Genetic and functional studies of the microphthalmia with linear skin defects (MLS) syndrome

MLS (microphthalmia with linear skin defects) syndrome is a rare, X-chromosomal neurocutaneous developmental disorder that usually manifests in females and is associated with male lethality in utero. Besides linear skin defects on face and neck, microphthalmia and sclerocornea other clinical features, such as congenital diaphragmatic hernia (CGH), can be present in affected individuals. The known disease genes HCCS, COX7B and NDUFB11, in which heterozygous mutations have been identified, code for proteins of the mitochondrial respiratory chain. Cells deficient of HCCS, COX7B or NDUFB11 show defects in assembly and activity of the respiratory chain enzymes as well as enhanced cell death. Downregulation of hccs in Medaka causes increased cell death via an apoptosome-independent caspase-9 activation which is triggered by reactive oxygen species (ROS).
In the second funding period, we will focus on work packages to elucidate further the genetic basis and pathomechanism underlying MLS syndrome. By sequencing and copy number analysis of the HCCS gene in patients with CGH, we will answer the question if HCCS mutations cause an isolated form of CGH. To test if detected HCCS variants affect respiratory chain activity, we will use a yeast complementation assay. By whole-exome sequencing (WES) in six patients affected by MLS syndrome, we aim to identify novel disease genes. We will determine pathogenicity of a single sequence variant or characterize consequences of different variants in the same gene by performing functional studies. By WES in a male patient with typical features of MLS syndrome, we identified the p.(A217V) variant in the X-chromosomal gene HDAC6 which encodes the histone deacetylase 6. We will use patient and control fibroblasts to biochemically analyse histone deacetylase 6 activity and protein-protein interactions. The effect of the HDAC6 variant on cell death and respiratory chain activity will be studied using annexin V staining, measurement of ROS levels, analysis of the mitochondrial membrane potential, cytochrome c release from mitochondria, and measurement of caspase activity. Similar experiments will be performed with stable, NDUFB11-deficient HeLa cells. We will generate zebrafish models for cox7b and ndufb11 by morpholino-induced knockdown and by using the CRISPR-Cas9 technology to detect and characterize developmental defects in the fishes. Zebrafish mutants will also be used to study the effect of the genetic defect on cell death, cell proliferation and oxidative phosphorylation.