MUTATION IN BRIEF

HUMAN MUTATION Mutation in Brief #530 (2002) Online

© 2002 WILEY-LISS, INC.DOI: 10.1002/humu.9059

Received 3 April 2002; accepted revised manuscript 26 June 2002.

Germline Mutations of BRCA1 and BRCA2 in KoreanBreast and/or Ovarian Cancer FamiliesHio Chung Kang2†, Il-Jin Kim2†, Jae-Hyun Park2, Hyuk-Jun Kwon2, Yong-Jin Won2, Seung ChulHeo3, Sang-Yon Lee2, Kyung-Hee Kim2, Yong Shin2, Dong Young Noh3, Dae-Hyun Yang4, Kuk JinChoe3, Bong Hwa Lee5, Soon Beom Kang6, and Jae-Gahb Park1,2,3*

1Familial Cancer Clinic, National Cancer Center, Goyang, Gyeonggi, Korea; 2Korean Hereditary Tumor Registry,Laboratory of Cell Biology, Cancer Research Center and Cancer Research Institute, Seoul National UniversityCollege of Medicine, Seoul, Korea; 3Department of Surgery, Seoul National University College of Medicine, Seoul,Korea; 4Department of Surgery, Kangnam General Hospital Public Corporation, Seoul, Korea; 5Department ofSurgery, Hallym University College of Medicine, Seoul, Korea; 6Department of Obstetrics and Gynecology, SeoulNational University College of Medicine, Seoul, Korea

†HC Kang and I-J Kim equally contributed to this work.

*Correspondence to: Dr. Jae-Gahb Park, National Cancer Center, 809 Madu-dong, Ilsan-gu, Goyang, Gyeonggi,411-764, Korea; Tel.:+82-31-920-1501; Fax:+82-31-920-1511; E-mail: jgpark@plaza.snu.ac.kr

Contract grant sponsor: National Cancer Center Research Grant; Contract grant number: Research Grant 2001 and2001 BK21 project for Medicine, Dentistry, and Pharmacy.

Communicated by Mark H. Paalman

Germline mutations in the BRCA1 and BRCA2 genes are responsible for the predispositionand development of familial breast and/or ovarian cancer. Most mutations of BRCA1 andBRCA2 associated with breast and/or ovarian cancer result in truncated proteins. Toinvestigate the presence of BRCA1 and BRCA2 germline mutations in Korean breast and/orovarian cancer families, we screened a total of 27 cases from 21 families including two ormore affected first- or second-degree relatives with breast and/or ovarian cancer. PTT,PCR-SSCP, and DHPLC analysis, followed by sequencing were used in the screeningprocess. In nine families, we found BRCA1 and BRCA2 germline mutations that comprisedfour frameshift mutations and five nonsense mutations. All nine mutations led to prematuretermination producing shortened proteins. Among the nine mutations, three novel BRCA1mutations (E1114X, Q1299X, 4159delGA) and two novel BRCA2 mutations (K467X,8945delAA) were identified in this work. © 2002 Wiley-Liss, Inc.

KEY WORDS: BRCA1; BRCA2; germline mutations; breast cancer; ovarian cancer; Korean

INTRODUCTION

Five to ten percent of all breast and/or ovarian cancers arise from an autosomal dominant pattern of inheritanceaccording to segregation analysis studies (Newman et al., 1988; Schildkraut et al., 1989). Since two highsusceptibility genes, BRCA1 (MIM# 113705) and BRCA2 (MIM# 600185), for familial breast and/or ovarian

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cancer were identified in 1994 and 1995 (Miki et al., 1994; Wooster et al., 1995), respectively, many researchersfrom different countries have reported hundreds of disease-associated BRCA1 and BRCA2 mutations. Among thosereported mutations, several founder mutations have been identified in specific ethnic groups such as Icelanders,Ashkenazi Jews, Russians, and Israelis. Due to these founder effects as well as other environmental andgeographical factors, the prevalence of BRCA1 and BRCA2 mutations is variable among different populations(Neuhausen, 1999; Loman et al., 2001). Most BRCA1 and BRCA2 mutations related to breast and/or ovarian cancerresult in truncation of the encoded protein and malfunction of protein activation (Wooster et al., 1995).

