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Association between the BRCA2N372H variant and male breast cancer risk: a population-based case-control study in Tuscany, Central Italy

Association between the BRCA2N372H variant and male breast cancer risk: a population-based... Background: Male breast cancer (MBC) is a rare disease and little is known about its aetiology. Germ- line mutations of BRCA2 and, at lower frequency, of BRCA1 are implicated in a relatively small proportion of MBC cases. Common polymorphic variants in BRCA1 and BRCA2 genes may represent breast cancer (BC) susceptibility alleles and could be associated with a modestly increased risk of MBC at population level. Considering the relevant role of BRCA2 in MBC, we investigated whether the BRCA2 N372H variant, representing the only common non-synonymous polymorphism in BRCA2, might modulate the risk of BC in male populations. Methods: A case-control study was performed comparing a population-based series of 99 MBC cases, characterized for BRCA1 and BRCA2 mutations, with 261 male population controls, all residing in Tuscany, Central Italy. All MBC cases and controls were genotyped for the BRCA2 N372H allele by TaqMan allelic discrimination assays. To evaluate the genotype specific risk of the BRCA2 N372H variant, MBC carriers of germ-line BRCA1/2 mutations were excluded from the analyses. Results: No association emerged in univariate and age-adjusted analyses. Age-specific analyses suggested an increased risk for the HH homozygous genotype in subjects younger than 60 years. A statistically significant interaction emerged between this genotype and age (p = 0.032). When analyses were restricted to MBC cases enrolled in the first 4 years following diagnosis, a recessive model showed a significantly increased risk of MBC in HH subjects younger than 60 years (OR = 5.63; 95% CI = 1.70;18.61). Conclusion: Overall, our findings, although based on a relatively small series, suggest that the BRCA2 HH homozygous genotype might be positively associated with an increased risk of MBC in men younger than 60 years. Page 1 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:170 http://www.biomedcentral.com/1471-2407/7/170 women [16]. To our knowledge, the effect of the BRCA2 Background Male breast cancer (MBC) is a rare disease and little is N372H polymorphism on BC risk has not yet been inves- known about its aetiology compared with female breast tigated in male populations. cancer (FBC). MBC represents less than 1% of all cancers in men and its incidence is increasing in younger men [1]. Considering the relevance of BRCA2 in MBC, we have per- In Italy, MBC accounts for 0.2% of all cancers in males [2]. formed a population-based case-control study to investi- gate the role of BRCA2 N372H variant on BC risk in men, Similar to FBC, a positive family history (FH) of breast comparing a series of MBC, characterized for BRCA1 and cancer (BC) is associated with increased risk of MBC and BRCA2 mutation status, with a control group from the approximately 15% to 20% of male patients with BC have same area of Central Italy (Florence, Tuscany). a positive FH [3]. The two major hereditary BC genes, BRCA1 (OMIM #113705) and, to a larger extent, BRCA2 Methods (OMIM # 600185), are implicated in MBC. Both genes are Study population and data collection estimated to be responsible respectively for up to 16% and In a previous study [17], we had identified a population- 76% of the MBCs in high-risk breast/ovarian cancer fami- based series of 25 MBC cases diagnosed in the area of Flor- lies [3,4]. The frequencies of BRCA1/BRCA2 mutations are ence (Tuscany, Central Italy) during the period 1990– sharply different in ethnically diverse population- and 1998. One of the problems in MBC studies is represented clinic-based series ranging from 0% to 4% for BRCA1 and by the rarity of the disease: in order to increase the from 4% to 40% for BRCA2 [5,6]. However, at population number of cases for meaningful statistical analyses it is level only a small proportion of all MBC cases are due to necessary to enrol patients diagnosed over a long period inherited mutations in BRCA1/BRCA2 genes. of time (or in a larger geographical area). Thus, using all available local sources (including Pathology Departments Low-penetrance polymorphisms in BC susceptibility and the Hospital Discharge database) the original series genes are present in a high percentage of individuals and has been expanded and 74 additional MBC cases diag- might account for BC risk at population level. In this nosed in the period 1991–2006 have been enrolled for a respect, common polymorphic variants in BRCA1/BRCA2 total of 99 MBCs available for this study, all residing in genes may represent BC susceptibility alleles and could be Tuscany. associated with a modest risk of MBC that would explain, on population basis, a large proportion of the disease. Overall, after exclusion of deceased and migrated patients, 85 additional unrelated MBC were traced and invited to The N372H (rs144848) polymorphism is the unique var- participate into the study. Eleven cases refused to partici- iant in BRCA2 gene that results in an amino acid change pate, mostly because of advanced age or severe illness. and has a rare allele frequency greater than 10%. Little is Confirming our previous response rate of about 80%, 74 known on the functional effect of this polymorphism, of these 85 MBC cases (87.1%) agreed to participate. although the substitution of asparagine (a neutral amino According to the original study protocol, each MBC acid) by histidine (a basic amino acid) may be expected to patient provided: 1) informed consent; 2) blood samples; affect BRCA2 structure and function as it falls in a region 3) detailed information on his personal and family his- that has been shown to interact with the histone acetyl- tory of cancer at any site, including all first- and second- transferase P/CAF prior to transcriptional activation of degree relatives of both genders; 4) a detailed smoking other genes [7]. Intriguingly, an excess of NH heterozy- and working history up to the date of BC diagnosis. Infor- gotes was observed in female controls and an effect of this mation has been validated by available sources (mainly variant on fetal survival in a sex-dependent manner has local Cancer and Mortality Registries). Procedures to been suggested as newborn females showed an excess of maintain