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. 2025 Jul;117(7):e2499.
doi: 10.1002/bdr2.2499.

Functional Annotation of De Novo Variants Found Near GWAS Loci Associated With Cleft Lip With or Without Cleft Palate

Sarah W Curtis  1 , Laura E Cook  2 , Kitt Paraiso  2 , Axel Visel  2   3 , Justin L Cotney  4 , Jeffrey C Murray  5 , Terri H Beaty  6 , Mary L Marazita  7   8 , Jenna C Carlson  8   9 , Elizabeth J Leslie-Clarkson  1
Affiliations

Functional Annotation of De Novo Variants Found Near GWAS Loci Associated With Cleft Lip With or Without Cleft Palate

Sarah W Curtis et al. Birth Defects Res. 2025 Jul.

Abstract

Background: Orofacial clefts (OFCs) are the most common craniofacial birth defects, affecting 1 in 700 births, and have a strong genetic basis with a high recurrence risk within families.

Aims: While many of the previous studies have associated common, noncoding genetic loci with OFCs, previous studies on de novo variants (DNVs) in OFC cases have focused on coding variants that could have a functional impact on protein structure, and the contribution of noncoding DNVs to the formation of OFCs has largely been ignored and is not well understood.

Materials and methods: We reanalyzed an existing dataset of DNVs from 1409 trios with OFCs that had undergone targeted sequencing of known OFC-associated loci. We then annotated these DNVs with information from datasets of predicted epigenetic function during human craniofacial development.

Results: Of the 66 DNVs called in the targeted regions in this study, 17 (25.7%) were within a predicted enhancer or promoter region. Two DNVs fell within the same enhancer region (hs1617), which is more than expected by chance (p = 0.0017). The sequence changes caused by these hs1617 DNVs are predicted to create binding sites not seen in the reference sequence for transcription factors PAX6 and ZBTB7A and to disrupt binding sites for STAT1 and STAT3.

Discussion: The hs1617 enhancer region is within the same topologically associated domain as HHAT, SERTAD4, and IRF6, all of which are involved in craniofacial development. All three genes are highly expressed in human neural crest cells. Knockout mice for Hhat and Irf6 have abnormal embryonic development including a cleft palate, and variants in and around IRF6 are associated with nonsyndromic and syndromic forms of OFCs in humans.

Conclusion: Taken together, this suggests that noncoding DNVs contribute to the genetic architecture of OFCs, with an excess of DNVs in OFC trios in enhancer regions near known OFC-associated genes. Overall, this adds to our understanding of the genetic mechanisms that underlie OFC formation.

Keywords: cleft lip and palate; enhancers; noncoding; rare variant; targeted sequencing; transcription factor.

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