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Journal of Clinical Oncology, Vol 25, No 15 (May 20), 2007: pp. 2027-2034 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.06.9443 Screening for a BRCA2 Rearrangement in High-Risk Breast/Ovarian Cancer Families: Evidence for a Founder Effect and Analysis of the Associated Phenotypes
From the Molecular Biology Department and Breast Cancer Risk Evaluation Clinic, Instituto Português de Oncologia de Lisboa, Francisco Gentil, Lisboa, Portugal Address reprint requests to Fátima Vaz, MD, Consulta de Risco Familiar de Cancro da Mama e Ovário, Serviço de Oncologia Médica–Instituto Português de Oncologia, de Francisco Gentil, Rua Prof Lima Basto, 1099-023 Lisboa, Portugal; e-mail: fvaz{at}ipolisboa.min-saude.pt
Purpose BRCA2 rearrangements are rare genetic events. A large BRCA2 genomic insertion was recurrently observed in our participants, and we sought to characterize it at the molecular and phenotypic level. Patients and Methods We studied 210 high-risk breast/ovarian cancer families. Fifty-three probands were fully screened for BRCA1/2 mutations, and three of 53 had a large insertion in exon 3 of BRCA2. This finding was analyzed by polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), and sequencing. An additional 157 consecutive families were screened for this mutation by a three-step PCR method. Phenotype and haplotype analysis was also performed. Results Sixteen BRCA mutations were observed in 19 of 53 patients (36% detection rate). A recurrent Alu motif insertion in position c.156_157 was observed after sequencing of an abnormal fragment obtained after the amplification of BRCA2 exon 3. RT-PCR revealed exon 3 skipping. Screening of this rearrangement identified 14 additional families (out of 157). In total, 17 (8%) of 210 high-risk families ascertained in our clinic were positive for this mutation. Segregation of a common haplotype (from D13S260 to D13S1695) confirmed a common origin, estimated to have occurred 2,400 to 2,600 years ago. The following four cancer phenotypes were observed in the 17 positive families: female breast (n = 9), male breast (n = 4), breast/ovarian (n = 2), and heterogeneous (n = 2). Male breast cancer was more frequently observed in c.156_157insAlu–positive families compared with negative families (23% v 12%, respectively), and 33% of all male breast cancer families with an identified BRCA mutation were c.156_157insAlu positive. Conclusion c.156_157insAlu is a founder mutation of Portuguese origin and is the most frequent BRCA2 rearrangement described to date.
Genomic rearrangements, or the wholesale movement of sequences from one position to another in genomic DNA,1 correspond to 8% to 40% of all mutations in the BRCA1 gene,2 but only seven rearrangements have been described in BRCA2-positive families.2-7 These include large deletions,3,4 duplications,4 deletions/insertions,5 and Alu insertions.6,7 Rarity of BRCA2 rearrangements may be a result of the fact that sensitive assays for rearrangement detection have only recently been added to the routine molecular diagnosis of breast/ovarian cancer predisposition.8,9 Furthermore, it has been reported that families negative for BRCA2 rearrangements were breast/ovarian cancer families or female breast cancer–only families. Male breast cancer families, which are mainly associated with BRCA2 mutations, could have a higher frequency of rearrangements in this gene.2 In fact, three of the described BRCA2 rearrangements were observed in male breast cancer families negative for BRCA1/2 point mutations.4 Although no founder effect has yet been described for BRCA2 rearrangements, BRCA1 genomic deletions have been found to represent major founder mutations in the Dutch population.10 Founder mutations are identified only in specific countries or ethnic groups, suggesting that they have spread from a single ancestor. Founder BRCA1/2 mutations have been described in the Ashkenazi Jewish population11 and in other countries such as Finland, the Netherlands, Norway, Russia, Latvia,12 and Spain.13 Identification of founder mutations and their ethnic and geographic origins allows a more rational and faster approach to mutational screening and genetic counseling in defined subpopulations because BRCA1/2 are large genes (each one spanning > 100 kb of genomic DNA) and do not have hot spots. Unless a fast screening possibility exists, full screening of all the exonic and exon-intron boundary sequences is necessary, which makes this diagnosis expensive and time consuming.14 The spectrum of BRCA1/2 mutations in Portugal includes few recurrent mutations, probably because the Portuguese genetic background is heterogeneous. In fact, peoples of different origins invaded the Iberian Peninsula, and Portuguese sailors and emigrants have been in contact with several peoples, in all continents, since the 15th century. During full gene BRCA1/2 screening of high-risk breast/ovarian cancer families, 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, which was previously observed in a Portuguese family.7 Because this large insertion was likely to represent a founder mutation, we optimized a three-step polymerase chain reaction (PCR) method to screen all new families ascertained in our clinic for BRCA1/2 genetic testing, and the founder effect hypothesis was further explored by haplotype analysis. Phenotypic characterization of positive families was carried out.
