Introduction

Germline mutations in the BRCA1 (MIM# 113705) and BRCA2 (MIM# 600185) genes can be detected in high risk breast/ovarian cancer 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 1990s [2]. Among Ashkenazi Jews (i.e., Jews of east European ancestry) three mutations in BRCA1 (185delAG and 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 [3–6]. There are only a handful of other, family specific, mutations in Jewish Ashkenazi high risk families [7, 8]. Notably, one of these seemingly private mutations (8075delAT*BRCA2) was reported in more than one Jewish Ashkenazi high risk family [9]. Among the non Ashkenazim (i.e., Jews from diverse ethnicities such as the Balkans, Iraq, Iran, North Africa, and Yemen) there are also a handful of recurring mutations in both genes: 185delAG Tyr978X (BRCA1) and 8765delAG (BRCA2) [10, 11]. Over the past few years, the mutational spectrum of BRCA1 and BRCA2 in high risk non Ashkenazi families has been reported [12] and few mutations have been reported in more than two families: A1708E*BRCA1 IVS2+ 1G > A*BRCA2 in Jews of Balkan origin [13]. In addition, anecdotal cases of additional mutations were noted in the course of genetic testing: 981delAT*BRCA1 (BIC; 1100delAT accession number 1006) in Jews of North African origin, the R2336P*BRCA2 (BIC; R2336P accession number 2111) in families of Balkan and Mediterranean origin.

Since the early 1990s, about one million immigrants came to Israel from the ex Soviet Union. A sizeable portion of these immigrants (estimated at 50%) are of Russian non Jewish origin. Two recurring mutations in BRCA1 in Russian and Baltic region high risk families have been reported: 5382InsC and 4153delA in BRCA1 [14–18]. In addition, the C61G is a mutation frequently detected in non Jewish Polish high risk families [14, 15] and rarely in non Ashkenazi Jews [19].

The aim of the study was to define the rate of these recurring germline mutations in both the BRCA1 BRCA2 genes in high risk Israeli individuals of Ashkenazi, non Ashkenazi, and Russian non Jewish origin.

Patients and methods

Participant identification and recruitment

The study population was recruited from among individuals counseled and tested at one of the two Oncogenetics services located at the Sheba Medical Center, Tel-Hashomer, or the Rivkah Ziv Medical center in Zefat, since January 1, 2000. Participants recruited were diagnosed with either breast or ovarian cancer [in the minority of cases (n = 664)] or tested individuals were asymptomatic women from “high risk breast/ovarian cancer families” based on well accepted criteria [20]. 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), following 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) and BRCA2 (6174delT) was carried out by a PCR directed mutagenesis assay to introduce a restriction site that distinguishes between the wild type and the mutant allele, as previously described [3, 10, 11, 21].

Mutation genotyping

BRCA1 mutation genotyping was performed for the A1708E, Tyr978X the C61G, and the 4153delA using a restriction enzyme digest of PCR amplified fragments targeting the specific mutation bearing DNA region, with subsequent analysis on agarose gels, as previously described [10, 13, 15, 19]. The 981delAT BRCA1 mutation was detected by PCR in the relevant genomic region using the following primers: Forward primer 5′-AGCCAGTTGGTTGATTTCCACCTC-3′ and reverse primer 5′-TTCAGTTACATGGCTTAAGTTGGG-3′. The amplified 867-bp fragment was subsequently digested by the XceI restriction enzyme (Fermentas, Kaunas, Lithuania) and analyzed on an agarose gel. The mutation resulted in a different fragment pattern than the wild type allele. All fragments displaying an abnormal digest pattern were sequenced in an independent PCR. In every experiment, a known mutation was included (= positive control). The 4075delAT, R2336P, and IVS2+ 1G > A mutations were genotyped by direct sequencing of the amplified PCR fragment.

Results

Participants’ characteristics

Overall, there were 4,192 participants (5,270 lab tests) in the study: 827 Ashkenazim, 2,777 non Ashkenazi Jews, and 193 non Jewish Caucasians. The ethnic origin of the non Ashkenazim was as follows: Balkans (i.e., Greece, Turkey, Bulgaria, and ex Yugoslavia) 804; North Africans (Moroccans, Tunisians, Libyans, and Algiers) 847, Asians (Iraqi, Iranian, and Afghans) 892. Of the participants, 2,484 women were diagnosed with breast cancer (mean age at diagnosis 47.2 ± 9.6 years), 324 women diagnosed with ovarian cancer (mean age at diagnosis 56.7 ± 12.2 years). In addition, 664 cancer-free women with a significant family history of breast/ovarian cancer participated, and the affected individual could not be tested. The mean age at genotyping of these women was 44.1 ± 11.4 years. Males (n = 236) were also included in the study, of whom 24 had breast cancer and 35 were diagnosed with pancreatic cancer. The other tested women participants (n = 484) had other types of cancer (mostly pancreas (n = 61), endometrial (n = 112), and gastric cancers (n = 137). The relevant features of the study participants are shown in table 1.

