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Phenotypic features and genetic characterization of male breast cancer families: identification of two recurrent BRCA2 mutations in north-east of Italy

Phenotypic features and genetic characterization of male breast cancer families: identification... Background: Breast cancer in men is an infrequent occurrence, accounting for ~1% of all breast tumors with an incidence of about 1:100,000. The relative rarity of male breast cancer (MBC) limits our understanding of the epidemiologic, genetic and clinical features of this tumor. Methods: From 1997 to 2003, 10 MBC patients were referred to our Institute for genetic counselling and BRCA1/2 testing. Here we report on the genetic and phenotypic characterization of 10 families with MBC from the North East of Italy. In particular, we wished to assess the occurrence of specific cancer types in relatives of MBC probands in families with and without BRCA2 predisposing mutations. Moreover, families with recurrent BRCA2 mutations were also characterized by haplotype analysis using 5 BRCA2-linked dinucleotide repeat markers and 8 intragenic BRCA2 polymorphisms. Results: Two pathogenic mutations in the BRCA2 gene were observed: the 9106C>T (Q2960X) and the IVS16-2A>G (splicing) mutations, each in 2 cases. A BRCA1 mutation of uncertain significance 4590C>G (P1491A) was also observed. In families with BRCA2 mutations, female breast cancer was more frequent in the first and second-degree relatives compared to the families with wild type BRCA1/2 (31.9% vs. 8.0% p = 0.001). Reconstruction of the chromosome phasing in three families and the analysis of three isolated cases with the IVS16-2A>G BRCA2 mutation identified the same haplotype associated with MBC, supporting the possibility that this founder mutation previously detected in Slovenian families is also present in the North East of our Country. Moreover, analysis of one family with the 9106C>T BRCA2 mutation allowed the identification of common haplotypes for both microsatellite and intragenic polymorphisms segregating with the mutation. Three isolated cases with the same mutation shared the same intragenic polymorphisms and three 5' microsatellite markers, but showed a different haplotype for 3' markers, which were common to all three cases. Conclusion: The 9106C>T and the IVS16-2A>G mutations constitute recurrent BRCA2 mutations in MBC cases from the North-East of Italy and may be associated with a founder effect. Knowledge of these two recurrent BRCA2 mutations predisposing to MBC may facilitate the analyses aimed at the identification of mutation carriers in our geographic area. Page 1 of 9 (page number not for citation purposes) BMC Cancer 2006, 6:156 http://www.biomedcentral.com/1471-2407/6/156 tions. Moreover, we also searched for the presence of Background Approximately 5–10% of breast and ovarian cancer cases recurrent mutations in this area due to a possible founder are hereditary, occurring mainly in women with germ-line effect. mutations in the BRCA1 or BRCA2 genes [1-4]. Although earlier estimates suggested that BRCA1 and BRCA2 muta- Methods tions were responsible for 75% of breast cancer families From 1997 to 2003, 197 patients with breast or ovarian [4-7] recent population-based studies indicate that these cancer with a family history of these tumors were referred rates may have been overestimated. In fact, the percentage for genetic counselling to the Centro di Riferimento of high-risk families associated with BRCA1 and BRCA2 Oncologico (National Cancer Institute, Aviano, Italy). mutations seems to be around 25% in all groups investi- The present study is part of an Institutional program on gated [8-12]. hereditary tumors approved by local Ethical Committee. Families were selected according to previously reported BRCA1 germ-line mutations are also associated with an criteria [22]. All consecutive male patients with a breast increased risk of other cancer types, including those affect- cancer, independently of age and family history, were ing colon, pancreas, stomach and fallopian tubes [13,14]. included and counselled for genetic testing for BRCA1 and Other possible sites of cancer for BRCA1 mutation carriers BRCA2 gene mutations. Ten families were ascertained by included cutaneous melanoma, basal cell carcinoma and a male index case; one patient with MBC (BR175) also sarcoma [15]. Although BRCA1 mutations have been developed prostatic cancer at the age of 66 (Table 1). One described also in men with breast cancer, the presence of additional male patient (BR331), showing an atypic intra- germ-line mutations in the BRCA1 gene does not seem to cystic papilloma of the breast together with a melanoma convey a significantly increased risk for MBC [16]. How- of the ear, was not included in the statistical analysis. All ever, this issue is still controversial, since a recent study individuals tested for BRCA1/2 mutations gave a signed carried out in 483 BRCA1 mutation carriers reported a 58- informed consent. fold increased risk [13]. Blood samples were obtained from each proband and Available evidence indicates that BRCA2 mutation carriers after the identification of a specific mutation the analyses have an increased risk of breast cancer in both males and were extended to 98 relatives. Screening for mutations in females, as well as of prostate and pancreatic carcinomas. the BRCA1 and BRCA2 genes was carried out by a combi- Carcinomas of the stomach, gallbladder bile ducts, cuta- nation of protein truncation test, single strand conforma- neous melanoma, and basal cell carcinoma are also tion polymorphism, and sequencing techniques (Table slightly more prevalent in carriers of BRCA2 mutations 1). Primer sequences and PCR conditions were previously [17,18]. The large majority of data accumulated so far described [22,23]. All mutations and genetic variants were indicates that, unlike what reported for female breast can- named according to Antonarakis et al. [24]. Missense cer (FBC), MBC is mainly linked to BRCA2 gene muta- mutations and mutations occurring within intronic tions. In fact, up to 30% of MBC were associated with regions whose clinical significance has not yet been BRCA2 mutations, whereas only less than 4% of the cases reported were defined as genetic unclassified variants. carried BRCA1 gene mutations, with variable percentages Germline rearrangements of BRCA1 and BRCA2 gene depending on founder mutations [19,20]. were also analysed by multiplex ligation dependent probe amplification (MLPA). To date, only few studies have attempted to estimate the risk of breast cancer in male carriers of a BRCA2 mutation. The haplotype associated with the 9106C>T BRCA2 muta- The analysis of two large families carried out by Easton et tion was analyzed using five BRCA2-linked di-nucleotide al. [21] resulted in a cumulative risk of MBC of 6.3% (95% repeat markers: D13S290, D13S260, D13S1698, CI 1.4% to 25.6%) by age 70. In contrast, the study by D13S171, and D13S1695, whereas for the IVS16-2A>G Thompson et al. [17], based on 59 cases, failed to detect a mutation the D13S290, D13S171, and D13S1695 mark- significantly higher cumulative risk of MBC by 70 years of ers were used. age (2.8%; 95% CI 0.6 – 13.0%). However, the risk increased to 6.9% (95% CI 1.2% to 38,6%) by 80 years, The first three markers were placed in 3' flanking of the 80-fold higher compared to the general population. The BRCA2 gene whereas the other two were placed in the 5' present study was carried out with the aim to characterize flanking of the BRCA2 gene. The order of markers from the genetic and phenotypic features of families with MBC centromere to telomere is as follows: cen-D13S290, from the North East of Italy. In particular, we wished to D13S260, D13S1698, D13S171, D13S1695-tel. PCR assess the occurrence of specific cancer subtypes in first, products were analyzed on the ABI Prism 3100 using the second and third degree relatives of MBC probands in POP4 matrix and fluorescently labelled primers. For these families with and without BRCA2 predisposing muta- markers, a heterozygosity of 46%, 78%, 63%, 72%, and Page 2 of 9 (page number not for citation purposes) BMC Cancer 2006, 6:156 http://www.biomedcentral.com/1471-2407/6/156 Table 1: Clinicopathologic and genetic characteristics of the 10 MBC cases analyzed for the presence of BRCA1/2 mutations. Family Patient Age TNM histology ER PR Method Mutation Effect Affected relatives 25 BR 22 40 N.A. N.A. N.A. N.A. PTT-SSCP 9106C>T Q2960X BC(31)s, BC(48)s, BC(44)s, BC(30)s, BC(65)s, BC(37)ptc. 103 BR 111 59 pT1N0 intraductal carcinoma N.A. N.A. PTT-SSCP MLPA w.t. BC(78)s, BC(75)pta, BC(55)ptc, BC(N.A.)ptc, BC(60)ptc, BC(60)ptc, BC(58)ptc, BC(60)ptc. 125 BR 138 62 pTis intraductal carcinoma + + PTT-SSCP MLPA w.t. BC(82)m. 127 BR 140 52 pT1cN0 infiltrating ductal + + PTT-SSCP MLPA w.t. BC(55)mta, carcinoma BC(47)mta, PC(61)mtu, BC(45)mtc, BCbil.