Participants Selection criteria for full BRCA1/2 screening were 25% of BRCA1/2 combined probability of a mutation,15,16 family history of male breast cancer, or diagnosis of breast cancer at less than 30 years of age. Pedigrees included at least three family generations, and all patients underwent genetic counseling and signed an informed consent form, according to procedures approved by the ethics committee of our institute. In patients found to be positive for the most frequent BRCA2 mutation, permission was obtained to disclose the results to relatives at risk and invite them for genetic screening and for haplotype analysis with polymorphic BRCA2-linked markers. The initial group of 53 patients reported in this study was the first to be analyzed for BRCA1/2 mutations in the context of a multidisciplinary group, and counseling occurred between July 2000 and July 2002. After complete BRCA1/2 screening and clarification of recurrent mutations of the first group, all consecutive index nonrelated patients counseled between September 2002 and March 2006 (a total of 157 patients) were prescreened for the two recurrent mutations observed. All samples from patients negative for the recurrent mutations were later included in the general BRCA1/2 mutation analysis.
General Mutation Analysis Samples negative for BRCA1/2 mutations were tested for BRCA1 rearrangements using the multiplex ligation-dependent probe amplification assay (MRCHolland, Amsterdam, the Netherlands), following the manufacturer's protocol. Amplification products were analyzed with an ABI Prism 310 automatic sequencer using the Genescan software (Applied Biosystems, Foster City, CA).
Identification of the c.156_157insAlu Recurrent Mutation in Exon 3 of BRCA2 To specifically amplify the allele with the c.156_157insAlu mutation, we performed a first PCR with primers 3F and 3R, followed by a nested PCR with the following primers: 3AluF: 5'CGGATCACGAGGTCAGGA-3'; and 3AluR: 5'-GGTTTGGTTCGTAATTGTTGTTT-3'. Primer 3AluF was designed to recognize a sequence in the Alu insertion, and primer 3AluR binds to exon 3. The nested PCR product (approximately 300 bp) was analyzed by agarose gel electrophoresis and detected only in the DNA of patients with the Alu insertion. PCR conditions are available upon request. RNA analysis. Total RNA was extracted from peripheral-blood leukocytes using TRIzol reagent (Invitrogen, Paisley, United Kingdom) according to the manufacturer's protocol. cDNA was synthesized using random primers (Roche, Mannheim, Germany) and Superscript II reverse transcriptase (Invitrogen) according to the manufacturers’ protocols. Primers (1FcDNA and 10RcDNA)3 were used to amplify the BRCA2 coding region from exon 1 through exon 10. Purification and sequencing analysis. PCR products were isolated and purified using QIAquick Gel extraction kit (Qiagen, Hilden, Germany). DNA sequencing was performed using the same primers for PCR and the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) in an automated sequencer ABI Prism 310 (Applied Biosystems).
