Founder BRCA1/2 mutations among male patients with breast cancer in Israel.

To the Editor: Germline mutations in the BRCA1 (MIM 113705) and BRCA2 (MIM 600185) genes are associated with a high risk of breast and ovarian cancer in women (Ford et al. 1998). The risk of male breast cancer is higher among carriers of BRCA2 mutations compared with carriers of BRCA1 mutations, although the absolute risk to male carriers is not well characterized (Wooster et al. 1995; Ford et al. 1998). Studies of BRCA2 in small population- and clinic-based series of male breast cancer patients from the United States and the United Kingdom have found carrier frequencies of 4%–14% (Couch et al. 1996; Friedman et al. 1997; Mavraki et al. 1997). Carrier frequencies for specific founder BRCA2 germline mutations have been higher: 21%–40% (Thorlacius et al. 1996; Haraldsson et al. 1998; Csokay et al. 1999). The number of cases analyzed has ranged from 18 to 54, however, and the confidence limits on the carrier frequencies are very large. Among predominantly Ashkenazi Jewish populations, three founder mutations in the BRCA1/2 genes are present in >2% of all individuals, and they may account for ∼80% of all the mutations in these genes (Struewing et al. 1997; Frank et al. 1998). This allows the relatively efficient analysis of larger numbers of cases. All male patients with breast cancer (n=165) diagnosed in five Israeli hospitals during 1980–97 were reviewed for inclusion in this study. Jewish patients were characterized as Ashkenazi or non-Ashkenazi, on the basis of either the recorded place of birth in the Israeli Population Registry or, if they were born in Israel, their father's recorded place of birth. Non-Ashkenazi Jews were born in Turkey, Syria, Iraq, Iran, Afghanistan, Morocco, or Libya. Israeli-born non-Jews were designated as Arab, Christian, or Druze. Samples were analyzed anonymously. Among the 122 histologically verified cases for whom adequate pathological material could be obtained, 1 was excluded, because analysis from multiple specimens differed with respect to mutation status and marker genotypes. Eighty-nine cases were Ashkenazi, 21 were non-Ashkenazi, and 14 were Arabs. Unstained 5-μm paraffin sections were scraped from slides and were digested in 100 μl of buffer containing 10 mM Tris (pH 8.0), 50 mM KCl, 10 mM EDTA, 2 mM MgCl2, 1% Tween-20 and 10 μg of Proteinase K at 57°C overnight. The samples were then heated to 100°C for 5 min and then 1–5 μl were used as template in the PCR reactions. A 20-μl multiplex PCR reaction was used to amplify the three segments containing the founder mutations. Each reaction contained 1 × PCR Buffer, 2.5 mM MgCl2, 100 μM each dNTP, 15% sucrose, and 0.75 U Taq Gold (PE Biosystems). Primers included 300 nM 2F103 (6FAM-tcgcgttgaagaagtacaaaatgtc), 300 nM 2R102 (caaattaatacactcttgtgctgacttac), 200 nM 20F101 (HEX-gtcaatggaagaaaccaccaaggtc), 200 nM 20R101 (tgcaaaggggagtggaatacacagt), 200 nM 11F101 (TET-tagggaagcttcataagtcagtctca), and 200 nM 11R101 (cttgcgttttgtaatgaagcatct). Cycling consisted of 94°C for 12 min, followed by 10 cycles of 92°C for 10 s, annealing at 68°C for 10 s, and 72°C for 20 s, with the annealing temperature being decreased 1.5°C per cycle, followed by 30 cycles of 92°C for 15 s, 55°C for 15 s, and 72°C for 30 s, followed by 72°C for 10 min. The PCR products were diluted 1:149 with water and then were diluted 1:6 with loading cocktail, according to the manufacturers' recommendations, and were electrophoresed on an ABI 310 capillary electrophoresis machine by use of GENESCAN software (PE Biosystems). Known mutant, wild-type, and no-DNA controls were run with each PCR reaction. Mutants were identified by visual inspection of the electropherograms, with the small-insertion or -deletion mutations resulting in an extra peak in the pattern. Primers to amplify the chromosome 13 markers D13S260, D13S1695, D13S1698, and D13S1701 were redesigned to result in shorter amplicons. All carriers of the 6174delT mutation and a reference family with this mutation were analyzed for these four markers, on the ABI 310 (primer sequences are available, on request, from the corresponding author.) The results of mutation testing are shown in table 1. Of the 19 mutation carriers, 17 were Ashkenazi and 2 were non-Ashkenazi Jews. Carrier frequencies were not statistically different between the two ethnic groups (P=.7). None of the Arab men was a carrier, and the 5382insC mutation was not detected in any patients. The mean ages at diagnosis of all 19 mutation carriers (64 years) and of the BRCA2 6174delT mutation carriers (66 years) were not significantly different than that of noncarriers (68 years). Reliable genotypes could not be obtained for all the markers for all carriers, but 14 of 14 carriers successfully genotyped at D13S1698 shared the same allele as was seen in the reference family; 12 of 12 carriers successfully genotyped at D13S260 shared the correct allele; 7 of 8 carriers successfully genotyped at D13S1701 shared the correct allele; and 13 of 15 carriers successfully genotyped at D13S1695 shared the correct allele. Table 1 Israeli Male-Breast-Cancer Mutation Testing Results These results suggest that ⩾17% of Jewish men diagnosed with breast cancer in Israel carry a mutation in BRCA1 or BRCA2, since only the three common founder mutations were screened. This study was based on the analysis of a small amount of pathological tissue, and the proportion of the tissue that was tumor was unknown. Our methods may have led both to false negatives, caused by the complete loss of the relevant genes in a tumor—although this might be expected to result in preferential loss of the wild-type and retention of the mutant allele—and false positives, caused by contamination of the specimens during processing and handling. Our observed carrier frequency is somewhat higher than those in most series studied in the United States and the United Kingdom but is lower than frequencies observed in several other populations with common founder mutations. Unlike that in female carriers, who have an earlier age at diagnosis of breast cancer, the age at diagnosis among male carriers was not significantly lower than among noncarriers. This may reflect the fact that most men carried a BRCA2 mutation, because the age at diagnosis for female breast cancer appears to be later for BRCA2 carriers than for BRCA1 carriers (Ford et al. 1998). The BRCA1 185delAG founder mutation has been observed in both Ashkenazi and non-Ashkenazi Jewish women with breast or ovarian cancer (Bar-Sade et al. 1998). We observed the 6174delT mutation in two non-Ashkenazi Jewish men with breast cancer, and this is the first published observation of this mutation in this population (Neuhausen et al. 1998). Our characterization of Jewish men as Ashkenazi or non-Ashkenazi was based on the place of birth as recorded in the Israeli Population Registry. Since ethnic origin was not determined in person or by biological testing, some misclassification could have occurred. If verified in other studies, however, this finding would suggest that this mutation is considerably older than previously estimated (Neuhausen et al. 1998). Genotyping of four microsatellite markers near BRCA2 was consistent with their sharing the same haplotype as was seen in Ashkenazi carriers of this mutation. Although the number of cases was small, we did not observe an earlier age at onset among mutation carriers, nor did we detect any of the three mutations in Arab men with breast cancer.