confidentiality for all the information collected heterozygotes and a deficit of homozygotes, whereas the have been developed and strictly applied. The study was opposite was observed in newborn males [8]. approved by the local Ethical Research Committee (Flor- ence Health Unit). Several studies have analysed the effect of the BRCA2 N372Hpolymorphism on the risk of breast cancer in Population controls female populations with inconsistent findings. The HH In the frame of a multi-site epidemiological project, two homozygous genotype was reported to be associated with series of healthy adults of both sexes have been randomly a 1.3- to 1.5-fold increased risk of breast and ovarian can- selected from the municipality lists of two areas in Tus- cer [8-11]. In contrast, other studies did not show any cany: the city of Florence and one rural area [18,19]. All effect of this polymorphism on breast or ovarian cancer participants signed an informed consent form and pro- risk [12-15]. A recent large pooled analysis, however, vided a blood sample. Out of 700 randomly selected sub- could not exclude an effect of this genotype in younger jects, 553 subjects (79%) accepted to participate into the Page 2 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:170 http://www.biomedcentral.com/1471-2407/7/170 study; males were 48.6% (269/553) and 261 samples expected genotype frequencies (based on observed qs) to were available for analyses (eight of the initial 269 con- observed genotype frequencies. The association between MBC risk and BRCA2 N372H was measured by the odds trol's samples (3%) yielded insufficient DNA). The blood samples have been processed at the study laboratory ratio (OR) and its corresponding 95% confidence interval (CSPO, Florence), in the same day of collection and and was estimated using unconditional logistic regression divided in specific aliquots (RBC, buffy coat, serum, after adjustment for age considered as dichotomous vari- plasma). The aliquots have been stored in a -80°C freezer able (<60 years, >60 years). The analysis were performed at the Biological Bank of the Molecular Epidemiology with four logistic regression models based on a co-domi- Unit at CSPO. nant, dominant, recessive and multiplicative codominant effect (inheritance model). In the dominant model, both Mutational analysis the heterozygous variant and rare homozygous variant Buffy coat aliquots from MBC cases and controls were were combined in a dummy variable. In the recessive anonymously shipped to the research laboratory (Experi- model, the variant was defined in a dummy variable as mental Medicine, Rome) where genomic DNA was only the rare homozygous genotype. In the co-dominant extracted using QIAamp DNA Mini Kit (Qiagen Inc., model both rare homozygous and heterozygous variant Charlesworth, CA). The entire BRCA1 and BRCA2 coding effects were estimated using two dummy variables while sequences, including intron-exon boundaries, were ana- in the multiplicative codominant model a dose-response lysed by using single strand conformation polymor- effect were tested on the variable counting the number of phisms (SSCP) combined with protein truncation test copies of the H allele. Analyses have also been carried out (PTT) and automatic sequencing analysis [17,20]. Muta- according to stratification by age at diagnosis for cases and tions were always verified by PCR direct sequencing on age at interview for controls (below/above 60 years) and two independent blood samples (reference sequence for restricted to MBC cases with a time interval after diagnosis BRCA1: Genbank, U14680; reference sequence for shorter than the median value of the series (4 years). Inter- BRCA2: Genbank, NM_000059). actions between age, modelled as dichotomous variable (<60 years, >60 years), and genotype under co-dominant, SNP genotyping dominant, recessive and multiplicative codominant mod- The BRCA2 N372H (rs144848) polymorphism was ana- els, were assessed using Likelihood Ratio test (LR) com- lysed by TaqMan allelic discrimination using Real-Time paring logistic regression models with and without PCR. Fluorescent hybridisation probes, labelled with 6- interaction term. carboxyfluorescin (FAM) to detect the 1342C (372H) allele and with Texas Red to detect the 1342A (372N) Results allele were specifically designed. TaqMan assays were per- In order to evaluate the putative influence of the BRCA2 formed in a reaction volume of 50 µl, comprising 100 ng N372H polymorphism on MBC risk, we carried out a pop- of DNA, 400 nM each probe, 400 nM each primer and 1 × ulation-based case-control study based on a total of 99 Universal Master Mix (Biorad). The following conditions MBC cases and 261 adult male controls from the same were used for amplification: 3 minutes at 95°C and 55 area of Central Italy (Florence, Tuscany). The mean age at cycles at 95°C for 10" and 63°C for 45". The Real-Time interview was 67.9 (SD 11.8) in cases and 55.9 (SD 6.9) PCR was accomplished on iCycler thermal cycler (Biorad) in controls (p < 0.0001). The mean age at diagnosis in the with 96 × 0.2 ml samples. The fluorescence was visualised MBC series was 63.4 (SD 12.1; median 65) and the time through iCycler Optical System (Biorad) during Real- interval between diagnosis and interview ranged from 0 to Time PCR and analysed by using the iCycler IQ Real-Time 26 years (median 4.0). A detailed FH for breast and ovar- Detection System (Biorad) 3.0 software. About 15% of the ian cancers diagnosed in first-degree relatives was col- genotyping results were confirmed by sequencing analy- lected for all MBC cases. Overall, 25.3% (25/99) of MBC sis. The genotype controls for the three possible genotypes patients reported a first degree FH positive for breast/ovar- plus "no template" controls were always included in each ian cancer. analysis. MBC cases were characterized for BRCA1 and BRCA2 Statistical analysis mutations. Overall, 8/99 (8.1%) MBC cases resulted to be Allele frequencies have been calculated as the number of mutated in BRCA1/BRCA2 genes: 6 cases (6.1%) carried alleles divided by the number of chromosomes. Genotype BRCA2 mutations and 2 cases (2.0%) carried BRCA1 frequencies have been calculated as the number of partic- mutations. All 99 MBC cases and 261 controls were ana- ipants with a particular genotype divided by the number lysed for the BRCA2 N372H allele and genotype frequen- of participants. Tests for Hardy-Weinberg equilibrium