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The occurrence of germline BRCA1 and BRCA2sequence alterations in Slovenian population

The occurrence of germline BRCA1 and BRCA2sequence alterations in Slovenian population Background: The BRCA1 and BRCA2 mutation spectrum and mutation detection rates according to different family histories were investigated in 521 subjects from 322 unrelated Slovenian cancer families with breast and/or ovarian cancer. Methods: The BRCA1 and BRCA2 genes were screened using DGGE, PTT, HRM, MLPA and direct sequencing. Results: Eighteen different mutations were found in BRCA1 and 13 in BRCA2 gene. Mutations in one or other gene were found in 96 unrelated families. The mutation detection rates were the highest in the families with at least one breast and at least one ovarian cancer - 42% for BRCA1 and 8% for BRCA2. The mutation detection rate observed in the families with at least two breast cancers with disease onset before the age of 50 years and no ovarian cancer was 23% for BRCA1 and 13% for BRCA2. The mutation detection rate in the families with at least two breast cancers and only one with the disease onset before the age of 50 years was 11% for BRCA1 and 8% for BRCA2. In the families with at least two breast cancers, all of them with disease onset over the age of 50 years, the detection rate was 5% for BRCA2 and 0% for BRCA1. Conclusion: Among the mutations detected in Slovenian population, 5 mutations in BRCA1 and 4 mutations in BRCA2 have not been described in other populations until now. The most frequent mutations in our population were c.181T > G, c.1687C > T, c.5266dupC and c.844_850dupTCATTAC in BRCA1 gene and c.7806-2A > G, c.5291C > G and c.3978insTGCT in BRCA2 gene (detected in 69% of BRCA1 and BRCA2 positive families). Background cancer in carriers [7-9]. The cumulative breast cancer Breast cancer represents 20.7% of all malignancies in risk for BRCA1 and BRCA2 mutation carriers at the age females and is the most frequent type of cancer in the of 70 was, in different studies, reported to be between Slovenian female population. In 2007, 1147 new breast 36% and up to 71% [10,11]. The studies of the Breast cancer cases were registered in Slovenia. The incidence Cancer Linkage consortium based on the families with rate among Slovenian women was 112/100000 and is four or more cancer cases reported an approximate slowly increasing. Breast and ovarian cancers together breast cancer risk of 80% at the age of 70 for mutation represent as much as 23.9% of all cancers in Slovenian carriers [7,8,12]. The reported cumulative risk for ovar- females [1]. The majority of these two types of cancer ian cancer in BRCA1 and BRCA2 carriers at the age of are sporadic and only a small proportion (5%-10%) is 70 was 39% to 60% and 22% to 27%, respectively known to be caused by dominantly inherited susceptibil- [8,13,14]. Up to now, several comprehensive studies performed ity genes [2]. The BRCA1 and BRCA2 genes were found to be mutated in a large number of families with multi- throughout Europe determined the mutation profiles of ple cases of an early onset of breast and ovarian cancer BRCA1 and BRCA2 in families with history of breast [3-6]. Mutations in BRCA1 and BRCA2 are highly pene- and ovarian cancer. One of the largest studies carried trant and confer an increased risk of breast and ovarian out by a German consortium included 989 unrelated families with a history of breast and ovarian cancer who were screened for mutations in BRCA1 and BRCA2. The * Correspondence: snovakovic@onko-i.si Department of Molecular Diagnostics, Institute of Oncology Ljubljana, frequency of detected mutations in this study varied from Zaloska 2, 1000 Ljubljana, Slovenia 14% to 50% according to different family histories [15]. Full list of author information is available at the end of the article © 2011 Stegel et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Stegel et al. BMC Medical Genetics 2011, 12:9 Page 2 of 11 http://www.biomedcentral.com/1471-2350/12/9 Another large study included 1010 unrelated families from Subgroup BA - families with at least one ovarian the Czech Republic with an overall mutation detection cancer and at least two breast cancers under the age rate of 29% for BRCA1 and BRCA2 [16]. A similar study of 50; was performed in the Dutch population which included Subgroup BC - families with at least one ovarian 517 unrelated families, where an overall mutation detec- cancer and at least two breast cancers, one under tion of 23% was reported [17]. In French, Swedish and the age of 50; Finnish populations, 100 to 200 families were screened Subgroup BD - Families with at least one ovarian for BRCA1 or BRCA2 mutations [18-20]. The mutation cancer and at least two breast cancers, all beyond the age of 50; detection rates in these studies varied from 10 to 25%. A higher mutation detection rate of 33% was observed in Subgroup 1B+1O - Families with only one ovarian the study which included 42 Belgian families [21,22]. cancer and one breast cancer in the family or only A comparably high overall mutation detection rate of one affected individual in the family, but having 30% was reported also among 102 Spanish families [23]. both types of cancer at the same time; In the Austrian population, the mutation detection rate Subgroup more O+1B - Families with at least two was reported to be 20% for BRCA1 among 86 breast and ovarian cancers and only one breast cancer; ovarian cancer families [24]. In Italy, several reports were Subgroup only O - Families with at least two ovarian published. The overall mutation detection rate for cancers and no breast cancer; BRCA1 and BRCA2 in these reports varied from 8 to 37% [25-29]. Group C: Families with two or more cases of breast Slovenia is a country located in the central European cancer among first-degree relatives, including one case area with approximately 2 million inhabitants. Since diagnosed before the age of 50 and no ovarian cancer. 1999, genetic counseling and testing for hereditary breast Group D: Families with two or more cases of breast and ovarian cancer have been offered at the Institute of cancer among first-degree relatives, all diagnosed Oncology Ljubljana. Until now, approximately 1100 indi- beyond the age of 50 and no ovarian cancer. viduals have attended genetic counseling and 521 indivi- Group E: Families with a single case of female breast duals from 322 families opted for genetic testing. cancer diagnosed under the age of 40. With this article, we wanted to present the BRCA1 Group MBR: Families with a single breast cancer and BRCA2 mutation spectrum and mutation detection among first-degree relatives being the male breast rates according to different family histories in Slovenian cancer. the population. Bilateral breast cancers were counted as two indepen- dent cases of breast cancer. Methods Controls: The control group consisted of healthy Tested individuals volunteers, females aged from 50 to 69 years without 521 persons from 322 Slovenian families with breast any familial and personal history of breast and/or ovar- and/or ovarian cancer underwent genetic testing. When ian cancer who were tested for the presence of all muta- possible, the proband tested for the presence of muta- tions, unclassified variants (UV) and polymorphisms tion in BRCA1 and BRCA2 genesinthe family wasthe found in patients. youngest individual affected by breast or ovarian cancer. All tested individuals provided written informed consent Mutation screening and attended genetic counseling sessions before and The DNA was isolated from peripheral blood using the after testing. DNA blood isolation kit (Qiagen, Hilden, Germany). All tested individuals were classified according to their Mutation screening in BRCA1 and BRCA2 genes was family history. The family history data were verified in performed by two laboratories - the Laboratory of Mole- the Slovenian state cancer registry established in 1950. cular Oncology at the Vrije University Brussels, Belgium The enrolled families were divided into 6 different and the Laboratory of Molecular Diagnostics at the groups according to the following criteria: Institute of Oncology Ljubljana, Slovenia. Group A: Families with two or more cases of breast All samples were, at the beginning, tested for six most cancer among first-degree relatives, including at least frequent mutations in the Slovenian population (c.7806- two cases with the disease onset under the age of 50, 2A > G in BRCA2, c.5266dupC, c.1687C > T, c.191G > with no ovarian cancer. A, c.181T > G, c.181T > A in BRCA1) [30,31]. Group B: Families with one or more cases of breast Inthe LaboratoryoftheVrijeUniversityBrussels, and at least one ovarian cancer among first-degree screening for six most frequent mutations was done by relatives. denaturing gradient gel electrophoresis (DGGE) for Stegel et al. BMC Medical Genetics 2011, 12:9 Page 3 of 11 http://www.biomedcentral.com/1471-2350/12/9 mutations c.5266dupC, c.191G >A, c.181T > G, c.181T > rate for BRCA1 and BRCA2 genes was 29.8% (96/322). Ain BRCA1 and c.7806-2A > G in BRCA2 and by protein Thus, in 226 families, we found no pathogenic mutation truncation test (PTT) for mutation c.1687C > T in in either of the two investigated genes. BRCA1. The additional screening of BRCA1/2 was per- formed with PTT (screening of exon 11 in BRCA1 and BRCA1 mutation spectrum exons 10 and 11 in BRCA2) and DGGE (the rest of exons In BRCA1, 18 different deleterious mutations were of BRCA1 and BRCA2) [21]. found in 68 families (Table 1). The four most common IntheLaboratoryoftheInstituteofOncologyLjubl- mutations (c.181T > G, c.1687C > T, c.5266dupC and jana, screening for six most frequent mutations was c.844_850dupTCATTAC) were detected in 44 families done using fluorescent hybridization probes on Light (65%). The additional 14 mutations in BRCA1 appeared Cycler 2.1 for c.5266dupC, c.1687C > T, c.191G > A, in other 24 families. c.181T > G in BRCA1, high-resolution melting - HRM Four of the mentioned 18 mutations are missense on Light Cycler 480 II (Roche Molecular Biochemicals, mutations affecting the 5’RING domain (c.116G > A, Mannheim, Germany) for c.181T > A in BRCA1 and c.181T > G, c.181T > A, c.191G > A), three are nonsense restriction length polymorphism for c.7806-2A > G in mutations (c.1687C > T, c.5251C > T and c.5377A > T), BRCA2 [30,31]. The additional screening of BRCA1/2 seven are frame-shift mutations (c.457_458delAG, was performed with DGGE. c.844_850dupTCATTAC, c.843_846delCTCA, c.2269- In both laboratories, the DGGE was performed using delG, c.3018_3021delTTCA, c.5177_5180delGAAA and primers designed by Ingeny and according to the manu- c.5266dupC) and four are deletions of the whole exons facturer’sinstructions(Ingeny InternationalBV, Goes, (Ex1-2 del, Ex5-7 del, Ex5-8 del and Ex5-10 del). Netherlands). The most common mutation found in BRCA1 gene Samples presenting migration abnormalities were was c.181T > G. It was detected in 15 families. The further sequenced on the automated sequence analyzer patients with ovarian cancer represented 36% of all ABI310 (Applied Biosystems, USA) (using the BDT v.1.1 breast and ovarian cancer cases in the families bearing and performing the labeling PCR reaction) according to the mutation c.181T > G. The average age of the manufacturer’s instructions. The samples were screened patients at the onset of ovarian cancer was 52.1 years for the presence of large deletions and duplications and the average age of the patients at the onset of breast using the multiplex ligation-dependent probe amplifica- cancer was 48.1 years in these families, respectively. tion MLPA kit (MRC Holland, Amsterdam, Nether- Breast cancer was, in these families, in 56% of cases lands). For BRCA1 gene, the MLPA analysis was detected before the age of 50. In five families, colorectal performed with probe set P002 and confirmed by probe cancer, and in two families, cancers of the uterus, were set P087, and for BRCA2 with probe set P045-B1. The also