Arnaud Vanlander

Mutation of the iron-sulfur cluster assembly gene IBA57 causes severe myopathy and encephalopathy

Two siblings from consanguineous parents died perinatally with a condition characterized by generalized hypotonia, respiratory insufficiency, arthrogryposis, microcephaly, congenital brain malformations and hyperglycinemia. Catalytic activities of the mitochondrial respiratory complexes I and II were deficient in skeletal muscle, a finding suggestive of an inborn error in mitochondrial biogenesis. Homozygosity mapping identified IBA57 located in the largest homozygous region on chromosome 1 as a culprit candidate gene. IBA57 is known to be involved in the biosynthesis of mitochondrial [4Fe-4S] proteins. Sequence analysis of IBA57 revealed the homozygous mutation c.941A > C, p.Gln314Pro. Severely decreased amounts of IBA57 protein were observed in skeletal muscle and cultured skin fibroblasts from the affected subjects. HeLa cells depleted of IBA57 showed biochemical defects resembling the ones found in patient-derived cells, including a decrease in various mitochondrial [4Fe-4S] proteins and in proteins covalently linked to lipoic acid (LA), a cofactor produced by the [4Fe-4S] protein LA synthase. The defects could be complemented by wild-type IBA57 and partially by mutant IBA57. As a result of the mutation, IBA57 protein was excessively degraded, an effect ameliorated by protease inhibitors. Hence, we propose that the mutation leads to partial functional impairment of IBA57, yet the major pathogenic impact is due to its proteolytic degradation below physiologically critical levels. In conclusion, the ensuing lethal complex biochemical phenotype of a novel metabolic syndrome results from multiple Fe/S protein defects caused by a deficiency in the Fe/S cluster assembly protein IBA57.

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.

Clinical, biochemical, and genetic spectrum of seven patients with NFU1 deficiency

Disorders of the mitochondrial energy metabolism are clinically and genetically heterogeneous. An increasingly recognized subgroup is caused by defective mitochondrial iron–sulfur (Fe–S) cluster biosynthesis, with defects in 13 genes being linked to human disease to date. Mutations in three of them, NFU1, BOLA3, and IBA57, affect the assembly of mitochondrial [4Fe–4S] proteins leading to an impairment of diverse mitochondrial metabolic pathways and ATP production. Patients with defects in these three genes present with lactic acidosis, hyperglycinemia, and reduced activities of respiratory chain complexes I and II, the four lipoic acid-dependent 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). To date, five different NFU1 pathogenic variants have been reported in 15 patients from 12 families. We report on seven new patients from five families carrying compound heterozygous or homozygous pathogenic NFU1 mutations identified by candidate gene screening and exome sequencing. Six out of eight different disease alleles were novel and functional studies were performed to support the pathogenicity of five of them. Characteristic clinical features included fatal infantile encephalopathy and pulmonary hypertension leading to death within the first 6 months of life in six out of seven patients. Laboratory investigations revealed combined defects of pyruvate dehydrogenase complex (five out of five) and respiratory chain complexes I and II+III (four out of five) in skeletal muscle and/or cultured skin fibroblasts as well as increased lactate (five out of six) and glycine concentration (seven out of seven). Our study contributes to a better definition of the phenotypic spectrum associated with NFU1 mutations and to the diagnostic workup of future patients.