cies. Genotype distribution was consistent with Hardy- among cases and controls have been assessed using Pear- Weinberg equilibrium (p = 0.529) among our population son's χ test with one degree of freedom comparing controls and cases (p = 0.067). In order to evaluate the Page 3 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:170 http://www.biomedcentral.com/1471-2407/7/170 genotype specific risk of the BRCA2 N372H polymor- both age at diagnosis and time interval after diagnosis dif- phism, MBC carriers of germ-line BRCA1/2 mutations (8 ferent from those identified a priori. These analyses con- cases) were excluded from the analyses. firmed the association, when older individuals (up to 65 years of age) or cases with a longer time interval (up to 6 As shown in Table 1, no statistically significant difference years) were included. in the distribution of the three specific BRCA2 N372H genotypes was observed between MBC cases and controls Discussion (p = 0.46). The age-adjusted analysis of the genotype-spe- We evaluated the effect of the BRCA2 N372H (1342A > C) cific risks showed that individuals with NH heterozygous polymorphism on MBC risk in a population based case- and HH homozygous genotypes were not at increased control study. Overall, a positive association between the MBC risk. These results were confirmed on dominant, HH genotype and an increased risk of MBC was suggested recessive and multiplicative codominant transmission in men younger than 60 years; this association was partic- models (data not shown). Separate analyses stratified ularly strong (with a five-fold increased risk) when analy- according to age (below/above 60 years) showed a ten- ses were restricted to 53 MBC cases enrolled in the study dency towards an inverse association between the H allele with a blood donation not later than 4 years after their and MBC risk in the older sub-group (age > 60 years), diagnosis. To our knowledge, the present study is the first although far from being statistically significant. On the reporting an analysis of the association between BC risk contrary, an increased MBC risk (OR = 3.12; 95%CI = and the BRCA2 N372H variant in men. 1.08; 9.03) was observed in younger subjects (age ≤ 60 years) on the basis of a recessive model (Table 2). A model The role of the BRCA2 N372H variant on BC risk has been specifically aimed to evaluate a possible effect modifica- investigated by several studies in women from different tion of age on the association between MBC risk and the populations with inconsistent findings that might reflect HH homozygous genotype found a statistically significant genetic differences across populations. In particular the interaction (LR test p = 0.02; OR = 0.17; 95%CI = HH genotype was associated with a 1.3- to 1.5-fold interaction 0.04; 0.76). increased risk of BC in series from Northern Europe [8], Australia [9] and in a selected series of radiologic technol- We carried out additional analyses taking into account the ogists from USA [10]. This association was not detected in length of the interval between diagnosis and blood dona- series from Central Europe [13], Japan [12] and in an tion among cases. Overall, no association between the H unselected series from USA [14]. allele and MBC risk was observed, when analyses were restricted to a comparison between the group of 53 MBC Here, we performed a population-based case control cases with an interval shorter than the median value of the study on male subjects all residing in the same area of whole series (≤4 years) and the series of controls (Table Central Italy (Tuscany). Our analyses based on a recessive 3). Additional analyses stratified by age, showed a transmission model suggested an association between the strongly increased MBC risk (OR = 5.63; 95%CI = 1.70; BRCA2 N372H variant and increased MBC risk in men 18.61) in younger subjects with the HH homozygous gen- younger than 60 years. This effect on MBC risk was partic- otype (Table 4), while no statistically significant result ularly evident when analyses were restricted to the sub- emerged in older individuals. Further statistical analyses group of MBC cases with an interval between diagnosis confirmed that the effect of the HH homozygous geno- and blood donation ≤ 4 years, in order to avoid a possible type on MBC risk tended to be modified by age also in selection bias related to the enrolment of MBC cases with these analyses restricted to MBC cases with a shorter time a longer survival in the attempt of expanding the series of interval between diagnosis and blood donation (LR test p patients affected with this rare disease. Overall, we had = 0.03; OR = 0.17 (95%CI = 0.04–0.83). Sensitiv- found a statistically significant interaction between interaction ity analyses were carried out based on cut-off points for N372H genotype and age: the effect on MBC risk of the Table 1: Distribution of 91 MBC cases not mutated in BRCA genes and 261 male population controls according to the BRCA2 N372H genotype frequencies. Genotype Cases n % Controls n % Co-dominant model OR 95% CI N/N 48 52.7 127 48.7 N/H 31 34.1 107 41.0 0.68 (0.40; 1.18) H/H 12 13.2 27 10.3 0.94 (0.43; 2.09) Total 91 100.0 261 100.0 Odds Ratios (OR) and 95% Confidence Intervals according to a co-dominant model, estimated by a logistic regression model after adjustment for age considered as a dichotomous variable (<60 years, >60 years). Page 4 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:170 http://www.biomedcentral.com/1471-2407/7/170 Table 2: Distribution of 91 MBC cases not mutated in BRCA genes and 261 male population controls according to the BRCA2 N372H genotype frequencies, stratified by age at diagnosis. Genotype Cases n % Controls n % Co-dominant Recessive model model OR 95% CI OR 95% CI ≤60 years N/N 1650.094 54.0 N/H 10 31.2 68 39.1 0.86 (0.37; 2.03) H/H 6 18.8 12 6.9 2.94 (0.94; 9.15) 3.12 (1.06; 9.16) Total 32 100.0 174 100.0 >60 years N/N 3254.233 37.9 N/H 21 35.6 39 44.8 0.56 (0.27; 1.15) H/H 6 10.2 15 17.2 0.41 (0.14; 1.22) 0.54 (0.20; 1.51) Total 59 100.0 87 100.0 Odds Ratios (OR) and 95% Confidence Intervals according to co-dominant and recessive models, estimated using logistic regression models. Interaction among between BRCA2 N372H genotype and age: OR = 0.17 (95%CI = 0.04–0.76); p = 0.02. interaction Table 3: Distribution of 53 MBC cases with a time interval between diagnosis and interview ≤4 years and 261 male population controls