BRCA2 splice site mutations in an Italian breast/ovarian cancer family.

We read with great interest the article by Pensabene et al. [1] who reported a case in which mutational analysis revealed two mutations in the BRCA2 gene in two members of an Italian breast/ovarian cancer family. One mutation was a variation which affects the last nucleotide of the exon 7 (c.859G>A) and entails an amino acid substitution in the protein sequence (p.V211I). The other mutation was an intronic change close to the exon 13 (IVS132A>T). Both variants are localised in critical splicing points. To verify their effects on messenger RNA (mRNA) maturation, total RNA from peripheral blood lymphocytes was analysed by RT–PCR with PCR amplification extended to adjacent exons in each case. Anomalous PCR products obtained revealed that both changes led to altered mRNAs, one lacking exon 7, with a premature stop codon in exon 9, and the other lacking exon 14, with an anomalous stop codon in exon 15. To evaluate if both mutations were in the same BRCA2 allele (cis), the authors carried out a retrotranscription analysis using a gene-specific primer in exon 14 and PCR amplification of the complementary DNA obtained using a forward primer in exon 6 and a reverse primer in exon 10. If the mutations were in cis, a 0.9-kb band corresponding to the wild-type allele should be obtained (because the allele without exons 7 and 14 would not be amplified), and if the mutations were in trans, only a 0.8-kb band corresponding to the allele lacking exon 7 (carrying the c.859G>A change) would be obtained. However, the authors say that two bands were obtained (0.9 and 0.8 kb), the former corresponding to a product containing exons 6–10 and the small one to a product lacking exon 7. No other explanation is given in the paper for this contradictory result, and there is no clear conclusion about the cis or trans position of both mutations, although this point is of most importance. There are a number of reasons that lead to assume a cis position for both mutations. While BRCA2 monoallelic truncating mutations mainly confer high risks of breast and ovarian cancers [2], biallelic BRCA2 mutation carriers may present with different severe forms of Fanconi anaemia (FA) subtype D1 [3] with high risks of childhood cancer, particularly Wilms’ tumours, brain tumours and acute myelogenous leukaemia [4]. On the contrary, breast and ovarian cancers have not been reported in biallelic mutation carriers, probably due to the very high mortality in childhood. The pedigree reported by Pensabene et al. showed multiple cases of breast cancer in firstand second-degree family members and other cancers (ovary, prostate and uterus) and no traces of FA-associated tumours. Furthermore, the authors refer that the family history of at least three generations was acquired by pedigree construction and only maternal family history of cancer is reported for both carriers, indicating that mutated alleles were inherited only through this branch. We agree with the authors that the two BRCA2 mutations identified affect the mRNA splicing fidelity and expression and lead to a truncated nonfunctioning BRCA2 protein. However, the issue of a possible alternative splicing, the possibility that in one of the mutated alleles the pre-mRNAs would be alternatively correctly spliced, giving both normal and abnormal mRNA products, and an explanation of a cis/trans transmission of both mutations should be better clarified in order to know the specific role of BRCA2 in the carcinogenesis in this family and the cancer spectrum phenotype related to each mutation.