A Combination of Variants in SEPTIN9 and MSX1 Genes Leads to the Formation of Orofacial Clefts

Nonsyndromic cleft lip with or without palate (nCL/P) is a common congenital anomaly with a complex genetic basis. Previous whole-exome sequencing of Malagasy case-parent trios identified variants in several craniofacial development genes, including SEPTIN9, SKI, WNT5B, GPC4, and MSX1, enriched on East Asian ancestry segments. To assess functional contributions, we used Xenopus laevis embryos to evaluate the effects of individual and combined perturbations of candidate genes. Neither single-gene depletion nor ectopic expression of candidate genes induced orofacial clefts, although some craniofacial malformations were observed. In contrast, double knockdown of SEPTIN9 and MSX1 reproducibly produced orofacial clefts. These defects were rescued by co-injection of wild type human SEPTIN9 and MSX1 mRNAs, but not by variant alleles such as SEPTIN9 p.Glu370Lys and MSX1 p.Glu84Val. Our findings reveal a functional interaction between SEPTIN9 and MSX1 in craniofacial morphogenesis and provide experimental evidence for digenic inheritance in nCL/P. This work underscores the importance of ancestry-enriched variant combinations in the etiology of complex congenital anomalies.