Functional classification of DNA variants by hybrid minigenes: Identification of 30 spliceogenic variants of BRCA2 exons 17 and 18.
Functional classification of DNA variants by hybrid minigenes: Identification of 30 spliceogenic variants of BRCA2 exons 17 and 18.
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DOI：
10.1371/journal.pgen.1006691
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发表时间：
2017-03
期刊：
影响因子：
4.5
通讯作者：
Velasco EA
中科院分区：
文献类型：
--
作者：
Fraile-Bethencourt E;Díez-Gómez B;Velásquez-Zapata V;Acedo A;Sanz DJ;Velasco EA
乳腺癌基因BRCA 1和BRCA 2的突变筛查鉴定了很大一部分临床意义不确定（VUS）的变体，其功能和临床解释对基因组医学构成了挑战。同样，越来越多的证据表明，遗传变异可能对前体mRNA剪接产生有害影响。我们的目标是研究一组BRCA 2外显子17和18的变体对剪接的影响，以评估它们在遗传性乳腺癌中的作用，并确定可能构成剪接变异热点的关键调控元件。在pSAD载体中构建具有BRCA 2外显子14至20的剪接报告小基因（MGBR 2_ex14 -20）。使用剪接预测程序选择52种候选变体，通过定点诱变引入MGBR 2_ex14 -20中，并在MCF-7细胞中一式三份测定。野生型MGBR 2_ex14 -20产生预期大小（1，806个核苷酸）和结构（V1-[BRCA 2_exons_14 -20]-V2）的稳定转录物。通过微缺失进行的功能定位揭示了外显子17的3'端（c.7944-7973）和外显子18的5'端（c.7979-7988，c.7999-8013）上的外显子识别的必需序列。52种所选变体中的30种在具有>16种不同异常转录物的小基因测定中诱导异常剪接，其中外显子跳跃是最常见的事件。广泛的剪接基序受到影响，包括典型的剪接位点（15个变体），新的替代位点（3个变体），多聚嘧啶段（3个变体）和增强子/沉默子（9个变体）。根据美国医学遗传学和基因组学学会（ACMG）的指南，20种变异可被归类为致病性（c.7806-2A>G，c.7806 - 1G>A，c.7806-1G>T，c.7806-1_7806-2dup，c.7976+1G>A，c.7977-3_7978del，c.7977-2A>T，c.7977 - 1G>T，c.7977 - 1G>C，c.8009C>A，c.8331+1G>T和c.8331+ 2 T>C）或可能致病（c.7806-9T>G，c.7976G>C，c.7976G>A，c.7977-7C>G，c.7985C>G，c.8023A>G，c.8035G>T和c.8331G>A），占BRCA Share数据库中外显子17-18的所有致病/可能致病变体的30.8%。其余8种变体（c.7975 A>G、c.7977- 6 T>G、c.7988 A>T、c.7992 T>A、c.8007 A>G、c.8009 C>T、c.8009 C>G和c.8072 C>T）诱导部分剪接异常，具有重要的全长转录本比例（≥70%），因此它们仍被归类为VUS。因此，异常剪接在BRCA 2外显子17和18中尤其普遍，这是由于存在参与外显子识别的活性ESE。使用小基因的剪接功能测定对于剪接结果的初始表征和任何疾病基因的变体的后续临床解释是有价值的策略，尽管这些结果应尽可能针对患者RNA进行检查。很大一部分遗传性疾病的致病突变损害剪接。遗传疾病的大规模测序项目产生了成千上万的序列变异，需要功能和临床解释。我们已经表明，剪接报告乳腺癌基因BRCA 1和BRCA 2的小基因是有用的工具，功能测试DNA变异。在这项工作中，我们构建了一个7外显子BRCA 2小基因（外显子14至20），我们映射关键剪接调控序列，并测试了52个选定的变体外显子17和18检测乳腺癌患者。我们精确地定位了两个外显子上的三个DNA片段，推测它们含有剪接增强子序列。我们观察到总共有30种任何类型的变异破坏了剪接模式，考虑到其后果的严重性，我们将其中20种归类为致病性或可能致病性。我们还表明，广泛的剪接元件受到影响，包括典型的和新的5'和3'剪接位点，多嘧啶道和增强子和沉默序列。我们的结论是，剪接畸变是常见的遗传性乳腺癌和卵巢癌，小基因是有价值的工具，功能分类的任何人类疾病基因的DNA变异的剪接的观点。
Mutation screening of the breast cancer genes BRCA1 and BRCA2 identifies a large fraction of variants of uncertain clinical significance (VUS) whose functional and clinical interpretations pose a challenge for genomic medicine. Likewise, an increasing amount of evidence indicates that genetic variants can have deleterious effects on pre-mRNA splicing. Our goal was to investigate the impact on splicing of a set of reported variants of BRCA2 exons 17 and 18 to assess their role in hereditary breast cancer and to identify critical regulatory elements that may constitute hotspots for spliceogenic variants. A splicing reporter minigene with BRCA2 exons 14 to-20 (MGBR2_ex14-20) was constructed in the pSAD vector. Fifty-two candidate variants were selected with splicing prediction programs, introduced in MGBR2_ex14-20 by site-directed mutagenesis and assayed in triplicate in MCF-7 cells. Wild type MGBR2_ex14-20 produced a stable transcript of the expected size (1,806 nucleotides) and