Strategic validation of variants of uncertain significance in ECHS1 genetic testing.

BACKGROUND: Enoyl-CoA hydratase short-chain 1 (ECHS1) is an enzyme involved in the metabolism of branched chain amino acids and fatty acids. Mutations in the ECHS1 gene lead to mitochondrial short-chain enoyl-CoA hydratase 1 deficiency, resulting in the accumulation of intermediates of valine. This is one of the most common causative genes in mitochondrial diseases. While genetic analysis studies have diagnosed numerous cases with ECHS1 variants, the increasing number of variants of uncertain significance (VUS) in genetic diagnosis is a major problem. METHODS: Here, we constructed an assay system to verify VUS function for ECHS1 gene. A high-throughput assay using ECHS1 knockout cells was performed to index these phenotypes by expressing cDNAs containing VUS. In parallel with the VUS validation system, a genetic analysis of samples from patients with mitochondrial disease was performed. The effect on gene expression in cases was verified by RNA-seq and proteome analysis. RESULTS: The functional validation of VUS identified novel variants causing loss of ECHS1 function. The VUS validation system also revealed the effect of the VUS in the compound heterozygous state and provided a new methodology for variant interpretation. Moreover, we performed multiomics analysis and identified a synonymous substitution p.P163= that results in splicing abnormality. The multiomics analysis complemented the diagnosis of some cases that could not be diagnosed by the VUS validation system. CONCLUSIONS: In summary, this study uncovered new ECHS1 cases based on VUS validation and omics analysis; these analyses are applicable to the functional evaluation of other genes associated with mitochondrial disease.

Antibody Deficiency in Patients with Biallelic KARS1 Mutations.

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.

Novel ITPA variants identified by whole genome sequencing and RNA sequencing.

Approximately 80% of rare diseases have a genetic cause, and an accurate genetic diagnosis is necessary for disease management, prognosis prediction, and genetic counseling. Whole-exome sequencing (WES) is a cost-effective approach for exploring the genetic cause, but several cases often remain undiagnosed. We combined whole genome sequencing (WGS) and RNA sequencing (RNA-seq) to identify the pathogenic variants in an unsolved case using WES. RNA-seq revealed aberrant exon 4 and exon 6 splicing of ITPA. WGS showed a previously unreported splicing donor variant, c.263+1G>A, and a novel heterozygous deletion, including exon 6. Detailed examination of the breakpoint indicated the deletion caused by recombination between Alu elements in different introns. The proband was found to have developmental and epileptic encephalopathies caused by variants in the ITPA gene. The combination of WGS and RNA-seq may be effective in diagnosing conditions in proband who could not be diagnosed using WES.

Impact of measuring heteroplasmy of a pathogenic mitochondrial DNA variant at the single-cell level in individuals with mitochondrial disease.

Pathogenic mitochondrial DNA heteroplasmy has mainly been assessed with bulk sequencing in individuals with mitochondrial disease. However, the distribution of heteroplasmy at the single-cell level in skin fibroblasts obtained from individuals, together with detailed clinical and biochemical information, remains to be investigated. We used the mitochondrial DNA single-cell assay for the transposase-accessible chromatin sequencing method. Skin fibroblasts were obtained from six individuals with mitochondrial disease and pathogenic m.3243A>G variants of differing severity. Different distributions of heteroplasmy at the single-cell level were identified in skin fibroblasts from all six individuals. Four individuals with different outcomes showed similar averaged heteroplasmy rates with normal mitochondrial respiratory chain enzyme activity, while the distribution of single-cell heteroplasmy patterns differed among the individuals. This study showed different heteroplasmy distribution patterns at the single-cell level in individuals with the m.3243A>G variant, who had a similar averaged heteroplasmy rates with normal mitochondrial respiratory chain enzyme activity. Whether such different heteroplasmy distribution patterns explain the different clinical outcomes should be assessed further in future studies. Measuring heteroplasmy of pathogenic mitochondrial DNA variants at the single-cell level could be important in individuals with mitochondrial disease.

Biallelic variants in LIG3 cause a novel mitochondrial neurogastrointestinal encephalomyopathy.

