Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ∼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS.

Background A chromosomal locus at 4q32.1 has been genome-wide significantly associated with coronary artery disease risk. The locus encompasses GUCY1A3, which encodes the α1-subunit of the soluble guanylyl cyclase (sGC), a key enzyme in the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway. The mechanism linking common variants in this region with coronary risk is not known. Methods Gene and protein expression were analyzed using quantitative polymerase chain reaction (qPCR) and immunoblotting, respectively. Putative allele-specific transcription factors were identified using in silico analyses and validated via allele-specific quantification of antibody-precipitated chromatin fractions. Regulatory properties of the lead risk variant region were analyzed using reporter gene assays. To assess the effect of ZEB1, siRNA-mediated knockdown as well as overexpression experiments were performed. Association of GUCY1A3 genotype and cellular phenotypes were analyzed using vascular smooth muscle cell (VSMC) migration assays and platelet aggregation analyses. Results Whole blood GUCY1A3 mRNA levels were significantly lower in individuals homozygous for the lead (rs7692387) risk variant. Likewise, reporter gene assays demonstrated significantly lower GUCY1A3 promoter activity for constructs carrying this allele. In silico analyses located a DNase I hypersensitivity site to rs7692387 and predicted binding of the transcription factor ZEB1 rather to the non-risk allele, which was confirmed experimentally. Knockdown of ZEB1 resulted in more profound reduction of non-risk allele promoter activity, as well as a significant reduction of endogenous GUCY1A3 expression. Ex vivo studied platelets from homozygous non-risk allele carriers displayed enhanced inhibition of adenosine diphosphate-induced platelet aggregation by the NO donor sodium nitroprusside and the phosphodiesterase 5 inhibitor sildenafil as compared to homozygous risk allele carriers. Moreover, pharmacologic stimulation of sGC led to reduced migration only in VSMC homozygous for the non-risk allele. In the Hybrid Mouse Diversity Panel higher levels of GUCY1A3 expression correlated with less atherosclerosis in the aorta. Conclusions Rs7692387 is located in an intronic site that modulates GUCY1A3 promoter activity. The transcription factor ZEB1 binds preferentially to the non-risk allele leading to an increase in GUCY1A3 expression, higher sGC levels, and higher sGC activity after stimulation. Finally, human and mouse data link augmented sGC expression to lower risk of atherosclerosis.