Damaging Mutations in AFDN Contribute to Risk of Nonsyndromic Cleft Lip With or Without Cleft Palate

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.

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.

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.

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.