Possible pathogenic mechanism of propofol infusion syndrome involves coenzyme Q

Background:Propofol is a short-acting intravenous anesthetic agent. In rare conditions, a life-threatening complication known as propofol infusion syndrome can occur. The pathophysiologic mechanism is still unknown. Some studies suggested that propofol acts as uncoupling agent, others suggested that it inhibits complex I or complex IV, or causes increased oxidation of cytochrome c and cytochrome aa3, or inhibits mitochondrial fatty acid metabolism. Although the exact site of interaction is not known, most hypotheses point to the direction of the mitochondria. Methods:Eight rats were ventilated and sedated with propofol up to 20 h. Sequential biopsy specimens were taken from liver and skeletal muscle and used for determination of respiratory chain activities and propofol concentration. Activities were also measured in skeletal muscle from a patient who died of propofol infusion syndrome. Results:In rats, authors detected a decrease in complex II+III activity starting at low tissue concentration of propofol (20 to 25 µM), further declining at higher concentrations. Before starting anesthesia, the complex II+III/citrate synthase activity ratio in liver was 0.46 (0.25) and in skeletal muscle 0.23 (0.05) (mean [SD]). After 20 h of anesthesia, the ratios declined to 0.17 (0.03) and 0.12 (0.02), respectively. When measured individually, the activities of complexes II and III remained normal. Skeletal muscle from one patient taken in the acute phase of propofol infusion syndrome also shows a selective decrease in complex II+III activity (z-score: −2.96). Conclusion:Propofol impedes the electron flow through the respiratory chain and coenzyme Q is the main site of interaction with propofol.

Mitochondria in peroxisome-deficient hepatocytes exhibit impaired respiration, depleted DNA, and PGC-1α independent proliferation

The tight interrelationship between peroxisomes and mitochondria is illustrated by their cooperation in lipid metabolism, antiviral innate immunity and shared use of proteins executing organellar fission. In addition, we previously reported that disruption of peroxisome biogenesis in hepatocytes severely impacts on mitochondrial integrity, primarily damaging the inner membrane. Here we investigated the molecular impairments of the dysfunctional mitochondria in hepatocyte selective Pex5 knockout mice. First, by using blue native electrophoresis and in-gel activity stainings we showed that the respiratory complexes were differentially affected with reduction of complexes I and III and incomplete assembly of complex V, whereas complexes II and IV were normally active. This resulted in impaired oxygen consumption in cultured Pex5(-/-) hepatocytes. Second, mitochondrial DNA was depleted causing an imbalance in the expression of mitochondrial- and nuclear-encoded subunits of the respiratory chain complexes. Third, mitochondrial membranes showed increased permeability and fluidity despite reduced content of the polyunsaturated fatty acid docosahexaenoic acid. Fourth, the affected mitochondria in peroxisome deficient hepatocytes displayed increased oxidative stress. Acute deletion of PEX5 in vivo using adeno-Cre virus phenocopied these effects, indicating that mitochondrial perturbations closely follow the loss of functional peroxisomes in time. Likely to compensate for the functional impairments, the volume of the mitochondrial compartment was increased several folds. This was not driven by PGC-1α but mediated by activation of PPARα, possibly through c-myc overexpression. In conclusion, loss of peroxisomal metabolism in hepatocytes perturbs the mitochondrial inner membrane, depletes mitochondrial DNA and causes mitochondrial biogenesis independent of PGC-1α.

Two Siblings with Homozygous Pathogenic Splice‐Site Variant in Mitochondrial Asparaginyl–tRNA Synthetase (NARS2)

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‐tRNAAsn 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.

Inborn oxidative phosphorylation defect as risk factor for propofol infusion syndrome

Propofol is an anesthetic agent widely used for induction and maintenance of anesthesia, and sedation in children. Although generally considered as reliable and safe, administration of propofol can occasionally induce a potentially fatal complication known as propofol infusion syndrome (PRIS). Mitochondrial dysfunction has been implicated in the pathogenesis of PRIS. We report on an adult patient with Leber hereditary optic neuropathy (LHON) who developed PRIS. He was a carrier of the m.3460G>A mutation, one of the major three pathogenic point mutations associated with LHON. The propositus was blind and underwent propofol sedation after severe head injury. Five days after start of propofol infusion, the patient died. The activity of complex I of the oxidative phosphorylation (OXPHOS) system was severely deficient in skeletal muscle. Our observation indicates that fulminate PRIS can occur in an adult patient with an inborn OXPHOS defect and corroborates the hypothesis that PRIS is caused by inhibition of the OXPHOS system.