reported in the family history (46% of c.181T > G deletion breakpoints were not characterized at the families have the HNPCC (hereditary non-polyposis sequence level. colon cancer) related cancers in their family history). The description of nucleotide sequence variations is in The second most common mutation in BRCA1 gene accordance with HGVS nomenclature, and in tables, in Slovenian families with breast and/or ovarian cancer also in accordance with BIC nomenclature [32,33]. was c.1687C > T. This mutation was observed in 13 According to HGVS nomenclature DNA variants are families. The patients with ovarian cancer represented numerated according to NCBI reference sequence 32% of all breast and ovarian cancer cases in the NM_007294.2 for BRCA1, NM_000059.3 for BRCA2. families bearing the mutation c.1687C > T. In these The first nucleotide of the start codon ATG is numer- families the average age of the patients at the onset of ated as 1. According to BIC nomenclature, DNA var- ovarian cancer was 52.5 years and the average age of iants are numerated according to NCBI reference the patients at the onset of breast cancer was 46.0. In sequence HSU14680 for mRNA of BRCA1, or U43746 these families, in 53% of cases, breast cancer was for mRNA of BRCA2. The first nucleotide of mRNA is detected before the age of 50. numerated as 1. The mutation c.5266dupC in BRCA1 gene was Statistical analysis was performed using the Chi-square detected in 8 families. The patients with ovarian cancer test. represented just 9% of all breast and ovarian cancer cases in the families bearing the c.5266dupC mutation Results since it was diagnosed in merely 2 cases (onset at 50 The study was performed on 521 individuals from 322 and 48 years, respectively). The average age of the Slovenian breast and/or ovarian cancer families. In 96 patients at the onset of breast cancer in these families was 37.3 years. In more than 89% of cases, breast cancer families, either the mutation in BRCA1 or the mutation was diagnosed before the age of 50. in BRCA2 was detected. The overall mutation detection Stegel et al. BMC Medical Genetics 2011, 12:9 Page 4 of 11 http://www.biomedcentral.com/1471-2350/12/9 Table 1 Mutations in BRCA1 gene in Slovenian population BIC HGVS Protein change Described in BIC Present in other than Slovenian No. of positive nomenclature* nomenclature** according to BIC database (No. of population (No. of families) *** Slovenian database quotations in BIC) families 235G > A c.116G > A C39Y yes (4) 3 300T > G c.181T > G C61G yes (202) Germany (30), Italy (?), Czech rep (20), 15 Austria (3), Slovakia (2) 300T > A c.181T > A C61S no 4 310G > A c.191G > A C64Y yes (20) Germany (1), Czech rep (1), Canada 1 (2) 576delAG c.457_458delAG 157X no 1 963-969 dup c.844_850dupTCATTAC 288X yes (2) Germany (2) 8 (969ins7) 962 del4 c.843_846delCTCA 297X yes (17) Germany (2), Italy (?), Czech rep (1), 2 Austria (2), Slovakia (2), Canada (2- Slavic origin) 1806C > T c.1687C > T Q563X yes (88) Germany (5), Czech rep (6), Austria (2) 13 2388delG c.2269delG 764X yes (7) 1 3137delTTCA c.3018_3021delTTCA 1022X yes (2) 1 5296 del4 c.5177_5180delGAAA 1728X yes (45) 1 (GAAA) 5370C > T c.5251C > T R1751X yes (30) Germany (1), 1 5382insC c.5266dupC 1829X yes (200) Germany (46), Italy (?), Czech rep. (75), 8 Netherlands (5), Austria (2), Slovakia (2) 5496A > T c.5377A > T K1793X no 2 Ex1-2del Ex1-2del no Germany (?),Czech (1) Canada, Spain 1 Ex5-10 del Ex5-10 del no 3 Ex5-8 del Ex5-8 del no 1 Ex5-7 del Ex5-7 del no Spain, Germany 2 Total BRCA1 68 Description of nucleotide variations is in accordance with *BIC nomenclature (DNA variants are numerated according to NCBI reference sequence HSU14680 for mRNA of BRCA1; the first nucleotide of mRNA is numerated as 1) or **HGVS nomenclature (DNA variants are numerated according to NCBI reference sequence NM_007294.2 for BRCA1; the first nucleotide of the start codon ATG is numerated as 1). *** References: Spain [23,44]. Czech Rep [16,45]. Germany [15,46]. Italy [15]. Netherlands [17]. Austria [24]. Slovakia [47]. Canada [48]. The mutation c.844_850dupTCATTAC in BRCA1 mutations or frame-shift mutations causing a truncation gene was noticed in 8 families. The patients with ovar- of the encoded protein. No large deletions were found ian cancer represented 30% of all breast and ovarian in the BRCA2 gene. cancers cases in the families bearing the mutation The c.7806-2A > G mutation was observed in 11 c.844_850dupTCATTAC. The average age of the families. The patients with ovarian cancer (three patients patients at the onset of ovarian cancer was 53.7 years, aged 50, 59 and 70 years at the onset of disease) repre- while the average age of the patients at the onset of sented only 6% of all breast and ovarian cancer cases in breast cancer was 47.5 years. In 69% of cases, breast the families bearing the mutation c.7806-2A > G. The cancer was diagnosed before the age of 50. average age of the patients at the onset of breast cancer was 50.9 years. Breast cancer was in 40% of cases dis- BRCA2 mutation spectrum covered before the age of 50. The analysis of the BRCA2 gene revealed 13 different The second most frequent mutation in BRCA2 gene mutations in 28 families (Table 2). The most common was c.5291C > G. It was detected in four families. The mutation in BRCA2 gene is a splice site mutation average age of the patients at the onset of breast cancer c.7806-2A > G. It was detected in 11 families (which in these families was 45.5 years. In 72% of cases, breast represents 39% of all BRCA2 positive families). The cancer was discovered before the age of 50 years. No remaining 12 BRCA2 mutations were much less fre- ovarian cancers were observed in these families. quent in the Slovenian population (each being discov- The third most common mutation in BRCA2 gene ered in 1 to 4 families) (Table 2). They are nonsense occurring in three families was c.3975_3978dupTGCT. Stegel et al. BMC Medical Genetics 2011, 12:9 Page 5 of 11 http://www.biomedcentral.com/1471-2350/12/9 Table 2 Mutations in BRCA2 gene in Slovenian population BIC HGVS Protein change Described in BIC Present in other than No. of positive nomenclature* nomenclature** according to BIC database (No. of Slovenian population (No. of Slovenian database quotations in BIC) families) *** families 1003A > T**** c.775A > T R259X no 1 1756G > T c.1528G > T E510X yes (1) 1 2041insA c.1813dupA 615X yes (97) Germany (5), Czech rep (2) 1 3493C > T c.3265C > T Q1089X yes (1) 1 4206 ins4 c.3978insTGCT 1330X yes 3 (TGCT) c.3975_3978dupTGCT 4203_4206dupl 5164delGAAA c.4936_4939delGAAA 1668X yes 1 5519C > G c.5291C > G S1764X no 4 5579insA c.5351insA c.5351dupA 1786X yes Netherland (6) 1 5837TC > AG c.5609_5610delTCinsAG F1870X yes (2) 1 6719delAGTT c.6491_6494delAGTT 2166X no 1 7531C > T c.7303C > T Q2435X no 1 IVS16-2A > G c.7806-2A > G aberrant splicing yes (5) Germany (2 ), Italy (2) 11 8064-2A > G 9514C > T c.9286C > T E3096X yes (1) no 1 Total 28 Description of nucleotide variations is in accordance with BIC nomenclature* (DNA variants are numerated according to NCBI reference sequence U43746 for mRNA of BRCA2; the first nucleotide of mRNA is numerated as 1) or HGVS nomenclature** (DNA variants are numerated according to NCBI reference NM_000059.3 for BRCA2; the first nucleotide of the start codon ATG is numerated as 1). ***References: Germany [15]. Italy [15]. Czech Rep [16,45]. Netherland [17]. ****Albanian origin - Kosovo. The mutation c.3975_3978dupTGCT is located in the familial breast and/or ovarian cancer history was also “ovarian cluster region” of BRCA2 gene [34,35]. The tested for the presence of all known mutations, UVs patients with ovarian cancer(two patientsaged40and and polymorphisms in BRCA1 and BRCA2 genes in 52 years at the onset of the disease) represented 29% of Slovenian population. The results are given in the all breast and ovarian cancer cases in these families. The Tables 2 and 3. No mutations were detected in the average age of the patients at the onset of breast cancer control group. All frequent polymorphisms observed in was 52.2 years. In 66% of cases, breast cancer was individuals with breast and ovarian cancer family his- detected before the age of 50 years. tory, with the allele frequency of more than 2%, were detected also in the control group with similar allele Polymorphisms and unclassified sequence variants in frequencies (Table 3). BRCA1 and BRCA2 genes All sequence variants which were not classified as muta- Mutation detection rates tions were categorized either as polymorphisms or as The mutation detection rates in different groups and unclassified variants (UVs) according to the BIC database subgroups defined according to the familial cancer his- (Breast cancer information core) (Tables 3, 4 and 5). When tory are presented in Tables 6 and 7. the sequence variant was not reported in the BIC database, To estimate the influence of number of breast cancer we classified it as a UV. Polymorphisms detected in indivi- cases in the family on the mutation detection rate duals with breast and ovarian cancer family history and among the families with only breast cancer history, we those found in the control group are listed in Table 3, subdivided Groups A, C and D according to the number while unclassified variants are given in Tables 4 and 5. of breast cancer cases in the family. The proportions of Unclassified variants were more common in BRCA2 (20 families with 2, 3 or 4 breast cancers were relatively different UVs) than in BRCA1 (12 different UV) (Tables 4 similar in all three groups (Table 8). and 5). Most UVs are missense mutations, yet two UVs in The frequency at which BRCA1 and BRCA2 gene muta- the BRCA2 gene cause frame-shift of the open reading frame. tions were detected in the families belonging to Group B was statistically significantly higher when compared to Controls Groups A, C, D, E and MBR (p-values: 0.034, 0.021, 0.008, The DNA of 40 healthy Slovenian women aged 0.006, 0.003, respectively). Also the frequency of mutations between 50 and 69 years without any personal and seen in Group A was statistically significantly different Stegel et al. BMC Medical Genetics 2011, 12:9 Page 6 of 11 http://www.biomedcentral.com/1471-2350/12/9 Table 3 Polymorphisms in BRCA1 and BRCA2 genes in Slovenian population BIC HGVS Protein Allele No. of alleles/all No. of alleles/all alleles of Described Clinical importance nomenclature* nomenclature** change alleles of tested tested healthy individuals in BIC entered in BIC probands (allele (allele frequencies) database database (allele frequencies) frequencies) BRCA1 710 C > T c.591C > T C197C T 1/380 (0.003) 0/80 (0.000) yes no (0.02) 1186A > G c.1067A > G Q356R G 25/380 (0.065) 6/80 (0.075) yes no (0.06) 2196G > A c.2077G > A D693N A 6/208 (0.029) 4/80 (0.050) yes no (0.08) 2201C > T c.2082C > T S694S T 66/208 (0.32) 25/80 (0.31) yes no (0.31) 2430T > C c.2311T > C L771L C 66/208 (0.32) 25/80 (0.31) yes no (0.31) 2731C > T c.2612C > T P871L T 66/208 (0.32) 25/80 (0.31) yes no (0.34) 3232A > G c.3113A > G E1038G G 66/208 (0.32) 25/80 (0.31) yes no (0.31) 3667A > G c.3548A > G K1183G G 66/208 (0.32) 25/80 (0.31) yes no (0.31) 4427T > C c.4308T > C F1436S C 66/208 (0.32) 25/80 (0.31) yes no (0.31) 4956A > G c.4837A > G S1613G G 66/208 (0.32) 25/80 (0.31) yes no (0.31) BRCA2 203G > A c.1-25G > A 5’-UTR A 66/208 (0.32) 25/80 (0.31) yes no (0.25) 1093A > C c.865A > C N289H C 18/380 (0.047) 3/80 (0.038) yes no (?) 1342C > A c.1114C > A H372N A 160/208 (0.77) 56/80 (0.70) yes no (0.72) 1379C > T c.1151C > T S384F T 1/208 (0.005) 0/80 (0.000) yes no (?) 1593A > G c.1365A > G S455S G 7/208 (0.034) 3/80 (0.038) yes no (0.01) 2020A > G c.1792A > G T598A G 4/208 (0.019) 0/80 (0.000) yes no (?) 2457T > C c.2229T > C H743H C 18/380 (0.047) 5/80 (0.063) yes no (?) 3199A > G c.2971A > G N991D G 7/208 (0.034) 3/80 (0.038) yes no (?) 3624A > G c.3396A > G L1132L G 73/208 (0.35) 38/80 (0.48) yes no (0.31) 4035T > C c.3807T > C V1269V C 27/208 (0.130) 18/80 (0.225) yes no (0.19) 4486G > T c.4258G > T D1420Y T 3/208 (0.014) 0/80 (0.000) yes no (?) 