Mutations of CASK cause an X-linked brain malformation phenotype with microcephaly and hypoplasia of the brainstem and cerebellum.

CASK is a multi-domain scaffolding protein that interacts with the transcription factor TBR1 and regulates expression of genes involved in cortical development such as RELN. Here we describe a previously unreported X-linked brain malformation syndrome caused by mutations of CASK. All five affected individuals with CASK mutations had congenital or postnatal microcephaly, disproportionate brainstem and cerebellar hypoplasia, and severe mental retardation.

Inhibition of aldehyde dehydrogenase type 2 attenuates vasodilatory action of nitroglycerin in human veins.

Recent studies suggest that the mitochondrial aldehyde dehydrogenase (ALDH)2 is involved in vascular bioactivation of nitroglycerin (GTN). However, neither expression of ALDH2 nor its functional role in GTN bioactivation has been reported for the main drug target in humans, namely capacitance vessels. We investigated whether ALDH2 is expressed in human veins and whether inhibition of the enzyme attenuates nitroglycerin effects in these vessels. We determined expression of ALDH2 and dehydrogenase activity in human veins by reverse transcriptase-polymerase chain reaction, Western blotting, and immunofluorescence microscopy. In vitro contraction experiments were performed in the presence or absence of the ALDH inhibitors chloral hydrate, cyanamide, and ethoxycyclopropanol. Concentration response curves were determined for the alpha-agonist phenylephrine, nitroglycerin, and the direct NO donor diethylamine NONOate (DEA-NONOate). ALDH2 expression was largely confined to smooth muscle cells as determined by confocal immunofluorescence microscopy. Contractile responses to phenylephrine were unaffected by all ALDH inhibitors tested. In clear contrast, the ALDH inhibitors significantly reduced the potency of nitroglycerin by approximately 1 order of magnitude (P < or = 0.01). Neither of the inhibitors affected the potency of the direct NO donor DEA-NONOate, which ruled out nonspecific effects on the NO signaling cascade. In human capacitance vessels, ALDH2 is a key enzyme in the biotransformation of the frequently used antianginal drug nitroglycerin.

HCCS loss-of-function missense mutation in a female with bilateral microphthalmia and sclerocornea: a novel gene for severe ocular malformations?

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.

Mother and daughter with a terminal Xp deletion: implication of chromosomal mosaicism and X-inactivation in the high clinical variability of the microphthalmia with linear skin defects (MLS) syndrome.

The microphthalmia with linear skin defects (MLS or MIDAS) syndrome is a rare X-linked dominant inherited disorder with male lethality, associated with segmental aneuploidy of the Xp22.2 region in most of the cases. However, we recently described heterozygous sequence alterations in a single gene, HCCS, in females with MLS. Beside the classical MLS phenotype, occasional features such as sclerocornea, agenesis of the corpus callosum, and congenital heart defects can occur. Although the majority of cases are sporadic, mother-to-daughter transmission has been observed and a high intra- and interfamilial phenotypic variability exists. We describe an asymptomatic mother and her daughter presenting with the typical features of MLS syndrome. By cytogenetic analysis both females were found to have a terminal Xp deletion with the breakpoint in Xp22.2, mapping near to or within the MSL3L1 gene which is located centromeric to HCCS. FISH analysis revealed that the mother is a mosaic with 45,X(11)/46,X,del(X)(p22.2)(89), while in all cells of the MLS-affected daughter a hybridization pattern consistent with a 46,X,del(X)(p22.2) karyotype was detected. By haplotype analysis we identified the paternal X chromosome of the mother to carry the terminal Xp deletion. X-inactivation studies showed a completely skewed pattern in mother and daughter with the deleted X chromosome to be preferentially inactivated in their peripheral blood cells. We suggest that both chromosomal mosaicism as well as functional X chromosome mosaicism could contribute to the lack of any typical MLS feature in individuals with a heterozygous MLS-associated mutation. The 45,X cell population, that most likely is also present in other tissues of the mother, might have protected her from developing MLS. Nonetheless, a non-random X-inactivation pattern in favor of activity of the wild-type X chromosome in the early blastocyte could also account for the apparent lack of any disease sign in this female.

Mutations of the mitochondrial holocytochrome c-type synthase in X-linked dominant microphthalmia with linear skin defects syndrome.

The microphthalmia with linear skin defects syndrome (MLS, or MIDAS) is an X-linked dominant male-lethal disorder almost invariably associated with segmental monosomy of the Xp22 region. In two female patients, from two families, with MLS and a normal karyotype, we identified heterozygous de novo point mutations--a missense mutation (p.R217C) and a nonsense mutation (p.R197X)--in the HCCS gene. HCCS encodes the mitochondrial holocytochrome c-type synthase that functions as heme lyase by covalently adding the prosthetic heme group to both apocytochrome c and c(1). We investigated a third family, displaying phenotypic variability, in which the mother and two of her daughters carry an 8.6-kb submicroscopic deletion encompassing part of the HCCS gene. Functional analysis demonstrates that both mutant proteins (R217C and Delta 197-268) were unable to complement a Saccharomyces cerevisiae mutant deficient for the HCCS orthologue Cyc3p, in contrast to wild-type HCCS. Moreover, ectopically expressed HCCS wild-type and the R217C mutant protein are targeted to mitochondria in CHO-K1 cells, whereas the C-terminal-truncated Delta 197-268 mutant failed to be sorted to mitochondria. Cytochrome c, the final product of holocytochrome c-type synthase activity, is implicated in both oxidative phosphorylation (OXPHOS) and apoptosis. We hypothesize that the inability of HCCS-deficient cells to undergo cytochrome c-mediated apoptosis may push cell death toward necrosis that gives rise to severe deterioration of the affected tissues. In summary, we suggest that disturbance of both OXPHOS and the balance between apoptosis and necrosis, as well as the X-inactivation pattern, may contribute to the variable phenotype observed in patients with MLS.