Screening of BRCA2 c.156_157insAlu
Haplotype Analysis The nine microsatellite markers used were localized in a 5.36-cM (2.9-Mb) region encompassing BRCA2 (locus order: cen-D13S1246-D13S1229-D13S260-D13S1699-D13S1698-D13S1701-D13S171-D13S1695-D13S1493-tel) on chromosome 13q12.3-13q13.2.19,20 Fluorescently labeled primers were used to amplify the microsatellite polymorphic regions (PCR conditions are available upon request). PCR products were analyzed in an automated sequencer ABI Prism 310 using the 310 Genescan 3.1.2 software (Applied Biosystems). Allele sizes are given as the size of the PCR amplicons containing the microsatellites. One internal BRCA2 polymorphism (H372N) was also screened through direct sequencing.
Estimation of Founder Mutation Age
Probands Characteristics of all probands submitted to genetic screening are listed in Table 1. In the first group, five different BRCA1 and 11 BRCA2 mutations were diagnosed in 19 of 53 patients, corresponding to a 35.9% detection rate. The following two BRCA2 mutations were recurrent: c.156_157insAlu (three nonrelated families) and c.7208_7211del4 (two nonrelated families; Table 2). The second mutation was only detected after CSGE analysis, but c.156_157insAlu was immediately identified by agarose gel electrophoresis (Fig 1). The 157 additional consecutive families were then screened for the recurring mutations observed in the first set.
Identification of the BRCA2 c.156_157insAlu Recurrent Mutation In probands with the c.156_157insAlu BRCA2 mutation, PCR amplification of exon 3 originated, besides the expected 425-bp product, an aberrant fragment with approximately 800 bp (Fig 1A). Sequencing of this fragment with primers 3F and 3R (Figs 1B and 1C) revealed an unknown sequence starting in nucleotide 156 of BRCA2 cDNA. Primers 3AluF and 3AluR were designed to sequence the whole fragment that, after a Basic Local Alignment Search Tool search,24 revealed an Alu motif insertion,25,26 subtype Ya5. Amplification of cDNAs from two c.156_157insAlu–positive probands with primers 1FcDNA and 10RcDNA revealed the expected 1,300-bp product and an abnormal 1,100-bp band (Fig 1D). Sequencing of this abnormal 1,100-bp product revealed the in frame deletion of exon 3, which resulted in the fusion of exon 2 with exon 4 (Fig 1E). Exon 3 of BRCA2 encodes a transcriptional activation domain,27 and its relevance in the tumor suppression function of BRCA2 has been previously described.3
Screening for the BRCA2 c.156_157insAlu Mutation Considering the three of 53 c.156_157insAlu–positive families initially observed and these additional 14 families (of 157), 8% of all families tested were positive for the BRCA2 rearrangement. The following four different phenotypes could be distinguished: female breast (nine families, 53%), male breast (four families, 24%), breast/ovarian (two families, 12%) and heterogeneous (two families, 12%). These latter families had no more than two female breast cancers and no ovarian or male breast cancer, but other neoplasias like gastric cancer (two members in family 108), head and neck cancer (one member in family 108 and two members in family 129), and multiple myeloma (one member in family 108) were observed. Examples of representative phenotypes are shown in Figure 2, and phenotypic characteristics of all index patients and their families are shown in Appendix Figure A1 (online only).
Phenotypes of c.156_157insAlu–positive and –negative families are listed in Table 3. Among the 210 families, 28 had male breast cancer, and 12 were diagnosed with a BRCA2 mutation (four families were positive for the c.156_157insAlu mutation, two were positive for the c.9098_9099insA mutation, and the remaining six families showed six different BRCA2 mutations: c.1423G>T; c.1786G>C; c.2808_2811del4; c.5063_5066del4; c.6468_6469del2; and c.7208_7211del4). These data demonstrate that 33% of all male breast cancer families with a BRCA mutation identified are c.156_157insAlu positive.
