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A common missense variant in BRCA2 predisposes to early onset breast cancer

A common missense variant in BRCA2 predisposes to early onset breast cancer Introduction Mutations in the BRCA2 gene are one of the two Results The variant was present in approximately 6% of the major causes of hereditary breast cancer. Protein-truncating Polish population. In the study, 13 women (11 cases and two mutations of BRCA2 are usually deleterious and increase the controls (OR = 4.7; p = 0.02)) were homozygous for the variant risk of breast cancer up to 80% over a lifetime. A few missense allele. The overall odds ratio for breast cancer in women with a mutations in BRCA2 are believed to have a similarly high single copy of the BRCA2 C5972T variant was 1.1 (p = 0.7); penetrance, apart from more common neutral polymorphisms. It however, the effect was significant for patients diagnosed at or is often difficult to classify a particular sequence variant as a before age 40 (OR = 1.4; p = 0.04). We reviewed the mutation or a polymorphism. For a deleterious variant, one would association between the BRCA2 variant in different histologic expect a greater allele frequency in breast cancer cases than in subgroups and found the effect most pronounced in women ethnic-matched controls. In contrast, neutral polymorphic who had ductal carcinoma in situ (DCIS) with micro-invasion variants should be equally frequent in the two groups. (OR = 2.8; p < 0.0001). Methods We genotyped 3,241 cases of breast cancer Conclusion The BRCA2 C5972T allele is a common variant in diagnosed at under 51 years of age, unselected for family Poland that increases the risk of DCIS with micro-invasion. The history, from 18 hospitals throughout Poland and 2,791 ethnic- homozygous state is rare but increases the risk of breast cancer matched controls for a single BRCA2 C5972T variant. five-fold. eral deleterious founder alleles have been identified in Introduction There are several approaches to identifying low-penetrance BRCA1, but to date, no founder mutation in BRCA2 has been candidate genes for breast cancer. In one approach, it is identified [1-3]. There are a few common variant alleles in assumed that missense variants of genes for which truncating BRCA2 in Poland; one of these (C5972T) changes the amino mutations are clearly pathogenic might also be deleterious. If acid sequence of BRCA2 from threonine to methionine at the missense allele demonstrates high penetrance (i.e. like codon 1915. It lies within the range of the BRC encoded by truncating mutations), then it will be relatively straightforward exon 11 that are thought to be involved in binding to RAD51 to establish the association when allele frequency is high. If the [4]. We sought to determine whether this common missense penetrance of the missense variant is low, however, then the BRCA2 variant plays a role in breast cancer susceptibility. association may be missed if only a small number of cancers is studied and the variant may falsely be classified as a neutral Materials and methods polymorphism. We are in the process of establishing a large Study subjects database of breast cancer cases and ethnic-matched controls The study population included prospectively ascertained in order to evaluate the pathogenicity of the common founder cases of invasive breast cancer diagnosed at 50 years of age alleles of the most important cancer susceptibility genes. Sev- or less at 18 treatment centers throughout Poland between DCIS = ductal carcinoma in situ; LOH = loss of heterozygosity; OR = odds ratio; RFLP-PCR = restriction fragment length polymorphism polymerase chain reaction. R1023 Breast Cancer Research Vol 7 No 6 Górski et al. 1997 and 2003. Invitation to participate was delivered during DNA sequencing hospital stay or by mail. During the interview, the goals of the A fragment of BRCA2 exon 11 was amplified by PCR using study were explained, informed consent was obtained, genetic b5972F/b5972R primers. The purified PCR products were counseling was offered, and a blood sample was taken for sequenced directly with a BigDye Terminator v3.0 DNA DNA analysis. A detailed family history of cancer was taken Sequencing Kit (Perkin Elmer, Foster City, CA, USA) and the (first-, second-, and third-degree relatives included) and a risk same reverse primer used previously for the amplification of factor questionnaire was completed. A total of 4,778 cases of exon 11 of the BRCA2 gene. Products of sequencing reac- early onset invasive breast cancer were identified at the 18 tions were separated and analysed on the ABI 377 DNA participating centers during the study period. Of these, 3,627 Sequencer (Perkin Elmer). women (76%) accepted to participate and provided a blood BRCA1 testing sample for DNA analyses, 114 died, and 16 refused to partic- ipate. The medical record and pathology report were reviewed. All breast cancer patients were tested as described previously In the end, we had DNA available for genotyping from 3,241 [4] for three mutations (5382insC, C61G, 4153delA) repre- patients. senting 80% to 90% of all high-risk BRCA1 changes in Poland. Controls Two control groups were combined. The first group consisted Statistical analysis of 2,000 unselected neonates from ten hospitals throughout Statistical analysis included a comparison of the prevalence of Poland born in 2003 and 2004. Samples of cord blood were the C5972T allele in cases and controls. Odds ratios (ORs) forwarded to the study center in Szczecin. The second control were generated from two-by-two tables and statistical signifi- group consisted of 1,000 adults from the region of Szczecin cance was assessed with the chi-square test. BRCA2 nucle- unselected for cancer family history, sex, or age. We geno- otides were identified according to the GeneBank U43746 typed 2,791 controls for the C5972T allele, 1,993 neonates sequence. and 798 adults. To ensure comparability of the control groups, C5729T allele frequencies were computed separately for the Results adult and neonatal control groups and compared. The study The C5972T variant was detected in 8.3% of unselected protocol was approved by the ethics committee of the Pomer- breast cancer patients diagnosed at age 40 years or below, in anian Medical University. 