In Korea, breast cancer is the second most common cancer after gastric cancer among women and the incidencein the general population is continuously increasing. To investigate the presence of BRCA1 and BRCA2 germlinemutations in Korean breast and/or ovarian cancer families, we performed mutational analysis through the entirecoding exons and exon/intron boundaries of the BRCA1 and BRCA2 genes using protein truncation test (PTT),polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), and denaturing highperformance liquid chromatography (DHPLC) analysis, followed by sequencing.

MATERIALS AND METHODS

SamplesBlood samples from 20 Korean breast and/or ovarian cancer families were collected from Seoul National

University Hospital and one further from Kangnam General Hospital Public Corporation after informed consentswere obtained from all participants. Families SNU-B4, SNU-1003, and KGH-B1001 have previously beenreported (Won et al., 1997; 1998). Criteria for family inclusion were at least two or more affected first- or second-degree relatives with breast and/or ovarian cancer, at least one of whom was diagnosed with cancer under age 50.A total 27 family members from 21 families were studied in this work.

DNA extractionPeripheral blood lymphocytes from the family members were isolated using Ficoll-Paque according to the

manufacturer’s instructions (Pharmacia Biotech, Uppsala, Sweden). Total genomic DNA was extracted using TRIreagent following the manufacturer’s instructions (Molecular Research Center, Cincinnati, OH, USA).

Mutation screeningThe protein truncation test was performed for exon 11 of BRCA1. The PCR primer pairs and PTT protocol were

previously described by Plummer et al. (1995). The remaining exons of BRCA1 and all BRCA2 exons werescreened by PCR-SSCP. The primer sources and approach protocol for PCR-SSCP have been described by Simardet al. (1994) and Lancaster et al. (1996). For DHPLC (WAVE, Transgenomic, Omaha, USA) analysis, the PCRprimer pairs used to amplify exons of BRCA1 and BRCA2 have been referred to by Wagner et al. (1999a; 1999b).

All samples showing abnormal patterns in PTT, PCR-SSCP, and DHPLC analysis were subsequently cloned,using the TA cloning system (Invitrogen, Carlsbad, CA, USA) and bi-directionally sequenced using the Taqdideoxy terminator cycle sequencing kit and ABI 377 DNA sequencers (Perkin-Elmer, Foster City, CA, USA).

RESULTS AND DISCUSSION

Mutational screenings of BRCA1 and BRCA2 were performed in 21 Korean breast and/or ovarian cancerfamilies by PTT, PCR-SSCP, and DHPLC analysis followed by cloning and sequencing. PTT and PCR-SSCPwere used as preceded detection methods, and the families remaining undetected following these tests were re-screened with DHPLC analysis.

In nine out of the 21 Korean breast and/or ovarian cancer families, 5 BRCA1 and 4 BRCA2 germline mutationsleading to prematurely terminated proteins were found (Table 1). Among these nine identified mutations, threeBRCA1 mutations and two BRCA2 mutations were novel, and have not yet been reported in the Breast CancerInformation Core, BIC (http://www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic).

SNU-B1009 and SNU-B1003 harbored frameshift mutations, 4159delGA and 5221delTG, which produced atruncated protein signal at codons 1354 and 1714 in BRCA1. In the case of SNU-B1009, the proband was affectedwith bilateral ovarian cancer featuring papillary serous cystadenocarcinoma, and was diagnosed at age 43. Three

BRCA1 and BRCA2 Germline Mutations in Korea 3

nonsense mutations in BRCA1, 3459G>T (E1114X), 4014C>T (Q1299X), and 5563G>A (W1815X), wereidentified in SNU-B1, SNU-OV6, and KGH-B1001, respectively. KGH-B1001 was diagnosed with bilateral breastcancer, on the left side at age 41, and on the right at age 45. SNU-OV6 and SNU-B1009 had truncating mutationsin the first two-thirds of the BRCA1 gene, an occurrence known to indicate a higher risk of ovarian cancer (Gaytheret al., 1995), and SNU-OV6 had more ovarian cancer cases than breast cancer cases. Among the above mutationsfound in BRCA1, three were novel: 3459G>T (E1114X), 4014C>T (Q1299X), and 4159delGA.

