Prioritization of non-coding elements involved in non-syndromic cleft lip with/without cleft palate through genome-wide analysis of de novo mutations

Non-syndromic cleft lip with/without cleft palate (nsCL/P) is a highly heritable facial disorder. To date, systematic investigations of the contribution of rare variants in non-coding regions to nsCL/P etiology are sparse. Here, we re-analyzed available whole-genome sequence (WGS) data from 211 European case-parent trios with nsCL/P and identified 13,522 de novo mutations (DNMs) in nsCL/P cases, 13,055 of which mapped to non-coding regions. We integrated these data with DNMs from a reference cohort, with results of previous genome-wide association studies (GWASs), and functional and epigenetic datasets of relevance to embryonic facial development. A sig-nificant enrichment of nsCL/P DNMs was observed at two GWAS risk loci (4q28.1 (p = 8 3 10-4) and 2p21 (p = 0.02)), suggesting a convergence of both common and rare variants at these loci. We also mapped the DNMs to 810 position weight matrices indicative of transcription factor (TF) binding, and quantified the effect of the allelic changes in silico. This revealed a nominally significant over-representation of DNMs (p = 0.037), and a stronger effect on binding strength, for DNMs located in the sequence of the core binding region of the TF Musculin (MSC). Notably, MSC is involved in facial muscle development, together with a set of nsCL/P genes located at GWAS loci. Supported by additional results from single-cell transcriptomic data and molecular binding assays, this suggests that varia-tion in MSC binding sites contributes to nsCL/P etiology. Our study describes a set of approaches that can be applied to increase the added value of WGS data.

Automated summary

In this study, whole-genome sequence data from 211 European individuals with non-syndromic cleft lip/palate were analyzed, and 13,522 de novo mutations were identified. These mutations were enriched at two genome-wide association study risk loci, suggesting a convergence of common and rare variants at these loci. Additionally, mutations in the binding region of the Musculin transcription factor were found to contribute to the etiology of cleft lip/palate.