Denise M. Kirby

High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency

Discovering the molecular basis of mitochondrial respiratory chain disease is challenging given the large number of both mitochondrial and nuclear genes that are involved. We report a strategy of focused candidate gene prediction, high-throughput sequencing and experimental validation to uncover the molecular basis of mitochondrial complex I disorders. We created seven pools of DNA from a cohort of 103 cases and 42 healthy controls and then performed deep sequencing of 103 candidate genes to identify 151 rare variants that were predicted to affect protein function. We established genetic diagnoses in 13 of 60 previously unsolved cases using confirmatory experiments, including cDNA complementation to show that mutations in NUBPL and FOXRED1 can cause complex I deficiency. Our study illustrates how large-scale sequencing, coupled with functional prediction and experimental validation, can be used to identify causal mutations in individual cases.

Biochemical assays of respiratory chain complex activity.

Publisher Summary This chapter focuses on the biochemical assays of the respiratory chain (RC) complex activity. It presents the preparation of mitochondrial fractions from tissues and cultured cells for RC enzymology; the measurement of activity of the individual complexes I, II, III, IV, V, the mitochondrial matrix marker enzyme citrate synthase; and the combined activity of complexes II + III. RC enzyme activities are frequently expressed relative to its activity of citrate synthase. Such ratios are more robust than absolute activities because of the variability inherent in cell culture conditions, such as passage number and the degree of confluence, and the proliferation of mitochondria seen in tissues of many patients with mitochondrial disease. The effects of postmortem delay on RC enzymes from liver samples frozen at varying times after death were analyzed to assess the stability of RC enzyme activities postmortem. There can be considerable loss of RC enzyme activity postmortem, particularly in liver, but the observations suggest that muscle collected and frozen at -70°C within 6 h of death and liver within 2 h remain suitable for RC enzyme analysis. The chapter presents the effects of tissue pathology by comparing RC enzymes in tissues from patients without RC complexes I–IV defects with normal controls. The possibility of secondary decreases in enzyme activity and the broadening of reference ranges in the presence of tissue pathology should be considered in interpreting RC enzyme profiles.

Respiratory chain complex I deficiency: an underdiagnosed energy generation disorder.

Objective: To define the spectrum of clinical and biochemical features in 51 children with isolated complex I deficiency. Background: Mitochondrial respiratory chain defects are one of the most commonly diagnosed inborn errors of metabolism. Until recently there have been technical problems with the diagnosis of respiratory chain complex I defects, and there is a lack of information about this underreported cause of respiratory chain dysfunction. Methods: A retrospective review of clinical features and laboratory findings was undertaken in all diagnosed patients who had samples referred over a 22-year period. Results: Presentations were heterogeneous, ranging from severe multisystem disease with neonatal death to isolated myopathy. Classic indicators of respiratory chain disease were not present in 16 of 42 patients in whom blood lactate levels were normal on at least one occasion, and in 23 of 37 patients in whom muscle morphology was normal or nonspecific. Ragged red fibers were present in only five patients. Tissue specificity was observed in 19 of 41 patients in whom multiple tissues were examined, thus the diagnosis may be missed if the affected tissue is not analyzed. Nine patients had only skin fibroblasts available, the diagnosis being based on enzyme assay and functional tests. Modes of inheritance include autosomal recessive (suggested in five consanguineous families), maternal (mitochondrial DNA point mutations in eight patients), and possibly X-linked (slight male predominance of 30:21). Recurrence risk was estimated as 20 to 25%. Conclusion: Heterogeneous clinical features, tissue specificity, and absence of lactic acidosis or abnormal mitochondrial morphology in many patients have resulted in underdiagnosis of respiratory chain complex I deficiency.

