Aminoacyl-tRNA synthetases (aaRSs) are indispensable components of all cellular protein translational machineries, and in humans they drive translation in both cytoplasm and mitochondria. Mutations in aaRSs have been implicated in a plethora of diseases including neurological conditions, metabolic disorders and cancer.

Joseph Jeffrey T, Innes A Micheil, Smith Amanda C, Vanstone Megan R, Schwartzentruber Jeremy A, Bulman Dennis E, Majewski Jacek, Daza Ray A, Hevner Robert F, Michaud Jean, Boycott Kym M, .

Pontocerebellar hypoplasia is a group of severe developmental disorders with prenatal onset affecting the growth and function of the brainstem and cerebellum. The rarity and genetic heterogeneity of this group of disorders can make molecular diagnosis challenging. We report 3 siblings who were born to nonconsanguineous parents, were hypotonic at birth, developed seizures, had repeated apneic spells, and died within 2 months of life. Neuroimaging showed that all had profound cerebellar hypoplasia and simplified cortical gyration. Genetic analysis by whole-exome sequencing demonstrated compound heterozygous mutations in the mitochondrial arginyl transfer RNA synthetase gene RARS2, indicating that the children had pontocerebellar hypoplasia type 6. Autopsies on the younger twin siblings revealed small and immature cerebella at an approximate developmental age of less than 18 weeks. The basis pontis showed regressive changes, and the medulla had marked inferior olivary hypoplasia. The brains of both twins were microencephalic and had simplified gyri; cortices were immature, and deep white matter had extensive astrocytosis. The findings suggest a near-normal embryologic period followed by midgestation developmental slowing or cessation and later regression in select anatomic regions. This is the first detailed description of neuropathologic findings associated with pontocerebellar hypoplasia type 6 and demonstrates the profound effects of RARS2 disruption during early neurodevelopment.

Schwartzentruber Jeremy, Buhas Daniela, Majewski Jacek, Sasarman Florin, Papillon-Cavanagh Simon, Thiffault Isabelle, Sheldon Katherine M, Massicotte Christine, Patry Lysanne, Simon Mariella, Zare Amir S, McKernan Kevin J, , Michaud Jacques, Boles Richard G, Deal Cheri L, Desilets Valerie, Shoubridge Eric A, Samuels Mark E.

Mutations in the nuclear-encoded mitochondrial aminoacyl-tRNA synthetases are associated with a range of clinical phenotypes. Here, we report a novel disorder in three adult patients with a phenotype including cataracts, short-stature secondary to growth hormone deficiency, sensorineural hearing deficit, peripheral sensory neuropathy, and skeletal dysplasia. Using SNP genotyping and whole-exome sequencing, we identified a single likely causal variant, a missense mutation in a conserved residue of the nuclear gene IARS2, encoding mitochondrial isoleucyl-tRNA synthetase. The mutation is homozygous in the affected patients, heterozygous in carriers, and absent in control chromosomes. IARS2 protein level was reduced in skin cells cultured from one of the patients, consistent with a pathogenic effect of the mutation. Compound heterozygous mutations in IARS2 were independently identified in a previously unreported patient with a more severe mitochondrial phenotype diagnosed as Leigh syndrome. This is the first report of clinical findings associated with IARS2 mutations.

DiVincenzo Christina, Elzinga Christopher D, Medeiros Adam C, Karbassi Izabela, Jones Jeremiah R, Evans Matthew C, Braastad Corey D, Bishop Crystal M, Jaremko Malgorzata, Wang Zhenyuan, Liaquat Khalida, Hoffman Carol A, York Michelle D, Batish Sat D, Lupski James R, Higgins Joseph J.

We report the frequency, positive rate, and type of mutations in 14 genes (PMP22, GJB1, MPZ, MFN2, SH3TC2, GDAP1, NEFL, LITAF, GARS, HSPB1, FIG4, EGR2, PRX, and RAB7A) associated with Charcot-Marie-Tooth disease (CMT) in a cohort of 17,880 individuals referred to a commercial genetic testing laboratory. Deidentified results from sequencing assays and multiplex ligation-dependent probe amplification (MLPA) were analyzed including 100,102 Sanger sequencing, 2338 next-generation sequencing (NGS), and 21,990 MLPA assays. Genetic abnormalities were identified in 18.5% (n = 3312) of all individuals. Testing by Sanger and MLPA (n = 3216) showed that duplications (dup) (56.7%) or deletions (del) (21.9%) in the PMP22 gene accounted for the majority of positive findings followed by mutations in the GJB1 (6.7%), MPZ (5.3%), and MFN2 (4.3%) genes. GJB1 del and mutations in the remaining genes explained 5.3% of the abnormalities. Pathogenic mutations were distributed as follows: missense (70.6%), nonsense (14.3%), frameshift (8.7%), splicing (3.3%), in-frame deletions/insertions (1.8%), initiator methionine mutations (0.8%), and nonstop changes (0.5%). Mutation frequencies, positive rates, and the types of mutations were similar between tests performed by either Sanger (n = 17,377) or NGS (n = 503). Among patients with a positive genetic finding in a CMT-related gene, 94.9% were positive in one of four genes (PMP22, GJB1, MPZ, or MFN2).

Klein Christopher J, Middha Sumit, Duan Xiaohui, Wu Yanhong, Litchy William J, Gu Weihong, Dyck P James B, Gavrilova Ralitza H, Smith David I, Kocher Jean-Pierre, Dyck Peter J.

Inherited polyneuropathies often go undiagnosed. We investigated whole exome sequencing (WES) in utility to identify the genetic causes of diverse forms of inherited polyneuropathies without genetic diagnosis.

Diodato Daria, Melchionda Laura, Haack Tobias B, Dallabona Cristina, Baruffini Enrico, Donnini Claudia, Granata Tiziana, Ragona Francesca, Balestri Paolo, Margollicci Maria, Lamantea Eleonora, Nasca Alessia, Powell Christopher A, Minczuk Michal, Strom Tim M, Meitinger Thomas, Prokisch Holger, Lamperti Costanza, Zeviani Massimo, Ghezzi Daniele.

By way of whole-exome sequencing, we identified a homozygous missense mutation in VARS2 in one subject with microcephaly and epilepsy associated with isolated deficiency of the mitochondrial respiratory chain (MRC) complex I and compound heterozygous mutations in TARS2 in two siblings presenting with axial hypotonia and severe psychomotor delay associated with multiple MRC defects. The nucleotide variants segregated within the families, were absent in Single Nucleotide Polymorphism (SNP) databases and are predicted to be deleterious. The amount of VARS2 and TARS2 proteins and valyl-tRNA and threonyl-tRNA levels were decreased in samples of afflicted patients according to the genetic defect. Expression of the corresponding wild-type transcripts in immortalized mutant fibroblasts rescued the biochemical impairment of mitochondrial respiration and yeast modeling of the VARS2 mutation confirmed its pathogenic role. Taken together, these data demonstrate the role of the identified mutations for these mitochondriopathies. Our study reports the first mutations in the VARS2 and TARS2 genes, which encode two mitochondrial aminoacyl-tRNA synthetases, as causes of clinically distinct, early-onset mitochondrial encephalopathies.

Lee Seo Jin, Seo Ah Jung, Park Byung Sun, Jo Hyun Woo, Huh Youngbuhm.

Charcot-Marie-Tooth disease (CMT) is the most common inherited motor and sensory neuropathy. Previous studies have found that, according to CMT patients, neuropathic pain is an occasional symptom of CMT. However, neuropathic pain is not considered to be a significant symptom associated with CMT and, as a result, no studies have investigated the pathophysiology underlying neuropathic pain in this disorder. Thus, the first animal model of neuropathic pain was developed by our laboratory using an adenovirus vector system to study neuropathic pain in CMT. To this end, glycyl-tRNA synthetase (GARS) fusion proteins with a FLAG-tag (wild type [WT], L129P and G240R mutants) were expressed in spinal cord and dorsal root ganglion (DRG) neurons using adenovirus vectors. It is known that GARS mutants induce GARS axonopathies, including CMT type 2D (CMT2D) and distal spinal muscular atrophy type V (dSMA-V). Additionally, the morphological phenotypes of neuropathic pain in this animal model of GARS-induced pain were assessed using several possible markers of pain (Iba1, pERK1/2) or a marker of injured neurons (ATF3). These results suggest that this animal model of CMT using an adenovirus may provide information regarding CMT as well as a useful strategy for the treatment of neuropathic pain.

Taylor Robert W, Pyle Angela, Griffin Helen, Blakely Emma L, Duff Jennifer, He Langping, Smertenko Tania, Alston Charlotte L, Neeve Vivienne C, Best Andrew, Yarham John W, Kirschner Janbernd, Schara Ulrike, Talim Beril, Topaloglu Haluk, Baric Ivo, Holinski-Feder Elke, Abicht Angela, Czermin Birgit, Kleinle Stephanie, Morris Andrew A M, Vassallo Grace, Gorman Grainne S, Ramesh Venkateswaran, Turnbull Douglass M, Santibanez-Koref Mauro, McFarland Robert, Horvath Rita, Chinnery Patrick F.

Mitochondrial disorders have emerged as a common cause of inherited disease, but their diagnosis remains challenging. Multiple respiratory chain complex defects are particularly difficult to diagnose at the molecular level because of the massive number of nuclear genes potentially involved in intramitochondrial protein synthesis, with many not yet linked to human disease.

van der Knaap Marjo S, Hamilton Eline M, van Berge Laura.

Pinto Wladimir Bocca Vieira de Rezende, de Souza Paulo Victor Sgobbi.

Dallabona Cristina, Diodato Daria, Kevelam Sietske H, Haack Tobias B, Wong Lee-Jun, Salomons Gajja S, Baruffini Enrico, Melchionda Laura, Mariotti Caterina, Strom Tim M, Meitinger Thomas, Prokisch Holger, Chapman Kim, Colley Alison, Rocha Helena, Ounap Katrin, Schiffmann Raphael, Salsano Ettore, Savoiardo Mario, Hamilton Eline M, Abbink Truus E M, Wolf Nicole I, Ferrero Ileana, Lamperti Costanza, Zeviani Massimo, Vanderver Adeline, Ghezzi Daniele, van der Knaap Marjo S.

