A molecular chaperone for mitochondrial complex I assembly is mutated in a progressive encephalopathy.

NADH:ubiquinone oxidoreductase (complex I) deficiency is a common cause of mitochondrial oxidative phosphorylation disease. It is associated with a wide range of clinical phenotypes in infants, including Leigh syndrome, cardiomyopathy, and encephalomyopathy. In at least half of patients, enzyme deficiency results from a failure to assemble the holoenzyme complex; however, the molecular chaperones required for assembly of the mammalian enzyme remain unknown. Using whole genome subtraction of yeasts with and without a complex I to generate candidate assembly factors, we identified a paralogue (B17.2L) of the B17.2 structural subunit. We found a null mutation in B17.2L in a patient with a progressive encephalopathy and showed that the associated complex I assembly defect could be completely rescued by retroviral expression of B17.2L in patient fibroblasts. An anti-B17.2L antibody did not associate with the holoenzyme complex but specifically recognized an 830-kDa subassembly in several patients with complex I assembly defects and coimmunoprecipitated a subset of complex I structural subunits from normal human heart mitochondria. These results demonstrate that B17.2L is a bona fide molecular chaperone that is essential for the assembly of complex I and for the normal function of the nervous system.

Mutations in the mitochondrial tRNA Ser(AGY) gene are associated with deafness, retinal degeneration, myopathy and epilepsy.

Although over 200 pathogenic mitochondrial DNA (mtDNA) mutations have been reported to date, determining the genetic aetiology of many cases of mitochondrial disease is still not straightforward. Here, we describe the investigations undertaken to uncover the underlying molecular defect(s) in two unrelated Caucasian patients with suspected mtDNA disease, who presented with similar symptoms of myopathy, deafness, neurodevelopmental delay, epilepsy, marked fatigue and, in one case, retinal degeneration. Histochemical and biochemical evidence of mitochondrial respiratory chain deficiency was observed in the patient muscle biopsies and both patients were discovered to harbour a novel heteroplasmic mitochondrial tRNA (mt-tRNA)(Ser(AGY)) (MTTS2) mutation (m.12264C>T and m.12261T>C, respectively). Clear segregation of the m.12261T>C mutation with the biochemical defect, as demonstrated by single-fibre radioactive RFLP, confirmed the pathogenicity of this novel variant in patient 2. However, unusually high levels of m.12264C>T mutation within both COX-positive (98.4 ± 1.5%) and COX-deficient (98.2 ± 2.1%) fibres in patient 1 necessitated further functional investigations to prove its pathogenicity. Northern blot analysis demonstrated the detrimental effect of the m.12264C>T mutation on mt-tRNA(Ser(AGY)) stability, ultimately resulting in decreased steady-state levels of fully assembled complexes I and IV, as shown by blue-native polyacrylamide gel electrophoresis. Our findings expand the spectrum of pathogenic mutations associated with the MTTS2 gene and highlight MTTS2 mutations as an important cause of retinal and syndromic auditory impairment.

The molecular basis for tissue specificity of the oxidative phosphorylation deficiencies in patients with mutations in the mitochondrial translation factor EFG1.

Defects in mitochondrial translation are associated with a remarkable, but unexplained diversity of clinical phenotypes. Here we have investigated the molecular basis for tissue specificity in patients with a fatal hepatopathy due to mutations in the mitochondrial translation elongation factor EFG1. Blue-native gel electrophoresis revealed unique, tissue-specific patterns in the nature and severity of the defect. Liver was the most severely affected tissue, with less than 10% residual assembly of complexes I and IV, and a 50% decrease in complex V. Skeletal muscle showed a 50% reduction in complex I, and complexes IV and V were 20% of control. In fibroblasts, complexes I and IV were 20% of control, and there was a 40-60% reduction in complexes III and V. In contrast, except for a 50% decrease in complex IV, all complexes were near normal in heart. The severity of the defect paralleled the steady-state level of the mutant EFG1 protein, which varied from 60% of control in heart to undetectable in liver. The ratio of translation elongation factors EFTu:EFTs increased from 1:6 to 1:2 in patient heart, whereas in liver it decreased from 1:1 to 1:4. Over-expression of either EFTu or EFTs in control and patient fibroblasts produced dominant negative effects, indicating that the relative abundance of these factors is an important determinant of translation efficiency. Our results demonstrate marked differences among tissues in the organization of the mitochondrial translation system and its response to dysfunction, and explain the severe hepatopathy, but normal cardiac function in EFG1 patients.

Contrasting phenotypes in three patients with novel mutations in mitochondrial tRNA genes.

We studied three patients, each harboring a novel mutation at a highly conserved position in a different mitochondrial tRNA gene. The mutation in patient 1 (T5543C) was associated with isolated mitochondrial myopathy, and occurred in the anticodon loop of tRNA(Trp). In patient 2, with mitochondrial myopathy and marked retinopathy, the mutation (G14710A) resulted in an anticodon swap (Glu to Lys) in tRNA(Glu). Patient 3, who manifested mitochondrial encephalomyopathy and moderate retinal dysfunction, harbored a mutation (C3287A) in the TpsiC loop of tRNA(Leu(UUR)). The mutations were heteroplasmic in muscle in all cases, and sporadic in two cases. PCR-RFLP analysis in all patients showed much higher amounts of mutated mtDNA in affected tissue (muscle) than unaffected tissue (blood), and significantly higher levels of mutated mtDNA in cytochrome c oxidase (COX)-negative muscle fibers than in COX-positive fibers, confirming the pathogenicity of these mutations. The mutation was also detected in single hair roots from all three patients, indicating that each mutation must have arisen early in embryonic development or in maternal germ cells. This suggests that individual hair root analyses may reflect a wider tissue distribution of mutated mtDNA than is clinically apparent, and might be useful in predicting prognosis and, perhaps, the risk of transmitting the mutation to offspring. Our data suggest a correlation between clinical phenotype and distribution of mutated mtDNA in muscle versus hair roots. Furthermore, the high threshold for phenotypic expression in single muscle fibers (92-96%) suggests that therapies may only need to increase the percentage of wild-type mtDNA by a small amount to be beneficial.