Human CIA30 is involved in the early assembly of mitochondrial complex I and mutations in its gene cause disease

In humans, complex I of the respiratory chain is composed of seven mitochondrial DNA (mtDNA)‐encoded and 38 nuclear‐encoded subunits that assemble together in a process that is poorly defined. To date, only two complex I assembly factors have been identified and how each functions is not clear. Here, we show that the human complex I assembly factor CIA30 (complex I intermediate associated protein) associates with newly translated mtDNA‐encoded complex I subunits at early stages in their assembly before dissociating at a later stage. Using antibodies we identified a CIA30‐deficient patient who presented with cardioencephalomyopathy and reduced levels and activity of complex I. Genetic analysis revealed the patient had mutations in both alleles of the NDUFAF1 gene that encodes CIA30. Complex I assembly in patient cells was defective at early stages with subunits being degraded. Complementing the deficiency in patient fibroblasts with normal CIA30 using a novel lentiviral system restored steady‐state complex I levels. Our results indicate that CIA30 is a crucial component in the early assembly of complex I and mutations in its gene can cause mitochondrial disease.

Mutation of C20orf7 Disrupts Complex I Assembly and Causes Lethal Neonatal Mitochondrial Disease

Complex I (NADH:ubiquinone oxidoreductase) is the first and largest multimeric complex of the mitochondrial respiratory chain. Human complex I comprises seven subunits encoded by mitochondrial DNA and 38 nuclear-encoded subunits that are assembled together in a process that is only partially understood. To date, mutations causing complex I deficiency have been described in all 14 core subunits, five supernumerary subunits, and four assembly factors. We describe complex I deficiency caused by mutation of the putative complex I assembly factor C20orf7. A candidate region for a lethal neonatal form of complex I deficiency was identified by homozygosity mapping of an Egyptian family with one affected child and two affected pregnancies predicted by enzyme-based prenatal diagnosis. The region was confirmed by microcell-mediated chromosome transfer, and 11 candidate genes encoding potential mitochondrial proteins were sequenced. A homozygous missense mutation in C20orf7 segregated with disease in the family. We show that C20orf7 is peripherally associated with the matrix face of the mitochondrial inner membrane and that silencing its expression with RNAi decreases complex I activity. C20orf7 patient fibroblasts showed an almost complete absence of complex I holoenzyme and were defective at an early stage of complex I assembly, but in a manner distinct from the assembly defects caused by mutations in the assembly factor NDUFAF1. Our results indicate that C20orf7 is crucial in the assembly of complex I and that mutations in C20orf7 cause mitochondrial disease.

NDUFS6 mutations are a novel cause of lethal neonatal mitochondrial complex I deficiency

complex I deficiency, the most common respiratory chain defect, is genetically heterogeneous: mutations in 8 nuclear and 7 mitochondrial DNA genes encoding complex I subunits have been described. However, these genes account for disease in only a minority of complex I-deficient patients. We investigated whether there may be an unknown common gene by performing functional complementation analysis of cell lines from 10 unrelated patients. Two of the patients were found to have mitochondrial DNA mutations. The other 8 represented 7 different (nuclear) complementation groups, all but 1 of which showed abnormalities of complex I assembly. It is thus unlikely that any one unknown gene accounts for a large proportion of complex I cases. The 2 patients sharing a nuclear complementation group had a similar abnormal complex I assembly profile and were studied further by homozygosity mapping, chromosome transfers, and microarray expression analysis. NDUFS6, a complex I subunit gene not previously associated with complex I deficiency, was grossly underexpressed in the 2 patient cell lines. Both patients had homozygous mutations in this gene, one causing a splicing abnormality and the other a large deletion. This integrated approach to gene identification offers promise for identifying other unknown causes of respiratory chain disorders.