The study was focused on leukoencephalopathies of unknown cause in order to define a novel, homogeneous phenotype suggestive of a common genetic defect, based on clinical and MRI findings, and to identify the causal genetic defect shared by patients with this phenotype.

Tylki-Szymanska Anna, Jurkiewicz Elzbieta, Zakharova Ekaterina Y, Bobek-Billewicz Barbara.

Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation is a rare disorder caused by mutations in the gene encoding a mitochondrial aspartyl-tRNA synthetase, DARS2. Clinical features include childhood or adolescent onset, slowly progressive ataxia, spasticity, and dorsal column dysfunction with or without mild cognitive decline. Magnetic resonance (MR) images show a characteristic leukoencephalopathy with multiple long tract involvement. MR spectroscopy shows elevated levels of lactate. We present strikingly different clinical courses and discrepant MR images findings for a pair of brothers. It cannot be excluded that in the cases presented, the different clinical outcome is related to a varicella infection in infancy in the younger brother.

van Berge Laura, Hamilton Eline M, Linnankivi Tarja, Uziel Graziella, Steenweg Marjan E, Isohanni Pirjo, Wolf Nicole I, Krägeloh-Mann Ingeborg, Brautaset Nils J, Andrews P Ian, de Jong Brigit A, al Ghamdi Malak, van Wieringen Wessel N, Tannous Bakhos A, Hulleman Esther, Würdinger Thomas, van Berkel Carola G M, Polder Emiel, Abbink Truus E M, Struys Eduard A, Scheper Gert C, van der Knaap Marjo S, .

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation is a disorder caused by recessive mutations in the gene DARS2, which encodes mitochondrial aspartyl-tRNA synthetase. Recent observations indicate that the phenotypic range of the disease is much wider than initially thought. Currently, no treatment is available. The aims of our study were (i) to explore a possible genotype-phenotype correlation; and (ii) to identify potential therapeutic agents that modulate the splice site mutations in intron 2 of DARS2, present in almost all patients. A cross-sectional observational study was performed in 78 patients with two DARS2 mutations in the Amsterdam and Helsinki databases up to December 2012. Clinical information was collected via questionnaires. An inventory was made of the DARS2 mutations in these patients and those previously published. An assay was developed to assess mitochondrial aspartyl-tRNA synthetase enzyme activity in cells. Using a fluorescence reporter system we screened for drugs that modulate DARS2 splicing. Clinical information of 66 patients was obtained. The clinical severity varied from infantile onset, rapidly fatal disease to adult onset, slow and mild disease. The most common phenotype was characterized by childhood onset and slow neurological deterioration. Full wheelchair dependency was rare and usually began in adulthood. In total, 60 different DARS2 mutations were identified, 13 of which have not been reported before. Except for 4 of 42 cases published by others, all patients were compound heterozygous. Ninety-four per cent of the patients had a splice site mutation in intron 2. The groups of patients sharing the same two mutations were too small for formal assessment of genotype-phenotype correlation. However, some combinations of mutations were consistently associated with a mild phenotype. The mitochondrial aspartyl-tRNA synthetase activity was strongly reduced in patient cells. Among the compounds screened, cantharidin was identified as the most potent modulator of DARS2 splicing. In conclusion, the phenotypic spectrum of leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation is wide, but most often the disease has a relatively slow and mild course. The available evidence suggests that the genotype influences the phenotype, but because of the high number of private mutations, larger numbers of patients are necessary to confirm this. The activity of mitochondrial aspartyl-tRNA synthetase is significantly reduced in patient cells. A compound screen established a 'proof of principle' that the splice site mutation can be influenced. This finding is promising for future therapeutic strategies.

Nakajima Junya, Eminoglu Tuba F, Vatansever Goksel, Nakashima Mitsuko, Tsurusaki Yoshinori, Saitsu Hirotomo, Kawashima Hisashi, Matsumoto Naomichi, Miyake Noriko.

Mitochondrial diseases are associated with defects of adenosine triphosphate production and energy supply to organs as a result of dysfunctions of the mitochondrial respiratory chain. Biallelic mutations in the YARS2 gene encoding mitochondrial tyrosyl-tRNA synthetase cause myopathy, lactic acidosis, and sideroblastic anemia 2 (MLASA2), a type of mitochondrial disease. Here, we report a consanguineous Turkish family with two siblings showing severe metabolic decompensation including recurrent hypoglycemia, lactic acidosis, and transfusion-dependent anemia. Using whole-exome sequencing of the proband and his parents, we identified a novel YARS2 mutation (c.1303A>G, p.Ser435Gly) that was homozygous in the patient and heterozygous in his parents. This mutation is located at the ribosomal protein S4-like domain of the gene, while other reported YARS2 mutations are all within the catalytic domain. Interestingly, the proband showed more severe symptoms and an earlier onset than previously reported patients, suggesting the functional importance of the S4-like domain in tyrosyl-tRNA synthetase.

McMillan Hugh J, Schwartzentruber Jeremy, Smith Amanda, Lee Suzie, Chakraborty Pranesh, Bulman Dennis E, Beaulieu Chandree L, Majewski Jacek, Boycott Kym M, Geraghty Michael T.

Glycyl-tRNA synthetase (GARS) is an aminoacyl-tRNA synthetase (ARS) that links the amino acid glycine to its corresponding tRNA prior to protein translation and is one of three bifunctional ARS that are active within both the cytoplasm and mitochondria. Dominant mutations in GARS cause rare forms of Charcot-Marie-Tooth disease and distal spinal muscular atrophy.

Kassem Hassan, Wafaie Ahmed, Abdelfattah Sherif, Farid Tarek.

Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) is a recently identified autosomal recessive disorder with early onset of symptoms and slowly progressive pyramidal, cerebellar and dorsal column dysfunction. LBSL is characterized by distinct white matter abnormalities and selective involvement of brainstem and spinal cord tracts. The purpose of this study is to assess the imaging features of the involved white matter tracts in cases of LBSL by MRI.

Almalki Abdulraheem, Alston Charlotte L, Parker Alasdair, Simonic Ingrid, Mehta Sarju G, He Langping, Reza Mojgan, Oliveira Jorge M A, Lightowlers Robert N, McFarland Robert, Taylor Robert W, Chrzanowska-Lightowlers Zofia M A.

Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNA(Phe) transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNA(Phe) as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNA(Phe).

Kawakami Nobuko, Komatsu Kenichi, Yamashita Hirofumi, Uemura Kengo, Oka Nobuyuki, Takashima Hiroshi, Takahashi Ryosuke.

Charcot-Marie-Tooth disease (CMT) is a hereditary peripheral neuropathy; symptoms include distal wasting and weakness, usually with some sensory impairment. The clinical course is typically benign and the disease is not life threatening; however, in some cases, severe phenotypes include serious respiratory distress.

Abbott Jamie A, Francklyn Christopher S, Robey-Bond Susan M.

Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA) genes are "hotspots" for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase), mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing). Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

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Riley Lisa G, Menezes Minal J, Rudinger-Thirion Joëlle, Duff Rachael, de Lonlay Pascale, Rotig Agnes, Tchan Michel C, Davis Mark, Cooper Sandra T, Christodoulou John.

Mutations in the mitochondrial tyrosyl-tRNA synthetase (YARS2) gene have previously been identified as a cause of the tissue specific mitochondrial respiratory chain (RC) disorder, Myopathy, Lactic Acidosis, Sideroblastic Anaemia (MLASA). In this study, a cohort of patients with a mitochondrial RC disorder for who anaemia was a feature, were screened for mutations in YARS2.

Kastrissianakis Katherina, Anand Geetha, Quaghebeur Gerardine, Price Sue, Prabhakar Prab, Marinova Jasmina, Brown Garry, McShane Tony.

Mutations in the recently described RARS2 gene encoding for mitochondrial arginyl-transfer RNA synthetase give rise to a disorder characterised by early onset seizures, progressive microcephaly and developmental delay. The disorder was named pontocerebellar hypoplasia type 6 (PCH6) based on the corresponding radiological findings observed in the original cases. We report two siblings with the RARS2 mutation who displayed typical clinical features of PCH6, but who had distinct neuroimaging features. Early scans showed marked supratentorial, rather than infratentorial, atrophy, and the pons remained preserved throughout. One sibling also had bilateral subdural effusions at presentation. The deceleration in head growth pointed to an evolving genetic/metabolic process giving rise to cerebral atrophy and secondary subdural effusions. RARS2 mutations should be considered in infants presenting with seizures, subdural effusions, decelerating head growth and evidence of cerebral atrophy even in the absence of pontocerebellar hypoplasia on imaging.

Fujiwara Tohru, Harigae Hideo.

Sideroblastic anemias are heterogeneous congenital and acquired disorders characterized by anemia and the presence of ringed sideroblasts in the bone marrow. Congenital sideroblastic anemia (CSA) is a rare disease caused by mutations of genes involved in heme biosynthesis, iron-sulfur [Fe-S] cluster biosynthesis, and mitochondrial protein synthesis. The most common form is X-linked sideroblastic anemia, due to mutations in the erythroid-specific δ-aminolevulinate synthase (ALAS2), which is the first enzyme of the heme biosynthesis pathway in erythroid cells. Other known etiologies include mutations in the erythroid specific mitochondrial transporter (SLC25A38), adenosine triphosphate (ATP) binding cassette B7 (ABCB7), glutaredoxin 5 (GLRX5), thiamine transporter SLC19A2, the RNA-modifying enzyme pseudouridine synthase (PUS1), and mitochondrial tyrosyl-tRNA synthase (YARS2), as well as mitochondrial DNA deletions. Due to its rarity, however, there have been few systematic pathophysiological and genetic investigations focusing on sideroblastic anemia. Therefore, a nationwide survey of sideroblastic anemia was conducted in Japan to investigate the epidemiology and pathogenesis of this disease. This review will cover the findings of this recent survey and summarize the current understanding of the pathophysiology and genetic mutations involved in CSA.

