Implication of novel variants of BMP2 in isolated congenital heart disease: Functional characterization by in silico and invitro approaches

Bone morphogenic protein2 (BMP2), a member of TGF-{beta} super-family, known to play a wide range of roles during embryonic development, particularly in the formation of bone/skeleton, differentiation of neurons, skeletal muscle, and development of cardiac valve septa and outflow tract. BMP2 haploinsufficiency is reported to cause multiple congenital malformations including cardiac defects mainly endocardial cushion formation and chamber specification. To investigate the functional relevance of BMP2 variations in isolated CHD cases, we performed genetic screening of BMP2 in 285 CHD probands along with 400 healthy controls by Sangers method. Five non-synonymous variants namely, an already known variant p.Ser37Ala in N-terminal region, one nonsense variant, p.Lys241X in pro-peptide region and three missense variants p.His321Leu, p.Glu328Lys and p.Ser351Cys in mature domain, were identified in 8 unrelated CHD cases. In vitro functional analysis by western blotting depicted an increase in phosphorylation of SMAD1/5 due to all five variants. Furthermore, overexpression of cardiac-specific downstream target genes namely Smad1, Smad4, Smad5, Nkx2.5, Gata4 and Irx4 of the BMP pathway was observed in response to all the variants. Luciferase assay also validated the enhanced expression of multiple downstream promoters Id1-luc, Id3-luc, Tlx2-luc, and p(SBE)4-luc. Additionally, computational analysis of RNA structural features and protein secondary and tertiary structural changes also highlighted the increased activity of mutants, possibly due to enhanced interactions of mutant proteins with their binding partners owing to more stable structures. Overall, this is the first study which characterized the functional association of BMP2 variants with the pathogenesis of CHD by in vitro and in silico methods.