Multiple Respiratory Chain Deficiencies Research Articles

Increased Diagnostic Yield by Reanalysis of Whole Exome Sequencing Data in Mitochondrial Disease.

The genetic diagnosis of mitochondrial disorders is complicated by its genetic and phenotypic complexity. Next generation sequencing techniques have much improved the diagnostic yield for these conditions. A cohort of individuals with multiple respiratory chain deficiencies, reported in the literature 10 years ago, had a diagnostic rate of 60% by whole exome sequencing (WES) but 40% remained undiagnosed. We aimed to identify a genetic diagnosis by reanalysis of the WES data for the undiagnosed arm of this 10-year-old cohort of patients with suspected mitochondrial disorders. The WES data was transferred and processed by the RD-Connect Genome-Phenome Analysis Platform (GPAP) using their standardized pipeline. Variant prioritisation was carried out on the RD-Connect GPAP. Singleton WES data from 14 individuals was reanalysed. We identified a possible or likely genetic diagnosis in 8 patients (8/14, 57%). The variants identified were in a combination of mitochondrial DNA (n = 1, MT-TN), nuclear encoded mitochondrial genes (n = 2, PDHA1, and SUCLA2) and nuclear genes associated with nonmitochondrial disorders (n = 5, PNPLA2, CDC40, NBAS and SLC7A7). Variants in both the NBAS and CDC40 genes were established as disease causing after the original cohort was published. We increased the diagnostic yield for the original cohort by 15% without generating any further genomic data. In the era of multiomics we highlight that reanalysis of existing WES data is a valid tool for generating additional diagnosis in patients with suspected mitochondrial disease, particularly when more time has passed to allow for new bioinformatic pipelines to emerge, for the development of new tools in variant interpretation aiding in reclassification of variants and the expansion of scientific knowledge on additional genes.

Nuclear genetic background influences the phenotype of the Drosophila tko25t mitochondrial protein-synthesis mutant.

The Drosophila tko25t point mutation in the gene encoding mitoribosomal protein S12 produces a complex phenotype of multiple respiratory chain deficiency, developmental delay, bang-sensitivity, impaired hearing, sugar and antibiotic sensitivity, and impaired male courtship. Its phenotypic severity was previously shown to be alleviated by inbreeding and to vary with mitochondrial genetic background. Here, we show similarly profound effects conferred by nuclear genetic background. We backcrossed tko25t into each of 2 standard nuclear backgrounds, Oregon R and w1118, the latter used as recipient line in many transgenic applications requiring selection for the white minigene marker. In the w1118 background, tko25t flies showed a moderate developmental delay and modest bang-sensitivity. In the Oregon R background, males showed longer developmental delay and more severe bang-sensitivity, and we were initially unable to produce homozygous tko25t females in sufficient numbers to conduct a meaningful analysis. When maintained as a balanced stock over 2 years, tko25t flies in the Oregon R background showed clear phenotypic improvement though were still more severely affected than in the w1118 background. Phenotypic severity did not correlate with the expression level of the tko gene. Analysis of tko25t hybrids between the 2 backgrounds indicated that phenotypic severity was conferred by autosomal, X-chromosomal, and parent-of-origin-dependent determinants. Although some of these effects may be tko25t specific, we recommend that, in order to minimize genetic drift and confounding background effects, the genetic background of nonlethal mutants should be controlled by regular backcrossing, even if stocks are usually maintained over a balancer chromosome.

Mutation of Aspartate 238 in FAD Synthase Isoform 6 Increases the Specific Activity by Weakening the FAD Binding.