Table 1. BRCA1 and BRCA2 Germline Mutations Identified in Korean Breast and/or Ovarian CancerFamilies

Family No. Exon Mutation Total No. of No. of Br No. of Br No. of OvCa cases cases cases<age 50 Cases

BRCA1

SNU-B1 11 3459G>T (E1114X)** 3 3 2 -SNU-OV6 11 4014C>T (Q1299X)** 3 1 1 2SNU-B1009 11 4159delGA** 3 2 1 1SNU-B1003* 18 5221delTG 2 2 2 -KGH-B1001* 23 5563G>A (W1815X) 2 1 1 1

BRCA2

SNU-B1006 10 1627A>T (K467X)** 3 3 3 -SNU-B4* 11 3972delTGAG 5 5 3 -SNU-B5 15 7708C>T (R2494X) 4 4 1 -SNU-B1005 21 8945delAA** 2 2 2 -

Ca: Cancer; Br: Breast Cancer; Ov: Ovarian Cancer.*: previously reported in J Korean Cancer Assoc (Won et al., 1997; 1998).**: novel mutation.

Four families carried BRCA2 mutations, comprising two frameshifts (3972delTGAG, 8945delAA) in SNU-B4and SNU-B1005, and two nonsense mutations [1627A>T (K467X), 7708C>T (R2494X)] in SNU-B1006 andSNU-B5. The former two resulted in stop codons at codons 1257 and 2917. In SNU-B1005 with the frameshitmutation 8945delAA, there were two male mutation carriers who were both young (15 and 19 years old) and havenot shown any distinct phenotype. However, their mother and mother’s sister were both predisposed to early-onsetbreast cancer diagnosed at age 39 and 30, respectively. It has been suggested that the risk of male breast cancerinvolving BRCA2 mutations is 6% by age 70 and that the inherited mutations in BRCA2 might be associated with15% of all male breast cancers (Szabo and King, 1995). Although the lifetime risk of male breast cancer withBRCA2 mutations is less significant, a close surveillance of these two male carriers is required.

The mutation (3972delTGAG) of SNU-B4 was in the OCCR (ovarian cancer clustering region, codon 936-2092)area of BRCA2. However, none of the five affected members has exhibited the ovarian cancer phenotype yet. Theyall had breast cancer and the proband was diagnosed with bilateral breast cancer.

A proband from SNU-B5 harboring 7708C>T (R2494X) in BRCA2 presented with a medullary breastcarcinoma; this being a rare feature of breast cancer. Although it has been suggested that medullary breastcarcinoma might be more preferentially associated with BRCA1 mutations (Eisinger et al., 1998; BCLC, 1997), thepatient with medullary breast carcinoma of SNU-B5 harbored a germline mutation in BRCA2. Two (1627A>T(K467X), 8945delAA) of the four BRCA2 mutations were novel types. The novel frameshift mutation, 8945delAA,was the third truncation mutation identified in exon 21 of BRCA2 according to the BIC database.

Among nine families with BRCA1 or BRCA2 mutations, the three breast-ovarian cancer families, all had BRCA1mutations. All four families with BRCA2 mutations were site-specific breast cancer families. This result isconsistent with a previous report that BRCA1 accounts for the majority of breast/ovarian cancer families, and thatBRCA2 is more likely linked to site-specific breast cancer families (Ford et al., 1998).

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Three out of the five BRCA1 mutations were located in exon 11, and the remaining two were in exons 18 and23. On the other hand, only one BRCA2 mutation was identified in exon 11, despite this being the largest codingregion, while remaining three were spread at exons 10, 15, and 21.

Since the mutational analysis was carried out within only 21 families, it is hard to estimate the overall frequencyof BRCA1and BRCA2 mutations contributing to Korean breast and/or ovarian cancer families and no comparisonwith the other ethnic groups can reasonably be made. The results, however, suggest that the germline mutations intwo major genes, BRCA1 and BRCA2, have an important influence on the predisposition and development offamilial breast and/or ovarian cancer in Korea.

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