Exome sequencing reveals the genetic architecture of non-syndromic orofacial clefts and identifies BOC as a novel causal gene

Nonsyndromic orofacial clefts (NSOFCs) are the most common human craniofacial defects. Genetic factors play a critical role in the pathogenesis of NSOFCs. However, known causal genes only explain a minority of the estimated heritability. The findings substantiate the genetic and allelic heterogeneity of NSOFCs and underscore the crucial role of dysregulation of OFC-related signaling pathways in the occurrence of NSOFCs. Besides, the candidate variants discovered provide a fruitful resource for further genetic studies. Particularly, three BOC missense variants (p.R407W, p.G436S and p.D1018N) are identified in three cases with cleft palate. In parallel, a BOC nonsense variant (p.R681X), co-segregating with a GLI2 missense variant (p.A543G), is identified in a multiplex family with microform cleft lip. Functional studies demonstrate while the four BOC variants are hypomorphic alleles, the GLI2 variant is a hypermorphic allele. The counteraction between BOC p.R681X allele and GLI2 p.A543G allele is likely to account for the mild phenotype in the multiplex family. Thus, this study establishes BOC as a novel causal gene and implicates a two-locus model of inheritance via the epistatic antagonism of two SHH pathway variants in NSOFCs. We divided the collected zebrafish embryos into two groups and performed microinjections with GLI2-WT mRNA and GLI2-A543G mRNA, respectively. After allowing the embryos to develop for 24 hours, we lysed them and extracted RNA from both groups. RNA sequencing was then performed, followed by a series of data analyses.