FAD synthase (FADS, or FMN:ATP adenylyl transferase) coded by the FLAD1 gene is the last enzyme in the pathway of FAD synthesis. The mitochondrial isoform 1 and the cytosolic isoform 2 are characterized by the following two domains: the C-terminal PAPS domain (FADSy) performing FAD synthesis and pyrophosphorolysis; the N-terminal molybdopterin-binding domain (FADHy) performing a Co++/K+-dependent FAD hydrolysis. Mutations in FLAD1 gene are responsible for riboflavin responsive and non-responsive multiple acyl-CoA dehydrogenases and combined respiratory chain deficiency. In patients harboring frameshift mutations, a shorter isoform (hFADS6) containing the sole FADSy domain is produced representing an emergency protein. With the aim to ameliorate its function we planned to obtain an engineered more efficient hFADS6. Thus, the D238A mutant, resembling the D181A FMNAT “supermutant” of C. glabrata, was overproduced and purified. Kinetic analysis of this enzyme highlighted a general increase of Km, while the kcat was two-fold higher than that of WT. The data suggest that the FAD synthesis rate can be increased. Additional modifications could be performed to further improve the synthesis of FAD. These results correlate with previous data produced in our laboratory, and point towards the following proposals (i) FAD release is the rate limiting step of the catalytic cycle and (ii) ATP and FMN binding sites are synergistically connected.

A novel pathogenic m.4412G>A MT-TM mitochondrial DNA variant associated with childhood-onset seizures, myopathy and bilateral basal ganglia changes

Mitochondrial DNA variants in the MT-TM (mt-tRNAMet) gene are rare, typically associated with myopathic phenotypes. We identified a novel MT-TM variant resulting in prolonged seizures with childhood-onset myopathy, retinopathy, short stature and elevated CSF lactate associated with bilateral basal ganglia changes on neuroimaging. Muscle biopsy confirmed multiple respiratory chain deficiencies and focal cytochrome c oxidase (COX) histochemical abnormalities. Next-generation sequencing of the mitochondrial genome revealed a novel m.4412G>A variant at high heteroplasmy levels in muscle that fulfils all accepted criteria for pathogenicity including segregation within single muscle fibres, thus broadening the genotypic and phenotypic landscape of mitochondrial tRNA-related disease.

Leigh syndrome caused by mutations in MTFMT is associated with a better prognosis.

ObjectivesMitochondrial methionyl‐tRNA formyltransferase (MTFMT) is required for the initiation of translation and elongation of mitochondrial protein synthesis. Pathogenic variants in MTFMT have been associated with Leigh syndrome (LS) and mitochondrial multiple respiratory chain deficiencies. We sought to elucidate the spectrum of clinical, neuroradiological and molecular genetic findings of patients with bi‐allelic pathogenic variants in MTFMT.MethodsRetrospective cohort study combining new cases and previously published cases.ResultsThirty‐eight patients with pathogenic variants in MTFMT were identified, including eight new cases. The median age of presentation was 14 months (range: birth to 17 years, interquartile range [IQR] 4.5 years), with developmental delay and motor symptoms being the most frequent initial manifestation. Twenty‐nine percent of the patients survived into adulthood. MRI headings in MTFMT pathogenic variants included symmetrical basal ganglia changes (62%), periventricular and subcortical white matter abnormalities (55%), and brainstem lesions (48%). Isolated complex I and combined respiratory chain deficiencies were identified in 31% and 59% of the cases, respectively. Reduction of the mitochondrial complex I and complex IV subunits was identified in the fibroblasts (13/13). Sixteen pathogenic variants were identified, of which c.626C>T was the most common. Seventy‐four percent of the patients were alive at their last clinical review (median 6.8 years, range: 14 months to 31 years, IQR 14.5 years).InterpretationPatients that harbour pathogenic variants in MTFMT have a milder clinical phenotype and disease progression compared to LS caused by other nuclear defects. Fibroblasts may preclude the need for muscle biopsy, to prove causality of any novel variant.

Recessive Mutations in TRMT10C Cause Defects in Mitochondrial RNA Processing and Multiple Respiratory Chain Deficiencies

