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Journal of Clinical Oncology, Vol 25, No 31 (November 1), 2007: pp. 5035-5036 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2007.13.4346
Caution Should Be Used When Interpreting Alterations Affecting the Exon 3 of the BRCA2 Gene in Breast/Ovarian Cancer FamiliesServei de Genética, Hospital de la Santa Creu i Sant Pau, Programa de Medicina Molecular i Genètica, Hospital Universitari Vall d'Hebron, Barcelona, Spain
Servei d'Oncologia Mèdica, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
Consulta d'alt Risc i Prevenció del Càncer, Servei d’Oncologia Mèdica, Hospital Universitari Vall d'Hebron, Barcelona, Spain
Unitat de Consell Genètic, Servei de Prevenció i Control del Cáncer, Institut Català d'Oncologia, Barcelona, Spain, To the Editor: We read with great interest the article by Machado et al1 on the mutational screening of BRCA1 and BRCA2 genes in Portuguese breast/ovarian cancer families. During full gene BRCA1/2 screening, a large insertion in exon 3 of BRCA2 was recurrently observed. Characterization of this event revealed an Alu insertion in nucleotide c.156_157 of BRCA2. The authors demonstrated that the c.156_157insAlu is a founder mutation of Portuguese origin and is the most frequent BRCA2 rearrangement described to date. Amplification of cDNAs from positive probands revealed the in-frame deletion of exon 3, which resulted in the fusion of exon 2 with exon 4. According to their results, this alteration is likely to be the cause of the high-risk phenotype in the carrying families, although the presence of the mutation was corroborated in more than one affected member in only one family. The authors also referred to a previous rearrangement affecting exon 3 of BRCA2 published by Nordling et al.2 These authors had detected a large deletion in exon 3 in four affected individuals of one family: the 5068-bp deletion at nucleotide 504 (codon 92 in exon 3) and an insertion of four bases, CCAT, at the junction site (nt504del5068insCCAT), which encompassed the 3' part of the exon 3 and most of the 5751-bp-long intron 3, including the 5' splicing donor site of intron 3. The deletion explains the skipping of exon 3 on mRNA. The report of Machado and colleagues give us the opportunity to add some comments on different alterations affecting exon 3 of BRCA2 that have an unclear meaning. Working on somatic BRCA2 mutations in replication error–positive endometrial carcinomas, Koul et al3 described a –7delT mutation in intron 2 present in four of 12 replication error positive tumors. By means of reverse transcriptase-polymerase chain reaction (RT-PCR), the authors demonstrated an in-frame skipping of the entire exon 3 in one tumor with the variant. In the analysis of 57 Italian breast and ovarian cancer families, Santarosa et al4 identified T > A substitution in the same position of the BRCA2 gene (IVS2-7T>A) in one patient diagnosed with breast and ovarian cancer. The variant was not present in 43 breast and/or ovarian cancer patients, in 17 healthy blood donors, or in six ovarian cancers.5 To verify the effect of IVS2-7delT on transcription Santarosa et al5 performed an RT-PCR (exons 2 to 5 of BRCA2) on mRNA from lymphocytes derived from 11 breast and/or ovarian cancer patients. All samples showed a normal and an altered transcript. The sequence of this one revealed that the entire exon 3 was skipped. Interestingly, the authors consequently performed an RT-PCR on mRNA of different types of carcinomas, normal ovarian tissue, cancer cell lines, and healthy blood donors; all but one sample displayed the abnormal product, questioning a deleterious effect. Independently, in the context of a screening for BRCA1/BRCA2 mutations in breast/ovarian Spanish families, we found the same IVS2-7T > A variation in two families and the IVS2-7_8TT > AA substitution, which had not previously been described. Both alterations affect the poly T(10) of the splicing acceptor site, and presumably lead to the same altered transcript. In one family, the presence of the IVS2-7T > A variant was confirmed in the two affected members: the proband, with breast cancer at 29 years of age and her father, affected by a tongue cancer and soft palate cancer at age 51 years. Unfortunately, no other cosegregation analysis could be done due to the lack of available samples in the other families. There are other genetic alterations in the exon 3 in BRCA2 gene, initially reported as pathogenic in breast/ovarian cancer patients that may also correspond to nonpathogenic variants, like the missense mutation Tyr42Cys (c.125A>G). According to Milner et al,6 exon 3 sequences at the amino terminus of BRCA2 (within a region highly conserved between human and mouse) show sequence similarity to the activation domain of c-Jun. The authors