Mutation analysis of BRCA1 and BRCA2 from 793 Korean patients with sporadic breast cancer

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1 Clin Genet 2006: 70: Printed in Singapore. All rights reserved Short Report # 2006 The Authors Journal compilation # 2006 Blackwell Munksgaard CLINICAL GENETICS doi: /j x Mutation analysis of BRCA1 and BRCA2 from 793 Korean patients with sporadic breast cancer Han S-H, Lee K-R, Lee D-G, Kim B-Y, Lee K-E, Chung W-S. Mutation analysis of BRCA1 and BRCA2 from 793 Korean patients with sporadic breast cancer. Clin Genet 2006: 70: # Blackwell Munksgaard, 2006 To investigate the role of BRCA1 and BRCA2 mutations in Korean patients with sporadic breast cancer, 793 breast cancer patients were analyzed by denaturing high performance liquid chromatography and direct sequencing. The 793 breast cancer patients enrolled in this study had no family history of affected first- or second-degree relatives with breast and/or ovarian cancer. Seventy-nine different sequence variations were identified, of which 34 were novel. Fifteen deleterious mutations were detected in 20 out of 793 patients (2.5%): 11 frameshift mutations and 4 nonsense mutations (seven in BRCA1 and eight in BRCA2), and no recurrent or founder mutations were observed in BRCA mutation screening. However, three mutations (K467X, 3972delTGAG, and R2494X in BRCA2) were identified in other studies of the Korean population. Of 793 patients, the clinicopathological information was obtained in 135 patients, who included 20 deleterious mutation-positive and 115 deleterious mutation-negative groups. The median age at diagnosis, histologic type, histologic grade and T stage did not show statistically significant difference between these two groups. BRCA-mutation-associated tumors showed lower estrogen receptor, progesterone receptor, and HER-2/neu but higher p53 expression. Although poor prognostic features were noted in BRCA-associated tumors, we did not find statistically significant differences. The present study will be helpful in the evaluation of the need for the genetic screening of germline BRCA mutations and reliable genetic counseling for sporadic breast cancer patients. S-H Han a, K-R Lee a, D-G Lee a, B-Y Kim a, K-E Lee b and W-S Chung c a Department of Molecular Genetics, Seoul Medical Science Institute, Seoul Clinical Laboratories, Seoul, Korea, b Division of Hematology and Oncology, Department of Medicine, and c Department of Laboratory Medicine, Ewha Woman s University School of Medicine, Ewha Mokdong Hospital, Seoul, Korea Key words: BRCA1 BRCA2 breast cancer DHPLC Korean sporadic Corresponding author: Wha-Soon Chung, MD, PhD, Department of Laboratory Medicine, Ewha Woman s University School of Medicine, Ewha Mokdong Hospital, Mok-dong, Yangchun-Gu, Seoul Korea. Tel.: ; fax: ; wschung@ewha.ac.kr Received 27 June 2006, revised and accepted for publication 27 September 2006 The incidence of breast cancer in Korea has been increasing in recent years. Although this cancer is still much less prevalent in Korea than Europe and the United States, it became the most common cancer in Korean women since 2001 (1). Remarkably, the peak age of Korean women diagnosed with breast cancer is in the 40s, which is years younger than that in the United States. Breast cancer is typically diagnosed at young age in Asian countries including Korea, which suggests the important role of racial differences and genetic variations (2). Germline mutations in BRCA1 and BRCA2 genes account for genetic predisposition and increased risk of breast and ovarian cancer in the majority of affected families (3 5). Most BRCA mutations result in truncation of the encoded protein and malfunction of protein activation (6). Up to date, more than 900 deleterious BRCA mutations, polymorphisms, and sequence variations with unclassified significance have been described in the Breast Cancer Information Core (BIC database; Many researchers from different countries have reported hundreds of disease-associated BRCA mutations. Among those reported mutations, several founder mutations have been identified in specific ethnic groups such as Icelanders, Ashkenazi Jews, Russians, and Israelis. Due to founder effects including environmental and geographical factors, the prevalence of BRCA1 and BRCA2 mutations is variable among different 496