Abnormal gut motility is a feature of several mitochondrial encephalomyopathies, and mutations in genes such as TYMP and POLG, have been linked to these rare diseases. The human genome encodes three DNA ligases, of which only one, ligase III (LIG3), has a mitochondrial splice variant and is crucial for mitochondrial health. We investigated the effect of reduced LIG3 activity and resulting mitochondrial dysfunction in seven patients from three independent families, who showed the common occurrence of gut dysmotility and neurological manifestations reminiscent of mitochondrial neurogastrointestinal encephalomyopathy. DNA from these patients was subjected to whole exome sequencing. In all patients, compound heterozygous variants in a new disease gene, LIG3, were identified. All variants were predicted to have a damaging effect on the protein. The LIG3 gene encodes the only mitochondrial DNA (mtDNA) ligase and therefore plays a pivotal role in mtDNA repair and replication. In vitro assays in patient-derived cells showed a decrease in LIG3 protein levels and ligase activity. We demonstrated that the LIG3 gene defects affect mtDNA maintenance, leading to mtDNA depletion without the accumulation of multiple deletions as observed in other mitochondrial disorders. This mitochondrial dysfunction is likely to cause the phenotypes observed in these patients. The most prominent and consistent clinical signs were severe gut dysmotility and neurological abnormalities, including leukoencephalopathy, epilepsy, migraine, stroke-like episodes, and neurogenic bladder. A decrease in the number of myenteric neurons, and increased fibrosis and elastin levels were the most prominent changes in the gut. Cytochrome c oxidase (COX) deficient fibres in skeletal muscle were also observed. Disruption of lig3 in zebrafish reproduced the brain alterations and impaired gut transit in vivo. In conclusion, we identified variants in the LIG3 gene that result in a mitochondrial disease characterized by predominant gut dysmotility, encephalopathy, and neuromuscular abnormalities.

Genome sequencing and RNA-seq analyses of mitochondrial complex I deficiency revealed Alu insertion-mediated deletion in NDUFV2.

Isolated complex I deficiency is the most common cause of pediatric mitochondrial disease. Exome sequencing (ES) has revealed many complex I causative genes. However, there are limitations associated with identifying causative genes by ES analysis. In this study, we performed multiomics analysis to reveal the causal variants. We here report two cases with mitochondrial complex I deficiency. In both cases, ES identified a novel c.580G>A (p.Glu194Lys) variant in NDUFV2. One case additionally harbored c.427C>T (p.Arg143*), but no other variants were observed in the other case. RNA sequencing showed aberrant exon splicing of NDUFV2 in the unsolved case. Genome sequencing revealed a novel heterozygous deletion in NDUFV2, which included one exon and resulted in exon skipping. Detailed examination of the breakpoint revealed that an Alu insertion-mediated rearrangement caused the deletion. Our report reveals that combined use of transcriptome sequencing and GS was effective for diagnosing cases that were unresolved by ES.

Mutations in TOP3A Cause a Bloom Syndrome-like Disorder.

Bloom syndrome, caused by biallelic mutations in BLM, is characterized by prenatal-onset growth deficiency, short stature, an erythematous photosensitive malar rash, and increased cancer predisposition. Diagnostically, a hallmark feature is the presence of increased sister chromatid exchanges (SCEs) on cytogenetic testing. Here, we describe biallelic mutations in TOP3A in ten individuals with prenatal-onset growth restriction and microcephaly. TOP3A encodes topoisomerase III alpha (TopIIIα), which binds to BLM as part of the BTRR complex, and promotes dissolution of double Holliday junctions arising during homologous recombination. We also identify a homozygous truncating variant in RMI1, which encodes another component of the BTRR complex, in two individuals with microcephalic dwarfism. The TOP3A mutations substantially reduce cellular levels of TopIIIα, and consequently subjects' cells demonstrate elevated rates of SCE. Unresolved DNA recombination and/or replication intermediates persist into mitosis, leading to chromosome segregation defects and genome instability that most likely explain the growth restriction seen in these subjects and in Bloom syndrome. Clinical features of mitochondrial dysfunction are evident in several individuals with biallelic TOP3A mutations, consistent with the recently reported additional function of TopIIIα in mitochondrial DNA decatenation. In summary, our findings establish TOP3A mutations as an additional cause of prenatal-onset short stature with increased cytogenetic SCEs and implicate the decatenation activity of the BTRR complex in their pathogenesis.