Németh Andrea H, Kwasniewska Alexandra C, Lise Stefano, Parolin Schnekenberg Ricardo, Becker Esther B E, Bera Katarzyna D, Shanks Morag E, Gregory Lorna, Buck David, Zameel Cader M, Talbot Kevin, de Silva Rajith, Fletcher Nicholas, Hastings Rob, Jayawant Sandeep, Morrison Patrick J, Worth Paul, Taylor Malcolm, Tolmie John, O'Regan Mary, , Valentine Ruth, Packham Emily, Evans Julie, Seller Anneke, Ragoussis Jiannis.

Many neurological conditions are caused by immensely heterogeneous gene mutations. The diagnostic process is often long and complex with most patients undergoing multiple invasive and costly investigations without ever reaching a conclusive molecular diagnosis. The advent of massively parallel, next-generation sequencing promises to revolutionize genetic testing and shorten the 'diagnostic odyssey' for many of these patients. We performed a pilot study using heterogeneous ataxias as a model neurogenetic disorder to assess the introduction of next-generation sequencing into clinical practice. We captured 58 known human ataxia genes followed by Illumina Next-Generation Sequencing in 50 highly heterogeneous patients with ataxia who had been extensively investigated and were refractory to diagnosis. All cases had been tested for spinocerebellar ataxia 1-3, 6, 7 and Friedrich's ataxia and had multiple other biochemical, genetic and invasive tests. In those cases where we identified the genetic mutation, we determined the time to diagnosis. Pathogenicity was assessed using a bioinformatics pipeline and novel variants were validated using functional experiments. The overall detection rate in our heterogeneous cohort was 18% and varied from 8.3% in those with an adult onset progressive disorder to 40% in those with a childhood or adolescent onset progressive disorder. The highest detection rate was in those with an adolescent onset and a family history (75%). The majority of cases with detectable mutations had a childhood onset but most are now adults, reflecting the long delay in diagnosis. The delays were primarily related to lack of easily available clinical testing, but other factors included the presence of atypical phenotypes and the use of indirect testing. In the cases where we made an eventual diagnosis, the delay was 3-35 years (mean 18.1 years). Alignment and coverage metrics indicated that the capture and sequencing was highly efficient and the consumable cost was ∼£400 (€460 or US$620). Our pathogenicity interpretation pathway predicted 13 different mutations in eight different genes: PRKCG, TTBK2, SETX, SPTBN2, SACS, MRE11, KCNC3 and DARS2 of which nine were novel including one causing a newly described recessive ataxia syndrome. Genetic testing using targeted capture followed by next-generation sequencing was efficient, cost-effective, and enabled a molecular diagnosis in many refractory cases. A specific challenge of next-generation sequencing data is pathogenicity interpretation, but functional analysis confirmed the pathogenicity of novel variants showing that the pipeline was robust. Our results have broad implications for clinical neurology practice and the approach to diagnostic testing.

Rivera Henry, Martín-Hernández Elena, Delmiro Aitor, García-Silva María Teresa, Quijada-Fraile Pilar, Muley Rafael, Arenas Joaquín, Martín Miguel A, Martínez-Azorín Francisco.

HUPRA syndrome is a rare mitochondrial disease characterized by hyperuricemia, pulmonary hypertension, renal failure in infancy and alkalosis. This syndrome was previously described in three patients with a homozygous mutation c.1169A > G (p.D390G) in SARS2, encoding the mitochondrial seryl-tRNA synthetase.

Shahni Rojeen, Wedatilake Yehani, Cleary Maureen A, Lindley Keith J, Sibson Keith R, Rahman Shamima.

Nuclear-encoded disorders of mitochondrial translation are clinically and genetically heterogeneous. Genetic causes include defects of mitochondrial aminoacyl-tRNA synthetases, and factors required for initiation, elongation and termination of protein synthesis as well as ribosome recycling. We report on a new case of myopathy, lactic acidosis and sideroblastic anemia (MLASA) syndrome caused by defective mitochondrial tyrosyl aminoacylation. The patient presented at 1 year with anemia initially attributed to iron deficiency. Bone marrow aspirate at 5 years revealed ringed sideroblasts but transfusion dependency did not occur until 11 years. Other clinical features included lactic acidosis, poor weight gain, hypertrophic cardiomyopathy and severe myopathy leading to respiratory failure necessitating ventilatory support. Long-range PCR excluded mitochondrial DNA rearrangements. Clinical diagnosis of MLASA prompted direct sequence analysis of the YARS2 gene encoding the mitochondrial tyrosyl-tRNA synthetase, which revealed homozygosity for a known pathogenic mutation, c.156C>G;p.F52L. Comparison with four previously reported cases demonstrated remarkable clinical homogeneity. First line investigation of MLASA should include direct sequence analysis of YARS2 and PUS1 (encoding a tRNA modification factor) rather than muscle biopsy. Early genetic diagnosis is essential for counseling and to facilitate appropriate supportive therapy. Reasons for segregation of specific clinical phenotypes with particular mitochondrial aminoacyl tRNA-synthetase defects remain unknown.

Martikainen Mika H, Ellfolk Ulla, Majamaa Kari.

Leukoencephalopathy with brainstem and spinal cord involvement and elevated lactate (LBSL) is clinically characterized by progressive pyramidal and cerebellar dysfunction, dorsal column dysfunction and sometimes with axonal neuropathy. Magnetic resonance imaging of brain and the spinal cord reveals characteristic findings. LBSL is caused by mutations in the DARS2 gene that encodes the mitochondrial aspartyl-tRNA synthetase. The presentation and clinical course of LBSL is not uniform, and there is lack of longitudinal data on these patients. In addition, the existing data on the prevalence and characteristics of cognitive abnormalities in patients with LBSL are scarce and somewhat conflicting. Here we report long-term data of neurological and cognitive functioning in three non-related adult patients with LBSL. Cognitive impairment seems to be common among patients with LBSL and DARS2 mutations. The cognitive profile in LBSL shares similarities with that reported in multiple sclerosis, as information-processing speed and working memory are especially affected. In addition, our results and the previously reported carrier frequencies of common pathogenic DARS2 mutations suggest that LBSL may be underdiagnosed in the population.

Santos-Cortez Regie Lyn P, Lee Kwanghyuk, Azeem Zahid, Antonellis Patrick J, Pollock Lana M, Khan Saadullah, Irfanullah , Andrade-Elizondo Paula B, Chiu Ilene, Adams Mark D, Basit Sulman, Smith Joshua D, , Nickerson Deborah A, McDermott Brian M, Ahmad Wasim, Leal Suzanne M.

Previously, DFNB89, a locus associated with autosomal-recessive nonsyndromic hearing impairment (ARNSHI), was mapped to chromosomal region 16q21-q23.2 in three unrelated, consanguineous Pakistani families. Through whole-exome sequencing of a hearing-impaired individual from each family, missense mutations were identified at highly conserved residues of lysyl-tRNA synthetase (KARS): the c.1129G>A (p.Asp377Asn) variant was found in one family, and the c.517T>C (p.Tyr173His) variant was found in the other two families. Both variants were predicted to be damaging by multiple bioinformatics tools. The two variants both segregated with the nonsyndromic-hearing-impairment phenotype within the three families, and neither mutation was identified in ethnically matched controls or within variant databases. Individuals homozygous for KARS mutations had symmetric, severe hearing impairment across all frequencies but did not show evidence of auditory or limb neuropathy. It has been demonstrated that KARS is expressed in hair cells of zebrafish, chickens, and mice. Moreover, KARS has strong localization to the spiral ligament region of the cochlea, as well as to Deiters' cells, the sulcus epithelium, the basilar membrane, and the surface of the spiral limbus. It is hypothesized that KARS variants affect aminoacylation in inner-ear cells by interfering with binding activity to tRNA or p38 and with tetramer formation. The identification of rare KARS variants in ARNSHI-affected families defines a gene that is associated with ARNSHI.

Lieber Daniel S, Calvo Sarah E, Shanahan Kristy, Slate Nancy G, Liu Shangtao, Hershman Steven G, Gold Nina B, Chapman Brad A, Thorburn David R, Berry Gerard T, Schmahmann Jeremy D, Borowsky Mark L, Mueller David M, Sims Katherine B, Mootha Vamsi K.

To evaluate the utility of targeted exome sequencing for the molecular diagnosis of mitochondrial disorders, which exhibit marked phenotypic and genetic heterogeneity.

Schicks Julia, Schöls Ludger, van der Knaap Marjo S, Synofzik Matthis.

Pierce Sarah B, Gersak Ksenija, Michaelson-Cohen Rachel, Walsh Tom, Lee Ming K, Malach Daniel, Klevit Rachel E, King Mary-Claire, Levy-Lahad Ephrat.

The genetic causes of premature ovarian failure (POF) are highly heterogeneous, and causative mutations have been identified in more than ten genes so far. In two families affected by POF accompanied by hearing loss (together, these symptoms compose Perrault syndrome), exome sequencing revealed mutations in LARS2, encoding mitochondrial leucyl-tRNA synthetase: homozygous c.1565C>A (p.Thr522Asn) in a consanguineous Palestinian family and compound heterozygous c.1077delT and c.1886C>T (p.Thr629Met) in a nonconsanguineous Slovenian family. LARS2 c.1077delT leads to a frameshift at codon 360 of the 901 residue protein. LARS2 p.Thr522Asn occurs in the LARS2 catalytic domain at a site conserved from bacteria through mammals. LARS2 p.Thr629Met occurs in the LARS2 leucine-specific domain, which is adjacent to a catalytic loop critical in all species but for which primary sequence is not well conserved. A recently developed method of detecting remote homologies revealed threonine at this site in consensus sequences derived from multiple-species alignments seeded by human and E. coli residues at this region. Yeast complementation indicated that LARS2 c.1077delT is nonfunctional and that LARS2 p.Thr522Asn is partially functional. LARS2 p.Thr629Met was functional in this assay but might be insufficient as a heterozygote with the fully nonfunctional LARS2 c.1077delT allele. A known C. elegans strain with the protein-truncating alteration LARS-2 p.Trp247Ter was confirmed to be sterile. After HARS2, LARS2 is the second gene encoding mitochondrial tRNA synthetase to be found to harbor mutations leading to Perrault syndrome, further supporting a critical role for mitochondria in the maintenance of ovarian function and hearing.

Konovalova Svetlana, Tyynismaa Henna.

