Global Evaluation of Congenital Heart Disease-Associated Non-Coding Variants

Pena-Martinez, E. G.; Sharma, S.; Medina-Feliciano, J. G.; Root, E.; Parks, L. G.; Granitto, M.; Pomales-Matos, D. A.; Messon-Bird, J. L.; Barreiro-Rosario, A. C.; Sanabria-Alberto, L.; Rivera-Madera, A.; Rodriguez-Rios, J. M.; Velazquez-Roig, R.; Figueroa-Rosado, J. A.; Noon, M.; Donmez, O. A.; Forney, C.; Hesse, H. K.; Dunn, K. A.; Chen, X.; Hass, M.; Lawson, L.; Weirauch, M. T.; Kottyan, L.; Reilly, S. K.; Bhimsaria, D.; Rodriguez-Martinez, J. A.

doi:10.64898/2025.12.02.691900

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bioRxiv · 10.64898/2025.12.02.691900

Global Evaluation of Congenital Heart Disease-Associated Non-Coding Variants

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Abstract

Abstract (Summary)Genome-wide association studies (GWAS) have mapped thousands of congenital heart disease (CHD)-associated variants within non-coding regions of the genome. Non-coding variants can alter regulatory mechanisms, such as transcription factor (TF) binding control of gene expression, potentially contributing human diseases. However, with the increasing number of disease-associated variants, comprehensive functional validation remains a significant challenge. In this work, we developed a novel method called SNP Bind-n-Seq to evaluate >3,000 CHD-risk variants for allelic binding for the cardiac TFs NKX2-5, GATA4, and TBX5 in a high-throughput manner. These binding affinity data sets were coupled with a massively parallel reporter assay (MPRA) to screen CHD-risk variant genotype-dependent regulatory activity. We identified 170 variants that exhibit allelic TF binding and 187 that modulate gene expression. Combining both approaches revealed three high-confidence variants with genotype-dependent TF binding, genotype-dependent transcriptional activity, and eQTL behavior in cardiac cells. Collectively, this study provides the first combined high-throughput biochemical and functional genomic evaluation of thousands of CHD-risk variants. HighlightsO_LIAllelic binding affinity measurements of [~]9,600 variants for NKX2-5, GATA4, and TBX5 C_LIO_LIEvaluaFon of >3,000 CHD-risk variants for genotype-dependent regulatory acFvity C_LIO_LIInteracFon networks idenFfy funcFonal variants and genes involving cardiac eQTLs C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/691900v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1f2dc96org.highwire.dtl.DTLVardef@1700929org.highwire.dtl.DTLVardef@696ec4org.highwire.dtl.DTLVardef@1e724d8_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Pena-Martinez, E. G., Sharma, S., Medina-Feliciano, J. G., Root, E., Parks, L. G., Granitto, M., Pomales-Matos, D. A., Messon-Bird, J. L., Barreiro-Rosario, A. C., Sanabria-Alberto, L., Rivera-Madera, A., Rodriguez-Rios, J. M., Velazquez-Roig, R., Figueroa-Rosado, J. A., Noon, M., Donmez, O. A., Forney, C., Hesse, H. K., Dunn, K. A., Chen, X., Hass, M., Lawson, L., Weirauch, M. T., Kottyan, L., Reilly, S. K., Bhimsaria, D., Rodriguez-Martinez, J. A.. 2025-12-05. Global Evaluation of Congenital Heart Disease-Associated Non-Coding Variants. https://doi.org/10.64898/2025.12.02.691900

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