2 Mutation analysis of BRCA1/2 from 793 Korean sporadic breast cancer patients populations (7, 8). There have been few data on the contribution of germline BRCA mutations to breast cancer in Asia. In this study, BRCA1 and BRCA2 germline mutations were screened in 793 samples of breast cancer patients with no family history of breast cancer and/or ovarian cancer as well as in 167 samples of normal controls with negative mammography to investigate the presence of BRCA1 and BRCA2 germline mutations of sporadic breast cancer patients in Korea. The mutational analysis was performed by denaturing high performance liquid chromatography (DHPLC) analysis through the entire coding exons and exon intron boundaries of the BRCA1 and BRCA2 genes followed by sequencing. Materials and methods Patients A total of 793 pathologically confirmed breast cancer patients were enrolled in this study. Family histories were examined by interviewing patients, and only cases without a family history of affected first- or second-degree relatives with breast and/or ovarian cancer were selected for this study. Additionally, 167 normal control women with normal mammography were analyzed, who had no family history of breast and/or ovarian cancer. Among the 793 breast cancer patients, the clinicopathological information could be acquired in 135 patients by medical record review, which was divided into deleterious mutation-positive (n ¼ 20) and deleterious mutation-negative group (n ¼ 115) for comparison between them. The clinicopathological characteristics such as the age at diagnosis, family history, histological subtype, histologic grade, T stage, and estrogen receptor (ER), progesterone receptor (PR), p53, and HER-2/ neu expression were obtained. This study was approved by an institutional board, and informed consent was obtained form all subjects. Denaturing high performance chromatography Genomic DNA was extracted from peripheral whole-blood samples, using standard techniques with the Gentra kit Systems (Gentra Inc., Minneapolis, MN). All samples were tested for small deletion, insertion, or point mutations on all exons of the BRCA1 and BRCA2 gene using DHPLC (Transgenomic Inc., San Jose, CA). DHPLC analysis was performed on a WAVE Maker System as previously described (9). DHPLC was carried out on automated HPLC instrumentation equipped with a DNASep column (Transgenomic Inc.). The column is packed with proprietary, 2-mm, nonporous, alkylated poly (styrene divinylbenzene) particles (10, 11). An alternative phase consisting of 3.5-mm-wide pore, alkylated silica beads (Eclipse dsdna Analysis Column, San Jose, CA) is available commercially from Hewlett Packard (Palo Alto, CA). The mobile phase was 0.1 M triethylammonium acetate buffer, ph 7.0 (PE Biosystems, San Jose, CA), containing 0.1 mm tetrasodium ethylenediamine-tetraacetic acid (Sigma, St Louis, MO). Crude polymerase chain reaction (PCR) products, subjected to an additional 95 C denaturing step for 5 min followed by gradual reannealing from 95 C to23 C over a period of 40 min prior to analysis, were eluted with a linear acetonitrile (J.T. Baker, Phillipsburg, NJ) gradient at a flow rate of 0.9 ml/min. The start- and end-points of the gradient were adjusted according to the size of the PCR products using an algorithm provided by the WAVE Maker System Control software. Generally, an analysis took 5 10 min, including column regeneration and re-equilibration to the starting condition. The temperature required for successful resolution of heteroduplex molecules was determined by the DHPLC