Complex I assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency

Acyl-CoA dehydrogenase 9 (ACAD9) is an assembly factor for mitochondrial respiratory chain Complex I (CI), and ACAD9 mutations are recognized as a frequent cause of CI deficiency. ACAD9 also retains enzyme ACAD activity for long-chain fatty acids in vitro, but the biological relevance of this function remains controversial partly because of the tissue specificity of ACAD9 expression: high in liver and neurons and minimal in skin fibroblasts. In this study, we hypothesized that this enzymatic ACAD activity is required for full fatty acid oxidation capacity in cells expressing high levels of ACAD9 and that loss of this function is important in determining phenotype in ACAD9-deficient patients. First, we confirmed that HEK293 cells express ACAD9 abundantly. Then, we showed that ACAD9 knockout in HEK293 cells affected long-chain fatty acid oxidation along with Cl, both of which were rescued by wild type ACAD9. Further, we evaluated whether the loss of ACAD9 enzymatic fatty acid oxidation affects clinical severity in patients with ACAD9 mutations. The effects on ACAD activity of 16 ACAD9 mutations identified in 24 patients were evaluated using a prokaryotic expression system. We showed that there was a significant inverse correlation between residual enzyme ACAD activity and phenotypic severity of ACAD9-deficient patients. These results provide evidence that in cells where it is strongly expressed, ACAD9 plays a physiological role in fatty acid oxidation, which contributes to the severity of the phenotype in ACAD9-deficient patients. Accordingly, treatment of ACAD9 patients should aim at counteracting both CI and fatty acid oxidation dysfunction

Mutations in SDHD lead to autosomal recessive encephalomyopathy and isolated mitochondrial complex II deficiency

BACKGROUND Defects of the mitochondrial respiratory chain complex II (succinate dehydrogenase (SDH) complex) are extremely rare. Of the four nuclear encoded proteins composing complex II, only mutations in the 70 kDa flavoprotein (SDHA) and the recently identified complex II assembly factor (SDHAF1) have been found to be causative for mitochondrial respiratory chain diseases. Mutations in the other three subunits (SDHB, SDHC, SDHD) and the second assembly factor (SDHAF2) have so far only been associated with hereditary paragangliomas and phaeochromocytomas. Recessive germline mutations in SDHB have recently been associated with complex II deficiency and leukodystrophy in one patient. METHODS AND RESULTS We present the clinical and molecular investigations of the first patient with biochemical evidence of a severe isolated complex II deficiency due to compound heterozygous SDHD gene mutations. The patient presented with early progressive encephalomyopathy due to compound heterozygous p.E69 K and p.*164Lext*3 SDHD mutations. Native polyacrylamide gel electrophoresis and western blotting demonstrated an impaired complex II assembly. Complementation of a patient cell line additionally supported the pathogenicity of the novel identified mutations in SDHD. CONCLUSIONS This report describes the first case of isolated complex II deficiency due to recessive SDHD germline mutations. We therefore recommend screening for all SDH genes in isolated complex II deficiencies. It further emphasises the importance of appropriate genetic counselling to the family with regard to SDHD mutations and their role in tumorigenesis

Lack of the mitochondrial protein acylglycerol kinase causes Sengers syndrome

Exome sequencing of an individual with congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, and lactic acidosis, all typical symptoms of Sengers syndrome, discovered two nonsense mutations in the gene encoding mitochondrial acylglycerol kinase (AGK). Mutation screening of AGK in further individuals with congenital cataracts and cardiomyopathy identified numerous loss-of-function mutations in an additional eight families, confirming the causal nature of AGK deficiency in Sengers syndrome. The loss of AGK led to a decrease of the adenine nucleotide translocator in the inner mitochondrial membrane in muscle, consistent with a role of AGK in driving the assembly of the translocator as a result of its effects on phospholipid metabolism in mitochondria

Complex I assembly function and fatty acid oxidation enzyme activity of ACAD9 both contribute to disease severity in ACAD9 deficiency

Acyl-CoA dehydrogenase 9 (ACAD9) is an assembly factor for mitochondrial respiratory chain Complex I (CI), and ACAD9 mutations are recognized as a frequent cause of CI deficiency. ACAD9 also retains enzyme ACAD activity for long-chain fatty acids in vitro, but the biological relevance of this function remains controversial partly because of the tissue specificity of ACAD9 expression: high in liver and neurons and minimal in skin fibroblasts. In this study, we hypothesized that this enzymatic ACAD activity is required for full fatty acid oxidation capacity in cells expressing high levels of ACAD9 and that loss of this function is important in determining phenotype in ACAD9-deficient patients. First, we confirmed that HEK293 cells express ACAD9 abundantly. Then, we showed that ACAD9 knockout in HEK293 cells affected long-chain fatty acid oxidation along with Cl, both of which were rescued by wild type ACAD9. Further, we evaluated whether the loss of ACAD9 enzymatic fatty acid oxidation affects clinical severity in patients with ACAD9 mutations. The effects on ACAD activity of 16 ACAD9 mutations identified in 24 patients were evaluated using a prokaryotic expression system. We showed that there was a significant inverse correlation between residual enzyme ACAD activity and phenotypic severity of ACAD9-deficient patients. These results provide evidence that in cells where it is strongly expressed, ACAD9 plays a physiological role in fatty acid oxidation, which contributes to the severity of the phenotype in ACAD9-deficient patients. Accordingly, treatment of ACAD9 patients should aim at counteracting both CI and fatty acid oxidation dysfunctions

