Mitochondrial complex I deficiency of nuclear origin I. Structural genes

Complex I (or NADH-ubiquinone oxidoreductase), is by far the largest respiratory chain complex with 38 subunits nuclearly encoded and 7 subunits encoded by the mitochondrial genome. Its deficiency is the most frequently encountered in mitochondrial disorders. Here, we summarize recent data obtained on architecture of complex I, and review the pathogenic mutations identified to date in nuclear structural complex I genes. The structural NDUFS1, NDUFS2, NDUFV1, and NDUFS4 genes are mutational hot spot genes for isolated complex I deficiency. The majority of the pathogenic mutations are private and the genotype-phenotype correlation is inconsistent in the rare recurrent mutations.     

Rapid screening for nuclear genes mutations in isolated respiratory chain complex I defects

Complex I or reduced nicotinamide adenine dinucleotide (NADH): ubiquinone oxydoreductase deficiency is the most common cause of respiratory chain defects. Molecular bases of complex I deficiencies are rarely identified because of the dual genetic origin of this multi-enzymatic complex (nuclear DNA and mitochondrial DNA) and the lack of phenotype–genotype correlation. We used a rapid method to screen patients with isolated complex I deficiencies for nuclear genes mutations by Surveyor nuclease digestion of cDNAs. Eight complex I nuclear genes, among the most frequently mutated (NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1 and NDUFV2), were studied in 22 cDNA fragments spanning their coding sequences in 8 patients with a biochemically proved complex I deficiency. Single nucleotide polymorphisms and missense mutations were detected in 18.7% of the cDNA fragments by Surveyor nuclease treatment. Molecular defects were detected in 3 patients. Surveyor nuclease screening is a reliable method for genotyping nuclear complex I deficiencies, easy to interpret, and limits the number of sequence reactions. Its use will enhance the possibility of prenatal diagnosis and help us for a better understanding of complex I molecular defects.     

Mutant NDUFS3 subunit of mitochondrial complex I causes Leigh syndrome

Respiratory chain complex I deficiency represents a genetically heterogeneous group of diseases resulting from mutations in mitochondrial or nuclear genes. Mutations have been reported in 13 of the 14 subunits encoding the core of complex I (seven mitochondrial and six nuclear genes) and these result in Leigh or Leigh-like syndromes or cardiomyopathy. In this study, a combination of denaturing high performance liquid chromatography and sequence analysis was used to study the NDUFS3 gene in a series of complex I deficient patients. Mutations found in this gene (NADH dehydrogenase iron-sulphur protein 3), coding for the seventh and last subunit of complex I core, were shown to cause late onset Leigh syndrome, optic atrophy, and complex I deficiency. A biochemical diagnosis of complex I deficiency on cultured amniocytes from a later pregnancy was confirmed through the identification of disease causing NDUFS3 mutations in these cells. While mutations in the NDUFS3 gene thus result in Leigh syndrome, a dissimilar clinical phenotype is observed in mutations in the NDUFV2 and NDUFS2 genes, resulting in encephalomyopathy and cardiomyopathy. The reasons for these differences are uncertain.     

A novel NDUFA1 mutation leads to a progressive mitochondrial complex I-specific neurodegenerative disease

Mitochondrial diseases have been shown to result from mutations in mitochondrial genes located in either the nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). Mitochondrial OXPHOS complex I has 45 subunits encoded by 38 nuclear and 7 mitochondrial genes. Two male patients in a putative X-linked pedigree exhibiting a progressive neurodegenerative disorder and a severe muscle complex I enzyme defect were analyzed for mutations in the 38 nDNA and seven mtDNA encoded complex I subunits. The nDNA X-linked NDUFA1 gene (MWFE polypeptide) was discovered to harbor a novel missense mutation which changed a highly conserved glycine at position 32 to an arginine, shown to segregate with the disease. When this mutation was introduced into a NDUFA1 null hamster cell line, a substantial decrease in the complex I assembly and activity was observed. When the mtDNA of the patient was analyzed, potentially relevant missense mutations were observed in the complex I genes. Transmitochondrial cybrids containing the patient’s mtDNA resulted in a mild complex I deficiency. Interestingly enough, the nDNA encoded MWFE polypeptide has been shown to interact with various mtDNA encoded complex I subunits. Therefore, we hypothesize that the novel G32R mutation in NDUFA1 is causing complex I deficiency either by itself or in synergy with additional mtDNA variants.     

