Functional characterization of novel mutations in the human cytochrome b gene

Abstract

The great variability of the human mitochondrial DNA (mtDNA) sequence induces many difficulties in the search for its deleterious mutations. We illustrate these pitfalls by the analysis of the cytochrome b gene of 21 patients affected with a mitochondrial disease. Eighteen different sequence variations were found, five of which were new mutations. Extensive analysis of the cytochrome b gene of 146 controls found 20 supplementary mutations, thus further demonstrating the high variability of the cytochrome b sequence. We fully evaluated the functional relevance of 36 of these 38 mutations using indirect criteria such as the nature of the mutation, its frequency in controls, or the phylogenetic conservation of the mutated amino acid. When appropriate, the mtDNA haplotype, the heteroplasmic state of the mutation, its tissue distribution or its familial transmission were also assessed. The molecular consequences of the mutations, which appeared possibly deleterious in that first step of evaluation, were evaluated on the complex III enzymological properties and protein composition using specific antibodies that we have generated against four of its subunits. Two original deleterious mutations were found in the group of seven patients with overt complex III defect. Both mutations (G15150A (W135X) and T15197C (S151P)) were heteroplasmic and restricted to muscle. They had significant consequences on the complex III structure. In contrast, only two homoplasmic missense mutations with dubious clinical relevance were found in the patients without overt complex III defect.

Detection of unrecognized low-level mtDNA heteroplasmy may explain the variable phenotypic expressivity of apparently homoplasmic mtDNA mutations

“…Cytochrome b plays a central role as a catalytic subunit in complex III of the respiratory chain [Fisher and Meunier, 2001]. Several point mutations in human MTCYB have been associated to several diseases, such as Leber hereditary optic neuropathy (LHON), mitochondrial myopathy, isolated complex III deficiency, and mitochondrial encephalopathy, all characterized by impaired complex III activity [Andreu et al, 1999;Legros et al, 2001]. Hearing impairment is the sole clinical symptom of m.15287T4C carriers; none of them presenting evidence of respiratory complex III deficiency.…”

Rapid screening of mtDNA coding region SNPs for the identification of west European Caucasian haplogroups

“…From publications on the phylogenetic history and geographical origin of human populations [16,17,18,19], we chose 16 candidate SNP sites, which in combination are useful for the characterization of a haplogroup and further have a reasonable frequency distribution in the populations with respect to all nine major European haplogroups (H, I, J, K, T, U, V, W, X) ( Table 3). Individuals can be assigned to haplogroups by following the branches of a bifurcating tree (Fig.…”

Human Disease-related Mutations in Cytochrome b Studied in Yeast

“…Several mutations in the hinge region of the ISP have been previously obtained as suppressors of the cytochrome b mutation A144F. 2 The mutation A144F affects the binding of quinol, presumably by altering the local structure of the Q o site. The reported suppressor mutations were likely to correct the positioning of the ISP head group on the mutant cytochrome b.…”

“…Gly-33 is located within transmembrane helix A, a hydrophobic environment at the Q i site and close to heme b h . S152P has also been reported in a patient with exercise intolerance (2). Ser-152 is located in a loop connecting helices cd1 and cd2 at the entry of the Q o binding pocket.…”