ONCOLOGY REPORTS 15: 565-569, 2006 565 Germline mutations of BRCA1 in two Korean hereditary breast / ovarian cancer families TAE-JOONG KIM, KYUNG-MEE LEE, CHEL HUN CHOI, JEONG-WON LEE, JE-HO LEE, DUK-SOO BAE and BYOUNG-GIE KIM Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Received September 19, 2005; Accepted November 11, 2005 Abstract. Testing for cancer susceptibility gene, in particular mutations in the BRCA1 gene in association with hereditary breast/ovarian cancer has been extensively studied. We investigated germline mutations in the BRCA1 gene from two Korean hereditary breast/ovarian cancer families using direct DNA sequencing. Blood samples of the thirteen family members were studied. We found three missense mutations; 3232 A→G, 2731 C→T, 3667 A→G. These mutations were involved in the altered coding of amino acids. According to the BIC database, clinical significance of these mutations is regarded as favor polymorphisms. Therefore, these genetic variations are not believed to be involved in the development of the disease, but may be associated with breast/ovarian cancers in another yet undefined way. For further clinical significance of these variations, additional study such as a case-controlled haplotyping study is needed. Introduction Breast cancer is the most common cancer in Korean women and the incidence of ovarian cancer is the ninth in order with increasing tendency (1). Although most cases of breast and ovarian cancer occur sporadically in women with no previous personal or family history of cancer, it is well known that 5-7% of breast cancers and 10% of ovarian cancers are hereditary with a pattern of autosomal-dominant inheritance (2). Testing for cancer susceptibility genes support a variety of clinical decisions by providing results that indicate risk for future disease, confirmation of diagnoses and more recently, therapeutic selection and prognosis. The BRCA1 associated with hereditary breast/ovarian cancer is the most extensively studied cancer susceptible gene to date. BRCA1 _________________________________________ Correspondence to: Dr Byoung-Gie Kim, Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Kangnam-Ku, Seoul 135-710, Korea E-mail: bgkim@smc.samsung.co.kr Key words: ovarian neoplasms, breast neoplasms, genes, BRCA1, Koreans was originally isolated using positional cloning techniques (3). Germline alterations in the gene result in a predisposition for developing early-onset breast and ovarian cancer with a penetrance as high as 85% and 65%, respectively (4). The protein products of the BRCA1 gene regulate, at least in part, transcriptional activation, DNA repair, cell-cycle checkpoint control and chromosomal re-modeling (5). Although about 1500 genetic variants of BRCA1 have been described in the Breast Cancer Information Core (BIC) (http://research.nhgri.nih.gov/bic/), there is little data on the contribution of germline BRCA1 mutations to breast and/or ovarian cancer in Korea (6,7). To detect mutations, a variety of mutation screening methods, including dHPLC, CSGE, DGGE, SSCP and direct DNA sequencing have been adopted in Korea (8). Direct DNA sequencing is regarded as the ‘gold standard’ for sensitivity (9). To further understand the implication of genetic variants, we performed mutational analysis on the BRCA1 gene in two families having two or more affected first- or second-degree relatives with breast and/or ovarian cancer using direct DNA sequencing. Materials and methods Subjects. We evaluated germline mutations of BRCA1 from 13 blood samples in two families including two or more affected first- or second-degree relatives with breast and/or ovarian cancer. One proband is a patient with ovarian cancer and the other one is a patient with breast cancer (Fig. 1). Genomic DNA isolation. Using an iNtRon blood genomic DNA purification kit (iNtRON Biotechnology, Seoul, Korea) and in accordance with the manufacturer's protocols, genomic DNA was extracted from peripheral