American Association for Cancer Research
10780432ccr121100-sup-ccr-12-1100fig1-2tab1.pdf (764.5 kB)

Supplementary Figures 1 - 3 and Table 1 from BRCA2 Deep Intronic Mutation Causing Activation of a Cryptic Exon: Opening toward a New Preventive Therapeutic Strategy

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journal contribution
posted on 2023-03-31, 17:07 authored by Olga Anczuków, Monique Buisson, Mélanie Léoné, Christine Coutanson, Christine Lasset, Alain Calender, Olga M. Sinilnikova, Sylvie Mazoyer

PDF file, 764K, BRCA2 exon 12 to exon 13 gene, transcript and protein sequences (Supp Fig 1); Enhancer motifs contained within the cryptic exon and the constitutive surrounding exons (Supp Fig 2); Conservation of the cryptic exon across species (Supp Fig 3); Primers used for BRCA1 and BRCA2 routine mRNA analyses (Supp Table 1).



Purpose: Diagnostic screening of the BRCA1/2 genes in breast cancer families is mostly done on genomic DNA. For families with a very strong family history and no mutation identified in the coding sequences or the exon–intron boundaries, BRCA1/2 transcripts' analysis is an efficient approach to uncover gene inversion and pre-mRNA splicing defaults missed by conventional DNA-based protocols.Experimental Design: We analyzed RNA from patients of negative BRCA families by reverse transcriptase PCR and identified an insertion in one family that we characterized by sequencing and by using a minigene splicing assay. More than 2,000 additional BRCA1/2 negative families were subsequently screened for this mutation using a dedicated PCR approach.Results: Nine families were found to harbor a BRCA2 mutant transcript containing a 95-nucleotide cryptic exon between exons 12 and 13. This cryptic exon results from a new mutation located deep into intron 12, c.6937+594T > G, which reinforces the strength of a preexisting 5′ splice site, turning it into a perfect consensus sequence. It is systematically included in transcripts produced by the mutant allele in cells from mutation carriers or produced by a mutant splicing reporter minigene. The inclusion of the cryptic exon was prevented when we cotransfected the minigene with antisense oligonucleotides complementary to the 3′ or mutated 5′ splice sites.Conclusion: This first deep intronic BRCA mutation emphasizes the importance of analyzing RNA to provide comprehensive BRCA1/2 diagnostic tests and opens the possibility of using antisense therapy in the future as an alternative strategy for cancer prevention. Clin Cancer Res; 18(18); 4903–9. ©2012 AACR.