132 Non-synonymous SMAD6 mutations impaired inhibition of bmp signalling in patients with congenital cardiovascular malformation

IntroductionCongenital cardiovascular malformation (CVM) exhibits familial predisposition but the specific genetic factors involved are unknown. Bone morphogenetic proteins (BMPs) regulate many processes during development, including cardiac development. Five genes of the BMP signalling were surveyed for novel variants predisposing to CVM risk. One of the genes, SMAD6, functions as an inhibitory SMAD which preferentially inhibits BMP signalling. The SMAD6 knockout mouse is characterised by cardiac valve and outflow tract defects, including aortic ossification. We hypothesised that rare functional variation in SMAD6 could predispose to congenital cardiovascular malformation (CVM).MethodsThe coding regions of BMP2, BMP4, BMPR1A, BMPR2 and SMAD6 were sequenced in 90 unrelated Caucasian cases of CVM. The MH2 domain of SMAD6 were further sequenced in additional 346 CVM patients. Functional effects of the wild-type and variant SMAD6 proteins were expressed in C2C12 cells and their capacity to inhibit ALK3 activated expression of a BMP-responsive reporter, or to inhibit osteogenic differentiation (using an alkaline phosphatase assay) was assessed.ResultsWe identified two novel non-synonymous variants, P415L and C484F, that were not present in 1000 ethnically-matched controls. P415L was identified in a patient with congenital aortic stenosis and C484F was identified in a patient with coarctation and calcification of the aorta. Both mutations are in evolutionarily conserved amino acid residues and are predicted to be damaging by in silico analysis. This was confirmed in functional assays as both SMAD6 variants failed to inhibit BMP signalling compared with wild-type SMAD6. The P415L mutant appeared to be hypomorphic whereas C484F appeared to be a null allele in the luciferase assay. The C484F mutant had a significantly (p