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.

中文翻译：

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IRF6 中的同义外显子剪接消音器变体作为非综合征性唇裂和腭裂的一个新的和隐秘的原因

干扰素调节因子 6 (IRF6) 中的错义、无义、剪接位点和调节区变体已被证明会导致唇裂和/或腭裂 (CL/P) 的综合征和非综合征形式。我们报告了一个复杂的多代洪都拉斯血统家族的诊断评估，其谱系结构与常染色体显性遗传一致，具有不完全外显率和可变表达性。先证者的祖母与两个伴侣生了孩子，每个血统的两边都有 CL/P 分离。通过对该家族 5 名成员的全外显子组测序，我们确定了一个共享的同义变异，位于 IRF6 外显子 7 的中间（p.Ser307Ser；g.209963979 G>A；c.921C>T）。该变异被证明在七个受影响的个体中分离，并通过三个未受影响的专性携带者分离，跨越该谱系的两侧。这种变异非常罕见，仅在 gnomAD 数据库的 251,352 个等位基因中的三个（所有拉丁裔血统）中发现。虽然该变体没有产生剪接受体/供体位点，但计算机分析预测它会影响外显子剪接消音元件和主要剪接调节因子的结合。体外剪接分析通过揭示多个异常剪接事件来支持这一点，估计影响> 60% 的等位基因转录本。对替代剪接产物的测序证明了变体 5' 6 个核苷酸的隐蔽剪接位点的暴露，以及内含子 6 中隐蔽剪接位点的可变利用。不同剪接调节蛋白的异位表达改变了剪接试验中异常剪接事件的比例，尽管这种改变取决于剪接因子。重要的是，预测每个选择性剪​​接的 mRNA 会导致移码和过早截断 IRF6 蛋白。这是第一项将同义变异确定为 NS-CL/P 可能原因的研究，并强调了实验室在考虑和解释变异时应注意的事项。