Mitochondrial disorders are clinically and genetically diverse, with mutations in mitochondrial or nuclear genes able to cause defects in mitochondrial gene expression. Recently, mutations in several genes encoding factors involved in mt-tRNA processing have been identified to cause mitochondrial disease. Using whole-exome sequencing, we identified mutations in TRMT10C (encoding the mitochondrial RNase P protein 1 [MRPP1]) in two unrelated individuals who presented at birth with lactic acidosis, hypotonia, feeding difficulties, and deafness. Both individuals died at 5 months after respiratory failure. MRPP1, along with MRPP2 and MRPP3, form the mitochondrial ribonuclease P (mt-RNase P) complex that cleaves the 5′ ends of mt-tRNAs from polycistronic precursor transcripts. Additionally, a stable complex of MRPP1 and MRPP2 has m1R9 methyltransferase activity, which methylates mt-tRNAs at position 9 and is vital for folding mt-tRNAs into their correct tertiary structures. Analyses of fibroblasts from affected individuals harboring TRMT10C missense variants revealed decreased protein levels of MRPP1 and an increase in mt-RNA precursors indicative of impaired mt-RNA processing and defective mitochondrial protein synthesis. The pathogenicity of the detected variants—compound heterozygous c.542G>T (p.Arg181Leu) and c.814A>G (p.Thr272Ala) changes in subject 1 and a homozygous c.542G>T (p.Arg181Leu) variant in subject 2—was validated by the functional rescue of mt-RNA processing and mitochondrial protein synthesis defects after lentiviral transduction of wild-type TRMT10C. Our study suggests that these variants affect MRPP1 protein stability and mt-tRNA processing without affecting m1R9 methyltransferase activity, identifying mutations in TRMT10C as a cause of mitochondrial disease and highlighting the importance of RNA processing for correct mitochondrial function.

TRMT5 Mutations Cause a Defect in Post-transcriptional Modification of Mitochondrial tRNA Associated with Multiple Respiratory-Chain Deficiencies

Deficiencies in respiratory-chain complexes lead to a variety of clinical phenotypes resulting from inadequate energy production by the mitochondrial oxidative phosphorylation system. Defective expression of mtDNA-encoded genes, caused by mutations in either the mitochondrial or nuclear genome, represents a rapidly growing group of human disorders. By whole-exome sequencing, we identified two unrelated individuals carrying compound heterozygous variants in TRMT5 (tRNA methyltransferase 5). TRMT5 encodes a mitochondrial protein with strong homology to members of the class I-like methyltransferase superfamily. Both affected individuals presented with lactic acidosis and evidence of multiple mitochondrial respiratory-chain-complex deficiencies in skeletal muscle, although the clinical presentation of the two affected subjects was remarkably different; one presented in childhood with failure to thrive and hypertrophic cardiomyopathy, and the other was an adult with a life-long history of exercise intolerance. Mutations in TRMT5 were associated with the hypomodification of a guanosine residue at position 37 (G37) of mitochondrial tRNA; this hypomodification was particularly prominent in skeletal muscle. Deficiency of the G37 modification was also detected in human cells subjected to TRMT5 RNAi. The pathogenicity of the detected variants was further confirmed in a heterologous yeast model and by the rescue of the molecular phenotype after re-expression of wild-type TRMT5 cDNA in cells derived from the affected individuals. Our study highlights the importance of post-transcriptional modification of mitochondrial tRNAs for faithful mitochondrial function.

Leukoencephalopathy with cysts and hyperglycinemia may result from NFU1 deficiency

Lipoic acid metabolism defects are new metabolic disorders that cause neurological, cardiomuscular or pulmonary impairment. We report on a patient that presented with progressive neurological regression suggestive of an energetic disease, involving leukoencephalopathy with cysts. Elevated levels of glycine in plasma, urine and CSF associated with intermittent increases of lactate were consistent with a defect in lipoic acid metabolism. Support for the diagnosis was provided by pyruvate dehydrogenase deficiency and multiple mitochondrial respiratory chain deficiency in skin fibroblasts, as well as no lipoylated protein by western blot. Two mutations in the NFU1 gene confirmed the diagnosis. The p.Gly208Cys mutation has previously been reported suggesting a founder effect in Europe.

