Mitochondrial respiratory chain disorders are a heterogeneous group of disorders in which the underlying genetic defect is often unknown. We have identified a pathogenic mutation (c.156C>G [p.F52L]) in YARS2, located at chromosome 12p11.21, by using genome-wide SNP-based homozygosity analysis of a family with affected members displaying myopathy, lactic acidosis, and sideroblastic anemia (MLASA). We subsequently identified the same mutation in another unrelated MLASA patient. The YARS2 gene product, mitochondrial tyrosyl-tRNA synthetase (YARS2), was present at lower levels in skeletal muscle whereas fibroblasts were relatively normal. Complex I, III, and IV were dysfunctional as indicated by enzyme analysis, immunoblotting, and immunohistochemistry. A mitochondrial protein-synthesis assay showed reduced levels of respiratory chain subunits in myotubes generated from patient cell lines. A tRNA aminoacylation assay revealed that mutant YARS2 was still active; however, enzyme kinetics were abnormal compared to the wild-type protein. We propose that the reduced aminoacylation activity of mutant YARS2 enzyme leads to decreased mitochondrial protein synthesis, resulting in mitochondrial respiratory chain dysfunction. MLASA has previously been associated with PUS1 mutations; hence, the YARS2 mutation reported here is an alternative cause of MLASA.

SLC39A8 variants have recently been reported to cause a type II congenital disorder of glycosylation (CDG) in patients with intellectual disability and cerebellar atrophy. Here we report a novel SLC39A8 variant in siblings with features of Leigh-like mitochondrial disease. Two sisters born to consanguineous Lebanese parents had profound developmental delay, dystonia, seizures and failure to thrive. Brain MRI of both siblings identified bilateral basal ganglia hyperintensities on T2-weighted imaging and cerebral atrophy. CSF lactate was elevated in patient 1 and normal in patient 2. Respiratory chain enzymology was only performed on patient 1 and revealed complex IV and II + III activity was low in liver, with elevated complex I activity. Complex IV activity was borderline low in patient 1 muscle and pyruvate dehydrogenase activity was reduced. Whole genome sequencing identified a homozygous Chr4(GRCh37):g.103236869C>G; c.338G>C; p.(Cys113Ser) variant in SLC39A8, located in one of eight regions identified by homozygosity mapping. SLC39A8 encodes a manganese and zinc transporter which localises to the cell and mitochondrial membranes. Patient 2 blood and urine manganese levels were undetectably low. Transferrin electrophoresis of patient 2 serum revealed a type II CDG defect. Oral supplementation with galactose and uridine led to improvement of the transferrin isoform pattern within 14 days of treatment initiation. Oral manganese has only recently been added to the treatment. These results suggest SLC39A8 deficiency can cause both a type II CDG and Leigh-like syndrome, possibly via reduced activity of the manganese-dependent enzymes β-galactosyltransferase and mitochondrial manganese superoxide dismutase.

Mitochondrial fatty acid synthesis (mtFAS) is an evolutionarily conserved pathway essential for the function of the respiratory chain and several mitochondrial enzyme complexes. We report here a unique neurometabolic human disorder caused by defective mtFAS. Seven individuals from five unrelated families presented with childhood-onset dystonia, optic atrophy, and basal ganglia signal abnormalities on MRI. All affected individuals were found to harbor recessive mutations in MECR encoding the mitochondrial trans-2-enoyl-coenzyme A-reductase involved in human mtFAS. All six mutations are extremely rare in the general population, segregate with the disease in the families, and are predicted to be deleterious. The nonsense c.855T>G (p.Tyr285), c.247_250del (p.Asn83Hisfs4), and splice site c.830+2_830+3insT mutations lead to C-terminal truncation variants of MECR. The missense c.695G>A (p.Gly232Glu), c.854A>G (p.Tyr285Cys), and c.772C>T (p.Arg258Trp) mutations involve conserved amino acid residues, are located within the cofactor binding domain, and are predicted by structural analysis to have a destabilizing effect. Yeast modeling and complementation studies validated the pathogenicity of the MECR mutations. Fibroblast cell lines from affected individuals displayed reduced levels of both MECR and lipoylated proteins as well as defective respiration. These results suggest that mutations in MECR cause a distinct human disorder of the mtFAS pathway. The observation of decreased lipoylation raises the possibility of a potential therapeutic strategy.

