Association of IRX4 synonymous variants with congenital heart disease: Leveraging in-silico approaches to predict the functional impact

Synonymous variants are often overlooked during genetic screening, however current reports forecasted their significant biological impact and inevitably considered pathogenic. These silent changes in genome significantly affect the mRNA structure and stability and hence, alter the protein expression and function. IRX4 is an essential transcription factor for cardiogenesis and reported to be associated with congenital heart disease (CHD). In our study, we have performed genetic screening of IRX4 in 205 isolated cases of CHD. Five synonymous variants c.90 A > C , c.240 G > A , c.381 A > G , c.1281 G > A , and c.1509 C > T , six intronic variants c.1-139G>A, c.21-107G>C, c.46-107G>C, c.297+6T>G, c.815-130C>A, c.1638+62C>T were identified. A computed analysis by diverse tools namely RNAfold, MutaRNA, Human Splicing Finder (HSF), and RNA22 was applied to predict the substantial effect on downstream function. RNAfold analysis indicated that all five variants impacted RNA structure and stability. Further, notable changes in the base-pairing probability and RNA accessibility were induced by c.90 A > C , c.240 G > A , c.381 A > G , c.1281 G > A , and c.1509 C > T variants as shown by MutaRNA. Moreover, the effect on the cis-acting regulatory element of splicing was speculated due to c.1281 G > A variant only. Likewise, various modes of the RNA22 tool indicated changes in miRNA binding sites, showing that 61.5% of targets were altered and 38.5% were completely lost as a result of the c.1281 G > A variant. Our findings provide an insight into the molecular effect on mRNA structure and stability, splicing and miRNA target binding sites that potentially impair the transcription and translation and consequently might be associated with the pathogenesis of CHD.