Genetic Modulation Of Irf6 Confers Nscl/P By Dna Methylation

By combining epigenetic and genome-wide association studies (GWAS), our study provides an in-depth analysis of the mechanisms regulating methylation dynamics and further revealing the etiological basis of non-syndromic cleft lip with or without cleft palate (NSCL/P). The samples for GWAS were obtained from 1,069 NSCL/P cases and 1,724 healthy controls collected by our group, peripheral blood and lip tissue samples from 105 NSCL/P patients were also collected for methylation sequencing. Ethics approval was obtained from our institution. This study integrated GWAS and methylation quantitative trait loci data to identify cg26035071, a methylation site in the IRF6 promoter region, as significantly correlated with cleft lip/palate risk. Through dual-luciferase reporter assays, rs6659367 was identified as a functional genetic variant regulating IRF6 expression. Correlation analysis predicted that the methylation transferase DNMT3B was significantly associated with IRF6 expression levels, subsequently validated by allele-specific chromatin immunoprecipitation and Sanger sequencing. These demonstrated that the rs6659367 T>G mutation enhances DNMT3B binding, elevates promoter methylation, suppresses IRF6 expression. Phase separation experiments revealed that DNMT3B forms liquid-liquid phase-separated condensates. Subsequently, we constructed the CRISPRoff system to precisely implement site methylation editing confirmed the functional correlation between cg26035071 and NSCL/P pathogenesis. In summary, the functional variant rs6659367 increases NSCL/P risk through enhancing DNMT3B recruitment and phase-separated condensate formation at the IRF6 promoter. This elevates cg26035071 methylation, suppresses IRF6 expression, and disrupts cellular functions.