5.7% of cases diagnosed from age 41 to 50 years, and in 5.8% of Polish controls. The overall OR for breast cancer with Laboratory methods a C5972T mutation was 1.1 (95% CI: 0.8–1.3); however, in All experiments were performed at the Department of Genet- women less than 40 years old the OR was 1.4 (95% CI: 1.0 – ics and Pathology, Pomeranian Medical University, Szczecin, 1.9) (Table 1). Poland. We found 13 C5972T homozygotes (11 patients and 2 con- DNA isolation trols). The OR associated with the homozygous state was 4.7 DNA samples were obtained from peripheral blood of adults or (95% CI: 1.1 to 21.4). Among 11 cases of breast cancer diag- from cord blood of neonates using the non-enzymatic rapid nosed in homozygotes, 10 were diagnosed between ages 40 method as described previously [5]. and 50 years. The 11 homozygotes had predominantly intra- ductal (ductal carcinoma in situ (DCIS) with micro-invasion, 4 cases), ductal G3 (4 cases), lobular (2 cases), and ductal G2 Analysis of the C5972T variant The C5972T variant (Thr1915Met) was analyzed by restriction (1 case) cancers. None of the first- or second-degree relatives fragment length polymorphism PCR using b5972F (5'-CTC of the homozygotes were diagnosed with breast or ovarian TCT AGA TAA TGA TGA ATG ATG CA) and b5972R (5'- cancer. CCA AAC TAA CAT CAC AAG GTG) primers. The forward primer introduces an artificial restriction site for the Mph1103I We then explored the characteristics of the breast cancers enzyme (Fermentas, St. Leon-Rot, Germany) that were identified in women with the BRCA2 C5972T allele (Table 2) and compared them with tumors in women without PCR products were digested in mutation positive cases. PCR the predisposing allele (women with BRCA1 mutations were products were separated in 3% agarose gel and visualized in excluded from all comparisons and women who received pre- ultraviolet light. For positive cases with RFLP-PCR (cleaved by operative chemotherapy were excluded from the histological Mph1103I), a DNA sample was sequenced to confirm the comparisons). Women with the BRCA2 variant were more presence of the mutation. Additionally, 72 randomly selected likely to have bilateral cancer (5% versus 3%) or multifocal samples negative with RFLP-PCR for the C5972T variant cancer (30% versus 23%) but these differences were not sta- were sequenced. The results of RFLP-PCR and of direct DNA tistically significant. However, a significant effect was sequencing were 100% concordant. observed with intraductal cancer. Among the 5972C/T R1024 Available online http://breast-cancer-research.com/content/7/6/R1023 Table 1 Frequencies of the BRCA2 C5972T variant allele in breast cancer patients and controls Variant Controls Cases Odds ratio p-value 95% CI All subjects CC 2,630 3,039 1.0 CT 159 191 1.0 0.74 [0.8–1.3 ] TT 2 11 4.8 0.02 [1.1–21.4] CT+TT 161 202 1.1 0.45 [0.9–1.3 ] Total 2,791 3,241 Cases under age 40 years CC 2,630 598 1.0 CT 159 51 1.4 0.04 [1.0–2.0 ] TT 2 1 2.2 0.46 [0.2–23.7] CT+TT 161 52 1.4 0.03 [1.0–1.9 ] Total 2,791 650 Cases 41+ years CC 2,630 2,441 1.0 CT 159 140 0.9 0.68 [0.8–1.2 ] TT 2 10 5.4 0.02 [1.2–24.6] CT+TT 161 150 1.0 0.97 [0.8–1.3 ] Total 2,791 2,591 CI, confidence interval. heterozygotes, 18 of 103 tumors were predominantly intra- age). To our knowledge, this has not been seen before and ductal (17%), compared to 7% in 5972C/C breast cancer there is no compelling explanation; however, we believe that patients (OR = 2.82; p ≤ 0.0001; 95% CI: 1.6–4.8). A heter- the sizes of these subgroups are small and require confirma- ozygous BRCA2 variant was present in 13% of the women tion in other studies. with predominantly intraductal cancer. Compared to the pop- ulation controls, the OR for intraductal cancer given a (hetero- Our study population consisted of DCIS cases with a micro- zygous) form of BRCA2 variant was 2.5 (p = 0.0005; 95% CI: invasive component only. To our knowledge, this is the first 1.5–4.1). For women under 40 years old, the association was genetic association reported for intraductal cancer with micro- stronger (OR = 4.1; p = 0.006; 95% CI: 1.2–8.3). invasion. Our data set did not include cases of DCIS alone and we were unable to assess the effect of the BRCA2 C5972T allele in women with predominantly invasive cancer and asso- Discussion The C5972T allele of the BRCA2 gene appears to be over- ciated DCIS. These topics will be the focus of future studies. represented in women with early onset breast cancer in We did not see other pathological features that distinguished Poland compared to controls. Although the effect was modest BRCA2-associated tumors from tumors in non-carriers. This in the overall data set, it was stronger for women who had pre- situation is not different from that described for BRCA2 carri- dominantly intraductal breast cancer and for women who were ers in general [8-10]. homozygous for the variant allele. Existing reports on the C5972T variant do not record any case of homozygote TT