Genome-wide assessment of rare protein-coding variants identifies associations with non-syndromic cleft lip/palate

Nonsyndromic cleft lip and palate (NSCLP) is a complex, multifactorial condition for which only about 25% of the genetic contribution has been identified. Although more than 60 genetic loci have been associated with NSCLP, a large portion of its heritability remains unexplained. Rare variants-often with stronger functional effects-are believed to account for some of this missing genetic risk. In this study, short-read whole-genome sequencing data from 786 NSCLP cases and 2149 controls were analyzed to identify genes enriched for rare protein-coding variants. Gene-based association testing, meta-analysis, functional prioritization, and effect-size estimation revealed several novel NSCLP candidate genes and confirmed associations with previously reported genes. The four novel candidate genes with the strongest association evidence were SCT, MALRD1, GPR156, and MYOCOS, all with p-values < 10⁻⁴. Replication of known genes was most robust for ARHGAP29, FGF8, SKI, and TP63. Pathway analyses showed significant enrichment of these genes in neuroactive ligand-receptor interactions, MAPK signaling, and other signal transduction pathways. Precise estimates of the risks conferred by the identified rare variants showed a substantial increase in NSCLP risk, with odds ratio point estimates between 2 and 4. These results provide strong support for a polygenic inheritance model in NSCLP, in which certain genes contribute varying levels of risk.