Clinical variability in neurohepatic syndrome due to combined mitochondrial DNA depletion and Gaucher disease

A 1-year-old girl born to consanguineous parents presented with unexplained liver failure, leading to transplantation at 19months. Subsequent partial splenectomy for persistent cytopenia showed the presence of foamy cells, and Gaucher disease was confirmed by homozygosity for the p.Leu483Pro mutation in the GBA gene. She was treated by enzyme replacement therapy (ERT). Clinical follow-up showed mild developmental delay, strabismus, nystagmus and oculomotor apraxia. Biochemical studies revealed multiple respiratory chain deficiencies and a mosaic pattern of deficient complex IV immunostaining in liver and fibroblast. Molecular analysis identified a mtDNA depletion syndrome due to the homozygous p.Pro98Leu mutation in MPV17. A younger sister unaffected by mtDNA depletion, presented with pancytopenia and hepatosplenomegaly. ERT for Gaucher disease resulted in visceral normalization without any neurological symptom. A third sister, affected by both conditions, had marked developmental delay, strabismus and ophthalmoplegia but no liver cirrhosis. In conclusion, intrafamilal variability occurs in MPV17-related disease. The combined pathological effect of Gaucher and mitochondrial diseases can negatively impact neurological and liver functions and influence the outcome in consanguineous families. The immunocytochemical staining of OXPHOS protein in tissues and cultured cells is a powerful tool revealing mosaic pattern of deficiency pointing to mtDNA-related mitochondrial disorders.

Mutations in mitochondrial ribosomal protein MRPL12 leads to growth retardation, neurological deterioration and mitochondrial translation deficiency

Multiple respiratory chain deficiencies represent a common cause of mitochondrial diseases and are associated with a wide range of clinical symptoms. We report a subject, born to consanguineous parents, with growth retardation and neurological deterioration. Multiple respiratory chain deficiency was found in muscle and fibroblasts of the subject as well as abnormal assembly of complexes I and IV. A microsatellite genotyping of the family members detected only one region of homozygosity on chromosome 17q24.2–q25.3 in which we focused our attention to genes involved in mitochondrial translation. We sequenced MRPL12, encoding the mitochondrial ribosomal protein L12 and identified a c.542C>T transition in exon 5 changing a highly conserved alanine into a valine (p.Ala181Val). This mutation resulted in a decreased steady-state level of MRPL12 protein, with altered integration into the large ribosomal subunit. Moreover, an overall mitochondrial translation defect was observed in the subject's fibroblasts with a significant reduction of synthesis of COXI, COXII and COXIII subunits. Modeling of MRPL12 shows Ala181 positioned in a helix potentially involved in an interface of interaction suggesting that the p.Ala181Val change might be predicted to alter interactions with the elongation factors. These results contrast with the eubacterial orthologues of human MRPL12, where L7/L12 proteins do not appear to have a selective effect on translation. Therefore, analysis of the mutated version found in the subject presented here suggests that the mammalian protein does not function in an entirely analogous manner to the eubacterial L7/L12 equivalent.

Screen for abnormal mitochondrial phenotypes in mouse embryonic stem cells identifies a model for succinyl-CoA ligase deficiency and mtDNA depletion.

ABSTRACTMutations in subunits of succinyl-CoA synthetase/ligase (SCS), a component of the citric acid cycle, are associated with mitochondrial encephalomyopathy, elevation of methylmalonic acid (MMA), and mitochondrial DNA (mtDNA) depletion. A FACS-based retroviral-mediated gene trap mutagenesis screen in mouse embryonic stem (ES) cells for abnormal mitochondrial phenotypes identified a gene trap allele of Sucla2 (Sucla2SAβgeo), which was used to generate transgenic mice. Sucla2 encodes the ADP-specific β-subunit isoform of SCS. Sucla2SAβgeo homozygotes exhibited recessive lethality, with most mutants dying late in gestation (e18.5). Mutant placenta and embryonic (e17.5) brain, heart and muscle showed varying degrees of mtDNA depletion (20–60%). However, there was no mtDNA depletion in mutant liver, where the gene is not normally expressed. Elevated levels of MMA were observed in embryonic brain. SCS-deficient mouse embryonic fibroblasts (MEFs) demonstrated a 50% reduction in mtDNA content compared with wild-type MEFs. The mtDNA depletion resulted in reduced steady state levels of mtDNA encoded proteins and multiple respiratory chain deficiencies. mtDNA content could be restored by reintroduction of Sucla2. This mouse model of SCS deficiency and mtDNA depletion promises to provide insights into the pathogenesis of mitochondrial diseases with mtDNA depletion and into the biology of mtDNA maintenance. In addition, this report demonstrates the power of a genetic screen that combines gene trap mutagenesis and FACS analysis in mouse ES cells to identify mitochondrial phenotypes and to develop animal models of mitochondrial dysfunction.