Mitochondrial aminoacyl-tRNA synthetases (mtARSs) are essential in the process of transferring genetic information from mitochondrial DNA to the complexes of the oxidative phosphorylation system. These synthetases perform an integral step in the initiation of mitochondrial protein synthesis by charging tRNAs with their cognate amino acids. All mtARSs are encoded by nuclear genes, nine of which have recently been described as disease genes for mitochondrial disorders. Unexpectedly, the clinical presentations of these diseases are highly specific to the affected synthetase. Encephalopathy is the most common manifestation but again with gene-specific outcomes. Other clinical presentations include myopathy with anemia, cardiomyopathy, tubulopathy and hearing loss with female ovarian dysgenesis. Here we review the described mutation types and the associated patient phenotypes. The identified mutation spectrum suggests that only mutation types that allow some residual tRNA-charging activity can result in the described mtARS diseases but the molecular mechanisms behind the selective tissue involvement are not currently understood.

Yamashita Sumimasa, Miyake Noriko, Matsumoto Naomichi, Osaka Hitoshi, Iai Mizue, Aida Noriko, Tanaka Yukichi.

We diagnosed three siblings from consanguineous east Asian parents with leukoencephalopathy with brainstem and spinal cord involvement and high lactate (LBSL) from characteristic MRI, MRS findings and a homozygous mutation in the DARS2 gene. The neurological symptoms of the three patients consisted of psychomotor developmental delay, cerebellar ataxia since infancy, spasticity in the initial phase and peripheral neuropathy in later stages. Their mental development was delayed, but did not deteriorate. MRI signal abnormalities included the same abnormalities reported previously but tended to be more extensive. Signal abnormalities in the cerebral and cerebellar white matter were homogeneous and confluent from early stages. In addition, other tract such as the central tegmental tract was involved. Furthermore, an atrophic change in the cerebral white matter was observed on follow-up in one case. Two of the patients were autopsied and neuropathological findings revealed characteristic vacuolar changes in the white matter of the cerebrum, cerebellum and the nerve tracts of the brain stem and spinal cord. The central myelin sheath showed intralamellar splitting by electron microscopy. These findings were consistent to a spongy degeneration in the diffuse white matter of the brain, or spongiform leukoencephalopathy. In addition, peripheral nerves showed both axonal degeneration and abnormal myelin structures. We discussed the relationship between deficits in mitochondrial aspartyl-tRNA synthetase activity and the neuropathology observed.

Cheng Fu-Bo, Shen Ping-Ping, Zhou Hong-Wei, Meng Hong-Mei, Yang Yu, Feng Jia-Chun.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a recently described disorder with an autosomal recessive mode of inheritance. We report a case of rare adult-onset LBSL with typical magnetic resonance imaging (MRI) features. The DARS2 gene mutation analysis has identified a c. 228-20_21delTTinsC (p.R76SfsX5) mutation and a c. 850G > A (p. 284E > K) mutation. With glucocorticosteroid treatment the patient has had improvement in bladder symptoms. This is the first reported adult-onset LBSL case in the Chinese Han population. A review of the literature suggests that brain lactate elevation in adult-onset LBSL is lower than early-onset cases (P < 0.01), and early-onset cases show mild intelligence and cognition decline. These observations suggest that age of onset and brain lactate levels probably influence the prognosis of LBSL.

van Berge Laura, Kevenaar Josta, Polder Emiel, Gaudry Agnès, Florentz Catherine, Sissler Marie, van der Knaap Marjo S, Scheper Gert C.

The autosomal recessive white matter disorder LBSL (leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation) is caused by mutations in DARS2, coding for mtAspRS (mitochondrial aspartyl-tRNA synthetase). Generally, patients are compound heterozygous for mutations in DARS2. Many different mutations have been identified in patients, including several missense mutations. In the present study, we have examined the effects of missense mutations found in LBSL patients on the expression, enzyme activity, localization and dimerization of mtAspRS, which is important for understanding the cellular defect underlying the pathogenesis of the disease. Nine different missense mutations were analysed and were shown to have various effects on mtAspRS properties. Several mutations have a direct effect on the catalytic activity of the enzyme; others have an effect on protein expression or dimerization. Most mutations have a clear impact on at least one of the properties of mtAspRS studied, probably resulting in a small contribution of the missense variants to the mitochondrial aspartylation activity in the cell.

Talim Beril, Pyle Angela, Griffin Helen, Topaloglu Haluk, Tokatli Aysegul, Keogh Michael J, Santibanez-Koref Mauro, Chinnery Patrick F, Horvath Rita.

Cassandrini Denise, Cilio Maria Roberta, Bianchi Marzia, Doimo Mara, Balestri Martina, Tessa Alessandra, Rizza Teresa, Sartori Geppo, Meschini Maria Chiara, Nesti Claudia, Tozzi Giulia, Petruzzella Vittoria, Piemonte Fiorella, Bisceglia Luigi, Bruno Claudio, Dionisi-Vici Carlo, D'Amico Adele, Fattori Fabiana, Carrozzo Rosalba, Salviati Leonardo, Santorelli Filippo M, Bertini Enrico.

Recessive mutations in the mitochondrial arginyl-transfer RNA synthetase (RARS2) gene have been associated with early onset encephalopathy with signs of oxidative phosphorylation defects classified as pontocerebellar hypoplasia 6. We describe clinical, neuroimaging and molecular features on five patients from three unrelated families who displayed mutations in RARS2. All patients rapidly developed a neonatal or early-infantile epileptic encephalopathy with intractable seizures. The long-term follow-up revealed a virtual absence of psychomotor development, progressive microcephaly, and feeding difficulties. Mitochondrial respiratory chain enzymes in muscle and fibroblasts were normal in two. Blood and CSF lactate was abnormally elevated in all five patients at early stages while appearing only occasionally abnormal with the progression of the disease. Cerebellar vermis hypoplasia with normal aspect of the cerebral and cerebellar hemispheres appeared within the first months of life at brain MRI. In three patients follow-up neuroimaging revealed a progressive pontocerebellar and cerebral cortical atrophy. Molecular investigations of RARS2 disclosed the c.25A>G/p.I9V and the c.1586+3A>T in family A, the c.734G>A/p.R245Q and the c.1406G>A/p.R469H in family B, and the c.721T>A/p.W241R and c.35A>G/p.Q12R in family C. Functional complementation studies in Saccharomyces cerevisiae showed that mutation MSR1-R531H (equivalent to human p.R469H) abolished respiration whereas the MSR1-R306Q strain (corresponding to p.R245Q) displayed a reduced growth on non-fermentable YPG medium. Although mutations functionally disrupted yeast we found a relatively well preserved arginine aminoacylation of mitochondrial tRNA. Clinical and neuroimaging findings are important clues to raise suspicion and to reach diagnostic accuracy for RARS2 mutations considering that biochemical abnormalities may be absent in muscle biopsy.

Lee Hye Jin, Park Jin, Nakhro Khriezanou, Park Jin Mo, Hur Yoon-Mi, Choi Byung-Ok, Chung Ki Wha.

Glycyl-tRNA synthetase (GARS), which encodes the enzyme responsible for charging tRNA(Gly) with glycine in both the cytoplasm and mitochondria, is implicated to Charcot-Marie-Tooth disease 2D (CMT2D) and distal hereditary motor neuropathy type V (dHMN-V). We performed whole exome sequencing (WES) to identify the genetic defects in the two dHMN families. WES revealed several decades of non-synonymous variants in the CMT and aminoacyl-tRNA synthetase genes. The subsequent capillary sequencing for family members and controls revealed two novel causative mutations, c.598G>A (D200N) and c.794C>T (S265F), in the GARS gene in each dHMN family. Both mutations were cosegregated with affected individuals in each family, and were not found in the 200 controls. The mutation sites were well conserved between the different species and in silico analysis predicted that both mutations may affect protein function. Therefore, we believe that these two novel GARS mutations are the underlying causes of the dHMN phenotype.

Elo Jenni M, Yadavalli Srujana S, Euro Liliya, Isohanni Pirjo, Götz Alexandra, Carroll Christopher J, Valanne Leena, Alkuraya Fowzan S, Uusimaa Johanna, Paetau Anders, Caruso Eric M, Pihko Helena, Ibba Michael, Tyynismaa Henna, Suomalainen Anu.

Next-generation sequencing has turned out to be a powerful tool to uncover genetic basis of childhood mitochondrial disorders. We utilized whole-exome analysis and discovered novel compound heterozygous mutations in FARS2 (mitochondrial phenylalanyl transfer RNA synthetase), encoding the mitochondrial phenylalanyl transfer RNA (tRNA) synthetase (mtPheRS) in two patients with fatal epileptic mitochondrial encephalopathy. The mutations affected highly conserved amino acids, p.I329T and p.D391V. Recently, a homozygous FARS2 variant p.Y144C was reported in a Saudi girl with mitochondrial encephalopathy, but the pathogenic role of the variant remained open. Clinical features, including postnatal onset, catastrophic epilepsy, lactic acidemia, early lethality and neuroimaging findings of the patients with FARS2 variants, resembled each other closely, and neuropathology was consistent with Alpers syndrome. Our structural analysis of mtPheRS predicted that p.I329T weakened ATP binding in the aminoacylation domain, and in vitro studies with recombinant mutant protein showed decreased affinity of this variant to ATP. Furthermore, p.D391V and p.Y144C were predicted to disrupt synthetase function by interrupting the rotation of the tRNA anticodon stem-binding domain from a closed to an open form. In vitro characterization indicated reduced affinity of p.D391V mutant protein to phenylalanine, whereas p.Y144C disrupted tRNA binding. The stability of p.I329T and p.D391V mutants in a refolding assay was impaired. Our results imply that the three FARS2 mutations directly impair aminoacylation function and stability of mtPheRS, leading to a decrease in overall tRNA charging capacity. This study establishes a new genetic cause of infantile mitochondrial Alpers encephalopathy and reports a new mitochondrial aminoacyl-tRNA synthetase as a cause of mitochondrial disease.

Sasarman Florin, Nishimura Tamiko, Thiffault Isabelle, Shoubridge Eric A.