Investigation of the complex I assembly chaperones B17.2L and NDUFAF1 in a cohort of CI deficient patients

Dysfunction of complex I (NADH:ubiquinone oxidoreductase; CI), the largest enzyme of the oxidative phosphorylation (OXPHOS) system, often results in severe neuromuscular disorders and early childhood death. Mutations in its seven mitochondrial and 38 nuclear DNA-encoded structural components can only partly explain these deficiencies. Recently, CI assembly chaperones NDUFAF1 and B17.2L were linked to CI deficiency, but it is still unclear by which mechanism. To better understand their requirement during assembly we have studied their presence in CI subcomplexes in a cohort of CI deficient patients using one- and two-dimensional blue-native PAGE. This analysis revealed distinct differences between their associations to subcomplexes in different patients. B17.2L occurred in a 830 kDa subcomplex specifically in patients with mutations in subunits NDUFV1 and NDUFS4. Contrasting with this seemingly specific requirement, the previously described NDUFAF1 association to 500-850 kDa intermediates did not appear to be related to the nature and severity of the CI assembly defect. Surprisingly, even in the absence of assembly intermediates in a patient harboring a mutation in translation elongation factor G1 (EFG1), NDUFAF1 remained associated to the 500-850 kDa subcomplexes. These findings illustrate the difference in mechanism between B17.2L and NDUFAF1 and suggest that the involvement of NDUFAF1 in the assembly process could be indirect rather than direct via the binding to assembly intermediates.     

Respiratory chain complex I deficiency caused by mitochondrial DNA mutations

Defects of the mitochondrial respiratory chain are associated with a diverse spectrum of clinical phenotypes, and may be caused by mutations in either the nuclear or the mitochondrial genome (mitochondrial DNA (mtDNA)). Isolated complex I deficiency is the most common enzyme defect in mitochondrial disorders, particularly in children in whom family history is often consistent with sporadic or autosomal recessive inheritance, implicating a nuclear genetic cause. In contrast, although a number of recurrent, pathogenic mtDNA mutations have been described, historically, these have been perceived as rare causes of paediatric complex I deficiency. We reviewed the clinical and genetic findings in a large cohort of 109 paediatric patients with isolated complex I deficiency from 101 families. Pathogenic mtDNA mutations were found in 29 of 101 probands (29%), 21 in MTND subunit genes and 8 in mtDNA tRNA genes. Nuclear gene defects were inferred in 38 of 101 (38%) probands based on cell hybrid studies, mtDNA sequencing or mutation analysis (nuclear gene mutations were identified in 22 probands). Leigh or Leigh-like disease was the most common clinical presentation in both mtDNA and nuclear genetic defects. The median age at onset was higher in mtDNA patients (12 months) than in patients with a nuclear gene defect (3 months). However, considerable overlap existed, with onset varying from 0 to >60 months in both groups. Our findings confirm that pathogenic mtDNA mutations are a significant cause of complex I deficiency in children. In the absence of parental consanguinity, we recommend whole mitochondrial genome sequencing as a key approach to elucidate the underlying molecular genetic abnormality.     

A novel mutation of the NDUFS7 gene leads to activation of a cryptic exon and impaired assembly of mitochondrial complex I in a patient with Leigh syndrome

Complex I deficiency is a frequent cause of mitochondrial disease as it accounts for one third of these disorders. By genotyping several putative disease loci using microsatellite markers we were able to describe a new NDUFS7 mutation in a consanguineous family with Leigh syndrome and isolated complex I deficiency. This mutation lies in the first intron of the NDUFS7 gene (c.17-1167 C>G) and creates a strong donor splice site resulting in the generation of a cryptic exon. This mutation is predicted to result in a shortened mutant protein of 41 instead of 213 amino acids containing only the first five amino acids of the normal protein. Analysis of the assembly state of the respiratory chain complexes under native condition revealed a marked decrease of fully assembled complex I while the quantity of the other complexes was not altered. These results report the first intronic NDUFS7 gene mutation and demonstrate the crucial role of NDUFS7 in the biogenesis of complex I.     

