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 protein-altering 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 some of which are based on available evidence, contribute to the risk of nsCL/P. These include novel protein-truncating 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. Many of these protein-altering DNMs were predicted to be pathogenic. Analysis using mouse transcriptomics data showed that some of these genes are expressed during the development of primary and secondary palate. Gene-set enrichment analysis of the protein-altering DNMs identified palatal development and neural crest migration among the few processes that were significantly enriched. These processes are directly involved in the etiopathogenesis of clefting. The analysis of the coding sequence in the WGS data provides more evidence of the opportunity for novel findings in the African genome.

Figure 1

Case-parent trios’ definition, cleft sub-types, and Data filtration pipeline. (A) Data filtration pipeline used to identify the high confidence de novo mutations (DNMs) that contribute to the risk of nsCL/P. (B) Details of the number of variants from each data filtering steps which resulted in 162 DNMs in protein-coding genes. (C) Pie chart showing the distribution of the effects of the de novo Variants. Majority of the DNMs (94%) cause amino acid changes which alters the protein structures and functions while 6% cause loss of function mutation in the protein-coding genes.

Figure 2

AlhpaFold predicted protein structures of the SHH (A,B). Highlighted in blue is the side-chain where the mutation occurred ((A) closed-up view in (B)). This is located within the Hedgehog domain that is critical for the hedgehog signaling. Table shows thermodynamic prediction of the effect of the p.Ser363Leu SHH DNM on the protein stability. The amino acid change resulted in a change in the folding free energy by 4.984 kcal/mol (± 0.221). This change is predicted to be disease causing.

Figure 3

Graph showing significantly enriched BP from GSEA. The palate development and neural crest migration are among the processes significantly enriched (p < 0.05).

Figure 4

SysFACE-based expression analysis of candidate genes in mouse facial development. Expression of candidate genes based on analysis using (A) GSE7759 microarray data generated on the Affymetrix Mouse Genome 430 2.0 Array platform, (B) FaceBase microarray data generated on the Affymetrix Mouse Gene 1.0 ST Array platform, and (C) GSE55965 microarray data generated on the Affymetrix Mouse Gene 1.0 ST Array platform. Heat-map denotes row-wise comparative expression of individual genes in different tissues at Embryonic (E) and/or postnatal (P) stages. Intensity of the color in the heat-map is representative of candidate gene expression and the average fluorescence signal intensity is shown.