Abstract
Missense, nonsense, splice site and regulatory region variants in interferon regulatory factor 6 (IRF6) have been shown to contribute to both syndromic and non-syndromic forms of cleft lip and/or palate (CL/P). We report the diagnostic evaluation of a complex multigeneration family of Honduran ancestry with a pedigree structure consistent with autosomal-dominant inheritance with both incomplete penetrance and variable expressivity. The proband’s grandmother bore children with two partners and CL/P segregates on both sides of each lineage. Through whole-exome sequencing of five members of the family, we identified a single shared synonymous variant, located in the middle of exon 7 of IRF6 (p.Ser307Ser; g.209963979 G>A; c.921C>T). The variant was shown to segregate in the seven affected individuals and through three unaffected obligate carriers, spanning both sides of this pedigree. This variant is very rare, only being found in three (all of Latino ancestry) of 251,352 alleles in the gnomAD database. While the variant did not create a splice acceptor/donor site, in silico analysis predicted it to impact an exonic splice silencer element and the binding of major splice regulatory factors. In vitro splice assays supported this by revealing multiple abnormal splicing events, estimated to impact >60% of allelic transcripts. Sequencing of the alternate splice products demonstrated the unmasking of a cryptic splice site six nucleotides 5′ of the variant, as well as variable utilization of cryptic splice sites in intron 6. The ectopic expression of different splice regulatory proteins altered the proportion of abnormal splicing events seen in the splice assay, although the alteration was dependent on the splice factor. Importantly, each alternatively spliced mRNA is predicted to result in a frame shift and prematurely truncated IRF6 protein. This is the first study to identify a synonymous variant as a likely cause of NS-CL/P and highlights the care that should be taken by laboratories when considering and interpreting variants.
Highlights
Clefts of the lip and/or palate (CL/P) are among the most common and recognizable inborn errors of development, presenting in one in 700 live births [1]
We performed whole exome sequencing (WES) on several family members from a large, multiplex Honduran family with NS-cleft lip and/or palate (CL/P), identifying a novel segregating synonymous variant in interferon regulatory factor 6 (IRF6). We demonstrate that this synonymous variant impacts an exonic splice silencer element that results in the utilization of multiple cryptic exonic and intronic splice sites, each of which is predicted to result in a frame shift and loss of function of IRF6
After exclusion of the 13 genes implicated in other diseases, as well as the remaining with incomplete penetrance was considered the best model and variants shared among affected variants located in introns and untranslated regions, we were left with seven initial candidate genes: individuals were assessed with greater scrutiny
Summary
Clefts of the lip and/or palate (CL/P) are among the most common and recognizable inborn errors of development, presenting in one in 700 live births [1]. Orofacial clefting often occurs as a component of various genetic syndromes and sequences, but approximately 70% of CL/P cases arise in apparent isolation [1] These individuals with non-syndromic CL/P (NS-CL/P) display complex epidemiology, varying in prevalence across geographic regions and ethnic groups, and complex etiology, with numerous genetic and environmental risk factors contributing to disease [1]. We performed whole exome sequencing (WES) on several family members from a large, multiplex Honduran family with NS-CL/P, identifying a novel segregating synonymous variant in IRF6. We demonstrate that this synonymous variant impacts an exonic splice silencer element that results in the utilization of multiple cryptic exonic and intronic splice sites, each of which is predicted to result in a frame shift and loss of function of IRF6. Similar types of mutations have been reported in other diseases, this represents the first example underlying the presentation of CL/P
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