Distinct IRF6 dysfunction mechanisms in syndromic orofacial clefts: Computational evidence for allosteric versus direct disruption

Orofacial clefts (OC) are a congenital anomaly typically classified as syndromic or non-syndromic. Among the syndromic cases, Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are primarily caused by pathogenic variants in <i>IRF6</i>, which encodes a transcriptional factor essential for orofacial development. Our aim is to identify and characterize <i>IRF6</i> variants in two syndromic OC cases and assess their structural and functional consequences through computational modeling. Sequencing of <i>IRF6</i> exons 3, 4, 7, and 9 was conducted in one proband with VWS and one proband with PPS, as well as their parents. Structural impacts were measured through comparative modeling based on the IRF4-DNA complex, followed by molecular dynamics simulations. Binding free energies and protein-DNA interactions were assessed using MM/GBSA calculations and hydrogen bond occupancy analysis. We identified two pathogenic variants: NP_006138.1:p.(Ala16Val) in a VWS proband and NP_006138.1:p.(Arg84Cys) in a PPS proband, both confirmed as <i>de novo</i>. Variants were classified as pathogenic by the American College of Medical Genetics and Genomics criteria, and by most pathogenicity predictors. Molecular dynamics simulations showed that NP_006138.1:p.(Ala16Val) induces long-range allosteric effects with increased structural fluctuations, while NP_006138.1:p.(Arg84Cys) directly disrupts critical DNA-binding interactions. MM/GBSA analysis demonstrated reduced DNA-binding affinity for both variants, with NP_006138.1:p.(Arg84Cys) showing the most severe electrostatic disruption. Our computational analyses suggest that pathogenic <i>IRF6</i> variants may impact DNA binding through distinct molecular mechanisms: NP_006138.1:p.(Ala16Val) potentially through allosteric conformational changes and NP_006138.1:p.(Arg84Cys) likely through direct disruption of the protein-DNA interface.