Table 1 Relevant clinical features of the study participants
Full size table

BRCA1 and BRCA2 genotyping results

Overall, 47 of the total of 4,192 genotyped individuals carried one of the eight pathogenic mutations (1.1% rate for mutations detected). The types and rates of mutations by ethnicity are shown in table 2.

Table 2 Mutations detected by ethnicity
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Discussion

In the present study, the rate of some seemingly recurring germline mutations in both the BRCA1 and BRCA2 genes in breast/ovarian high risk Israeli women of diverse ethnic origin were determined. For the mostly Ashkenazi Jewish high risk families, none of the 500 individuals from seemingly independent families was found to harbor the 4075delAG*BRCA2 mutation. These data are consistent with data reported by us [12] and by the Memorial Sloan Kettering Cancer Center in New York [7], where the rate of any “private mutation” in both BRCA1 and BRCA2 in Jewish Ashkenazi high risk families is 4–5% if there are ovarian cancer cases, and less than 2% in families with site specific breast cancer. Thus, it seems that specific genotyping for the 4075delAG*BRCA2 mutation is of no added value as a routine test for Ashkenazi Jewish high risk families.

Among non Ashkenazi Jewish high risk cases, three mutations were detected at rates that may justify adding these mutations to the panel of tested mutations in BRCA1 and BRCA2 in the Israeli population: the Tyr978X mutation was detected in 2.6% of individuals, and was exclusively confined to Jews of Asian (i.e., Iraqi, Iranian, and Afghan) origin. The inability to detect this mutation in a large number of non Ashkenazi high risk families of non Asian origin, confirms the prevailing notion being practiced in oncogenetic clinics in Israel, to offer this test only to Jews of Iraqi, Iranian, and Afghan origin.

In Jewish high risk families of North African origin, there is a paucity of mutations in high risk families: our own data [12] show that none of 27 high risk North African women carried either a BRCA1 or a BRCA2 mutations when full analysis of both genes is applied. Noteworthy, in the present study, 7/264 individuals of North African ancestry carried the same mutation—a rate of 2.65%. The mutational spectrum of BRCA1 and BRCA2 mutations in non Jewish families of North African origin is also limited, and in none of these reports the 981delAT*BRCA1 mutation is noted [22, 23]. Noteworthy, this mutation was reported six times in the BIC database [2], with three of these reported cases is of Italian origin, one of British origin, one of American–German origin, and one from Australia, with no assignment of ethnicity. Based on the Jewish way of life in North Africa and the recurring nature of this mutation in the Jewish North African population, it seems plausible that the 981delAT*BRCA1 mutation is seemingly a founder mutation in North African Jewish families.

The origin of Jews from the Balkan is attributed for the most part to Jews expelled by the inquisition from Spain in 1492 and Portugal in 1497 [24]. However, these Jewish populations also exhibit admixtures with the local communities, as well as some influence of the original Ashkenazim originating from Italy [25, 26]. In that ethnically mixed population, two of the three genotyped mutations (A1708E*BRCA1 and R2336P*BRCA2) were noted at rates of 2.8 and 2.2%, respectively. These mutations were detected in other ethnically diverse populations [9, 27], and therefore in all likelihood do not represent founder Jewish mutations but rather mutations that are prevalent in the geographical region where these Jewish communities resided. Regardless of the origin of these mutations, the rates of these recurring mutations in the present study justify adding at least the A1708E*BRCA1 and R2336P*BRCA2 to the panel of germline mutations routinely tested in Israeli high risk families of the relevant ethnic origin.

In the 1990s, Israel experienced a wave of migration that added about 1,000,000 people to a country of six million. A sizeable portion of these immigrants form the ex Soviet Union are non Jews from Russia and east Europe. For high risk families of this origin, the rates of the predominant mutations in BRCA1 in the present study are 1.1% (C61G) and 0.7% (4153delA). These data are in line with data reported on non Jewish high risk families from Russia [16] Poland [14, 15] and the Baltic republics [17, 18]. Thus, for these unique non Jewish high risk families, these two mutations should be offered in the course of oncogenetics counseling and testing in Israel, prior to offering full mutational analysis of both genes.

This is a study that encompassed high risk families from two medical centers in Israel, and thus the representation of the high risk families and the non Jewish populations may have been suboptimal, as these may under represent some sects in Israel. Hence, the study may have been underpowered to detect some of the mutations, e.g., the 4153delA*BRCA1) due to the small number of tested individuals. We have not formally tested for the “founder origin” of the 981delAT*BRCA1, primarily as a result of testing only one affected individual from a family, thus limiting the ability to accurately phase mutations carriers. So, this attribution-founder mutation should be used cautiously.

In conclusion, there are few recurring mutations in the BRCA1 (981delAT, A1708E, and 4153delA) and BRCA2 (R2336P) genes among high risk non Ashkenazi Jews and non Jewish Caucasians in Israel. These mutations should be added to the panel of germline mutations offered during oncogenetic testing in high risk individuals of the relevant ethnic origin in Israel.