Introduction

Germline mutations in the BRCA1 (MIM # 113705) and BRCA2 (MIM# 600185) genes can be detected in high risk breast/ovarian families, and serve to estimate the lifetime risk for developing these neoplasms in mutation carriers and the consequential recommendations for early detection and risk reducing surgeries [1]. More than 3,000 pathogenic mutations and sequence alterations have been reported within both genes since they were identified in the mid 1990’s (http://research.nhgri.nih.gov/bic/). In the majority of world populations, mutations in both genes are family specific, with no obvious clustering to defined gene regions. Yet, in several populations, the spectrum of mutations is rather limited, reflecting a “founder mutation”: the Icelandic [2], the Polish [3], Russian [4], and the Norwegian [5] populations. Notably, among Ashkenazi Jews (i.e., Jews of east European ancestry) three mutations in BRCA1 (185delAG, 5382InsC) and BRCA2 (6174delT) occur frequently. These three mutations can be detected in the overwhelming majority of high risk Jewish Ashkenazi families, in about 35% of consecutive ovarian cancer cases and even in 2.5% of the general Jewish Ashkenazi population [6–9]. There are only a handful of other, family specific, mutations in Jewish Ashkenazi high risk families [10, 11]. Among the non-Ashkenazim (i.e., Jews from diverse ethnicities such as the Balkans, Iraq, Iran, North Africa, Yemen) there are also a handful of recurring mutations in both genes: 185delAG Tyr978X (BRCA1) and 8765delAG (BRCA2) [12, 13]. Yet these mutations account for only a minority of high risk, non Ashkenazi Jewish families.

While the rates of breast cancer among Arab and Druze women in Israel are lower than those for Jewish women, there is a reported increase in the rate of breast cancer diagnosis among non Jewish women in Israel since the mid 1990’s: the age standardized rate (ASR) for Jewish women was 71.1/100,000 in 1979–1981 and increased by 45.7% to 103.6/100,000 in 2000–2002. These rates for non Jewish women in Israel were 14.1/100,000 and 42.6/100,000, respectively, for the same time period, a threefold increase [14]. Furthermore, the age of diagnosis of breast cancer in Arab women is substantially younger than Jewish women in Israel, with the majority of Arab women (79%) diagnosed premenopausally and 11% being diagnosed under the age of 35 years [15]. This pattern of breast cancer morbidity of Arab women is also reported in Saudi Arabia [16]. Early age at diagnosis is suggestive of an inherited predisposition to breast cancer. However, there are only few reports on the spectrum of BRCA1 and BRCA2 mutations among Arab-Moslem women [17–26]. Furthermore, given the Moslem Arab traditions and practices, it seemed plausible that several founder mutations may underlie inherited predisposition in that population. Even less data is available on the mutational spectra in both genes among the Druze population, a specific sect that diverged from Islam in 1017 AD and has a unique social structure hallmarked by a high rate of consanguinity, inter marriage, and an inability to join the sect or leave it [27].

The aim of the study was to define the spectrum of germline mutations in both the BRCA1 BRCA2 genes in a large set of high risk Israeli individuals of Ashkenazi, non Ashkenazi, Moslem and Christian Arab, and Druze origin.

Patients and methods

Participant identification and recruitment

The study population was recruited from among individuals counseled and tested at one of three Oncogenetics services located at the Sheba Medical Center, Tel-Hashomer, the Rambam Medical Center, Haifa, or the Rivkah Ziv Medical center in Zefat, since January 1, 2000. Participants recruited were diagnosed with either breast or ovarian cancer or [in the minority of cases (n = 12), tested individuals were asymptomatic women from “high risk breast/ovarian cancer families” based on well accepted criteria] [28]. All study participants were unrelated to each other (i.e., only one patient per family was included). The study was approved by the IRB, and each patient signed an informed consent.

DNA extraction

Peripheral blood leukocyte DNA was extracted using the PUREgene kit (Gentra Inc., Minneapolis, MN), using the manufacturer’s recommended protocol.