Mutated NDUFS6 is the cause of fatal neonatal lactic acidemia in Caucasus Jews

NADH:ubiquinone oxidoreductase (complex I; EC 1.6.5.3), the largest respiratory chain complex is composed of 45 proteins and is located at the mitochondrial inner membrane. Defects in complex I are associated with energy generation disorders, of which the most severe is congenital lactic acidosis. We report on four infants from two unrelated families of Jewish Caucasus origin with fatal neonatal lactic acidemia due to isolated complex I deficiency. Whole genome homozygosity mapping, identified a 2.6 Mb region of identical haplotype in the affected babies. Sequence analysis of the nuclear gene encoding for the NDUFS6 mitochondrial complex I subunit located within this region identified the c.344G>A homozygous mutation resulting in substitution of a highly evolutionary conserved cysteine residue by tyrosine. This is the second report of NDUFS6 mutation in humans. Both reports describe three diverse homozygous mutations with variable consequential NDUFS6 protein defects that result in similar phenotype. Our study further emphasizes that NDUFS6 sequence should be analyzed in patients presenting with lethal neonatal lactic acidemia due to isolated complex I deficiency.     

Complex I deficiency: clinical features, biochemistry and molecular genetics

Complex I deficiency is the most frequent mitochondrial disorder presenting in childhood, accounting for up to 30% of cases. As with many mitochondrial disorders, complex I deficiency is characterised by marked clinical and genetic heterogeneity, leading to considerable diagnostic challenges for the clinician, not least because of the involvement of two genomes. The most prevalent clinical presentations include Leigh syndrome, leukoencephalopathy and other early-onset neurodegenerative disorders; fatal infantile lactic acidosis; hypertrophic cardiomyopathy; and exercise intolerance. Causative genetic defects may involve the seven mitochondrial-encoded or 38 nuclear-encoded subunits of the enzyme, or any of an increasing number of assembly factors implicated in the correct biosynthesis of complex I within the inner mitochondrial membrane. In this review, we discuss recent advances in knowledge of the structure, function and assembly of complex I and how these advances, together with new high-throughput genetic screening techniques, have translated into improved genetic diagnosis for affected patients and their families. Approximately 25% of cases have mitochondrial DNA mutations, while a further ～25% have mutations in a nuclear subunit or in one of nine known assembly factors. We also present a systematic review of all published cases of nuclear-encoded complex I deficiency, including 117 cases with nuclear subunit mutations and 55 with assembly factor mutations, and highlight clinical, radiological and biochemical clues that may expedite genetic diagnosis.     

Mitochondrial DNA background modulates the assembly kinetics of OXPHOS complexes in a cellular model of mitochondrial disease

Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disorder, is mostly due to three mitochondrial DNA (mtDNA) mutations in respiratory chain complex I subunit genes: 3460/ND1, 11778/ND4 and 14484/ND6. Despite considerable clinical evidences, a genetic modifying role of the mtDNA haplogroup background in the clinical expression of LHON remains experimentally unproven. We investigated the effect of mtDNA haplogroups on the assembly of oxidative phosphorylation (OXPHOS) complexes in transmitochondrial hybrids (cybrids) harboring the three common LHON mutations. The steady-state levels of respiratory chain complexes appeared normal in mutant cybrids. However, an accumulation of low molecular weight subcomplexes suggested a complex I assembly/stability defect, which was further demonstrated by reversibly inhibiting mitochondrial protein translation with doxycycline. Our results showed differentially delayed assembly rates of respiratory chain complexes I, III and IV amongst mutants belonging to different mtDNA haplogroups, revealing that specific mtDNA polymorphisms may modify the pathogenic potential of LHON mutations by affecting the overall assembly kinetics of OXPHOS complexes.     

A study of 133 Chinese children with mitochondrial respiratory chain complex I deficiency

To investigate the clinical, enzymological and mitochondrial gene profiles of complex I deficiency in Chinese, clinical and laboratory data of the patients (79 boys, 54 girls) were retrospectively assessed. Activities of mitochondrial respiratory chain complexes in peripheral leucocytes were spectrophotometrically measured. The entire mitochondrial DNA (mtDNA) sequence was analyzed in 62 patients. Restriction fragment length polymorphism and gene sequencing analyses were performed in 15 families. Ninety‐one patients had isolated complex I deficiency; 42 had combined deficiencies of complex I and other complexes. The main clinical presentations were neuromuscular disorders (107 patients) and non‐neurological dysfunction (hepatopathy, renal damage and cardiomyopathy; 26 patients). In 32 of 62 patients who underwent mtDNA sequencing, 24 mutations were identified in 15 mitochondrial genes. The 12338T>C, 4833A>G and 14502T>C mutations were found in 12.9%, 11.3% and 4.8% patients, respectively. Seven patients had multiple mutations. Three novel mutations were identified. Chinese patients with complex I deficiency presented heterogeneous phenotypes and genotypes. Twenty‐four mutations were identified in 15 mitochondrial genes in 51.6% patients. mtDNA mutations were more common in isolated complex I deficiency than in combined complex deficiencies. The 12338T>C, 4833A>G and 14502T>C mutations were common.     

