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. 2003 Jan;72(1):101-14.
doi: 10.1086/345489. Epub 2002 Dec 9.

Mutations in COX15 produce a defect in the mitochondrial heme biosynthetic pathway, causing early-onset fatal hypertrophic cardiomyopathy

Hana Antonicka  1 , Andre Mattman, Christopher G Carlson, D Moira Glerum, Kristen C Hoffbuhr, Scot C Leary, Nancy G Kennaway, Eric A Shoubridge
Affiliations

Mutations in COX15 produce a defect in the mitochondrial heme biosynthetic pathway, causing early-onset fatal hypertrophic cardiomyopathy

Hana Antonicka et al. Am J Hum Genet. 2003 Jan.

Abstract

Deficiencies in the activity of cytochrome c oxidase (COX), the terminal enzyme in the respiratory chain, are a frequent cause of autosomal recessive mitochondrial disease in infants. These patients are clinically and genetically heterogeneous, and all defects so far identified in this group have been found in genes coding for accessory proteins that play important roles in the assembly of the COX holoenzyme complex. Many patients, however, remain without a molecular diagnosis. We have used a panel of retroviral vectors expressing human COX assembly factors in these patients to identify the molecular basis for the COX deficiency by functional complementation. Here we show that overexpression of COX15, a protein involved in the synthesis of heme A, the heme prosthetic group for COX, can functionally complement the isolated COX deficiency in fibroblasts from a patient with fatal, infantile hypertrophic cardiomyopathy. Mutation analysis of COX15 in the patient identified a missense mutation (C700T) on one allele, changing a conserved arginine to tryptophan (R217W), and a splice-site mutation in intron 3 on the other allele (C447-3G), resulting in a deletion of exon 4. This splicing error introduces a frameshift and a premature stop codon, resulting in an unstable mRNA and, likely, a null allele. Mitochondrial heme A content was reduced in the patient's heart and fibroblast mitochondria, and levels of heme O were increased in the patient's heart. COX activity and the total amount of fully assembled enzyme were reduced by 50%-70% in patient fibroblasts. Expression of COX15 increased heme A content and rescued COX activity VSports手机版. These results suggest that reduced availability of heme A stalls the assembly of COX. This study establishes COX15 as an additional cause, along with SCO2, of fatal infantile, hypertrophic cardiomyopathy associated with isolated COX deficiency. .

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Figures

Figure 1
Figure 1
Abnormal ultrastructure of cardiac muscle in the patient. Electron micrograph of left ventricular cardiac tissue in the patient demonstrating replacement of myofibils with large numbers of morphologically abnormal mitochondria (×14,000).
Figure 2
Figure 2
Blue-Native PAGE analysis of COX levels in patient heart mitochondria. Heart mitochondria (10 μg protein) from two control subjects (C1 and C2) and the patient (N) were separated by BN-PAGE, and the content of COX, Complex I (Co I), Complex II (Co II), and Complex III (Co III) were determined by immunoblot analysis using antibodies against COX subunits I and IV (COX I and COX IV), the 39-kDa subunit of complex I, the 70-kDa subunit of complex II, and the Core1 protein of complex III. Patient heart mitochondria show a specific reduction in the total amount of fully assembled COX but no evidence of any subcomplexes. The arrow indicates the small amount of unassembled COX IV in the patient. The migration of molecular mass standards is indicated on the left.
Figure 3
Figure 3
Rescue of COX content and activity in patient fibroblasts. Mitoplasts (20 μg protein) isolated from patient (N) and control (C) fibroblasts and patient and control fibroblasts overexpressing COX15 (N+COX15 and C+COX15, respectively) were analyzed by BN-PAGE. A, Western blot analysis using antibodies against COX subunits I and IV and against the 70-kDa subunit of Complex II (Co II) and the Core1 protein of Complex III (Co III). Overexpression of COX15 rescues COX levels in patient mitochondria. B, In-gel activity staining of COX and Complex I demonstrating restoration of COX activity in patient fibroblasts overexpressing COX15. The migration of molecular mass standards is indicated on the left.
Figure 4
Figure 4
Sequence analysis of COX15. DNA sequence analysis of COX15 cDNA (A) and genomic DNA (C) from patient fibroblasts identifies the two pathogenic mutations: a C700T transition mutation in exon 5 and a C447-3G mutation in the splice acceptor of intron 3. The mutations shown are on the sense strand. The C700T mutation was confirmed by RFLP analysis with StyI (B). When the mutation is present, the enzyme digests a 598-bp PCR product once, producing two fragments of 402 and 196 bp. The wild-type PCR product remains uncut. A PCR utilizing a mismatch reverse primer to create a ScrfI site in wild-type DNA was used to confirm the C447-3G mutation (D). ScrfI digests wild-type DNA to yield 162-bp and 30-bp fragments (latter not shown). The intron C447-3G mutation abolishes the ScrfI site. Panels B and D, C = control, N = patient N, U = undigested PCR product.
Figure 5
Figure 5
Genomic structure of COX15, predicted protein sequences of human COX15 variants, and location of patient mutations. A, Genomic structure of COX15. The solid vertical bars represent the 9 exons, and the open boxes represent the 5′ and 3′ untranslated regions of the gene. Horizontal lines represent introns. Two-splice variants of the human gene have been described (COX15.1 and COX15.2) that differ in the 3′ end of the mRNA. The splice-site mutation in patient N results in skipping of exon 4 in the mature transcript. The missense mutation occurs in exon 5 and is indicated with an asterisk. B, The predicted protein sequence of the human COX15.1 and COX15.2 variants are shown in single amino acid code. The size of predicted proteins is shown in parentheses. The C-terminal region in which the COX15.2 species diverges from COX15.1 is indicated in bold italics. The missense mutation (R217W) and the predicted C-terminal end of the truncated protein resulting from the splice-site allele in patient N are shown in bold. Exon 4, which is deleted by the splice-site mutation, is boxed. The predicted (MITOPROT II) canonical mitochondrial targeting sequence is underlined, and the predicted transmembrane domains (ENSEMBL) are indicated with wavy lines. C, The short evolutionarily conserved sequence containing the missense mutation is shown.
Figure 6
Figure 6
Analysis of mitochondrial hemes in heart tissue and fibroblasts. Total hemes were extracted from isolated mitochondria with acidic acetone and separated by reverse phase HPLC. The heme B, heme A, and heme O peaks were identified from the elution times of known standards. The areas under the heme B, heme A, and heme O peaks were quantified and are reported in arbitrary units (in parentheses). Chromatograms are shown from heart mitochondria (A) and from fibroblast mitochondria (B) isolated from cells with and without overexpression of COX15. The insert shows that no heme O was detectable in the extract from patient fibroblasts.
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References

Electronic-Database Information

    1. dbSNP, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=SNP (for the T1171C polymorphism [rs2231687])
    1. GenBank, V体育平台登录 - http://www.ncbi.nlm.nih.gov/Genbank/ (for the COX15.1 variant mRNA sequence [NM_078470] and COX15.2 variant mRNA sequence [NM_004376])
    1. Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for cytochrome c oxidase deficiency [MIM 220110], SCO2 mutations [MIM 602472 and MIM 604377], and COX15 [MIM 603646])

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