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Abstract
Background This study investigated a girl with Leigh syndrome born to first-cousin parents of Pakistani descent with an isolated respiratory chain complex I deficiency in muscle and fibroblasts. Her early development was delayed, and from age 2 years she started losing motor abilities. Cerebral MRI showed basal ganglia lesions typical of Leigh syndrome.
Methods and results A genome-wide search for homozygosity was performed with the Affymetrix GeneChip 50K Xba array. The analysis revealed several homozygous regions. Three candidate genes were identified, and in one of the genes, NDUFA12, a homozygous c.178C→T mutation leading to a premature stop codon (p.Arg60X) was found. Western blot analysis showed absence of NDUFA12 protein in patient fibroblasts and functional complementation by a baculovirus system showed restoration of complex I activity.
Conclusion NDUFA12 mutations are apparently not a frequent cause of complex I deficiency, since mutations were not found by screening altogether 122 complex I deficient patients in two different studies. NDUFA12 encodes an accessory subunit of complex I and is a paralogue of NDUFAF2. Despite the complete absence of NDUFA12 protein, a fully assembled and enzymatically active complex I could be found, albeit in reduced amounts. This suggests that NDUFA12 is required either at a late step in the assembly of complex I, or in the stability of complex I.
- Metabolic disorders
- molecular genetics
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Introduction
Leigh syndrome (MIM 256000) is a progressive, neurodegenerative disorder characterised by focal, bilateral lesions in the central nervous system, including the basal ganglia, thalamus, cerebellum, brainstem, and spinal cord. The onset is early, usually before age 12 months, with loss of motor milestones, hypotonia with poor head control, recurrent vomiting, and a movement disorder. Pyramidal and extrapyramidal signs, nystagmus, breathing disorders, ophthalmoplegia, and peripheral neuropathy are usually seen later. Lifespan is shortened. Often a defect in oxidative phosphorylation (OXPHOS) is found, with deficiency of one or more of the mitochondrial respiratory chain (RC) complexes. The disorder is genetically heterogeneous and can be caused by mutations in mitochondrial DNA (mtDNA), and in nuclear genes involved in the OXPHOS and pyruvate dehydrogenase system. The most frequent deficiency of the RC is in complex I (NADH-ubiquinone oxidoreductase) (MIM 252010).1 Complex I catalyses the first step in the mitochondrial RC, where electrons are transferred from NADH to ubiquinone (coenzyme Q), which is accompanied by the translocation of protons across the inner mitochondrial membrane. This contributes to the proton electrochemical gradient used to synthesise ATP by complex V, the ATP synthase. Human complex I is the largest (∼950 kDa) of the RC complexes, composed of seven subunits encoded by mtDNA and around 39 nuclear encoded subunits.2 Apart from a minimal set of 14 core subunits, present in bacteria, that are required to fulfil the enzymatic function of the enzyme, the mammalian complex I has acquired another 32 subunits, which are called accessory subunits. These accessory subunits are proposed to play a role in the regulation, assembly, stability, and protection of the catalytic core; however, experimental evidence for such functions is still lacking.
Pathogenic mutations in the core subunits have been identified in all seven mtDNA subunit genes, and in seven nuclear subunit genes: NDUFS1 (MIM 157655),3 NDUFS2 (MIM 602985),4 NDUFS3 (MIM 603846),5 NDUFS7 (MIM 601825),6 NDUFS8 (MIM 602141),7 NDUFV1 (MIM 161015),3 and NDUFV2 (MIM 600532).8 In addition, mutations have been identified in genes encoding six accessory subunits, NDUFS4 (MIM 602694),9 NDUFS6 (MIM 603848),10 NDUFA1 (MIM 300078),11 NDUFA11 (MIM 612638),12 NDUFA2 (MIM 602137),13 and NDUFA10 (MIM 603835)14; in eight assembly or putative assembly factors, NDUFAF2 (B17.2L)15 (MIM 609653), NDUFAF1 (CIA30) (MIM 606934),16 C20orf7 (MIM 612360),17 NDUFAF3 (MIM 612911),18 NDUFAF4 (MIM 611776),19 NUBPL (MIM 613621),20 C8orf38 (MIM 612392),21 and ACAD9,22 and in a gene that may link complex I and amino acid metabolism, FOXRED1 (MIM 613622).20
Clinical data
We investigated a 10-year-old girl with complex I deficiency and Leigh syndrome. She was the first child of healthy first-cousin parents of Pakistani descent. A younger brother is healthy. The pregnancy was uncomplicated, and the patient was born at term with a birth weight of 2230 g. The early motor development was delayed, whereas the mental development was normal. She walked with support at age 9–10 months, and could walk unsupported at 20 months. From age 2 years, she started losing motor abilities, especially in the right side of her body, and her balance was affected. She had a scoliosis, and dystonia, which progressed over the following years and was treated with botulinum toxin injections of the affected muscles. She had growth retardation with height and weight around the 3rd centile. At age 10 years, she uses a wheelchair, but can stand on her feet with support. She attends special school and has learned to read and write. Her vision and hearing is normal. She has severe muscular atrophy, hypotonia and severe dystonia. Hypertrichosis is present on the legs and, to a lesser degree, on the arms and back.
