PURPOSE: To analyze if mutations in HCCS, encoding the mitochondrial holocytochrome c-type synthase, are associated with phenotypes other than the microphthalmia with linear skin defects (MLS) syndrome, including severe eye malformations such as microphthalmia and/or anophthalmia. In addition, we investigated the impact of the p.E159K missense mutation on sorting of HCCS to mitochondria and its functional integrity. METHODS: In a cohort of 27 females obtained from a population-based study on infants and fetuses with congenital eye malformations we performed mutation analysis of HCCS by PCR amplification of the coding exons and direct sequencing. The X-inactivation pattern was determined by analyzing the methylation pattern at the AR locus in one patient. For functional analysis of the identified missense mutation, we transfected CHO-K1 cells with wild-type HCCS or HCCS E159K mutant construct and analyzed subcellular localization of the expressed proteins by immunofluorescence analysis and confocal microscopy. Functional integrity of the mutated HCCS protein was investigated by complementation studies in yeast. Therefore, we ectopically expressed HCCS wild type and the E159K mutant in the S. cerevisiae strain B-8025, carrying a deletion of the HCCS ortholog CYC3, and analyzed the capacity of the yeast strain to grow on nonfermentable carbon sources. RESULTS: We detected the heterozygous c.475G>A mutation in exon 5 of HCCS, predicting an amino acid substitution of the highly conserved glutamate at position 159 by lysine, in a female presenting with bilateral microphthalmia and sclerocornea. This point mutation was not found on more than 460 X chromosomes. We identified a skewed X-inactivation in the patient's peripheral blood cells. Similar to HCCS wild type, ectopically expressed HCCS E159K was targeted to mitochondria in CHO-K1 cells. In contrast, expression of HCCS E159K did not complement respiratory growth of the CYC3-deficient yeast strain B-8025, while wild-type HCCS and the yeast heme lyase Cyc3p could rescue growth on nonfermentable carbon sources. CONCLUSIONS: Identification of the novel missense mutation p.E159K of HCCS, which leads to loss-of-function of the encoded holocytochrome c-type synthase, in a sporadic female patient with microphthalmia of both eyes and bilateral sclerocornea may suggest HCCS as candidate for severe ocular manifestations.