Several previous studies have shown that DCIS is a character- genotypes [6,7]. It is of interest that the heterozygote state istic feature of cancers in BRCA2 carriers [11-14]. In the case was associated with early onset breast cancer and the of BRCA2-associated breast cancers, it appears that loss of homozygote state with later onset cancer (above 40 years of heterozygosity (LOH) occurs as a means of silencing the sec- R1025 Breast Cancer Research Vol 7 No 6 Górski et al. Table 2 Characteristics of breast cancers associated with 5972C/T or TT variants and wild-type BRCA2 sequence 5972C/T or TT Wild type OR p-value 95% CI N% N % Histology Medullary 7 6 122 7 0.86 0.71 [0.4–1.9] Ductal 41 36 607 35 1.04 0.83 [0.7–1.5] Tubular or tubulo-lobular 3 3 104 6 0.42 0.13 [0.1–1.3] Lobular 22 19 364 21 0.90 0.70 [0.5–1.5] DCIS+micro-invasion 22 19 121 7 3.18 <0.0001 [1.9–5.2] Other 19 17 416 24 0.63 0.09 [0.4–1.0] Multifocal 19 30 211 23 1.47 0.17 [0.8–2.6] Bilateral 7 5 63 3 1.71 0.18 [0.7–3.8] ER+ 50 67 586 62 1.24 0.40 [0.7–2.0] Family history+ All patients 32 21 569 26 0.72 0.14 [0.5–1.2] Diagnosed at <40 years 4 10 97 24 0.36 0.06 [0.1–1.1] 00 0 p-value 95% CI Mean age (years) 43.9 44.4 0.5 0.34 [-0.5–1.5] Mean tumor size (cm) 2.0 2.1 0.1 0.41 [-0.1–0.3] Odds ratios (ORs) for individual variables represent the odds of being a mutation carrier for a woman with a particular characteristic, compared to all other women who did not have the characteristic. For histology the comparisons were made for one histological group to all other histologies. b c Family history+ indicates the occurrence of one or more breast or ovarian cancers among first and second degree relatives. For age and tumor size comparisons, p-values and confidence intervals refer to the difference of means. Data were missing from the following categories and these cases were excluded from the specific calculations: histology (909 cases), multi-focality (1,774 cases); bilaterality (744 cases), estrogen receptor status (1,746 cases), family history (756 cases), family history in group diagnosed <40 (150 cases). Patients who received neo-adjuvant chemotherapy were excluded from all analyses with the exception of family history. 184 patients with BRCA1 mutations were excluded from all analyses. ER+, estrogen receptor expression positive. ond allele. In one family with a germline BRCA2 mutation, Actually, at least some of the 5972C/T heterozygotes may be three cases of invasive breast cancer and one case of DCIS compound heterozygotes, with the second allele being were observed [13]. LOH at the BRCA2 locus was demon- another BRCA2 variant. strated in all four cases. The authors suggest that DCIS and LOH in the BRCA2 locus may be common early steps in the We now provide epidemiological and clinical evidence dem- development of invasive breast cancer in BRCA2 carriers. The onstrating the deleterious nature of the C5972T allele. To our LOH data support the concept that BRCA2 behaves as a knowledge, functional studies have not been performed on tumor suppressor gene. In support of this hypothesis are our this variant. Few other deleterious missense variants in observations of a greater breast cancer risk in women with two BRCA2 have been identified. inherited 5972T variants and a lower frequency of positive family history among 5972C/T heterozygotes. The HH genotype of the non-conservative amino acid substi- tution polymorphism N372H in the BRCA2 gene was In our study, we observed no association between the exam- reported to be associated with a 1.3-fold to 1.5-fold increase ined variant and increased family history. This may be in the risk of both breast and ovarian cancer [15-18], but there explained by the modest association of the examined variant have been negative studies as well [19,20]. with breast cancer risk and probable recessive pattern of inheritance (the highest OR values were observed in homozy- Our control group was drawn both from the newborns of 10 gotes, all of them with negative breast cancer family history). Polish cities and from the adult population in the region of Szc- zecin; however, the frequency of alleles was similar in the newborn (5.7%) and adult (5.8%) populations. There was no R1026 Available online http://breast-cancer-research.com/content/7/6/R1023 al.: A high proportion of founder BRCA1 mutations in Polish evidence that the genotype frequencies of the C5972T vari- breast cancer families. Int J Cancer 2004, 110:683-686. ants deviated from those expected from the Hardy-Weinberg 3. Górski B, Cybulski C, Huzarski T, Byrski T, Gronwald J, equilibrium for the combined control group (p = 0.79) or for Jakubowska A, Stawicka M, Gozdecka-Grodecka S, Szwiec M, Urbański K, et al.: Breast cancer predisposing alleles in Poland. newborn and adult control groups (p = 0.59 and p = 0.73). Breast Cancer Res Treat 2005, 92(1):19-24. There was no statistical difference in the C5972T allele fre- 4. Bork P, Blomberg N, Nilges M: Internal repeats in the BRCA2 quencies in newborns recruited from the Szczecin metropoli- protein sequece. Nature Genet 1996, 13:22-23. 