5427C > T c.5199C > T S1733S T 4/208 (0.019) 4/80 (0.050) yes no (?) 7470A > G c.7242A > G S2414S G 57/208 (0.27) 21/80 (0.26) yes no (0.21) 8034-14C > T c.7806-14C > T intron T 95/208 (0.46) 33/80 (0.41) yes no (0.50) 8410G > A c.8182G > A V2728I A 1/208 (0.005) 1/80 (0.013) yes no (?) 10323delCins11 10095delCins11 3369X del 3/380 (0.008) 0/80 (0.000) yes no (?) Description of nucleotide variations is in accordance with BIC nomenclature* (DNA variants are numerated according to NCBI reference sequence HSU14680 for mRNA of BRCA1,or U43746 for mRNA of BRCA2; the first nucleotide of mRNA is numerated as 1) or HGVS nomenclature** (DNA variants are numerated according to NCBI reference sequence NM_007294.2 for BRCA1, NM_000059.3 for BRCA2; the first nucleotide of the start codon ATG is numerated as 1). UTR - untranslated region. (?)-data unknown. from those observed in Groups C, D, E and MBR, respec- rate in our series was 21.1% for BRCA1 and 8.7% for tively (p-values < 0.001 in all four cases) (Table 6). BRCA2; for both genes, it was 29.8%. In general, the mutation detection rates for BRCA1 Discussion and BRCA2 genes in the members of Slovenian families Genetic counseling and testing of individuals from with breast and/or ovarian cancer are comparable with families with an increased risk of breast and/or ovarian the mutation frequencies in these two genes reported cancer in Slovenia has been available at the Institute of for other countries [15,17,25]. When comparing the Oncology Ljubljana since 1999. From then and until study groups, the highest mutation detection rate in Slo- January 2009, we screened 322 families and we detected venian population was observed in the families with at ahighlypenetrant BRCA1 and BRCA2 mutation in least one breast and at least one ovarian cancer (Group B) 96 of them. In this study, we are summarizing the most - 42% for BRCA1 and 8% for BRCA2 or 50% for both significant features of hereditary breast and ovarian can- genes (Table 6). Similar mutation detection rates for cer in Slovenian families. The overall mutation detection families with the same characteristics were reported in Stegel et al. BMC Medical Genetics 2011, 12:9 Page 7 of 11 http://www.biomedcentral.com/1471-2350/12/9 Table 4 Unclassified sequence variants in BRCA1 gene in Slovenian population BIC HGVS Protein Allele No. of alleles/all alleles No. of alleles/all alleles of Described Clinical nomenclature* nomenclature** change of tested probands tested healthy individuals in BIC importance (allele frequencies) (allele frequencies) database entered in BIC database Missense changes 212C > G c.93C > G I31M G 3/380 (0.008) 0/80 (0.000) yes unknown 462C > A c.343C > A P115S A 1/208 (0.005) 0/80 (0.000) no 1112G > C c.993G > C R331S C 2/208 (0.010) 0/80 (0.000) yes unknown 3238G > A c.3119G > A S1040N A 5/208 (0.024) 0/80 (0.000) yes unknown 3421G > A c.3302G > A S1101N A 1/208 (0.005) 0/80 (0.000) yes unknown 3573G > A c.3454G > A D1152N A 1/208 (0.005) 0/80 (0.000) yes unknown 3768T > C c.3649T > C S1217P C 1/208 (0.005) 0/80 (0.000) no 4158A > G c.4039A > G R1347G G 2/208 (0.010) 0/80 (0.000) yes unknown 4384G > A c.4265G > A G1422E A 1/208 (0.005) 0/80 (0.000) no 4675A > G c.4556A > G N1519S G 1/208 (0.005) 0/80 (0.000) no 5075G > A c.4956G > A M1652I A 3/208 (0.014) 0/80 (0.000) yes unknown 5124G > T c.5005G > T A1669S T 1/208 (0.005) 0/80 (0.000) yes unknown Description of nucleotide variations is in accordance with BIC nomenclature* (DNA variants are numerated according to NCBI reference sequence HSU14680 for mRNA of BRCA1; the first nucleotide of mRNA is numerated as 1) or HGVS nomenclature** (DNA variants are numerated according to NCBI reference sequence NM_007294.2 for BRCA1; the first nucleotide of the start codon ATG is numerated as 1). UV- unclassified variant. other populations - 53% in the German population, 43.7% hereditary ovarian cancers [36]. It is well known that in the Italian population, 43% in the Spanish population, hereditary ovarian cancer can be associated also with Lynch syndrome and with mutations in mismatch repair and 52% in the Dutch population [15,17,23,25]. genes [35]. However, with the additional subdividing of groups in our study, even higher mutation detection rate (of 85%) If we gather all families with at least two breast can- was observed in the subgroup of families with two or cers in their family history regardless of the age at the more ovarian cancers and only one breast cancer (Sub- disease onset and no ovarian cancer, the mutation group - more O+1B). On the other hand, the proportion detection rate in the Slovenian population is 24%. For of detected mutations in BRCA1 and BRCA2 genes fell comparison, the mutation detection rate for BRCA1 and to 36% when we investigated the subgroup of families BRCA2 in similar groups of families in the Dutch, with at least two ovarian cancers and no breast cancer German, Italian and Spanish population is 13%, 24%, in their family history (Subgroup - only O)(Table7). 18.5% and 15%, respectively [15,17,23,25]. In the families Similar findings were perceived by Ramus et al. In their with two or more breast cancer cases and at least two of studies, the frequency of BRCA1 and BRCA2 mutations them diagnosed under the age of 50 and no ovarian in the families with site-specific ovarian cancer was cancer (Group A), the mutation detection rate for lower than in the families with breast and ovarian can- BRCA1 is 23% and for BRCA2 is 13% or 36% for both cer. The families with three or more ovarian cancers genes in the Slovenian population, 27% in the Dutch and at least one breast cancer had mutations in BRCA1 population and 37% in the German population [15,17]. and BRCA2 in 81% of cases (similar to our results) In the group of families with no ovarian cancer but at while in the families with three or more ovarian cancers least two or more breast cancer cases with only one of and no breast cancer mutations in BRCA1 or BRCA2 them diagnosed before the age of 50 (Group C), the were detected in only 63% (which is still much higher mutation detection rate for BRCA1 was 11% and for than in our study) [34,35]. In the Dutch population, the BRCA2 8% or 19% for both genes. These detection rates mutation detection rate in the families with at least two are slightly higher than the detection rates reported by the German study group (7% and 3% for BRCA1 and ovarian cancers and one or more breast cancers was 82%, whereas in the families with only ovarian cancer BRCA2, respectively) [15]. In the Dutch population, the history, it was 36%, which is quite similar to our detec- mutation detection rate for both BRCA1 and BRCA2 in tion rates [17]. this group of families was 15% [17]. This lower mutation detection rate in the families with When considering our families with at least two breast ovarian cancers only indicates the existence of other cancers and all of them diagnosed after age of 50 and no (different from BRCA1/2) predisposing genes in ovarian cancer (Group D), the mutation detection rate Stegel et al. BMC Medical Genetics 2011, 12:9 Page 8 of 11 http://www.biomedcentral.com/1471-2350/12/9 Table 5 Unclassified sequence variants in BRCA2 gene in Slovenian population BIC HGVS Protein Allele No. of alleles/all alleles No. of alleles/all alleles of Described Clinical nomenclature* nomenclature** change of tested probands tested healthy individuals in BIC importance (allele frequencies) (allele frequencies) database entered in BIC database Missense changes 2032G > A c.1804G > A G602R A 1/208 (0.005) 0/80 (0.000) yes unknown 3747C > T c.3519C > T S1152L T 1/208 (0.005) 0/80 (0.000) no 5078G > A c.4850G > A S1617N A 1/208 (0.005) 0/80 (0.000) no 5972C > T c.5744C > T T1915M T 14/208 (0,067) 3/80 (0.038) yes unknown 8107A > T c.7879A > T I2627F T 1/380 (0.003) 0/80 (0.000) yes unknown 8482A > T c.8254A > T I2752F T 1/208 (0.005) 0/80 (0.000) yes unknown 8579G > A c.8351G > A R2784Q A 2/380 (0.005) 0/80 (0.000) yes unknown 9133G > A c.8905G > A V2969M A 2/380 (0.005) 0/80 (0.000) yes unknown 9599A > T c.9371A > T N3124I AT 1/380 (0.003) 0/80 (0.000) yes unknown 10462A > G 10234A > G I3412V G 1/208 (0.005) 0/80 (0.000) yes unknown Frameshift changes 4091delTAA c.3863delTAA del288N del 2/208 (0.010) 0/80 (0.000) no 9512dupl12 c.9284_9295dup12 dup 1/380 (0.003) 0/80 (0.000) no Intron changes IVS2-7A > T c.68-7A > T intron T 1/208 (0.005) 1/80 (0.013) yes unknown Synonymous changes 1395G > A c.1167G > A P389P A 1/208 (0.005) 0/80 (0.000) no 1977G > A c.1749G > A L583L A 1/208 (0.005) 0/80 (0.000) no 4296G > A c.4068G > A L1356L A 3/208 (0.014) 0/80 (0.000) yes unknown 5013G > A c.4785G > A Q1595Q A 1/208 (0.005) 0/80 (0.000) no 5985G > A c.5757G > A L1919L G 0/208 (0.000) 1/80 (0.013) no 10338G > A 10110G > A R3370R A 2/380 (0.005) 0/80 (0.000) yes unknown 10431G > A 10203G > A T3401T A 1/208 (0.005) 0/80 (0.000) no Description of nucleotide variations is in accordance with BIC nomenclature* (DNA variants are numerated according to NCBI reference sequence U43746 for mRNA of BRCA2.; the first nucleotide of mRNA is numerated as 1) or HGVS nomenclature** (DNA variants are numerated according to NCBI reference sequence NM_000059.3 for BRCA2; the first nucleotide of the start codon ATG is numerated as 1). was 5% (only one mutation discovered in BRCA2). In the influence the BRCA1/2 mutation detection rate (Table 8). Dutch population, the reported mutation detection rate A rather low mutation detection rate in the group with in similar group of families was 6% and for the German breast cancers diagnosed at the age over 50 years sug- population 10% [15,17]. Since the mutation detection gests that other potential predisposing genes confer an rate in the families with the same number of breast can- increased risk of postmenopausal breast cancer [37]. cer cases is higher in the group with earlier disease onset The families with a single male breast cancer case and (Group A) than in the groups with later disease onset no other breast/ovarian cancers among first degree rela- (Groups C and D), the age at the disease onset seems to tives (Group MBR) had the mutation detection rate of Table 6 Incidence of BRCA1 or BRCA2 mutations among different groups of Slovenian families defined according to family history Group A B C D E MBR No. of mutation positive families (%)* BRCA1 positive 18/77 (23%) 42/100 (42%) 8/75 (11%) 0/21 (0%) 2/24(8%) 0/25 (0%) BRCA2 positive 10/77 (13%) 8/100 (8%) 6/75 (8%) 1/21 (5%) 0/24(0%) 1/25 (4%) *number of positive families/number of all tested families in the group (the proportion of positive families). Group A - families with two or more cases of breast cancer including at least two cases with the disease onset under the age of 50 and with no ovarian cancer. Group B - families with one or more cases of breast and at least one ovarian cancer. Group C - families with two or more cases of breast cancer including one case diagnosed before the age of 50 and with no ovarian cancer. Group D - families with two or more cases of breast cancer all of them diagnosed beyond the age of 50 and with no ovarian cancer. Group E - families with a single case of breast cancer diagnosed at the age under 40 years. Group MBR - families with a single male breast cancer. Bilateral breast cancers were considered as two separate cases of cancer. Stegel et al. BMC Medical Genetics 2011, 12:9 Page 9 of 11 http://www.biomedcentral.com/1471-2350/12/9 Table 7 Incidence of BRCA1 or BRCA2 mutations among different sub-groups of Slovenian families with breast and ovarian cancer Sub-group BA BC BD 1B+1O MoreO+1B Only O No. of mutation positive families (%)* BRCA1 positive 10/19 (53%) 14/21 (67%) 2/7 (29%) 8/32 (25%) 6/7 (86%) 4/14 (29%) BRCA2 positive 1/19 (5%) 2/21 (10%) 2/7 (29%) 3/32 (9%) 0/7 (0%) 1/14 (7%) *number of positive families/number of all tested families in the group (the proportion of positive families). Group B (families with one or more cases of breast and at least one ovarian cancer) was further subdivided into sub-groups: BA - families with at least one ovarian cancer and at least two breast cancers diagnosed under the age of 50; BC - families with at least one ovarian cancer and at least two breast cancers, one of them diagnosed under the age of 50; BD - families with at least one ovarian cancer and at least two breast cancers all of them diagnosed beyond the age of 50; 1B+1O - families with only one ovarian cancer and one breast cancer in the family or only one affected individual in the family, but having both types of cancer at the same time; more O+1B - families with at least two ovarian cancers and only one breast cancer; only O - families with at least two ovarian cancers and no breast cancer. 