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

Abstract

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.

Rare variants found in multiplex families with orofacial clefts: Does expanding the phenotype make a difference?

“…In addition, we identified two variants in AFDN in two separate families, including a novel missense variant in Family 8 (NM_001386888.1: c.3545A > G; NP_001373817.1: p.Asn1182Ser) and an extremely rare splice donor variant in Family 22 (NM_001386888.1: c.1222 + 1G > T). Awotoye et al (2022a) recently reported rare heterozygous and compound heterozygous mutations in AFDN . C onditional deletion of Afdn in the palatal epithelium in mice causes a highly penetrant cleft palate (Lough et al, 2020).…”

Connecting the dots towards precision orthodontics

“…In the former category, much work using whole-genome and whole-exome sequencing approaches in patients with CF anomalies have identified several rare genetic variants that contribute to these defects. [63][64][65][66][67] Such genotypedriven diagnosis enables peri-natal risk assessment, early rendering of treatment and enables assessment of disease predilection and future risks. Importantly, the knowledge emerging from studying these rare genetic variants also helps us understand normal and abnormal CF development and provides a mechanistic framework for CF morphogenesis.…”

“…For example, genes harbouring deleterious variants that result in the described anomalies have been shown to play an essential role in the development of primary and secondary palate, neural crest migration, vasculogenesis, Meckel's cartilage development, retinoic acid pathway and suture fusion. [63][64][65][66][67] As noted in Section 3.2, the utilization of facial surface 3D imaging and computational clustering methods have also facilitated genome-wide association studies (GWAS) linking genetic loci to specific facial features. [54][55][56][57]68,69 To date, several genes have been implicated in skeletal Class II and Class III malocclusions (19 and 53 genes, respectively); these genes are involved in conserved signalling pathways playing a role in bone and cartilage development.…”

“…The phenotype of orthodontic patients lies on a spectrum; at one end are individuals with major structural defects such as cleft lip and palate, craniofacial (CF) microsomia and craniosynostosis, whereas at the other end are routine orthodontic patients with variations in jaw size, face shape, and malposition and space availability for teeth contributing to their dental malocclusions. In the former category, much work using whole‐genome and whole‐exome sequencing approaches in patients with CF anomalies have identified several rare genetic variants that contribute to these defects 63–67 . Such genotype‐driven diagnosis enables peri‐natal risk assessment, early rendering of treatment and enables assessment of disease predilection and future risks.…”

“…Importantly, the knowledge emerging from studying these rare genetic variants also helps us understand normal and abnormal CF development and provides a mechanistic framework for CF morphogenesis. For example, genes harbouring deleterious variants that result in the described anomalies have been shown to play an essential role in the development of primary and secondary palate, neural crest migration, vasculogenesis, Meckel's cartilage development, retinoic acid pathway and suture fusion 63–67 . As noted in Section 3.2, the utilization of facial surface 3D imaging and computational clustering methods have also facilitated genome‐wide association studies (GWAS) linking genetic loci to specific facial features 54–57,68,69 .…”