Identification and Characterization of Genetic Variants of TGFB1 in Patients with Congenital Heart Disease

Background: Congenital heart diseases (CHDs) are believed to be caused by abnormal gene functioning during embryonic heart development. Transforming growth factor-beta1 (TGFB1) is known to express in the early embryonic heart and regulates heart development. Methods: In this study, the coding region of TGFB1 was screened for 238 CHD patients by Sanger sequencing. Case-control association study was performed to identify the risk allele for CHD. In silico and in vitro approaches were used to elucidate the role of rare missense variant of TGFB1 using P19 cell line. Results: We identified a rare missense variant (c.29C > G; p.P10R) in the signal peptide of the TGFB1 in two cases (MAF = 0.0042017), which was absent in 200 healthy controls. Although this variation is reported in the gnomAD (rs1800470, MAF =0.0002386) and the ExAC database (MAF = 0.00064), it is not reported in INDEXdb and GenomeAsia 100K databases. We also found three polymorphisms, namely c.29C > T; p.P10L, c.74G > C; p.R25P and c.788C > T; p.T263I. Case-control studies revealed that c.29C > T (rs1800470) variation is a risk factor, significantly associated with the CHD phenotype (OR = 1.4361, P = 0.0083). However, c.74G > C (rs1800471) and c.788C > T (rs1800472) alleles are not associated with the disease. Additionally, two rare synonymous variations, i.e. c.348C > T; p.T116T (MAF = 0.0042017) and c.501C > T; p.H167H (MAF = 0.00210084) were also identified in two and one cases, respectively. These were absent in the 200 controls. In silico analysis showed that missense variation p.P10R enhances the formation of the alpha-helix in the signal peptide, which possibly increases the TGFB1 secretion. The luciferase-reporter assay demonstrated significantly increased activity of p(SBE)(4) (P = 0.016) and p(CAGA)(12) (P = 0.0004) promoters in response to p.P10R mutant versus wild-type TGFB1. Conclusion: The p.P10R variant of TGFB1 implicated a gain-of-function activity which is potentially deleterious, while the c.29C > T variation is a risk factor associated with the CHD.