(35)mtc, BC(42)mtc. 139 BR 173 58 pT2N1b infiltrating ductal + + PTT-SSCP MLPA w.t. / iii carcinoma 140 BR 175 58/ PT4bN0 infiltrating ductal + + PTT-SSCP 9106C>T Q2960X BCbil.(30)s, 66P carcinoma BC(40)pta, C BC(48)pta. 161 BR 195 50 N.A. N.A. N.A. N.A. PTT-SSCP IVS16- exon BC(38)f, BC(57)pta, 2A>G skipping PC(N.A.)ptu, BC(50)ptgm, BC(N.A.)ptggf, BC(N.A.)ptga, BC(N.A.)ptga. 190 BR 243 70 pT1c infiltrating ductal + + PTT-SSCP IVS16- exon BC(36)d, carcinoma 2A>G skipping BC(46)OC(N.A.)m, BC(N.A.)(OC)(N.A.) mta, BC(N.A.)mtc. 192 BR 247 38 pT4bN1 infiltrating ductal + + PTT-SSCP MLPA w.t. PC(75)f. biiiM0 carcinoma 217 BR 282 46 pT1aiii infiltrating ductal + + PTT-Direct 4590C>G P1491A BC(N.A.)pta, BC N0 carcinoma Sequencing (N.A.)ma. MLPA Number between parentheses indicate the age at diagnosis; N.A.: not available; w.t.: wild-type; PTT: protein truncation test; SSCP: single strand conformation polymorphism; MLPA: multiplex ligation-dependent probe amplification; mt: maternal; pt: paternal; m: mother; f: father; s: sister; d: daughter; gm: grandmother; ggf: great-grandfather; ga: great-aunt; c: cousin; u: uncle; a: aunt. BC: breast cancer; BCbil: bilateral breast cancer; OC: ovarian cancer; PC: prostate cancer. 79% has been reported in the Genome Data Base, respec- history of breast and/or ovarian cancer and came from the tively [25]. Alleles were numbered according to the size of same geographic area. each microsatellite repeat marker. The disease-associated haplotypes were deduced from allelic segregation in Statistical analyses known carriers by inspection of segregating genotypes in The first and second degree relatives of the male index case the analyzed families. Moreover, for the haplotype analy- of BRCA2 positive families were compared to first and sec- sis of the 9106C>T mutation, eight internal BRCA2 poly- ond degree relatives of families without a BRCA2 muta- morphisms were analyzed: 203G>A (exon 2), tion but with or without familial history for breast cancer, IVS8+56C>T (intron 8), 1342A>C (exon 10), 3624A>G using chi-square analyses [26]. The family with a BRCA1 (exon 11), 4035T>C (exon 11), IVS11+80del4 (intron mutation with unclear significance was excluded from this 11), 7470A>G (exon 14), IVS16-14T>C (intron 16). For comparison. Odds Ratios (OR), and their corresponding the haplotype analysis only, besides the 4 families ascer- 95% Confidence Interval (CI), was computed using tained by a MBC index case, 6 additional families were unconditional logistic regression model [27] in order to also analyzed, two carrying of the 9106C>T mutation and calculate the odds to develop a tumor in the first and sec- 4 with the IVS16-2A>G mutation. All these families were ond degree relatives conditional on the BRCA2 status of ascertained by a FBC index case, showed a positive family the proband. Page 3 of 9 (page number not for citation purposes) BMC Cancer 2006, 6:156 http://www.biomedcentral.com/1471-2407/6/156 In one IVS16-2A>G positive family, a first-degree relative Results The 10 MBC probands had a mean age at the time of diag- (the father of the BR195 proband) was affected by a MBC nosis of 53.3 years (range 38–70 years). 3/10 patients diagnosed at 38 years. When this case was also included in were younger than 50 years, whereas 7 were ≥50 years. the analysis, the median age of MBC onset in carriers of Only one patient (BR247) developed a breast cancer the IVS16-2A>G mutation decreased to 52.7 years. before the age of 40. Four of the 10 patients (40%) carried a predisposing mutation in the BRCA2 gene (Table 1). In 8/10 cases the histologic characterization of the tumor Five of the remaining six cases carried wild-type (w.t.) was available. Invasive ductal carcinoma (75%) was the BRCA1 and BRCA2 genes whereas in one case (BR282) a most frequent histologic subtype followed by intraductal missense BRCA1 mutation 4590C>G (P1491A), causing a carcinoma (25%). Another case, diagnosed as atypic intra- Pro>Ala replacement in position 1491 with unclear signif- cystic papilloma, which showed also a melanoma of the icance was observed (Table 1). The family history of this ear, was found negative for BRCA1 and BRCA2 mutations. latter case showed two second-degree female relatives This case was not included in the statistical analysis. All affected by breast cancer, one belonging to the maternal the 6 assessable cases were oestrogen and progesterone side and the other to the paternal side. This mutation was receptors positive by immunohistochemistry (Table 1), not reported in the Breast Cancer Information Core data- confirming previous findings indicating that MBC have a base [28]. Although mutations in the BRCA1 gene do not higher rate of positivity to hormone receptors than do seem to be associated with increased risk of MBC, further FBC (90% vs. 77%) [30]. However, when MBC were com- studies are required to assess the possible predisposing pared with cancers from postmenopausal women, the role of this mutation in case BR282. It was not possible to positivity rates were similar. All our BRCA2 positive cases assess whether this variant was present in other affected showed a positive family history for breast and/or ovarian relatives because none of them was alive. cancer (Table 1). The six cases negative for point mutations were also ana- Two out of three cases of prostatic cancers were observed lyzed for BRCA1 and BRCA2 genes rearrangements by at 61 and 75 years, respectively, in w.t. families. In the four MLPA. None of these cases showed structural alterations BRCA2 mutation carrier families, breast cancer was more of BRCA1 and BRCA2 genes. The median age of diagnosis frequent in the first and second-degree relatives than in was 54.5 years for the 4 BRCA2 mutation carriers and 53.8 the five w.t. families. In fact, as compared with w.t. fami- years for the other cases, excluding the unclassified vari- lies, in BRCA2 mutation carrier families there was a signif- ant. The 4 BRCA2 positive cases carried only two distinct icant higher rate of affected first and second degree mutations: the 9106C>T, involving exon 22 and leading relatives (31.9% vs. 8.0% p = 0.001). These results indicate to a premature protein truncation (cases BR22 and that first and second degree relatives of index cases with BR175), and the IVS16-2A>G, involving intron 16 and BRCA2 mutation carrier families have a five times greater leading to a partial or total skipping of exon 17 (cases risk to develop a breast cancer (Table 2). The two MBC BR195 and BR243) [29]. Both these mutations were local- cases without family history of cancer resulted negative for ized outside the Ovarian Cluster Cancer Region and were BRCA1/2 mutations. In three of the four families with a previously reported also by others in the BIC database BRCA2 gene