Isolated complex I deficiency in children: clinical, biochemical and genetic aspects

We retrospectively examined clinical and biochemical characteristics of 27 patients with isolated enzymatic complex I deficiency (established in cultured skin fibroblasts) in whom common pathogenic mtDNA point mutations and major rearrangements were absent. Clinical phenotypes present in this group are Leigh syndrome (n = 7), Leigh-like syndrome (n = 6), fatal infantile lactic acidosis (n = 3), neonatal cardiomyopathy with lactic acidosis (n = 3), macrocephaly with progressive leukodystrophy (n = 2), and a residual group of unspecified encephalomyopathy (n = 6) subdivided into progressive (n = 4) and stable (n = 2) variants. Isolated complex I deficiency is one of the most frequently observed disturbance of the OXPHOS system. Respiratory chain enzyme assays performed in cultured fibroblasts and skeletal muscle tissue in general reveal similar results, but for complete diagnostics we recommend enzyme measurements performed in at least two different tissues to minimize the possibility of overlooking the enzymatic diagnosis. Lactate levels in blood and CSF and cerebral CT/MRI studies are highly informative, although normal findings do not exclude complex I deficiency. With the discovery of mutations in nuclear encoded complex I subunits, adequate pre- and postnatal counseling becomes available. Finally, considering information currently available, isolated complex I deficiency in children seems to be caused in the majority by mutations in nuclear DNA.     

A nonsense mutation in the NDUFS4 gene encoding the 18 kDa (AQDQ) subunit of complex I abolishes assembly and activity of the complex in a patient with Leigh-like syndrome

Sequence analysis of mitochondrial and nuclear candidate genes of complex I in children with deficiency of this complex and exhibiting Leigh-like syndrome has revealed, in one of them, a novel mutation in the NDUFS4 gene encoding the 18 kDa subunit. Phosphorylation of this subunit by cAMP-dependent protein kinase has previously been found to activate the complex. The present mutation consists of a homozygous G-->A transition at nucleotide position +44 of the coding sequence of the gene, resulting in the change of a tryptophan codon to a stop codon. Such mutation causes premature termination of the protein after only 14 amino acids of the putative mitochondrial targeting peptide. Fibroblast cultures from the patient exhibited severe reduction of the rotenone-sensitive NADH-->UQ oxidoreductase activity of complex I, which was insensitive to cAMP stimulation. Two-dimensional electrophoresis showed the absence of detectable normally assembled complex I in the inner mitochondrial membrane. These findings show that the expression of the NDUFS4 gene is essential for the assembly of a functional complex I.     

NDUFA10 mutations cause complex I deficiency in a patient with Leigh disease

Mitochondrial complex I deficiency is the most common defect of the oxidative phosphorylation system. We report a patient with Leigh syndrome who showed a complex I deficiency expressed in cultured fibroblasts and muscle tissue. To find the genetic cause of the complex I deficiency, we screened the mitochondrial DNA and the nuclear-encoded subunits of complex I. We identified compound-heterozygous mutations in the NDUFA10 gene, encoding an accessory subunit of complex I. The first mutation disrupted the start codon and the second mutation resulted in an amino acid substitution. The fibroblasts of the patient displayed decreased amount and activity, and a disturbed assembly of complex I. These results indicate that NDUFA10 is a novel candidate gene to screen for disease-causing mutations in patients with complex I deficiency.     

