A Synonymous Exonic Splice Silencer Variant in IRF6 as a Novel and Cryptic Cause of Non-Syndromic Cleft Lip and Palate

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.

Analysis of FUS, PFN2, TDP-43, and PLS3 as potential disease severity modifiers in spinal muscular atrophy

ObjectiveTo investigate mutations in genes that are potential modifiers of spinal muscular atrophy (SMA) severity.MethodsWe performed a hypothesis-based search into the presence of variants in fused in sarcoma (FUS), transactive response DNA-binding protein 43 (TDP-43), plastin 3 (PLS3), and profilin 2 (PFN2) in a cohort of 153 patients with SMA types 1–4, including 19 families. Variants were detected with targeted next-generation sequencing and confirmed with Sanger sequencing. Functional effects of the identified variants were analyzed in silico and for PLS3, by analyzing expression levels in peripheral blood.ResultsWe identified 2 exonic variants in FUS exons 5 and 6 (p.R216C and p.S135N) in 2 unrelated patients, but clinical effects were not evident. We identified 8 intronic variants in PLS3 in 33 patients. Five PLS3 variants (c.1511+82T>C; c.748+130 G>A; c.367+182C>T; c.891-25T>C (rs145269469); c.1355+17A>G (rs150802596)) potentially alter exonic splice silencer or exonic splice enhancer sites. The variant c.367+182C>T, but not RNA expression levels, corresponded with a more severe phenotype in 1 family. However, this variant or level of PLS3 expression did not consistently correspond with a milder or more severe phenotype in other families or the overall cohort. We found 3 heterozygous, intronic variants in PFN2 and TDP-43 with no correlation with clinical phenotype or effects on splicing.ConclusionsPLS3 and FUS sequence variants do not modify SMA severity at the population level. Specific variants in individual patients or families do not consistently correlate with disease severity.