according to the BRCA2 N372H genotype frequencies. Genotype Cases n % Controls n % Co-dominant model OR 95% CI N/N 24 45.2 127 48.7 N/H 18 34.0 107 41.0 0.79 (0.40; 1.58) H/H 11 20.8 27 10.3 1.67 (0.70; 3.97) Total 53 100.0 261 100.0 Odds Ratios (OR) and 95% Confidence Intervals according to a co-dominant model, estimated using logistic regression models after adjustment for age considered as a dichotomous variable (<60 years, >60 years). Table 4: Distribution of 53 MBC with a time interval between diagnosis and interview ≤4 years and 261 male population controls according to the BRCA2 N372H genotype frequencies, stratified by age at diagnosis. Genotype Cases n % Controls n % Co-dominant Recessive model model OR 95% CI OR 95% CI ≤60 years N/N 6 35.3 94 54.0 N/H 6 35.3 68 39.1 1.38 (0.43; 4.47) H/H 5 29.4 12 6.9 6.53 (1.73; 24.69) 5.63 (1.70;18.61) Total 17 100.0 174 100.0 >60 years N/N 1850.0 3337.9 N/H 12 33.3 39 44.8 0.56 (0.24;1.34) H/H 6 16.7 15 17.2 0.73 (0.24;2.22) 0.96 (0.34;2.71) Total 36 100.0 87 100.0 Odds Ratios (OR) and 95% Confidence Intervals according to co-dominant and recessive models estimated using logistic regression models. Interaction between BRCA2 N372H genotype and age: OR = 0.17 (95%CI = 0.04–0.83); p = 0.03. interaction HH homozygous genotype was significantly modified by It could be speculated that the effect of the BRCA2 N372H age. In this respect, it is noteworthy that a recent pooled variant on BC risk could be modulated by the interactions analysis with over 15,000 BC cases and 15,000 controls with hormonal factors. In males, the hormonal back- provided some evidence of an interaction of BRCA2 ground is not influenced by reproductive factors as in N372H with age, suggesting a possibly increased BC risk females and sex steroid levels decline with age [21]. in women younger than 40 years of age [16]. Intriguingly, an association of the BRCA2 N372H variant with idiopathic male infertility, a condition possibly Page 5 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:170 http://www.biomedcentral.com/1471-2407/7/170 related to increased sensitivity to estrogens, has been Acknowledgements The authors wish to thank all study participants for their availability, Prof. recently suggested [22]. Here, we found that the BRCA2 Simonetta Bianchi (Pathology Department, University of Florence), Dr. N372H variant increased MBC risk in younger men that Carmelo Urso and Dr. Federica Zolfanelli (Pathology Departments, ASL 10 may show higher steroid hormones levels. Florence), Dr. Mauro Biancalani (Pathology Department, ASL 11 Empoli), Dr. Augusto Giannini (Pathology Department, ASL 4 Prato), Dr.s Francesco On the other hand, MBC risk could be influenced by inter- Mirri and Vincenzo Sforza (Pathology Departments, ASL 8 Arezzo) for their actions between genetic and environmental factors. help with histologic confirmation, Dr Mariella Muraca (CSPO, Florence) Recently, we reported a possible modifying effect on MBC and other local clinicians and general practitioners for their help in contact- risk of an occupational exposure to chemicals, as polycy- ing patients. The study was carried out in the frame of the activities of the Cancer Family Program of Tuscany. The study was supported by a grant clic aromatic hydrocarbon, in subjects carrying BRCA1/2 from Associazione Italiana per la Ricerca sul Cancro (AIRC) to Laura Ottini. germ-line mutations [23]. References It has been also suggested that the BRCA2 N372H variant 1. 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Br J uted to study design and statistical analyses. MC, MAC Cancer 2004, 90:1989-1994. 14. Cox DG, Hankinson SE, Hunter DJ: No association between and IZ collected samples, clinical data and performed sta- BRCA2 N372H and breast cancer risk. Cancer Epidemiol Biomar- tistical analyses. RL, CDA, PR performed BRCA1 and kers Prev 2005, 14:1353-1354. BRCA2 mutational analysis. FS coordinated statistical 15. Wenham RM, Schildkraut JM, McLean K, Calingaert B, Bentley RC, Marks J, Berchuck A: Polymorphisms in BRCA1 and BRCA2 analyses. LO participated in study design and supervision and risk of epithelial ovarian cancer. Clin Cancer Res 2003, of experimental conduct and analysis, interpretation of 9:4396-4403. 16. Breast Cancer Association Consortium: Commonly studied sin- results, drafting and revision of the manuscript, and gle-nucleotide polymorphisms and breast cancer: results approved the final version. from the Breast Cancer Association Consortium. 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Palli D, Saieva C, Gemma S, Masala G, Gomez-Miguel MJ, Luzzi I, D'Errico M, Matullo G, Ozzola G, Manetti R, Nesi G, Sera F, Zanna I, Dogliotti E, Testai E: GSTT1 and GSTM1 gene polymorphisms and gastric cancer in a high-risk italian population. Int J Cancer 2005, 115:284-289. 20. Ottini L, D'Amico C, Noviello C, Lauro S, Lalle M, Fornarini G, Col- antuoni OA, Pizzi C, Cortesi E, Carlini S, Guadagni F, Bianco AR, Frati L, Contegiacomo A, Mariani-Costantini R: BRCA1 and BRCA2 mutations in central and southern Italian patients. Breast Can- cer Res 2000, 2:307-310. 21. Orwoll E, Lambert LC, Marshall LM, Phipps K, Blank J, Barrett-Con- nor E, Cauley J, Ensrud K, Cummings S: Testosterone and estra- diol among older men. J Clin Endocrinol Metab 2006, 91:1336-1344. 22. Zhoucun A, Zhang S, Yang Y, Ma Y, Zhang W, Lin L: The common variant N372H in BRCA2 gene may be associated with idio- pathic male infertility with azoospermia or severe oligo- zoospermia. Eur J Obstet Gynecol Reprod Biol 2006, 124:61-64. 23. Palli D, Masala G, Mariani-Costantini R, Zanna I, Saieva C, Sera F, Decarli A, Ottini L: A gene-environment interaction between occupation and BRCA1/BRCA2 mutations in male breast cancer? Eur J Cancer 2004, 40:2474-2479. 24. Teare MD, Cox A, Shorto J, Anderson C, Bishop DT, Cannings C: Heterozygote excess is repeatedly observed in females at the BRCA2 locus N372H. J Med Genet 2004, 41:523-528. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/7/170/pre pub Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." 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Association between the BRCA2N372H variant and male breast cancer risk: a population-based case-control study in Tuscany, Central Italy

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Springer Journals
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Copyright © 2007 by Palli et al; licensee BioMed Central Ltd.