Mutations in the mitochondrial aminoacyl-tRNA synthetases (ARSs) are associated with a strikingly broad range of clinical phenotypes, the molecular basis for which remains obscure. Here, we report a novel missense mutation (c.137G>A, p.Gly46Asp) in the catalytic domain of YARS2, which codes for the mitochondrial tyrosyl-tRNA synthetase, in a subject with myopathy, lactic acidosis, and sideroblastic anemia (MLASA). YARS2 was undetectable by immunoblot analysis in subject myoblasts, resulting in a generalized mitochondrial translation defect. Retroviral expression of a wild-type YARS2 complementary DNA completely rescued the translation defect. We previously demonstrated that the respiratory chain defect in this subject was only present in fully differentiated muscle, and we show here that this likely reflects an increased requirement for YARS2 as muscle cells differentiate. An additional, heterozygous mutation was detected in TRMU/MTU1, a gene encoding the mitochondrial 2-thiouridylase. Although subject myoblasts and myotubes contained half the normal levels of TRMU, thiolation of mitochondrial tRNAs was normal. YARS2 eluted as part of high-molecular-weight complexes of ∼250 kDa and 1 MDa by gel filtration. This study confirms mutations in YARS2 as a cause of MLASA and shows that, like some of the cytoplasmic ARSs, mitochondrial ARSs occur in high-molecular-weight complexes.

Steenweg Marjan E, Ghezzi Daniele, Haack Tobias, Abbink Truus E M, Martinelli Diego, van Berkel Carola G M, Bley Annette, Diogo Luisa, Grillo Eugenio, Te Water Naudé Johann, Strom Tim M, Bertini Enrico, Prokisch Holger, van der Knaap Marjo S, Zeviani Massimo.

In the large group of genetically undetermined infantile-onset mitochondrial encephalopathies, multiple defects of mitochondrial DNA-related respiratory-chain complexes constitute a frequent biochemical signature. In order to identify responsible genes, we used exome-next-generation sequencing in a selected cohort of patients with this biochemical signature. In an isolated patient, we found two mutant alleles for EARS2, the gene encoding mitochondrial glutamyl-tRNA synthetase. The brain magnetic resonance imaging of this patient was hallmarked by extensive symmetrical cerebral white matter abnormalities sparing the periventricular rim and symmetrical signal abnormalities of the thalami, midbrain, pons, medulla oblongata and cerebellar white matter. Proton magnetic resonance spectroscopy showed increased lactate. We matched this magnetic resonance imaging pattern with that of a cohort of 11 previously selected unrelated cases. We found mutations in the EARS2 gene in all. Subsequent detailed clinical and magnetic resonance imaging based phenotyping revealed two distinct groups: mild and severe. All 12 patients shared an infantile onset and rapidly progressive disease with severe magnetic resonance imaging abnormalities and increased lactate in body fluids and proton magnetic resonance spectroscopy. Patients in the 'mild' group partially recovered and regained milestones in the following years with striking magnetic resonance imaging improvement and declining lactate levels, whereas those of the 'severe' group were characterized by clinical stagnation, brain atrophy on magnetic resonance imaging and persistent lactate increases. This new neurological disease, early-onset leukoencephalopathy with thalamus and brainstem involvement and high lactate, is hallmarked by unique magnetic resonance imaging features, defined by a peculiar biphasic clinical course and caused by mutations in a single gene, EARS2, expanding the list of medically relevant defects of mitochondrial DNA translation.

Rahman Shamima.

Mitochondrial respiratory chain disorders are relatively common inborn errors of energy metabolism, with a combined prevalence of one in 5000. These disorders typically affect tissues with high energy requirements, and cerebral involvement occurs frequently in childhood, often manifesting in seizures. Mitochondrial diseases are genetically heterogeneous; to date, mutations have been reported in all 37 mitochondrially encoded genes and more than 80 nuclear genes. The major genetic causes of mitochondrial epilepsy are mitochondrial DNA mutations (including those typically associated with the mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes [MELAS] and myoclonic epilepsy with ragged red fibres [MERRF] syndromes); mutations in POLG (classically associated with Alpers syndrome but also presenting as the mitochondrial recessive ataxia syndrome [MIRAS], spinocerebellar ataxia with epilepsy [SCAE], and myoclonus, epilepsy, myopathy, sensory ataxia [MEMSA] syndromes in older individuals) and other disorders of mitochondrial DNA maintenance; complex I deficiency; disorders of coenzyme Q(10) biosynthesis; and disorders of mitochondrial translation such as RARS2 mutations. It is not clear why some genetic defects, but not others, are particularly associated with seizures. Epilepsy may be the presenting feature of mitochondrial disease but is often part of a multisystem clinical presentation. Mitochondrial epilepsy may be very difficult to manage, and is often a poor prognostic feature. At present there are no curative treatments for mitochondrial disease. Individuals with mitochondrial epilepsy are frequently prescribed multiple anticonvulsants, and the role of vitamins and other nutritional supplements and the ketogenic diet remain unproven.

Glamuzina Emma, Brown Ruth, Hogarth Kieran, Saunders Dawn, Russell-Eggitt Isabelle, Pitt Matthew, de Sousa Carlos, Rahman Shamima, Brown Garry, Grunewald Stephanie.

Pontocerebellar hypoplasia type 6 (PCH6) (MIM #611523) is a recently described disorder caused by mutations in RARS2 (MIM *611524), the gene encoding mitochondrial arginyl-transfer RNA (tRNA) synthetase, a protein essential for translation of all mitochondrially synthesised proteins. This case confirms that progressive cerebellar and cerebral atrophy with microcephaly and complex epilepsy are characteristic features of PCH6. Additional features of PCH subtypes 2 and 4, including severe dystonia, optic atrophy and thinning of the corpus callosum, are demonstrated. Congenital lactic acidosis can be present, but respiratory chain dysfunction may be mild or absent, suggesting that disordered mitochondrial messenger RNA (mRNA) translation may not be the only mechanism of impairment or that a secondary mechanism exists to allow some translation. We report two novel mutations and expand the phenotypic spectrum of this likely underdiagnosed PCH variant, where recognition of the characteristic neuroradiological phenotype could potentially expedite genetic diagnosis and limit invasive investigations.

Jenkinson Emma M, Clayton-Smith Jill, Mehta Sarju, Bennett Christopher, Reardon Willie, Green Andrew, Pearce Simon H S, De Michele Giuseppe, Conway Gerard S, Cilliers Deirdre, Moreton Natalie, Davis Julian R E, Trump Dorothy, Newman William G.

Shamseldin Hanan E, Alshammari Muneera, Al-Sheddi Tarfa, Salih Mustafa A, Alkhalidi Hisham, Kentab Amal, Repetto Gabriela M, Hashem Mais, Alkuraya Fowzan S.

To investigate the utility of autozygome analysis and exome sequencing in a cohort of patients with suspected or confirmed mitochondrial encephalomyopathy.

Wong Lee-Jun C.

White matter involvement has recently been recognized as a common feature in patients with multisystem mitochondrial disorders that may be caused by molecular defects in either the mitochondrial genome or the nuclear genes. It was first realized in classical mitochondrial syndromes associated with mitochondrial DNA (mtDNA) mutations, such as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), Leigh's disease, and Kearns-Sayre's syndrome. Deficiencies in respiratory chain complexes I, II, IV, and V often cause Leigh's disease; most of them are due to nuclear defects that may lead to severe early-onset leukoencephalopathies. Defects in a group of nuclear genes involved in the maintenance of mtDNA integrity may also affect the white matter; for example, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) caused by thymidine phosphorylase deficiency, Navajo neurohepatopathy (NNH) due to MPV17 mutations, and Alpers syndrome due to defects in DNA polymerase gamma (POLG). More recently, leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) has been reported to be caused by autosomal recessive mutations in a mitochondrial aspartyl-tRNA synthetase, DARS2 gene. A patient with leukoencephalopathy and neurologic complications in addition to a multisystem involvement warrants a complete evaluation for mitochondrial disorders. A definite diagnosis may be achieved by molecular analysis of candidate genes based on the biochemical, clinical, and imaging results.

van Berge Laura, Dooves Stephanie, van Berkel Carola G M, Polder Emiel, van der Knaap Marjo S, Scheper Gert C.

LBSL (leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation) is an autosomal recessive white matter disorder with slowly progressive cerebellar ataxia, spasticity and dorsal column dysfunction. Magnetic resonance imaging shows characteristic abnormalities in the cerebral white matter and specific brain stem and spinal cord tracts. LBSL is caused by mutations in the gene DARS2, which encodes mtAspRS (mitochondrial aspartyl-tRNA synthetase). The selective involvement of specific white matter tracts in LBSL is striking since this protein is ubiquitously expressed. Almost all LBSL patients have one mutation in intron 2 of DARS2, affecting the splicing of the third exon. Using a splicing reporter construct, we find cell-type-specific differences in the sensitivity to these mutations: the mutations have a larger effect on exon 3 exclusion in neural cell lines, especially neuronal cell lines, than in non-neural cell lines. Furthermore, correct inclusion of exon 3 in the normal mtAspRS mRNA occurs less efficiently in neural cells than in other cell types, and this effect is again most pronounced in neuronal cells. The combined result of these two effects may explain the selective vulnerability of specific white matter tracts in LBSL patients.

Tzoulis Charalampos, Tran Gia Tuong, Gjerde Ivar Otto, Aasly Jan, Neckelmann Gesche, Rydland Jana, Varga Viktoria, Wadel-Andersen Pia, Bindoff Laurence A.

Leukoencephalopathy with brainstem and spinal cord involvement and elevated lactate (LBSL) is a rare, autosomal recessive disorder caused by mutations in the gene encoding a mitochondrial aspartyl-tRNA synthetase, DARS2. The disease is characterized by progressive spastic ataxia and magnetic resonance imaging (MRI) shows a highly characteristic leukoencephalopathy with multiple long tract involvement. We describe the clinical and radiological features of two new cases of LBSL and report a novel pathogenic mutation in the DARS2 gene. Both patients had typical clinical and radiological findings, although no elevated lactate was found. The severity of MRI changes did not correlate with clinical course and severity suggesting that, although of highly specific diagnostic value, MRI does not necessarily reflect clinical activity and should not be used to assess disease severity or prognosis in LBSL.

