Severe infantile encephalomyopathy caused by a mutation in COX6B1, a nucleus-encoded subunit of cytochrome c oxidase

Abstract

Cytochrome c oxidase (COX) deficiency, one of the most common respiratory-chain defects in humans, has been associated with mutations in either mitochondrial DNA genes or nucleus-encoded proteins that are not part in but promote the biogenesis of COX. Mutations of nucleus-encoded structural subunits were sought for but never found in COX-defective patients, leading to the conjecture that they may be incompatible with extra-uterine survival. We report a disease-associated mutation in one such subunit, COX6B1. Nuclear-encoded COX genes should be reconsidered and included in the diagnostic mutational screening of human disorders related to COX deficiency.

Figure 1

Brain MRI Panel (A) Patient AK at 9 years of age. Bilateral, symmetrical signal abnormalities affect the frontal, parietal, and occipital white matter, as well as the splenium and the genu of the corpus callosum. Numerous cystic lesions are also present, particularly in the anterior periventricular regions, with ex vacuo dilation of the lateral ventricles. (B) Patient AW at 6 years of age presented symmetrical white matter hyperintensity in the periventricular region, with cystic vacuolation. The splenium of the corpus callosum is also involved.

Figure 2

Genetic, Clinical, and Biochemical Analysis (A) Family pedigree and haplotype analysis of the COX6B1 locus. Patients (#15, patient AK; #16, patient AW) are indicated by black symbols. (B) COX histoenzymatic staining of muscle biopsy. The reaction is diffusely low in the muscle from patient AW, compared with an age-matched control (inset). (C) Electropherogram of the c.221G→A mutation in exon 2 of the COX6B1 gene. The alignment of the protein sequence in different species is shown below. The numeration of the amino acid residues corresponds to the mature COX6B1 as the first M is cleaved in the import process of the precursor into mitochondria. The R19 residue and the conserved RFP motif are shown in red and yellow, respectively. (D) Modeling of the bovine COX dimer. The COX6B1 and MT-CO2 subunits are in red/blue and yellow, respectively. The mutant R19 residue and the adjacent D17 and D35 residues are also indicated. Relevant nitrogen atoms in R19 and oxygen atoms in D17 and D35 are shown in blue and red, respectively (see main text for details).

Figure 3

RNA Interference Assays in HeLa Cells (A) Quantification by RT-PCR of the COX6B1 mRNA levels in the HeLa cells stable expression of the shRNAs corresponding to the sequences number 1 and number 14 with respect to the mock-transfected cells (pSuper.puro) and the nontransfected HeLa cells. (B) COX/CS activity measured in the HeLa cells stable expression of the shRNAs corresponding to sequences number 1 and number 14 with respect to the mock-transfected cells (pSuper.puro) and the nontransfected HeLa cells. The plotted values are expressed as the percentage of the mean value obtained for the nontransfected HeLa cells ± SD (n = 3). (C) Western-blot analysis to determine the protein levels of the COX6B1 subunit and other COX subunits. The antibody against the SDH 30 kDa subunit (complex II) was used for normalization purposes.

Figure 4

Western-Blot Analysis of COX Subunits (A) Muscle homogenates. An antibody against the SDH 30 kDa subunit (complex II) was used for normalization. Assembled complex IV holocomplex and individual COX subunits are both decreased. (B) Mitochondrial fractions from immortalized fibroblasts (see text for details). Arrows 1 and 2 indicate the band doublet corresponding to assembled COX species. S3 corresponds to a COX assembly intermediate.

Figure 5

Complementation Studies (A) Growth of S. cerevisiae recombinant ΔCOX12 strains on 2% ethanol-YNB medium transfected with COX6B1^R20H cDNA, COX6B1^wild-type cDNA, and the empty pYEX expression vector. (B) Cytochemical reaction to COX in fibroblast cell lines. (C) COX/CS activities, expressed as percentage of the mean control value ± SD, in immortalized fibroblasts and recombinant derivatives, transfected as indicated (A.K. & A.W. pBabe + pcDNA3: n = 13; A.K. & A.W. pBabe + COX6B1^wild-type: n = 15; A.K. & A.W. pBabe + COX6B1^R19H: n = 6). (D) BNGE-WB analysis of COX in transfected cells. Gel gradient was 7%–10% for better separation of the COX holocomplex doublet (CIV 1 and CIV 2).