BRCA1 and BRCA2 (BRCA1/2) are the main responsible genes of hereditary breast and ovarian cancers (HBOC). Genetic testing of BRCA1/2 for diagnostic purposes, comprises only DNA coding sequences and exon-intron boundaries, leaving behind unscreened regions that may harbour cancer-associated mutations. In this work we hypothesized that BRCA1/2 deep intronic regions may contain mutations that could have an impact on splicing and explain part of the missing HBOC susceptibility.
BRCA1/2 deep intronic regions were massively sequenced in DNA from blood samples of 192 patients with strong HBOC family history. Rare genetic variants (minor allelic frequency of <0.4%) were selected. We next used bioinformatics prediction tools to prioritize those variants that may have a deleterious impact on splicing. The predicted effects were validated with patient RNA.
We identified 32 BRCA1 and 41 BRCA2 deep intronic variants with potential deleterious effects. However, further analysis with patient RNA only confirmed one BRCA1 variant as being cancer-associated by generating aberrant splicing. RNA assays revealed the inclusion of 114 intronic nucleotides upstream the variant, creating a new exon in the coding sequence that leads to a truncated protein. Quantitative techniques showed BRCA1 haploinsufficiency in the patient sample, confirming its association with breast/ovarian cancer risk. We additionally screened this variant in 1030 HBOC patients, but we did not identify any other carrier individual.
Here we describe the first deep intronic cancer-associated variant occurring at BRCA1 locus. Although the frequency of deleterious variants in BRCA1/2 deep intronic regions appears to be low in our population, our findings highlight that non-coding regions may contain pathogenic variants and that RNA analysis complements genetic diagnosis of familial breast and ovarian cancers.