found that sequences spanning exon 3 have the potential to activate transcription in yeast, and also have potent activation activity in two different mammalian cell lines. The presence of this tyrosine-to-cysteine mutation (p.Y42C) severely compromised the activation potential of the region. Furthermore, the tyrosine residue is conserved in chicken and the change from a tyrosine to a cysteine is one of the most severe changes. However, the change p.Y42C has been observed in more than 100 families,7 some of which also carried a known BRCA2 deleterious mutation in trans. Goldgar et al.8 analyzed 17 pedigrees with this variant. On the basis of the co-occurrence and the pedigree cosegregation data they concluded that the odds are overwhelming against Y42C being a deleterious BRCA2 allele (1,500,000:1). We found the same Y42C variant in one breast/ovarian cancer Spanish family and the analysis of four affected family members also showed no cosegregation with cancer. In summary, although point mutations and large rearrangements in the BRCA2 gene giving rise to exon 3 skipping at the mRNA level might be associated to the abnormal phenotype in breast/ovarian cancer families, the skipping of exon 3 does not alter the reading frame, and the Rad51 binding sites and nuclear localization signals in the 3' region of BRCA2 still remain. According to some authors,5 this alternative splicing could play a role in regulating the biologic functions of BRCA2. Hence, given that different molecular findings with contradictory results have been described on the exon 3 of BRCA2 gene, caution should be taken when interpreting these alterations in breast/ovarian cancer families. More investigations are needed, including the pathological and clinical analysis of more families carrying mutations or variants in this region of the BRCA2 gene, the more accurate analysis of RNA, and the analysis of disease-free population controls. As a side note, in their article, Machado et al compiled previous works and state that only seven rearrangements have been described in BRCA2-positive families. However, during the review process of the manuscript other studies identifying germline large rearrangements in BRCA2 have been published on French,9 Australian,10 Danish,11 and Spanish12 breast/ovarian cancer families, among others. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The author(s) indicated no potential conflicts of interest. REFERENCES
1. Machado PM, Brandao RD, Cavaco MB, et al: Screening for a BRCA2 rearrangement in high-risk breast/ovarian cancer families: Evidence for a founder effect and analysis of the associated phenotypes. J Clin Oncol 25
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-2034, 2007 2. Nordling M, Karlsson P, Wahlstrom J, et al: A large deletion disrupts the exon 3 transcription activation domain of the BRCA2 gene in a breast/ovarian cancer family. Cancer Res 58
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-1375, 1998 3. Koul A, Nilbert M, Borg A: A somatic BRCA2 mutation in RER+ endometrial carcinomas that specifically deletes the amino-terminal transactivation domain. Genes Chromosomes Cancer 24 : 207 -212, 1999[CrossRef][Medline] 4. Santarosa M, Dolcetti R, Magri MD, et al: BRCA1 and BRCA2 genes: Role in hereditary breast and ovarian cancer in Italy. Int J Cancer 83 : 5 -9, 1999[CrossRef][Medline] 5. Santarosa M, Viel A, Boiocchi M: Splice variant lacking the transactivation domain of the BRCA2 gene and mutations in the splice acceptor site of intron 2. Genes Chromosomes Cancer 26 : 381 -382, 1999[CrossRef][Medline] 6. Milner J, Ponder B, Hughes-Davies L, et al: Transcriptional activation functions in BRCA2. Nature 386 : 772 -773, 1997[CrossRef][Medline] 7. Breast Cancer Information Core: http://www.nhgri.nih.gov/Intramural_reserach/Lab_transfer/Bic/ 8. Goldgar DE, Easton DF, Deffenbaugh AM, et al: Integrated evaluation of DNA sequence variants of unknown clinical significance: Application to BRCA1 and BRCA2. Am J Hum Genet 75 : 535 -544, 2004[CrossRef][Medline] 9. Casilli F, Tournier I, Sinilnikova OM, et al: The contribution of germline rearrangements to the spectrum of BRCA2 mutations. J Med Genet 43
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, 2005 11. Thomassen M, Gerdes AM, Cruger D, et al: Low frequency of large genomic rearrangements of BRCA1 and BRCA2 in western Denmark. Cancer Genet Cytogenet 168 : 168 -171, 2006[CrossRef][Medline] 12. Gutiérrez-Enríquez S, de la Hoya M, Martínez-Bouzas C, et al: Screening for large rearrangements of the BRCA2 gene in Spanish families with breast/ovarian cancer. Breast Cancer Res Treat 103 : 103 –107, 2007[CrossRef][Medline]
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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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