Huang Qiong-hui, Xiao Jiang-xi, Wang Jing-min, Jiang Yu-wu, Wu Ye.

Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) is a rare autosomal recessive disease. Affected individuals are invariably compound heterozygous for two mutations in DARS2. No reports of LBSL patients have been published in the mainland of China. The aim of this study was to explore the clinical and genetic features of a family with LBSL, which may contribute to definite diagnosis, genetic counseling and prenatal diagnosis of this rare disease in China.

Bayat Vafa, Thiffault Isabelle, Jaiswal Manish, Tétreault Martine, Donti Taraka, Sasarman Florin, Bernard Geneviève, Demers-Lamarche Julie, Dicaire Marie-Josée, Mathieu Jean, Vanasse Michel, Bouchard Jean-Pierre, Rioux Marie-France, Lourenco Charles M, Li Zhihong, Haueter Claire, Shoubridge Eric A, Graham Brett H, Brais Bernard, Bellen Hugo J.

An increasing number of genes required for mitochondrial biogenesis, dynamics, or function have been found to be mutated in metabolic disorders and neurological diseases such as Leigh Syndrome. In a forward genetic screen to identify genes required for neuronal function and survival in Drosophila photoreceptor neurons, we have identified mutations in the mitochondrial methionyl-tRNA synthetase, Aats-met, the homologue of human MARS2. The fly mutants exhibit age-dependent degeneration of photoreceptors, shortened lifespan, and reduced cell proliferation in epithelial tissues. We further observed that these mutants display defects in oxidative phosphorylation, increased Reactive Oxygen Species (ROS), and an upregulated mitochondrial Unfolded Protein Response. With the aid of this knowledge, we identified MARS2 to be mutated in Autosomal Recessive Spastic Ataxia with Leukoencephalopathy (ARSAL) patients. We uncovered complex rearrangements in the MARS2 gene in all ARSAL patients. Analysis of patient cells revealed decreased levels of MARS2 protein and a reduced rate of mitochondrial protein synthesis. Patient cells also exhibited reduced Complex I activity, increased ROS, and a slower cell proliferation rate, similar to Drosophila Aats-met mutants.

Steenweg Marianne E, Pouwels Petra J W, Wolf Nicole I, van Wieringen Wessel N, Barkhof Frederik, van der Knaap Marjo S.

Leucoencephalopathy with brainstem and spinal cord involvement and elevated lactate is a white matter disorder caused by DARS2 mutations. The pathology is unknown. We observed striking discrepancies between improvement on longitudinal conventional magnetic resonance images and clinical deterioration and between large areas of high signal on diffusion-weighted imaging and small areas with low apparent diffusion coefficient values. These observations prompted a longitudinal and quantitative magnetic resonance imaging study. We investigated eight patients (two males, mean age 27 years). Maps of T(2) relaxation times, fractional anisotropy, apparent diffusion coefficients, signal on diffusion-weighted imaging, and axial and radial diffusivities were generated. Brain metabolites, obtained by chemical shift imaging, were quantified. Data analysis focused on: (i) white matter with low apparent diffusion coefficient; (ii) white matter with high T(2) values; (iii) white matter with intermediate T(2) values; and (iv) normal-appearing white matter. The areas were compared with similarly located areas in eight matched controls. In five patients, T(2)-weighted images, spectroscopy, apparent diffusion coefficient maps and diffusion-weighted imaging maps were compared with those obtained 5-7 years ago. In white matter with low apparent diffusion coefficient, axial and radial diffusivities were decreased and fractional anisotropy was high. T(2) values were intermediate. These areas with truly restricted diffusion were small and often observed at the periphery of areas with high T(2) values. In the white matter with high and intermediate T(2) values, apparent diffusion coefficients and axial and radial diffusivities were increased and fractional anisotropy decreased. The signal on diffusion-weighted imaging was highest in white matter with high T(2) values, an effect of T(2) shinethrough. Chemical shift imaging in both white matter types showed increased lactate, increased myo-inositol and decreased N-acetylaspartate, most pronounced in white matter with high T(2) values. Normal-appearing white matter was comparable with white matter of control subjects. Over time, mild decreases in T(2) signal intensities, signal on diffusion-weighted imaging and in extent of the low apparent diffusion coefficient areas were seen. In conclusion, the disease process in leucoencephalopathy with brainstem and spinal cord involvement and elevated lactate is extremely slow. We hypothesize that diffusion restriction is the first stage of the disease caused by intramyelinic water accumulation, followed by slow shift and then loss of the surplus of water. On conventional T(2) images this leads to improvement. We hypothesize that it is loss of water rather than structural restoration that causes the change in T(2) signal intensity, which would be in better agreement with the slow clinical deterioration.

Synofzik Matthis, Schicks Julia, Lindig Tobias, Biskup Saskia, Schmidt Thorsten, Hansel Jochen, Lehmann-Horn Frank, Schöls Ludger.

Leukoencephalopathy with brain stem and spinal cord involvement and brain lactate elevation (LBSL) was recently shown to be caused by mutations in the DARS2 gene, encoding a mitochondrial aspartyl-tRNA synthetase. So far, affected individuals were invariably compound heterozygous for two mutations in DARS2, and drug treatments have remained elusive.

Mierzewska Hanna, van der Knaap Marjo S, Scheper Gert C, Bekiesinska-Figatowska Monika, Szczepanik Elzbieta, Jurkiewicz Elzbieta.

Leukoencephalopathy with brain stem and spinal cord involvement and elevated white matter lactate (LBSL) is a very rare autosomal recessive mitochondrial disorder. Clinically patients have slowly progressive ataxia, pyramidal syndrome and dorsal column dysfunction. The disease is defined on the basis of characteristic abnormalities observed on magnetic resonance imaging such as inhomogeneous, spotty involvement of the cerebral white matter, selective involvement of brain stem and spinal cord tracts as well as lactate elevation in the affected white matter on spectroscopy. We present the first identified Polish patient suffering from LBSL confirmed molecularly.

Miyake N, Yamashita S, Kurosawa K, Miyatake S, Tsurusaki Y, Doi H, Saitsu H, Matsumoto N.

Orcesi Simona, La Piana Roberta, Uggetti Carla, Tonduti Davide, Pichiecchio Anna, Pasin Moreno, Viselner Gisela, Comi Giacomo P, Del Bo Roberto, Ronchi Dario, Bastianello Stefano, Balottin Umberto.

Spinal cord calcifications are an unusual finding in pediatric neurology. We here describe a young child who presented severe psychomotor delay, tetraplegia, deafness, and anemia. Neuroradiological investigations revealed severe leukodystrophy and unusual calcifications in the cerebral white matter and all along the medullary pathways. Common infectious and metabolic diseases were ruled out. A mild reduction in the activity of several respiratory chain complexes was documented on muscle biopsy. Of interest, we found an intronic variant in DARS2, a gene involved in mitochondrial DNA translation, responsible for the syndrome of leukoencephalopathy with brainstem and spinal cord involvement and high brain lactate. In our opinion, our case, and probably 2 previously reported Japanese siblings with a picture very similar to that of our patient, could represent a new, progressive leukoencephalomyelopathy.

Sharma Suvasini, Sankhyan Naveen, Kumar Atin, Scheper Gert C, van der Knaap Marjo S, Gulati Sheffali.

A 17-year-old Indian boy with gradually progressive ataxia with onset at 12 years of age is described. Magnetic resonance imaging (MRI) of the brain revealed extensive, inhomogeneous signal abnormalities in the cerebral white matter, with involvement of selected tracts in the brain stem and spinal cord. The imaging findings were characteristic of leukoencephalopathy with brain stem and spinal cord involvement and high lactate, a recently described leukodystrophy. Interestingly, magnetic resonance spectroscopy of the abnormal white matter did not reveal elevated lactate. The patient was compound heterozygous for 2 new mutations in DARS2, genetically confirming the diagnosis.

Götz Alexandra, Tyynismaa Henna, Euro Liliya, Ellonen Pekka, Hyötyläinen Tuulia, Ojala Tiina, Hämäläinen Riikka H, Tommiska Johanna, Raivio Taneli, Oresic Matej, Karikoski Riitta, Tammela Outi, Simola Kalle O J, Paetau Anders, Tyni Tiina, Suomalainen Anu.

Infantile cardiomyopathies are devastating fatal disorders of the neonatal period or the first year of life. Mitochondrial dysfunction is a common cause of this group of diseases, but the underlying gene defects have been characterized in only a minority of cases, because tissue specificity of the manifestation hampers functional cloning and the heterogeneity of causative factors hinders collection of informative family materials. We sequenced the exome of a patient who died at the age of 10 months of hypertrophic mitochondrial cardiomyopathy with combined cardiac respiratory chain complex I and IV deficiency. Rigorous data analysis allowed us to identify a homozygous missense mutation in AARS2, which we showed to encode the mitochondrial alanyl-tRNA synthetase (mtAlaRS). Two siblings from another family, both of whom died perinatally of hypertrophic cardiomyopathy, had the same mutation, compound heterozygous with another missense mutation. Protein structure modeling of mtAlaRS suggested that one of the mutations affected a unique tRNA recognition site in the editing domain, leading to incorrect tRNA aminoacylation, whereas the second mutation severely disturbed the catalytic function, preventing tRNA aminoacylation. We show here that mutations in AARS2 cause perinatal or infantile cardiomyopathy with near-total combined mitochondrial respiratory chain deficiency in the heart. Our results indicate that exome sequencing is a powerful tool for identifying mutations in single patients and allows recognition of the genetic background in single-gene disorders of variable clinical manifestation and tissue-specific disease. Furthermore, we show that mitochondrial disorders extend to prenatal life and are an important cause of early infantile cardiac failure.

Abe Akiko, Numakura Chikahiko, Kijima Kazuki, Hayashi Makiko, Hashimoto Taeko, Hayasaka Kiyoshi.

