Comprehensive analysis of de novo variants across 2,497 orofacial cleft trios reveals novel genetic drivers of disease

Background Orofacial clefts (OFCs) and other palate abnormalities (PAs) are among the most common birth defects worldwide and are characterized by the abnormal formation of the lip and/or palate. Genetic studies have traditionally classified OFC cases as either syndromic, involving OFCs alongside other congenital anomalies, or nonsyndromic, which represent the majority of cases and occur in isolation. Emerging genomic evidence indicates that genes traditionally associated with syndromic forms of OFC can also harbor variants contributing to isolated cases, challenging the notion of a strict dichotomy between these categories and supporting their integration for gene discovery. Methods In this study, we applied multiple analytic approaches to characterize the genetic architecture of OFC and PAs by integrating genomic data from 2,497 trios with an OFC (n=2080) and PA (n=417) affected proband. We compared these findings across OFC subtypes and syndromic status with those from 5,515 control trios to identify enriched biological pathways and mechanisms and to prioritize candidate genes using variant burden testing. Results We observed a significant enrichment of de novo protein-truncating and damaging missense variants in cases compared to controls (OR = 2.17, p = 1.21x10-32), with particularly strong signals in biologically relevant gene sets involving OFC-associated, constrained, Mendelian disorder, and mouse candidate genes. Variant burden testing identified 39 OFC risk genes at FDR [≤] 0.05, which we then integrated with 593 established OFC genes to interrogate the functional underpinnings of OFC via network analysis. This analysis revealed 309 high-order interactor genes not previously associated with OFC. Notably, this OFC network clustered into ten distinct biological pathways, with nucleosome-associated genes showing significant enrichment among cases in our cohort (OR = 14.8, p = 8.1x10-4). In a final integrative step, we combined evidence across all analyses to nominate 231 candidate genes, 32 of which contained at least two deleterious de novo variants in our cohort. Conclusions These findings underscore the value of integrating diverse OFC and PA subtypes, syndromic status, and variant classes to refine the genetic architecture of these disorders, highlighting both phenotypic expansion of known disease genes and the emergence of novel gene-phenotype associations.