Université d'Angers, Inserm, CRCINA, 49000, Angers, France.
Institut de Cancérologie de l'Ouest - Paul Papin, 49000, Angers, France.
Mol Diagn Ther. 2020 Apr;24(2):233-243. doi: 10.1007/s40291-020-00452-z.
The identification of pretherapeutic somatic BRCA variants can have considerable clinical impact given that they affect response to the new poly (ADP-ribose) polymerase (PARP)-targeted therapy. One major issue with this type of testing is the identification of splicing variants of uncertain significance (VUS) on degraded somatic messenger RNA. It is therefore important to be able to quickly characterize these splice variants.
As part of PARP inhibitor targeted therapy, we have investigated a method for the direct confirmation of potential pathogenic somatic splice variants of BRCA1 found in fixed tumor samples. Previously these VUS have commonly only been tested by in silico analysis.
Five BRCA1 variants affecting splicing were characterized from formalin-fixed, paraffin-embedded (FFPE) ovarian carcinoma tissues by next-generation sequencing (NGS). Three patient samples had already been functionally characterized and were used as controls. Total somatic RNA from samples was extracted, reverse-transcribed, and amplified with several primer pairs encompassing the target exon. The polymerase chain reaction (PCR) products were analyzed by capillary gel electrophoresis to assess possible changes in size due to splicing alterations. Finally, we confirmed our results by cloning, followed by Sanger sequencing, and analyzed the expression of the aberrant forms.
Our molecular approach made it possible to visualize the splicing outcomes of three variants (c.5194-2A>G, c.5434C>G, and c.547+1G>A) already identified and present in databases and/or identified with prediction tools (ClinVar, UMD, ARUP Utah database, and Human Splice Finder splices sites prediction) and to confirm their exon skipping consequences, their expression in tumors, and thus their pathogenicity. The c.4484+5G>A variant was not found in databases and was predicted to have no impact on splicing, but was found to display altered processing in tumor tissue. This variant also had a major detrimental impact on transcriptional expression.
In a break from purely in silico approaches, we propose a simple and rapid pretherapeutic functional analysis of somatic BRCA1 variants potentially involved in splicing alterations. This approach will allow more ovarian cancer patients to benefit from new therapies targeting PARP.
由于治疗前的体细胞 BRCA 变异会影响对新型多聚（ADP-核糖）聚合酶（PARP）靶向治疗的反应，因此其具有重要的临床意义。此类检测的一个主要问题是鉴定降解的体细胞信使 RNA 上不确定意义的剪接变异体（VUS）。因此，能够快速鉴定这些剪接变体非常重要。
作为 PARP 抑制剂靶向治疗的一部分，我们研究了一种用于直接确认在固定肿瘤样本中发现的潜在致病性体细胞 BRCA1 剪接变体的方法。以前，这些 VUS 通常仅通过计算机分析进行测试。
通过下一代测序（NGS）对来自福尔马林固定、石蜡包埋（FFPE）卵巢癌组织的 5 个影响剪接的 BRCA1 变体进行了特征描述。其中 3 个患者样本已经进行了功能鉴定，用作对照。从样本中提取总体细胞 RNA，反转录，并使用几对包含靶外显子的引物进行扩增。通过毛细管凝胶电泳分析聚合酶链反应（PCR）产物，以评估由于剪接改变导致的大小变化。最后，我们通过克隆、随后的 Sanger 测序来确认我们的结果，并分析异常形式的表达。
我们的分子方法使得可视化三个已经在数据库中或通过预测工具（ClinVar、UMD、ARUP Utah 数据库和 Human Splice Finder 剪接位点预测）识别和鉴定的变体（c.5194-2A>G、c.5434C>G 和 c.547+1G>A）的剪接结果成为可能，并确认它们的外显子跳跃后果、在肿瘤中的表达，从而确定其致病性。c.4484+5G>A 变体未在数据库中找到，预测其对剪接无影响，但在肿瘤组织中发现其处理方式发生改变。该变体还对转录表达产生重大不利影响。
与纯粹的计算机方法不同，我们提出了一种简单快速的治疗前体细胞 BRCA1 变体功能分析方法，该方法可能涉及剪接改变。这种方法将使更多的卵巢癌患者受益于靶向 PARP 的新疗法。