Methods and results
Muscle histology showed type 1 fibre atrophy and fibre type disproportion. Cerebral MRI and MRS showed bilateral hyperintense signals in the globus pallidus, and elevated lactate in the entire cerebrum. Routine metabolic workup showed normal urine screening and normal amino acids in cerebrospinal fluid. Plasma lactate was 4.9 mmol/l (reference <2.1 mmol/l) and spinal fluid lactate 2.4 mmol/l (reference 1.1–1.8 mmol/l).
RC enzyme analyses in muscle and fibroblasts were performed essentially as described before.23–25 In both tissues an isolated complex I deficiency was found, with muscle and fibroblast complex I enzymatic activities of 11% and 60% of control values, respectively (figures 1A and 2A).
Muscle respiratory chain (RC) enzyme activity, SDS-PAGE of fibroblast protein, and sequencing analysis of the patient with an NDUFA12 mutation. (A) Enzyme activity of complexes I–IV normalised to citrate synthase in muscle from the patient (black bars) and mean of control values (n=53) (grey bars) ± SD. (B) Sequencing analysis showing the homozygous c.178C→T mutation in NDUFA12 in the patient (top panel), and the wildtype (bottom panel). (C) Immunoblot analysis of fibroblasts with an anti-NDUFA12 antibody showing the complete absence of full length NDUFA12 in patient fibroblasts (P) compared with controls (C). Anti-porin antibody was used as a loading control.
Rescue of the complex I deficiency and expression and incorporation of GFP tagged protein in patient fibroblasts. (A) Complex I and IV enzyme activity related to citrate synthase activity. Cells transduced with COX8 were used as a control and shows the decrease in complex I activity in the patient, which is rescued when transduced with NDUFA12 cDNA. (B) Blue native (BN)-PAGE analysis of the complex I assembly defect in the patient cells transduced with COX8 shows a reduction in the activity of the enzyme as measured by in-gel activity (IGA) assay and in the amount of holoenzyme as measured by immunoblot analysis of the BN gel using an anti-NDUFA9 antibody. Transduction with wild-type NDUFA12 (A12) cDNA rescued the biochemical defect of the patient cells. The 70 kDa subunit of complex II was used as a loading control. (C) Immunoblot of a BN gel showing a much higher incorporation of GFP tagged protein into complex I in the patient compared to control. (D) GFP fluorogram of the same BN gel as in figure 2C.
DNA was extracted from muscle and fibroblasts using standard methods. Mutation analysis of complex I subunit encoding mtDNA genes and all mtDNA tRNA genes revealed no pathogenic mutations. Due to the consanguinity of the parents, a genome-wide search for homozygosity was performed with the Affymetrix GeneChip 50K Xba array (Affymetrix Inc, Santa Clara, California, USA), as described.26 The analysis revealed several homozygous regions (supplemental table S1). The homozygous regions contained three genes that were either known to be involved in complex I biogenesis or candidate genes for complex I biogenesis.21 No pathogenic variants were identified in LACTB or NDUFC1, whereas sequencing analysis of NDUFA12 revealed a homozygous mutation leading to a stop codon, c.178C→T (p.Arg60X) (figure 1B).
To study the effect of the mutation on protein expression, SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gelelectrophoresis) analysis was performed with a mitochondria enriched fraction from cultured fibroblasts followed by immunoblotting with an antibody against NDUFA12 (Proteintech, Chicago, Illinois, USA). The analysis showed a complete absence of NDUFA12 protein in patient fibroblasts (figure 1C).