Haplotype Analysis and Estimation of Mutation Age Most of the families positive for the c.156_157insAlu mutation come from Central Portugal (Appendix Fig A2, online only), and haplotype analysis was performed in 37 individuals (14 index patients and 23 of their relatives). The results of c.156_157insAlu screening in relatives of representative families are shown in Figure 2. We observed a conserved haplotype cosegregating with the mutation (Table 4) and absent in noncarriers of these families (Fig 2). Encompassing BRCA2, it represents a 1.09-Mb interval from D13S260 to D13S1695, and its random population frequency is approximately one in 8,600. In two of 14 families, recombination events, either centromeric (marker D13S1698) or telomeric (marker D13S1701) to BRCA2, reduced the shared haplotype region to 0.63 Mb and 0.54 Mb, respectively (Table 4). Demonstration of a shared haplotype between all c.156_157insAlu families provides evidence for a common ancestry among these families.
The linkage disequilibrium measures (  ) between the BRCA2 c.156_157insAlu mutation and each of the recombinant microsatellite markers D13S1698 and D13S1701 were calculated as = 0.7857 and = 0.8137, respectively. The recombination fraction  was determined from the physical distances between markers D13S1698 and D13S1701 and the BRCA2 gene. The distance for D13S1698 is 0.185 Mb (approximately 0.185 cM), and the distance for D13S1701 is 0.171 Mb (approximately 0.171 cM),19,29 assuming that 1 cM is equivalent to approximately 1 Mb.28,29 Given these genetic distances, we estimated that the c.156_157insAlu mutation occurred between 120 and 130 generations ago, that is, approximately 2,400 to 2,600 years ago, assuming 20 years per generation.
In this study, we demonstrate that the insertion of an Alu fragment in position 156 of BRCA2 cDNA is a founder mutation of Portuguese origin. To date, only seven BRCA2 rearrangements have been detected (including c.156_157insAlu) in unique families.2-7 Therefore, besides the relevant implications in the genetic screening of breast/ovarian cancer families of Portuguese ancestry, our finding demonstrates that the c.156_157insAlu mutation is the most frequent BRCA2 rearrangement described to date. In our target population, including all of South Portugal, c.156_157insAlu has been observed in 8% of screened families, and taking into account families that have already been fully screened for BRCA1/2, it corresponds, approximately, to one in every six BRCA1/2 mutations identified. It is interesting to note that, in our families most extensively studied, not only was this rearrangement the most frequent genetic event observed, but also other rearrangements were diagnosed in negative families by CSGE. This observation reinforces the need to search for rearrangements in high-risk families negative for point mutations. The contribution of Alu insertions as disease-causing mutations in humans has been estimated as approximately one mutation in 600.30 Before the c.156_157insAlu mutation, which was first reported in one family of Portuguese origin,7 only one BRCA1/2 Alu insertion was described.6 Our initial finding of c.156_157insAlu in three of 53 families raised the hypothesis of a founder effect in our population, and we implemented the prescreen of all high-risk consecutive families ascertained in our clinic for this mutation. Although the first description of this mutation, in a Portuguese family, was obtained by Southern blotting7 after negative PCR-based screening, we optimized a three-step PCR for a quick and effective way to screen and confirm the presence of this rearrangement. A simple PCR reaction is enough to detect a positive individual. The Alu fragment is sequenced, and the pathogenic effect of the mutation (exon 3 skipping) is confirmed by RT-PCR. The clinical relevance of this strategy is that 8% of our families obtain a quick and less expensive result, without the need to wait for full screening. All c.156_157insAlu–negative families must be fully screened for BRCA1/2 genes. Using the three-step PCR, 157 consecutive families were screened for the c.156_157insAlu mutation, and an additional 14 nonrelated positive families were observed. Fifteen of these families came from the central part of Portugal, further supporting the possibility of a common ancestor. This hypothesis was confirmed through the observation of a conserved haplotype surrounding the BRCA2 locus that was found to segregate with the mutation in 12 of 14 index patients. In the remaining two families, recombination events reduced the shared haplotype region. Estimation of the age of the mutation suggested that the founder event occurred 2,400 to 2,600 years ago, that is, before the invasion of the Iberian Peninsula by the Romans and the Germanic warriors. At that time, the Lusitanians31 inhabited the territory that now