Rare variants found in clinical gene panels illuminate the genetic and allelic architecture of orofacial clefting

Abstract

Orofacial clefts (OFCs) are common craniofacial birth defects including cleft lip (CL), cleft lip with cleft palate (CLP), and cleft palate (CP). The etiological heterogeneity of OFCs complicates clinical diagnostics as it is not always readily apparent if the cause is Mendelian, environmental, or multifactorial. Although sequencing could aid diagnosis, it is not commonly used for the 60-70% of OFC cases that appear isolated or lack a strong family history. We aimed to estimate the diagnostic yield evaluating 503 genes using whole-genome sequencing data from 841 cases and 294 controls. After curating variants, we evaluated them according to the American College of Medical Genetics pathogenicity criteria blinded to case-control status. We found 'likely pathogenic' variants in 9.04% of cases and 1.36% of controls (p<0.0001), which was almost exclusively driven by dominant variants in autosomal genes. The yield was highest in CP (17.6%) and CLP (9.09%) cases, while CL (2.80%) cases were not significantly different from controls. We identified 'likely pathogenic' variants in cases in 39 genes, underscoring the genetic heterogeneity of OFCs. Notably, nine genes, including CTNND1, IRF6, and COL2A1, were recurrently mutated, accounting for more than half of the yield (occurring in 4.64% of OFC cases). Most reviewed variants (60.6%) were 'variants of uncertain significance' (VUS) and were more frequent in cases (p=7.31 x 10-4), but no individual gene showed a significant excess of VUS. Cumulatively, these results underscore the etiological heterogeneity of OFCs and suggest clinical sequencing could help reduce the diagnostic gap in OFC etiology.

Functional analysis ofESRP1/2gene variants andCTNND1isoforms in orofacial cleft pathogenesis

“…We and others demonstrated that Esrp1 and Esrp2 regulate the alternative splicing of Ctnnd1, generating isoforms that differ between epithelial and mesenchymal cell types (20, 60, 66, 73), making Ctnnd1 an interesting Esrp1/2 target that has also been implicated in CL/P (61).…”

“…Pathogenic variants in CTNND1 accounted for 0.8% of the cohort. Only 10% of the cohort had a pathogenic variant in 500 genes implicated in OFC that we analyzed, making CTNND1 the mostly frequently mutated variant in this cohort (61). In the gene-based burden test, rare variants were nominally over-transmitted to affected children (p=0.06); de novo variants are enriched in CTNND1 (p=0.005 for loss-of-function de novo variants; 0.001 for protein-altering de novo variants).…”

“…ClinVar variants were accessed in 2021, we note that new variants have been uploaded to ClinVar for ESRP1 and ESRP2, but these new variants did not include relevant clinical phenotype information so were not included in this study. OFC associated genes were based on a previously published study that curated a list of approximately 500 genes based on known clinical syndromes and association results from GWAS (61).…”

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

“…For models considering variants in OFC genes, we utilized a gene list comprised of 418 genes previously associated with craniofacial development and OFCs. These were compiled from four sources, including OMIM (Amberger et al, 2015), the PreventionGenetics Cleft Lip/Cleft Palate Panel (PreventionGenetics, Marshfield, WI), the Genomics England PanelApp Clefting Panel (v2.2, March 2020; (Martin et al, 2019)), and an additional set of literature-curated genes (more information on this gene list can be found in Diaz Perez et al (2022). We considered a significance level for both the “all genes” and “OFC genes” at P < 0.05.…”

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

“…OMIM clinical synopses search terms included: “cleft lip,” “cleft palate,” “oral cleft,” “orofacial cleft,” and “cleft lip and/or palate;”, (b) the PreventionGenetics Cleft Lip/Cleft Palate clinical genetic testing panel (PreventionGenetics, Marshfield, WI), (c) the Genomics England PanelApp cleft panel (v2.2), an expert‐curated list of genes for familial cleft lip and/or cleft palate, familial isolated clefting, and syndromic clefting (Martin et al, 2019), and (d) literature‐curated genes. More information on this gene list can be found in Diaz Perez et al (2023). For the mixed model analyses, we considered a significance level for both the “all genes” and “OFC genes” at p < 0.05.…”