Reply: MFN2, a new gene responsible for mitochondrial DNA depletion

Sir, we recently showed in Brain that the MFN2 gene, typically responsible for autosomal dominant axonal Charcot–Marie–Tooth disease, is a novel gene associated with ‘mitochondrial DNA breakage’ syndrome (Rouzier et al. , 2012). We described a novel MFN2 heterozygous missense mutation in Tunisian patients who presented with optic atrophy beginning in early childhood, axonal neuropathy and mitochondrial myopathy in adult life with accumulation of mitochondrial DNA deletions in muscle. Renaldo et al. (2012) confirm that MFN2 is involved in mitochondrial DNA maintenance disorders. Indeed, they show that this gene is responsible for a severe phenotype in a child who presented a developmental delay with hypotonia and failure to thrive at the age of 6 months. Evolution was marked by the gradual appearance of ataxia, areflexia, abnormal movements and axonal sensorimotor neuropathy. At the age of 9 years, weight, height and head circumference were <3 SD. The child presented a bilateral optic atrophy and was deaf. Brain MRI was normal. A mitochondrial myopathy, with cytochrome c-oxidase negative fibres, was diagnosed on muscle biopsy and a multiple respiratory chain deficiency was found both in muscle and fibroblasts. Furthermore, Renaldo et al. (2012) found mitochondrial DNA depletion in the patient’s muscle with a low amount of mitochondrial DNA (28% of controls). The association of optic atrophy and axonal neuropathy led the authors to analyse the MFN2 gene and to identify a novel missense heterozygous mutation (p.D210Y) that affects the same aspartic acid residue modified in the Tunisian family that we described previously (p.D210V). A defect in the maintenance of mitochondrial genome can lead to a reduction of mitochondrial DNA copy number and/or multiple mitochondrial DNA deletions. In mouse models, conditional inactivation of Mfn2 in muscle leads to a mitochondrial myopathy with a severe mitochondrial DNA depletion above …

Toward genotype phenotype correlations in GFM1 mutations

Multiple respiratory chain deficiencies represent a common cause of mitochondrial diseases. We report two novel GFM1 mutations in two unrelated patients with encephalopathy and liver failure respectively. The first patient had intrauterine growth retardation, seizures, encephalopathy and developmental delay. Brain MRI showed hypoplasia of the vermis and severe pontine atrophy of the brainstem that were similar to those reported in patients with mitochondrial translation deficiencies. The second patient had liver failure with hypoglycemia. Respiratory chain analysis showed a complex IV deficiency in muscle of both patients. A 10K SNP genotyping detected several regions of homozygosity in the two patients. In vitro translation deficiency prompted us to study genes involved in mitochondrial translation. Therefore, we sequenced the GFM1 gene, encoding the mitochondrial translation factor EFG1, included in a shared homozygous region and identified two different homozygous mutations (R671C and L398P). Modeling studies of EFG1 protein suggested that the R671C mutation disrupts an inter-subunit interface and could locally destabilize the mutant protein. The second mutation (L398P) disrupted the H-bond network in a rich-beta-sheet domain, and may have a dramatic effect on local structure. GFM1 mutations have been seldom reported and are associated with different clinical presentation. By modeling the structure of the protein and the position of the various mutations we suggest that the clinical phenotypes of the patients could be related to the localization of the mutations.

Exome sequencing identifies MRPL3 mutation in mitochondrial cardiomyopathy

By combining exome sequencing in conjunction with genetic mapping, we have identified the first mutation in large mitochondrial ribosomal protein MRPL3 in a family of four sibs with hypertrophic cardiomyopathy, psychomotor retardation, and multiple respiratory chain deficiency. Affected sibs were compound heterozygotes for a missense MRPL3 mutation (P317R) and a large-scale deletion, inherited from the mother and the father, respectively. These mutations were shown to alter ribosome assembly and cause a mitochondrial translation deficiency in cultured skin fibroblasts resulting in an abnormal assembly of several complexes of the respiratory chain. This observation gives support to the view that exome sequencing combined with genetic mapping is a powerful approach for the identification of new genes of mitochondrial disorders.