4% for BRCA2 and 0% for BRCA1. Yet, when taken described also in other European populations (Table 1), together, all families with male breast cancer in their but not the mutation c.844_850dupTCATTAC. This family history had the mutation detection rate of 11.5% mutation has been previously described only twice in (3 positive families from 26 tested families) [38]. The the German population, while it was found in as many mutations detected in male breast cancer were c.7806-2A as 8 families in the Slovenian population. Another rare > G and c.3975_3978dupTGCT in BRCA2 gene. In com- mutation in BRCA1 - the mutation c.181T > A has also parison, the mutation detection rates in the families with been previously described only in the Slovenian popula- tion [39]. male breast cancer in the German population were 25%, In BRCA2, the most frequent mutations were c.7806- (12 positive cases from 47 tested families) and as high as 42% in Italian (3 positive cases from 7 tested families) 2A > G, c.5291C > G and c.3975_3978dupTGCT. The and in the Dutch population (5 positive cases from 12 mutation c.7806-2A > G has been already reported by tested families), which is much higher than in our popu- our group [31]. It is a Slovenian founder mutation, lation [15,17,25]. In this place, it must definitely be reported before also by others [15,40,41]. The mutation stressed that, in our study, not all samples of male breast c.5291C > G, however, according to BIC database, has cancer have been fully screened up till now and that only not been described in any other populations, other then tests for the most frequent mutations in the Slovenian the Slovenian one. population have been performed. This might be one of Different mutations in BRCA1 and BRCA2 genes have the possible reasons for a lower mutation detection rate a different penetrance for breast and for ovarian cancer in this group of families in our population. [35]. This has been observed also in our population. Nevertheless, a low detection rate has also been per- The families affected by one mutation actually differ ceived in another group of families with a single case of from the families affected by another mutation by female breast cancer (Group E) where the mutation means of the proportion of ovarian cancers among all detection rates for BRCA1 and for BRCA2 were 8% and breast and ovarian cancers in the family. These differ- 0%, respectively. In the German population, the detec- ences can be due to the variations in the biology of the tion rates in this group are reported to be 8% in BRCA1 translated protein having a different effect on the nor- and 4% in BRCA2 [15]. mal breast or ovarian tissue. The difference could also Themajorityofcommonmutations in BRCA1 be associated with other than BRCA1 and BRCA2 com- (c.181T > G, c.1687C > T, c.5266dupC) observed in mon moderate/low penetrancegenes inthepopulation [35,42,43]. the Slovenian population have been frequently Table 8 Incidence of BRCA1 or BRCA2 mutations among Slovenian families with only breast cancer history (no ovarian cancer) subdivided according to the number of breast cancer cases in the family Group 2 breast cancers in family 3 breast cancers in family 4 breast cancers in family more then 5 breast cancers in family history* history* history* history* A 9/29 (31%) 5/21 (24%) 7/16 (43%) 7/11 (64%) C 5/37 (13%) 3/25 (12%) 4/9 (44%) 2/4 (50%) D 1/10 (10%) 0/6 (0%) 0/5 (0%) 0/0 *number of positive families/number of all tested families in the group (the proportion of positive families). Group A - families with two or more cases of breast cancer including at least two of them diagnosed under the age of 50 and with no ovarian cancer. Group C - families with two or more cases of breast cancer including one case diagnosed before the age of 50 and with no ovarian cancer. Group D - families with two or more cases of breast cancer all of them diagnosed beyond the age of 50 years and with no ovarian cancer. Stegel et al. BMC Medical Genetics 2011, 12:9 Page 10 of 11 http://www.biomedcentral.com/1471-2350/12/9 2. Claus EB, Risch N, Thompson WD: Genetic analysis of breast cancer in the Conclusion cancer and steroid hormone study. Am J Hum Genet 1991, 48(2):232-42. With the introduction of the genetic councelling and 3. 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As well, the mutation detection rate Eccles DM, Tang N, Olah E, Anton-Culver H, Warner E, Lubinski J, might further be influenced in the future - it might Gronwald J, Gorski B, Tulinius H, Thorlacius S, Eerola H, Nevanlinna H, Syrjäkoski K, Kallioniemi OP, Thompson D, Evans C, Peto J, Lalloo F, namely increase when the character of some of the Evans DG, Easton DF: Average risks of breast and ovarian cancer unclassified variants will be clarified as pathogenic. associated with BRCA1 or BRCA2 mutations detected in case Series unselected for family history: a combined analysis of 22 studies. Am J Hum Genet 2003, 72(5):1117-30. Acknowledgements 11. King MC, Marks JH, Mandell JB, New York Breast Cancer Study Group: The authors gratefully thank the genetic counselors and nurses as well as Breast and ovarian cancer risks due to inherited mutations in BRCA1 laboratory technicians who helped to recruit patients, collect their clinical and BRCA2. Science 2003, 302(5645):643-6. data and perform the analyses, Nikola Bešič, Aleš Vakselj, Cvetka Bilban- 12. Narod SA, Ford D, Devilee P, Barkardottir RB, Lynch HT, Smith SA, Jakopin, Kristijana Hertl, Alenka Vrečar, Katarina Lokar, and Simona Traven. Ponder BA, Weber BL, Garber JE, Birch JM, Cornelis RS, Kelsell DP, Spurr NK, Authors also thank Barbara Jezeršek-Novaković for reviewing the article. Smyth E, Haites N, Sobol H, Bignon YJ, Chang-Claude J, Hamann U, Lindblom A, Borg AM, Steven Piver MS, Gallion HH, Struewing JP, Author details Whittemore A, Tonin P, Goldgar DE, Easton DF: An evaluation of genetic Department of Molecular Diagnostics, Institute of Oncology Ljubljana, heterogeneity in 145 breast-ovarian cancer families. Am J Hum Genet Zaloska 2, 1000 Ljubljana, Slovenia. Unit of Genetic Counseling, Institute of 1995, 56(1):254-64, Breast Cancer Linkage Consortium. Oncology Ljubljana, Zaloska 2, 1000 Ljubljana, Slovenia. Department of 13. Chen S, Iversen ES, Friebel T, Finkelstein D, Weber BL, Eisen A, Peterson LE, Surgical Oncology, Institute of Oncology Ljubljana, Zaloska 2, 1000 Ljubljana, Schildkraut JM, Isaacs C, Peshkin BN, Corio C, Leondaridis L, Tomlinson G, Slovenia. Laboratory of Molecular Oncology, Oncologisch Centrum UZ Dutson D, Kerber R, Amos CI, Strong LC, Berry DA, Euhus DM, Parmigiani G: Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium. Characterization of BRCA1 and BRCA2 mutations in a large United States sample. J Clin Oncol 2006, 24(6):863-71. Authors’ contributions 14. Ford D, Easton DF, Stratton M, Narod S, Goldgar D, Devilee P, Bishop DT, VS, MK, JŽ, MH, ET, JG and SN designed the study, collected and analyzed Weber B, Lenoir G, Chang-Claude J, Sobol H, Teare MD, Struewing J, the data and wrote the paper. 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Breast Screpanti I, Gulino A: Novel BRCA1 and BRCA2 germline mutations and Cancer Res Treat 2010, 121(1):147-56. assessment of mutation spectrum and prevalence in Italian breast and/ 44. Palanca Suela S, Esteban Cardeñosa E, Barragán González E, Oltra Soler S, de or ovarian cancer families. Breast Cancer Res Treat 2006, 100(1):83-91. Juan Jiménez I, Chirivella González I, Segura Huerta A, Guillén Ponce C, 26. Capalbo C, Ricevuto E, Vestri A, Ristori E, Sidoni T, Buffone O, Adamo B, Martínez de Dueñas E, Bolufer Gilabert P: Identification of a novel BRCA1 Cortesi E, Marchetti P, Scambia G, Tomao S, Rinaldi C, Zani M, Ferraro S, large genomic rearrangement in a Spanish breast/ovarian cancer family. Frati L, Screpanti I, Gulino A, Giannini G: BRCA1 and BRCA2 genetic testing Breast Cancer Res Treat 2008, 112(1):63-7. in Italian breast and/or ovarian cancer families: mutation spectrum and 45. Vasickova P, Machackova E, Lukesova M, Damborsky J, Horky O, Pavlu H, prevalence and analysis of mutation prediction models. Ann Oncol 2006, Kuklova J, Kosinova V, Navratilova M, Foretova L: High occurrence of 17(Suppl 7):34-40, vii. BRCA1 intragenic rearrangements in hereditary breast and ovarian 27. Ottini L, Rizzolo P, Zanna I, Falchetti M, Masala G, Ceccarelli K, Vezzosi V, cancer syndrome in the Czech Republic. BMC Med Genet 2007, 11:8:32. Gulino A, Giannini G, Bianchi S, Sera F, Palli D: BRCA1/BRCA2 mutation 46. Hartmann C, John AL, Klaes R, Hofmann W, Bielen R, Koehler R, Janssen B, status and clinical-pathologic features of 108 male breast cancer cases Bartram CR, Arnold N, Zschocke J: Large BRCA1 gene deletions are found from Tuscany: a population-based study in central Italy. Breast Cancer Res in 3% of German high-risk breast cancer families. Hum Mutat 2004, Treat 2009, 116(3):577-86. 24(6):534. 28. Stuppia L, Di Fulvio P, Aceto G, Pintor S, Veschi S, Gatta V, Colosimo A, 47. Konecny M, Vizvaryova M, Weismanova E, Ilencikova D, Mlkva I, Cianchetti E, Cama A, Mariani-Costantini R, Battista P, Palka G: BRCA1 and Weismann P, Machackova G, Kausitz J: The spectrum and incidence of BRCA2 mutations in breast/ovarian cancer patients from central Italy. BRCA1 pathogenic mutations in Slovak breast/ovarian cancer families. Hum Mutat 2003, 22(2):178-9. Neoplasma 2007, 54(2):137-42. 29. Ottini L, D’Amico C, Noviello C, Lauro S, Lalle M, Fornarini G, Colantuoni OA, 48. Risch HA, McLaughlin JR, Cole DE, Rosen B, Bradley L, Fan I, Tang J, Li S, Pizzi C, Cortesi E, Carlini S, Guadagni F, Bianco AR, Frati L, Contegiacomo A, Zhang S, Shaw PA, Narod SA: Population BRCA1 and BRCA2 mutation Mariani-Costantini R: BRCA1 and BRCA2 mutations in central and frequencies and cancer penetrances: a kin-cohort study in Ontario, southern Italian patients. Breast Cancer Res 2000, 2(4):307-10. Canada. J Natl Cancer Inst 2006, 98(23):1694-706. 30. Novaković S, Stegel V: Rapid detection of most frequent Slovenian germ- line mutations in BRCA1 gene using real-time PCR and melting curve Pre-publication history analysis. Radiol Oncol 2005, 39:147-152. The pre-publication history for this paper can be accessed here: 31. Krajc M, De Grève J, Goelen G, Teugels E: BRCA2 founder mutation in http://www.biomedcentral.com/1471-2350/12/9/prepub Slovenian breast cancer families. Eur J Hum Genet 2002, 10(12):879-82. doi:10.1186/1471-2350-12-9 32. Human Genome Variation Society internet web site: [http://www.hgvs.org/ Cite this article as: Stegel et al.: The occurrence of germline BRCA1 and mutnomen/]. BRCA2 sequence alterations in Slovenian population. BMC Medical 33. Breast Cancer Information Core internet web site: [http://research.nhgri.nih. Genetics 2011 12:9. gov/bic/]. 34. Ramus SJ, Harrington PA, Pye C, DiCioccio RA, Cox MJ, Garlinghouse- Jones K, Oakley-Girvan I, Jacobs IJ, Hardy RM, Whittemore AS, Ponder BA, http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Medical Genetics Springer Journals

The occurrence of germline BRCA1 and BRCA2sequence alterations in Slovenian population

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Springer Journals
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Copyright © 2011 by Stegel et al; licensee BioMed Central Ltd.