Bile Acid Synthesis Disorders in Japan: Long-Term Outcome and Chenodeoxycholic Acid Treatment.

BACKGROUND: We encountered 7 Japanese patients with bile acid synthesis disorders (BASD) including 3ß-hydroxy-Δ5-C27-steroid dehydrogenase/isomerase (3ß-HSD) deficiency (n = 3), Δ4-3-oxosteroid 5ß-reductase (5ß-reductase) deficiency (n = 3), and oxysterol 7α-hydroxylase deficiency (n = 1) over 21 years between 1996 and 2017. AIM: We aimed to clarify long-term outcome in the 7 patients with BASD as well as long-term efficacy of chenodeoxycholic acid (CDCA) treatment in the 5 patients with 3ß-HSD deficiency or 5ß-reductase deficiency. METHODS: Diagnoses were made from bile acid and genetic analyses. Bile acid analysis in serum and urine was performed using gas chromatography-mass spectrometry. Clinical and laboratory findings and bile acid profiles at diagnosis and most recent visit were retrospectively obtained from medical records. Long-term outcome included follow-up duration, treatments, growth, education/employment, complications of treatment, and other problems. RESULTS: Medians with ranges of current patient ages and duration of CDCA treatment are 10 years (8 to 43) and 10 years (8 to 21), respectively. All 7 patients, who had homozygous or compound heterozygous mutations in the HSD3B7, SRD5B1, or CYP7B1 gene, are currently in good health without liver dysfunction. In the 5 patients with CDCA treatment, hepatic function gradually improved following initiation. No adverse effects were noted. CONCLUSIONS: We concluded that CDCA treatment is effective in 3ß-HSD deficiency and 5ß-reductase deficiency, as cholic acid has been in other countries. BASD carry a good prognosis following early diagnosis and initiation of long-term CDCA treatment.

Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies.

Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure. Although C1QBP has been reported to exert pleiotropic effects on many cellular processes, we report here four individuals from unrelated families where biallelic mutations in C1QBP cause a defect in mitochondrial energy metabolism. Infants presented with cardiomyopathy accompanied by multisystemic involvement (liver, kidney, and brain), and children and adults presented with myopathy and progressive external ophthalmoplegia. Multiple mitochondrial respiratory-chain defects, associated with the accumulation of multiple deletions of mitochondrial DNA in the later-onset myopathic cases, were identified in all affected individuals. Steady-state C1QBP levels were decreased in all individuals' samples, leading to combined respiratory-chain enzyme deficiency of complexes I, III, and IV. C1qbp-/- mouse embryonic fibroblasts (MEFs) resembled the human disease phenotype by showing multiple defects in oxidative phosphorylation (OXPHOS). Complementation with wild-type, but not mutagenized, C1qbp restored OXPHOS protein levels and mitochondrial enzyme activities in C1qbp-/- MEFs. C1QBP deficiency represents an important mitochondrial disorder associated with a clinical spectrum ranging from infantile lactic acidosis to childhood (cardio)myopathy and late-onset progressive external ophthalmoplegia.

A homozygous variant in NDUFA8 is associated with developmental delay, microcephaly, and epilepsy due to mitochondrial complex I deficiency.

Mitochondrial complex I deficiency is caused by pathogenic variants in mitochondrial and nuclear genes associated with complex I structure and assembly. We report the case of a patient with　NDUFA8-related mitochondrial disease. The patient presented with developmental delay, microcephaly, and epilepsy. His fibroblasts showed apparent biochemical defects in mitochondrial complex I. Whole-exome sequencing revealed that the patient carried a homozygous variant in NDUFA8. His fibroblasts showed a reduction in the protein expression level of not only NDUFA8, but also the other complex I subunits, consistent with assembly defects. The enzyme activity of complex I and oxygen consumption rate were restored by reintroducing wild-typeNDUFA8 cDNA into patient fibroblasts. The functional properties of the variant in NDUFA8 were also investigated using NDUFA8 knockout cells expressing wild-type or mutated NDUFA8 cDNA. These experiments further supported the pathogenicity of the variant in complex I assembly. This is the first report describing that the loss of NDUFA8, which has not previously been associated with mitochondrial disease, causes severe defect in the assembly of mitochondrial complex I, leading to progressive neurological and developmental abnormalities.