Subject
Biomedicine; Cancer Research; Oncology; Surgical Oncology; Health Promotion and Disease Prevention; Biomedicine general; Medicine/Public Health, general
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1471-2407
DOI
10.1186/1471-2407-7-170
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17767707
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Abstract

Background: Male breast cancer (MBC) is a rare disease and little is known about its aetiology. Germ- line mutations of BRCA2 and, at lower frequency, of BRCA1 are implicated in a relatively small proportion of MBC cases. Common polymorphic variants in BRCA1 and BRCA2 genes may represent breast cancer (BC) susceptibility alleles and could be associated with a modestly increased risk of MBC at population level. Considering the relevant role of BRCA2 in MBC, we investigated whether the BRCA2 N372H variant, representing the only common non-synonymous polymorphism in BRCA2, might modulate the risk of BC in male populations. Methods: A case-control study was performed comparing a population-based series of 99 MBC cases, characterized for BRCA1 and BRCA2 mutations, with 261 male population controls, all residing in Tuscany, Central Italy. All MBC cases and controls were genotyped for the BRCA2 N372H allele by TaqMan allelic discrimination assays. To evaluate the genotype specific risk of the BRCA2 N372H variant, MBC carriers of germ-line BRCA1/2 mutations were excluded from the analyses. Results: No association emerged in univariate and age-adjusted analyses. Age-specific analyses suggested an increased risk for the HH homozygous genotype in subjects younger than 60 years. A statistically significant interaction emerged between this genotype and age (p = 0.032). When analyses were restricted to MBC cases enrolled in the first 4 years following diagnosis, a recessive model showed a significantly increased risk of MBC in HH subjects younger than 60 years (OR = 5.63; 95% CI = 1.70;18.61). Conclusion: Overall, our findings, although based on a relatively small series, suggest that the BRCA2 HH homozygous genotype might be positively associated with an increased risk of MBC in men younger than 60 years. Page 1 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:170 http://www.biomedcentral.com/1471-2407/7/170 women [16]. To our knowledge, the effect of the BRCA2 Background Male breast cancer (MBC) is a rare disease and little is N372H polymorphism on BC risk has not yet been inves- known about its aetiology compared with female breast tigated in male populations. cancer (FBC). MBC represents less than 1% of all cancers in men and its incidence is increasing in younger men [1]. Considering the relevance of BRCA2 in MBC, we have per- In Italy, MBC accounts for 0.2% of all cancers in males [2]. formed a population-based case-control study to investi- gate the role of BRCA2 N372H variant on BC risk in men, Similar to FBC, a positive family history (FH) of breast comparing a series of MBC, characterized for BRCA1 and cancer (BC) is associated with increased risk of MBC and BRCA2 mutation status, with a control group from the approximately 15% to 20% of male patients with BC have same area of Central Italy (Florence, Tuscany). a positive FH [3]. The two major hereditary BC genes, BRCA1 (OMIM #113705) and, to a larger extent, BRCA2 Methods (OMIM # 600185), are implicated in MBC. Both genes are Study population and data collection estimated to be responsible respectively for up to 16% and In a previous study [17], we had identified a population- 76% of the MBCs in high-risk breast/ovarian cancer fami- based series of 25 MBC cases diagnosed in the area of Flor- lies [3,4]. The frequencies of BRCA1/BRCA2 mutations are ence (Tuscany, Central Italy) during the period 1990– sharply different in ethnically diverse population- and 1998. One of the problems in MBC studies is represented clinic-based series ranging from 0% to 4% for BRCA1 and by the rarity of the disease: in order to increase the from 4% to 40% for BRCA2 [5,6]. However, at population number of cases for meaningful statistical analyses it is level only a small proportion of all MBC cases are due to necessary to enrol patients diagnosed over a long period inherited mutations in BRCA1/BRCA2 genes. of time (or in a larger geographical area). Thus, using all available local sources (including Pathology Departments Low-penetrance polymorphisms in BC susceptibility and the Hospital Discharge database) the original series genes are present in a high percentage of individuals and has been expanded and 74 additional MBC cases diag- might account for BC risk at population level. In this nosed in the period 1991–2006 have been enrolled for a respect, common polymorphic variants in BRCA1/BRCA2 total of 99 MBCs available for this study, all residing in genes may represent BC susceptibility alleles and could be Tuscany. associated with a modest risk of MBC that would explain, on population basis, a large proportion of the disease. Overall, after exclusion of deceased and migrated patients, 85 additional unrelated MBC were traced and invited to The N372H (rs144848) polymorphism is the unique var- participate into the study. Eleven cases refused to partici- iant in BRCA2 gene that results in an amino acid change pate, mostly because of advanced age or severe illness. and has a rare allele frequency greater than 10%. Little is Confirming our previous response rate of about 80%, 74 known on the functional effect of this polymorphism, of these 85 MBC cases (87.1%) agreed to participate. although the substitution of asparagine (a neutral amino According to the original study protocol, each MBC acid) by histidine (a basic amino acid) may be expected to patient provided: 1) informed consent; 2) blood samples; affect BRCA2 structure and function as it falls in a region 3) detailed information on his personal and family his- that has been