blood lymphocytes. PCR program. DNA was amplified by PCR for the 24 exons of the BRCA1 gene using previously published primer sets (10) that yielded 169- to 400-base pair products. Because of its size, exon 11 was screened using 19 overlapping primer sets (Table I). PCRs were performed with genomic DNA containing 50 ng of genomic DNA, 1 µl of each primer at 5 pmol/µl, 2 µl of a mixture of dNTPs (each at 2.5 mM), 2.5 µl of 10X PCR buffer, 1 µl of Taq polymerase, and distilled water was added to a final volume of 25 µl. For amplification, 566 KIM et al: BRCA1 IN KOREAN CANCER FAMILIES Table I. BRCA1 primers used for amplification and SSCP analysis of exon 11. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Exon Sense Antisense Product length (bp) ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– 11.0 5'-GTGAATTTTCTGAGACGGATGTA 5'-GAGCTGGCATGAGTATTTGTG 169 11.1 5'-AGAGGCATCCAGAAAAGTATCAGG 5'-GGGAGTCCGCCTATCATTACA 239 11.2 5'-ACAGCCTGGCTTAGCAAGGAG 5'-CCCCATCATGTGAGTCATCAGA 278 11.3 5'-AGAAACTGCCATGCTCAGAGAATC 5'-ATGAGGATCACTGGCCAGTAAGTC 245 11.4 5'-TGTATTGGACGTTCTAAATGAGGT 5'-TTGTGAGGGGACGCTCTTGTA 266 11.5 5'-GCATTTGTTACTGAGCCACAGATA 5'-TCTATTGGGTTAGGATTTTTCTCA 263 11.6 5'-CAAACGGAGCAGAATGGTCA 5'-GCCTGGTAGAAGACTTCCTCCTC 244 11.7 5'-TCCACAATTCAAAAGCACCTAAAA 5'-CTCTGGGAAAGTATCGCTGTCAT 299 11.8 5'-GCAACTGGAGCCAAGAAGAGTAAC 5'-TTTGCAAAACCCTTTCTCCACTTA 256 11.9 5'-TTGTCAATCCTAGCCTTCCAAGAG 5'-TTTTGCCTTCCCTAGAGTGCTAAC 224 11.10 5'-TATGGCACTCAGGAAAGTATCTCG 5'-GCGCTTTGAAACCTTGAATGTAT 270 11.11 5'-ACAGTCGGGAAACAAGCATAGAA 5'-TTTGGCATTATCAACTGGCTTATC 314 11.12 5'-AGGCTTTCCTGTGGTTGGT 5'-TTACGGCTAATTGTGCTCACTG 306 11.13 5'-AACATTCCAAGTACAGTGAGCACA 5'-AGATGCATGACTACTTCCCATAGG 378 11.14 5'-TCCTGGAAGTAATTGTAAGCATCC 5'-GGCCCCTCTTCGGTAACC 325 11.15 5'-TCCTAGCCCTTTCACCCATACA 5'-AGATGCCTTTGCCAATATTACCTG 274 11.16 5'-TGCTACCGAGTGTCTGTCTAAGAA 5'-AGAAAGGATCCTGGGTGTTTGTAT 209 11.17 5'-GCTAGCTTGTTTTCTTCACAGTGC 5'-AAGTTTGAATCCATGCTTTGCTCT 218 11.18 5'-CAGGGAGTTGGTCTGAGTGAC 5'-GCTCCCCAAAAGCATAAAC 181 ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– each sample was denatured at 94˚C for 2 min and subjected to 35 cycles of PCR (94˚C for 30 sec, 55˚C for 15 sec, 60˚C for 15 sec, and extension at 72˚C for 1 min on Applied Biosystems DNA thermal cycler); this was followed by incubation at 72˚C for 10 min. SSCP analysis. For SSCP analysis, the PCR products were mixed with an equal volume of formamide loading dye. The mixture was heated at 95˚C for 5 min to denature the DNA. After denaturation, the sample was chilled on ice for a few minutes. The samples were then run on an acrylamide gel under non-denaturing conditions in an electrophoresis apparatus. Electrophoresis was carried out in 0.5X TBE for 3-5 h at 4˚C. Direct sequencing. All sequence variants were confirmed by using the PCR products of each sequence variant and Big Dye on an ABI3100 DNA sequencer (Applied Biosystems). Results In two breast and/or ovarian cancer families, 13 blood samples were evaluated for germline mutations in BRCA1. SSCP analysis using PCR products of the whole BRCA1 gene revealed that there were band shifts in only exon 11 compared to the wild-type BRCA1 gene (Fig. 2). All members of Park family showed a band shift in the location of the exon 11.13, except for a son of the proband who did not have the genetic mutation (Fig. 1). Also, all members of the Moon family had two genetic mutations in SSCP. The locations identified were exon 11.11 and exon 11.14. Direct DNA sequencing of exon 11, which is known as a ‘hot spot’, found three missense mutations as follows: 3232 A →G, 2731 C →T, 3667 A →G (Fig. 3). These mutations altered coding of amino acids in the study family. To understand the clinical significance of these missense mutations, we referred to the BIC database, which has about 1500 genetic variations registered. All of the genetic mutations that we found in two Korean families have already been entered into the BIC database. They are identified as