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

Background Tetralogy of Fallot (TOF) is the most common cyanotic form of congenital heart disease (CHD). The myocyte enhancer factor 2C (MEF2C ) 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 MEF2C 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. Methods Targeted sequencing of the MEF2C 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. Results Five variants (g.24599 G&gt;A, g.24918 T&gt;G, g.25270 T&gt;A (rs904793941), g.25701 C&gt;A and g.25769 T&gt;C (rs886060871)) were identified exclusively in TOF patients. Four of the five variants significantly reduced transcriptional activity at a conservative threshold of p&lt;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. Conclusion This study provides the first functional evidence that MEF2C promoter variants are associated with TOF and affect transcriptional regulation, offering new insights into the potential genetic mechanisms of TOF.