shown to interact with the histone acetyl- tory of cancer at any site, including all first- and second- transferase P/CAF prior to transcriptional activation of degree relatives of both genders; 4) a detailed smoking other genes [7]. Intriguingly, an excess of NH heterozy- and working history up to the date of BC diagnosis. Infor- gotes was observed in female controls and an effect of this mation has been validated by available sources (mainly variant on fetal survival in a sex-dependent manner has local Cancer and Mortality Registries). Procedures to been suggested as newborn females showed an excess of maintain confidentiality for all the information collected heterozygotes and a deficit of homozygotes, whereas the have been developed and strictly applied. The study was opposite was observed in newborn males [8]. approved by the local Ethical Research Committee (Flor- ence Health Unit). Several studies have analysed the effect of the BRCA2 N372Hpolymorphism on the risk of breast cancer in Population controls female populations with inconsistent findings. The HH In the frame of a multi-site epidemiological project, two homozygous genotype was reported to be associated with series of healthy adults of both sexes have been randomly a 1.3- to 1.5-fold increased risk of breast and ovarian can- selected from the municipality lists of two areas in Tus- cer [8-11]. In contrast, other studies did not show any cany: the city of Florence and one rural area [18,19]. All effect of this polymorphism on breast or ovarian cancer participants signed an informed consent form and pro- risk [12-15]. A recent large pooled analysis, however, vided a blood sample. Out of 700 randomly selected sub- could not exclude an effect of this genotype in younger jects, 553 subjects (79%) accepted to participate into the Page 2 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:170 http://www.biomedcentral.com/1471-2407/7/170 study; males were 48.6% (269/553) and 261 samples expected genotype frequencies (based on observed qs) to were available for analyses (eight of the initial 269 con- observed genotype frequencies. The association between MBC risk and BRCA2 N372H was measured by the odds trol's samples (3%) yielded insufficient DNA). The blood samples have been processed at the study laboratory ratio (OR) and its corresponding 95% confidence interval (CSPO, Florence), in the same day of collection and and was estimated using unconditional logistic regression divided in specific aliquots (RBC, buffy coat, serum, after adjustment for age considered as dichotomous vari- plasma). The aliquots have been stored in a -80°C freezer able (<60 years, >60 years). The analysis were performed at the Biological Bank of the Molecular Epidemiology with four logistic regression models based on a co-domi- Unit at CSPO. nant, dominant, recessive and multiplicative codominant effect (inheritance model). In the dominant model, both Mutational analysis the heterozygous variant and rare homozygous variant Buffy coat aliquots from MBC cases and controls were were combined in a dummy variable. In the recessive anonymously shipped to the research laboratory (Experi- model, the variant was defined in a dummy variable as mental Medicine, Rome) where genomic DNA was only the rare homozygous genotype. In the co-dominant extracted using QIAamp DNA Mini Kit (Qiagen Inc., model both rare homozygous and heterozygous variant Charlesworth, CA). The entire BRCA1 and BRCA2 coding effects were estimated using two dummy variables while sequences, including intron-exon boundaries, were ana- in the multiplicative codominant model a dose-response lysed by using single strand conformation polymor- effect were tested on the variable counting the number of phisms (SSCP) combined with protein truncation test copies of the H allele. Analyses have also been carried out (PTT) and automatic sequencing analysis [17,20]. Muta- according to stratification by age at diagnosis for cases and tions were always verified by PCR direct sequencing on age at interview for controls (below/above 60 years) and two independent blood samples (reference sequence for restricted to MBC cases with a time interval after diagnosis BRCA1: Genbank, U14680; reference sequence for shorter than the median value of the series (4 years). Inter- BRCA2: Genbank, NM_000059). actions between age, modelled as dichotomous variable (<60 years, >60 years), and genotype under co-dominant, SNP genotyping dominant, recessive and multiplicative codominant mod- The BRCA2 N372H (rs144848) polymorphism was ana- els, were assessed using Likelihood Ratio test (LR) com- lysed by TaqMan allelic discrimination using Real-Time paring logistic regression models with and without PCR. Fluorescent hybridisation probes, labelled with 6- interaction term. carboxyfluorescin (FAM) to detect the 1342C (372H) allele and with Texas Red to detect the 1342A (372N) Results allele were specifically designed. TaqMan assays were per- In order to evaluate the putative influence of the BRCA2 formed in a reaction volume of 50 µl, comprising 100 ng N372H polymorphism on MBC risk, we carried out a pop- of DNA, 400 nM each probe, 400 nM each primer and 1 × ulation-based case-control study based on a total of 99 Universal Master Mix (Biorad). The following conditions MBC cases and 261 adult male controls from the same were used for amplification: 3 minutes at 95°C and 55 area of Central Italy (Florence, Tuscany). The mean age at cycles at 95°C for 10" and 63°C for 45". The Real-Time interview was 67.9 (SD 11.8) in cases and 55.9 (SD 6.9) PCR was accomplished on iCycler thermal cycler (Biorad) in controls (p < 0.0001). The mean age at diagnosis in the with 96 × 0.2 ml samples. The fluorescence was visualised MBC series was 63.4 (SD 12.1; median 65) and the time through iCycler Optical System (Biorad) during Real- interval between diagnosis and interview ranged from 0 to Time PCR