High Frequency of Germ-Line BRCA2 Mutations among Hungarian Male Breast Cancer Patients without Family History

Introduction

Male breast cancer is a rare disease, comprising <1% of all malignancies in men in the western world; however, in some developing areas, it is more frequent, accounting for 6–15% of all breast cancers. 3 In continental Europe, the Hungarians have the highest male breast cancer mortality rates (1) . The biggest risk factor for breast cancer in both genders seems to be inherited predisposition. Approximately 5–10% of female breast cancer cases are due to inheritance of autosomal dominant susceptibility genes (2) . In the vast majority of inherited cases, the recently isolated genes BRCA1 and BRCA2 are thought to be responsible for the disease (3) . BRCA1 is estimated to account for most female breast/ovarian cancer families, but it can be infrequently detected in families with male breast cancer cases. However, breast cancer-prone families in whom male breast cancer occurred have been shown to be linked to or have mutation in BRCA2 (4) . Our earlier results did not reveal BRCA2 carrier male breast cancer patients in Hungarian breast cancer families, but two individuals without family history were found to carry BRCA2 mutations (5) .

Very little is yet known about the genetic background of male breast cancer. Somatic mutations of the p53 gene were identified in 41% of male breast cancer patients studied (6) . Defects in only a few genes have been associated with predisposition to the development of this disease. Genetic abnormalities of the androgen receptor have been reported in association with male breast cancer cases (7) . There are some reports of BRCA1 mutation involvement in male breast cancer (8, 9, 10) , but in all cases the patients with male breast cancer were members of families carrying BRCA1 mutation. There is much more evidence that inherited BRCA2 mutations increase the risk for developing male breast cancer. Several families that also contained male breast cancer patients were reported to carry BRCA2 mutations (4 , 11, 12, 13) . Men who carry a germ-line BRCA2 mutation have an increased risk of breast cancer (14) .

Although men with breast cancer also often have gynecomastia, it is still unknown whether gynecomastia per se predisposes the male breast to malignant disease. Some authors report that as many as 20–36% of the male breast cancer patients in their studies have a history of gynecomastia (15 , 16) .

In the present study, we have determined the contribution of germ-line mutations in the BRCA1 and BRCA2 genes to the pathogenesis of male breast cancer in Hungary by screening a series of 18 unselected male breast cancer patients and three patients with gynecomastia.

Materials and Methods

Patients.

Blood samples were collected from all consenting patients with male breast cancer or gynecomastia at the National Institute of Oncology (Budapest, Hungary) in the period of 1996–1998. The histopathological typing of the cases was completed by board-certified pathologists. This panel represents consecutive cases who were not selected for family history of breast/ovarian cancer; however, information on family history was available for all patients. The patients with gynecomastia did not have a medical history of Klinefelter syndrome.

BRCA1 and BRCA2 Mutation Screening.

Genomic DNA was extracted by standard methods from peripheral lymphocytes from all patients. PCR amplification of 160–550-bp DNA fragments covering the entire BRCA2 coding region and splice junctions was carried out by using a set of 44 primer pairs (5 , 11) . Similarly, all BRCA1 exons with the exception of exon 11 were amplified with a set of 23 primer pairs; exon 11 was screened in three overlapping protein truncation test fragments as reported previously (5) . Mutational screening was performed in germ-line DNA by combined multiple heteroduplex analysis and/or by single strand conformation analysis. Mutation screening in both genes was performed irrespective of identification of a mutation in one gene, because it is possible that mutations of both BRCA1 and BRCA2 may be segregating in a family, as we reported in a Hungarian breast/ovarian cancer patient (17) . PCR products from variant conformers were purified using Wizard PCR Prep DNA Purification System (Promega Corp.) and sequenced using the Thermosequenase Cyclo Sequencing kit (USB-Amersham).

Results

The entire coding regions and intronic splice sites of both BRCA1 and BRCA2 were screened using multiple heteroduplex analysis/single-strand conformation analysis and protein truncation test, followed by direct sequencing of abnormalities. Six of the 18 male breast cancer cases (33%) were observed to carry truncating mutations in the BRCA2 gene (Table 1) ⇓ . Four of the six truncating mutations were novel; the other two alterations (277delAC and 9326insA) were reported earlier (5 , 18 , 19) . A review was conducted of the clinical and family history of the mutation carriers. None of them reported a family history for breast/ovarian cancer. Two patients (11%) had a family history of breast/ovarian cancer in at least one first-degree relative and another two (11%) in at least one second-degree relative in this unselected study; however, no BRCA2 mutation was observed among them.

No germ-line BRCA1 mutations were detected in the male breast cancer cases, nor was a mutation identified in either BRCA1 or BRCA2 in the three cases of gynecomastia; however, one patient (G2) from this group carried an unclassified BRCA2 variant allele with unclear significance (Table 2) ⇓ . There was no difference between mutation carriers and noncarriers with respect to clinicopathological features nor age at diagnosis (Table 3) ⇓ . The average age at diagnosis was 62 years; there was only an insignificant difference between the mean age of onset of carriers (58 years) and noncarriers (64 years).

Discussion

In the first studies of BRCA2 analysis (4 , 11) , the subjects of scrutiny were mostly large families that sometimes also contained members with male breast cancer. In more recent reports (20, 21, 22) , sets of population-based male breast cancer cases were studied for the frequency of germ-line BRCA2 carriers. Here we analyzed male breast cancer patients from a hospital-based study to determine the contribution of BRCA1 and BRCA2 mutations to the pathogenesis of male breast cancer in Hungary. Frequencies reported of BRCA2 mutations in male breast cancer vary between 4% (21) and 40% (23) , the latter results are possibly influenced by a strong founder effect in that particular population. In contrast to reports by Friedman et al. (21) and Mavraki et al. (20) , who found 4 and 7% of patients carrying germ-line BRCA2 mutations, respectively, in the present study a large proportion of cases was found to carry BRCA2 mutations (6 of 18; 33%). These results are even higher than the recent reports of Haraldsson et al. (22) , who identified 21% of all cases to be BRCA2 mutation carriers.