mutation, the molecular analysis was (three times for the 9106C>T mutation and four times for extended to ten non-affected relatives, (six males and four the IVS16-2A>G mutation in all). The carriers of the females), allowing the identification of 5 additional IVS16-2A>G mutation and those with the 9106C>T muta- BRCA2 mutation carriers (2 males and 3 females). tion showed a median age of breast cancer onset of 60 and 49 years, respectively. Table 2: Distribution of FBC in relatives of MBC cases belonging to w.t. and BRCA2 mutation carrier families. w.t. families (n = 5) BRCA2 mutation carrier families (n = 4) I+II degree relatives I+II degree relatives N (%) N (%) OR 95% CI Non-affected 58 (92) 32 (68.1) 1 Affected 5 (8) 15 (31.9) 5.43 (1.81–16.34) Total 63 47 X = 10.40; p = 0.001; w.t.: wild-type. Page 4 of 9 (page number not for citation purposes) BMC Cancer 2006, 6:156 http://www.biomedcentral.com/1471-2407/6/156 Besides the four MBC cases carrying BRCA2 mutations, fore, all isolated cases had the same 5,1,1,6,6 genotype, genotype analysis was also carried out in six additional which is in part consistent with the disease causing haplo- unrelated cases enrolled for the presence of FBC in the type detected in family 25. However, the analysis of 8 index case: two with the 9106C>T BRCA2 mutation and internal BRCA2 polymorphisms showed that the same A- four with the IVS16-2A>G BRCA2 mutation. On the C-A-G-T-(-4)-G-C haplotype was present in all the whole, genotype analysis was performed in four cases 9106C>T mutation carriers investigated (Table 4). The with the 9106C>T BRCA2 mutation, (one family and analysis of 55 healthy donors for 4 of these intragenic pol- three isolated cases), and in six IVS16-2A>G BRCA2 muta- ymorphisms (203G>A, IVS8+56C>T, 7470A>G, IVS16- tion cases (three families and three isolated cases). Recon- 14T>C) showed that, without phase reconstructing, only struction of the chromosome phasing was possible in four 15 of them (27%) carried the same haplotype detected in families. The analysis carried out in family 25, carrying the BRCA2 mutation carriers. On these grounds, it is highly 9106C>T BRCA2 mutation, allowed the identification of unlikely that these four polymorphisms are simultane- a common haplotype (5-6-2-6-6) shared by all mutation ously present in the four index cases by chance alone carriers belonging to this family (Figure 1). This haplotype (p = 0.0085, two-tailed Fisher's exact test). encompassed a distance of about 3.8 cM between the D13S290 to D13S1695 markers, and was not detected in The haplotype analysis by the reconstruction of the chro- non-carrier relatives. The isolated cases showed the same mosome phasing of three IVS16-2A>G BRCA2 mutation allelic pattern for the D13S290, D13S171, and D13S1695 families, showed that all mutation carriers had the same markers, whereas the D13S260 and D13S1698 markers, 5-4-2 haplotype for the D13S290, D13S171, and located within the 3' region flanking the BRCA2 gene, D13S1695 markers, whereas all the non-carrier relatives showed an allelic pattern (1,1) different from that of displayed a different allelic pattern for the three markers mutation carriers belonging to family 25 (Table 3). There- investigated (Table 5). Pedigree of Figure 1 the family 25 with the reconstruction of the chromosome phasing Pedigree of the family 25 with the reconstruction of the chromosome phasing. The type of cancer and age at diagnosis are indi- cated. Affected individuals are indicated by a red symbol and proband is denoted by arrow. The shared haplotype segregating with 9106C>T mutation is boxed. Page 5 of 9 (page number not for citation purposes) BMC Cancer 2006, 6:156 http://www.biomedcentral.com/1471-2407/6/156 Table 3: Genotype analysis of flanking microsatellites associated with the 9106C>T BRCA2 mutation. Markers carrier (+)/ non carrier (-) Probands D13S290 D13S260 D13S1698 D13S171 D13S1695 Family 25 BR22 5-5 6-5 2-2 6-6 6-5 + BR23 5-5 5-3 2-5 6-1 5-8 - BR99 5-3 2-2 4–6 2-2 5–7 - BR100 5-4 6-2 2-4 6-2 6-5 + BR101 5-5 6-1 2-5 6-2 6-2 + BR102 5-4 6-2 2-4 6-2 6-5 + BR103 5-5 5-32–5 6-15–8 - Isolated cases BR175 5,4 1,4 1,1 6,2 6,5 + BR342 5,3 1,3 1,3 6,2 6,6 + TBL38 5,5 1,5 1,5 6,2 6,6 + Numbers in boldface type indicate the 9106C>T mutation alleles. Haplotype segregating with the 9106C>T mutation alleles of phased alleles are separated by a dash. Genotypes of unphased alleles are separated by a comma. The mean age of breast cancer onset in males with and Discussion MBC accounts for less than 1% of all breast cancers and without BRCA2 mutations is still a controversial issue little is known on the epidemiologic, genetic and clinico- [16,32,35]. In the present study, we found a median age pathologic features of this malignancy [31-34]. Available of diagnosis of 54.5 years for the BRCA2 mutation carriers data indicate that the prevalence of BRCA2 mutations in (range 40–70 years) and of 53.8 years (range 38–62 years) MBC ranges between 4% and 40% [32-34]. These differ- for the w.t. type cases, supporting the findings reported by ences are probably related to the genetic features of the others [32] indicating that BRCA2 mutation carriers and families, with higher rates of BRCA2 mutations in men noncarriers have a similar age at the time of diagnosis. with a strong family history of cancer, particularly of those belonging to the BRCA2 spectrum. It has been reported that about 15–20% of MBC patients had a first-degree relative with breast carcinoma, support- In our series, the high prevalence of BRCA2 mutation ing the notion that a positive family history of breast can- (about 40%) correlated with the high number of relatives cer is associated with increased risk of MBC [36,37]. In our with breast cancer present in these families. In fact, survey, all MBC patients carrying deleterious BRCA2 excluding from the analysis the two apparently sporadic mutations had a family history of breast cancer. This was MBC, the frequency of BRCA2 mutations rises up to 50%. not due to a selection bias, since the eligibility criteria Table 4: Haplotype analysis of intragenic BRCA2 polymorphisms associated with the 9106C>T BRCA2 mutation. Polymorphisms Family 25 Isolated cases Position Nucleotide change BR22 BR23 BR99 BR100 BR101 BR102 BR103 BR175 BR342 TBL 38 M w.t. w.t.M M M w.t.M M M EX2 203G>A G>A G-G G>A A-A A-A A-A G-G G>A G>A G>A INTR8 IVS8+56C>T C-C C-C C>T C>T C-C C-C C-C C>T C-C C>T EX10B 1342A>C A>C C-C A-A A-A A-A A-A C-C A-A A-A A-A EX11-J 3624A>G A>G A-A A>G G-G G-G G-G A-A A>G A>G A>G EX11K 4035T>C T-T T-T T>C T-T T-T T-T T-T T-T T>C T-T INTR11Q IVS11+80del4 -4/4 4/4 -4/4 -4/-4 -4/4 -4/-4 4/4 -4/4 -4/4 -4/4 EX14A 7470A>G A>G A-A A>G G-G G-G G-G A-A A>G A>G A>G INTR17 IVS16-14T>C T>C T>C T>C C-C C-C C-C T>C C-C T>C T>C M: mutated; w.t.: wild-type; numbers in bold indicate the shared allele. Page 6 of 9 (page number not for citation purposes) BMC Cancer 2006, 6:156 http://www.biomedcentral.com/1471-2407/6/156 Table 5: Genotype analysis of flanking microsatellites associated with the IVS16-2A>G BRCA2 mutation. Markers Carrier (+)/Non carrier (-) Probands D13S290 D13S171 D13S1695 Family 14 BR6 5-5 4-2 2-5 + BR73 5-1 2-2 5–6 - BR82 5-1 4-2 2-6 + Family 161 BR195 5-5 4-2 2-4 + BR285 5-5 4-2 2-4 + BR289 5-2 2-1 4–6 - Family 190 BR243 5-3 4-4 2-4 + BR290 5-5 4-5 2-6 + BR291 3–5 4–5 4–5 - Isolated cases BR60 5,5 4,4 2,1 + BR85 5,5 4,2 2,5 + BR312 5,5 4,2 2,3 + Numbers in boldface type indicate the IVS16-2A>G mutation alleles. Haplotype segregating with the IVS16-2A>G mutation alleles of phased alleles are separated by a dash. Genotypes of unphased alleles are separated by a comma. used for MBC did not require the presence of a positive BRCA2 mutations including two mutations recurring in family history. These findings are in keeping with those central Italy (BRCA1 3345delAG and BRCA2 6696delTC). reported by others