melting algorithm. Details of the temperature conditions and the algorithm can be obtained form the WAVE Maker System software. The DHPLC gradients and temperatures were determined by WAVE Maker System software. Heterozygous profiles were identified by visual inspection of the chromatograms on the basis of additional earlier eluting peaks compared with one peak in corresponding homozygous profiles. Sequencing of PCR products Once subset of PCR products with an aberrant DHPLC pattern had been identified, its PCR products were amplified using the same primers as used for DHPLC, and DNA was sequenced on a ABI 3100 sequencer (Perkin Elmer Applied Biosystems, Foster City, CA) in both directions. The sequence was compared with the Breast Cancer Information Core (BIC database; research.nhgri.nih.gov/bic), Human Genome Mutation Database ( ac/index.php) and the National Center for Biotechnology Information database ( ncbi.nlm.nih.gov). Statistical analysis Clinicopathologic characteristics and BRCA mutation results were analyzed using SPSS (Statistical Package for the Social Sciences, 497

3 Han et al. Chicago, IL) 12.0 for Windows. Differences in categorical variables between deleterious mutation-positive and deleterious mutation-negative group were compared using chi-square analysis or Fisher s exact test. p value of,0.05 was considered as significant. Results A total of 79 sequence variations in BRCA1 and BRCA2 were identified in 793 patients, which consisted of 38 missense, 26 polymorphism, and 15 deleterious (11 frameshift and 4 nonsense) mutations, by mutation type. The novel sequence variations were identified in 34 out of 79 sequence variations (43%). Out of 79 sequence variations, 15 deleterious mutations were detected in 20 out of 793 patients (2.5%), which were not recurrent or founder mutation. Seven out of 15 deleterious mutations (1047C.T, 2167delA, 2478delG, and 5616del10 in BRCA1; 2487delT, 8662delGGA, and 10378C.T in BRCA2) were novel. The mutational spectrum is summarized in Tables 1 and 2. In BRCA1, seven deleterious mutations (1047C.T, 2167delA, 2478delG, 2552delC, 3746insA, 4184delTCAA, and 5616del10) produced a truncated protein signal at codons 310, 700, 791, 811, 1218, 1364, and 1839, respectively. Four of these seven BRCA1 deleterious mutations (1047C.T, 2167delA, 2478delG, and 5616del10) were novel. In BRCA2, four frameshift mutations (1537delAAAG, 2487delT, 3423delTAAT, and 3972delTGAG), one in frame deletion (8662delGGA), and three nonsense mutations (1627A.T, 7708C.T, and 10378C.T) were identified. Among the eight deleterious mutations in BRCA2, three novel mutations (2487delT, 8662delGGA, and 10378C.T) were identified. Table 1. Sequence alterations detected in BRCA1 Exon Nucleotide change Amino acid change Mutation type Cases Patients (n a ¼ 793) Normal controls (n a ¼ 167) BIC entries Deleterious mutations C.T b Q310X NS 1 0 None delA b 700X FS 1 0 None delG b 791X FS 1 0 None delC 811X FS insA 1218X FS delTCAA 1364X FS del10 b 1839X FS 1 0 None Unclassified variants 5 273C.T L52F MS G.A V271M MS A.C b E827D MS 2 3 None G.A b V990I MS 2 0 None C.G b S1241C MS 2 0 None T.C S1577P MS A.C E1581D MS T.C M1628T MS T.C L1780P MS Polymorphisms 3 233G.A K38K P C.T c S694S P T.C c L771L P T.C Y856H MS C.T c P871L MS A.G c E1038G MS A.G c K1183R MS G.A b P871P P 2 0 None T.C c S1436S P A.G b T1449T P 4 0 None A.G Q1604Q P T.C L1605L P A.G c S1613G MS G.A b V1832V P 2 3 None BIC, the Breast Cancer Information Core (last modified: Tuesday, 17 January 2006); FS, frameshift; MS, missense; NS, nonsense; P, polymorphism. a Number of subjects. b Novel mutations. c Mutations show a high frequency in both patients and normal controls. 498