favor polymorphisms. In addition, they have reference SNP numbers (Table II). Discussion The BRCA1 gene is located on chromosome 17q21 and has a total length of about 100 kb. This gene consists of 24 exons and the coding region starts at the middle of exon 2. The size of the mRNA is 7.8 kb and BRCA1 product, localized in the nucleus (11), is a phosphorylated protein that consists of 1863 amino acids and has a molecular weight of 220 kDa. The phosphorylation is dependent on the cell cycle (12). BRCA1 is known to have several functions. One is repair of DNA damage by interacting with Rad51 and participation in homologous recombination (13). Another is regulation of gene expression by performing as a cofactor of many transcription factors. For instance, BRCA1 functions as a coactivator in p53- ONCOLOGY REPORTS 15: 565-569, 2006 567 Figure 1. Korean breast and breast-ovarian cancer families with germline BRCA1 missense mutations. Cancer types, age at diagnosis and mutation status are shown. Confirmed from blood sample to carry mutation; ^Confirmed from blood sample not to carry mutation; Circles, patients with cancer; Br, breast cancer; Ov, ovarian cancer; Cx, cervical cancer. * Figure 2. Single-strand conformational polymorphism (SSCP) assay for germline mutation in BRCA1 exon 11. Lanes 1, 2, exon 11.13 PCR products; Lanes 3, 4, exon 11.11 PCR products; Lanes 5, 6, exon 11.14 PCR products; Wt, wild-type; Mt, mutant type. dependent transcription (14) and as a corepressor in estrogen response transcription (15). Therefore, BRCA1 is believed to regulate cell growth, apoptosis and cell function by regulating gene expression. The other function is to maintain chromosomal stability. BRCA1-deficient cultured cells show decreased cell growth and hypersensitivity to ionizing radiation, resulting in DNA damage associated with chromosomal abnormalities (16-18). Overall, BRCA1 in its non-mutated form function as a tumor suppressor gene. The process of cancer development in individuals with germline mutations in the BRCA1 gene requires somatic inactivation of the remaining wild-type allele. The BRCA1 gene is very large and hundreds of specific different mutations of BRCA1 have been described. Most of the mutations are unique to the family in which they occur (19). Myriad Genetic Laboratories, Inc., where most variants in BRCA1 were discovered and classified, classify genetic variants as deleterious, suspected deleterious, uncertain clinical significance, favor polymorphism and polymorphism/ neutral (20). Mutations that result in a truncated protein can be assumed to be deleterious. The most common deleterious mutations for BRCA1 are non-sense or frame-shift mutations that begin prior to, or at, codon 1853. In addition, some specific missense mutations and splice mutations are recognized as deleterious. Although many variations that are not deleterious mutations have been found, many remain to be identified. Ethnicity may affect genetic mutations. Founder mutations have been identified in various ethnic groups including Ashkenazi Jews, Icelanders, Russians, and Israelis (21-23). However, it is still necessary to screen the entire sequences of BRCA1 genes in Asian women. Most studies of BRCA1 mutations in Japan reported many frameshift or non-sense mutations as well as polymorphisms and unclassified variants in BRCA1 gene, but there appears not to be specific Japanese ‘hot spots’ for BRCA1 mutations (24). A previous study in 21 Korean hereditary breast/ovarian cancer families identified only 5 deleterious mutations in BRCA1 gene; 2 frameshift and 3 non-sense mutations, without polymorphisms or unclassified variants. They used the protein truncation test for exon 11 of BRCA1 as a screening method (7). The present study found three genetic variations in BRCA1 in two Korean families with hereditary breast and/or ovarian cancer (Table II). All mutations identified were also reported in the Japanese 568 KIM et al: BRCA1 IN KOREAN CANCER FAMILIES Figure 3. Missense mutations identified by direct sequencing. Direct PCR based sequencing was performed on all samples in which a shift band pattern was observed in SSCP analysis. (a) Park family: Left, wild-type sequence of a portion of exon 11; Right, missense mutation sequence of a portion of exon 11 (3232 A→G). (b) and (c) Moon family: Left, wild-type sequence of a portion of exon 11; Right, missense mutation sequences of a portion of exon 11 (2731 C→T), (3667 A→G). Table II. Polymorphisms in BRCA1 gene. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Family Exon Codon Nucleotide changea Amino acid change Reference SNP (rs) number BICb ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Park 11 1038 3232 A>G Glu to Gly 16941 11 871 2731 C>T Pro to Leu 799917 Moon 11 1183 3667 A>G Lys to Arg 16942 ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– aNucleotide number are based on U14680; bChange entered in the BIC database are marked as -. ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– studies (24). According to the BIC database, three variations are all regarded as favor polymorphisms. They are given reference SNP (rs) numbers (http://www.ncbi.nlm.nih.gov). Therefore, these genetic variations are not believed to be causative mutations for cancer, but may be associated with the disease in another yet undefined manner. For a more detailed understanding of the clinical significance, further study such as a case-controlled haplotyping is needed. Here we may not have found any deleterious mutations reported in the Korean cancer families, because of the small number of samples studied. Instead, we identified polymorphisms, not found in a previous study for Korean women, this difference could have resulted from different screening methods. At first, we used a single-strand conformational polymorphism (SSCP), which showed a band-shift in exon 11. Then direct DNA sequencing was performed. Many studies have used SSCP or denaturing high-performance liquid chromatography (DHPLC), but direct DNA sequencing is considered optimal and as the ‘gold standard’. BRCA1-related breast cancers can be associated with a poor prognosis, but BRCA1-related ovarian cancers are associated with a high frequency of serous adenocarcinoma and a good outcome (24). Among patients with FIGO stage III ovarian cancer, the 5-year survival rate of BRCA1-mutation positive patients is reported as 78.6%, as compared with only 30.3% in control patients, with other etiologies for their ovarian cancer (25). The clinicopathologic features of one proband with ovarian cancer were consistent with the familial ovarian cancer with a BRCA1 mutation. The proband had primary surgery with adjuvant chemotherapy 14 years ago. Pathologic diagnosis was stage IIIc serous adenocarcinoma. Recurrence developed six years after the primary treatment and she had cytoreductive surgery and palliative chemotherapy, total of 50 cycles to date. She has been very sensitive to platinumbased chemotherapy. One possible mechanism supporting the favorable outcome in BRCA carriers with ovarian cancer is the increased sensitivity to platinum induced DNA damage in BRCA heterozygotes (26). Although this study investigated a small number of samples of the Korean BRCA1 families, it identified genetic variations of BRCA1 genes through direct DNA sequencing methods screening the entire sequences. All identified ONCOLOGY REPORTS 15: 565-569, 2006 variations were favor polymorphisms and are therefore not believed to be causative mutations in the development of the breast/ovarian cancer. Because only a limited number of cases have been analyzed in Korea, for further understanding of the genetic variations in the BRCA1 gene in the Korean population, additional study is required. References 1. Ministry of Health and Welfare RoK: Annual report of cancer registry programme in the Republic of Korea (2000.1-2000.12), 2003. 2. 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