and analysed by using the iCycler IQ Real-Time 26 years (median 4.0). A detailed FH for breast and ovar- Detection System (Biorad) 3.0 software. About 15% of the ian cancers diagnosed in first-degree relatives was col- genotyping results were confirmed by sequencing analy- lected for all MBC cases. Overall, 25.3% (25/99) of MBC sis. The genotype controls for the three possible genotypes patients reported a first degree FH positive for breast/ovar- plus "no template" controls were always included in each ian cancer. analysis. MBC cases were characterized for BRCA1 and BRCA2 Statistical analysis mutations. Overall, 8/99 (8.1%) MBC cases resulted to be Allele frequencies have been calculated as the number of mutated in BRCA1/BRCA2 genes: 6 cases (6.1%) carried alleles divided by the number of chromosomes. Genotype BRCA2 mutations and 2 cases (2.0%) carried BRCA1 frequencies have been calculated as the number of partic- mutations. All 99 MBC cases and 261 controls were ana- ipants with a particular genotype divided by the number lysed for the BRCA2 N372H allele and genotype frequen- of participants. Tests for Hardy-Weinberg equilibrium cies. Genotype distribution was consistent with Hardy- among cases and controls have been assessed using Pear- Weinberg equilibrium (p = 0.529) among our population son's χ test with one degree of freedom comparing controls and cases (p = 0.067). In order to evaluate the Page 3 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:170 http://www.biomedcentral.com/1471-2407/7/170 genotype specific risk of the BRCA2 N372H polymor- both age at diagnosis and time interval after diagnosis dif- phism, MBC carriers of germ-line BRCA1/2 mutations (8 ferent from those identified a priori. These analyses con- cases) were excluded from the analyses. firmed the association, when older individuals (up to 65 years of age) or cases with a longer time interval (up to 6 As shown in Table 1, no statistically significant difference years) were included. in the distribution of the three specific BRCA2 N372H genotypes was observed between MBC cases and controls Discussion (p = 0.46). The age-adjusted analysis of the genotype-spe- We evaluated the effect of the BRCA2 N372H (1342A > C) cific risks showed that individuals with NH heterozygous polymorphism on MBC risk in a population based case- and HH homozygous genotypes were not at increased control study. Overall, a positive association between the MBC risk. These results were confirmed on dominant, HH genotype and an increased risk of MBC was suggested recessive and multiplicative codominant transmission in men younger than 60 years; this association was partic- models (data not shown). Separate analyses stratified ularly strong (with a five-fold increased risk) when analy- according to age (below/above 60 years) showed a ten- ses were restricted to 53 MBC cases enrolled in the study dency towards an inverse association between the H allele with a blood donation not later than 4 years after their and MBC risk in the older sub-group (age > 60 years), diagnosis. To our knowledge, the present study is the first although far from being statistically significant. On the reporting an analysis of the association between BC risk contrary, an increased MBC risk (OR = 3.12; 95%CI = and the BRCA2 N372H variant in men. 1.08; 9.03) was observed in younger subjects (age ≤ 60 years) on the basis of a recessive model (Table 2). A model The role of the BRCA2 N372H variant on BC risk has been specifically aimed to evaluate a possible effect modifica- investigated by several studies in women from different tion of age on the association between MBC risk and the populations with inconsistent findings that might reflect HH homozygous genotype found a statistically significant genetic differences across populations. In particular the interaction (LR test p = 0.02; OR = 0.17; 95%CI = HH genotype was associated with a 1.3- to 1.5-fold interaction 0.04; 0.76). increased risk of BC in series from Northern Europe [8], Australia [9] and in a selected series of radiologic technol- We carried out additional analyses taking into account the ogists from USA [10]. This association was not detected in length of the interval between diagnosis and blood dona- series from Central Europe [13], Japan [12] and in an tion among cases. Overall, no association between the H unselected series from USA [14]. allele and MBC risk was observed, when analyses were restricted to a comparison between the group of 53 MBC Here, we performed a population-based case control cases with an interval shorter than the median value of the study on male subjects all residing in the same area of whole series (≤4 years) and the series of controls (Table Central Italy (Tuscany). Our analyses based on a recessive 3). Additional analyses stratified by age, showed a transmission model suggested an association between the strongly increased MBC risk (OR = 5.63; 95%CI = 1.70; BRCA2 N372H variant and increased MBC risk in men 18.61) in younger subjects with the HH homozygous gen- younger than 60 years. This effect on MBC risk was partic- otype (Table 4), while no statistically significant result ularly evident when analyses were restricted to the sub- emerged in older individuals. Further statistical analyses group of MBC cases with an interval between diagnosis confirmed that the effect of the HH homozygous geno- and blood donation ≤ 4 years, in order to avoid a possible type on MBC risk tended to be modified by age also in selection bias related to the enrolment of MBC cases with these analyses restricted to MBC cases with a shorter time a longer survival in the attempt of expanding the series of interval between diagnosis and blood donation (LR test p patients affected with this rare disease. Overall, we had = 0.03; OR = 0.17 (95%CI = 0.04–0.83). Sensitiv- found a statistically significant interaction between interaction ity analyses were