5. Lahiri DK, Schnabel B: DNA isolation by rapid method from tan region compared to other Polish cities (data not shown). human blood samples: effects of MgCl , EDTA, storage time, According to existing literature data, the frequency of the and temperature on DNA yield and quality. Biochem Genet 1993, 31:321-328. C5972T variant is similar in other populations such as North 6. Sigurdson A, Hauptmann M, Chatterjee N, Alexander B, Doody M, American, Austrian and Oceanian [6,7]. Rutter J, Struewing J: Kin-cohort estimates for familial breast cancer risk in relation to variants in DNA base excision repair, BRCA1 interacting and growth factor genes. BMC Cancer Conclusion 2004, 4:9. The heterozygosity of the BRCA2 C5972T variant appears to 7. Wagner TM, Hirtenlehner K, Shen P, Moeslinger R, Muhr D, Fleis- chmann E, Concin H, Doeller W, Haid A, Lang AH, et al.: Global be a neutral polymorphism in women above the age of 40 but sequence diversity of BRCA2: analysis of 71 breast cancer may be deleterious prior to age 40. The risk associated with families and 95 control individuals of worldwide populations. this allele was modest (OR = 1.4) in young women. Strong Hum Mol Genet 1999, 8:413-423. 8. Lakhani SR, Jacquemier J, Sloane JP, Gusterson BA, Anderson TJ, associations were not observed until cases were subclassified van de Vijver MJ, Farid LM, Venter D, Antoniou A, Storfer-Isser A, by their histological appearance or by homozygous state. It is et al.: Multifactorial analysis of differences between sporadic important to confirm these observations in other populations. breast cancers and cancers involving BRCA1 and BRCA2 mutations. J Natl Cancer Inst 1998, 90:1138-1145. Large, well-controlled studies are needed to establish the full 9. Narod SA, Foulkes WD: BRCA1 and BRCA2: 1994 and beyond. range of risks associated with other BRCA2 alleles before Nat Rev Cancer 2004, 4:665-676. 10. Honrado E, Benitez J, Palacios J: The pathology of hereditary they can definitely be identified as neutral. Identification of breast cancer. Hered Cancer Clin Practice 2004, 2:131-138. breast cancer susceptibility genes that are associated with 11. Armes JE, Egan AJ, Southey MC, Dite GS, McCredie MR, Giles modest penetrance requires very large association studies, GG, Hopper JL, Venter DJ: The histologic phenotypes of breast carcinoma occurring before age 40 years in women with and unless the high-risk alleles are very common. without BRCA1 or BRCA2 germline mutations: a population- based study. Cancer 1998, 83:2335-2345. 12. Marcus JN, Watson P, Page DL, Narod SA, Tonin P, Lenoir GM, Competing interests Serova O, Lynch HT: BRCA2 hereditary breast cancer The authors declare that they have no competing interests. pathophenotype. Breast Cancer Res Treat 1997, 44:275-277. 13. Thomassen M, Kruse TA, Olsen KE, Borg A, Gerdes AM: Loss of heterozygosity at BRCA2 in a ductal carcinoma in situ and Authors' contributions three invasive breast carcinomas in a family with a germline BG designed and performed lab assays. BG and JL con- BRCA2 mutation. Breast Cancer Res Treat 2004, 87:273-6. ducted data analysis and drafted the manuscript. BG, JL and 14. Claus EB, Petruzella S, Matloff E, Carter D: Prevalence of BRCA1 and BRCA2 mutations in women diagnosed with ductal carci- SN contributed to interpretation of results and revision of the noma in situ. J Am Med Assoc 2005, 293:964-969. manuscript. 15. Healey CS, Dunning AM, Teare MD, Chase D, Parker L, Burn J, Chang-Claude J, Mannermaa A, Kataja V, Huntsman DG, et al.: A common variant in BRCA2 is associated with both breast can- Acknowledgements cer risk and prenatal viability. Nat Genet 2000, 26:362-364. We thank Tomasz Huzarski, Tomasz Byrski, Jacek Gronwald, Olga 16. Spurdle AB, Hopper JL, Chen X, Dite GS, Cui J, McCredie MR, Haus, Hanna Janiszewska, Tomasz Mierzwa, Andrzej Mackiewicz, Anna Giles GG, Ellis-Steinborner S, Venter DJ, Newman B, Southey MC, Chenevix-Trench G: The BRCA2 372 HH genotype is asso- Przybyła, Katarzyna Lamperska, Malgorzata Stawicka, Dariusz God- ciated with risk of breast cancer in Australian women under lewski, Sylwia Grodecka-Gozdecka, Marek Bębenek, Andrzej Wojnar, age 60 years. Cancer Epidemiol Biomarkers Prev 2002, Ewa Grzybowska, Wioletta Pękala, Jolanta Pamula, Stanislaw Niepsuj, 11:413-416. Karol Gugała, Stanislaw Góźdź, Jacek Sygut, Monika Siołek, Boguslaw 17. Auranen A, Spurdle AB, Chen X, Lipscombe J, Purdie DM, Hopper JL, Green A, Healey CS, Redman K, Dunning AM, et al.: BRCA2 Musiatowicz, Michal Posmyk, Radzislaw Kordek, Maria Błasińska- Arg372 hispolymorphism and epithelial ovarian cancer risk. Morawiec, Oscar Zambrano, Bernard Was´ko, Ludmila Fudali, Dariusz Int J Cancer 2003, 103:427-430. Surdyka, Krzysztof Urbański, Marek Szwiec, Jan Koc, Elzbieta Marczyk, 18. Goode EL, Ulrich CM, Potter JD: Polymorphisms in DNA repair Janusz Rys´, Andrzej Rozmiarek, Tadeusz Dębniak, Ireneusz Dziuba, genes and associations with cancer risk. Cancer Epidemiol Biomarkers Prev 2002, 11:1513-1530. Cezary Szczylik, Agnieszka Synowiec, Wojciech Kozłowski, Piotr 19. Cox DG, Hankinson SE, Hunter DJ: No association between Wandzel, Beata Czeszyńska, and Wenancjusz Domagała for recruiting BRCA2 N372H and breast cancer risk. Cancer Epidemiol subjects and collecting clinical data. The study was supported with Biomarkers Prev 2005, 14:1353-1354. grant no. PBZ-54/01 from the State Committee for Scientific Research. 20. Wenham RM, Schildkraut JM, McLean K, Calingaert B, Bentley RC, Marks J, Berchuck A: Polymorphisms in BRCA1 and BRCA2 and risk of epithelial ovarian cancer. Clin Cancer Res 2003, References 9:4396-4403. 1. Górski B, Byrski T, Huzarski T, Jakubowska A, Menkiszak J, Gron- wald J, Pluzanska A, Bebenek M, Fischer-Maliszewska L, Grzy- bowska E, et al.: Founder mutations in the BRCA1 gene in Polish families with breast-ovarian cancer. Am J Hum Genet 2000, 66:1963-1968. 2. Górski B, Jakubowska A, Huzarski T, Byrski T, Gronwald J, Grzy- bowska E, Mackiewicz A, Stawicka M, Bębenek M, Sorokin D, et R1027 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Breast Cancer Research Springer Journals

A common missense variant in BRCA2 predisposes to early onset breast cancer