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Biomedicine; Human Genetics; Cytogenetics; Gene Function
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1471-2350
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10.1186/1471-2350-12-9
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21232165
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Abstract

Background: The BRCA1 and BRCA2 mutation spectrum and mutation detection rates according to different family histories were investigated in 521 subjects from 322 unrelated Slovenian cancer families with breast and/or ovarian cancer. Methods: The BRCA1 and BRCA2 genes were screened using DGGE, PTT, HRM, MLPA and direct sequencing. Results: Eighteen different mutations were found in BRCA1 and 13 in BRCA2 gene. Mutations in one or other gene were found in 96 unrelated families. The mutation detection rates were the highest in the families with at least one breast and at least one ovarian cancer - 42% for BRCA1 and 8% for BRCA2. The mutation detection rate observed in the families with at least two breast cancers with disease onset before the age of 50 years and no ovarian cancer was 23% for BRCA1 and 13% for BRCA2. The mutation detection rate in the families with at least two breast cancers and only one with the disease onset before the age of 50 years was 11% for BRCA1 and 8% for BRCA2. In the families with at least two breast cancers, all of them with disease onset over the age of 50 years, the detection rate was 5% for BRCA2 and 0% for BRCA1. Conclusion: Among the mutations detected in Slovenian population, 5 mutations in BRCA1 and 4 mutations in BRCA2 have not been described in other populations until now. The most frequent mutations in our population were c.181T > G, c.1687C > T, c.5266dupC and c.844_850dupTCATTAC in BRCA1 gene and c.7806-2A > G, c.5291C > G and c.3978insTGCT in BRCA2 gene (detected in 69% of BRCA1 and BRCA2 positive families). Background cancer in carriers [7-9]. The cumulative breast cancer Breast cancer represents 20.7% of all malignancies in risk for BRCA1 and BRCA2 mutation carriers at the age females and is the most frequent type of cancer in the of 70 was, in different studies, reported to be between Slovenian female population. In 2007, 1147 new breast 36% and up to 71% [10,11]. The studies of the Breast cancer cases were registered in Slovenia. The incidence Cancer Linkage consortium based on the families with rate among Slovenian women was 112/100000 and is four or more cancer cases reported an approximate slowly increasing. Breast and ovarian cancers together breast cancer risk of 80% at the age of 70 for mutation represent as much as 23.9% of all cancers in Slovenian carriers [7,8,12]. The reported cumulative risk for ovar- females [1]. The majority of these two types of cancer ian cancer in BRCA1 and BRCA2 carriers at the age of are sporadic and only a small proportion (5%-10%) is 70 was 39% to 60% and 22% to 27%, respectively known to be caused by dominantly inherited susceptibil- [8,13,14]. Up to now, several comprehensive studies performed ity genes [2]. The BRCA1 and BRCA2 genes were found to be mutated in a large number of families with multi- throughout Europe determined the mutation profiles of ple cases of an early onset of breast and ovarian cancer BRCA1 and BRCA2 in families with history of breast [3-6]. Mutations in BRCA1 and BRCA2 are highly pene- and ovarian cancer. One of the largest studies carried trant and confer an increased risk of breast and ovarian out by a German consortium included 989 unrelated families with a history of breast and ovarian cancer who were screened for mutations in BRCA1 and BRCA2. The * Correspondence: snovakovic@onko-i.si Department of Molecular Diagnostics, Institute of Oncology Ljubljana, frequency of detected mutations in this study varied from Zaloska 2, 1000 Ljubljana, Slovenia 14% to 50% according to different family histories [15]. Full list of author information is available at the end of the article © 2011 Stegel et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Stegel et al. BMC Medical Genetics 2011, 12:9 Page 2 of 11 http://www.biomedcentral.com/1471-2350/12/9 Another large study included 1010 unrelated families from Subgroup BA - families with at least one ovarian the Czech Republic with an overall mutation detection cancer and at least two breast cancers under the age rate of 29% for BRCA1 and BRCA2 [16]. A similar study of 50; was performed in the Dutch population which included Subgroup BC - families with at least one ovarian 517 unrelated families, where an overall mutation detec- cancer and at least two breast cancers, one under tion of 23% was reported [17]. In French, Swedish and the age of 50; Finnish populations, 100 to 200 families were screened Subgroup BD - Families with at least one ovarian for BRCA1 or BRCA2 mutations [18-20]. The mutation cancer and at least two breast cancers, all beyond the age of 50; detection rates in these studies varied from 10 to 25%. A higher mutation detection rate of 33% was observed in Subgroup 1B+1O - Families with only one ovarian the study which included 42 Belgian families [21,22]. cancer and one breast cancer in the family or only A comparably high overall mutation detection rate of one affected individual in the family, but having 30% was reported also among 102 Spanish families [23]. both types of cancer at the same time; In the Austrian population, the mutation detection rate Subgroup more O+1B - Families with at least two was reported to be 20% for BRCA1 among 86 breast and ovarian cancers and only one breast cancer; ovarian cancer families [24]. In Italy, several reports were Subgroup only O - Families with at least two ovarian published. The overall mutation detection rate for cancers and no breast cancer; BRCA1 and BRCA2 in these reports varied from 8 to 37% [25-29]. Group C: Families with two or more cases of breast Slovenia is a country located in the central European cancer among first-degree relatives, including one case area with approximately 2 million inhabitants. Since diagnosed before the age of 50 and no ovarian cancer. 1999, genetic counseling and testing for hereditary breast Group D: Families with two or more cases of breast and ovarian cancer have been offered at the Institute of cancer among first-degree relatives, all diagnosed Oncology Ljubljana. Until now, approximately 1100 indi- beyond the age of 50 and no ovarian cancer. viduals have attended genetic counseling and 521 indivi- Group E: Families with a single case of female breast duals from 322 families opted for genetic testing. cancer diagnosed under the age of 40. With this article, we wanted to present the BRCA1 Group MBR: Families with a single breast cancer and BRCA2 mutation spectrum and mutation detection among first-degree relatives being the male breast rates according to different family histories in Slovenian cancer. the population. Bilateral breast cancers were counted as two indepen- dent cases of breast cancer. Methods Controls: The control group consisted of healthy Tested individuals volunteers, females aged from 50 to 69 years without 521 persons from 322 Slovenian families with breast any familial and personal history of breast and/or ovar- and/or ovarian cancer underwent genetic testing. When ian cancer who were tested for the presence of all muta- possible, the proband tested for the presence of muta- tions, unclassified variants (UV) and polymorphisms tion in BRCA1 and BRCA2 genesinthe family wasthe found in patients. youngest individual affected by breast or ovarian cancer. All tested individuals provided written informed consent Mutation screening and attended genetic counseling sessions before and The DNA was isolated from peripheral blood using the after testing. DNA blood isolation kit (Qiagen, Hilden, Germany). All tested individuals were classified according to their Mutation screening in BRCA1 and BRCA2 genes was family history. The family history data were verified in performed by two laboratories - the Laboratory of Mole- the Slovenian state cancer registry established in 1950. cular Oncology at the Vrije University Brussels, Belgium The enrolled families were divided into 6 different and the Laboratory of Molecular Diagnostics at the groups according to the following criteria: Institute of Oncology Ljubljana, Slovenia. Group A: Families with two or more cases of breast All samples were, at the beginning, tested for six most cancer among first-degree relatives, including at least frequent mutations in the Slovenian population (c.7806- two cases with the disease onset under the age of 50, 2A > G in BRCA2, c.5266dupC, c.1687C > T, c.191G > with no ovarian cancer. A, c.181T > G, c.181T > A in BRCA1) [30,31]. Group B: Families with one or more cases of breast Inthe LaboratoryoftheVrijeUniversityBrussels, and at least one ovarian cancer among first-degree screening for six most frequent mutations was done by relatives. denaturing gradient gel electrophoresis (DGGE) for Stegel et al. BMC Medical Genetics 2011, 12:9 Page 3 of 11 http://www.biomedcentral.com/1471-2350/12/9 mutations c.5266dupC, c.191G >A, c.181T > G, c.181T > rate for BRCA1 and BRCA2 genes was 29.8% (96/322). Ain BRCA1 and c.7806-2A > G in BRCA2 and by protein Thus, in 226 families, we found no pathogenic mutation truncation test (PTT) for mutation c.1687C > T in in either of the two investigated genes. BRCA1. The additional screening of BRCA1/2 was per- formed with PTT (screening of exon 11 in BRCA1 and BRCA1 mutation spectrum exons 10 and 11 in BRCA2) and DGGE (the rest of exons In BRCA1, 18 different deleterious mutations were of BRCA1 and BRCA2) [21]. found in 68 families (Table 1). The four most common IntheLaboratoryoftheInstituteofOncologyLjubl- mutations (c.181T > G, c.1687C > T, c.5266dupC and jana, screening for six most frequent mutations was c.844_850dupTCATTAC) were detected in 44 families done using fluorescent hybridization probes on Light (65%). The additional 14 mutations in BRCA1 appeared Cycler 2.1 for c.5266dupC, c.1687C > T, c.191G > A, in other 24 families. c.181T > G in BRCA1, high-resolution melting - HRM Four of the mentioned 18 mutations are missense on Light Cycler 480 II (Roche Molecular Biochemicals, mutations affecting the 5’RING domain (c.116G > A, Mannheim, Germany) for c.181T > A in BRCA1 and c.181T > G, c.181T > A, c.191G > A), three are nonsense restriction length polymorphism for c.7806-2A > G in mutations (c.1687C > T, c.5251C > T and c.5377A > T), BRCA2 [30,31]. The additional screening of BRCA1/2 seven are frame-shift mutations (c.457_458delAG, was performed with DGGE. c.844_850dupTCATTAC, c.843_846delCTCA, c.2269- In both laboratories, the DGGE was performed using delG, c.3018_3021delTTCA, c.5177_5180delGAAA and primers designed by Ingeny and according to the manu- c.5266dupC) and four are deletions of the whole exons facturer’sinstructions(Ingeny InternationalBV, Goes, (Ex1-2 del, Ex5-7 del, Ex5-8 del and Ex5-10 del). Netherlands). The most common mutation found in BRCA1 gene Samples presenting migration abnormalities were was c.181T > G. It was detected in 15 families. The further sequenced on the automated sequence analyzer patients with ovarian cancer represented 36% of all ABI310 (Applied Biosystems, USA) (using the BDT v.1.1 breast and ovarian cancer cases in the families bearing and performing the labeling PCR reaction) according to the mutation c.181T > G. The average age of the manufacturer’s instructions. The samples were screened patients at the onset of ovarian cancer was 52.1 years for the presence of large deletions and duplications and the average age of the patients at