carried out based on cut-off points for N372H genotype and age: the effect on MBC risk of the Table 1: Distribution of 91 MBC cases not mutated in BRCA genes and 261 male population controls according to the BRCA2 N372H genotype frequencies. Genotype Cases n % Controls n % Co-dominant model OR 95% CI N/N 48 52.7 127 48.7 N/H 31 34.1 107 41.0 0.68 (0.40; 1.18) H/H 12 13.2 27 10.3 0.94 (0.43; 2.09) Total 91 100.0 261 100.0 Odds Ratios (OR) and 95% Confidence Intervals according to a co-dominant model, estimated by a logistic regression model after adjustment for age considered as a dichotomous variable (<60 years, >60 years). Page 4 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:170 http://www.biomedcentral.com/1471-2407/7/170 Table 2: Distribution of 91 MBC cases not mutated in BRCA genes and 261 male population controls according to the BRCA2 N372H genotype frequencies, stratified by age at diagnosis. Genotype Cases n % Controls n % Co-dominant Recessive model model OR 95% CI OR 95% CI ≤60 years N/N 1650.094 54.0 N/H 10 31.2 68 39.1 0.86 (0.37; 2.03) H/H 6 18.8 12 6.9 2.94 (0.94; 9.15) 3.12 (1.06; 9.16) Total 32 100.0 174 100.0 >60 years N/N 3254.233 37.9 N/H 21 35.6 39 44.8 0.56 (0.27; 1.15) H/H 6 10.2 15 17.2 0.41 (0.14; 1.22) 0.54 (0.20; 1.51) Total 59 100.0 87 100.0 Odds Ratios (OR) and 95% Confidence Intervals according to co-dominant and recessive models, estimated using logistic regression models. Interaction among between BRCA2 N372H genotype and age: OR = 0.17 (95%CI = 0.04–0.76); p = 0.02. interaction Table 3: Distribution of 53 MBC cases with a time interval between diagnosis and interview ≤4 years and 261 male population controls according to the BRCA2 N372H genotype frequencies. Genotype Cases n % Controls n % Co-dominant model OR 95% CI N/N 24 45.2 127 48.7 N/H 18 34.0 107 41.0 0.79 (0.40; 1.58) H/H 11 20.8 27 10.3 1.67 (0.70; 3.97) Total 53 100.0 261 100.0 Odds Ratios (OR) and 95% Confidence Intervals according to a co-dominant model, estimated using logistic regression models after adjustment for age considered as a dichotomous variable (<60 years, >60 years). Table 4: Distribution of 53 MBC with a time interval between diagnosis and interview ≤4 years and 261 male population controls according to the BRCA2 N372H genotype frequencies, stratified by age at diagnosis. Genotype Cases n % Controls n % Co-dominant Recessive model model OR 95% CI OR 95% CI ≤60 years N/N 6 35.3 94 54.0 N/H 6 35.3 68 39.1 1.38 (0.43; 4.47) H/H 5 29.4 12 6.9 6.53 (1.73; 24.69) 5.63 (1.70;18.61) Total 17 100.0 174 100.0 >60 years N/N 1850.0 3337.9 N/H 12 33.3 39 44.8 0.56 (0.24;1.34) H/H 6 16.7 15 17.2 0.73 (0.24;2.22) 0.96 (0.34;2.71) Total 36 100.0 87 100.0 Odds Ratios (OR) and 95% Confidence Intervals according to co-dominant and recessive models estimated using logistic regression models. Interaction between BRCA2 N372H genotype and age: OR = 0.17 (95%CI = 0.04–0.83); p = 0.03. interaction HH homozygous genotype was significantly modified by It could be speculated that the effect of the BRCA2 N372H age. In this respect, it is noteworthy that a recent pooled variant on BC risk could be modulated by the interactions analysis with over 15,000 BC cases and 15,000 controls with hormonal factors. In males, the hormonal back- provided some evidence of an interaction of BRCA2 ground is not influenced by reproductive factors as in N372H with age, suggesting a possibly increased BC risk females and sex steroid levels decline with age [21]. in women younger than 40 years of age [16]. Intriguingly, an association of the BRCA2 N372H variant with idiopathic male infertility, a condition possibly Page 5 of 7 (page number not for citation purposes) BMC Cancer 2007, 7:170 http://www.biomedcentral.com/1471-2407/7/170 related to increased sensitivity to estrogens, has been Acknowledgements The authors wish to thank all study participants for their availability, Prof. recently suggested [22]. Here, we found that the BRCA2 Simonetta Bianchi (Pathology Department, University of Florence), Dr. N372H variant increased MBC risk in younger men that Carmelo Urso and Dr. Federica Zolfanelli (Pathology Departments, ASL 10 may show higher steroid hormones levels. Florence), Dr. Mauro Biancalani (Pathology Department, ASL 11 Empoli), Dr. Augusto Giannini (Pathology Department, ASL 4 Prato), Dr.s Francesco On the other hand, MBC risk could be influenced by inter- Mirri and Vincenzo Sforza (Pathology Departments, ASL 8 Arezzo) for their actions between genetic and environmental factors. help with histologic confirmation, Dr Mariella Muraca (CSPO, Florence) Recently, we reported a possible modifying effect on MBC and other local clinicians and general practitioners for their help in contact- risk of an occupational exposure to chemicals, as polycy- ing patients. The study was carried out in the frame of the activities of the Cancer Family Program of Tuscany. The study was supported by a grant clic aromatic hydrocarbon, in subjects carrying BRCA1/2 from Associazione Italiana per la Ricerca sul Cancro (AIRC) to Laura Ottini. germ-line mutations [23]. References It has been also suggested that the BRCA2 N372H variant 1. 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Eur J Obstet Gynecol Reprod Biol 2006, 124:61-64. 23. Palli D, Masala G, Mariani-Costantini R, Zanna I, Saieva C, Sera F, Decarli A, Ottini L: A gene-environment interaction between occupation and BRCA1/BRCA2 mutations in male breast cancer? Eur J Cancer 2004, 40:2474-2479. 24. Teare MD, Cox A, Shorto J, Anderson C, Bishop DT, Cannings C: Heterozygote excess is repeatedly observed in females at the BRCA2 locus N372H. J Med Genet 2004, 41:523-528. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/7/170/pre pub Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." 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Published: Sep 3, 2007

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