Background Genetic analysis of BRCA1 and BRCA2 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 BRCA1/2 deep intronic regions to identify potential spliceogenic variants that could explain part of the missing HBOC susceptibility.
Methods We analysed BRCA1/2 deep intronic regions by targeted gene sequencing in 192 high-risk HBOC families testing negative for BRCA1/2 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.
Results A novel transcript was detected in BRCA1 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.
Conclusion Screening of BRCA1/2 intronic regions has identified the first BRCA1 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.
- hereditary breast and ovarian cancer
- deep intronic regions
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Contributors GM, OD and SG-E designed the study. GM conducted RNA experiments and drafted and edited the manuscript. GM, SB, AT, AG-B and VB performed experiments and procedures. AM-F performed bioinformatics analysis. EC, AL-F, NS and JB provided samples and patient data. OD and SG-E supervised experiments. All authors read and reviewed the manuscript.
Funding This work was supported by Spanish Instituto de Salud Carlos III (ISCIII) funding, an initiative of the Spanish Ministry of Economy and Innovation partially supported by European Regional Development FEDER Funds: FIS PI12/02585 and PI15/00355 (to O Diez), PI13/01711 and PI16/01218 (to SG-E). SG-E and SB are supported by the Miguel Servet Program (CP16/00034) and Asociación Española Contra el Cáncer (AECC) contract, respectively.
Competing interests None declared.
Patient consent Not required.
Ethics approval Clinical Research Ethics Committee (CEIC), Vall d’Hebron Research Institute (VHIR).
Provenance and peer review Not commissioned; internally peer reviewed.
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