Functional characterization of QT interval associated SCN5A enhancer variants identify combined additive effects

Area of Science:

• Genetics
• Molecular Biology
• Cardiology

Background:

• Multiple enhancers regulate gene expression, and sequence variations in enhancers contribute to gene expression variability.
• Enhancers and their regulatory variations are often unknown, especially their combined effects on gene expression.

Purpose of the Study:

• To investigate the combined effects of five SCN5A enhancers and their variants on cardiac gene expression.
• To identify transcription factors (TFs) involved in SCN5A enhancer activity and QT interval association.

Main Methods:

• Episomal reporter assays in mouse cardiomyocyte cell lines to assess enhancer activity.
• Oligonucleotide-based pulldown assays to identify transcription factors.
• Reporter assays with synthetic constructs and transient enhancer assays in zebrafish embryos.

Main Results:

• SCN5A variants and flanking sequences are critical for individual enhancer activities, likely involving TF binding sites.
• Specific TFs were identified as driving allele-specific enhancer activities.
• Combined additive effects of five bi-allelic enhancers and their variants were observed.
• Four regulatory elements demonstrated enhancer activity in vivo in zebrafish embryos.

Conclusions:

• Identified TFs driving enhancer activity for SCN5A regulatory variants associated with QT interval.
• Demonstrated additive effects of allelic combinations of SCN5A regulatory variants.
• Confirmed the in vivo enhancer potential of these SCN5A regulatory elements.