4 Table 2. Sequence alterations detected in BRCA2 Mutation analysis of BRCA1/2 from 793 Korean sporadic breast cancer patients Cases Exon Nucleotide change Amino acid change Mutation type Patients (n a ¼ 793) Normal controls (n a ¼ 167) BIC entries Deleterious mutations delAAAG 458X FS A.T b K467X NS delT c 771X FS 1 0 None delTAAT 1075X FS delTGAG b 1257X FS C.T b R2494X NS delGGA c G2813del IFD 1 0 None C.T c R3384X NS 1 0 None Unclassified variants 2 281G.A R18H MS A.G Y42C MS G.A c C616Y MS 2 0 None A.G M784V MS G.A c C822Y MS 2 0 None T.G c L1114R MS 2 0 None A.G M1272V MS A.T c M1272L MS 2 0 None C.T T1915M MS A.G I1929V MS A.C c D1990A MS 1 0 None G.T G2044V MS A.C c K2150Q MS 2 0 None G.T c V2166L MS 1 0 None T.G c V2280G MS 2 0 None C.G A2351G MS A.G Y2363C MS T.C I2490T MS G.A c C2605Y MS 1 0 None T.C c W2626R MS 2 0 None A.G I3412V MS Polymorphisms 2 203G.A d 5#UTR P A.C d N289H MS C.A d H372N MS G.A c P389P P 6 3 None C.G c A406A P 2 0 None A.G c E425E P 16 0 None A.G d S455S P G.C c L709L P 4 0 None T.C d H743H P C.T c N830N P 2 0 None A.G d N991D MS A.G c L1072L P 4 0 None A.G d K1132K P T.C c S1140S P 2 0 None C.G c T1154T P 2 0 None T.C d V1269V P A.G c Q1931Q P 8 0 None G.A c L2396L P 2 0 None A.G d S2414S P A.G c S3351S P 2 0 None BIC, the Breast Cancer Information Core (last modified: Tuesday, 17 January 2006); FS, frameshift; IFD, in frame deletion; MS, missense; NS, nonsense; P, polymorphism; UTR, untranslated region. a Number of subjects. b Mutations identified independently in previous studies on Korean population. c Novel mutations. d Mutations show a high frequency in both patients and normal controls. Three out of eight BRCA2 deleterious mutations (1627A.T, 3972delTGAG, and 7708C.T) were detected in 7 of 20 patients, which were also identified in previous studies on Korean population (12 15). A family study was not performed for patients with deleterious mutations. 499

5 Han et al. Out of 38 missense mutations in BRCA1 and BRCA2, 22 were located in exon 11. The remaining 11 were spread at exons 5, 16, and 22 in BRCA1 and exons 2, 3, 10, 14 15, 17, and 27 in BRCA2. Among the 38 missense mutations, 3 novel BRCA1 and 10 novel BRCA2 mutations were identified in this study. A total of 30 sequence variations were identified in 167 normal controls, which consisted of 18 missense and 12 polymorphisms by mutation type. No deleterious mutations were detected in normal controls. Among the 13 novel missense mutations, 12 (2 in BRCA1 and 10 in BRCA2) mutations were not found in 167 normal controls (334 chromosomes). Seven BRCA1 sequence variations (2201T, 2430C, 2731T, 3232G, 3667G, 4427C, and 4956G) and nine BRCA2 sequence variations (203A, 1093C, 1342A, 1593G, 2457C, 3199G, 3624G, 4035C, and 7470G) appeared at a high frequency in 167 normal controls. They also showed similar frequency in 793 breast cancer patients (Tables 1 and 2). The clinicopathological information was obtained in 135 patients, who included 20 deleterious mutation-positive and 115 deleterious mutation-negative patients. The median age at diagnosis of both the BRCA deleterious mutationpositive and deleterious mutation-negative group was similar, 43 and 48 years, respectively. Infiltrating ductal carcinoma was the predominant histologic subtype in both groups. The cases with deleterious BRCA mutations showed lower ER, PR, and HER-2/neu and higher p53 expression compared with mutation-negative cases, of which difference was not statistically significant. Discussion In this study, we aimed to evaluate the role of BRCA1 and BRCA2 germline mutations in Korean patients with sporadic breast cancer. Whereas the majority of studies on BRCA gene mutations have focused on the western population with a family history or ovarian cancer, only a relatively small number of investigations on the role of the BRCA genes have been undertaken in Asian sporadic breast cancer populations (2). In this study, BRCA mutation analysis of 793 breast cancer patients showed 34 novel variants out of 79 sequence variations, which may be of Korean-specific origin. Fifteen deleterious mutations (11 frameshift and 4 nonsense mutations) in BRCA1 and BRCA2 that led to premature termination producing shortened