Functional Characterization of QT Interval Associated SCN5A Enhancer Variants Identify Combined Additive Effects

Authors

Lavanya Gunamalai
Parul Singh
Brian Berg
Leilei Shi
Ernesto Sanchez
Alexa Smith
Ghislain Breton
Mark T Bedford
Darius Balciunas
Ashish Kapoor

Publication Date

1-9-2025

Journal

Human Genetics and Genomics Advances

Abstract

Several empirical and theoretical studies suggest the presence of multiple enhancers per gene that collectively regulate gene expression, and that common sequence variation impacting on the activities of these enhancers is a major source of inter-individual gene expression variability. However, for the vast majority of genes, enhancers and the underlying regulatory variation remains unknown. Even for the genes with well-characterized enhancers, the nature of the combined effects from multiple enhancers and their variants, when known, on gene expression regulation remains unexplored. Here, we have evaluated the combined effects from five SCN5A enhancers and their regulatory variants that are known to collectively correlate with SCN5A cardiac expression and underlie QT interval association in the general population. Using small deletions centered at the regulatory variants in episomal reporter assays in a mouse cardiomyocyte cell line, we demonstrate that the variants and their flanking sequences play critical role in individual enhancer activities, likely being a transcription factor (TF) binding site. By oligonucleotide-based pulldown assays on predicted TFs, we identify the TFs likely driving allele-specific enhancer activities. Using all 32 possible allelic synthetic constructs in reporter assays, representing the five bi-allelic enhancers, we demonstrate combined additive effects on overall enhancer activities. Using transient enhancer assays in zebrafish embryos we demonstrate that four elements act as enhancers in vivo. Together, these studies uncover the TFs driving the enhancer activities of QT interval associated SCN5A regulatory variants, reveal the additive effects from allelic combinations of these regulatory variants, and prove their potential to act as enhancers in vivo.

Keywords

NAV1.5 Voltage-Gated Sodium Channel, Enhancer Elements, Genetic, Animals, Mice, Humans, Myocytes, Cardiac, Gene Expression Regulation, Long QT Syndrome, Zebrafish, Transcription Factors, Cell Line, Alleles, SCN5A, Enhancer, Variant, QT interval, Additive, Transcription factor

DOI

10.1016/j.xhgg.2024.100358

PMID

39354714

PMCID

PMC11532988

PubMedCentral® Posted Date

9-30-2024

PubMedCentral® Full Text Version

Post-print

Published Open-Access

yes