indicating that a positive history of breast cancer is more frequent in BRCA2 gene mutation The three BRCA2 mutations identified in this study were carriers, with values ranging from 13 to 80% [36-38], however different from those we observed. Notably, the although opposite results have been also reported [32]. In MBC cases from our series were characterized by the recur- our 8 families with a positive history of breast cancer, the rence of a restricted set of BRCA2 mutations detected in presence of a deleterious BRCA2 mutation was not associ- families coming from a limited geographic area (North ated with a different occurrence of specific types of cancers East of Italy), suggesting the possible existence of a as compared to BRCA2-negative families. A significantly founder effect. Indeed, it has been previously reported higher frequency of breast cancers was observed in the that all carriers of the IVS16-2A>G mutation shared a first- and second-degree relatives of mutation carriers, common haplotype, indicating a likely founder effect whereas the prevalence of breast cancers in third degree mainly confined to the Slovenian population [39]. Con- relatives was higher in w.t. families. sistently, our haplotype analysis carried out in six Italian families with the IVS16-2A>G BRCA2 mutation allowed Similarly to what reported in other studies [33], also the the detection of a common haplotype shared by all muta- majority (75%) of our MBC were infiltrating ductal or tion carriers. These findings, taken together, support the intraductal carcinomas. One case was classified as intra- possible occurrence of a founder effect involving the 5' cystic atypic papilloma a relatively infrequent subtype region flanking the BRCA2 gene in both North-East of accounting for about 2–5% of all MBC [33]. The patient Italy and the neighbouring Slovenia, suggesting that the carrying this tumor was negative to the molecular screen- IVS16-2A>G BRCA2 mutations probably recognize a com- ing, as those reported in other series [16], ruling out the mon ancestral origin. The IVS16-2A>G mutation was pre- possibility that BRCA1/2 mutations may confer a signifi- viously reported by Krajc et al. [39] in three breast cancer- cantly increased risk of developing this peculiar MBC his- only Slovenian families, whereas it was not identified in totype. three families with ovarian cancer, suggesting that the can- cer phenotype associated with this mutation is limited to Available data on the prevalence of BRCA1 and BRCA2 breast cancer. Conversely, our pedigree's analysis of six mutations in Italian MBC patients are limited. In a previ- Italian families with the IVS16-2A>G mutation disclosed ous study [16], Ottini et al. reported one BRCA1 and three the presence of one family in which two ovarian carcino- Page 7 of 9 (page number not for citation purposes) BMC Cancer 2006, 6:156 http://www.biomedcentral.com/1471-2407/6/156 mas were present along with breast cancer, indicating thus carriers in North East of Italy. In fact, the IVS16-2A>G and that the risk conferred by this mutation is not restricted to the 9106C>T mutations were detected in about 4% of the breast cancer. Moreover, in the six families with the IVS16- families we have investigated so far, whereas the recurrent 2A>G mutation of our database, two additional MBC and putative founder mutations account for about 12% of were detected (relatives of patients BR195 and BR312), the total. This would support the practical value of starting further supporting the increased risk of developing breast the mutational analysis of BRCA2 in the North-East Ital- cancer in males conferred by this mutation. Since the ian population with the search for these particular muta- IVS16-2A>G mutation can result in either a partial or total tions. This may enhance the collection of larger numbers skipping of exon 17 [29], the increased risk for breast can- of families with the same predisposing BRCA2 mutations, cer in males due to this mutation may be related to these providing thus the basis for studies aimed at identifying effects on BRCA2 gene transcription. Studies are under genetic modifying factors that may modulate the pheno- way in our laboratory to elucidate this issue. typic manifestations of MBC. With regard to the 9106C>T mutation, the data obtained Competing interests The author(s) declare that they have no competing inter- by haplotype evaluation coupled with the reconstruction of chromosome phasing suggest but do not conclusively ests. prove the occurrence of a possible founder effect also for this mutation. In fact, all the mutation carriers of Family Authors' contributions 25 showed the same haplotype for the markers D13S290- GMM performed haplotype analyses and drafted the man- D13S260-D13S1698-D13S171-D13S1695; however, the uscript; AVi performed molecular genetic studies and con- three isolated cases showed a different allelic pattern for tributed to interpretation of data; MS, and LDP performed the markers D13S260 and D13S1698 located within the BRCA tests and haplotype analyses; CDG, MGT, and AVe 3' region flanking the BRCA2 gene. These results may collected and analyzed the clinical data; EB performed the underlie the occurrence of a possible recombination event statistical analyses; RD was involved in drafting and revis- involving these two markers in the 3' region flanking the ing the paper, and participated in design and coordina- BRCA2 gene as also suggested by the finding that all muta- tion of the study. All Authors read and approved the final tion carriers of Family 25 and the three isolated cases manuscript. shared the same allelic pattern 6-6 for the 5' markers D13S171 and D13S1695. Acknowledgements This study was supported by Alleanza Contro il Cancro. In support of this possibility, the analysis of 8 different References intragenic BRCA2 gene polymorphisms demonstrated 1. Hall JM, Lee MK, Newman B, Morrow JE, Anderson LA, Huey B, King that all carriers of the 9106C>T mutation shared the same MC: Linkage of early-onset familial breast cancer to chromo- haplotype. It can be hypothesized that the common hap- some 17q21. Science 1990, 250:1684-1689. 2. Easton DF, Ford D, Bishop DT: Breast and ovarian cancer inci- lotype shared by all mutation carriers could be comprised dence in BRCA1-mutation carriers. Breast Cancer Linkage within a region starting from the 3' end of the gene and Consortium. Am J Hum Genet 1995, 56:265-271. extending to the 5' flanking microsatellite markers. 3. Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tav- tigian S, Liu Q, Cochran C, Bennett LM, Ding W: A strong candi- date for the breast and ovarian cancer susceptibility gene The analysis of larger series of families is however required BRCA1. Science 1994, 266:66-71. 4. Wooster R, Bignell G, Lancaster J, Swift S, Seal S, Mangion J, Collins to conclusively assess whether the 9106C>T mutation is N, Gregory S, Gumbs C, Micklem G: Identification of the breast associated with a founder effect in the North East of Italy, cancer susceptibility gene BRCA2. 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Shih HA, Couch FJ, Nathanson KL, Blackwood MA, Rebbeck TR, Although carried out on a limited series, the present study Armstrong KA, Calzone K, Stopfer J, Seal S, Stratton MR, Weber BL: BRCA1 and BRCA2 mutation frequency in women evalu- confirms that BRCA2 mutations are associated with a sig- ated in a breast cancer risk evaluation clinic. J Clin Oncol 2002, nificant fraction of breast cancer cases in men. Moreover, 20:994-999. 9. Hakansson S, Johannsson O, Johansson U, Sellberg G, Loman N, Ger- the identification of two recurrent BRCA2 mutations pre- des AM, Holmberg E, Dahl N, Pandis N, Kristoffersson U, Olsson H, disposing to MBC may be of practical relevance to opti- Borg A: Moderate frequency of BRCA1 and BRCA2 germ-line mize the molecular analyses aimed at detecting mutation mutations in Scandinavian familial breast cancer. Am J Hum Genet 1997, 60:1068-1078. 