proteins were detected. Among the 15 mutations, 7 (47%) had not been published before. The incidence of novel mutation in this study (47%) was similar to three previous studies in Korean populations ( %) (12 14). The spectrum of BRCA1 and BRCA2 mutations in the Korean population appears different from those of Ashkenazi Jew and other populations. None of three founder mutations identified in Ashkenazi Jewish descent (185delAG and 5382 insc for BRCA1, and 6174delT for BRCA2) or 999del5 mutation for BRCA2 common to the Icelandic population was detected in this study. This study did not show any founder mutations in BRCA1 and BRCA2 in Korean breast cancer patients. However, the presence of three mutations in BRCA2 (1627A.T, 3972delTGAG, and 7708C.T) in 7 of 20 patients was found in this study, as well as in other previous studies (12 15). Although more population-based study is required, these findings suggest that the above three mutations might be a recurrent mutation and have important implication for screening strategies for breast cancer among Korean population. The prevalence of germline deleterious mutations in BRCA1 and BRCA2 was estimated to be 5.1 and 6.7% in previous studies, and phenotypes were variable by ethnicity (2, 16). This study showed a lower prevalence of BRCA mutations (2.5%), which was similar to previous studies on BRCA1 and BRCA2 mutations in Korean patients with sporadic breast cancer ( %) (14, 15). Further studies on large numbers of cases may give a more accurate estimation of prevalence and variations between Asian populations. The true contribution of BRCA genes to sporadic breast cancer remains controversial for a number of reasons. First, missense mutations with an unknown significance could have a pathogenic effect. Second, in addition to missense mutations, silent polymorphisms and intronic variants may affect splicing mechanisms. However, these variants cannot be classified as associated with disease in the absence of a good functional assay system for BRCA gene. Once a functional assay becomes available, it could be important to elucidate the relevance of such variations with currently unknown significance. Moreover, it should be noted that no currently available technique can guarantee detection of all disease-associated mutations in the BRCA genes. In this study, the BRCA mutation analysis for 167 normal controls with normal mammography was performed to compare the frequency of BRCA mutation with the breast cancer patient group. Deleterious mutations were not found in normal controls. Also, 12 novel missense mutations detected in this study were not found in normal controls (334 control chromosomes), which 500

6 Mutation analysis of BRCA1/2 from 793 Korean sporadic breast cancer patients could have a pathogenic effect. The sequence variations listed in Results showed high frequency in normal controls. They also showed similar frequency in 793 breast cancer patients. It is speculated that they are not likely to be disease-associated mutations in the BRCA1 and BRCA2 genes. Although haplotype analysis was not performed, the above BRCA mutations with high allele frequency might have high possibility of linkage disequilibrium. In this study, scanning for the presence of sequence variations was performed by mutational analysis through the entire coding exons and exon intron boundaries of the BRCA1 and BRCA2 genes using DHPLC analysis. DHPLC offers the advantage of distinct elution profiles that differ not only between different sequence variants but also between combinations of them. In this study, to verify the sensitivity of DHPLC screening, 960 fragmentsweretestedinablindmannerbydirect DNA sequencing, and no discrepancies were found (data not shown). As shown in Tables 1 and 2, many sequence variations including novel variants and single substitutions were detected using this system, which also demonstrates the efficiency of DHPLC as a scanning method. Results