Abstract：
Splicing is controlled by a large set of regulatory elements (SREs) including splicing enhancers and silencers, which are involved in exon recognition. Variants at these motifs may dysregulate splicing and trigger loss-of-function transcripts associated with disease. Our goal here was to study the alternatively spliced exons 8 and 10 of the breast cancer susceptibility gene CHEK2. For this purpose, we used a previously published minigene with exons 6-10 that produced the expected minigene full-length transcript and replicated the naturally occurring events of exon 8 [Δ(E8)] and exon 10 [Δ(E10)] skipping. We then introduced 12 internal microdeletions of exons 8 and 10 by mutagenesis in order to map SRE-rich intervals by splicing assays in MCF-7 cells. We identified three minimal (10-, 11-, 15-nt) regions essential for exon recognition: c.863_877del [ex8, Δ(E8): 75%] and c.1073_1083del and c.1083_1092del [ex10, Δ(E10): 97% and 62%, respectively]. Then 87 variants found within these intervals were introduced into the wild-type minigene and tested functionally. Thirty-eight of them (44%) impaired splicing, four of which (c.883G>A, c.883G>T, c.884A>T, and c.1080G>T) induced negligible amounts (<5%) of the minigene full-length transcript. Another six variants (c.886G>A, c.886G>T, c.1075G>A, c.1075G>T, c.1076A>T, and c.1078G>T) showed significantly strong impacts (20-50% of the minigene full-length transcript). Thirty-three of the 38 spliceogenic variants were annotated as missense, three as nonsense, and two as synonymous, underlying the fact that any exonic change is capable of disrupting splicing. Moreover, c.883G>A, c.883G>T, and c.884A>T were classified as pathogenic/likely pathogenic variants according to ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based criteria. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
摘要：
剪接由大量的调控元件（SRE）控制，包括剪接增强剂和消音器，参与外显子识别。这些基序的变体可能会失调剪接并引发与疾病相关的功能丧失转录本。我们的目标是研究乳腺癌易感基因CHEK2的可变剪接外显子8和10。为此,我们使用了先前发表的具有外显子6-10的小基因，该小基因产生了预期的小基因全长转录本，并复制了外显子8[Δ（E8）]和外显子10[Δ（E10）]跳跃的自然发生事件。然后，我们通过诱变引入了外显子8和10的12个内部微缺失，以便通过剪接测定法在MCF-7细胞中定位富含SRE的间隔。我们确定了三个最小（10-，11-，15-nt）外显子识别所必需的区域：c.863_877del[ex8，Δ（E8）：75％]和c.1073_1083del和c.1083_1092del[ex10，Δ（E10）：97％和62％，分别]。然后将在这些区间内发现的87种变体引入野生型小基因中并进行功能测试。其中38例（44%）剪接受损，其中四个(c.883G>A，c.883G>T,c.884A>T，和c.1080G>T）诱导的小基因全长转录物的量可忽略不计（<5％）。另外六个变体（c.886G>A，c.886G>T，c.1075G>A，c.1075G>T，c.1076A>T，和c.1078G>T）显示出明显的强烈影响（小基因全长转录物的20-50％）。38个剪接变体中有33个被注释为错义，三是胡说八道，两个是同义词，任何外显子变化都能破坏剪接。此外，c.883G>A,c.883G>T,根据ACMG/AMP（美国医学遗传学和基因组学学院/分子病理学协会）的标准，c.884A>T被分类为致病性/可能的致病性变异。©2024作者由JohnWiley＆SonsLtd代表英国和爱尔兰病理学会出版的病理学杂志。
