Yoshihito Kishita

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.

Mutations in GTPBP3 Cause a Mitochondrial Translation Defect Associated with Hypertrophic Cardiomyopathy, Lactic Acidosis, and Encephalopathy

Respiratory chain deficiencies exhibit a wide variety of clinical phenotypes resulting from defective mitochondrial energy production through oxidative phosphorylation. These defects can be caused by either mutations in the mtDNA or mutations in nuclear genes coding for mitochondrial proteins. The underlying pathomechanisms can affect numerous pathways involved in mitochondrial physiology. By whole-exome and candidate gene sequencing, we identified 11 individuals from 9 families carrying compound heterozygous or homozygous mutations in GTPBP3, encoding the mitochondrial GTP-binding protein 3. Affected individuals from eight out of nine families presented with combined respiratory chain complex deficiencies in skeletal muscle. Mutations in GTPBP3 are associated with a severe mitochondrial translation defect, consistent with the predicted function of the protein in catalyzing the formation of 5-taurinomethyluridine (τm(5)U) in the anticodon wobble position of five mitochondrial tRNAs. All case subjects presented with lactic acidosis and nine developed hypertrophic cardiomyopathy. In contrast to individuals with mutations in MTO1, the protein product of which is predicted to participate in the generation of the same modification, most individuals with GTPBP3 mutations developed neurological symptoms and MRI involvement of thalamus, putamen, and brainstem resembling Leigh syndrome. Our study of a mitochondrial translation disorder points toward the importance of posttranscriptional modification of mitochondrial tRNAs for proper mitochondrial function.

Deficiency of ECHS1 causes mitochondrial encephalopathy with cardiac involvement.

Short‐chain enoyl‐CoA hydratase (ECHS1) is a multifunctional mitochondrial matrix enzyme that is involved in the oxidation of fatty acids and essential amino acids such as valine. Here, we describe the broad phenotypic spectrum and pathobiochemistry of individuals with autosomal‐recessive ECHS1 deficiency.

Loss of Trx-2 enhances oxidative stress-dependent phenotypes in Drosophila.

Overexpression of thioredoxin (TRX) confers oxidative stress resistance and extends lifespan in mammals and insects. However, less is known about phenotypes associated with loss of TRX. We investigated loss‐of‐function phenotypes of Trx‐2 in Drosophila, and found that the mutant flies are hyper‐susceptible to paraquat, a free radical generator, but not to hydrogen peroxide. They contain a high amount of protein carbonyl, which dramatically increases with age. Trx‐2 mutants express high levels of anti‐oxidant genes, such as superoxide dismutase, catalase, and glutathione synthetase. This is the first demonstration of biochemical and physiological consequences caused by loss of Trx‐2 in Drosophila.

Diagnosis and molecular basis of mitochondrial respiratory chain disorders: Exome sequencing for disease gene identification

Mitochondrial disorders have the highest incidence among congenital metabolic diseases, and are thought to occur at a rate of 1 in 5000 births. About 25% of the diseases diagnosed as mitochondrial disorders in the field of pediatrics have mitochondrial DNA abnormalities, while the rest occur due to defects in genes encoded in the nucleus. The most important function of the mitochondria is biosynthesis of ATP. Mitochondrial disorders are nearly synonymous with mitochondrial respiratory chain disorder, as respiratory chain complexes serve a central role in ATP biosynthesis. By next-generation sequencing of the exome, we analyzed 104 patients with mitochondrial respiratory chain disorders. The results of analysis to date were 18 patients with novel variants in genes previously reported to be disease-causing, and 27 patients with mutations in genes suggested to be associated in some way with mitochondria, and it is likely that they are new disease-causing genes in mitochondrial disorders. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.

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.

Impaired energy metabolism in a Drosophila model of mitochondrial aconitase deficiency.

Aconitase catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid (TCA) cycle, and its deficiency in humans is associated with an infantile neurodegenerative disorder affecting mainly the cerebellum and retina. Here we investigated the effect of gene knockout and knockdown of the mitochondrial aconitase Acon in Drosophila. Acon-knockout flies were homozygous lethal, indicating that Acon is essential for viability. RNA interference-generated Acon-knockdown flies exhibited a variety of phenotypes, such as reduced locomotor activity, a shortened lifespan, and increased cell death in the developing brain. Metabolomic analysis revealed that acetyl-CoA, citrate/isocitrate, and cis-aconitate were significantly increased, while most metabolites of glycolysis and the TCA cycle were reduced. Reduced triacylglyceride and increased acetyl-CoA suggested that lipids were used as an energy source because of the impaired glycolysis and TCA cycle. The Acon-knockdown model should facilitate further understanding of the pathophysiology of m-aconitase deficiency in humans.

Impaired fatty acid oxidation in a Drosophila model of mitochondrial trifunctional protein (MTP) deficiency.

Mitochondrial trifunctional protein (MTP), which consists of the MTPα and MTPβ subunits, catalyzes long-chain fatty acid β-oxidation. MTP deficiency in humans results in Reye-like syndrome. Here, we generated Drosophila models of MTP deficiency by targeting two genes encoding Drosophila homologs of human MTPα and MTPβ, respectively. Both Mtpα(KO) and Mtpβ(KO) flies were viable, but demonstrated reduced lifespan, defective locomotor activity, and reduced fecundity represented by the number of eggs laid by the females. The phenotypes of Mtpα(KO) flies were generally more striking than those of Mtpβ(KO) flies. Mtpα(KO) flies were hypersensitive to fasting, and retained lipid droplets in their fat body cells as in non-fasting conditions. The amount of triglyceride was also unchanged upon fasting in Mtpα(KO) flies, suggesting that lipid mobilization was disrupted. Finally, we showed that both Mtpα(KO) and Mtpβ(KO) flies accumulated acylcarnitine and hydroxyacylcarnitine, diagnostic markers of MTP deficiencies in humans. Our results indicated that both Mtpα(KO) and Mtpβ(KO) flies were impaired in long-chain fatty acid β-oxidation. These flies should be useful as a model system to investigate the molecular pathogenesis of MTP deficiency.

Clinical validity of biochemical and molecular analysis in diagnosing Leigh syndrome: a study of 106 Japanese patients

Leigh syndrome (LS) is a progressive neurodegenerative disorder of infancy and early childhood. It is clinically diagnosed by typical manifestations and characteristic computed tomography (CT) or magnetic resonance imaging (MRI) studies. Unravelling mitochondrial respiratory chain (MRC) dysfunction behind LS is essential for deeper understanding of the disease, which may lead to the development of new therapies and cure. The aim of this study was to evaluate the clinical validity of various diagnostic tools in confirming MRC disorder in LS and Leigh-like syndrome (LL). The results of enzyme assays, molecular analysis, and cellular oxygen consumption rate (OCR) measurements were examined. Of 106 patients, 41 were biochemically and genetically verified, and 34 had reduced MRC activity but no causative mutations. Seven patients with normal MRC complex activities had mutations in the MT-ATP6 gene. Five further patients with normal activity in MRC were identified with causative mutations. Conversely, 12 out of 60 enzyme assays performed for genetically verified patients returned normal results. No biochemical or genetic background was confirmed for 19 patients. OCR was reduced in ten out of 19 patients with negative enzyme assay results. Inconsistent enzyme assay results between fibroblast and skeletal muscle biopsy samples were observed in 33% of 37 simultaneously analyzed cases. These data suggest that highest diagnostic rate is reached using a combined enzymatic and genetic approach, analyzing more than one type of biological materials where suitable. Microscale oxygraphy detected MRC impairment in 50% cases with no defect in MRC complex activities.

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

Abstract 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.