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Multiple sequence variants of BRCA2 exon 7 alter splicing regulation
  1. Pascaline Gaildrat1,2,3,
  2. Sophie Krieger1,4,5,
  3. Daniela Di Giacomo1,2,
  4. Julie Abdat1,
  5. Françoise Révillion6,
  6. Sandrine Caputo7,
  7. Dominique Vaur1,4,
  8. Estelle Jamard4,
  9. Elodie Bohers1,
  10. Danielle Ledemeney4,
  11. Jean-Philippe Peyrat6,
  12. Claude Houdayer8,
  13. Etienne Rouleau7,
  14. Rosette Lidereau7,
  15. Thierry Frébourg1,2,3,
  16. Agnès Hardouin1,4,
  17. Mario Tosi1,2,
  18. Alexandra Martins1,2
  1. 1Inserm U1079, Rouen, France
  2. 2Institute for Research and Innovation in Biomedicine, University of Rouen, Rouen, France
  3. 3Department of Genetics, Rouen University Hospital, Rouen, France
  4. 4Laboratoire de Biologie Clinique et Oncologique, Centre François Baclesse, Caen, France
  5. 5University of Caen, EA4656, France
  6. 6Laboratoire d'Oncologie Moléculaire Humaine, Centre Oscar Lambret, Lille, France
  7. 7Service d'Oncogénétique, Institut Curie, Hôpital René Huguenin, St Cloud, France
  8. 8Service de Génétique, Institut Curie et Université Paris Descartes, Paris, France
  1. Correspondence to Dr Pascaline Gaildrat, Inserm UMR 1079, Faculté de Médecine, 22 boulevard Gambetta, Rouen Cedex 1 76183, France; pascaline.gaildrat{at}


Background Exonic variants of unknown biological significance (VUS) identified in patients can affect mRNA splicing, either by changing 5′ or 3′ splice sites or by modifying splicing regulatory elements. Bioinformatic predictions of these elements are still inaccurate and only few such elements have been functionally mapped in BRCA2. We studied the effect on splicing of eight exon 7 VUS, selected from the French UMD-BRCA2 mutation database.

Methods We performed splicing minigene assays and analyses of patient RNA. We also developed a pyrosequencing-based quantitative assay, to measure, in patient RNA, the relative contribution of each allele to the production of exon 7-containing transcripts. Moreover, an exonic splicing enhancer (ESE)-dependent minigene assay was used to evaluate the splicing regulatory properties of wild-type and mutant segments.

Results Six out of the eight variants induced splicing defects. In the minigene assay, c.517G>T and c.631G>A altered the natural splice sites, c.572A>G created a new 5′ splice site, and c.520C>T, c.587G>A and c.617C>G induced exon 7 skipping (66%, 25% and 46%, respectively). Pyrosequencing of patient RNA confirmed these levels of exon skipping for c.520C>T and c.617C>G. Results from the ESE-dependent minigene assay indicated that c.520C>T and c.587G>A disturb splicing regulatory elements.

Conclusions BRCA2 exon 7 splicing is regulated by multiple exonic elements and is sensitive to disease-associated sequence variations. Measurements of allelic imbalance in patient-derived RNA and/or quantitative analyses using minigene assays provide valuable estimates of the extent of partial splicing defects. Assessment of pathogenicity of variants with partial splicing effect awaits additional evidence and especially the completion of segregation analyses.

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