While de novo mutations (DNMs) are known to increase risk of congenital defects, DNMs have not been fully explored regarding orofacial clefts (OFCs), one of the most common human birth defects. Therefore, whole-genome sequencing of 756 case-parent trios of European, Colombian, and Taiwanese ancestry was performed to determine the contributions of coding DNMs to OFC risk. Overall, we identified a significant excess of loss-of-function DNMs in genes highly expressed in craniofacial tissues, as well as genes associated with known autosomal dominant OFC syndromes. This analysis also revealed roles for zinc-finger homeobox domain and SOX2-interacting genes in OFC etiology.

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.

Whole-exome sequencing (WES) is now a relatively straightforward process to identify causal variants in Mendelian disorders. However, the same is not true for WES in families where the inheritance patterns are less clear, and a complex etiology is suspected. Orofacial clefts (OFCs) are highly heritable birth defects with both Mendelian and complex etiologies. The phenotypic spectrum of OFCs may include overt clefts and several subclinical phenotypes, such as discontinuities in the orbicularis oris muscle (OOM) in the upper lip, velopharyngeal insufficiency (VPI), microform clefts or bifid uvulas. We hypothesize that expanding the OFC phenotype to include these phenotypes can clarify inheritance patterns in multiplex families, making them appear more Mendelian. We performed whole-exome sequencing to find rare, likely causal genetic variants in 31 multiplex OFC families, which included families with multiple individuals with OFCs and individuals with subclinical phenotypes. We identified likely causal variants in COL11A2, IRF6, KLF4, SHROOM3, SMC3, TP63, and TBX3 in seven families. Although we did not find clear evidence supporting the subclinical phenotype hypothesis, our findings support a role for rare variants in the etiology of OFCs.