Molecular diagnosis in mitochondrial complex I deficiency using exome sequencing

BACKGROUND: Next generation sequencing has become the core technology for gene discovery in rare inherited disorders. However, the interpretation of the numerous sequence variants identified remains challenging. We assessed the application of exome sequencing for diagnostics in complex I deficiency, a disease with vast genetic heterogeneity. METHODS: Ten unrelated individuals with complex I deficiency were selected for exome sequencing and sequential bioinformatic filtering. Cellular rescue experiments were performed to verify pathogenicity of novel disease alleles. RESULTS: The first filter criterion was 'Presence of known pathogenic complex I deficiency variants'. This revealed homozygous mutations in NDUFS3 and ACAD9 in two individuals. A second criterion was 'Presence of two novel potentially pathogenic variants in a structural gene of complex I', which discovered rare variants in NDUFS8 in two unrelated individuals and in NDUFB3 in a third. Expression of wild-type cDNA in mutant cell lines rescued complex I activity and assembly, thus providing a functional validation of their pathogenicity. Using the third criterion 'Presence of two potentially pathogenic variants in a gene encoding a mitochondrial protein', loss-of-function mutations in MTFMT were discovered in two patients. In three patients the molecular genetic correlate remained unclear and follow-up analysis is ongoing. CONCLUSION: Appropriate in silico filtering of exome sequencing data, coupled with functional validation of new disease alleles, is effective in rapidly identifying disease-causative variants in known and new complex I associated disease genes

Phenotypic spectrum of eleven patients and five novel MTFMT mutations identified by exome sequencing and candidate gene screening

Defects of mitochondrial oxidative phosphorylation (OXPHOS) are associated with a wide range of clinical phenotypes and time courses. Combined OXPHOS deficiencies are mainly caused by mutations of nuclear genes that are involved in mitochondrial protein translation. Due to their genetic heterogeneity it is almost impossible to diagnose OXPHOS patients on clinical grounds alone. Hence next generation sequencing (NGS) provides a distinct advantage over candidate gene sequencing to discover the underlying genetic defect in a timely manner. One recent example is the identification of mutations in MTFMT that impair mitochondrial protein translation through decreased formylation of Met-tRNA(Met). Here we report the results of a combined exome sequencing and candidate gene screening study. We identified nine additional MTFMT patients from eight families who were affected with Leigh encephalopathy or white matter disease, microcephaly, mental retardation, ataxia, and muscular hypotonia. In four patients, the causal mutations were identified by exome sequencing followed by stringent bioinformatic filtering. In one index case, exome sequencing identified a single heterozygous mutation leading to Sanger sequencing which identified a second mutation in the non-covered first exon. High-resolution melting curve-based MTFMT screening in 350 OXPHPOS patients identified pathogenic mutations in another three index cases. Mutations in one of them were not covered by previous exome sequencing. All novel mutations predict a loss-of-function or result in a severe decrease in MTFMT protein in patients' fibroblasts accompanied by reduced steady-state levels of complex I and IV subunits. Being present in 11 out of 13 index cases the c.626C>T mutation is one of the most frequent disease alleles underlying OXPHOS disorders. We provide detailed clinical descriptions on eleven MTFMT patients and review five previously reported cases

<em>ADCK4</em> mutations promote steroid-resistant nephrotic syndrome through CoQ₁₀ biosynthesis disruption.

Identification of single-gene causes of steroid-resistant nephrotic syndrome (SRNS) has furthered the understanding of the pathogenesis of this disease. Here, using a combination of homozygosity mapping and whole human exome resequencing, we identified mutations in the aarF domain containing kinase 4 (ADCK4) gene in 15 individuals with SRNS from 8 unrelated families. ADCK4 was highly similar to ADCK3, which has been shown to participate in coenzyme Q(10) (CoQ(10)) biosynthesis. Mutations in ADCK4 resulted in reduced COQ(10). levels and reduced mitochondrial respiratory enzyme activity in cells isolated from individuals with SRNS and transformed lymphoblasts. Knockdown of adck4 in zebrafish and Drosophila recapitulated nephrotic syndrome-associated phenotypes. Furthermore, ADCK4 was expressed in glomerular podocytes and partially localized to podocyte mitochondria and foot processes in rat kidneys and cultured human podocytes. In human podocytes, ADCK4 interacted with members of the CoQ(10) biosynthesis pathway, including COQ6, which has been linked with SRNS and COQ7. Knockdown of ADCK4 in podocytes resulted in decreased migration, which was reversed by CoQ(10) addition. Interestingly, a patient with SRNS with a homozygous ADCK4 frameshift mutation had partial remission following CoQ(10) treatment. These data indicate that individuals with SRNS with mutations in ADCK4 or other genes that participate in CoQ(10) biosynthesis may be treatable with CoQ(10)