Mortality of Japanese patients with Leigh syndrome: Effects of age at onset and genetic diagnosis.

Leigh syndrome is a major phenotype of mitochondrial diseases in children. With new therapeutic options being proposed, assessing the mortality and clinical condition of Leigh syndrome patients is crucial for evaluating therapeutics. As data are scarce in Japan, we analysed the mortality rate and clinical condition of Japanese Leigh syndrome patients that we diagnosed since 2007. Data from 166 Japanese patients diagnosed with Leigh syndrome from 2007 to 2017 were reviewed. Patients' present status, method of ventilation and feeding, and degree of disability as of April 2018 was analysed. Overall, 124 (74.7%) were living, 40 (24.1%) were deceased, and 2 (1.2%) were lost to follow-up. Median age of living patients was 8 years (1-39 years). Median length of disease course was 91 months for living patients and 23.5 months for deceased patients. Nearly 90% of deaths occurred by age 6. Mortality rate of patients with onset before 6 months of age was significantly higher than that of onset after 6 months. All patients with neonatal onset were either deceased or bedridden. MT-ATP6 deficiency caused by m.8993T>G mutation and MT-ND5 deficiency induced a severe form of Leigh syndrome. Patients with NDUFAF6, ECHS1, and SURF1 deficiency had relatively mild symptoms and better survival. The impact of onset age on prognosis varied across the genetic diagnoses. The clinical condition of many patients was poor; however, few did not require mechanical ventilation or tube-feeding and were not physically dependent. Early disease onset and genetic diagnosis may have prognostic value.

Metabolic and chemical regulation of tRNA modification associated with taurine deficiency and human disease.

Modified uridine containing taurine, 5-taurinomethyluridine (τm5U), is found at the anticodon first position of mitochondrial (mt-)transfer RNAs (tRNAs). Previously, we reported that τm5U is absent in mt-tRNAs with pathogenic mutations associated with mitochondrial diseases. However, biogenesis and physiological role of τm5U remained elusive. Here, we elucidated τm5U biogenesis by confirming that 5,10-methylene-tetrahydrofolate and taurine are metabolic substrates for τm5U formation catalyzed by MTO1 and GTPBP3. GTPBP3-knockout cells exhibited respiratory defects and reduced mitochondrial translation. Very little τm5U34 was detected in patient's cells with the GTPBP3 mutation, demonstrating that lack of τm5U results in pathological consequences. Taurine starvation resulted in downregulation of τm5U frequency in cultured cells and animal tissues (cat liver and flatfish). Strikingly, 5-carboxymethylaminomethyluridine (cmnm5U), in which the taurine moiety of τm5U is replaced with glycine, was detected in mt-tRNAs from taurine-depleted cells. These results indicate that tRNA modifications are dynamically regulated via sensing of intracellular metabolites under physiological condition.

Nationwide epidemiological survey of holoprosencephaly in Japan.

BACKGROUND: Holoprosencephaly (HPE) is a congenital malformation with an estimated prevalence of 0.10-6.06 per 10 000 births but with no nationwide data specific to Japan. METHODS: This nationwide retrospective questionnaire survey was conducted from 2011 to 2013. All 467 training hospitals for perinatal and neonatal care certified by the Japan Society of Perinatal and Neonatal Medicine were contacted. The birth prevalence rate (BPR) was assessed from the primary survey and clinical characteristics from the secondary survey. RESULTS: We received valid responses from 253 hospitals in the primary survey (54.6%). Of 390 342 live births, 60 were diagnosed with HPE (23 males and 37 females), resulting in an actual BPR of 1.54 per 10 000 live births. The point estimate for HPE cases was 100 (95% confidence interval [CI]: 80.7-120), and the estimated BPR of HPE was calculated to be 0.32 per 10 000 live births (95% CI: 0.26-0.38) based on 3 117 853 live births according to Japanese national statistics during the study period. In the secondary survey, we obtained data for 49 cases (19 males and 30 females). Of these, 20 were alobar (40.8%), 20 were semilobar (40.8%), five were lobar (10.4%), and four were of unknown type. Genetic examination was performed in 37 of the 49 HPE patients and revealed that chromosomes 13, 18, and 7 were affected in eight, six, and four patients, respectively. CONCLUSIONS: This is the most extensive survey on holoprosencephaly to date in Japan. The estimated BPR was consistent with that reported in previous research.