A homozygous variant in NDUFA8 is associated with developmental delay,microcephaly, and epilepsy due to mitochondrial complex I deficiency

Mitochondrial complex I deficiency is caused by pathogenic variants in mitochondrial and nuclear genes associated with complex I structure and assembly. We report the case of a patient with NDUFA8-related mitochondrial disease. The patient presented with developmental delay, microcephaly, and epilepsy. His fibroblasts showed apparent biochemical defects in mitochondrial complex I. Whole-exome sequencing revealed that the patient carried a homozygous variant in NDUFA8. His fibroblasts showed a reduction in the protein expression level of not only NDUFA8, but also the other complex I subunits, consistent with assembly defects. The enzyme activity of complex I and oxygen consumption rate were restored by reintroducing wild-typeNDUFA8 cDNA into patient fibroblasts. The functional properties of the variant in NDUFA8 were also investigated using NDUFA8 knockout cells expressing wild-type or mutated NDUFA8 cDNA. These experiments further supported the pathogenicity of the variant in complex I assembly. This is the first report describing that the loss of NDUFA8, which has not previously been associated with mitochondrial disease, causes severe defect in the assembly of mitochondrial complex I, leading to progressive neurological and developmental abnormalities.     

Mitochondrial complex I deficiency of nuclear origin II. Non-structural genes

Complex I deficiency is the most frequent cause of respiratory chain diseases. This large multiprotein complex is composed in human of 45 structural subunits, of which 7 are mitochondrial-encoded and 38 are nuclear-encoded. Most of the pathological mutations responsible for complex I deficiencies have been identified to date in complex I structural subunits. Numerous studies from last decade gave some insight into the biogenesis of this huge multi subunit complex of double genetic origin. A sequential incorporation of the structural subunits as well as ten complex I assembly factors has been described. Here, we present a short overview of the human complex I biogenesis and we review the pathological mutations identified to date in eight of the ten known complex I assembly factors.     

Nuclear genes of human complex I of the mitochondrial electron transport chain: state of the art

The mitochondrial electron transport chain (mtETC) consists of four multi-subunit enzyme complexes. Complex I or NADH:ubiquinone oxidoreductase, the largest mtETC multisubunit complex, consists of approximately 41 subunits. Seven of these subunits are encoded by the mitochondrial genome, the remainder by the nuclear genome. Among the mitochondriocytopathies, complex I deficiencies are encountered frequently. Although some complex I deficiencies have been associated with mitochondrial DNA mutations, the genetic defect has not been elucidated in the majority of complex I-deficient patients. It is expected that many of these patients have mutations in the nuclear- encoded subunits of this complex, so vital for cellular energy production. After a brief summary of the current knowledge of complex I from cow, bacteria and fungi, this review presents the state of the art of the knowledge of the human nuclear-encoded complex I genes which, in the last 18 months, has made enormous progress. At present, the complete gene structure of four subunits and the cDNA structure of 18 of the 34 complex I nuclear-encoded subunits are known. Mapping of these subunits shows a random distribution over the chromosomes. The chromosomal localization is known for 14 complex I genes. Recently, the first mutation, a 5 bp duplication in the 18 kDa (AQDQ) subunit, has been reported. We expect that within 1 year all human nuclear-encoded complex I subunits will be cloned. Mutational analysis of these subunits is warranted in complex I-deficient patients and will not only be important for genetic counselling but will also extend the knowledge regarding the functional properties of the individual human complex I subunits.      

Novel non‐neutral mitochondrial DNA mutations found in childhood acute lymphoblastic leukemia

Mitochondria produce adenosine triphosphate (ATP) for energy requirements via the mitochondrial oxidative phosphorylation (OXPHOS) system. One of the hallmarks of cancer is the energy shift toward glycolysis. Low OXPHOS activity and increased glycolysis are associated with aggressive types of cancer. Mitochondria have their own genome (mitochondrial DNA [mtDNA]) encoding for 13 essential subunits of the OXPHOS enzyme complexes. We studied mtDNA in childhood acute lymphoblastic leukemia (ALL) to detect potential pathogenic mutations in OXPHOS complexes. The whole mtDNA from blood and bone marrow samples at diagnosis and follow‐up from 36 ALL patients were analyzed. Novel or previously described pathogenic mtDNA mutations were identified in 8 out of 36 patients. Six out of these 8 patients had died from ALL. Five out of 36 patients had an identified poor prognosis genetic marker, and 4 of these patients had mtDNA mutations. Missense or nonsense mtDNA mutations were detected in the genes encoding subunits of OXPHOS complexes, as follows: MT‐ND1, MT‐ND2, MT‐ND4L and MT‐ND6 of complex I; MT‐CO3 of complex IV; and MT‐ATP6 and MT‐ATP8 of complex V. We discovered mtDNA mutations in childhood ALL supporting the hypothesis that non‐neutral variants in mtDNA affecting the OXPHOS function may be related to leukemic clones.