Van der Woude syndrome (VWS) is the most common syndromic form of orofacial clefts (OFCs). The heritability of VWS is largely (~70%) explained by variants in the IRF6 gene. Experimental studies to confirm genetic variant effects are essential but are time intensive and expensive. Advancing next-generation sequencing techniques has made it possible to identify genetic variation quicker than their causal effects in disease processes can be ascertained. Therefore, employing computational tools to reliably predict their causal effects is crucial for advancing translational research. We conducted Sanger sequencing of the IRF6 gene in 46 VWS cases across 34 families to identify likely causal missense variants. We then assessed structure-informed stability changes to the IRF6 protein when these variants were incorporated into the protein, using DDGun3D. We compared DDGun3D predictions to results from previous experimental studies. Among four variants previously shown to prevent functional rescue in zebrafish, only the variant IRF6(NM_006147.4):c.881T>C, shown to cause rupture of zebrafish embryo, exhibited a high free energy change (∆∆G_fold = 3.5 kcal/mol), indicating that substantial predicted thermodynamic destabilization does not capture all disruptive function of IRF6. Our study demonstrates the utility of a protein structure-based computational approach for functional variant interpretation, providing a complementary method for studying genetic variation. Integrating computational predictions with experimental data can enhance our understanding of genetic conditions. However, success depends on fully elucidating protein structures, which is an active area of research.

Monozygotic (MZ) twins are expected to share nearly identical genomes, yet a substantial proportion is discordant for complex diseases like nonsyndromic orofacial clefts (nsOFC). This suggests a role for postzygotic discordant genetic variations and other developmental mechanisms. In this study, we performed whole genome sequencing of two MZ twin pairs of African ancestry who were discordant for nsOFC to identify genetic variants that may contribute to phenotypic discordance. Following quality control and genotype‐level filtering, we identified 37,271 variants that were present exclusively in the affected twins. Of these, 431 variants were predicted by CADD to have deleterious effects. Among these, 66 were in protein‐coding regions, including 2 predicted protein‐altering missense variants and splice‐site variants. The remaining 365 variants were in noncoding regions including putative craniofacial enhancers. Some of these variants are rare (MAF < 0.01) and were predicted to alter transcription factor binding or disrupt local chromatin architecture within putative craniofacial enhancers located near genes with established roles in craniofacial development, including COL27A1 , MPHOSPH8 , and SPTBN1 . In addition, some common variants previously associated with noncraniofacial traits were also predicted to affect regulatory activity of putative craniofacial enhancers near craniofacial genes. This raises the possibility of pleiotropic regulatory effects that warrant further investigation. Overall, this study identified a set of candidate coding and regulatory variants in MZ twins discordant for nsOFC,providing targets for future functional investigations. We also proposed an overarching genetic mechanism for the prioritized discordant variants.