Mitochondrial ribosomal protein PTCD3 mutations cause oxidative phosphorylation defects with Leigh syndrome.

Pentatricopeptide repeat domain proteins are a large family of RNA-binding proteins involved in mitochondrial RNA editing, stability, and translation. Mitochondrial translation machinery defects are an expanding group of genetic diseases in humans. We describe a patient who presented with low birth weight, mental retardation, and optic atrophy. Brain MRI showed abnormal bilateral signals at the basal ganglia and brainstem, and the patient was diagnosed as Leigh syndrome. Exome sequencing revealed two potentially loss-of-function variants [c.415-2A>G, and c.1747_1748insCT (p.Phe583Serfs*3)] in PTCD3 (also known as MRPS39). PTCD3, a member of the pentatricopeptide repeat domain protein family, is a component of the small mitoribosomal subunit. The patient had marked decreases in mitochondrial complex I and IV levels and activities, oxygen consumption and ATP biosynthesis, and generalized mitochondrial translation defects in fibroblasts. Quantitative proteomic analysis revealed decreased levels of the small mitoribosomal subunits. Complementation experiments rescued oxidative phosphorylation complex I and IV levels and activities, ATP biosynthesis, and MT-RNR1 rRNA transcript level, providing functional validation of the pathogenicity of identified variants. This is the first report of an association of PTCD3 mutations with Leigh syndrome along with combined oxidative phosphorylation deficiencies caused by defects in the mitochondrial translation machinery.

Biallelic IARS Mutations Cause Growth Retardation with Prenatal Onset, Intellectual Disability, Muscular Hypotonia, and Infantile Hepatopathy.

tRNA synthetase deficiencies are a growing group of genetic diseases associated with tissue-specific, mostly neurological, phenotypes. In cattle, cytosolic isoleucyl-tRNA synthetase (IARS) missense mutations cause hereditary weak calf syndrome. Exome sequencing in three unrelated individuals with severe prenatal-onset growth retardation, intellectual disability, and muscular hypotonia revealed biallelic mutations in IARS. Studies in yeast confirmed the pathogenicity of identified mutations. Two of the individuals had infantile hepatopathy with fibrosis and steatosis, leading in one to liver failure in the course of infections. Zinc deficiency was present in all affected individuals and supplementation with zinc showed a beneficial effect on growth in one.

Recent topics: the diagnosis, molecular genesis, and treatment of mitochondrial diseases.

Mitochondrial diseases are inherited metabolic diseases based on disorders of energy production. The expansion of exome analyses has led to the discovery of many pathogenic nuclear genes associated with these diseases, and research into the pathogenesis of metabolic diseases has progressed. In cases of Leigh syndrome, it is desirable to perform both biochemical and genetic analyses, and pathogenic gene mutations have been identified in over half of the cases analyzed this way. Tandem mass screening and organic acid analyses of urine can sometimes provide important information that leads to the identification of pathogenic genes. Our comprehensive gene analyses have led to the discovery of several novel genes for mitochondrial diseases. Indeed, we reported that GTPBP3 and QRSL1 are involved in mitochondrial DNA maturation. In 2017, as a result of international collaboration, we also identified that mutations in ATAD3 and C1QBP cause mitochondrial disease. Given the varied pathogeneses, treatments for mitochondrial diseases should be specifically tailored to the mutated gene. Clinical trials of sodium pyruvate, 5-aminolevulinic acid with sodium ferrous citrate, and taurine as a treatment for mitochondrial disease have begun in Japan. Given that some mitochondrial diseases may respond well to certain treatments if the pathogenic gene can be identified, an early genetic diagnosis is crucial. Additionally, in Japan, prenatal diagnoses for mitochondrial diseases caused by nuclear genes have been achieved for genes shown to be pathogenic. Treatment and management approaches, including prenatal diagnoses, specifically tailored to the various phenotypes and pathologies of mitochondrial diseases are expected to become increasingly available.

Mutations in COA7 cause spinocerebellar ataxia with axonal neuropathy.