To confirm the pathogenic role of the p.Arg60X NDUFA12 mutation, fibroblasts were transduced with a baculovirus construct expressing the wild-type NDUFA12 cDNA with wildtype COX8 as a negative control, as previously described.13 The NDUFA12 gene was cloned in frame with a GFP tag, which allowed us to specifically check the expression of the transduced protein. This tag did not interfere with the incorporation of NDUFA12 into complex I. Rescue of the complex I assembly defect was tested by RC enzyme analysis, blue native PAGE (BN-PAGE) and complex I catalytic activity using an in-gel assay.27 Analysis of enzyme activity showed a deficiency of complex I to around 60% of control values in fibroblasts transduced with COX8, whereas transduction with NDUFA12 led to a rescue of the defect (figure 2A). The in-gel activity assay showed a slightly decreased activity of complex I in the patient fibroblasts (71% of the control), with partial restoration of the activity after complementation (83% of the control) (figure 2B). Using an antibody against NDUFA9, we found a decreased amount of fully assembled complex I in patient fibroblasts, which was restored after transduction with NDUFA12.
When studying the expression of the GFP-tagged NDUFA12 transgene, we found that the incorporation into complex I was much higher in the patient compared to control, indicating that the transgene is more required in the patient cells, because the endogenous protein is absent (figure 2C,D). SDS-PAGE western blot incubated with anti-GFP antibody showed that in both the control cells and in the patient all transduced GFP tagged proteins are expressed (supplemental figure S1). Two dimensional BN- and SDS-PAGE immunodetection (supplemental figure S2) of patient fibroblasts showed a similar distribution of peripheral arm (NDUFS3) and membrane arm (NDUFS9) assembly intermediates28 as compared to control fibroblasts.
Discussion
To our knowledge, this is the first report of an NDUFA12 mutation, which was identified in a single patient with Leigh syndrome and an isolated complex I deficiency from a consanguineous family of Pakistani descent. We believe the mutation is pathogenic for the following reasons: (1) the mutation introduces a premature stop codon and leads to complete absence of NDUFA12 protein in patient fibroblasts; (2) patient fibroblasts showed a decrease in complex I amount and activity as revealed by immunoblotting and biochemical and in-gel activity measurements; and (3) transduction with a baculoviral vector containing the wild-type NDUFA12 gene specifically restored both complex I amount and activity.
NDUFA12 mutations are apparently not a frequent cause of complex I deficiency, since no NDUFA12 mutations were found in a screening of 19 patients with isolated complex I deficiency, where NDUFA12 and other genes encoding proteins in the flavoprotein subunit of complex I were screened for mutations.29 In another study, sequencing of 103 candidate genes for complex I deficiency was performed in a group of 103 patients, resulting in the identification of a genetic defect in 43 patients20: mtDNA mutations were found in 25 patients, and 18 patients had mutations in 10 different nuclear genes, but none were identified in NDUFA12.
The catalytic core of complex I comprise 14 evolutionary conserved subunits that are essential for catalysis of electron transfer from NADH to ubiquinone and for generation of the proton gradient. NDUFA12 encodes one of the accessory subunits of complex I, and it is a paralogue of NDUFAF2 (B17.2L), which is also associated with Leigh syndrome or a Leigh-like disorder.15 The function of the accessory subunits is unclear, but it has been suggested that they have general roles, including protection of the complexes against oxidative stress or improvement of their structural stability, but they may also play specific roles in regulation of activity, or in the assembly of complex I.30 So far, all investigated accessory subunits have been shown to be essential for complex I function, and pathogenic mutations have now been reported in genes encoding seven accessory subunits, including the present patient with NDUFA12 mutations. The complex I deficiency is differently expressed in muscle and fibroblast, which is often seen in mitochondrial disease. Analysis of patient fibroblasts shows that absence of NDUFA12 protein (figure 1C) still allows assembly of an almost complete and active complex I. However, the reduction in the amount and activity of complex I in the patient shows that the accessory subunit NDUFA12 is required for proper expression and function of this enzyme. Further research is required to elucidate the precise role of this subunit.
Web resources
The URLs for data presented herein are as follows: Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/
Acknowledgments
We thank the family for their cooperation, and Herman Swarts for production of the baculovirus particles.
References
Footnotes
Funding This work was supported by a grant from the Danish Medical Research Council.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.