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Springer Journals
Copyright
2005 Górski et al.; licensee BioMed Central Ltd.
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1465-542X
DOI
10.1186/bcr1338
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Abstract

Introduction Mutations in the BRCA2 gene are one of the two Results The variant was present in approximately 6% of the major causes of hereditary breast cancer. Protein-truncating Polish population. In the study, 13 women (11 cases and two mutations of BRCA2 are usually deleterious and increase the controls (OR = 4.7; p = 0.02)) were homozygous for the variant risk of breast cancer up to 80% over a lifetime. A few missense allele. The overall odds ratio for breast cancer in women with a mutations in BRCA2 are believed to have a similarly high single copy of the BRCA2 C5972T variant was 1.1 (p = 0.7); penetrance, apart from more common neutral polymorphisms. It however, the effect was significant for patients diagnosed at or is often difficult to classify a particular sequence variant as a before age 40 (OR = 1.4; p = 0.04). We reviewed the mutation or a polymorphism. For a deleterious variant, one would association between the BRCA2 variant in different histologic expect a greater allele frequency in breast cancer cases than in subgroups and found the effect most pronounced in women ethnic-matched controls. In contrast, neutral polymorphic who had ductal carcinoma in situ (DCIS) with micro-invasion variants should be equally frequent in the two groups. (OR = 2.8; p < 0.0001). Methods We genotyped 3,241 cases of breast cancer Conclusion The BRCA2 C5972T allele is a common variant in diagnosed at under 51 years of age, unselected for family Poland that increases the risk of DCIS with micro-invasion. The history, from 18 hospitals throughout Poland and 2,791 ethnic- homozygous state is rare but increases the risk of breast cancer matched controls for a single BRCA2 C5972T variant. five-fold. eral deleterious founder alleles have been identified in Introduction There are several approaches to identifying low-penetrance BRCA1, but to date, no founder mutation in BRCA2 has been candidate genes for breast cancer. In one approach, it is identified [1-3]. There are a few common variant alleles in assumed that missense variants of genes for which truncating BRCA2 in Poland; one of these (C5972T) changes the amino mutations are clearly pathogenic might also be deleterious. If acid sequence of BRCA2 from threonine to methionine at the missense allele demonstrates high penetrance (i.e. like codon 1915. It lies within the range of the BRC encoded by truncating mutations), then it will be relatively straightforward exon 11 that are thought to be involved in binding to RAD51 to establish the association when allele frequency is high. If the [4]. We sought to determine whether this common missense penetrance of the missense variant is low, however, then the BRCA2 variant plays a role in breast cancer susceptibility. association may be missed if only a small number of cancers is studied and the variant may falsely be classified as a neutral Materials and methods polymorphism. We are in the process of establishing a large Study subjects database of breast cancer cases and ethnic-matched controls The study population included prospectively ascertained in order to evaluate the pathogenicity of the common founder cases of invasive breast cancer diagnosed at 50 years of age alleles of the most important cancer susceptibility genes. Sev- or less at 18 treatment centers throughout Poland between DCIS = ductal carcinoma in situ; LOH = loss of heterozygosity; OR = odds ratio; RFLP-PCR = restriction fragment length polymorphism polymerase chain reaction. R1023 Breast Cancer Research Vol 7 No 6 Górski et al. 1997 and 2003. Invitation to participate was delivered during DNA sequencing hospital stay or by mail. During the interview, the goals of the A fragment of BRCA2 exon 11 was amplified by PCR using study were explained, informed consent was obtained, genetic b5972F/b5972R primers. The purified PCR products were counseling was offered, and a blood sample was taken for sequenced directly with a BigDye Terminator v3.0 DNA DNA analysis. A detailed family history of cancer was taken Sequencing Kit (Perkin Elmer, Foster City, CA, USA) and the (first-, second-, and third-degree relatives included) and a risk same reverse primer used previously for the amplification of factor questionnaire was completed. A total of 4,778 cases of exon 11 of the BRCA2 gene. Products of sequencing reac- early onset invasive breast cancer were identified at the 18 tions were separated and analysed on the ABI 377 DNA participating centers during the study period. Of these, 3,627 Sequencer (Perkin Elmer). women (76%) accepted to participate and provided a blood BRCA1 testing sample for DNA analyses, 114 died, and 16 refused to partic- ipate. The medical record and pathology report were reviewed. All breast cancer patients were tested as described previously In the end, we had DNA available for genotyping from 3,241 [4] for three mutations (5382insC, C61G, 4153delA) repre- patients. senting 80% to 90% of all high-risk BRCA1 changes in Poland. Controls Two control groups were combined. The first group consisted Statistical analysis of 2,000 unselected neonates from ten hospitals throughout Statistical analysis included a comparison of the prevalence of Poland born in 2003 and 2004. Samples of cord blood were the C5972T allele in cases and controls. Odds ratios (ORs) forwarded to the study center in Szczecin. The second control were generated from two-by-two tables and statistical signifi- group consisted of 1,000 adults from the region of Szczecin cance was assessed with the chi-square test. BRCA2 nucle- unselected for cancer family history, sex, or age. We geno- otides were identified according to the GeneBank U43746 typed 2,791 controls for the C5972T allele, 1,993 neonates sequence. and 798 adults. To ensure comparability of the control groups, C5729T allele frequencies were computed separately for the Results adult and neonatal control groups and compared. The study The C5972T variant was detected in 8.3% of unselected protocol was approved by the ethics committee of the Pomer- breast cancer patients diagnosed at age 40 years or below, in anian Medical University. 