Analysis for the predominant Jewish mutations in BRCA1 BRCA2 genes

Analysis for the predominant Jewish mutations in BRCA1 (185delAG 5382InsC, Tyr978X) and BRCA2 (6174delT, 8765delAG) was carried out using a PCR directed mutagenesis assay to introduce a restriction site that distinguishes between the wild type and the mutant allele, as previously described [6, 12, 13, 29].

BRCA1 genotyping

BRCA1 genotyping was performed by exon-specific amplification using flanking intronic primers and primers designed to generate slightly overlapping fragments from exon 11, as previously described [30]. No analysis for large genomic rearrangements of BRCA1 was performed.

BRCA2 genotyping

BRCA2 genotyping was performed by exon-specific amplification using flanking intronic primers and primers designed to generate slightly overlapping fragments from exon 11, as previously described [30]. In a subset of participants (some of the non Ashkenazim, the Moslem and Druze participants) BRCA2 mutational analysis was performed using heteroduplex analysis (HDA) using enhanced mismatch mutation analysis (EMMA) technique supplemented by sequencing of abnormally migrating fragments, as previously described [31]. No analysis for large genomic rearrangements of BRCA2 was performed.

Results

Participants’ characteristics

Overall, there were 250 participants in the study: 72 Ashkenazim, 90 non Ashkenazi Jews, 45 Moslem Arabs, 21 non Moslem Arabs, 17 Druze, and 5 non Jewish Caucasians. There were 219 women diagnosed with breast cancer (mean age at diagnosis 43.6 ± 10.85), 19 women diagnosed with ovarian cancer (mean age at diagnosis 57.5 ± 10.7 years). Twelve women were asymptomatic with a significant family history of breast/ovarian cancer, where the affected individual could not be tested. The mean age at genotyping of these women was 45.7 ± 9.5 years. There were 133 first degree relatives with breast cancer and 16 first degree relatives with ovarian cancer. The features of the study participants are shown in Table 1.

Table 1 Characteristics of study participants
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BRCA1 and BRCA2 genotyping results

Overall, 22 individuals carried 19 clearly pathogenic mutations (19/250—6.8% rate for unique mutations detected and 22/250—8.8% for overall detection rate). There were 10 mutations in BRCA1 (including three pathogenic missense mutations) and 9 in BRCA2 (including two pathogenic missense mutations). Three novel mutations were detected (Q1721X in BRCA1, 6855del8 and 9256ins4 in BRCA2), whereas the other pathogenic mutations were previously reported. The rates of mutation carriers by ethnic origin was as follows: 3/72 among Ashkenazim (4.2%), 8/90 among non Ashkenazim (8.9%), 7/45 Moslems (15.5%), 2/17 Druze (11.7%), and 2/5 non Jewish Caucasians (40%). Three single mutations were detected in seemingly unrelated families: A1708E (BRCA1) and C61G (BRCA1) in two non Ashkenazi families, 9256ins4 (BRCA2) in 2 Arab-Moslem families. Additionally, the putatively pathogenic combination of the N550H, F486L, Y179C missense mutations (BRCA1) were detected in two non Ashkenazi families and one Moslem family. The precise mutations, their novelty and predicted effects on protein function, clinical characteristics of the proband and family history of cancer are shown in Table 2 (BRCA1) and Table 3 (BRCA2). Additional sequence alterations and their pathogenicity status, are also shown in the same tables.

Table 2 Pathogenic and missense mutations in BRCA1 by ethnic origin
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Table 3 Pathogenic and missense mutations in BRCA2 by ethnic origin
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Discussion