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International Agency for Research on Cancer, Lyon, France; 1980. "BioMed Central will be the most significant development for 28. Breast Cancer Information Core [http://research.nhgri.nih.gov/ disseminating the results of biomedical researc h in our lifetime." bic/] Sir Paul Nurse, Cancer Research UK 29. Santarosa M, Dolcetti R, Magri MD, Crivellari D, Tibiletti MG, Gallo A, Tumolo S, Della Puppa L, Furlan D, Boiocchi M, Viel A: BRCA1 Your research papers will be: and BRCA2 genes: role in hereditary breast and ovarian can- available free of charge to the entire biomedical community cer in Italy. Int J Cancer 1999, 83:5-9. 30. Meijer-van Gelder ME, Look MP, Bolt-de Vries J, Peters HA, Klijn JG, peer reviewed and published immediately upon acceptance Foekens JA: Clinical relevance of biologic factors in male cited in PubMed and archived on PubMed Central breast cancer. Breast Cancer Res Treat 2001, 68:249-60. 31. Parker SL, Tong T, Bolden S, Wingo PA: Cancer statistics. CA Can- yours — you keep the copyright cer J Clin 1997, 47:5-27. BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 9 of 9 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Cancer Springer Journals

Phenotypic features and genetic characterization of male breast cancer families: identification of two recurrent BRCA2 mutations in north-east of Italy

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Springer Journals
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Copyright © 2006 by Miolo et al; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Oncology; Surgical Oncology; Health Promotion and Disease Prevention; Biomedicine general; Medicine/Public Health, general
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10.1186/1471-2407-6-156
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16764716
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Abstract

Background: Breast cancer in men is an infrequent occurrence, accounting for ~1% of all breast tumors with an incidence of about 1:100,000. The relative rarity of male breast cancer (MBC) limits our understanding of the epidemiologic, genetic and clinical features of this tumor. Methods: From 1997 to 2003, 10 MBC patients were referred to our Institute for genetic counselling and BRCA1/2 testing. Here we report on the genetic and phenotypic characterization of 10 families with MBC from the North East of Italy. In particular, we wished to assess the occurrence of specific cancer types in relatives of MBC probands in families with and without BRCA2 predisposing mutations. Moreover, families with recurrent BRCA2 mutations were also characterized by haplotype analysis using 5 BRCA2-linked dinucleotide repeat markers and 8 intragenic BRCA2 polymorphisms. Results: Two pathogenic mutations in the BRCA2 gene were observed: the 9106C>T (Q2960X) and the IVS16-2A>G (splicing) mutations, each in 2 cases. A BRCA1 mutation of uncertain significance 4590C>G (P1491A) was also observed. In families with BRCA2 mutations, female breast cancer was more frequent in the first and second-degree relatives compared to the families with wild type BRCA1/2 (31.9% vs. 8.0% p = 0.001). Reconstruction of the chromosome phasing in three families and the analysis of three isolated cases with the IVS16-2A>G BRCA2 mutation identified the same haplotype associated with MBC, supporting the possibility that this founder mutation previously detected in Slovenian families is also present in the North East of our Country. Moreover, analysis of one family with the 9106C>T BRCA2 mutation allowed the identification of common haplotypes for both microsatellite and intragenic polymorphisms segregating with the mutation. Three isolated cases with the same mutation shared the same intragenic polymorphisms and three 5' microsatellite markers, but showed a different haplotype for 3' markers, which were common to all three cases. Conclusion: The 9106C>T and the IVS16-2A>G mutations constitute recurrent BRCA2 mutations in MBC cases from the North-East of Italy and may be associated with a founder effect. Knowledge of these two recurrent BRCA2 mutations predisposing to MBC may facilitate the analyses aimed at the identification of mutation carriers in our geographic area. Page 1 of 9 (page number not for citation purposes) BMC Cancer 2006, 6:156 http://www.biomedcentral.com/1471-2407/6/156 tions. Moreover, we also searched for the presence of Background Approximately 5–10% of breast and ovarian cancer cases recurrent mutations in this area due to a possible founder are hereditary, occurring mainly in women with germ-line effect. mutations in the BRCA1 or BRCA2 genes [1-4]. Although earlier estimates suggested that BRCA1 and BRCA2 muta- Methods tions were responsible for 75% of breast cancer families From 1997 to 2003, 197 patients with breast or ovarian [4-7] recent population-based studies indicate that these cancer with a family history of these tumors were referred rates may have been overestimated. In fact, the percentage for genetic counselling to the Centro di Riferimento of high-risk families associated with BRCA1 and BRCA2 Oncologico (National Cancer Institute, Aviano, Italy). mutations seems to be around 25% in all groups investi- The present study is part of an Institutional program on gated [8-12]. hereditary tumors approved by local Ethical Committee. Families were selected according to previously reported BRCA1 germ-line mutations are also associated with an criteria [22]. All consecutive male patients with a breast increased risk of other cancer types, including those affect- cancer, independently of age and family history, were ing colon, pancreas, stomach and fallopian tubes [13,14]. included and counselled for genetic testing for BRCA1 and Other possible sites of cancer for BRCA1 mutation carriers BRCA2 gene mutations. Ten families were ascertained by included cutaneous melanoma, basal cell carcinoma and a male index case; one patient with MBC (BR175) also sarcoma [15]. Although BRCA1 mutations have been developed prostatic cancer at the age of 66 (Table 1). One described also in men with breast cancer, the presence of additional male patient (BR331), showing an atypic intra- germ-line mutations in the BRCA1 gene does not seem to cystic papilloma of the breast together with a melanoma convey a significantly increased risk for MBC [16]. How- of the ear, was not included in the statistical analysis. All ever, this issue is still controversial, since a recent study individuals tested for BRCA1/2 mutations gave a signed carried out in 483 BRCA1 mutation carriers reported a 58- informed consent. fold increased risk [13]. Blood samples were obtained from each proband and Available evidence indicates that BRCA2 mutation carriers after the identification of a specific mutation the analyses have an increased risk of breast cancer in both males and were extended to 98 relatives. Screening for mutations in females, as well as of prostate and pancreatic carcinomas. the BRCA1 and BRCA2 genes was carried out by a combi- Carcinomas of the stomach, gallbladder bile ducts, cuta- nation of protein truncation test, single strand conforma- neous melanoma, and basal cell carcinoma are also tion polymorphism, and sequencing techniques (Table slightly more prevalent in carriers of BRCA2 mutations 1). Primer sequences and PCR conditions were previously [17,18]. The large majority of data accumulated so far described [22,23]. All mutations and genetic variants were indicates that, unlike what reported for female breast can- named according to Antonarakis et al. [24]. Missense cer (FBC), MBC is mainly linked to BRCA2 gene muta- mutations and mutations occurring within intronic tions. In fact, up to 