Whole-genome Sequencing Reveals De-novo Mutations Associated with Nonsyndromic Cleft Lip/Palate

Abstract

The majority (85%) of nonsyndromic cleft lip with or without cleft palate (nsCL/P) cases occur sporadically, suggesting a role for de novo mutations (DNMs) in the etiology of nsCL/P. To identify high impact DNMs that contribute to the risk of nsCL/P, we conducted whole genome sequencing (WGS) analyses in 130 African case-parent trios (affected probands and unaffected parents). We identified 162 high confidence protein-altering DNMs that contribute to the risk of nsCL/P. These include novel loss-of-function DNMs in the ACTL6A, ARHGAP10, MINK1, TMEM5 and TTN genes; as well as missense variants in ACAN, DHRS3, DLX6, EPHB2, FKBP10, KMT2D, RECQL4, SEMA3C, SEMA4D, SHH, TP63, and TULP4. Experimental evidence showed that ACAN, DHRS3, DLX6, EPHB2, FKBP10, KMT2D, MINK1, RECQL4, SEMA3C, SEMA4D, SHH, TP63, and TTN genes contribute to facial development and mutations in these genes could contribute to CL/P. Association studies have identified TULP4 as a potential cleft candidate gene, while ARHGAP10 interacts with CTNNB1 to control WNT signaling. DLX6, EPHB2, SEMA3C and SEMA4D harbor novel damaging DNMs that may affect their role in neural crest migration and palatal development. This discovery of pathogenic DNMs also confirms the power of WGS analysis of trios in the discovery of potential pathogenic variants.

Damaging Mutations in AFDN Contribute to Risk of Nonsyndromic Cleft Lip With or Without Cleft Palate

“…Our results from the de novo analysis reported novel genetic variants that contribute to the risk of nsCL ± P in the African population. 22 To identify other genes, we analyzed for novel or rare inherited protein-altering variants in genes reported to contribute to craniofacial development. These analyses are premised on the fact that some risk genes have low penetrance where parents carry the variant but not the nsCL ± P phenotype, and proband has both variant and phenotype.…”

“…Details of these processes have been previously published. 22 Briefly, following the shipment of the samples to the US laboratory, DNA was extracted using the Oragene DNA extraction protocol. Quantifications of the extracted DNA were done using Qubit (http://www.invitrogen.com/site/us/en/home/brands/Product-Brand/Qubit.html; Thermo Fisher Scientific).…”