Although in some large BRCA1 families male breast cancer patients have been shown to have germ-line BRCA1 mutations (8, 9, 10) , in the present study, as in the study of Friedman et al. (21) , no BRCA1 mutations were found. This indicates that BRCA1 germ-line mutations do not contribute substantially to male breast cancer.

In the present study, six BRCA2 mutations were identified among the male breast cancer patients. The mutations were all frameshifts predicted to result in truncated proteins. This is in agreement with previous findings, that >85% of BRCA mutations identified cause protein truncation (21) . Four of six truncating mutations were novel; the other two alterations have been reported earlier. The 277delAC mutation was reported from the United States in a large family that also contained three cases of male breast cancer (18) . The other recurrent mutation (9326insA) was first identified in Hungary (5) and has also been observed in a Hungarian male breast cancer patient living in Sweden (22) and in a French breast-ovarian cancer family that did not contain male breast cancer (19) . Several additional Hungarian breast cancer families were identified to carry 9326insA, all without male breast cancer cases. 4 In a recent international collaborative study, we identified a common founding haplotype for this mutation (26) , and we have indication for a common origin of 9326insA from the Central-Eastern European region. 5

The mutations we found in this study are located in exons 2, 7, 11, 16, and 23 (Table 1 ⇓ and Fig. 1 ⇓ ). This is not notably different from the distribution of either BRCA2 mutation spectra in male breast cancers from other studies (Fig. 1) ⇓ or of all BRCA2 mutations. 6 Thus, there is no evidence to date that mutations in specific domains of BRCA2 may be associated with male breast cancer.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1.

BRCA2 mutation spectrum in male breast cancer patients. ▴, mutations found in this study (exons 2, 11, 16, and 23). ▵, mutations found by Wooster et al. (4) ; Couch et al. (11) ; Phelan et al. (12) ; Tavtigian et al. (18) ; Thorlacius et al. (23) ; Friedman et al. (21) ; Mavraki et al. (20) ; Serova et al. (19) ; and Haraldsson et al. (22) . Only names of recurrent mutations are indicated.

The 18 breast cancer patients screened for germ-line BRCA mutations were selected without any regard for family history. However, information on family history was available for all patients (Table 3) ⇓ . In the present study, the proportion of patients with family history of breast/ovarian cancer in at least one first-degree relative was 11% (2 of 18), and in at least one second-degree relative, also 11% (2/18). This is in agreement with the studies of Rosenblatt et al. (24) and Friedman et al. (21) , who found similar proportions in their studies (18–10% and 17–13%, respectively). Surprisingly, no BRCA2 mutation was identified among the cases with family history; neither did the mutation carriers report family histories of breast/ovarian cancer. This is in contrast with the results of Couch et al. (11) , who identified germ-line BRCA2 mutations in 7 of 50 (14%) male breast cancer cases, 6 of 7 (85%) of whom had a family history of breast cancer. Friedman et al. (21) found two mutation carriers in their panel, one of whom had a family history. Similarly to our results, Haraldsson et al. (22) reported recently that only one of seven BRCA2 mutation carriers had a positive family history of breast cancer.

No evidence for differences in clinicopathological features was seen between mutation carriers and noncarriers. Most patients had invasive ductal carcinoma (17 of 18; 95%), including all germ-line BRCA2 mutation carriers (Table 3) ⇓ . This is consistent with literature data that invasive ductal carcinomas account for 84–93% of cases (25) . The average age of men at diagnosis of breast cancer is close to 65, about 5 years older than the average age for women. 3 In our studies, the average age at diagnosis in carriers was not significantly lower than that of noncarriers. Compared with other BRCA2 mutations, 9326insA appears to be linked with younger age of onset in female breast cancer (26) and possibly also in male breast cancer (22) .

No mutation was identified in either of the genes in the three cases of gynecomastia; however, one patient (G2) from this group carried a missense BRCA2 variant allele (7081A→G) with unclear significance. This alteration was reported eight times in the Breast Cancer Information Core as an unclassified variant and lies in a conserved region of the gene in human, rat, and mouse; however, expression analysis would be required to elucidate the nature of this type of alteration.

The data presented here imply that germ-line mutations in the BRCA2 gene are involved in the development of one-third of male breast cancer cases in Hungary. There may be other genetic factors that are responsible for some of the male breast cancers that cannot be explained by mutations in the BRCA2 gene. There is no evidence for a male breast cancer-specific cluster region in the BRCA2 gene thus far, but because the majority of BRCA2 mutations found in male breast cancer are unique and not identified in female breast cancer cases, it is possible that at least some BRCA2 mutations may predispose to male rather than female breast cancer. On the other hand, certain mutations (e.g., 9326insA) result in both male and female breast cancers and can be associated with various phenotypes in different families. This could be explained by modifying factors in disease development different in males and females. The first evidence of a modifying genetic factor of BRCA2 in male breast cancer was provided recently (27) , reporting that an interstitial tandem duplication on 9p increases the risk of breast cancer in male patients conferred by BRCA2. The lack of a positive family history in all carriers suggests that at least some BRCA2 mutations may have a lower penetrance than others, and selection of putative BRCA2 mutation carriers among male breast cancer patients cannot be based on the family background of the disease. Our data provide further evidence that in certain populations there is a high genetic component in the development of this disease, and in these populations all patients with male breast cancer, regardless of their family history, should be screened for BRCA2 mutations.