of medical record review for the clinicopathological information about 20 deleterious mutation-positive and 115 deleterious mutation-negative groups are similar to that of the other studies: BRCA-associated tumors showed lower ER, PR, and HER-2/neu and higher p53 expression, findings in accordance with previous studies. Although poor prognostic features were noted in BRCA-associated tumors, the differences were not statistically significant. These data suggest that neither hormone receptors nor HER-2/neu stimulation seems to be important in the pathogenesis of cancers arising in mutation carriers. A reasonable explanation for these results is that p53 loss rescues the proliferative deficiency of BRCA mutant cells at the expense of genetic instability (17). In conclusion, a total of 793 Korean patients with sporadic breast cancer were analyzed for germline mutations in the BRCA1 and BRCA2 genes, using a combination of DHPLC and direct sequencing. The present study revealed 79 different sequence variations including 15 deleterious mutations, 34 of which have not been reported previously and may be of Koreanspecific origin. The genetic testing of BRCA mutations remains too expensive and laborious and thus is restricted to high-risk families. The present study allows a better evaluation of the need for the genetic screening of germline BRCA mutations and reliable genetic counseling for sporadic breast cancer patients. References 1. Korean Breast Cancer Society. Clinical Characteristics of Breast Cancer Patients in Korea in Arch Surg 2004: 139: De Leon Matsuda ML, Liede A, Kwan E. BRCA1 and BRCA2 mutations among breast cancer patients from the Philippines. Int J Cancer 2002: 98: Easton DF, Bishop DT, Ford D, Crockford GP. Genetic linkage analysis in familial breast and ovarian cancer results from 214 families. The Breast Cancer Linkage Consortium. Am J Hum Genet 1993: 52: Frank TS, Manley SA, Olopade OI et al. Sequence analysis of BRCA1 and BRCA2: correlation of mutations with family history and ovarian cancer risk. J Clin Oncol 1998: 16: Hall JM, Lee MK, Newman B et al. Linkage of early-onset familial breast cancer to chromosome 17q21. Science 1990: 250: Wooster R, Bignell G, Lancaster J et al. Identification of the breast cancer susceptibility gene BRCA2. Nature 1995: 378: Loman N, Johannsson O, Kristoffersson U, Olsson H, Borg A. Family history of breast and ovarian cancers and BRCA1 and BRCA2 mutations in a population-based series of earlyonset breast cancer. J Natl Cancer Inst 2001: 93: Neuhausen SL. Ethnic differences in cancer risk resulting from genetic variation. Cancer 1999: 86: Gross E, Amold N, Pfeifer K, Bandick K, Kiechle M. Identification of specific BRCA1 and BRCA 2 variants by DHPLC. Hum Mutat 2000: 16: Huber CG, Oefner PJ, Bonn GK. High-resolution liquid chromatography of oligonucleotides on nonporous alkylated styrene-divinylbenzene copolymers. Anal Biochem 1993: 212: Huber CG, Oefer PJ, Bonn GK. Rapid and accurate sizing of DNA fragments by ion-pair chromatography on alkylated nonporous poly (styrene-divinylbenzene) particles. Anal Chem 1995: 67: Kang HC, Kim IJ, Park JH et al. Germline mutations of BRCA1 and BRCA2 in Korean breast and/or ovarian cancer families. Hum Mutat 2002: 20: Choi DH, Lee MH, Bale AE, Carter D, Haffty BG. Incidence of BRCA1 and BRCA2 mutations in young Korean breast cancer patients. J Clin Oncol 2004: 22: Seo JH, Cho DY, Ahn SH et al. BRCA1 and BRCA2 germline mutations in Korean patients with sporadic breast cancer. Hum Mutat 2004: 24: Ahn SH, Hwang UK, Kwak BS et al. Prevalence of BRCA1 and BRCA2 mutations in Korean breast cancer patients. J Korean Med Sci 2004: 19: Liede A, Malik IA, Aziz Z et al. Contribution of BRCA1 and BRCA2 mutations to breast and ovarian cancer in Pakistan. Am J Hum Genet 2002: 71: Xu X, Qiao W, Linke SP et al. Genetic interactions between tumor suppressors Brca1 and p53 in apoptosis, cell cycle and tumorigenesis. Nat Genet 2001: 28: