Article Text

Download PDFPDF
Original article
Screening of BRCA1/2 deep intronic regions by targeted gene sequencing identifies the first germline BRCA1 variant causing pseudoexon activation in a patient with breast/ovarian cancer
  1. Gemma Montalban1,
  2. Sandra Bonache1,
  3. Alejandro Moles-Fernández1,
  4. Alexandra Gisbert-Beamud1,
  5. Anna Tenés2,
  6. Vanessa Bach1,
  7. Estela Carrasco3,
  8. Adrià López-Fernández3,
  9. Neda Stjepanovic3,4,
  10. Judith Balmaña3,4,
  11. Orland Diez1,2,
  12. Sara Gutiérrez-Enríquez1
  1. 1 Oncogenetics Group, Vall d’Hebron Institut d’Oncologia, Barcelona, Spain
  2. 2 Area of Clinical and Molecular Genetics, University Hospital of Vall d’Hebron, Barcelona, Spain
  3. 3 High Risk and Cancer Prevention Group, Vall d’Hebron Institut d’Oncologia, Barcelona, Spain
  4. 4 Medical Oncology Department, University Hospital of Vall d’Hebron, Barcelona, Spain
  1. Correspondence to Dr. Orland Diez and Dr. Sara Gutiérrez-Enríquez, Oncogenetics Group, Vall d’Hebron Institute of Oncology (VHIO), Barcelona, 08035, Spain; odiez{at}, sgutierrez{at}


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.

  • Brca1/2
  • hereditary breast and ovarian cancer
  • deep intronic regions
  • splicing
  • pseudoexon

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.


  • 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.