Variants in CALD1, ESRP1, and RBFOX1 are associated with orofacial cleft risk

Nonsyndromic orofacial clefts (OFCs) are common, heritable birth defects caused by both genetic and environmental risk factors. Despite the identification of many genetic loci harboring OFC-risk variants, there are many unknown genetic determinants of OFC. Furthermore, while the process of embryonic facial development is well characterized, the molecular mechanisms that underly it are not. This represents a major hurdle in understanding how disruptions in these biological processes result in OFC. Thus, we sought to identify novel OFC-risk loci through a genome-wide multi-ancestry study of five nested OFC phenotypes (isolated cleft lip [CLO], isolated cleft palate [CPO], cleft lip and palate [CLP], cleft lip with/without cleft palate [CL/P], and any cleft [ANY]) representing distinct cleft subtypes to identify subtype-specific signals and grouped types to maximize power to detect shared genetic effects. We performed genome-wide meta-analyses of these five OFC phenotypes from three cohorts totaling >14,000 individuals using METAL. In addition to replicating 13 known OFC-risk loci, we observed novel association in three regions: the 1p36.32 locus (lead variant rs584402, an intergenic variant, pCLO = 3.14e-8), the 7q33 locus (lead variant rs17168118, an intronic variant in CALD1, pCLP = 9.17e-9), and the 16p13.3 locus (lead variant rs77075754, an intronic variant in RBFOX1, pCL/P = 1.53e-9, pANY = 1.93e-9). We also observed a novel association within the known risk locus 8q22.1 that was independent of the previously reported signal (lead variant rs4735314, an intronic variant in ESRP1, pCLP = 1.07e-9, pCL/P = 3.88e-8). Next, we performed multi-tissue TWAS with s-MulTiXcan and identified four overlapping genes with significant genetically predicted transcription associated with OFC risk. These genes also overlapped the genome-wide significant association signals from the meta-analysis, including CALD1 and ESRP1 and known OFC-risk genes TANC2 and NTN1. Each of the newly reported loci has potential regulatory effects, including evidence of craniofacial enhancer activity, that offer new clues as to the molecule mechanisms underlying embryonic facial development.