Mutations in the mitochondrial cysteinyl-tRNA synthase gene, CARS2, lead to a severe epileptic encephalopathy and complex movement disorder

Background 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. Methods and results We identified a proband with an epileptic encephalopathy, complex movement disorder and a combined mitochondrial respiratory chain enzyme deficiency. The child presented with neurological regression, complex movement disorder and intractable seizures. A combined deficiency of mitochondrial complexes I, III and IV was noted in liver tissue, along with increased mitochondrial DNA content in skeletal muscle. Incomplete assembly of complex V, using blue native polyacrylamide gel electrophoretic analysis and complex I, using western blotting, suggested a disorder of mitochondrial transcription or translation. Exome sequencing identified compound heterozygous mutations in CARS2, a mitochondrial aminoacyl-tRNA synthetase. Both mutations affect highly conserved amino acids located within the functional ligase domain of the cysteinyl-tRNA synthase. A specific decrease in the amount of charged mt-tRNACys was detected in patient fibroblasts compared with controls. Retroviral transfection of the wild-type CARS2 into patient skin fibroblasts led to the correction of the incomplete assembly of complex V, providing functional evidence for the role of CARS2 mutations in disease aetiology. Conclusions Our findings indicate that mutations in CARS2 result in a mitochondrial translational defect as seen in individuals with mitochondrial epileptic encephalopathy.

Analysis of the whole mitochondrial genome: translation of the Ion Torrent Personal Genome Machine system to the diagnostic bench?

Next-generation sequencing (NGS), an innovative sequencing technology that enables the successful analysis of numerous gene sequences in a massive parallel sequencing approach, has revolutionized the field of molecular biology. Although NGS was introduced in a rather recent past, the technology has already demonstrated its potential and effectiveness in many research projects, and is now on the verge of being introduced into the diagnostic setting of routine laboratories to delineate the molecular basis of genetic disease in undiagnosed patient samples. We tested a benchtop device on retrospective genomic DNA (gDNA) samples of controls and patients with a clinical suspicion of a mitochondrial DNA disorder. This Ion Torrent Personal Genome Machine platform is a high-throughput sequencer with a fast turnaround time and reasonable running costs. We challenged the chemistry and technology with the analysis and processing of a mutational spectrum composed of samples with single-nucleotide substitutions, indels (insertions and deletions) and large single or multiple deletions, occasionally in heteroplasmy. The output data were compared with previously obtained conventional dideoxy sequencing results and the mitochondrial revised Cambridge Reference Sequence (rCRS). We were able to identify the majority of all nucleotide alterations, but three false-negative results were also encountered in the data set. At the same time, the poor performance of the PGM instrument in regions associated with homopolymeric stretches generated many false-positive miscalls demanding additional manual curation of the data.

Biallelic Mutations in TMEM126B Cause Severe Complex I Deficiency with a Variable Clinical Phenotype

Complex I deficiency is the most common biochemical phenotype observed in individuals with mitochondrial disease. With 44 structural subunits and over 10 assembly factors, it is unsurprising that complex I deficiency is associated with clinical and genetic heterogeneity. Massively parallel sequencing (MPS) technologies including custom, targeted gene panels or unbiased whole-exome sequencing (WES) are hugely powerful in identifying the underlying genetic defect in a clinical diagnostic setting, yet many individuals remain without a genetic diagnosis. These individuals might harbor mutations in poorly understood or uncharacterized genes, and their diagnosis relies upon characterization of these orphan genes. Complexome profiling recently identified TMEM126B as a component of the mitochondrial complex I assembly complex alongside proteins ACAD9, ECSIT, NDUFAF1, and TIMMDC1. Here, we describe the clinical, biochemical, and molecular findings in six cases of mitochondrial disease from four unrelated families affected by biallelic (c.635G>T [p.Gly212Val] and/or c.401delA [p.Asn134Ilefs∗2]) TMEM126B variants. We provide functional evidence to support the pathogenicity of these TMEM126B variants, including evidence of founder effects for both variants, and establish defects within this gene as a cause of complex I deficiency in association with either pure myopathy in adulthood or, in one individual, a severe multisystem presentation (chronic renal failure and cardiomyopathy) in infancy. Functional experimentation including viral rescue and complexome profiling of subject cell lines has confirmed TMEM126B as the tenth complex I assembly factor associated with human disease and validates the importance of both genome-wide sequencing and proteomic approaches in characterizing disease-associated genes whose physiological roles have been previously undetermined.