A genetic investigation of the bone morphogenetic protein signalling pathway in congenital cardiovascular malformation

Abstract

Background Congenital cardiovascular malformations (CVM) affects approximately 7 in every 1000 live births and exhibits familial predisposition but the specific genetic factors involved are unknown. It was hypothsised that rare variants in genes of the bone morphogenetic protein (BMP) signaling pathway, which is known to play a key role in cardiac development, could influence the risk of CVM. Therefore, five genes of the BMP signaling pathway were surveyed for novel variants predisposing to CVM risk. Methods and Results The exonic, splice site and untranslated regions of the BMP2, BMP4, BMPR1A, BMPR2, and SMAD6 genes were sequenced in 90 unrelated Caucasian cases of CVM. One nonsynonymous variant (p.C484F) in the MH2 domain of SMAD6 was identified with predicted impaired protein function. Sequencing of the MH2 domain of SMAD6 in an additional 348 cases showed two further non-synonymous variants, one of which (p.P415L) was also predicted to affect protein function. Both non-synonymous variants were absent in 1000 controls. SMAD6 is an intracellular inhibitor of BMP signaling and functional effects of both variants were investigated using BRE-luciferase transcriptional reporter and alkaline phosphatase assays. Both SMAD6 variants had significantly (p<0.05) lower activity than wild-type SMAD6 in inhibiting BMP signaling in the BRE-luciferase assay. In addition, the p.C484F variant had a significantly (p<0.05) lower capacity to inhibit alkaline phosphatase generation in response to BMP signaling. This was consistent with the phenotype of the patient carrying the p.C484F variant, which featured aortic ossification with coarctation. Conclusions Inadequate inhibition of BMP signaling due to genetic variation in SMAD6 can be a cause of CVM in man. The potential significant SMAD6 variants were identified in approximately 1% of the CVM patients or 6% in patients with coarctation and/or aortic stenosis. These findings show that SMAD6 mutations in the MH2 domain may be important in abnormal cardiac development.