A novel variant in COX16 causes cytochrome c oxidase deficiency, severe fatal neonatal lactic acidosis, encephalopathy, cardiomyopathy, and liver dysfunction.

COX16 is involved in the biogenesis of cytochrome-c-oxidase (complex IV), the terminal complex of the mitochondrial respiratory chain. We present the first report of two unrelated patients with the homozygous nonsense variant c.244C>T(p. Arg82*) in COX16 with hypertrophic cardiomyopathy, encephalopathy and severe fatal lactic acidosis, and isolated complex IV deficiency. The absence of COX16 protein expression leads to a complete loss of the holo-complex IV, as detected by Western blot in patient fibroblasts. Lentiviral transduction of patient fibroblasts with wild-type COX16 complementary DNA rescued complex IV biosynthesis. We hypothesize that COX16 could play a role in the copper delivery route of the COX2 module as part of the complex IV assembly. Our data provide clear evidence for the pathogenicity of the COX16 variant as a cause for the observed clinical features and the isolated complex IV deficiency in these two patients and that COX16 deficiency is a cause for mitochondrial disease.

TMEM70 functions in the assembly of complexes I and V.

Protein complexes from the oxidative phosphorylation (OXPHOS) system are assembled with the help of proteins called assembly factors. We here delineate the function of the inner mitochondrial membrane protein TMEM70, in which mutations have been linked to OXPHOS deficiencies, using a combination of BioID, complexome profiling and coevolution analyses. TMEM70 interacts with complex I and V and for both complexes the loss of TMEM70 results in the accumulation of an assembly intermediate followed by a reduction of the next assembly intermediate in the pathway. This indicates that TMEM70 has a role in the stability of membrane-bound subassemblies or in the membrane recruitment of subunits into the forming complex. Independent evidence for a role of TMEM70 in OXPHOS assembly comes from evolutionary analyses. The TMEM70/TMEM186/TMEM223 protein family, of which we show that TMEM186 and TMEM223 are mitochondrial in human as well, only occurs in species with OXPHOS complexes. Our results validate the use of combining complexome profiling with BioID and evolutionary analyses in elucidating congenital defects in protein complex assembly.

NDUFS4 deletion triggers loss of NDUFA12 in Ndufs4-/- mice and Leigh syndrome patients: A stabilizing role for NDUFAF2.

Mutations in NDUFS4, which encodes an accessory subunit of mitochondrial oxidative phosphorylation (OXPHOS) complex I (CI), induce Leigh syndrome (LS). LS is a poorly understood pediatric disorder featuring brain-specific anomalies and early death. To study the LS pathomechanism, we here compared OXPHOS proteomes between various Ndufs4-/- mouse tissues. Ndufs4-/- animals displayed significantly lower CI subunit levels in brain/diaphragm relative to other tissues (liver/heart/kidney/skeletal muscle), whereas other OXPHOS subunit levels were not reduced. Absence of NDUFS4 induced near complete absence of the NDUFA12 accessory subunit, a 50% reduction in other CI subunit levels, and an increase in specific CI assembly factors. Among the latter, NDUFAF2 was most highly increased. Regarding NDUFS4, NDUFA12 and NDUFAF2, identical results were obtained in Ndufs4-/- mouse embryonic fibroblasts (MEFs) and NDUFS4-mutated LS patient cells. Ndufs4-/- MEFs contained active CI in situ but blue-native-PAGE highlighted that NDUFAF2 attached to an inactive CI subcomplex (CI-830) and inactive assemblies of higher MW. In NDUFA12-mutated LS patient cells, NDUFA12 absence did not reduce NDUFS4 levels but triggered NDUFAF2 association to active CI. BN-PAGE revealed no such association in LS patient fibroblasts with mutations in other CI subunit-encoding genes where NDUFAF2 was attached to CI-830 (NDUFS1, NDUFV1 mutation) or not detected (NDUFS7 mutation). Supported by enzymological and CI in silico structural analysis, we conclude that absence of NDUFS4 induces near complete absence of NDUFA12 but not vice versa, and that NDUFAF2 stabilizes active CI in Ndufs4-/- mice and LS patient cells, perhaps in concert with mitochondrial inner membrane lipids.

Bi-allelic Mutations in the Mitochondrial Ribosomal Protein MRPS2 Cause Sensorineural Hearing Loss, Hypoglycemia, and Multiple OXPHOS Complex Deficiencies.

Biogenesis of the mitochondrial oxidative phosphorylation system, which produces the bulk of ATP for almost all eukaryotic cells, depends on the translation of 13 mtDNA-encoded polypeptides by mitochondria-specific ribosomes in the mitochondrial matrix. These mitoribosomes are dual-origin ribonucleoprotein complexes, which contain mtDNA-encoded rRNAs and tRNAs and ∼80 nucleus-encoded proteins. An increasing number of gene mutations that impair mitoribosomal function and result in multiple OXPHOS deficiencies are being linked to human mitochondrial diseases. Using exome sequencing in two unrelated subjects presenting with sensorineural hearing impairment, mild developmental delay, hypoglycemia, and a combined OXPHOS deficiency, we identified mutations in the gene encoding the mitochondrial ribosomal protein S2, which has not previously been implicated in disease. Characterization of subjects' fibroblasts revealed a decrease in the steady-state amounts of mutant MRPS2, and this decrease was shown by complexome profiling to prevent the assembly of the small mitoribosomal subunit. In turn, mitochondrial translation was inhibited, resulting in a combined OXPHOS deficiency detectable in subjects' muscle and liver biopsies as well as in cultured skin fibroblasts. Reintroduction of wild-type MRPS2 restored mitochondrial translation and OXPHOS assembly. The combination of lactic acidemia, hypoglycemia, and sensorineural hearing loss, especially in the presence of a combined OXPHOS deficiency, should raise suspicion for a ribosomal-subunit-related mitochondrial defect, and clinical recognition could allow for a targeted diagnostic approach. The identification of MRPS2 as an additional gene related to mitochondrial disease further expands the genetic and phenotypic spectra of OXPHOS deficiencies caused by impaired mitochondrial translation.

A Heterozygous NDUFV1 Variant Aggravates Mitochondrial Complex I Deficiency in a Family with a Homoplasmic ND1 Variant.

We demonstrate that a heterozygous nuclear variant in the gene encoding mitochondrial complex I subunit NDUFV1 aggravates the cellular phenotype in the presence of a mitochondrial DNA variant in complex I subunit ND1. Our findings suggest that heterozygous variants could be more significant in inherited mitochondrial diseases than hitherto assumed.

NDUFAF4 variants are associated with Leigh syndrome and cause a specific mitochondrial complex I assembly defect.

Mitochondrial respiratory chain complex I consists of 44 different subunits and can be subgrouped into three functional modules: the Q-, the P- and the N-module. NDUFAF4 (C6ORF66) is an assembly factor of complex I that associates with assembly intermediates of the Q-module. Via exome sequencing, we identified a homozygous missense variant in a complex I-deficient patient with Leigh syndrome. Supercomplex analysis in patient fibroblasts revealed specifically altered stoichiometry. Detailed assembly analysis of complex I, indicative of all of its assembly routes, showed an accumulation of parts of the P- and the N-module but not the Q-module. Lentiviral complementation of patient fibroblasts with wild-type NDUFAF4 rescued complex I deficiency and the assembly defect, confirming the causal role of the variant. Our report on the second family affected by an NDUFAF4 variant further characterizes the phenotypic spectrum and sheds light into the role of NDUFAF4 in mitochondrial complex I biogenesis.

Mutation in mitochondrial complex IV subunit COX5A causes pulmonary arterial hypertension, lactic acidemia, and failure to thrive.

COX5A is a nuclear-encoded subunit of mitochondrial respiratory chain complex IV (cytochrome c oxidase). We present patients with a homozygous pathogenic variant in the COX5A gene. Clinical details of two affected siblings suffering from early-onset pulmonary arterial hypertension, lactic acidemia, failure to thrive, and isolated complex IV deficiency are presented. We show that the variant lies within the evolutionarily conserved COX5A/COX4 interface domain, suggesting that it alters the interaction between these two subunits during complex IV biogenesis. In patient skin fibroblasts, the enzymatic activity and protein levels of complex IV and several of its subunits are reduced. Lentiviral complementation rescues complex IV deficiency. The monomeric COX1 assembly intermediate accumulates demonstrating a function of COX5A in complex IV biogenesis. A potential therapeutic lead is demonstrated by showing that copper supplementation leads to partial rescue of complex IV deficiency in patient fibroblasts.

Mutations in mitochondrial complex I assembly factor NDUFAF3 cause Leigh syndrome.

NDUFAF3 is an assembly factor of mitochondrial respiratory chain complex I. Variants in NDUFAF3 have been identified as a cause of severe multisystem mitochondrial disease. In a patient presenting with Leigh syndrome, which has hitherto not been described as a clinical feature of NDUFAF3 deficiency, we identified a novel homozygous variant and confirmed its pathogenicity in patient fibroblasts studies. Furthermore, we present an analysis of complex I assembly routes representative of each functional module and, thereby, link NDUFAF3 to a specific step in complex I assembly. Therefore, our report expands the phenotype of NDUFAF3 deficiency and further characterizes the role of NDUFAF3 in complex I biogenesis.

Mutations in Complex I Assembly Factor TMEM126B Result in Muscle Weakness and Isolated Complex I Deficiency.

Mitochondrial complex I deficiency results in a plethora of often severe clinical phenotypes manifesting in early childhood. Here, we report on three complex-I-deficient adult subjects with relatively mild clinical symptoms, including isolated, progressive exercise-induced myalgia and exercise intolerance but with normal later development. Exome sequencing and targeted exome sequencing revealed compound-heterozygous mutations in TMEM126B, encoding a complex I assembly factor. Further biochemical analysis of subject fibroblasts revealed a severe complex I deficiency caused by defective assembly. Lentiviral complementation with the wild-type cDNA restored the complex I deficiency, demonstrating the pathogenic nature of these mutations. Further complexome analysis of one subject indicated that the complex I assembly defect occurred during assembly of its membrane module. Our results show that TMEM126B defects can lead to complex I deficiencies and, interestingly, that symptoms can occur only after exercise.

Mutations in COA6 cause cytochrome c oxidase deficiency and neonatal hypertrophic cardiomyopathy.

COA6/C1ORF31 is involved in cytochrome c oxidase (complex IV) biogenesis. We present a new pathogenic COA6 variant detected in a patient with neonatal hypertrophic cardiomyopathy and isolated complex IV deficiency. For the first time, clinical details about a COA6-deficient patient are given and patient fibroblasts are functionally characterized: COA6 protein is undetectable and steady-state levels of complex IV and several of its subunits are reduced. The monomeric COX1 assembly intermediate accumulates. Using pulse-chase experiments, we demonstrate an increased turnover of mitochondrial encoded complex IV subunits. Although monomeric complex IV is decreased in patient fibroblasts, the CI/CIII2 /CIVn -supercomplexes remain unaffected. Copper supplementation shows a partial rescue of complex IV deficiency in patient fibroblasts. We conclude that COA6 is required for complex IV subunit stability. Furthermore, the proposed role in the copper delivery pathway to complex IV subunits is substantiated and a therapeutic lead for COA6-deficient patients is provided.

A mutation in the human CBP4 ortholog UQCC3 impairs complex III assembly, activity and cytochrome b stability.

Complex III (cytochrome bc1) is a protein complex of the mitochondrial inner membrane that transfers electrons from ubiquinol to cytochrome c. Its assembly requires the coordinated expression of mitochondrial-encoded cytochrome b and nuclear-encoded subunits and assembly factors. Complex III deficiency is a severe multisystem disorder caused by mutations in subunit genes or assembly factors. Sequence-profile-based orthology predicts C11orf83, hereafter named UQCC3, to be the ortholog of the fungal complex III assembly factor CBP4. We describe a homozygous c.59T>A missense mutation in UQCC3 from a consanguineous patient diagnosed with isolated complex III deficiency, displaying lactic acidosis, hypoglycemia, hypotonia and delayed development without dysmorphic features. Patient fibroblasts have reduced complex III activity and lower levels of the holocomplex and its subunits than controls. They have no detectable UQCC3 protein and have lower levels of cytochrome b protein. Furthermore, in patient cells, cytochrome b is absent from a high-molecular-weight complex III. UQCC3 is reduced in cells depleted for the complex III assembly factors UQCC1 and UQCC2. Conversely, absence of UQCC3 in patient cells does not affect UQCC1 and UQCC2. This suggests that UQCC3 functions in the complex III assembly pathway downstream of UQCC1 and UQCC2 and is consistent with what is known about the function of Cbp4 and of the fungal orthologs of UQCC1 and UQCC2, Cbp3 and Cbp6. We conclude that UQCC3 functions in complex III assembly and that the c.59T>A mutation has a causal role in complex III deficiency.

A Patient with Complex I Deficiency Caused by a Novel ACAD9 Mutation Not Responding to Riboflavin Treatment.

Here we report a patient with a new pathogenic mutation in ACAD9. Shortly after birth she presented with respiratory insufficiency and a high lactate level. At age 7 weeks, she was diagnosed with severe hypertrophic cardiomyopathy and she suffered from muscle weakness and hypotonia. Her condition deteriorated during intercurrent illnesses and she died at 6 months of age in cardiogenic shock. Analysis of respiratory chain activities in muscle and fibroblasts revealed an isolated complex I deficiency. A genome-wide screen for homozygosity revealed several homozygous regions. Four candidate genes were found and sequencing revealed a homozygous missense mutation in ACAD9. The mutation results in an Ala220Val amino acid substitution located near the catalytic core of ACAD9. SDS and BN-PAGE analysis showed severely decreased ACAD9 and complex I protein levels, and lentiviral complementation of patient fibroblasts partially rescued the complex I deficiency. Riboflavin supplementation did not ameliorate the complex I deficiency in patient fibroblasts. More than a dozen ACAD9 patients with complex I deficiency have been identified in the last 3 years, indicating that ACAD9 is important for complex I assembly, and that ACAD9 mutations are a relatively frequent cause of complex I deficiency.

Mutations in the UQCC1-interacting protein, UQCC2, cause human complex III deficiency associated with perturbed cytochrome b protein expression.

Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis.

A mutation in the FAM36A gene, the human ortholog of COX20, impairs cytochrome c oxidase assembly and is associated with ataxia and muscle hypotonia.

The mitochondrial respiratory chain complex IV (cytochrome c oxidase) is a multi-subunit enzyme that transfers electrons from cytochrome c to molecular oxygen, yielding water. Its biogenesis requires concerted expression of mitochondria- and nuclear-encoded subunits and assembly factors. In this report, we describe a homozygous missense mutation in FAM36A from a patient who displays ataxia and muscle hypotonia. The FAM36A gene is a remote, putative ortholog of the fungal complex IV assembly factor COX20. Messenger RNA (mRNA) and protein co-expression analyses support the involvement of FAM36A in complex IV function in mammals. The c.154A>C mutation in the FAM36A gene, a mutation that is absent in sequenced exomes, leads to a reduced activity and lower levels of complex IV and its protein subunits. The FAM36A protein is nearly absent in patient's fibroblasts. Cells affected by the mutation accumulate subassemblies of complex IV that contain COX1 but are almost devoid of COX2 protein. We observe co-purification of FAM36A and COX2 proteins, supporting that the FAM36A defect hampers the early step of complex IV assembly at the incorporation of the COX2 subunit. Lentiviral complementation of patient's fibroblasts with wild-type FAM36A increases the complex IV activity as well as the amount of holocomplex IV and of individual subunits. These results establish the function of the human gene FAM36A/COX20 in complex IV assembly and support a causal role of the gene in complex IV deficiency.

Identification and functional analysis of mitochondrial complex I assembly factor homologues in C. elegans.

The biogenesis of mitochondrial NADH:ubiquinone oxidoreductase (complex I) requires several assembly chaperones. These so-called complex I assembly factors have emerged as a new class of human disease genes. Here, we identified putative assembly factor homologues in Caenorhabditis elegans. We demonstrate that two candidates (C50B8.3/NUAF-1, homologue of NDUFAF1 and R07H5.3/NUAF-3, homologue of NDUFAF3) clearly affect complex I function. Assembly factor deficient worms were shorter, showed a diminished brood size and displayed reduced fat content. Our results suggest that mitochondrial complex I biogenesis is evolutionarily conserved. Moreover, Caenorhabditis elegans appears to be a promising model organism to study assembly factor related human diseases.

Iterative orthology prediction uncovers new mitochondrial proteins and identifies C12orf62 as the human ortholog of COX14, a protein involved in the assembly of cytochrome c oxidase.

BACKGROUND: Orthology is a central tenet of comparative genomics and ortholog identification is instrumental to protein function prediction. Major advances have been made to determine orthology relations among a set of homologous proteins. However, they depend on the comparison of individual sequences and do not take into account divergent orthologs. RESULTS: We have developed an iterative orthology prediction method, Ortho-Profile, that uses reciprocal best hits at the level of sequence profiles to infer orthology. It increases ortholog detection by 20% compared to sequence-to-sequence comparisons. Ortho-Profile predicts 598 human orthologs of mitochondrial proteins from Saccharomyces cerevisiae and Schizosaccharomyces pombe with 94% accuracy. Of these, 181 were not known to localize to mitochondria in mammals. Among the predictions of the Ortho-Profile method are 11 human cytochrome c oxidase (COX) assembly proteins that are implicated in mitochondrial function and disease. Their co-expression patterns, experimentally verified subcellular localization, and co-purification with human COX-associated proteins support these predictions. For the human gene C12orf62, the ortholog of S. cerevisiae COX14, we specifically confirm its role in negative regulation of the translation of cytochrome c oxidase. CONCLUSIONS: Divergent homologs can often only be detected by comparing sequence profiles and profile-based hidden Markov models. The Ortho-Profile method takes advantage of these techniques in the quest for orthologs.

C7orf30 specifically associates with the large subunit of the mitochondrial ribosome and is involved in translation.

In a comparative genomics study for mitochondrial ribosome-associated proteins, we identified C7orf30, the human homolog of the plant protein iojap. Gene order conservation among bacteria and the observation that iojap orthologs cannot be transferred between bacterial species predict this protein to be associated with the mitochondrial ribosome. Here, we show colocalization of C7orf30 with the large subunit of the mitochondrial ribosome using isokinetic sucrose gradient and 2D Blue Native polyacrylamide gel electrophoresis (BN-PAGE) analysis. We co-purified C7orf30 with proteins of the large subunit, and not with proteins of the small subunit, supporting interaction that is specific to the large mitoribosomal complex. Consistent with this physical association, a mitochondrial translation assay reveals negative effects of C7orf30 siRNA knock-down on mitochondrial gene expression. Based on our data we propose that C7orf30 is involved in ribosomal large subunit function. Sequencing the gene in 35 patients with impaired mitochondrial translation did not reveal disease-causing mutations in C7orf30.

A catalytic defect in mitochondrial respiratory chain complex I due to a mutation in NDUFS2 in a patient with Leigh syndrome.

In this study, we investigated the pathogenicity of a homozygous Asp446Asn mutation in the NDUFS2 gene of a patient with a mitochondrial respiratory chain complex I deficiency. The clinical, biochemical, and genetic features of the NDUFS2 patient were compared with those of 4 patients with previously identified NDUFS2 mutations. All 5 patients presented with Leigh syndrome. In addition, 3 out of 5 showed hypertrophic cardiomyopathy. Complex I amounts in the patient carrying the Asp446Asn mutation were normal, while the complex I activity was strongly reduced, showing that the NDUFS2 mutation affects complex I enzymatic function. By contrast, the 4 other NDUFS2 patients showed both a reduced amount and activity of complex I. The enzymatic defect in fibroblasts of the patient carrying the Asp446Asn mutation was rescued by transduction of wild type NDUFS2. A 3-D model of the catalytic core of complex I showed that the mutated amino acid residue resides near the coenzyme Q binding pocket. However, the K(M) of complex I for coenzyme Q analogs of the Asp446Asn mutated complex I was similar to the K(M) observed in other complex I defects and in controls. We propose that the mutation interferes with the reduction of coenzyme Q or with the coupling of coenzyme Q reduction with the conformational changes involved in proton pumping of complex I.

Mitochondrial complex III stabilizes complex I in the absence of NDUFS4 to provide partial activity.

Mitochondrial complex I (CI) is a multi-subunit enzyme that forms the major entry point of nicotinamide adenine dinucleotide (NADH) electrons into the respiratory chain. Mutations in the NDUFS4 gene, encoding an accessory subunit of this complex, cause a Leigh-like phenotype in humans. To study the nature and penetrance of the CI defect in different tissues, we investigated the role of NDUFS4 in mice with fatal mitochondrial encephalomyopathy, caused by a systemic inactivation of the Ndufs4 gene. We report that the absence of NDUFS4 in different mouse tissues results in decreased activity and stability of CI. This CI instability leads to an increased disconnection of electron influx of the NADH dehydrogenase module from the holo-complex. However, the formation of respiratory supercomplexes still allows formation of active CI in these Ndufs4 knock-out mice. These results reveal the importance of these supramolecular interactions not only for stabilization but also for the assembly of CI, which becomes especially relevant in pathological conditions.

New mitochondrial tRNA HIS mutation in a family with lactic acidosis and stroke-like episodes (MELAS).

We report a new mutation in m.12146 A>G in the mt-tRNA(His) in a family with a remarkable clinical history having different degrees of lactic acidosis and stroke-like episodes. Biochemical measurements of a muscle biopsy established an isolated complex IV deficiency, while similar analysis of fibroblasts showed a combined complex I,III and IV deficiency. Transmitochondrial cybrid analysis proved that this tRNA(His) mutation causes the enzymatic deficiency. This family illustrates the complexity of the clinical, biochemical and genetic characteristics of a novel mtDNA encoded disorder, as well as the challenge to prove its pathogenicity.