the onset of breast using the multiplex ligation-dependent probe amplifica- cancer was 48.1 years in these families, respectively. tion MLPA kit (MRC Holland, Amsterdam, Nether- Breast cancer was, in these families, in 56% of cases lands). For BRCA1 gene, the MLPA analysis was detected before the age of 50. In five families, colorectal performed with probe set P002 and confirmed by probe cancer, and in two families, cancers of the uterus, were set P087, and for BRCA2 with probe set P045-B1. The also reported in the family history (46% of c.181T > G deletion breakpoints were not characterized at the families have the HNPCC (hereditary non-polyposis sequence level. colon cancer) related cancers in their family history). The description of nucleotide sequence variations is in The second most common mutation in BRCA1 gene accordance with HGVS nomenclature, and in tables, in Slovenian families with breast and/or ovarian cancer also in accordance with BIC nomenclature [32,33]. was c.1687C > T. This mutation was observed in 13 According to HGVS nomenclature DNA variants are families. The patients with ovarian cancer represented numerated according to NCBI reference sequence 32% of all breast and ovarian cancer cases in the NM_007294.2 for BRCA1, NM_000059.3 for BRCA2. families bearing the mutation c.1687C > T. In these The first nucleotide of the start codon ATG is numer- families the average age of the patients at the onset of ated as 1. According to BIC nomenclature, DNA var- ovarian cancer was 52.5 years and the average age of iants are numerated according to NCBI reference the patients at the onset of breast cancer was 46.0. In sequence HSU14680 for mRNA of BRCA1, or U43746 these families, in 53% of cases, breast cancer was for mRNA of BRCA2. The first nucleotide of mRNA is detected before the age of 50. numerated as 1. The mutation c.5266dupC in BRCA1 gene was Statistical analysis was performed using the Chi-square detected in 8 families. The patients with ovarian cancer test. represented just 9% of all breast and ovarian cancer cases in the families bearing the c.5266dupC mutation Results since it was diagnosed in merely 2 cases (onset at 50 The study was performed on 521 individuals from 322 and 48 years, respectively). The average age of the Slovenian breast and/or ovarian cancer families. In 96 patients at the onset of breast cancer in these families was 37.3 years. In more than 89% of cases, breast cancer families, either the mutation in BRCA1 or the mutation was diagnosed before the age of 50. in BRCA2 was detected. The overall mutation detection Stegel et al. BMC Medical Genetics 2011, 12:9 Page 4 of 11 http://www.biomedcentral.com/1471-2350/12/9 Table 1 Mutations in BRCA1 gene in Slovenian population BIC HGVS Protein change Described in BIC Present in other than Slovenian No. of positive nomenclature* nomenclature** according to BIC database (No. of population (No. of families) *** Slovenian database quotations in BIC) families 235G > A c.116G > A C39Y yes (4) 3 300T > G c.181T > G C61G yes (202) Germany (30), Italy (?), Czech rep (20), 15 Austria (3), Slovakia (2) 300T > A c.181T > A C61S no 4 310G > A c.191G > A C64Y yes (20) Germany (1), Czech rep (1), Canada 1 (2) 576delAG c.457_458delAG 157X no 1 963-969 dup c.844_850dupTCATTAC 288X yes (2) Germany (2) 8 (969ins7) 962 del4 c.843_846delCTCA 297X yes (17) Germany (2), Italy (?), Czech rep (1), 2 Austria (2), Slovakia (2), Canada (2- Slavic origin) 1806C > T c.1687C > T Q563X yes (88) Germany (5), Czech rep (6), Austria (2) 13 2388delG c.2269delG 764X yes (7) 1 3137delTTCA c.3018_3021delTTCA 1022X yes (2) 1 5296 del4 c.5177_5180delGAAA 1728X yes (45) 1 (GAAA) 5370C > T c.5251C > T R1751X yes (30) Germany (1), 1 5382insC c.5266dupC 1829X yes (200) Germany (46), Italy (?), Czech rep. (75), 8 Netherlands (5), Austria (2), Slovakia (2) 5496A > T c.5377A > T K1793X no 2 Ex1-2del Ex1-2del no Germany (?),Czech (1) Canada, Spain 1 Ex5-10 del Ex5-10 del no 3 Ex5-8 del Ex5-8 del no 1 Ex5-7 del Ex5-7 del no Spain, Germany 2 Total BRCA1 68 Description of nucleotide variations is in accordance with *BIC nomenclature (DNA variants are numerated according to NCBI reference sequence HSU14680 for mRNA of BRCA1; the first nucleotide of mRNA is numerated as 1) or **HGVS nomenclature (DNA variants are numerated according to NCBI reference sequence NM_007294.2 for BRCA1; the first nucleotide of the start codon ATG is numerated as 1). *** References: Spain [23,44]. Czech Rep [16,45]. Germany [15,46]. Italy [15]. Netherlands [17]. Austria [24]. Slovakia [47]. Canada [48]. The mutation c.844_850dupTCATTAC in BRCA1 mutations or frame-shift mutations causing a truncation gene was noticed in 8 families. The patients with ovar- of the encoded protein. No large deletions were found ian cancer represented 30% of all breast and ovarian in the BRCA2 gene. cancers cases in the families bearing the mutation The c.7806-2A > G mutation was observed in 11 c.844_850dupTCATTAC. The average age of the families. The patients with ovarian cancer (three patients patients at the onset of ovarian cancer was 53.7 years, aged 50, 59 and 70 years at the onset of disease) repre- while the average age of the patients at the onset of sented only 6% of all breast and ovarian cancer cases in breast cancer was 47.5 years. In 69% of cases, breast the families bearing the mutation c.7806-2A > G. The cancer was diagnosed before the age of 50. average age of the patients at the onset of breast cancer was 50.9 years. Breast cancer was in 40% of cases dis- BRCA2 mutation spectrum covered before the age of 50. The analysis of the BRCA2 gene revealed 13 different The second most frequent mutation in BRCA2 gene mutations in 28 families (Table 2). The most common was c.5291C > G. It was detected in four families. The mutation in BRCA2 gene is a splice site mutation average age of the patients at the onset of breast cancer c.7806-2A > G. It was detected in 11 families (which in these families was 45.5 years. In 72% of cases, breast represents 39% of all BRCA2 positive families). The cancer was discovered before the age of 50 years. No remaining 12 BRCA2 mutations were much less fre- ovarian cancers were observed in these families. quent in the Slovenian population (each being discov- The third most common mutation in BRCA2 gene ered in 1 to 4 families) (Table 2). They are nonsense occurring in three families was c.3975_3978dupTGCT. Stegel et al. BMC Medical Genetics 2011, 12:9 Page 5 of 11 http://www.biomedcentral.com/1471-2350/12/9 Table 2 Mutations in BRCA2 gene in Slovenian population BIC HGVS Protein change Described in BIC Present in other than No. of positive nomenclature* nomenclature** according to BIC database (No. of Slovenian population (No. of Slovenian database quotations in BIC) families) *** families 1003A > T**** c.775A > T R259X no 1 1756G > T c.1528G > T E510X yes (1) 1 2041insA c.1813dupA 615X yes (97) Germany (5), Czech rep (2) 1 3493C > T c.3265C > T Q1089X yes (1) 1 4206 ins4 c.3978insTGCT 1330X yes 3 (TGCT) c.3975_3978dupTGCT 4203_4206dupl 5164delGAAA c.4936_4939delGAAA 1668X yes 1 5519C > G c.5291C > G S1764X no 4 5579insA c.5351insA c.5351dupA 1786X yes Netherland (6) 1 5837TC > AG c.5609_5610delTCinsAG F1870X yes (2) 1 6719delAGTT c.6491_6494delAGTT 2166X no 1 7531C > T c.7303C > T Q2435X no 1 IVS16-2A > G c.7806-2A > G aberrant splicing yes (5) Germany (2 ), Italy (2) 11 8064-2A > G 9514C > T c.9286C > T E3096X yes (1) no 1 Total 28 Description of nucleotide variations is in accordance with BIC nomenclature* (DNA variants are numerated according to NCBI reference sequence U43746 for mRNA of BRCA2; the first nucleotide of mRNA is numerated as 1) or HGVS nomenclature** (DNA variants are numerated according to NCBI reference NM_000059.3 for BRCA2; the first nucleotide of the start codon ATG is numerated as 1). ***References: Germany [15]. Italy [15]. Czech Rep [16,45]. Netherland [17]. ****Albanian origin - Kosovo. The mutation c.3975_3978dupTGCT is located in the familial breast and/or ovarian cancer history was also “ovarian cluster region” of BRCA2 gene [34,35]. The tested for the presence of all known mutations, UVs patients with ovarian cancer(two patientsaged40and and polymorphisms in BRCA1 and BRCA2 genes in 52 years at the onset of the disease) represented 29% of Slovenian population. The results are given in the all breast and ovarian cancer cases in these families. The Tables 2 and 3. No mutations were detected in the average age of the patients at the onset of breast cancer control group. All frequent polymorphisms observed in was 52.2 years. In 66% of cases, breast cancer was individuals with breast and ovarian cancer family his- detected before the age of 50 years. tory, with the allele frequency of more than 2%, were detected also in the control group with similar allele Polymorphisms and unclassified sequence variants in frequencies (Table 3). BRCA1 and BRCA2 genes All sequence variants which were not classified as muta- Mutation detection rates tions were categorized either as polymorphisms or as The mutation detection rates in different groups and unclassified variants (UVs) according to the BIC database subgroups defined according to the familial cancer his- (Breast cancer information core) (Tables 3, 4 and 5). When tory are presented in Tables 6 and 7. the sequence variant was not reported in the BIC database, To estimate the influence of number of breast cancer we classified it as a UV. Polymorphisms detected in indivi- cases in the family on the mutation detection rate duals with breast and ovarian cancer family history and among the families with only breast cancer history, we those found in the control group are listed in Table 3, subdivided Groups A, C and D according to the number while unclassified variants are given in Tables 4 and 5. of breast cancer cases in the family. The proportions of Unclassified variants were more common in BRCA2 (20 families with 2, 3 or 4 breast cancers were relatively different UVs) than in BRCA1 (12 different UV) (Tables 4 similar in all three groups (Table 8). and 5). Most UVs are missense mutations, yet two UVs in The frequency at which BRCA1 and BRCA2 gene muta- the BRCA2 gene cause frame-shift of the open reading frame. tions were detected in the families belonging to Group B was statistically significantly higher when compared to Controls Groups A, C, D, E and MBR (p-values: 0.034, 0.021, 0.008, The DNA of 40 healthy Slovenian women aged 0.006, 0.003, respectively). Also the frequency of mutations between 50 and 69 years without any personal and seen in Group A was statistically significantly different Stegel et al. BMC Medical Genetics 2011, 12:9 Page 6 of 11 http://www.biomedcentral.com/1471-2350/12/9 Table 3 Polymorphisms in BRCA1 and BRCA2 genes in Slovenian population BIC HGVS Protein Allele No. of alleles/all No. of alleles/all alleles of Described Clinical importance nomenclature* nomenclature** change alleles of tested tested healthy individuals in BIC entered in BIC probands (allele (allele frequencies) database database (allele frequencies) frequencies) BRCA1 710 C > T c.591C > T C197C T 1/380 (0.003) 0/80 (0.000) yes no (0.02) 1186A > G c.1067A > G Q356R G 25/380 (0.065) 6/80 (0.075) yes no (0.06) 2196G > A c.2077G > A D693N A 6/208 (0.029) 4/80 (0.050) yes no (0.08) 2201C > T c.2082C > T S694S T 66/208 (0.32) 25/80 (0.31) yes no (0.31) 2430T > C c.2311T > C L771L C 66/208 (0.32) 25/80 (0.31) yes no (0.31) 2731C > T c.2612C > T P871L T 66/208 (0.32) 25/80 (0.31) yes no (0.34) 3232A > G c.3113A > G E1038G G 66/208 (0.32) 25/80 (0.31) yes no (0.31) 3667A > G c.3548A > G K1183G G 66/208 (0.32) 25/80 (0.31) yes no (0.31) 4427T > C c.4308T > C F1436S C 66/208 (0.32) 25/80 (0.31) yes no (0.31) 4956A > G c.4837A > G S1613G G 66/208 (0.32) 25/80 (0.31) yes no (0.31) BRCA2 203G > A c.1-25G > A 5’-UTR A 66/208 (0.32) 25/80 (0.31) yes no (0.25) 1093A > C c.865A > C N289H C 18/380 (0.047) 3/80 (0.038) yes no (?) 