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Journal of Clinical Oncology, Vol 25, No 31 (November 1), 2007: pp. 5036-5038 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2007.13.5442
In ReplyMolecular Biology Department, Instituto Português de Oncologia de Lisboa, Francisco Gentil, Portugal
Breast Cancer Risk Evaluation Clinic, Instituto Português de Oncologia de Lisboa, Francisco Gentil, Portugal
Molecular Biology Department and Breast Cancer Risk Evaluation Clinic, Instituto Português de Oncologia de Lisboa, Francisco Gentil, Portugal We thank Dr Díez and his collaborators for their interest in our study.1 In their letter, they make two major comments that could raise concerns about our results; the first refers to the fact that the rearrangement we screened (the insertion of an Alu fragment in position c.156_157 of BRCA2 causing exon 3 skipping at the mRNA level) was confirmed in more than one affected member in only one family. The second comment questions the functional implications of exon 3 skipping for the BRCA2 protein. When our article was submitted for publication, the confirmation of the mutation in further affected members, besides the index, had only been done in one family (family 40); however, a common haplotype segregated with this mutation in all positive individuals from 14 families and was absent in noncarriers. This observation is a compelling piece of evidence to establish the c.156_157insAlu as the cause of cancer predisposition in these families, and we now have more data on segregation analysis that reinforces this statement. Since all new high-risk registered patients counseled in our clinic are first screened for the Portuguese founder mutation, we have diagnosed seven new, apparently nonrelated c.156_157insAlu–positive families since the publication of our study. The family history of these additional positive patients was exhaustively analyzed to investigate any link to the families already described. Indeed, two (patients 385 and 502) of the seven patients belonged to families 52 and 40 described in our article. Families 40 and 52 had the most striking pedigrees identified as c.156_157insAlu positive, because only one generation in each was affected by cancer and it was not possible to determine which family side carried the mutation. The addition of new family information clearly shows the autosomic dominant pattern of cancer predisposition inheritance, from the paternal side for family 40, and from the maternal side for family 52 (Fig 1). In total, we have now identified 22 nonrelated c.156_157insAlu positive families and have tested 80 relatives for the family mutation. Fifty five relatives, 30 of which affected with cancer, were found to be positive for c.156_157insAlu. More than one affected member has been tested in eight families; in the remaining pedigrees the other affected patients were either deceased or did not consent on genetic screening.
Regarding the second comment, we agree that BRCA2 exon 3 skipping does not imply the existence of a pathogenic mutation in this gene. When we implemented our three-step polymerase chain reaction (PCR) method for the diagnosis and confirmation of c.156_157insAlu, different reverse transcriptase (RT)-PCR primers were tested. Similarly to Santarosa et al,2 when using primers to amplify exons 2 to 5 of BRCA2, we also observed an extra RT-PCR band, consistent with exon 3 skipping. This extra band was observed not only in our mutation-positive individuals, but also in c.156_157insAlu negative individuals, in normal controls, in patients with other known pathogenic BRCA1/2 mutations and also in adenomatosis polyposis coli (APC) patients (Fig 2). However, analyzing the same set of samples by RT-PCR, using oligonucleotides to amplify exons 2 to 9 (primers 1FcDNA and 10RcDNA as referred to in our article),3 exon 3 skipping was only detected in c.156_157insAlu–positive patients. In agreement with Santarosa et al,2 our observations suggested the existence of an ubiquitous alternative BRCA2 splicing product that lacks exon 3 and most probably exon 10 (since it is not amplified when using primer 10RcDNA, that binds to exon 10 of BRCA2). Therefore, this pair of primers that amplify exons 2 to 9 are the correct option to confirm exon 3 skipping resulting from the insertion of an Alu fragment in BRCA2 exon 3. In fact, these oligonucleotides amplify part of the main BRCA2 transcript and not the alternative one. Consequently, to definitely confirm positive BRCA2 individuals with the Portuguese founder mutation, we included in our diagnostic PCR methodology both the DNA confirmation of the mutational event (we directly sequence the Alu fragment insertion) and the RT-PCR confirmation of the Alu-mediated exon 3 skipping using primers 1FcDNA and 10RcDNA.
The occurrence of BRCA2 alternative splicing originates transcripts with exon 3 skipping2,4 and other isoforms.5,6 Products of BRCA2 alternative splicing are observed in healthy controls,2,6 have been described in different tumor specimens2,5-7 and have probably been erroneously associated with intronic variants.7 Therefore, the intronic variants referred to by Díez et al may not be the cause of exon 3 skipping and most probably represent BRCA2 polymorphisms without pathogenic relevance. Exon 3 skipping in a BRCA2 alternative transcript results from a physiological splicing mechanism whose relevance for cellular function is still unknown. In contrast, the c.156_157insAlu-mediated exon 3 skipping occurs in the main 27-exon BRCA2 transcript with the resulting BRCA2 protein lacking the two transactivation domains. This loss is predicted to cause the BRCA2 protein to be dysfunctional as in the case of the other rearrangement that also mediates exon 3 skipping in the main BRCA2 transcript.3 Although c.156_157insAlu is still the most frequent BRCA2 rearrangement described due to its founder effect in the Portuguese population, the identification of more BRCA2 rearrangements8-10 is clearly important, since it will definitely contribute to the genetic screening of breast cancer risk families without identified cancer predisposition mutations. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The author(s) indicated no potential conflicts of interest.
REFERENCES
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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