30% of MBC were associated with regions whose clinical significance has not yet been BRCA2 mutations, whereas only less than 4% of the cases reported were defined as genetic unclassified variants. carried BRCA1 gene mutations, with variable percentages Germline rearrangements of BRCA1 and BRCA2 gene depending on founder mutations [19,20]. were also analysed by multiplex ligation dependent probe amplification (MLPA). To date, only few studies have attempted to estimate the risk of breast cancer in male carriers of a BRCA2 mutation. The haplotype associated with the 9106C>T BRCA2 muta- The analysis of two large families carried out by Easton et tion was analyzed using five BRCA2-linked di-nucleotide al. [21] resulted in a cumulative risk of MBC of 6.3% (95% repeat markers: D13S290, D13S260, D13S1698, CI 1.4% to 25.6%) by age 70. In contrast, the study by D13S171, and D13S1695, whereas for the IVS16-2A>G Thompson et al. [17], based on 59 cases, failed to detect a mutation the D13S290, D13S171, and D13S1695 mark- significantly higher cumulative risk of MBC by 70 years of ers were used. age (2.8%; 95% CI 0.6 – 13.0%). However, the risk increased to 6.9% (95% CI 1.2% to 38,6%) by 80 years, The first three markers were placed in 3' flanking of the 80-fold higher compared to the general population. The BRCA2 gene whereas the other two were placed in the 5' present study was carried out with the aim to characterize flanking of the BRCA2 gene. The order of markers from the genetic and phenotypic features of families with MBC centromere to telomere is as follows: cen-D13S290, from the North East of Italy. In particular, we wished to D13S260, D13S1698, D13S171, D13S1695-tel. PCR assess the occurrence of specific cancer subtypes in first, products were analyzed on the ABI Prism 3100 using the second and third degree relatives of MBC probands in POP4 matrix and fluorescently labelled primers. For these families with and without BRCA2 predisposing muta- markers, a heterozygosity of 46%, 78%, 63%, 72%, and Page 2 of 9 (page number not for citation purposes) BMC Cancer 2006, 6:156 http://www.biomedcentral.com/1471-2407/6/156 Table 1: Clinicopathologic and genetic characteristics of the 10 MBC cases analyzed for the presence of BRCA1/2 mutations. Family Patient Age TNM histology ER PR Method Mutation Effect Affected relatives 25 BR 22 40 N.A. N.A. N.A. N.A. PTT-SSCP 9106C>T Q2960X BC(31)s, BC(48)s, BC(44)s, BC(30)s, BC(65)s, BC(37)ptc. 103 BR 111 59 pT1N0 intraductal carcinoma N.A. N.A. PTT-SSCP MLPA w.t. BC(78)s, BC(75)pta, BC(55)ptc, BC(N.A.)ptc, BC(60)ptc, BC(60)ptc, BC(58)ptc, BC(60)ptc. 125 BR 138 62 pTis intraductal carcinoma + + PTT-SSCP MLPA w.t. BC(82)m. 127 BR 140 52 pT1cN0 infiltrating ductal + + PTT-SSCP MLPA w.t. BC(55)mta, carcinoma BC(47)mta, PC(61)mtu, BC(45)mtc, BCbil.(35)mtc, BC(42)mtc. 139 BR 173 58 pT2N1b infiltrating ductal + + PTT-SSCP MLPA w.t. / iii carcinoma 140 BR 175 58/ PT4bN0 infiltrating ductal + + PTT-SSCP 9106C>T Q2960X BCbil.(30)s, 66P carcinoma BC(40)pta, C BC(48)pta. 161 BR 195 50 N.A. N.A. N.A. N.A. PTT-SSCP IVS16- exon BC(38)f, BC(57)pta, 2A>G skipping PC(N.A.)ptu, BC(50)ptgm, BC(N.A.)ptggf, BC(N.A.)ptga, BC(N.A.)ptga. 190 BR 243 70 pT1c infiltrating ductal + + PTT-SSCP IVS16- exon BC(36)d, carcinoma 2A>G skipping BC(46)OC(N.A.)m, BC(N.A.)(OC)(N.A.) mta, BC(N.A.)mtc. 192 BR 247 38 pT4bN1 infiltrating ductal + + PTT-SSCP MLPA w.t. PC(75)f. biiiM0 carcinoma 217 BR 282 46 pT1aiii infiltrating ductal + + PTT-Direct 4590C>G P1491A BC(N.A.)pta, BC N0 carcinoma Sequencing (N.A.)ma. MLPA Number between parentheses indicate the age at diagnosis; N.A.: not available; w.t.: wild-type; PTT: protein truncation test; SSCP: single strand conformation polymorphism; MLPA: multiplex ligation-dependent probe amplification; mt: maternal; pt: paternal; m: mother; f: father; s: sister; d: daughter; gm: grandmother; ggf: great-grandfather; ga: great-aunt; c: cousin; u: uncle; a: aunt. BC: breast cancer; BCbil: bilateral breast cancer; OC: ovarian cancer; PC: prostate cancer. 79% has been reported in the Genome Data Base, respec- history of breast and/or ovarian cancer and came from the tively [25]. Alleles were numbered according to the size of same geographic area. each microsatellite repeat marker. The disease-associated haplotypes were deduced from allelic segregation in Statistical analyses known carriers by inspection of segregating genotypes in The first and second degree relatives of the male index case the analyzed families. Moreover, for the haplotype analy- of BRCA2 positive families were compared to first and sec- sis of the 9106C>T mutation, eight internal BRCA2 poly- ond degree relatives of families without a BRCA2 muta- morphisms were analyzed: 203G>A (exon 2), tion but with or without familial history for breast cancer, IVS8+56C>T (intron 8), 1342A>C (exon 10), 3624A>G using chi-square analyses [26]. The family with a BRCA1 (exon 11), 4035T>C (exon 11), IVS11+80del4 (intron mutation with unclear significance was excluded from this 11), 7470A>G (exon 14), IVS16-14T>C (intron 16). For comparison. Odds Ratios (OR), and their corresponding the haplotype analysis only, besides the 4 families ascer- 95% Confidence Interval (CI), was computed using tained by a MBC index case, 6 additional families were unconditional logistic regression model [27] in order to also analyzed, two carrying of the 9106C>T mutation and calculate the odds to develop a tumor in the first and sec- 4 with the IVS16-2A>G mutation. All these families were ond degree relatives conditional on the BRCA2 status of ascertained by a FBC index case, showed a positive family the proband. Page 3 of 9 (page number not for citation purposes) BMC Cancer 2006, 6:156 http://www.biomedcentral.com/1471-2407/6/156 In one IVS16-2A>G positive family, a first-degree relative Results The 10 MBC probands had a mean age at the time of diag- (the father of the BR195 proband) was affected by a MBC nosis of 53.3 years (range 38–70 years). 3/10 patients diagnosed at 38 years. When this case was also included in were younger than 50 years, whereas 7 were ≥50 years. the analysis, the median age of MBC onset in carriers of Only one patient (BR247) developed a breast cancer the IVS16-2A>G mutation decreased to 52.7 years. before the age of 40. Four of the 10 patients (40%) carried a predisposing mutation in the BRCA2 gene (Table 1). In 8/10 cases the histologic characterization of the tumor Five of the remaining six cases carried wild-type (w.t.) was available. Invasive ductal carcinoma (75%) was the BRCA1 and BRCA2 genes whereas in one case (BR282) a most frequent histologic subtype followed by intraductal missense BRCA1 mutation 4590C>G (P1491A), causing a carcinoma (25%). Another case, diagnosed as atypic intra- Pro>Ala replacement in position 1491 with unclear signif- cystic papilloma, which showed also a melanoma of the icance was observed (Table 1). The family history of this ear, was found negative for BRCA1 and BRCA2 mutations. latter case showed two second-degree female relatives This case was not included in the statistical analysis. All affected by breast cancer, one belonging to the maternal the 6 assessable cases were oestrogen and progesterone side and the other to the paternal side. This mutation was receptors positive by immunohistochemistry (Table 1), not reported in the Breast Cancer Information Core data- confirming previous findings indicating that MBC have a base [28]. Although mutations in the BRCA1 gene do not higher rate of positivity to hormone receptors than do seem to be associated with increased risk of MBC, further FBC (90% vs. 77%) [30]. However, when MBC were com- studies are required to assess the possible predisposing pared with cancers from postmenopausal women, the role of this mutation in case BR282. It was not possible to positivity rates were similar. All our BRCA2 