includes the districts of origin of most of these families. All migrations that involved Portuguese people in the following centuries were all outward of this territory, either to the western part of the country or abroad. This may explain the higher prevalence of the c.156_157insAlu mutation in that area and also allows us to speculate that the two families from the north were also originated from the same founder. This historical hypothesis is still acceptable even if the age of the mutation is being overestimated, either because of the fact that mutation rates of the microsatellite markers were not taken into account or because recombination events in two families were considered. Breast and prostate cancer were the malignancies most frequently observed in c.156_157insAlu families, and the most frequent phenotype was female breast cancer, with a mean age at diagnosis of 48 years. Four male breast cancer families were also registered, and it is remarkable that 33% of all our male breast cancer families with BRCA2 mutations harbor the founder mutation. This may reflect a particular phenotype associated to this mutation, or it may be the result of the high frequency of this genetic event in our BRCA2 families. Prostate cancer was the most frequent cancer diagnosis in men belonging to c.156_157insAlu families. Two of the male breast cancer probands had also been diagnosed with prostate cancer, and three of the eight prostate cancer patients registered were observed in only one family, with a mean age at diagnosis of 65 years (family 24). The association of BRCA2 mutations with prostate cancer is well known,32,33 not only in relatives of women with breast and ovarian cancer, but also in men unselected for family history but with early-onset disease.34 The clustering of early-onset prostate cancer in families may be a result not only of the BRCA2 mutation described but also of unknown modifier factors that may affect its penetrance in men. Two families had heterogeneous phenotypes, with only two cases of breast cancers each but with two cases of head and neck and gastric cancers in one of the families and a case of multiple myeloma and head and neck cancer in the other family. Because the probands included in this study were the only affected patients alive, we cannot conclude that all of these tumors are related to the mutation, even though gastric cancer35 and multiple myeloma36 have been associated with BRCA2. Only two women were diagnosed with ovarian cancer, although they were diagnosed at an early age (23 and 38 years old). This observation may be explained by the fact that c.156_157insAlu is at the 5' region of the BRCA2 gene.37 Further follow-up of these families will clarify ovarian cancer incidence and age at diagnosis in positive women. In conclusion, the results of this study suggest that screening of the c.156_157insAlu mutation should be extended to all high-risk breast/ovarian cancer families with Portuguese ancestry. Because most patients were from the central and southern part of the country, we cannot rule out the possibility that this mutation can be found in other regions of the country and also in other areas of the world, where Portuguese sailors, traders, and emigrants have settled since the 15th century to the present. Targeting the initial screening to the founder mutation in these families will clarify the global incidence of this mutation in a fast and inexpensive manner. More importantly, it will help to clarify the risk for several cancers in high-risk individuals and, through vigilance, preventive attitudes, or inclusion in clinical studies, help to modify the incidence and mortality by cancer in these families.
Author's Note.
The authors indicated no potential conflicts of interest.
Conception and design: Patrícia M. Machado, Rita D. Brandão, Branca M. Cavaco, Fátima Vaz Financial support: Patrícia M. Machado, Fátima Vaz Provision of study materials or patients: Sandra Bento, Mónica Nave, Aires Fernandes, Fátima Vaz Collection and assembly of data: Patrícia M. Machado, Rita D. Brandão, Joana Eugénio, Paula Rodrigues, Fátima Vaz Data analysis and interpretation: Patrícia M. Machado, Rita D. Brandão, Branca M. Cavaco, Fátima Vaz Manuscript writing: Patrícia M. Machado, Rita D. Brandão, Branca M. Cavaco, Aires Fernandes, Fátima Vaz Final approval of manuscript: Branca M. Cavaco, Aires Fernandes, Fátima Vaz
We are grateful to all families for their cooperation and to Valeriano Leite, MD, PhD, and Sylvie Mazoyer, PhD, for their comments on this article. We thank the support Jorge Soares, MD, PhD, gave us on the implementation of the genetic screening of BRCA1/2 mutations in our center, as well as Barbara L. Weber, MD, and Arupa Ganguly, PhD, whose advice was crucial for the development of the Breast Cancer Risk Evaluation Clinic and the methodologies of mutation screening.