1342C > A c.1114C > A H372N A 160/208 (0.77) 56/80 (0.70) yes no (0.72) 1379C > T c.1151C > T S384F T 1/208 (0.005) 0/80 (0.000) yes no (?) 1593A > G c.1365A > G S455S G 7/208 (0.034) 3/80 (0.038) yes no (0.01) 2020A > G c.1792A > G T598A G 4/208 (0.019) 0/80 (0.000) yes no (?) 2457T > C c.2229T > C H743H C 18/380 (0.047) 5/80 (0.063) yes no (?) 3199A > G c.2971A > G N991D G 7/208 (0.034) 3/80 (0.038) yes no (?) 3624A > G c.3396A > G L1132L G 73/208 (0.35) 38/80 (0.48) yes no (0.31) 4035T > C c.3807T > C V1269V C 27/208 (0.130) 18/80 (0.225) yes no (0.19) 4486G > T c.4258G > T D1420Y T 3/208 (0.014) 0/80 (0.000) yes no (?) 5427C > T c.5199C > T S1733S T 4/208 (0.019) 4/80 (0.050) yes no (?) 7470A > G c.7242A > G S2414S G 57/208 (0.27) 21/80 (0.26) yes no (0.21) 8034-14C > T c.7806-14C > T intron T 95/208 (0.46) 33/80 (0.41) yes no (0.50) 8410G > A c.8182G > A V2728I A 1/208 (0.005) 1/80 (0.013) yes no (?) 10323delCins11 10095delCins11 3369X del 3/380 (0.008) 0/80 (0.000) yes no (?) Description of nucleotide variations is in accordance with BIC nomenclature* (DNA variants are numerated according to NCBI reference sequence HSU14680 for mRNA of BRCA1,or U43746 for mRNA of BRCA2; the first nucleotide of mRNA is numerated as 1) or HGVS nomenclature** (DNA variants are numerated according to NCBI reference sequence NM_007294.2 for BRCA1, NM_000059.3 for BRCA2; the first nucleotide of the start codon ATG is numerated as 1). UTR - untranslated region. (?)-data unknown. from those observed in Groups C, D, E and MBR, respec- rate in our series was 21.1% for BRCA1 and 8.7% for tively (p-values < 0.001 in all four cases) (Table 6). BRCA2; for both genes, it was 29.8%. In general, the mutation detection rates for BRCA1 Discussion and BRCA2 genes in the members of Slovenian families Genetic counseling and testing of individuals from with breast and/or ovarian cancer are comparable with families with an increased risk of breast and/or ovarian the mutation frequencies in these two genes reported cancer in Slovenia has been available at the Institute of for other countries [15,17,25]. When comparing the Oncology Ljubljana since 1999. From then and until study groups, the highest mutation detection rate in Slo- January 2009, we screened 322 families and we detected venian population was observed in the families with at ahighlypenetrant BRCA1 and BRCA2 mutation in least one breast and at least one ovarian cancer (Group B) 96 of them. In this study, we are summarizing the most - 42% for BRCA1 and 8% for BRCA2 or 50% for both significant features of hereditary breast and ovarian can- genes (Table 6). Similar mutation detection rates for cer in Slovenian families. The overall mutation detection families with the same characteristics were reported in Stegel et al. BMC Medical Genetics 2011, 12:9 Page 7 of 11 http://www.biomedcentral.com/1471-2350/12/9 Table 4 Unclassified sequence variants in BRCA1 gene in Slovenian population BIC HGVS Protein Allele No. of alleles/all alleles No. of alleles/all alleles of Described Clinical nomenclature* nomenclature** change of tested probands tested healthy individuals in BIC importance (allele frequencies) (allele frequencies) database entered in BIC database Missense changes 212C > G c.93C > G I31M G 3/380 (0.008) 0/80 (0.000) yes unknown 462C > A c.343C > A P115S A 1/208 (0.005) 0/80 (0.000) no 1112G > C c.993G > C R331S C 2/208 (0.010) 0/80 (0.000) yes unknown 3238G > A c.3119G > A S1040N A 5/208 (0.024) 0/80 (0.000) yes unknown 3421G > A c.3302G > A S1101N A 1/208 (0.005) 0/80 (0.000) yes unknown 3573G > A c.3454G > A D1152N A 1/208 (0.005) 0/80 (0.000) yes unknown 3768T > C c.3649T > C S1217P C 1/208 (0.005) 0/80 (0.000) no 4158A > G c.4039A > G R1347G G 2/208 (0.010) 0/80 (0.000) yes unknown 4384G > A c.4265G > A G1422E A 1/208 (0.005) 0/80 (0.000) no 4675A > G c.4556A > G N1519S G 1/208 (0.005) 0/80 (0.000) no 5075G > A c.4956G > A M1652I A 3/208 (0.014) 0/80 (0.000) yes unknown 5124G > T c.5005G > T A1669S T 1/208 (0.005) 0/80 (0.000) yes unknown Description of nucleotide variations is in accordance with BIC nomenclature* (DNA variants are numerated according to NCBI reference sequence HSU14680 for mRNA of BRCA1; the first nucleotide of mRNA is numerated as 1) or HGVS nomenclature** (DNA variants are numerated according to NCBI reference sequence NM_007294.2 for BRCA1; the first nucleotide of the start codon ATG is numerated as 1). UV- unclassified variant. other populations - 53% in the German population, 43.7% hereditary ovarian cancers [36]. It is well known that in the Italian population, 43% in the Spanish population, hereditary ovarian cancer can be associated also with Lynch syndrome and with mutations in mismatch repair and 52% in the Dutch population [15,17,23,25]. genes [35]. However, with the additional subdividing of groups in our study, even higher mutation detection rate (of 85%) If we gather all families with at least two breast can- was observed in the subgroup of families with two or cers in their family history regardless of the age at the more ovarian cancers and only one breast cancer (Sub- disease onset and no ovarian cancer, the mutation group - more O+1B). On the other hand, the proportion detection rate in the Slovenian population is 24%. For of detected mutations in BRCA1 and BRCA2 genes fell comparison, the mutation detection rate for BRCA1 and to 36% when we investigated the subgroup of families BRCA2 in similar groups of families in the Dutch, with at least two ovarian cancers and no breast cancer German, Italian and Spanish population is 13%, 24%, in their family history (Subgroup - only O)(Table7). 18.5% and 15%, respectively [15,17,23,25]. In the families Similar findings were perceived by Ramus et al. In their with two or more breast cancer cases and at least two of studies, the frequency of BRCA1 and BRCA2 mutations them diagnosed under the age of 50 and no ovarian in the families with site-specific ovarian cancer was cancer (Group A), the mutation detection rate for lower than in the families with breast and ovarian can- BRCA1 is 23% and for BRCA2 is 13% or 36% for both cer. The families with three or more ovarian cancers genes in the Slovenian population, 27% in the Dutch and at least one breast cancer had mutations in BRCA1 population and 37% in the German population [15,17]. and BRCA2 in 81% of cases (similar to our results) In the group of families with no ovarian cancer but at while in the families with three or more ovarian cancers least two or more breast cancer cases with only one of and no breast cancer mutations in BRCA1 or BRCA2 them diagnosed before the age of 50 (Group C), the were detected in only 63% (which is still much higher mutation detection rate for BRCA1 was 11% and for than in our study) [34,35]. In the Dutch population, the BRCA2 8% or 19% for both genes. These detection rates mutation detection rate in the families with at least two are slightly higher than the detection rates reported by the German study group (7% and 3% for BRCA1 and ovarian cancers and one or more breast cancers was 82%, whereas in the families with only ovarian cancer BRCA2, respectively) [15]. In the Dutch population, the history, it was 36%, which is quite similar to our detec- mutation detection rate for both BRCA1 and BRCA2 in tion rates [17]. this group of families was 15% [17]. This lower mutation detection rate in the families with When considering our families with at least two breast ovarian cancers only indicates the existence of other cancers and all of them diagnosed after age of 50 and no (different from BRCA1/2) predisposing genes in ovarian cancer (Group D), the mutation detection rate Stegel et al. BMC Medical Genetics 2011, 12:9 Page 8 of 11 http://www.biomedcentral.com/1471-2350/12/9 Table 5 Unclassified sequence variants in BRCA2 gene in Slovenian population BIC HGVS Protein Allele No. of alleles/all alleles No. of alleles/all alleles of Described Clinical nomenclature* nomenclature** change of tested probands tested healthy individuals in BIC importance (allele frequencies) (allele frequencies) database entered in BIC database Missense changes 2032G > A c.1804G > A G602R A 1/208 (0.005) 0/80 (0.000) yes unknown 3747C > T c.3519C > T S1152L T 1/208 (0.005) 0/80 (0.000) no 5078G > A c.4850G > A S1617N A 1/208 (0.005) 0/80 (0.000) no 5972C > T c.5744C > T T1915M T 14/208 (0,067) 3/80 (0.038) yes unknown 8107A > T c.7879A > T I2627F T 1/380 (0.003) 0/80 (0.000) yes unknown 8482A > T c.8254A > T I2752F T 1/208 (0.005) 0/80 (0.000) yes unknown 8579G > A c.8351G > A R2784Q A 2/380 (0.005) 0/80 (0.000) yes unknown 9133G > A c.8905G > A V2969M A 2/380 (0.005) 0/80 (0.000) yes unknown 9599A > T c.9371A > T N3124I AT 1/380 (0.003) 0/80 (0.000) yes unknown 10462A > G 10234A > G I3412V G 1/208 (0.005) 0/80 (0.000) yes unknown Frameshift changes 4091delTAA c.3863delTAA del288N del 2/208 (0.010) 0/80 (0.000) no 9512dupl12 c.9284_9295dup12 dup 1/380 (0.003) 0/80 (0.000) no Intron changes IVS2-7A > T c.68-7A > T intron T 1/208 (0.005) 1/80 (0.013) yes unknown Synonymous changes 1395G > A c.1167G > A P389P A 1/208 (0.005) 0/80 (0.000) no 1977G > A c.1749G > A L583L A 1/208 (0.005) 0/80 (0.000) no 4296G > A c.4068G > A L1356L A 3/208 (0.014) 0/80 (0.000) yes unknown 5013G > A c.4785G > A Q1595Q A 1/208 (0.005) 0/80 (0.000) no 5985G > A c.5757G > A L1919L G 0/208 (0.000) 1/80 (0.013) no 10338G > A 10110G > A R3370R A 2/380 (0.005) 0/80 (0.000) yes unknown 10431G > A 10203G > A T3401T A 1/208 (0.005) 0/80 (0.000) no Description of nucleotide variations is in accordance with BIC nomenclature* (DNA variants are numerated according to NCBI reference sequence U43746 for mRNA of BRCA2.; the first nucleotide of mRNA is numerated as 1) or HGVS nomenclature** (DNA variants are numerated according to NCBI reference sequence NM_000059.3 for BRCA2; the first nucleotide of the start codon ATG is numerated as 1). was 5% (only one mutation discovered in BRCA2). In the influence the BRCA1/2 mutation detection rate (Table 8). Dutch population, the reported mutation detection rate A rather low mutation detection rate in the group with in similar group of families was 6% and for the German breast cancers diagnosed at the age over 50 years sug- population 10% [15,17]. Since the mutation detection gests that other potential predisposing genes confer an rate in the families with the same number of breast can- increased risk of postmenopausal breast cancer [37]. cer cases is higher in the group with earlier disease onset The families with a single male breast cancer case and (Group A) than in the groups with later disease onset no other breast/ovarian cancers among first degree rela- (Groups C and D), the age at the disease onset seems to tives (Group MBR) had the mutation detection rate of Table 6 Incidence of BRCA1 or BRCA2 mutations among different groups of Slovenian families defined according to family history Group A B C D E MBR No. of mutation positive families (%)* BRCA1 positive 18/77 (23%) 42/100 (42%) 8/75 (11%) 0/21 (0%) 2/24(8%) 0/25 (0%) BRCA2 positive 10/77 (13%) 8/100 (8%) 6/75 (8%) 1/21 (5%) 0/24(0%) 1/25 (4%) *number of positive families/number of all tested families in the group (the proportion of positive families). Group A - families with two or more cases of breast cancer including at least two cases with the disease onset under the age of 50 and with no