Whole-Exome Sequencing Identifies Novel Variants for Tooth Agenesis

Abstract

Tooth agenesis is a common craniofacial abnormality in humans and represents failure to develop 1 or more permanent teeth. Tooth agenesis is complex, and variations in about a dozen genes have been reported as contributing to the etiology. Here, we combined whole-exome sequencing, array-based genotyping, and linkage analysis to identify putative pathogenic variants in candidate disease genes for tooth agenesis in 10 multiplex Turkish families. Novel homozygous and heterozygous variants in LRP6, DKK1, LAMA3, and COL17A1 genes, as well as known variants in WNT10A, were identified as likely pathogenic in isolated tooth agenesis. Novel variants in KREMEN1 were identified as likely pathogenic in 2 families with suspected syndromic tooth agenesis. Variants in more than 1 gene were identified segregating with tooth agenesis in 2 families, suggesting oligogenic inheritance. Structural modeling of missense variants suggests deleterious effects to the encoded proteins. Functional analysis of an indel variant (c.3607+3_6del) in LRP6 suggested that the predicted resulting mRNA is subject to nonsense-mediated decay. Our results support a major role for WNT pathways genes in the etiology of tooth agenesis while revealing new candidate genes. Moreover, oligogenic cosegregation was suggestive for complex inheritance and potentially complex gene product interactions during development, contributing to improved understanding of the genetic etiology of familial tooth agenesis.

Keywords: WNT signaling pathway; array genotyping; gene; hypodontia; next generation sequencing; oligodontia.

Figure 1.

Clinical and genetic findings in families in which 1 candidate gene was identified as pathogenic for isolated and suspected syndromic tooth agenesis. Probands’ panoramic radiographs and schematics of missing teeth, familial segregation analyses, and corresponding Sanger sequencing chromatograms are shown. Missing teeth are indicated by red stars in radiographs and filled boxes in schematic maxillary (MAX) and mandibular (MAN) arches. Probands are indicated by arrows in each pedigree. (A) Families with isolated tooth agenesis. Previously reported mutations in WNT10A (c.697G>T, c.682T>A, and c.433G>A) were identified segregating with tooth agenesis in 5 families (TF-1, TF-2, and TF-6 presented here; TF-4 and TF-5 presented in the Appendix). A novel likely pathogenic heterozygous potential splicing mutation in LRP6 (c.3607+3_6del: p.?) was identified in 1 family (TF-7). Quantitative polymerase chain reaction was performed to detect LRP6 gene expression on 3 affected individuals (II-1, II-2, and III-2) and 1 unrelated unaffected control individual. LRP6 expression was decreased in all affected individuals as compared with the control individual. Sanger sequencing confirmed the presence of only wild-type message between exons 16 and 17. (B) Families with suspected syndromic tooth agenesis (TF-8 and TF-9). Variants in KREMEN1 (c.146C>G and c.773_778del) were identified in the probands with tooth agenesis and mild ectodermal features. Family members presented with tooth agenesis and no ectodermal features. Of note, in TF-8, the unaffected father (II-12) and brother (III-3) are carriers for the heterozygous mutation, while in TF-9, both affected (mother, II-4) and unaffected (maternal grandfather, I-2, and sister, III-1) individuals were heterozygotes.

Figure 2.

Clinical and genetic findings in families in which oligogenic inheritance was proposed in isolated tooth agenesis. Variants in WNT10A (c.682T>A) and LAMA3 (c.1097G>A) were identified as potentially pathogenic in TF-3, whereas variants in DKK1 (c.548-4G>T), LAMA3 (c.2798G>T) and COL17A1 (c.3277+3G>C) were identified in TF-10.

Figure 3.

Structural modeling of individual mutations. WNT10A is colored blue, and its binding partner is colored green. Disulfide bridges are highlighted in yellow, and modification sites are colored magenta. (A) WNT10A F228I variant is predicted to disrupt protein structure by destabilizing disulfide bridges. (B) WNT10A V145M is predicted to be probably deleterious by MutationTaster, or neutral by VIPUR. (C) KREMEN1 T49R is predicted to be deleterious by VIPUR due to an unfavorable backbone conformation. The domains of KREMEN1 are colored blue (Kringle domain), cyan (WSC domain), and green (CUB domain), with disulfide bridges colored in yellow and modification sites in magenta. (D) LAMA3 is colored in blue, and R366H falls into a region of LAMA3 that is missing in isoforms 3 and 4 (colored in cyan). (E) LAMA3 G933V is predicted to be deleterious by VIPUR because of poor packing. G933 occurs in the first laminin G–like domain of LAMA3 (laminin G–like domains are colored in cyan).