Deciphering the functional association of novel variants of BMP7 in isolated congenital heart disease by integrating in vitro and in silico approaches

Abstract Bone morphogenetic protein7 (BMP7), an important member of the TGFβ superfamily, is known to be vital for embryonic growth and development. To decipher the role of BMP7 in congenital cardiac malformations, genetic screening of 285 CHD cases along with 400 healthy controls individuals was performed by Sanger sequencing method. Five missense variants were identified in 5 unrelated CHD probands with distinct phenotypes. Three novel missense (p.D85V, p.R175W, and p.A283T) variants in the pro-peptide region and two other variants (p.M315I and p.N321S) in the mature domain were documented. In vitro functional analysis revealed WT as well as all five mutant BMP7 proteins localized within ER compartment, which was confirmed by ER-Tracker and SERCA2 staining. Western blot analysis demonstrated enhanced phosphorylation of SMAD1/5 associated with these variants. Furthermore, transactivation assays showed increased activity of BMP-responsive promoters-Id1-luc, Id3-luc, Tlx2-luc, and p(SBE)4-luc with a synergistic effect observed upon AKL2 co-expression. Additionally, overexpression of downstream targets, namely Bmp2, Bmp7, Nkx2.5, Gata4, Irx4, Smad1, Smad4, and Smad5, alongside downregulation of left-right patterning genes (Nodal and Pitx2) and the BMP antagonist Chordin, was observed. Likewise, EdU staining revealed increased cellular proliferation associated with all these variants. Moreover, modeling of secondary and tertiary structures also suggested that these variants might induce conformational changes in BMP7, potentially strengthening its binding affinity with the receptor and thereby amplifying SMAD signaling. Collectively, in vitro and in silico analyses suggest that these BMP7 variants exhibit gain-of-function (GoF) activity, disrupting normal BMP signaling pathways and thereby contributing to the development of CHD. Competing Interest Statement The authors have declared no competing interest. Data Availability Raw data and derived data supporting the findings of this study are available from the corresponding author on request.