Mutation in the mitochondrial translation elongation factor EFTs results in severe infantile liver failure

Multiple respiratory chain deficiencies represent a common cause of mitochondrial diseases and often result in hepatic failure. A significant fraction of patients present mitochondrial DNA depletion but a number of cases remain unexplained. The aim of our study was to identify the disease causing gene in a kindred with intrauterine growth retardation, neonatal lactic acidosis, liver dysfunction and multiple respiratory chain deficiency in muscle. Homozygosity mapping was performed by 50K SNP genotyping and candidate genes were successively analyzed by direct sequencing on genomic DNA of the family members. SNP genotyping detected several regions of homozygosity in which we focused our attention to genes involved in mitochondrial translation. We sequenced the TSFM gene, encoding the mitochondrial translation factor EFTs and identified a homozygous mutation changing a highly conserved arginine into a tryptophan (R312W). This mutation has been previously reported in two unrelated kindred presenting two distinct syndromes (fatal mitochondrial encephalomyopathy and hypertrophic cardiomyopathy respectively). The description of a third syndrome associated with a same TSFM mutation gives support to the broad clinical and genetic heterogeneity of mitochondrial translation deficiencies in human. It suggests that mitochondrial translation deficiency represents a growing cause of hepatic failure of mitochondrial origin in infants.

Liver hepatoblastoma and multiple OXPHOS deficiency in the follow-up of a patient with methylmalonic aciduria

A boy who was diagnosed with methylmalonic aciduria (MMA) at the age of 10 days developed persistent hepatomegaly and raised transaminases from the age of 4 years. He was subsequently diagnosed with Leigh syndrome and required a kidney transplantation for end-stage renal failure. A massive hepatoblastoma led to his death by the age of 11 years. Methylmalonyl-CoA mutase activity was undetectable on both cultured skin fibroblasts and kidney biopsy and multiple respiratory chain deficiency was demonstrated in the kidney. Mitochondrial dysfunction and/or post-transplant immunosuppressive therapy should be considered as a possible cause of liver cancer in this patient.

Mitochondrial DNA Depletion is a Prevalent Cause of Multiple Respiratory Chain Deficiency in Childhood

To determine the actual incidence of mitochondrial DNA (mtDNA) depletion syndrome in multiple respiratory chain deficiency. We carried out a real-time polymerase chain reaction quantification of mtDNA in liver or muscle tissue of 100 children with unexplained multiple oxidative phosphorylation enzyme deficiency. A reduction of mtDNA copy number to <35% of control values was found in liver and/or muscle in half of the children (50/100). Most of these patients (32/50; 64%) presented with severe neonatal onset liver involvement; 7 (14%) had Alpers syndrome, and 11 (22%) exhibited various forms of neurologic involvement. Deoxyguanosine kinase or polymerase gamma (POLG) mutations could be identified in 11 of 32 patients with liver involvement, and POLG mutations were consistently found in all 7 patients with Alpers syndrome. Homozygous thymidine kinase 2 and MPV17 gene mutations were found in 2 patients. Our findings show that mtDNA depletion is a prevalent cause of multiple respiratory chain deficiency in infancy.

Sequence and structure of the human OXA1L gene and its upstream elements

The genes encoding proteins involved in respiratory chain assembly represent candidate genes for nuclearly-encoded multiple respiratory chain deficiency. Using the long PCR amplification procedure, we have characterized the organization and complete sequence of OXA1L, a gene involved in the assembly of several complexes of the mitochondrial respiratory chain. The OXA1L gene (5 kb) is composed of 10 exons and 9 introns and contains a 24 N-terminal amino-acid stretch is characteristic of a mitochondrial presequence. The screening of OXA1L mutation in patients with multiple respiratory chain deficiency is now feasible.