To study the genetic background of Japanese Charcot-Marie-Tooth disease (CMT) patients, we analyzed qualitative and quantitative changes in the disease-causing genes mainly by denaturing high performance liquid chromatography and multiplex ligation-dependent probe analysis in 227 patients with demyelinating CMT and 127 patients with axonal CMT. In demyelinating CMT, we identified 53 patients with PMP22 duplication, 10 patients with PMP22 mutations, 20 patients with MPZ mutations, eight patients with NEFL mutations, 19 patients with GJB1 mutations, one patient with EGR2 mutation, five patients with PRX mutations and no mutations in 111 patients. In axonal CMT, we found 14 patients with MFN2 mutations, one patient with GARS mutation, five patients with MPZ mutations, one patient with GDAP1 mutation, six patients with GJB1 mutations and no mutations in 100 patients. Most of the patients carrying PMP22, MPZ, NEFL, PRX and MFN2 mutations showed early onset, whereas half of the patients carrying PMP22 duplication and all patients with GJB1 or MPZ mutations showing axonal phenotype were adult onset. Our data showed that a low prevalence of PMP22 duplication and high frequency of an unknown cause are features of Japanese CMT. Low prevalence of PMP22 duplication is likely associated with the mild symptoms due to genetic and/or epigenetic modifying factors.

Pierce Sarah B, Chisholm Karen M, Lynch Eric D, Lee Ming K, Walsh Tom, Opitz John M, Li Weiqing, Klevit Rachel E, King Mary-Claire.

Perrault syndrome is a genetically heterogeneous recessive disorder characterized by ovarian dysgenesis and sensorineural hearing loss. In a nonconsanguineous family with five affected siblings, linkage analysis and genomic sequencing revealed the genetic basis of Perrault syndrome to be compound heterozygosity for mutations in the mitochondrial histidyl tRNA synthetase HARS2 at two highly conserved amino acids, L200V and V368L. The nucleotide substitution creating HARS2 p.L200V also created an alternate splice leading to deletion of 12 codons from the HARS2 message. Affected family members thus carried three mutant HARS2 transcripts. Aminoacylation activity of HARS2 p.V368L and HARS2 p.L200V was reduced and the deletion mutant was not stably expressed in mammalian mitochondria. In yeast, lethality of deletion of the single essential histydyl tRNA synthetase HTS1 was fully rescued by wild-type HTS1 and by HTS1 p.L198V (orthologous to HARS2 p.L200V), partially rescued by HTS1 p.V381L (orthologous to HARS2 p.V368L), and not rescued by the deletion mutant. In Caenorhabditis elegans, reduced expression by RNAi of the single essential histydyl tRNA synthetase hars-1 severely compromised fertility. Together, these data suggest that Perrault syndrome in this family was caused by reduction of HARS2 activity. These results implicate aberrations of mitochondrial translation in mammalian gonadal dysgenesis. More generally, the relationship between HARS2 and Perrault syndrome illustrates how causality may be demonstrated for extremely rare inherited mutations in essential, highly conserved genes.

Kasher Paul R, Namavar Yasmin, van Tijn Paula, Fluiter Kees, Sizarov Aleksander, Kamermans Maarten, Grierson Andrew J, Zivkovic Danica, Baas Frank.

Pontocerebellar hypoplasia (PCH) represents a group (PCH1-6) of neurodegenerative autosomal recessive disorders characterized by hypoplasia and/or atrophy of the cerebellum, hypoplasia of the ventral pons, progressive microcephaly and variable neocortical atrophy. The majority of PCH2 and PCH4 cases are caused by mutations in the TSEN54 gene; one of the four subunits comprising the tRNA-splicing endonuclease (TSEN) complex. We hypothesized that TSEN54 mutations act through a loss of function mechanism. At 8 weeks of gestation, human TSEN54 is expressed ubiquitously in the brain, yet strong expression is seen within the telencephalon and metencephalon. Comparable expression patterns for tsen54 are observed in zebrafish embryos. Morpholino (MO) knockdown of tsen54 in zebrafish embryos results in loss of structural definition in the brain. This phenotype was partially rescued by co-injecting the MO with human TSEN54 mRNA. A developmental patterning defect was not associated with tsen54 knockdown; however, an increase in cell death within the brain was observed, thus bearing resemblance to PCH pathophysiology. Additionally, N-methyl-N-nitrosourea mutant zebrafish homozygous for a tsen54 premature stop-codon mutation die within 9 days post-fertilization. To determine whether a common disease pathway exists between TSEN54 and other PCH-related genes, we also monitored the effects of mitochondrial arginyl-tRNA synthetase (rars2; PCH1 and PCH6) knockdown in zebrafish. Comparable brain phenotypes were observed following the inhibition of both genes. These data strongly support the hypothesis that TSEN54 mutations cause PCH through a loss of function mechanism. Also we suggest that a common disease pathway may exist between TSEN54- and RARS2-related PCH, which may involve a tRNA processing-related mechanism.

Belostotsky Ruth, Ben-Shalom Efrat, Rinat Choni, Becker-Cohen Rachel, Feinstein Sofia, Zeligson Sharon, Segel Reeval, Elpeleg Orly, Nassar Suheir, Frishberg Yaacov.

An uncharacterized multisystemic mitochondrial cytopathy was diagnosed in two infants from consanguineous Palestinian kindred living in a single village. The most significant clinical findings were tubulopathy (hyperuricemia, metabolic alkalosis), pulmonary hypertension, and progressive renal failure in infancy (HUPRA syndrome). Analysis of the consanguineous pedigree suggested that the causative mutation is in the nuclear DNA. By using genome-wide SNP homozygosity analysis, we identified a homozygous identity-by-descent region on chromosome 19 and detected the pathogenic mutation c.1169A>G (p.Asp390Gly) in SARS2, encoding the mitochondrial seryl-tRNA synthetase. The same homozygous mutation was later identified in a third infant with HUPRA syndrome. The carrier rate of this mutation among inhabitants of this Palestinian isolate was found to be 1:15. The mature enzyme catalyzes the ligation of serine to two mitochondrial tRNA isoacceptors: tRNA(Ser)(AGY) and tRNA(Ser)(UCN). Analysis of amino acylation of the two target tRNAs, extracted from immortalized peripheral lymphocytes derived from two patients, revealed that the p.Asp390Gly mutation significantly impacts on the acylation of tRNA(Ser)(AGY) but probably not that of tRNA(Ser)(UCN). Marked decrease in the expression of the nonacylated transcript and the complete absence of the acylated tRNA(Ser)(AGY) suggest that this mutation leads to significant loss of function and that the uncharged transcripts undergo degradation.

Labauge Pierre, Dorboz Imen, Eymard-Pierre Eleonore, Dereeper Olivier, Boespflug-Tanguy Odile.

Messmer Marie, Florentz Catherine, Schwenzer Hagen, Scheper Gert C, van der Knaap Marjo S, Maréchal-Drouard Laurence, Sissler Marie.

Mutations in the nuclear gene coding for the mitochondrial aspartyl-tRNA synthetase, a key enzyme for mitochondrial translation, are correlated with leukoencephalopathy. A Ser⁴⁵ to Gly⁴⁵ mutation is located in the predicted targeting signal of the protein. We demonstrate in the present study, by in vivo and in vitro approaches, that this pathology-related mutation impairs the import process across mitochondrial membranes.

Namavar Yasmin, Barth Peter G, Kasher Paul R, van Ruissen Fred, Brockmann Knut, Bernert Günther, Writzl Karin, Ventura Karen, Cheng Edith Y, Ferriero Donna M, Basel-Vanagaite Lina, Eggens Veerle R C, Krägeloh-Mann Ingeborg, De Meirleir Linda, King Mary, Graham John M, von Moers Arpad, Knoers Nine, Sztriha Laszlo, Korinthenberg Rudolf, , Dobyns William B, Baas Frank, Poll-The Bwee Tien.

Pontocerebellar hypoplasia is a group of autosomal recessive neurodegenerative disorders with prenatal onset. The common characteristics are cerebellar hypoplasia with variable atrophy of the cerebellum and the ventral pons. Supratentorial involvement is reflected by variable neocortical atrophy, ventriculomegaly and microcephaly. Mutations in the transfer RNA splicing endonuclease subunit genes (TSEN54, TSEN2, TSEN34) were found to be associated with pontocerebellar hypoplasia types 2 and 4. Mutations in the mitochondrial transfer RNA arginyl synthetase gene (RARS2) were associated with pontocerebellar hypoplasia type 6. We studied a cohort of 169 patients from 141 families for mutations in these genes, of whom 106 patients tested positive for mutations in one of the TSEN genes or the RARS2 gene. In order to delineate the neuroradiological and clinical phenotype of patients with mutations in these genes, we compared this group with 63 patients suspected of pontocerebellar hypoplasia who were negative on mutation analysis. We found a strong correlation (P < 0.0005) between TSEN54 mutations and a dragonfly-like cerebellar pattern on magnetic resonance imaging, in which the cerebellar hemispheres are flat and severely reduced in size and the vermis is relatively spared. Mutations in TSEN54 are clinically associated with dyskinesia and/or dystonia and variable degrees of spasticity, in some cases with pure generalized spasticity. Nonsense or splice site mutations in TSEN54 are associated with a more severe phenotype of more perinatal symptoms, ventilator dependency and early death. In addition, we present ten new mutations in TSEN54, TSEN2 and RARS2. Furthermore, we show that pontocerebellar hypoplasia type 1 together with elevated cerebrospinal fluid lactate may be caused by RARS2 mutations.

Lin Jaime, Chiconelli Faria Eliete, Da Rocha Antônio José, Rodrigues Masruha Marcelo, Pereira Vilanova Luiz Celso, Scheper Gert C, Van der Knaap Marjo S.

Leukoencephalopathy with brainstem and spinal cord involvement and elevated brain lactate diagnosis is based on its highly characteristic pattern of abnormalities observed by magnetic resonance imaging and spectroscopy. Clinically, affected patients develop slowly progressive cerebellar ataxia, spasticity, and dorsal column dysfunction, sometimes with a mild cognitive deficit or decline. In 2007, the pathophysiology of this disorder was elucidated with the discovery of mutations in the DARS2 gene, which encodes mitochondrial aspartyl-tRNA synthetase, in affected individuals. Here, the authors present a case of leukoencephalopathy with brainstem and spinal cord involvement with normal brain lactate, in which genetic analysis revealed a new mutation in the DARS2 gene not previously described.