5.7% of cases diagnosed from age 41 to 50 years, and in 5.8% of Polish controls. The overall OR for breast cancer with Laboratory methods a C5972T mutation was 1.1 (95% CI: 0.8–1.3); however, in All experiments were performed at the Department of Genet- women less than 40 years old the OR was 1.4 (95% CI: 1.0 – ics and Pathology, Pomeranian Medical University, Szczecin, 1.9) (Table 1). Poland. We found 13 C5972T homozygotes (11 patients and 2 con- DNA isolation trols). The OR associated with the homozygous state was 4.7 DNA samples were obtained from peripheral blood of adults or (95% CI: 1.1 to 21.4). Among 11 cases of breast cancer diag- from cord blood of neonates using the non-enzymatic rapid nosed in homozygotes, 10 were diagnosed between ages 40 method as described previously [5]. and 50 years. The 11 homozygotes had predominantly intra- ductal (ductal carcinoma in situ (DCIS) with micro-invasion, 4 cases), ductal G3 (4 cases), lobular (2 cases), and ductal G2 Analysis of the C5972T variant The C5972T variant (Thr1915Met) was analyzed by restriction (1 case) cancers. None of the first- or second-degree relatives fragment length polymorphism PCR using b5972F (5'-CTC of the homozygotes were diagnosed with breast or ovarian TCT AGA TAA TGA TGA ATG ATG CA) and b5972R (5'- cancer. CCA AAC TAA CAT CAC AAG GTG) primers. The forward primer introduces an artificial restriction site for the Mph1103I We then explored the characteristics of the breast cancers enzyme (Fermentas, St. Leon-Rot, Germany) that were identified in women with the BRCA2 C5972T allele (Table 2) and compared them with tumors in women without PCR products were digested in mutation positive cases. PCR the predisposing allele (women with BRCA1 mutations were products were separated in 3% agarose gel and visualized in excluded from all comparisons and women who received pre- ultraviolet light. For positive cases with RFLP-PCR (cleaved by operative chemotherapy were excluded from the histological Mph1103I), a DNA sample was sequenced to confirm the comparisons). Women with the BRCA2 variant were more presence of the mutation. Additionally, 72 randomly selected likely to have bilateral cancer (5% versus 3%) or multifocal samples negative with RFLP-PCR for the C5972T variant cancer (30% versus 23%) but these differences were not sta- were sequenced. The results of RFLP-PCR and of direct DNA tistically significant. However, a significant effect was sequencing were 100% concordant. observed with intraductal cancer. Among the 5972C/T R1024 Available online http://breast-cancer-research.com/content/7/6/R1023 Table 1 Frequencies of the BRCA2 C5972T variant allele in breast cancer patients and controls Variant Controls Cases Odds ratio p-value 95% CI All subjects CC 2,630 3,039 1.0 CT 159 191 1.0 0.74 [0.8–1.3 ] TT 2 11 4.8 0.02 [1.1–21.4] CT+TT 161 202 1.1 0.45 [0.9–1.3 ] Total 2,791 3,241 Cases under age 40 years CC 2,630 598 1.0 CT 159 51 1.4 0.04 [1.0–2.0 ] TT 2 1 2.2 0.46 [0.2–23.7] CT+TT 161 52 1.4 0.03 [1.0–1.9 ] Total 2,791 650 Cases 41+ years CC 2,630 2,441 1.0 CT 159 140 0.9 0.68 [0.8–1.2 ] TT 2 10 5.4 0.02 [1.2–24.6] CT+TT 161 150 1.0 0.97 [0.8–1.3 ] Total 2,791 2,591 CI, confidence interval. heterozygotes, 18 of 103 tumors were predominantly intra- age). To our knowledge, this has not been seen before and ductal (17%), compared to 7% in 5972C/C breast cancer there is no compelling explanation; however, we believe that patients (OR = 2.82; p ≤ 0.0001; 95% CI: 1.6–4.8). A heter- the sizes of these subgroups are small and require confirma- ozygous BRCA2 variant was present in 13% of the women tion in other studies. with predominantly intraductal cancer. Compared to the pop- ulation controls, the OR for intraductal cancer given a (hetero- Our study population consisted of DCIS cases with a micro- zygous) form of BRCA2 variant was 2.5 (p = 0.0005; 95% CI: invasive component only. To our knowledge, this is the first 1.5–4.1). For women under 40 years old, the association was genetic association reported for intraductal cancer with micro- stronger (OR = 4.1; p = 0.006; 95% CI: 1.2–8.3). invasion. Our data set did not include cases of DCIS alone and we were unable to assess the effect of the BRCA2 C5972T allele in women with predominantly invasive cancer and asso- Discussion The C5972T allele of the BRCA2 gene appears to be over- ciated DCIS. These topics will be the focus of future studies. represented in women with early onset breast cancer in We did not see other pathological features that distinguished Poland compared to controls. Although the effect was modest BRCA2-associated tumors from tumors in non-carriers. This in the overall data set, it was stronger for women who had pre- situation is not different from that described for BRCA2 carri- dominantly intraductal breast cancer and for women who were ers in general [8-10]. homozygous for the variant allele. Existing reports on the C5972T variant do not record