ovarian cancer. Group B - families with one or more cases of breast and at least one ovarian cancer. Group C - families with two or more cases of breast cancer including one case diagnosed before the age of 50 and with no ovarian cancer. Group D - families with two or more cases of breast cancer all of them diagnosed beyond the age of 50 and with no ovarian cancer. Group E - families with a single case of breast cancer diagnosed at the age under 40 years. Group MBR - families with a single male breast cancer. Bilateral breast cancers were considered as two separate cases of cancer. Stegel et al. BMC Medical Genetics 2011, 12:9 Page 9 of 11 http://www.biomedcentral.com/1471-2350/12/9 Table 7 Incidence of BRCA1 or BRCA2 mutations among different sub-groups of Slovenian families with breast and ovarian cancer Sub-group BA BC BD 1B+1O MoreO+1B Only O No. of mutation positive families (%)* BRCA1 positive 10/19 (53%) 14/21 (67%) 2/7 (29%) 8/32 (25%) 6/7 (86%) 4/14 (29%) BRCA2 positive 1/19 (5%) 2/21 (10%) 2/7 (29%) 3/32 (9%) 0/7 (0%) 1/14 (7%) *number of positive families/number of all tested families in the group (the proportion of positive families). Group B (families with one or more cases of breast and at least one ovarian cancer) was further subdivided into sub-groups: BA - families with at least one ovarian cancer and at least two breast cancers diagnosed under the age of 50; BC - families with at least one ovarian cancer and at least two breast cancers, one of them diagnosed under the age of 50; BD - families with at least one ovarian cancer and at least two breast cancers all of them diagnosed beyond the age of 50; 1B+1O - families with only one ovarian cancer and one breast cancer in the family or only one affected individual in the family, but having both types of cancer at the same time; more O+1B - families with at least two ovarian cancers and only one breast cancer; only O - families with at least two ovarian cancers and no breast cancer. 4% for BRCA2 and 0% for BRCA1. Yet, when taken described also in other European populations (Table 1), together, all families with male breast cancer in their but not the mutation c.844_850dupTCATTAC. This family history had the mutation detection rate of 11.5% mutation has been previously described only twice in (3 positive families from 26 tested families) [38]. The the German population, while it was found in as many mutations detected in male breast cancer were c.7806-2A as 8 families in the Slovenian population. Another rare > G and c.3975_3978dupTGCT in BRCA2 gene. In com- mutation in BRCA1 - the mutation c.181T > A has also parison, the mutation detection rates in the families with been previously described only in the Slovenian popula- tion [39]. male breast cancer in the German population were 25%, In BRCA2, the most frequent mutations were c.7806- (12 positive cases from 47 tested families) and as high as 42% in Italian (3 positive cases from 7 tested families) 2A > G, c.5291C > G and c.3975_3978dupTGCT. The and in the Dutch population (5 positive cases from 12 mutation c.7806-2A > G has been already reported by tested families), which is much higher than in our popu- our group [31]. It is a Slovenian founder mutation, lation [15,17,25]. In this place, it must definitely be reported before also by others [15,40,41]. The mutation stressed that, in our study, not all samples of male breast c.5291C > G, however, according to BIC database, has cancer have been fully screened up till now and that only not been described in any other populations, other then tests for the most frequent mutations in the Slovenian the Slovenian one. population have been performed. This might be one of Different mutations in BRCA1 and BRCA2 genes have the possible reasons for a lower mutation detection rate a different penetrance for breast and for ovarian cancer in this group of families in our population. [35]. This has been observed also in our population. Nevertheless, a low detection rate has also been per- The families affected by one mutation actually differ ceived in another group of families with a single case of from the families affected by another mutation by female breast cancer (Group E) where the mutation means of the proportion of ovarian cancers among all detection rates for BRCA1 and for BRCA2 were 8% and breast and ovarian cancers in the family. These differ- 0%, respectively. In the German population, the detec- ences can be due to the variations in the biology of the tion rates in this group are reported to be 8% in BRCA1 translated protein having a different effect on the nor- and 4% in BRCA2 [15]. mal breast or ovarian tissue. The difference could also Themajorityofcommonmutations in BRCA1 be associated with other than BRCA1 and BRCA2 com- (c.181T > G, c.1687C > T, c.5266dupC) observed in mon moderate/low penetrancegenes inthepopulation [35,42,43]. the Slovenian population have been frequently Table 8 Incidence of BRCA1 or BRCA2 mutations among Slovenian families with only breast cancer history (no ovarian cancer) subdivided according to the number of breast cancer cases in the family Group 2 breast cancers in family 3 breast cancers in family 4 breast cancers in family more then 5 breast cancers in family history* history* history* history* A 9/29 (31%) 5/21 (24%) 7/16 (43%) 7/11 (64%) C 5/37 (13%) 3/25 (12%) 4/9 (44%) 2/4 (50%) D 1/10 (10%) 0/6 (0%) 0/5 (0%) 0/0 *number of positive families/number of all tested families in the group (the proportion of positive families). Group A - families with two or more cases of breast cancer including at least two of them diagnosed under the age of 50 and with no ovarian cancer. Group C - families with two or more cases of breast cancer including one case diagnosed before the age of 50 and with no ovarian cancer. Group D - families with two or more cases of breast cancer all of them diagnosed beyond the age of 50 years and with no ovarian cancer. Stegel et al. BMC Medical Genetics 2011, 12:9 Page 10 of 11 http://www.biomedcentral.com/1471-2350/12/9 2. Claus EB, Risch N, Thompson WD: Genetic analysis of breast cancer in the Conclusion cancer and steroid hormone study. Am J Hum Genet 1991, 48(2):232-42. With the introduction of the genetic councelling and 3. 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In our study, 96 families Science 1994, 266(5182):66-71. bearing the BRCA1 or BRCA2 mutation were identified. 4. Wooster R, Bignell G, Lancaster J, Swift S, Seal S, Mangion J, Collins N, Gregory S, Gumbs C, Micklem G: Identification of the breast cancer The mutation detection rates for BRCA1 and BRCA2 susceptibilitygene BRCA2. Nature 1995, 378(6559):789-92. genes in different groups of families with different family 5. Smith SA, Easton DF, Evans DG, Ponder BA: Allele losses in the region history were similar to the mutation detection rates in 17q12-21 in familial breast and ovarian cancer involve the wild-type chromosome. Nat Genet 1992, 2(2):128-31. other European populations. The most common muta- 6. Collins N, McManus R, Wooster R, Mangion J, Seal S, Lakhani SR, tions in the BRCA1 gene in the families with an Ormiston W, Daly PA, Ford D, Easton DF, Stratton MR: Consistent loss of increased risk of breast and/or ovarian cancer in Slove- the wild type allele in breast cancers from a family linked to the BRCA2 gene on chromosome 13q12-13. Oncogene 1995, 10(8):1673-5. nia are c.181T > G, c.1687C > T, c.5266dupC, 7. Ford D, Easton DF, Bishop DT, Narod SA, Goldgar DE: Risks of cancer in c.844_850dupTCATTAC and c.181T > A. The most fre- BRCA1-mutation carriers. Lancet 1994, 343(8899):692-5, Breast Cancer quent mutations in BRCA2 are c.7806-2A > G, c.5291C Linkage Consortium. 8. Easton DF, Ford D, Bishop DT: Breast and ovarian cancer incidence in > G and c.3975_3978dupTGCT. The cumulative share BRCA1-mutation carriers. Am J Hum Genet 1995, 56(1):265-71, Breast of these frequent mutations in BRCA1 and BRCA2 Cancer Linkage Consortium. genes in Slovenian families affected with mutations is 9. Couch FJ, Farid LM, DeShano ML, Tavtigian SV, Calzone K, Campeau L, Peng Y, Bogden B, Chen Q, Neuhausen S, Shattuck-Eidens D, Godwin AK, 68.7%. In the Slovenian population, the mutation detec- Daly M, Radford DM, Sedlacek S, Rommens J, Simard J, Garber J, Merajver S, tion rate seems to be influenced by the presence of Weber BL: BRCA2 germline mutations in male breast cancer cases and ovarian cancer history, the age at the diagnosis of breast breast cancer families. Nat Genet 1996, 13(1):123-5. 10. Antoniou A, Pharoah PD, Narod S, Risch HA, Eyfjord JE, Hopper JL, cancers in the family and the number of breast cancer Loman N, Olsson H, Johannsson O, Borg A, Pasini B, Radice P, Manoukian S, cases in the family. As well, the mutation detection rate Eccles DM, Tang N, Olah E, Anton-Culver H, Warner E, Lubinski J, might further be influenced in the future - it might Gronwald J, Gorski B, Tulinius H, Thorlacius S, Eerola H, Nevanlinna H, Syrjäkoski K, Kallioniemi OP, Thompson D, Evans C, Peto J, Lalloo F, namely increase when the character of some of the Evans DG, Easton DF: Average risks of breast and ovarian cancer unclassified variants will be clarified as pathogenic. associated with BRCA1 or BRCA2 mutations detected in case Series unselected for family history: a combined analysis of 22 studies. Am J Hum Genet 2003, 72(5):1117-30. Acknowledgements 11. King MC, Marks JH, Mandell JB, New York Breast Cancer Study Group: The authors gratefully thank the genetic counselors and nurses as well as Breast and ovarian cancer risks due to inherited mutations in BRCA1 laboratory technicians who helped to recruit patients, collect their clinical and BRCA2. Science 2003, 302(5645):643-6. data and perform the analyses, Nikola Bešič, Aleš Vakselj, Cvetka Bilban- 12. Narod SA, Ford D, Devilee P, Barkardottir RB, Lynch HT, Smith SA, Jakopin, Kristijana Hertl, Alenka Vrečar, Katarina Lokar, and Simona Traven. Ponder BA, Weber BL, Garber JE, Birch JM, Cornelis RS, Kelsell DP, Spurr NK, Authors also thank Barbara Jezeršek-Novaković for reviewing the article. Smyth E, Haites N, Sobol H, Bignon YJ, Chang-Claude J, Hamann U, Lindblom A, Borg AM, Steven Piver MS, Gallion HH, Struewing JP, Author details Whittemore A, Tonin P, Goldgar DE, Easton DF: An evaluation of genetic Department of Molecular Diagnostics, Institute of Oncology Ljubljana, heterogeneity in 145 breast-ovarian cancer families. Am J Hum Genet Zaloska 2, 1000 Ljubljana, Slovenia. Unit of Genetic Counseling, Institute of 1995, 56(1):254-64, Breast Cancer Linkage Consortium. Oncology Ljubljana, Zaloska 2, 1000 Ljubljana, Slovenia. Department of 13. Chen S, Iversen ES, Friebel T, Finkelstein D, Weber BL, Eisen A, Peterson LE, Surgical Oncology, Institute of Oncology Ljubljana, Zaloska 2, 1000 Ljubljana, Schildkraut JM, Isaacs C, Peshkin BN, Corio C, Leondaridis L, Tomlinson G, Slovenia. Laboratory of Molecular Oncology, Oncologisch Centrum UZ Dutson D, Kerber R, Amos CI, Strong LC, Berry DA, Euhus DM, Parmigiani G: Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium. Characterization of BRCA1 and BRCA2 mutations in a large United States sample. J Clin Oncol 2006, 24(6):863-71. Authors’ contributions 14. Ford D, Easton DF, Stratton M, Narod S, Goldgar D, Devilee P, Bishop DT, VS, MK, JŽ, MH, ET, JG and SN designed the study, collected and analyzed Weber B, Lenoir G, Chang-Claude J, Sobol H, Teare MD, Struewing J, the data and wrote the paper. 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Ramus SJ, Harrington PA, Pye C, DiCioccio RA, Cox MJ, Garlinghouse- Jones K, Oakley-Girvan I, Jacobs IJ, Hardy RM, Whittemore AS, Ponder BA,

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