Several genes related to mitochondrial functions have been identified as causative genes of neuropathy or ataxia. Cytochrome c oxidase assembly factor 7 (COA7) may have a role in assembling mitochondrial respiratory chain complexes that function in oxidative phosphorylation. Here we identified four unrelated patients with recessive mutations in COA7 among a Japanese case series of 1396 patients with Charcot-Marie-Tooth disease (CMT) or other inherited peripheral neuropathies, including complex forms of CMT. We also found that all four patients had characteristic neurological features of peripheral neuropathy and ataxia with cerebellar atrophy, and some patients showed leukoencephalopathy or spinal cord atrophy on MRI scans. Validated mutations were located at highly conserved residues among different species and segregated with the disease in each family. Nerve conduction studies showed axonal sensorimotor neuropathy. Sural nerve biopsies showed chronic axonal degeneration with a marked loss of large and medium myelinated fibres. An immunohistochemical assay with an anti-COA7 antibody in the sural nerve from the control patient showed the positive expression of COA7 in the cytoplasm of Schwann cells. We also observed mildly elevated serum creatine kinase levels in all patients and the presence of a few ragged-red fibres and some cytochrome c oxidase-negative fibres in a muscle biopsy obtained from one patient, which was suggestive of subclinical mitochondrial myopathy. Mitochondrial respiratory chain enzyme assay in skin fibroblasts from the three patients showed a definitive decrease in complex I or complex IV. Immunocytochemical analysis of subcellular localization in HeLa cells indicated that mutant COA7 proteins as well as wild-type COA7 were localized in mitochondria, which suggests that mutant COA7 does not affect the mitochondrial recruitment and may affect the stability or localization of COA7 interaction partners in the mitochondria. In addition, Drosophila COA7 (dCOA7) knockdown models showed rough eye phenotype, reduced lifespan, impaired locomotive ability and shortened synaptic branches of motor neurons. Our results suggest that loss-of-function COA7 mutation is responsible for the phenotype of the presented patients, and this new entity of disease would be referred to as spinocerebellar ataxia with axonal neuropathy type 3.

A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies.

Mitochondrial disorders have the highest incidence among congenital metabolic disorders characterized by biochemical respiratory chain complex deficiencies. It occurs at a rate of 1 in 5,000 births, and has phenotypic and genetic heterogeneity. Mutations in about 1,500 nuclear encoded mitochondrial proteins may cause mitochondrial dysfunction of energy production and mitochondrial disorders. More than 250 genes that cause mitochondrial disorders have been reported to date. However exact genetic diagnosis for patients still remained largely unknown. To reveal this heterogeneity, we performed comprehensive genomic analyses for 142 patients with childhood-onset mitochondrial respiratory chain complex deficiencies. The approach includes whole mtDNA and exome analyses using high-throughput sequencing, and chromosomal aberration analyses using high-density oligonucleotide arrays. We identified 37 novel mutations in known mitochondrial disease genes and 3 mitochondria-related genes (MRPS23, QRSL1, and PNPLA4) as novel causative genes. We also identified 2 genes known to cause monogenic diseases (MECP2 and TNNI3) and 3 chromosomal aberrations (6q24.3-q25.1, 17p12, and 22q11.21) as causes in this cohort. Our approaches enhance the ability to identify pathogenic gene mutations in patients with biochemically defined mitochondrial respiratory chain complex deficiencies in clinical settings. They also underscore clinical and genetic heterogeneity and will improve patient care of this complex disorder.

Intra-mitochondrial Methylation Deficiency Due to Mutations in SLC25A26.

S-adenosylmethionine (SAM) is the predominant methyl group donor and has a large spectrum of target substrates. As such, it is essential for nearly all biological methylation reactions. SAM is synthesized by methionine adenosyltransferase from methionine and ATP in the cytoplasm and subsequently distributed throughout the different cellular compartments, including mitochondria, where methylation is mostly required for nucleic-acid modifications and respiratory-chain function. We report a syndrome in three families affected by reduced intra-mitochondrial methylation caused by recessive mutations in the gene encoding the only known mitochondrial SAM transporter, SLC25A26. Clinical findings ranged from neonatal mortality resulting from respiratory insufficiency and hydrops to childhood acute episodes of cardiopulmonary failure and slowly progressive muscle weakness. We show that SLC25A26 mutations cause various mitochondrial defects, including those affecting RNA stability, protein modification, mitochondrial translation, and the biosynthesis of CoQ10 and lipoic acid.