any case of homozygote TT Several previous studies have shown that DCIS is a character- genotypes [6,7]. It is of interest that the heterozygote state istic feature of cancers in BRCA2 carriers [11-14]. In the case was associated with early onset breast cancer and the of BRCA2-associated breast cancers, it appears that loss of homozygote state with later onset cancer (above 40 years of heterozygosity (LOH) occurs as a means of silencing the sec- R1025 Breast Cancer Research Vol 7 No 6 Górski et al. Table 2 Characteristics of breast cancers associated with 5972C/T or TT variants and wild-type BRCA2 sequence 5972C/T or TT Wild type OR p-value 95% CI N% N % Histology Medullary 7 6 122 7 0.86 0.71 [0.4–1.9] Ductal 41 36 607 35 1.04 0.83 [0.7–1.5] Tubular or tubulo-lobular 3 3 104 6 0.42 0.13 [0.1–1.3] Lobular 22 19 364 21 0.90 0.70 [0.5–1.5] DCIS+micro-invasion 22 19 121 7 3.18 <0.0001 [1.9–5.2] Other 19 17 416 24 0.63 0.09 [0.4–1.0] Multifocal 19 30 211 23 1.47 0.17 [0.8–2.6] Bilateral 7 5 63 3 1.71 0.18 [0.7–3.8] ER+ 50 67 586 62 1.24 0.40 [0.7–2.0] Family history+ All patients 32 21 569 26 0.72 0.14 [0.5–1.2] Diagnosed at <40 years 4 10 97 24 0.36 0.06 [0.1–1.1] 00 0 p-value 95% CI Mean age (years) 43.9 44.4 0.5 0.34 [-0.5–1.5] Mean tumor size (cm) 2.0 2.1 0.1 0.41 [-0.1–0.3] Odds ratios (ORs) for individual variables represent the odds of being a mutation carrier for a woman with a particular characteristic, compared to all other women who did not have the characteristic. For histology the comparisons were made for one histological group to all other histologies. b c Family history+ indicates the occurrence of one or more breast or ovarian cancers among first and second degree relatives. For age and tumor size comparisons, p-values and confidence intervals refer to the difference of means. Data were missing from the following categories and these cases were excluded from the specific calculations: histology (909 cases), multi-focality (1,774 cases); bilaterality (744 cases), estrogen receptor status (1,746 cases), family history (756 cases), family history in group diagnosed <40 (150 cases). Patients who received neo-adjuvant chemotherapy were excluded from all analyses with the exception of family history. 184 patients with BRCA1 mutations were excluded from all analyses. ER+, estrogen receptor expression positive. ond allele. In one family with a germline BRCA2 mutation, Actually, at least some of the 5972C/T heterozygotes may be three cases of invasive breast cancer and one case of DCIS compound heterozygotes, with the second allele being were observed [13]. LOH at the BRCA2 locus was demon- another BRCA2 variant. strated in all four cases. The authors suggest that DCIS and LOH in the BRCA2 locus may be common early steps in the We now provide epidemiological and clinical evidence dem- development of invasive breast cancer in BRCA2 carriers. The onstrating the deleterious nature of the C5972T allele. To our LOH data support the concept that BRCA2 behaves as a knowledge, functional studies have not been performed on tumor suppressor gene. In support of this hypothesis are our this variant. Few other deleterious missense variants in observations of a greater breast cancer risk in women with two BRCA2 have been identified. inherited 5972T variants and a lower frequency of positive family history among 5972C/T heterozygotes. The HH genotype of the non-conservative amino acid substi- tution polymorphism N372H in the BRCA2 gene was In our study, we observed no association between the exam- reported to be associated with a 1.3-fold to 1.5-fold increase ined variant and increased family history. This may be in the risk of both breast and ovarian cancer [15-18], but there explained by the modest association of the examined variant have been negative studies as well [19,20]. with breast cancer risk and probable recessive pattern of inheritance (the highest OR values were observed in homozy- Our control group was drawn both from the newborns of 10 gotes, all of them with negative breast cancer family history). Polish cities and from the adult population in the region of Szc- zecin; however, the frequency of alleles was similar in the newborn (5.7%) and adult (5.8%) populations. There was no R1026 Available online http://breast-cancer-research.com/content/7/6/R1023 al.: A high proportion of founder BRCA1 mutations in Polish evidence that the genotype frequencies of the C5972T vari- breast cancer families. Int J Cancer 2004, 110:683-686. ants deviated from those expected from the Hardy-Weinberg 3. Górski B, Cybulski C, Huzarski T, Byrski T, Gronwald J, equilibrium for the combined control group (p = 0.79) or for Jakubowska A, Stawicka M, Gozdecka-Grodecka S, Szwiec M, Urbański K, et al.: Breast cancer predisposing alleles in Poland. newborn and adult control groups (p = 0.59 and p = 0.73). Breast Cancer Res Treat 2005, 92(1):19-24. There was no statistical difference in the C5972T allele fre- 4. Bork P, Blomberg N, Nilges M: Internal repeats in the BRCA2 quencies in newborns recruited from the Szczecin metropoli- protein sequece. Nature Genet 1996, 13:22-23. 