positive cases assess whether this variant was present in other affected showed a positive family history for breast and/or ovarian relatives because none of them was alive. cancer (Table 1). The six cases negative for point mutations were also ana- Two out of three cases of prostatic cancers were observed lyzed for BRCA1 and BRCA2 genes rearrangements by at 61 and 75 years, respectively, in w.t. families. In the four MLPA. None of these cases showed structural alterations BRCA2 mutation carrier families, breast cancer was more of BRCA1 and BRCA2 genes. The median age of diagnosis frequent in the first and second-degree relatives than in was 54.5 years for the 4 BRCA2 mutation carriers and 53.8 the five w.t. families. In fact, as compared with w.t. fami- years for the other cases, excluding the unclassified vari- lies, in BRCA2 mutation carrier families there was a signif- ant. The 4 BRCA2 positive cases carried only two distinct icant higher rate of affected first and second degree mutations: the 9106C>T, involving exon 22 and leading relatives (31.9% vs. 8.0% p = 0.001). These results indicate to a premature protein truncation (cases BR22 and that first and second degree relatives of index cases with BR175), and the IVS16-2A>G, involving intron 16 and BRCA2 mutation carrier families have a five times greater leading to a partial or total skipping of exon 17 (cases risk to develop a breast cancer (Table 2). The two MBC BR195 and BR243) [29]. Both these mutations were local- cases without family history of cancer resulted negative for ized outside the Ovarian Cluster Cancer Region and were BRCA1/2 mutations. In three of the four families with a previously reported also by others in the BIC database BRCA2 gene mutation, the molecular analysis was (three times for the 9106C>T mutation and four times for extended to ten non-affected relatives, (six males and four the IVS16-2A>G mutation in all). The carriers of the females), allowing the identification of 5 additional IVS16-2A>G mutation and those with the 9106C>T muta- BRCA2 mutation carriers (2 males and 3 females). tion showed a median age of breast cancer onset of 60 and 49 years, respectively. Table 2: Distribution of FBC in relatives of MBC cases belonging to w.t. and BRCA2 mutation carrier families. w.t. families (n = 5) BRCA2 mutation carrier families (n = 4) I+II degree relatives I+II degree relatives N (%) N (%) OR 95% CI Non-affected 58 (92) 32 (68.1) 1 Affected 5 (8) 15 (31.9) 5.43 (1.81–16.34) Total 63 47 X = 10.40; p = 0.001; w.t.: wild-type. Page 4 of 9 (page number not for citation purposes) BMC Cancer 2006, 6:156 http://www.biomedcentral.com/1471-2407/6/156 Besides the four MBC cases carrying BRCA2 mutations, fore, all isolated cases had the same 5,1,1,6,6 genotype, genotype analysis was also carried out in six additional which is in part consistent with the disease causing haplo- unrelated cases enrolled for the presence of FBC in the type detected in family 25. However, the analysis of 8 index case: two with the 9106C>T BRCA2 mutation and internal BRCA2 polymorphisms showed that the same A- four with the IVS16-2A>G BRCA2 mutation. On the C-A-G-T-(-4)-G-C haplotype was present in all the whole, genotype analysis was performed in four cases 9106C>T mutation carriers investigated (Table 4). The with the 9106C>T BRCA2 mutation, (one family and analysis of 55 healthy donors for 4 of these intragenic pol- three isolated cases), and in six IVS16-2A>G BRCA2 muta- ymorphisms (203G>A, IVS8+56C>T, 7470A>G, IVS16- tion cases (three families and three isolated cases). Recon- 14T>C) showed that, without phase reconstructing, only struction of the chromosome phasing was possible in four 15 of them (27%) carried the same haplotype detected in families. The analysis carried out in family 25, carrying the BRCA2 mutation carriers. On these grounds, it is highly 9106C>T BRCA2 mutation, allowed the identification of unlikely that these four polymorphisms are simultane- a common haplotype (5-6-2-6-6) shared by all mutation ously present in the four index cases by chance alone carriers belonging to this family (Figure 1). This haplotype (p = 0.0085, two-tailed Fisher's exact test). encompassed a distance of about 3.8 cM between the D13S290 to D13S1695 markers, and was not detected in The haplotype analysis by the reconstruction of the chro- non-carrier relatives. The isolated cases showed the same mosome phasing of three IVS16-2A>G BRCA2 mutation allelic pattern for the D13S290, D13S171, and D13S1695 families, showed that all mutation carriers had the same markers, whereas the D13S260 and D13S1698 markers, 5-4-2 haplotype for the D13S290, D13S171, and located within the 3' region flanking the BRCA2 gene, D13S1695 markers, whereas all the non-carrier relatives showed an allelic pattern (1,1) different from that of displayed a different allelic pattern for the three markers mutation carriers belonging to family 25 (Table 3). There- investigated (Table 5). Pedigree of Figure 1 the family 25 with the reconstruction of the chromosome phasing Pedigree of the family 25 with the reconstruction of the chromosome phasing. The type of cancer and age at diagnosis are indi- cated. Affected individuals are indicated by a red symbol and proband is denoted by arrow. The shared haplotype segregating with 9106C>T mutation is boxed. Page 5 of 9 (page number not for citation purposes) BMC Cancer 2006, 6:156 http://www.biomedcentral.com/1471-2407/6/156 Table 3: Genotype analysis of flanking microsatellites associated with the 9106C>T BRCA2 mutation. Markers carrier (+)/ non carrier (-) Probands D13S290 D13S260 D13S1698 D13S171 D13S1695 Family 25 BR22 5-5 6-5 2-2 6-6 6-5 + BR23 5-5 5-3 2-5 6-1 5-8 - BR99 5-3 2-2 4–6 2-2 5–7 - BR100 5-4 6-2 2-4 6-2 6-5 + BR101 5-5 6-1 2-5 6-2 6-2 + BR102 5-4 6-2 2-4 6-2 6-5 + BR103 5-5 5-32–5 6-15–8 - Isolated cases BR175 5,4 1,4 1,1 6,2 6,5 + BR342 5,3 1,3 1,3 6,2 6,6 + TBL38 5,5 1,5 1,5 6,2 6,6 + Numbers in boldface type indicate the 9106C>T mutation alleles. Haplotype segregating with the 9106C>T mutation alleles of phased alleles are separated by a dash. Genotypes of unphased alleles are separated by a comma. The mean age of breast cancer onset in males with and Discussion MBC accounts for less than 1% of all breast cancers and without BRCA2 mutations is still a controversial issue little is known on the epidemiologic, genetic and clinico- [16,32,35]. In the present study, we found a median age pathologic features of this malignancy [31-34]. Available of diagnosis of 54.5 years for the BRCA2 mutation carriers data indicate that the prevalence of BRCA2 mutations in (range 40–70 years) and of 53.8 years (range 38–62 years) MBC ranges between 4% and 40% [32-34]. These differ- for the w.t. type cases, supporting the findings reported by ences are probably related to the genetic features of the others [32] indicating that BRCA2 mutation carriers and families, with higher rates of BRCA2 mutations in men noncarriers have a similar age at the time of diagnosis. with a strong family history of cancer, particularly of those belonging to the BRCA2 spectrum. It has been reported that about 15–20% of MBC patients had a first-degree relative with breast carcinoma, support- In our series, the high prevalence of BRCA2 mutation ing the notion that a positive family history of breast can- (about 40%) correlated with the high number of relatives cer is associated with increased risk of MBC [36,37]. In our with breast cancer present in these families. In fact, survey, all MBC patients carrying deleterious BRCA2 excluding from the analysis the two apparently sporadic mutations had a family history of breast cancer. This was MBC, the frequency of BRCA2 mutations rises up to 50%. not due to a selection bias, since the eligibility criteria Table 4: Haplotype analysis of intragenic BRCA2 polymorphisms associated with