PT  - JOURNAL ARTICLE
AU  - Edwin G. Peña-Martínez
AU  - Alejandro Rivera-Madera
AU  - Diego A. Pomales-Matos
AU  - Leandro Sanabria-Alberto
AU  - Brittany M. Rosario-Cañuelas
AU  - Jessica M. Rodríguez-Ríos
AU  - Emmanuel A. Carrasquillo-Dones
AU  - José A. Rodríguez-Martínez
TI  - Disease-Associated Non-Coding Variants Alter NKX2-5 DNA-Binding Affinity
AID  - 10.1101/2022.12.02.518772
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.12.02.518772
4099  - http://biorxiv.org/content/early/2022/12/02/2022.12.02.518772.short
4100  - http://biorxiv.org/content/early/2022/12/02/2022.12.02.518772.full
AB  - 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.Competing Interest StatementThe authors have declared no competing interest.