Functional characterisation and pathological significance of variants of <i>MEF2C</i> promoter in tetralogy of Fallot.

Abstract

Tetralogy of Fallot (TOF) is the most common cyanotic form of congenital heart disease (CHD). The myocyte enhancer factor 2C <i>(MEF2C</i>) transcription factor is a crucial regulator of cardiac development, and variants in the coding region of this gene are a known cause of CHD. However, a significant gap exists in understanding the role of non-coding regulatory variants, specifically those within the <i>MEF2C</i> promoter. This study seeks to fill this gap by investigating whether such promoter variants contribute to TOF pathogenesis and providing functional evidence for their impact. Targeted sequencing of the <i>MEF2C</i> promoter was performed in 305 TOF patients and 306 controls. Identified rare variants were functionally characterised using dual-luciferase reporter assays in AC16 cardiomyocytes. Bioinformatic predictions and electrophoretic mobility shift assays (EMSA) were applied to assess the impact on transcription factor binding. Five variants (g.24599 G>A, g.24918 T>G, g.25270 T>A (rs904793941), g.25701 C>A and g.25769 T>C (rs886060871)) were identified exclusively in TOF patients. Four of the five variants significantly reduced transcriptional activity at a conservative threshold of p<0.01, with reductions ranging from 56.5% to 87.8% of wild-type. Bioinformatic analysis and EMSA confirmed that these variants alter key transcription factor binding sites (eg, for ETS1, GATA3, NFATC2) and disrupt DNA-protein interactions. This study provides the first functional evidence that <i>MEF2C</i> promoter variants are associated with TOF and affect transcriptional regulation, offering new insights into the potential genetic mechanisms of TOF.