Supported by Grant No. 47320 from Serviço de Saúde e Desenvolvimento da Fundação Calouste Gulbenkian, Lisboa, and by grants (2000/2001 and 2004/2006) from Terry Fox, Núcleo Regional Sul da Liga Portuguesa Contra o Cancro, Lisboa, Portugal. P.M.M. and R.D.B. contributed equally to this study. Presented in part at the 53rd Annual Meeting of the American Society of Human Genetics, November 4-8, 2003, Los Angeles, CA, and at the European Human Genetics Conference, May 7-10, 2005, Prague, Czech Republic. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
1. Melcher U: Molecular genetics by Ulrich Melcher at Oklahoma State University: DNA rearrangements. http://opbs.okstate.edu/  melcher/MG/MGW3/MG32.html2. Mazoyer S: Genomic rearrangements in the BRCA1 and BRCA2 genes. Hum Mutat 25: 415-422, 2005[CrossRef][Medline] 3. 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: 1372-1375, 1998 4. Tournier I, Paillerets BB, Sobol H, et al: Significant contribution of germline BRCA2 rearrangements in male breast cancer families. Cancer Res 64: 8143-8147, 2004 5. Wang T, Lerer I, Gueta Z, et al: A deletion/insertion mutation in the BRCA2 gene in a breast cancer family: A possible role of the Alu-polyA tail in the evolution of the deletion. Genes Chromosomes Cancer 31: 91-95, 2001[CrossRef][Medline] 6. Miki Y, Katagiri T, Kasumi F, et al: Mutation analysis in the BRCA2 gene in primary breast cancers. Nat Genet 13: 245-247, 1996[CrossRef][Medline] 7. Teugels E, De Brakeleer S, Goelen G, et al: De novo Alu element insertions targeted to a sequence common to the BRCA1 and BRCA2 genes. Hum Mutat 26: 284, 2005[Medline] 8. Casilli F, Di Rocco ZC, Gad S, et al: Rapid detection of novel BRCA1 rearrangements in high-risk breast-ovarian cancer families using multiplex PCR of short fluorescent fragments. Hum Mutat 20: 218-226, 2002[CrossRef][Medline] 9. Hogervorst FB, Nederlof PM, Gille JJ, et al: Large genomic deletions and duplications in the BRCA1 gene identified by a novel quantitative method. Cancer Res 63: 1449-1453, 2003 10. Petrij-Bosch A, Peelen T, van Vliet M, et al: BRCA1 genomic deletions are major founder mutations in Dutch breast cancer patients. Nat Genet 17: 341-345, 1997[CrossRef][Medline] 11. Levy-Lahad E, Catane R, Eisenberg S, et al: Founder BRCA1 and BRCA2 mutations in Ashkenazi Jews in Israel: Frequency and differential penetrance in ovarian cancer and breast-ovarian cancer families. Am J Hum Genet 60: 1059-1067, 1997[CrossRef][Medline] 12. Neuhausen S: Founder populations and their uses for breast cancer genetics. Breast Cancer Res 2: 77-78, 2000[CrossRef][Medline] 13. Vega A, Campos B, Bressac-de-Paillerets B, et al: The R71G BRCA1 is a founder Spanish mutation and leads to aberrant splicing of the transcript. Hum Mutat 17: 520-521, 2001[Medline] 14. Welcsh PL, King MC: BRCA1 and BRCA2 and the genetics of breast and ovarian cancer. Hum Mol Genet 10: 705-713, 2001 15. Parmigiani G, Berry DA, Aguilar O: Determining carrier probabilities for breast cancer-susceptibility genes BRCA1 and BRCA2. Am J Hum Genet 62: 145-158, 1998[CrossRef][Medline] 16. Frank TS, Manley SA, Olopade OI, et al: Sequence analysis of BRCA1 and BRCA2: Correlation of mutations with family history and ovarian cancer risk. J Clin Oncol 16: 2417-2425, 1998[Abstract] 17. The Breast Cancer Information Core Database. http://research.nhgri.nih.gov/bic/ 18. Ganguly A, Rock MJ, Prockop