structure (V1-[BRCA2_exons_14–20]–V2). Functional mapping by microdeletions revealed essential sequences for exon recognition on the 3’ end of exon 17 (c.7944-7973) and the 5’ end of exon 18 (c.7979-7988, c.7999-8013). Thirty out of the 52 selected variants induced anomalous splicing in minigene assays with >16 different aberrant transcripts, where exon skipping was the most common event. A wide range of splicing motifs were affected including the canonical splice sites (15 variants), novel alternative sites (3 variants), the polypyrimidine tract (3 variants) and enhancers/silencers (9 variants). According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), 20 variants could be classified as pathogenic (c.7806-2A>G, c.7806-1G>A, c.7806-1G>T, c.7806-1_7806-2dup, c.7976+1G>A, c.7977-3_7978del, c.7977-2A>T, c.7977-1G>T, c.7977-1G>C, c.8009C>A, c.8331+1G>T and c.8331+2T>C) or likely pathogenic (c.7806-9T>G, c.7976G>C, c.7976G>A, c.7977-7C>G, c.7985C>G, c.8023A>G, c.8035G>T and c.8331G>A), accounting for 30.8% of all pathogenic/likely pathogenic variants of exons 17–18 at the BRCA Share database. The remaining 8 variants (c.7975A>G, c.7977-6T>G, c.7988A>T, c.7992T>A, c.8007A>G, c.8009C>T, c.8009C>G, and c.8072C>T) induced partial splicing anomalies with important ratios of the full-length transcript (≥70%), so that they remained classified as VUS. Aberrant splicing is therefore especially prevalent in BRCA2 exons 17 and 18 due to the presence of active ESEs involved in exon recognition. Splicing functional assays with minigenes are a valuable strategy for the initial characterization of the splicing outcomes and the subsequent clinical interpretation of variants of any disease-gene, although these results should be checked, whenever possible, against patient RNA. A significant proportion of disease-causing mutations of inherited disorders impair splicing. Massive sequencing projects of genetic diseases generate thousands of sequence variations that require functional and clinical interpretations. We have shown that splicing reporter minigenes of the breast cancer genes BRCA1 and BRCA2 are useful tools to functionally test DNA variants. In this work, we have constructed a 7-exon BRCA2 minigene (exons 14 to 20) where we mapped critical splicing regulatory sequences and tested 52 selected variants of exons 17 and 18 detected in breast cancer patients. We finely located three DNA segments on both exons that presumably contain splicing enhancer sequences. We observed that a total of 30 variants of any type disrupted the splicing patterns and, given the severity of their outcomes, we classified 20 of them as pathogenic or likely pathogenic. We also showed that a wide range of splicing elements were affected including canonical and novel 5’ and 3’ splice sites, the polypyrimidine tract and enhancer and silencer sequences. We concluded that splicing aberrations are frequent in Hereditary Breast and Ovarian Cancer and that minigenes are valuable tools to functionally classify DNA variants of any human disease gene under the splicing viewpoint.