BRCA1 and BRCA2 whole cDNA analysis in unsolved hereditary breast/ovarian cancer patients

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 del 21 and BRCA2 Delta 8q_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 del 20A, Delta 22 and del 20A+ Delta 22 transcripts. In silico analysis and semi-quantitative data identified the polymorphism BRCA2 c.8755-66T>C as a potential modifier of Delta 22 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. (C) 2021 Elsevier Inc. All rights reserved.

Automated summary

Germline pathogenic variants in BRCA1 and BRCA2 genes explain hereditary breast/ovarian cancer cases. Analysis of deep intronic variants in a large cohort identified rare mRNA alterations in Spanish population. RNA analysis complements DNA-based strategies for detecting potential alterations.