DJ: Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: Evidence for solvent-induced bends in DNA heteroduplexes. Proc Natl Acad Sci U S A 90: 10325-10329, 1993 19. Ensembl Genome Browser. Homepage. http://www.ensembl.org 20. The GDB Human Genome Database. Homepage. http://www.gdb.org/ 21. Risch N, de Leon D, Ozelius L, et al: Genetic analysis of idiopathic torsion dystonia in Ashkenazi Jews and their recent descent from a small founder population. Nat Genet 9: 152-159, 1995[CrossRef][Medline] 22. BRCA1 Exon 13 Duplication Screening Group: The exon 13 duplication in the BRCA1 gene is a founder mutation present in geographically diverse populations. Am J Hum Genet 67: 207-212, 2000[CrossRef][Medline] 23. den Dunnen JT, Antonarakis SE: Mutation nomenclature extensions and suggestions to describe complex mutations: A discussion. Hum Mutat 15: 7-12, 2000[CrossRef][Medline] 24. National Center for Biotechnology Information: Basic Local Alignment Search Tool (BLAST). http://ncbi.nlm.nih.gov/BLAST 25. Shaikh TH, Joomyeong K, Bennett L, et al: Sequence diversity and chromosomal distribution of "young" Alu repeats. Gene 163: 273-278, 1995[CrossRef][Medline] 26. Batzer MA, Deininger PL: Alu repeats and human genomic diversity. Nat Rev Genet 3: 370-379, 2002[CrossRef][Medline] 27. Milner JB, Ponder B, Hughes-Davies L, et al: Transcriptional activation functions in BRCA2. Nature 386: 772-773, 1997[CrossRef][Medline] 28. National Human Genome Research Institute: Talking glossary of genetic terms. http://www.genome.gov/10002096 29. Wajnrajch MP, Gertner JM, Sokoloff AS, et al: Haplotype analysis of the growth hormone releasing hormone receptor locus in three apparently unrelated kindreds from the Indian subcontinent with the identical mutation in the GHRH receptor. Am J Med Genet A 120: 77-83, 2003[Medline] 30. Kazazian HH Jr: An estimated frequency of endogenous insertional mutations in humans. Nat Genet 22: 130, 1999[Medline] 31. de Motes JM: Western Europe before the colonisations: The Iberian Peninsula and Southern Galia, in Saraiva JH (ed): Historia Universal - O Mundo Mediterrânico e a Europa Continental da Pré-História ao Século III. Volume I. Lisboa, Portugal, Publicações Alfa, 1985, pp 215-226 32. Breast Cancer Linkage Consortium: Cancer risks in BRCA2 mutation carriers. J Natl Cancer Inst 91: 1310-1316, 1999 33. Liede A, Karlan BY, Narod SA: Cancer risks for male carriers of germline mutations in BRCA1 or BRCA2: A review of the literature. J Clin Oncol 22: 735-742, 2004 34. Edwards SM, Kote-Jarai Z, Meitz J, et al: Two percent of men with early-onset prostate cancer harbor germline mutations in the BRCA2 gene. Am J Hum Genet 72: 1-12, 2003[CrossRef][Medline] 35. Jakubowska A, Nej K, Huzarski T, et al: BRCA2 gene mutations in families with aggregations of breast and stomach cancers. Br J Cancer 87: 888-891, 2002[CrossRef][Medline] 36. Struewing JP, Hartge P, Wacholder S, et al: The risk of cancer associated with specific mutations of BRCA1 and BRCA2 among Ashkenazi Jews. N Engl J Med 336: 1401-1408, 1997 37. Thompson D, Easton D: Variation in cancer risks, by mutation position, in BRCA2 mutation carriers. Am J Hum Genet 68: 410-419, 2001[CrossRef][Medline] Submitted April 13, 2006; accepted February 20, 2007.
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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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ABSTRACT
INTRODUCTION
