Mis-splicing and breast cancer: systematic analysis of splicing variants of BRCA2 exons 2-9 by minigene assays

Paper minigene BRCA2 exons 2-9"Mis-splicing in breast cancer: identification of pathogenic BRCA2 variants by systematic minigene assays"<b>Abstract </b><br>Splicing disruption is a common mechanism of gene inactivation associated with germline variants of susceptibility genes. To study the role of BRCA2 mis-splicing in hereditary breast/ovarian cancer (HBOC), we performed a comprehensive analysis of variants from BRCA2 exons 2 to 9, as well as the initial characterization of the regulatory mechanisms of such exons. A pSAD-based minigene with exons 2-9 was constructed and validated in MCF-7 cells, producing the expected transcript (1,016-nt/V1-BRCA2_exons_2-9-V2). DNA variants from mutational databases were analyzed by NNSplice and Human Splicing Finder softwares. To refine ESE-variant prediction, we mapped the regulatory regions through a functional strategy whereby 26 exonic microdeletions were introduced into the minigene and tested in MCF-7 cells. Thus, we identified nine spliceogenic ESE-rich intervals where ESE-variants may be located. Combining bioinformatics and microdeletion assays, 83 variants were selected and genetically engineered in the minigene. Fifty-three changes impaired splicing: 28 variants disrupted the canonical sites, four created new ones, 10 abrogated enhancers, eight created silencers and three caused a double-effect. Notably, nine spliceogenic-ESE variants were located within ESE-containing intervals. Capillary electrophoresis and sequencing revealed more than 23 aberrant transcripts, where exon skipping was the most common event. Interestingly, variant c.67G&gt;A triggered the usage of a non-canonical GC-donor 4-nt upstream. Thirty-six variants that induced severe anomalies (&gt;60% aberrant transcripts) were analyzed according to the ACMG guidelines. Thus, 28 variants were classified as pathogenic, five as likely pathogenic and three as variants of uncertain significance. Interestingly, 13 VUS were reclassified as pathogenic or likely pathogenic variants.<br>In conclusion, a large fraction of BRCA2 variants (~64%) provoked splicing anomalies lending further support to the high prevalence of this disease-mechanism. The low accuracy of ESE-prediction algorithms may be circumvented by functional ESE-mapping that represents an optimal strategy to identify spliceogenic ESE-variants. Finally, systematic functional assays by minigenes depict a valuable tool for the initial characterization of splicing anomalies and the clinical interpretation of variants.<br><br>

《乳腺癌中的错误剪接：通过系统性迷你基因实验鉴定致病性BRCA2变异》——BRCA2外显子2-9迷你基因（minigene）研究
**摘要**
剪接紊乱是与易感基因生殖系变异相关的基因失活的常见机制。为研究BRCA2错误剪接在遗传性乳腺癌/卵巢癌（hereditary breast/ovarian cancer, HBOC）中的作用，我们对BRCA2外显子2至9的变异开展了全面分析，并初步解析了这些外显子的调控机制。我们构建了携带有外显子2-9的基于pSAD的迷你基因，并在MCF-7细胞中进行了验证，得到了预期的转录本（transcript）(1016 nt/V1-BRCA2_exons_2-9-V2)。我们通过NNSplice和Human Splicing Finder两款软件对突变数据库中的DNA变异进行了分析。为优化外显子剪接增强子（exonic splicing enhancer, ESE）变异的预测效果，我们通过功能实验策略绘制了调控区域图谱：将26个外显子微缺失引入迷你基因，并在MCF-7细胞中进行检测。由此我们鉴定出9个富含ESE的剪接致病区间，ESE变异可能位于此类区间内。结合生物信息学分析与微缺失实验，我们筛选出83个变异，并在迷你基因中进行了基因工程改造。其中53个变异会损害剪接功能：28个变异破坏了经典剪接位点，4个变异产生了新的剪接位点，10个变异消除了剪接增强子，8个变异产生了剪接沉默子，另有3个变异同时产生双重效应。值得注意的是，9个剪接致病ESE变异位于包含ESE的区间内。毛细管电泳与测序结果显示存在超过23种异常转录本，其中以外显子跳跃最为常见。有趣的是，变异c.67G>A会激活上游4个核苷酸位置的非经典GC供体剪接位点。我们依据美国医学遗传学与基因组学学会（American College of Medical Genetics and Genomics, ACMG）指南，对36个会引发严重剪接异常（异常转录本占比＞60%）的变异开展了分析。据此，28个变异被归类为致病性变异，5个为可能致病性变异，3个为意义未明变异（variants of uncertain significance, VUS）。值得关注的是，13个VUS被重新归类为致病性或可能致病性变异。

综上，大部分BRCA2变异（约64%）会引发剪接异常，进一步印证了该致病机制在疾病发生中的高占比。ESE预测算法的准确性较低这一问题可通过功能ESE图谱分析得以解决，该方法是鉴定剪接致病ESE变异的最优策略。最后，基于迷你基因的系统性功能实验可为剪接异常的初步解析以及变异的临床解读提供极具价值的研究工具。