Iron-sulfur clusters (ISCs) are important prosthetic groups that define the functions of many proteins. Proteins with ISCs (called iron-sulfur or Fe-S proteins) are present in mitochondria, the cytosol, the endoplasmic reticulum and the nucleus. They participate in various biological pathways including oxidative phosphorylation (OXPHOS), the citric acid cycle, iron homeostasis, heme biosynthesis and DNA repair. Here, we report a homozygous mutation in LYRM4 in two patients with combined OXPHOS deficiency. LYRM4 encodes the ISD11 protein, which forms a complex with, and stabilizes, the sulfur donor NFS1. The homozygous mutation (c.203G>T, p.R68L) was identified via massively parallel sequencing of >1000 mitochondrial genes (MitoExome sequencing) in a patient with deficiency of complexes I, II and III in muscle and liver. These three complexes contain ISCs. Sanger sequencing identified the same mutation in his similarly affected cousin, who had a more severe phenotype and died while a neonate. Complex IV was also deficient in her skeletal muscle. Several other Fe-S proteins were also affected in both patients, including the aconitases and ferrochelatase. Mutant ISD11 only partially complemented for an ISD11 deletion in yeast. Our in vitro studies showed that the l-cysteine desulfurase activity of NFS1 was barely present when co-expressed with mutant ISD11. Our findings are consistent with a defect in the early step of ISC assembly affecting a broad variety of Fe-S proteins. The differences in biochemical and clinical features between the two patients may relate to limited availability of cysteine in the newborn period and suggest a potential approach to therapy.

BackgroundMutations in the mitochondrial tyrosyl-tRNA synthetase (YARS2) gene have previously been identified as a cause of the tissue specific mitochondrial respiratory chain (RC) disorder, Myopathy, Lactic Acidosis, Sideroblastic Anaemia (MLASA). In this study, a cohort of patients with a mitochondrial RC disorder for who anaemia was a feature, were screened for mutations in YARS2.MethodsTwelve patients were screened for YARS2 mutations by Sanger sequencing. Clinical data were compared. Functional assays were performed to confirm the pathogenicity of the novel mutations and to investigate tissue specific effects.ResultsPathogenicYARS2 mutations were identified in three of twelve patients screened. Two patients were found to be homozygous for the previously reported p.Phe52Leu mutation, one severely and one mildly affected. These patients had different mtDNA haplogroups which may contribute to the observed phenotypic variability. A mildly affected patient was a compound heterozygote for two novel YARS2 mutations, p.Gly191Asp and p.Arg360X. The p.Gly191Asp mutation resulted in a 38-fold loss in YARS2 catalytic efficiency and the p.Arg360X mutation did not produce a stable protein. The p.Phe52Leu and p.Gly191Asp/p.Arg360X mutations resulted in more severe RC deficiency of complexes I, III and IV in muscle cells compared to fibroblasts, but had relatively normal YARS2 protein levels. The muscle-specific RC deficiency can be related to the increased requirement for RC complexes in muscle. There was also a failure of mtDNA proliferation upon myogenesis in patient cells which may compound the RC defect. Patient muscle had increased levels of PGC1-α and TFAM suggesting mitochondrial biogenesis was activated as a potential compensatory mechanism.ConclusionIn this study we have identified novel YARS2 mutations and noted marked phenotypic variability among YARS2 MLASA patients, with phenotypes ranging from mild to lethal, and we suggest that the background mtDNA haplotype may be contributing to the phenotypic variability. These findings have implications for diagnosis and prognostication of the MLASA and related phenotypes.

Functional defects of the mitochondrial translation machinery, as a result of mutations in nuclear-encoded genes, have been associated with combined oxidative phosphorylation (OXPHOS) deficiencies. We report siblings with congenital sensorineural deafness and lactic acidemia in association with combined respiratory chain (RC) deficiencies of complexes I, III and IV observed in fibroblasts and liver. One of the siblings had a more severe phenotype showing progressive hepatic and renal failure. Whole-exome sequencing revealed a homozygous mutation in the gene encoding mitochondrial ribosomal protein S7 (MRPS7), a c.550A>G transition that encodes a substitution of valine for a highly conserved methionine (p.Met184Val) in both affected siblings. MRPS7 is a 12S ribosomal RNA-binding subunit of the small mitochondrial ribosomal subunit, and is required for the assembly of the small ribosomal subunit. Pulse labeling of mitochondrial protein synthesis products revealed impaired mitochondrial protein synthesis in patient fibroblasts. Exogenous expression of wild-type MRPS7 in patient fibroblasts rescued complexes I and IV activities, demonstrating the deleterious effect of the mutation on RC function. Moreover, reduced 12S rRNA transcript levels observed in the patient's fibroblasts were also restored to normal levels by exogenous expression of wild-type MRPS7. Our data demonstrate the pathogenicity of the identified MRPS7 mutation as a novel cause of mitochondrial RC dysfunction, congenital sensorineural deafness and progressive hepatic and renal failure.

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