Abstract
Objectives Novel or rare damaging mutations have been implicated in the developmental pathogenesis of nonsyndromic cleft lip with or without cleft palate (nsCL +/- P). Thus, we investigated the human genome for high-impact mutations that could explain the risk of nsCL +/- P in our cohorts. Materials and Methods: We conducted next-generation sequencing (NGS) analysis of 130 nsCL +/- P case-parent African trios to identify pathogenic variants that contribute to the risk of clefting. We replicated this analysis using whole-exome sequence data from a Brazilian nsCL +/- P cohort. Computational analyses were then used to predict the mechanism by which these variants could result in increased risks for nsCL +/- P. Results We discovered damaging mutations within the AFDN gene, a cell adhesion molecule (CAMs) that was previously shown to contribute to cleft palate in mice. These mutations include p.Met1164Ile, p.Thr453Asn, p.Pro1638Ala, p.Arg669Gln, p.Ala1717Val, and p.Arg1596His. We also discovered a novel splicing p.Leu1588Leu mutation in this protein. Computational analysis suggests that these amino acid changes affect the interactions with other cleft-associated genes including nectins (PVRL1, PVRL2, PVRL3, and PVRL4) CDH1, CTNNA1, and CTNND1. Conclusion This is the first report on the contribution of AFDN to the risk for nsCL +/- P in humans. AFDN encodes AFADIN, an important CAM that forms calcium-independent complexes with nectins 1 and 4 (encoded by the genes PVRL1 and PVRL4). This discovery shows the power of NGS analysis of multiethnic cleft samples in combination with a computational approach in the understanding of the pathogenesis of nsCL +/- P.