Screening of <i>BRCA1/2</i> deep intronic regions by targeted gene sequencing identifies the first germline <i>BRCA1</i> variant causing pseudoexon activation in a patient with breast/ovarian cancer.

Abstract

Genetic analysis of <i>BRCA1</i> and <i>BRCA2</i> for the diagnosis of hereditary breast and ovarian cancer (HBOC) is commonly restricted to coding regions and exon-intron boundaries. Although germline pathogenic variants in these regions explain about ~20% of HBOC cases, there is still an important fraction that remains undiagnosed. We have screened <i>BRCA1/2</i> deep intronic regions to identify potential spliceogenic variants that could explain part of the missing HBOC susceptibility. We analysed <i>BRCA1/2</i> deep intronic regions by targeted gene sequencing in 192 high-risk HBOC families testing negative for <i>BRCA1/2</i> during conventional analysis. Rare variants (MAF <0.005) predicted to create/activate splice sites were selected for further characterisation in patient RNA. The splicing outcome was analysed by RT-PCR and Sanger sequencing, and allelic imbalance was also determined when heterozygous exonic loci were present. A novel transcript was detected in <i>BRCA1</i> c.4185+4105C>T variant carrier. This variant promotes the inclusion of a pseudoexon in mature mRNA, generating an aberrant transcript predicted to encode for a non-functional protein. Quantitative and allele-specific assays determined haploinsufficiency in the variant carrier, supporting a pathogenic effect for this variant. Genotyping of 1030 HBOC cases and 327 controls did not identify additional carriers in Spanish population. Screening of <i>BRCA1/2</i> intronic regions has identified the first <i>BRCA1</i> deep intronic variant associated with HBOC by pseudoexon activation. Although the frequency of deleterious variants in these regions appears to be low, our study highlights the importance of studying non-coding regions and performing comprehensive RNA assays to complement genetic diagnosis.