the 9106C>T BRCA2 mutation. Polymorphisms Family 25 Isolated cases Position Nucleotide change BR22 BR23 BR99 BR100 BR101 BR102 BR103 BR175 BR342 TBL 38 M w.t. w.t.M M M w.t.M M M EX2 203G>A G>A G-G G>A A-A A-A A-A G-G G>A G>A G>A INTR8 IVS8+56C>T C-C C-C C>T C>T C-C C-C C-C C>T C-C C>T EX10B 1342A>C A>C C-C A-A A-A A-A A-A C-C A-A A-A A-A EX11-J 3624A>G A>G A-A A>G G-G G-G G-G A-A A>G A>G A>G EX11K 4035T>C T-T T-T T>C T-T T-T T-T T-T T-T T>C T-T INTR11Q IVS11+80del4 -4/4 4/4 -4/4 -4/-4 -4/4 -4/-4 4/4 -4/4 -4/4 -4/4 EX14A 7470A>G A>G A-A A>G G-G G-G G-G A-A A>G A>G A>G INTR17 IVS16-14T>C T>C T>C T>C C-C C-C C-C T>C C-C T>C T>C M: mutated; w.t.: wild-type; numbers in bold indicate the shared allele. Page 6 of 9 (page number not for citation purposes) BMC Cancer 2006, 6:156 http://www.biomedcentral.com/1471-2407/6/156 Table 5: Genotype analysis of flanking microsatellites associated with the IVS16-2A>G BRCA2 mutation. Markers Carrier (+)/Non carrier (-) Probands D13S290 D13S171 D13S1695 Family 14 BR6 5-5 4-2 2-5 + BR73 5-1 2-2 5–6 - BR82 5-1 4-2 2-6 + Family 161 BR195 5-5 4-2 2-4 + BR285 5-5 4-2 2-4 + BR289 5-2 2-1 4–6 - Family 190 BR243 5-3 4-4 2-4 + BR290 5-5 4-5 2-6 + BR291 3–5 4–5 4–5 - Isolated cases BR60 5,5 4,4 2,1 + BR85 5,5 4,2 2,5 + BR312 5,5 4,2 2,3 + Numbers in boldface type indicate the IVS16-2A>G mutation alleles. Haplotype segregating with the IVS16-2A>G mutation alleles of phased alleles are separated by a dash. Genotypes of unphased alleles are separated by a comma. used for MBC did not require the presence of a positive BRCA2 mutations including two mutations recurring in family history. These findings are in keeping with those central Italy (BRCA1 3345delAG and BRCA2 6696delTC). reported by others indicating that a positive history of breast cancer is more frequent in BRCA2 gene mutation The three BRCA2 mutations identified in this study were carriers, with values ranging from 13 to 80% [36-38], however different from those we observed. Notably, the although opposite results have been also reported [32]. In MBC cases from our series were characterized by the recur- our 8 families with a positive history of breast cancer, the rence of a restricted set of BRCA2 mutations detected in presence of a deleterious BRCA2 mutation was not associ- families coming from a limited geographic area (North ated with a different occurrence of specific types of cancers East of Italy), suggesting the possible existence of a as compared to BRCA2-negative families. A significantly founder effect. Indeed, it has been previously reported higher frequency of breast cancers was observed in the that all carriers of the IVS16-2A>G mutation shared a first- and second-degree relatives of mutation carriers, common haplotype, indicating a likely founder effect whereas the prevalence of breast cancers in third degree mainly confined to the Slovenian population [39]. Con- relatives was higher in w.t. families. sistently, our haplotype analysis carried out in six Italian families with the IVS16-2A>G BRCA2 mutation allowed Similarly to what reported in other studies [33], also the the detection of a common haplotype shared by all muta- majority (75%) of our MBC were infiltrating ductal or tion carriers. These findings, taken together, support the intraductal carcinomas. One case was classified as intra- possible occurrence of a founder effect involving the 5' cystic atypic papilloma a relatively infrequent subtype region flanking the BRCA2 gene in both North-East of accounting for about 2–5% of all MBC [33]. The patient Italy and the neighbouring Slovenia, suggesting that the carrying this tumor was negative to the molecular screen- IVS16-2A>G BRCA2 mutations probably recognize a com- ing, as those reported in other series [16], ruling out the mon ancestral origin. The IVS16-2A>G mutation was pre- possibility that BRCA1/2 mutations may confer a signifi- viously reported by Krajc et al. [39] in three breast cancer- cantly increased risk of developing this peculiar MBC his- only Slovenian families, whereas it was not identified in totype. three families with ovarian cancer, suggesting that the can- cer phenotype associated with this mutation is limited to Available data on the prevalence of BRCA1 and BRCA2 breast cancer. Conversely, our pedigree's analysis of six mutations in Italian MBC patients are limited. In a previ- Italian families with the IVS16-2A>G mutation disclosed ous study [16], Ottini et al. reported one BRCA1 and three the presence of one family in which two ovarian carcino- Page 7 of 9 (page number not for citation purposes) BMC Cancer 2006, 6:156 http://www.biomedcentral.com/1471-2407/6/156 mas were present along with breast cancer, indicating thus carriers in North East of Italy. In fact, the IVS16-2A>G and that the risk conferred by this mutation is not restricted to the 9106C>T mutations were detected in about 4% of the breast cancer. Moreover, in the six families with the IVS16- families we have investigated so far, whereas the recurrent 2A>G mutation of our database, two additional MBC and putative founder mutations account for about 12% of were detected (relatives of patients BR195 and BR312), the total. This would support the practical value of starting further supporting the increased risk of developing breast the mutational analysis of BRCA2 in the North-East Ital- cancer in males conferred by this mutation. Since the ian population with the search for these particular muta- IVS16-2A>G mutation can result in either a partial or total tions. This may enhance the collection of larger numbers skipping of exon 17 [29], the increased risk for breast can- of families with the same predisposing BRCA2 mutations, cer in males due to this mutation may be related to these providing thus the basis for studies aimed at identifying effects on BRCA2 gene transcription. Studies are under genetic modifying factors that may modulate the pheno- way in our laboratory to elucidate this issue. typic manifestations of MBC. With regard to the 9106C>T mutation, the data obtained Competing interests The author(s) declare that they have no competing inter- by haplotype evaluation coupled with the reconstruction of chromosome phasing suggest but do not conclusively ests. prove the occurrence of a possible founder effect also for this mutation. In fact, all the mutation carriers of Family Authors' contributions 25 showed the same haplotype for the markers D13S290- GMM performed haplotype analyses and drafted the man- D13S260-D13S1698-D13S171-D13S1695; however, the uscript; AVi performed molecular genetic studies and con- three isolated cases showed a different allelic pattern for tributed to interpretation of data; MS, and LDP performed the markers D13S260 and D13S1698 located within the BRCA tests and haplotype analyses; CDG, MGT, and AVe 3' region flanking the BRCA2 gene. These results may collected and analyzed the clinical data; EB performed the underlie the occurrence of a possible recombination event statistical analyses; RD was involved in drafting and revis- involving these two markers in the 3' region flanking the ing the paper, and participated in design and coordina- BRCA2 gene as also suggested by the finding that all muta- tion of the study. All Authors read and approved the final tion carriers of Family 25 and the three isolated cases manuscript. shared the same allelic pattern 6-6 for the 5' markers D13S171 and D13S1695. Acknowledgements This study was supported by Alleanza Contro il Cancro. In support of this possibility, the analysis of 8 different References intragenic BRCA2 gene polymorphisms demonstrated 1. Hall JM, Lee MK, Newman B, Morrow JE, Anderson LA, Huey B, King that all carriers of the 9106C>T mutation shared the same MC: Linkage of early-onset familial breast cancer to chromo- haplotype. 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