Germline pathogenic variants in BRCA1 and BRCA2 genes (BRCA1/2) explain an important fraction of hereditary breast/ovarian cancer (HBOC) cases. Genetic testing generally involves examining coding regions and exon/intron boundaries, thus the frequency of deleterious variants in non-coding regions is unknown. Here we analysed BRCA1/2 whole cDNA in a large cohort of 320 unsolved high-risk HBOC cases in order to identify potential splicing alterations explained by variants in BRCA1/2 deep intronic regions. Whole RNA splicing profiles were analysed by RT-PCR using Sanger sequencing or high-resolution electrophoresis in a QIAxcel instrument.

Known predominant BRCA1/2 alternative splicing events were detected, together with two novel events BRCA1 ▼21 and BRCA2 Δ18q_27p. BRCA2 exon 3 skipping was detected in one patient (male) affected with breast cancer, caused by a known Portuguese founder mutation (c.156_157insAluYa5). An altered BRCA2 splicing pattern was detected in three patients, consisting in the up-regulation of ▼20A, Δ22 and ▼20A+Δ22 transcripts. In silico analysis and semi-quantitative data identified the polymorphism BRCA2 c.8755-66T>C as a potential modifier of Δ22 levels.

Our findings suggest that mRNA alterations in BRCA1/2 caused by deep intronic variants are rare in Spanish population. However, RNA analysis complements DNA-based strategies allowing the identification of alterations that could go undetected by conventional testing.

BRCA1和BRCA2基因 ( BRCA1/2 ) 中的种系致病变异解释了遗传性乳腺癌/卵巢癌 (HBOC) 病例的重要部分。基因检测通常涉及检查编码区和外显子/内含子边界，因此非编码区有害变异的频率是未知的。在这里，我们分析了 320 个未解决的高风险 HBOC 病例的大型队列中的BRCA1/2全 cDNA，以确定由BRCA1/2深内含子区域中的变异解释的潜在剪接改变。在 QIAxcel 仪器中使用 Sanger 测序或高分辨率电泳通过 RT-PCR 分析全 RNA 剪接图谱。

检测到已知的主要BRCA1/2可变剪接事件，以及两个新事件BRCA1 ▼21 和BRCA2 Δ18q_27p。在一名患有乳腺癌的患者（男性）中检测到BRCA2外显子 3 跳跃，这是由已知的葡萄牙创始人突变（c.156_157insAluYa5）引起的。在三名患者中检测到改变的BRCA2剪接模式，包括 ▼20A、Δ22 和 ▼20A+Δ22 转录物的上调。计算机分析和半定量数据确定多态性BRCA2 c.8755-66T>C 作为 Δ22 水平的潜在调节剂。

我们的研究结果表明，由深内含子变异引起的BRCA1/2 mRNA 改变在西班牙人群中很少见。然而，RNA 分析补充了基于 DNA 的策略，允许识别传统测试无法检测到的改变。