5. Lahiri DK, Schnabel B: DNA isolation by rapid method from tan region compared to other Polish cities (data not shown). human blood samples: effects of MgCl , EDTA, storage time, According to existing literature data, the frequency of the and temperature on DNA yield and quality. Biochem Genet 1993, 31:321-328. C5972T variant is similar in other populations such as North 6. Sigurdson A, Hauptmann M, Chatterjee N, Alexander B, Doody M, American, Austrian and Oceanian [6,7]. Rutter J, Struewing J: Kin-cohort estimates for familial breast cancer risk in relation to variants in DNA base excision repair, BRCA1 interacting and growth factor genes. BMC Cancer Conclusion 2004, 4:9. The heterozygosity of the BRCA2 C5972T variant appears to 7. Wagner TM, Hirtenlehner K, Shen P, Moeslinger R, Muhr D, Fleis- chmann E, Concin H, Doeller W, Haid A, Lang AH, et al.: Global be a neutral polymorphism in women above the age of 40 but sequence diversity of BRCA2: analysis of 71 breast cancer may be deleterious prior to age 40. The risk associated with families and 95 control individuals of worldwide populations. this allele was modest (OR = 1.4) in young women. Strong Hum Mol Genet 1999, 8:413-423. 8. Lakhani SR, Jacquemier J, Sloane JP, Gusterson BA, Anderson TJ, associations were not observed until cases were subclassified van de Vijver MJ, Farid LM, Venter D, Antoniou A, Storfer-Isser A, by their histological appearance or by homozygous state. It is et al.: Multifactorial analysis of differences between sporadic important to confirm these observations in other populations. breast cancers and cancers involving BRCA1 and BRCA2 mutations. J Natl Cancer Inst 1998, 90:1138-1145. Large, well-controlled studies are needed to establish the full 9. Narod SA, Foulkes WD: BRCA1 and BRCA2: 1994 and beyond. range of risks associated with other BRCA2 alleles before Nat Rev Cancer 2004, 4:665-676. 10. Honrado E, Benitez J, Palacios J: The pathology of hereditary they can definitely be identified as neutral. Identification of breast cancer. Hered Cancer Clin Practice 2004, 2:131-138. breast cancer susceptibility genes that are associated with 11. Armes JE, Egan AJ, Southey MC, Dite GS, McCredie MR, Giles modest penetrance requires very large association studies, GG, Hopper JL, Venter DJ: The histologic phenotypes of breast carcinoma occurring before age 40 years in women with and unless the high-risk alleles are very common. without BRCA1 or BRCA2 germline mutations: a population- based study. Cancer 1998, 83:2335-2345. 12. Marcus JN, Watson P, Page DL, Narod SA, Tonin P, Lenoir GM, Competing interests Serova O, Lynch HT: BRCA2 hereditary breast cancer The authors declare that they have no competing interests. pathophenotype. Breast Cancer Res Treat 1997, 44:275-277. 13. Thomassen M, Kruse TA, Olsen KE, Borg A, Gerdes AM: Loss of heterozygosity at BRCA2 in a ductal carcinoma in situ and Authors' contributions three invasive breast carcinomas in a family with a germline BG designed and performed lab assays. BG and JL con- BRCA2 mutation. Breast Cancer Res Treat 2004, 87:273-6. ducted data analysis and drafted the manuscript. BG, JL and 14. Claus EB, Petruzella S, Matloff E, Carter D: Prevalence of BRCA1 and BRCA2 mutations in women diagnosed with ductal carci- SN contributed to interpretation of results and revision of the noma in situ. J Am Med Assoc 2005, 293:964-969. manuscript. 15. Healey CS, Dunning AM, Teare MD, Chase D, Parker L, Burn J, Chang-Claude J, Mannermaa A, Kataja V, Huntsman DG, et al.: A common variant in BRCA2 is associated with both breast can- Acknowledgements cer risk and prenatal viability. Nat Genet 2000, 26:362-364. We thank Tomasz Huzarski, Tomasz Byrski, Jacek Gronwald, Olga 16. Spurdle AB, Hopper JL, Chen X, Dite GS, Cui J, McCredie MR, Haus, Hanna Janiszewska, Tomasz Mierzwa, Andrzej Mackiewicz, Anna Giles GG, Ellis-Steinborner S, Venter DJ, Newman B, Southey MC, Chenevix-Trench G: The BRCA2 372 HH genotype is asso- Przybyła, Katarzyna Lamperska, Malgorzata Stawicka, Dariusz God- ciated with risk of breast cancer in Australian women under lewski, Sylwia Grodecka-Gozdecka, Marek Bębenek, Andrzej Wojnar, age 60 years. Cancer Epidemiol Biomarkers Prev 2002, Ewa Grzybowska, Wioletta Pękala, Jolanta Pamula, Stanislaw Niepsuj, 11:413-416. Karol Gugała, Stanislaw Góźdź, Jacek Sygut, Monika Siołek, Boguslaw 17. Auranen A, Spurdle AB, Chen X, Lipscombe J, Purdie DM, Hopper JL, Green A, Healey CS, Redman K, Dunning AM, et al.: BRCA2 Musiatowicz, Michal Posmyk, Radzislaw Kordek, Maria Błasińska- Arg372 hispolymorphism and epithelial ovarian cancer risk. Morawiec, Oscar Zambrano, Bernard Was´ko, Ludmila Fudali, Dariusz Int J Cancer 2003, 103:427-430. Surdyka, Krzysztof Urbański, Marek Szwiec, Jan Koc, Elzbieta Marczyk, 18. Goode EL, Ulrich CM, Potter JD: Polymorphisms in DNA repair Janusz Rys´, Andrzej Rozmiarek, Tadeusz Dębniak, Ireneusz Dziuba, genes and associations with cancer risk. Cancer Epidemiol Biomarkers Prev 2002, 11:1513-1530. Cezary Szczylik, Agnieszka Synowiec, Wojciech Kozłowski, Piotr 19. Cox DG, Hankinson SE, Hunter DJ: No association between Wandzel, Beata Czeszyńska, and Wenancjusz Domagała for recruiting BRCA2 N372H and breast cancer risk. Cancer Epidemiol subjects and collecting clinical data. The study was supported with Biomarkers Prev 2005, 14:1353-1354. grant no. PBZ-54/01 from the State Committee for Scientific Research. 20. Wenham RM, Schildkraut JM, McLean K, Calingaert B, Bentley RC, Marks J, Berchuck A: Polymorphisms in BRCA1 and BRCA2 and risk of epithelial ovarian cancer. Clin Cancer Res 2003, References 9:4396-4403. 1. Górski B, Byrski T, Huzarski T, Jakubowska A, Menkiszak J, Gron- wald J, Pluzanska A, Bebenek M, Fischer-Maliszewska L, Grzy- bowska E, et al.: Founder mutations in the BRCA1 gene in Polish families with breast-ovarian cancer. Am J Hum Genet 2000, 66:1963-1968. 2. Górski B, Jakubowska A, Huzarski T, Byrski T, Gronwald J, Grzy- bowska E, Mackiewicz A, Stawicka M, Bębenek M, Sorokin D, et R1027

Journal

Breast Cancer ResearchSpringer Journals

Published: Dec 1, 2005

Keywords: Cancer Research; Oncology; Surgical Oncology

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