Mutations in COX15 produce a defect in the mitochondrial heme biosynthetic pathway, causing early-onset fatal hypertrophic cardiomyopathy.

Deficiencies in the activity of cytochrome c oxidase (COX), the terminal enzyme in the respiratory chain, are a frequent cause of autosomal recessive mitochondrial disease in infants. These patients are clinically and genetically heterogeneous, and all defects so far identified in this group have been found in genes coding for accessory proteins that play important roles in the assembly of the COX holoenzyme complex. Many patients, however, remain without a molecular diagnosis. We have used a panel of retroviral vectors expressing human COX assembly factors in these patients to identify the molecular basis for the COX deficiency by functional complementation. Here we show that overexpression of COX15, a protein involved in the synthesis of heme A, the heme prosthetic group for COX, can functionally complement the isolated COX deficiency in fibroblasts from a patient with fatal, infantile hypertrophic cardiomyopathy. Mutation analysis of COX15 in the patient identified a missense mutation (C700T) on one allele, changing a conserved arginine to tryptophan (R217W), and a splice-site mutation in intron 3 on the other allele (C447-3G), resulting in a deletion of exon 4. This splicing error introduces a frameshift and a premature stop codon, resulting in an unstable mRNA and, likely, a null allele. Mitochondrial heme A content was reduced in the patient's heart and fibroblast mitochondria, and levels of heme O were increased in the patient's heart. COX activity and the total amount of fully assembled enzyme were reduced by 50%-70% in patient fibroblasts. Expression of COX15 increased heme A content and rescued COX activity. These results suggest that reduced availability of heme A stalls the assembly of COX. This study establishes COX15 as an additional cause, along with SCO2, of fatal infantile, hypertrophic cardiomyopathy associated with isolated COX deficiency.

Mutations in COX10 result in a defect in mitochondrial heme A biosynthesis and account for multiple, early-onset clinical phenotypes associated with isolated COX deficiency.

Deficiencies in the activity of cytochrome c oxidase (COX) are an important cause of autosomal recessive respiratory chain disorders. Patients with isolated COX deficiency are clinically and genetically heterogeneous, and mutations in several different assembly factors have been found to cause specific clinical phenotypes. Two of the most common clinical presentations, Leigh Syndrome and hypertrophic cardiomyopathy, have so far only been associated with mutations in SURF1 or SCO2 and COX15, respectively. Here we show that expression of COX10 from a retroviral vector complements the COX deficiency in a patient with anemia and Leigh Syndrome, and in a patient with anemia, sensorineural deafness and fatal infantile hypertrophic cardiomyopathy. A partial rescue was also obtained following microcell-mediated transfer of mouse chromosomes into patient fibroblasts. COX10 functions in the first step of the mitochondrial heme A biosynthetic pathway, catalyzing the conversion of protoheme (heme B) to heme O via the farnesylation of a vinyl group at position C2. Heme A content was reduced in mitochondria from patient muscle and fibroblasts in proportion to the reduction in COX enzyme activity and the amount of fully assembled enzyme. Mutation analysis of COX10 identified four different missense alleles, predicting amino acid substitutions at evolutionarily conserved residues. A topological model places these residues in regions of the protein shown to have important catalytic functions by mutation analysis of a prokaryotic ortholog. Mutations in COX10 have previously been reported in a single family with tubulopathy and leukodystrophy. This study shows that mutations in this gene can cause nearly the full range of clinical phenotypes associated with early onset isolated COX deficiency.

Identification and characterization of a common set of complex I assembly intermediates in mitochondria from patients with complex I deficiency.

Deficiencies in the activity of complex I (NADH: ubiquinone oxidoreductase) are an important cause of human mitochondrial disease. Complex I is composed of at least 46 structural subunits that are encoded in both nuclear and mitochondrial DNA. Enzyme deficiency can result from either impaired catalytic efficiency or an inability to assemble the holoenzyme complex; however, the assembly process remains poorly understood. We have used two-dimensional Blue-Native/SDS gel electrophoresis and a panel of 11 antibodies directed against structural subunits of the enzyme to investigate complex I assembly in the muscle mitochondria from four patients with complex I deficiency caused by either mitochondrial or nuclear gene defects. Immunoblot analyses of second dimension denaturing gels identified seven distinct complex I subcomplexes in the patients studied, five of which could also be detected in nondenaturing gels in the first dimension. Although the abundance of these intermediates varied among the different patients, a common constellation of subcomplexes was observed in all cases. A similar profile of subcomplexes was present in a human/mouse hybrid fibroblast cell line with a severe complex I deficiency due to an almost complete lack of assembly of the holoenzyme complex. The finding that diverse causes of complex I deficiency produce a similar pattern of complex I subcomplexes suggests that these are intermediates in the assembly of the holoenzyme complex. We propose a possible assembly pathway for the complex, which differs significantly from that proposed for Neurospora, the current model for complex I assembly.