Disease-Associated Non-Coding Variants Alter NKX2-5 DNA-Binding Affinity

Abstract

1.Genome-wide association studies (GWAS) have mapped over 90% of disease- or trait-associated variants within the non-coding genome, like cis-regulatory elements (CREs). Non-coding single nucleotide polymorphisms (SNPs) are genomic variants that can change how DNA-binding regulatory proteins, like transcription factors (TFs), interact with the genome and regulate gene expression. NKX2-5 is a TF essential for proper heart development, and mutations affecting its function have been associated with congenital heart diseases (CHDs). However, establishing a causal mechanism between non-coding genomic variants and human disease remains challenging. To address this challenge, we identified 8,475 SNPs predicted to alter NKX2-5 DNA- binding using a position weight matrix (PWM)-based predictive model. Five variants were prioritized for in vitro validation; four of them are associated with traits and diseases that impact cardiovascular health. The impact of these variants on NKX2-5 binding was evaluated with electrophoretic mobility shift assay (EMSA) using recombinantly expressed and purified human NKX2-5 homeodomain. Binding curves were constructed to determine changes in binding between variant and reference alleles. Variants rs7350789, rs7719885, rs747334, and rs3892630 increased binding affinity, whereas rs61216514 decreased binding by NKX2-5 when compared to the reference genome. Our findings suggest that differential TF-DNA binding affinity can be key in establishing a causal mechanism of pathogenic variants. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/518772v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@c5091dorg.highwire.dtl.DTLVardef@1d96d0forg.highwire.dtl.DTLVardef@18723eborg.highwire.dtl.DTLVardef@147517a_HPS_FORMAT_FIGEXP M_FIG C_FIG