Functional analysis of novel genetic variants of NKX2‐5 associated with nonsyndromic congenital heart disease

Abstract NKX2‐5, a master cardiac regulatory transcription factor was the first known genetic cause of congenital heart diseases (CHDs). To further investigate its role in CHD pathogenesis, we performed mutational screening of 285 CHD probands and 200 healthy controls. Five coding sequence variants were identified in six CHD cases (2.1%), including three in the N‐terminal region (p.A61G, p.R95L, and p.E131K) and one each in homeodomain (HD) (p.A148E) and tyrosine‐rich domain (p.P247A). Variant‐p.A148E showed tertiary structure changes and differential DNA binding affinity of mutant compared to wild type. Two N‐terminal variants—p.A61G and p.E131K along with HD variant p.A148E demonstrated significantly reduced transcriptional activity of Nppa and Actc1 promoters in dual luciferase promoter assay supported by their reduced expression in qRT‐PCR. Nonetheless, variant p.R95L affected the synergy of NKX2‐5 with serum response factor and TBX5 leading to significantly decreased Actc1 promoter activity depicting a distinctive role of this region. The aberrant expression of other target genes— Irx4 , Mef2c , Bmp10 , Myh6 , Myh7 , and Myocd is also observed in response to NKX2‐5 variants, possibly due to the defective gene regulatory network. Severely impaired downstream promoter activities and abnormal expression of target genes due to N‐terminal variants supports the emerging role of this region during cardiac‐developmental pathways.