Contribution of bioinformatics predictions and of functional splicing assays to the interpretation of unclassified variants of the BRCA genes

Jean Christophe Théry (1) , Sophie Krieger (2) , Pascaline Gaildrat (1) , Françoise Révillon (3) , Marie-Pierre Buisine (1) , Audrey Killian (1) , Christiane Duponchel (1) , Antoine Rousselin (2) , Dominique Vaur (2) , Jean François Peyrat (3) , Pascaline Berthet (4) , Thierry Frébourg (1, 5) , Alexandra Martins (1) , Agnès Hardouin (2) , Mario Tosi (1)

1 Génétique médicale et fonctionnelle du cancer et des maladies neuropsychiatriques
2 Laboratoire de biologie clinique et oncologique
3 Laboratoire d'Oncologie Moléculaire Humaine
4 Consultation d'Oncogénétique
5 Service de génétique [Rouen]

A large fraction of sequence variants of unknown significance (VUS) of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 may induce splicing defects. We analyzed 53 VUS of BRCA1 or BRCA2, detected in consecutive molecular screenings, by using 5 splicing prediction programs and we classified them into two groups according to the strength of the predictions. In parallel, we tested them by using functional splicing assays. Ten VUS were predicted by two or more programs to induce a significant reduction of splice site strength, or activation of cryptic splice sites, or generation of new splice sites. Minigene-based splicing assays confirmed 4 of these predictions. Five additional VUS, all at internal exon positions, were not predicted to induce alterations of splice sites, but revealed variable levels of exon skipping, most likely induced by the modification of exonic splicing regulatory elements. We provide new data in favor of the pathogenic nature of the variants BRCA1 c.212+3A>G and BRCA1 c.5194-12G>A, which induced aberrant out-of-frame mRNA forms. Moreover, the novel variant BRCA2 c.7977-7C>G induced in frame inclusion of 6 nt from the 3' end of intron 17. The novel variants BRCA2 c.520C>T and BRCA2 c7992T>A induced incomplete skipping of exons 7 and 18, respectively. This work highlights the contribution of splicing minigene assays to the assessment of pathogenicity, not only when patient RNA is not available, but also as a tool to improve the accuracy of bioinformatics predictions.

Mots clés: sequence variants of unknown significance; splicing defects; BRCA1 and BRCA2

Jean Christophe Théry, Sophie Krieger, Pascaline Gaildrat, Françoise Révillon, Marie-Pierre Buisine, et al.. Contribution of bioinformatics predictions and of functional splicing assays to the interpretation of unclassified variants of the BRCA genes: variants of unknown significance of the BRCA genes. European Journal of Human Genetics, 2011, ⟨10.1038/ejhg.2011.100⟩. ⟨hal-00652134⟩