McLaughlin Heather M, Sakaguchi Reiko, Liu Cuiping, Igarashi Takao, Pehlivan Davut, Chu Kristine, Iyer Ram, Cruz Pedro, Cherukuri Praveen F, Hansen Nancy F, Mullikin James C, , Biesecker Leslie G, Wilson Thomas E, Ionasescu Victor, Nicholson Garth, Searby Charles, Talbot Kevin, Vance Jeffrey M, Züchner Stephan, Szigeti Kinga, Lupski James R, Hou Ya-Ming, Green Eric D, Antonellis Anthony.

Charcot-Marie-Tooth (CMT) disease comprises a genetically and clinically heterogeneous group of peripheral nerve disorders characterized by impaired distal motor and sensory function. Mutations in three genes encoding aminoacyl-tRNA synthetases (ARSs) have been implicated in CMT disease primarily associated with an axonal pathology. ARSs are ubiquitously expressed, essential enzymes responsible for charging tRNA molecules with their cognate amino acids. To further explore the role of ARSs in CMT disease, we performed a large-scale mutation screen of the 37 human ARS genes in a cohort of 355 patients with a phenotype consistent with CMT. Here we describe three variants (p.Leu133His, p.Tyr173SerfsX7, and p.Ile302Met) in the lysyl-tRNA synthetase (KARS) gene in two patients from this cohort. Functional analyses revealed that two of these mutations (p.Leu133His and p.Tyr173SerfsX7) severely affect enzyme activity. Interestingly, both functional variants were found in a single patient with CMT disease and additional neurological and non-neurological sequelae. Based on these data, KARS becomes the fourth ARS gene associated with CMT disease, indicating that this family of enzymes is specifically critical for axon function.

Rankin Julia, Brown Ruth, Dobyns William B, Harington Judith, Patel Jay, Quinn Michael, Brown Garry.

Six subtypes of autosomal recessive pontocerebellar hypoplasia (PCH) have been identified and the genetic basis of four of these (PCH1, PCH2, PCH4, and PCH6) is known. PCH6 is associated with cerebral atrophy and multiple but variable respiratory chain defects in muscle and has been reported in one consanguineous Sephardic Jewish family. It is caused by mutations in the RARS2 gene which encodes mitochondrial arginine-transfer RNA synthetase. Here we describe a female patient born to nonconsanguineous British parents. She presented in the neonatal period with increased respiratory rate, poor feeding and transiently elevated blood and CSF lactate levels. She went on to manifest profound developmental delay and severe microcephaly. Edema of the hands, feet, and face were suggestive of a PEHO-like condition (progressive encephalopathy, edema, hypsarrhythmia and optic atrophy), although optic atrophy and hypsarrhythmia were absent. Cranial MRI at age 14 months showed generalized cerebral atrophy, thinning of the pons and gross atrophy and flattening of the cerebellar hemispheres. Muscle biopsies on two occasions were normal with normal respiratory chain studies. Despite the absence of respiratory chain defects, the phenotype was felt to be consistent with PCH6 and indeed two novel pathogenic RARS2 mutations were identified. Ours is the second report of PCH6 due to RARS2 mutations and demonstrates that respiratory chain abnormalities are not obligatory, whereas some features of PEHO might be present.

Riley Lisa G, Cooper Sandra, Hickey Peter, Rudinger-Thirion Joëlle, McKenzie Matthew, Compton Alison, Lim Sze Chern, Thorburn David, Ryan Michael T, Giegé Richard, Bahlo Melanie, Christodoulou John.

Mitochondrial respiratory chain disorders are a heterogeneous group of disorders in which the underlying genetic defect is often unknown. We have identified a pathogenic mutation (c.156C>G [p.F52L]) in YARS2, located at chromosome 12p11.21, by using genome-wide SNP-based homozygosity analysis of a family with affected members displaying myopathy, lactic acidosis, and sideroblastic anemia (MLASA). We subsequently identified the same mutation in another unrelated MLASA patient. The YARS2 gene product, mitochondrial tyrosyl-tRNA synthetase (YARS2), was present at lower levels in skeletal muscle whereas fibroblasts were relatively normal. Complex I, III, and IV were dysfunctional as indicated by enzyme analysis, immunoblotting, and immunohistochemistry. A mitochondrial protein-synthesis assay showed reduced levels of respiratory chain subunits in myotubes generated from patient cell lines. A tRNA aminoacylation assay revealed that mutant YARS2 was still active; however, enzyme kinetics were abnormal compared to the wild-type protein. We propose that the reduced aminoacylation activity of mutant YARS2 enzyme leads to decreased mitochondrial protein synthesis, resulting in mitochondrial respiratory chain dysfunction. MLASA has previously been associated with PUS1 mutations; hence, the YARS2 mutation reported here is an alternative cause of MLASA.

Hamaguchi Ayumi, Ishida Chiho, Iwasa Kazuo, Abe Akiko, Yamada Masahito.

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Isohanni P, Linnankivi T, Buzkova J, Lönnqvist T, Pihko H, Valanne L, Tienari P J, Elovaara I, Pirttilä T, Reunanen M, Koivisto K, Marjavaara S, Suomalainen A.

Leucoencephalopathy with brain stem and spinal cord involvement and high brain lactate (LBSL) was first defined by characteristic magnetic resonance imaging and spectroscopic findings. The clinical features include childhood or juvenile onset slowly progressive ataxia, spasticity, and dorsal column dysfunction, occasionally accompanied by learning difficulties. Mutations in DARS2, encoding mitochondrial aspartyl-tRNA synthetase, were recently shown to cause LBSL. The signs and symptoms show some overlap with the most common leucoencephalopathy of young adults, multiple sclerosis (MS).

Mikhaĭlova S V, Zakharova E Iu, Banin A V, Demushkina A A, Petrukhin A S.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a recently described disorder with autosomal recessive model of inheritance. Mutations in the DARS2 gene, which encode mitochondrial aspartyl-tRNA synthetase, have been found. We present 31 cases with characteristic clinical and neuroimaging findings of this disorder. Patients have been stratified into two groups (early and late forms) by age-at-onset and clinical symptoms. The early form was characterized clinically by progressive pyramidal dysfunction, cerebellar and intellectual problems appeared later. Patients with the late form had cerebellar and sensitive ataxia, disturbances of muscle tonus, spastic type, mostly in the low extremities, polyneuropathic and rarely - psychoorganic syndrome. The brain MRI of all patients was characterized by inhomogeneous T2W signal abnormalities in the periventricular and deep white matter and a strikingly selective involvement of certain brainstem and spinal tracts. Most of the patients were compound-heterozygous for common mutations in the DARS2. We found 4 new mutations associated with LBSL. This is the first clinical and molecular-genetic investigation of this rare leukoencephalopathy in Russia.

Uluc Kayihan, Baskan Ozdil, Yildirim Kadriye Agan, Ozsahin Selda, Koseoglu Mesrure, Isak Baris, Scheper G C, Gunal Dilek Ince, van der Knaap M S.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a recently described disorder with autosomal recessive mode of inheritance. Lately, mutations in the DARS2 gene, which encodes mitochondrial aspartyl-tRNA synthetase, have been found as the underlying defect. We report a 19-year-old male patient with cerebellar, pyramidal and dorsal column dysfunctions and specific magnetic resonance imaging (MRI) and characteristic magnetic resonance spectroscopy (MRS) abnormalities. The patient was compound-heterozygous for two mutations in DARS2. MRI showed selective involvement of cerebral and cerebellar white matter and superior and inferior cerebellar peduncles, without contrast enhancement. The U-fibers were spared. The sensory and the pyramidal tracts were affected over their entire length. Involvement of the intraparenchymal trajectories of the trigeminal nerves and mesencephalic trigeminal tracts was demonstrated. In the spinal cord, signal abnormalities were identified in the dorsal columns and the lateral corticospinal tracts. Proton-MRS of the frontal and cerebellar white matter showed elevated lactate, reduced N-acetylaspartate, increased myoinositol and mildly elevated choline. In LBSL, distinct MRI findings should lead to the diagnosis, which can be confirmed by the analysis of the disease gene DARS2.

Edvardson Simon, Shaag Avraham, Kolesnikova Olga, Gomori John Moshe, Tarassov Ivan, Einbinder Tom, Saada Ann, Elpeleg Orly.

Homozygosity mapping was performed in a consanguineous Sephardic Jewish family with three patients who presented with severe infantile encephalopathy associated with pontocerebellar hypoplasia and multiple mitochondrial respiratory-chain defects. This resulted in the identification of an intronic mutation in RARS2, the gene encoding mitochondrial arginine-transfer RNA (tRNA) synthetase. The mutation was associated with the production of an abnormally short RARS2 transcript and a marked reduction of the mitochondrial tRNA(Arg) transcript in the patients' fibroblasts. We speculate that missplicing mutations in mitochondrial aminoacyl-tRNA synthethase genes preferentially affect the brain because of a tissue-specific vulnerability of the splicing machinery.

Scheper Gert C, van der Klok Thom, van Andel Rob J, van Berkel Carola G M, Sissler Marie, Smet Joél, Muravina Tatjana I, Serkov Sergey V, Uziel Graziella, Bugiani Marianna, Schiffmann Raphael, Krägeloh-Mann Ingeborg, Smeitink Jan A M, Florentz Catherine, Van Coster Rudy, Pronk Jan C, van der Knaap Marjo S.

Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) has recently been defined based on a highly characteristic constellation of abnormalities observed by magnetic resonance imaging and spectroscopy. LBSL is an autosomal recessive disease, most often manifesting in early childhood. Affected individuals develop slowly progressive cerebellar ataxia, spasticity and dorsal column dysfunction, sometimes with a mild cognitive deficit or decline. We performed linkage mapping with microsatellite markers in LBSL families and found a candidate region on chromosome 1, which we narrowed by means of shared haplotypes. Sequencing of genes in this candidate region uncovered mutations in DARS2, which encodes mitochondrial aspartyl-tRNA synthetase, in affected individuals from all 30 families. Enzyme activities of mutant proteins were decreased. We were surprised to find that activities of mitochondrial complexes from fibroblasts and lymphoblasts derived from affected individuals were normal, as determined by different assays.