Expanding the Genetic Spectrum of Non-Syndromic Cleft Lip and Palate Through Whole-Exome Sequencing

Non-syndromic cleft lip with or without cleft palate (ns-CL/P) is one of the most common craniofacial anomalies with a multifactorial etiology. To investigate the contribution of rare variants to disease risk, we performed whole-exome sequencing (WES) in 58 patients with ns-CL/P from a homogeneous Polish population, excluding from analysis 423 previously investigated cleft candidate genes. After stringent filtering, prioritization, and segregation analysis, we identified 31 likely pathogenic (LP) variants across 30 genes, significantly enriched in categories related to developmental processes. Notably, 29% of variants occurred in genes not previously linked to clefting, including AGO1, ARID1A, ATP1A1, FOXA2, GDF7, HOXB3, LRP5, MAML1, and ZNF319. Three were de novo: FOXA2_p.Arg260Pro, MAML1_p.Gln65Ter, and ZNF319_p.Gln64Ter. Most of the remaining variants were inherited from unaffected parents, suggesting incomplete penetrance and possible modifier effects consistent with the heterogeneous etiology of ns-CL/P. Additionally, analysis of common variants in the 30 loci harboring rare LP variants revealed nominal associations with ns-CL/P for NXN, EXT1, MAML1, and TP53BP2 loci. These results support the candidacy of these genes and suggest contributions from both rare and common variants. In conclusion, we report novel LP variants expanding the spectrum of candidate genes and providing new insights into the genetic landscape of orofacial clefts.