Genetic Heterogeneity in Cleft Lip and/or Palate With Alveolar Cleft: A Targeted Analysis of NHEK-Active Enhancer Variants

Objective To identify susceptibility loci for nonsyndromic cleft lip and/or palate with cleft alveolus involvement and to elucidate subtype-specific regulatory mechanisms in a Chinese population. Design Subtype-stratified case-control targeted association analysis. Setting Tertiary medical center for orofacial cleft treatment in western China. Patients, Participants A total of 3009 unrelated Chinese individuals from two previously generated Genome-wide association study datasets, comprising 445 cases with cleft lip extending through the alveolus without palatal involvement (CLCA), 309 cases with cleft lip and palate extending through the alveolus (CLPCA), and 754 combined CL/P with cleft alveolus (CL/PCA) cases, along with unaffected controls. Interventions Not applicable. Main Outcome Measure(s) Single nucleotide polymorphisms associations with CLCA, CLPCA, and combined CL/PCA phenotypes at Bonferroni-corrected significance (P < 3.65 × 10 −6 ), with functional annotation integrating ENCODE chromatin states, Hi-C interactions, and GTEx eQTL data. Results Three SNPs (rs10863790, rs2064163, rs4880224) reached study-wide significance in the combined CL/PCA and CLCA subgroups but not in CLPCA. rs10863790 showed the highest population attributable risk in CLCA (29.63%). All three variants overlap NHEK-active enhancers and regulate craniofacial development genes ( IRF6, UTP25, TRAF3IP3, BNIP3, JAKMIP3, PPP2R2D ) through chromatin looping, pluripotency factor binding, and FOX family transcription factor recruitment. Conclusions These findings reveal pronounced genetic heterogeneity between CLCA and CLPCA subtypes, underscoring the necessity of subtype-stratified approaches for precision medicine in orofacial cleft management.