De novo mutations in the mitochondrial ND3 gene as a cause of infantile mitochondrial encephalopathy and complex I deficiency

Both nuclear and mitochondrial DNA mutations can cause energy generation disorders. Respiratory chain complex I deficiency is the most common energy generation disorder and a frequent cause of infantile mitochondrial encephalopathies such as Leighs disease and lethal infantile mitochondrial disease. Most such cases have been assumed to be caused by nuclear gene defects, but recently an increasing number have been shown to be caused by mutations in the mitochondrially encoded complex I subunit genes ND4, ND5, and ND6. We report the first four cases of infantile mitochondrial encephalopathies caused by mutations in the ND3 subunit gene. Three unrelated children have the same novel heteroplasmic mutation (T10158C), only the second mutation reported in ND3, and one has the previously identified T10191C mutation. Both mutations cause disproportionately greater reductions in enzyme activity than in the amount of fully assembled complex I, suggesting the ND3 subunit plays an unknown but important role in electron transport, proton pumping, or ubiquinone binding. Three cases appear to have a de novo mutation, with no mutation detected in maternal relatives. Mitochondrial DNA disease may be considerably more prevalent in the pediatric population than currently predicted and should be considered in patients with infantile mitochondrial encephalopathies and complex I deficiency.

Mutations of the mitochondrial ND1 gene as a cause of MELAS

Complex I is the largest of the mitochondrial respiratory chain enzyme complexes, consisting of at least 46 subunits, seven of which are encoded by mtDNA. Deficiency of complex I is the most common respiratory chain defect, and can be caused by mutations in both nuclear and mtDNA encoded genes. It has a wide range of clinical presentations, from lethal infantile mitochondrial disease to isolated myopathy.1–3 Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) is one of the syndromes associated with complex I deficiency and in approximately 80% of cases is caused by a mutation, 3243A→G, in the mitochondrial tRNALeu(UUR) gene ( MTTL1 ). Other mutations in MTTL1 and other transfer RNA genes ( MTTF , MTTV , MTTQ ) account for most of the remainder of cases.4 However, a number of mutations in the mitochondrial MTND subunit genes of complex I have also been reported to cause MELAS, most notably in MTND5 5 and to a lesser extent in MTND6 .6 In stark contrast, there are presently no mutations in the MTND1 subunit gene associated with MELAS. There are several mutations in MTND1 associated with Leber’s hereditary optic neuropathy (LHON) which may be pathogenic, but only one, the 3460G→A mutation, that has robust evidence, including cell biology studies, for pathogenicity.7–9 Here we report three unrelated patients with MELAS and isolated complex I deficiency in skeletal muscle and cultured fibroblasts due to previously unreported mutations in the MTND1 gene. Evidence confirming the pathogenic nature of these mutations includes data from cell fusion experiments and blue native polyacrylamide gel electrophoresis (BN-PAGE), the latter confirming a crucial role for the ND1 subunit in the assembly of complex I holoenzyme.10 ### Patient 1 Patient 1, a white male, presented at 4 years of age with a 3 month history of increasing tiredness, clumsiness, …

POLG mutations and Alpers syndrome

Alpers–Huttenlocher syndrome (AHS) an autosomal recessive hepatocerebral syndrome of early onset, has been associated with mitochondrial DNA (mtDNA) depletion and mutations in polymerase gamma gene (POLG). We have identified POLG mutations in four patients with hepatocerebral syndrome and mtDNA depletion in liver, who fulfilled criteria for AHS. All were compound heterozygous for the G848S and W748S mutations, previously reported in patients with progressive external ophtalmoplegia or ataxia. We conclude that AHS should be included in the clinical spectrum of mtDNA depletion and is often associated with POLG mutations, which can cause either multiple mtDNA deletions or mtDNA depletion. Ann Neurol 2005;57:921–924

Leigh disease caused by the mitochondrial DNA G14459A mutation in unrelated families

Leigh disease can be caused by defects of both nuclear and mitochondrially encoded genes. One mitochondrial DNA mutation, G14459A, has been associated with both respiratory chain complex I deficiency and Lebers hereditary optic neuropathy, with or without dystonia. Here, we report the occurrence of this mutation in 3 complex I–deficient patients from 2 separate pedigrees who presented with Leigh disease, with no evidence or family history of Lebers hereditary optic neuropathy or dystonia. Ann Neurol 2000;48:102–104