In the present study, the spectrum of germline mutations in both the BRCA1 and BRCA2 genes in breast/ovarian high risk Israeli women of diverse ethnic origin were determined. The overall rate was 8.8% for all tested individuals. For Ashkenazi Jews it seems apparent that the number of family specific mutations in both genes other than the three predominant mutations is limited. There were only 3 “private” mutations in this ethnic subset of high risk families of Ashkenazi origin, a rate of 4.2%. The most accurate factor in predicting the ability to detect these private mutations in Ashkenazim is the existence of ovarian cancer. Notably, there were no “private” mutations in Ashkenazi families even if there were multiple (up to 6) breast cancer cases diagnosed at an early age in more than one generation. These data are in line with data previously published from the Memorial Sloan Kettering Cancer Center in New York, where private mutations in both genes among Ashkenazim were detected in 3 of 70 high risk Ashkenazi families (4.3%) if there were ovarian cancer cases and 1.4% for breast cancer only cases [10]. Thus, it seems reasonable to recommend full testing of the BRCA1 BRCA2 genes in Ashkenazim to high risk families with at least one case of ovarian cancer, after exclusion of the existence of the founder mutations. Clearly an autosomal dominant mode of transmission is the most likely inheritance pattern in “BRCA mutation negative” families in the present study. Thus, these families should be targeted in the attempts to find novel breast cancer susceptibility genes.

Among non Ashkenazi Jews, there were six pathogenic mutations in BRCA1 (n = 3) and BRCA2 (n = 3) genes with two mutations detected in two families—overall a mutation detection rate of 8.9%. These data on the rate of BRCA1 and BRCA2 mutations in non Ashkenazim are in line with our own published data [29] as well as those of Palma and coworkers [11], but well below the report of Frank and co-workers of ~21.6% among Jewish non carriers of the predominant mutations [32]. Thus, although full mutational analysis of BRCA1 BRCA2 in non Ashkenazi Jews is warranted, clearly other genes underlie the majority of inherited predisposition to breast/ovarian cancer in this subset of individuals.

Of the 19 pathogenic mutations in BRCA1 and BRCA2 described herein, 6 were detected among in 7 individuals of 45 Arab Moslem (Palestinian) women (15.5%) and 2 among 17 Druze individuals (11.7%). This is the most comprehensive analysis published to date on high risk families of these origins. Overall, there are only a handful of germline mutations among Moslem women that were reported world-wide, with a paucity of data about the Palestinian population: an inactivating mutation (E1373X) in BRCA1 [18] another clearly pathogenic mutation (2482delGACT) in BRCA2 [17] and several missense mutations of unknown pathogenic significance in our own previously published series [29].

The data presented in this study, combined with these previous reports among Palestinians, signify that despite theoretical predictions and assumptions that the spectrum of mutations in BRCA1 BRCA2 among Palestinians is limited, the reality is that like most world populations full analysis of both genes is warranted in the appropriate clinical setting. Notably, all but one of the mutations was detected only once. This lack of an apparent founder mutation in both BRCA1 and BRCA2 genes in the Arab (Moslem, Christian, and Druze) population is intriguing. Despite the fact that the Druze sect is a clear example of a genetic isolate, there are no predominant or recurring mutations among high risk families of Druze (or Moslem) origin. Several reasons could account for the low rate of detection of BRCA1 and BRCA2 mutations in these ethnic populations: low threshold of family history at selection, inadequate selection criteria, phenocopies that were analyzed or the reduced rate of BRCA1 BRCA2 carriership predicted in societies where consanguineous marriages have been practiced [33].

The limitations of the present study should be outlined: the methodology for detecting BRCA2 mutations (EMMA) may have missed existing mutations. Notably EMMA has been shown to be a similar sensitivity and provide the same mutation rate and accuracy as DHPLC in a large series (n = 1525) of genotyped individuals [34]. The existence of large genomic rearrangement was not excluded. However, at least among Ashkenazim, the contribution of such genomic events to the overall burden of inherited predisposition to breast cancer is limited [11, 35]. Moreover, the representation of the non Jewish populations may have been suboptimal, as these represent families from three medical centers, which may under represent some sects in the non Jewish populations in Israel.

In conclusion, there are no recurring mutations in BRCA1 and BRCA2 genes among high risk Moslem Israeli, Druze and non Ashkenazi Jews. Thus, in order to evaluate the putative contribution of both genes to inherited predisposition to breast cancer of individuals from these ethnicities, full mutational analysis is warranted. The existence of novel breast cancer genes is supported by the extreme paucity of “private” mutations in BRCA1 BRCA2 among high risk Ashkenazi families.