Thirty patients with early onset breast cancer or familial breast cancer from Malaysia were analysed for germline mutation in the early onset breast cancer I gene (BRCA1). Direct sequencing of the entire coding region of BRCA1 identified a frameshift mutation, c.5447-5448insC (insC5447) (codon 1776 of exon 21) in a patient aged 32 of the Malay ethnic origin, who had no family history of breast and/or ovarian cancer. Eight polymorphisms (2201C > T, 2430T > C, P871L, E1038G, K1183R, 4427T > C, S1613G and IVS8-57delT) were identified in the samples tested

A. S. B. Khoo

H. Abdullah

J. A. M. A. Tan

L. Volpi

P. Balraj

S. Nair

AIR Universita degli studi di Milano

Mutation Analysis of the BRCA1 Genein Malaysian Breast Cancer PatientsP Balraj, A S B Khoo, L Volpi, J A M A Tan, S Nair, H AbdullahDivision ofMolecular PathologyInstitute forMedical ResearchJalan Pahang50588 Kuala LumpurMalaysiaP Balraj, BSc (Hons),MMSc (Mal)Research OfficerA S B Khoo, MBBS,MPH (Mal), MRCP(UK), AM (Mal) (#)Research (Medical)SpecialistL Volpi, PhD (Milan)Visiting ScientistDepartment ofAllied SciencesFaculty of Medicine,University of MalayaKuala LumpurMalaysiaJ A M A Tan,BSc (Hons), MSc,PhD (Mal)Associate ProfessorGleneagles HospitalKuala LumpurMalaysiaS Nair, MMBS,FRCS (Eng),FRCS (Edin)Consultant SurgeonDivision of SurgeryKuala LumpurHospitalKuala LumpurMalaysiaH Abdullah,MD, MS (Mal)Consultant SurgeonCorrespondence to:Dr AlanSoo Beng KhooTel: (603) 4040 2421Fax: (603) 2693 8219Email: alankhoo@imr.gov.mySingapore Med J 2002 Vol 43(4) : 194-197O r i g i n a l  A r t i c l eABSTRACTThirty patients with early onset breast canceror familial breast cancer from Malaysia wereanalysed for germline mutation in the early onsetbreast cancer 1 gene (BRCA1). Direct sequencingof the entire coding region of BRCA1 identifieda frameshift mutation, c.5447-5448insC (insC5447)(codon 1776 of exon 21) in a patient aged 32of the Malay ethnic origin, who had no familyhistory of breast and/or ovarian cancer. Eightpolymorphisms (2201C>T, 2430T>C, P871L,E1038G, K1183R, 4427T>C, S1613G and IVS8-57delT) were identified in the samples tested.Keywords: BRCA1, Breast cancer, Ethnic, Mutation,MalaysianSingapore Med J 2002 Vol 43(4):194-197INTRODUCTIONBreast cancer is a common malignancy affectingwomen around the world, including Malaysia.In 1995, 9.6% of the admissions for cancer in thegovernment hospitals in Malaysia was for breastcancer(1) and 20% of 317 women who died of breastcancer were below the age of 40(2). Early onsetbreast cancer susceptibility gene (BRCA1) has beenlinked to 52% of families with breast cancer(3,4).Germline mutations of BRCA1 in families are estimatedto increase the risk of developing breast cancer for thefirst time for BRCA1 carriers at 73% by the age of50 and 87% by 70 years(4). There is also a 29% riskfor breast cancer patients to develop ovarian cancerby 50 years and 44% by 70 years(4).Germline BRCA1 mutations have been identifiedin young-onset breast cancer patients without a strongfamily history of breast cancer. Greenman et al(5)reported BRCA1 mutations in 18% patients (five outof 27) with families with one to three relatives withbreast or ovarian cancer. Another study identified15 mutations from 208 breast cancer patients belowthe age of 45 who had first degree breast cancerfamily history (affected mother and/or sister withbreast cancer)(6). These studies show that a largeproportion of women with first degree breast cancerfamily history may not carry germline mutations.However, in studies with four or more first degreerelatives with breast and/or ovarian cancer, a higherprevalence of mutations have been reported. Couchet al(7) identified that 45% of families carried BRCA1mutations and similar findings were reported in30 Canadian families(8).Other breast cancer predisposing genes associatedwith familial breast cancer are the breast cancer 2gene (BRCA2) and Phosphatase and Tensin Homolog(PTEN). BRCA2 was found to be linked to 32% infamilies with breast cancer. However, the risk ofdeveloping breast cancer associated with BRCA2is lower compared to BRCA1(4). In addition, BRCA2mutations among male carriers confer a higher riskof male breast cancer compared to BRCA1(4). PTENmutations have been associated with Cowdensyndrome, characterised by multiple hamartomatouslesions of the skin, mucous membranes, intestinalpolyps and increased risk of breast and thyroidcancer. Although PTEN mutations have been found insporadic and familial breast cancer cases, it is not amajor determinant of non-BRCA1/BRCA2 cases(9,10).Currently, most reported BRCA1 mutations arefrom those of Caucasian origin and the type ofmutations in Asians could be different. Li et al(11)screened for BRCA1 mutations using single strandconformational polymorphism (SSCP) in patientswith familial breast cancer and identified a novelmutation. BRCA1 mutation analysis in tumours ofsporadic breast cancer cases unselected for age andearly onset breast cancer cases in Chinese fromHong Kong identified 589delCT as a potentialcandidate founder BRCA1 mutation among theChinese(12). In Singapore, the study of 43 breast cancerpatients found 2846insA in two unrelated Malayfamilies(13). Japanese studies found BRCA1 mutationsto be unique to the country(14,15). These reports alsoobserved that the prevalence of BRCA1 mutationsamong their patients was similar to those reported inUnited States. SSCP was used in all the studiesreported above.Singapore Med J 2002 Vol 43(4) : 195Malaysia is a country in South East Asia withthe main ethnic groups being the Malays, Chinese andIndians. Information on BRCA1 mutations in theMalay ethnic group, a major ethnic group in SouthEast Asia is still lacking. In this study, we carried adetailed analysis of the BRCA1 gene in patients inMalaysia regardless of their ethnicity. The entirecoding region of BRCA1 gene was analysed by directsequencing, as this approach would cause the least biasin the type of mutation identified while theoreticallybeing the most sensitive method. This is important asthe information on the mutation spectra of BRCA1among breast cancer patients (particularly amongthose with early onset sporadic breast cancer) in thisregion is limited. We performed the comprehensivescreening for BRCA1 mutations in Malaysian patientswith early onset breast cancer and those with two ormore relatives with breast and/or ovarian cancers.MATERIALS AND METHODSWe screened BRCA1 mutations in two clinicallyselected groups for whom testing might be indicated.The first group comprised of histologically confirmedcases of breast cancer aged below 35 years (i.e. earlyonset) and the second group comprised of histologicallyconfirmed cases of breast cancer with two or more firstor second degree relatives with breast and/or ovariancancers regardless of age (i.e. familial breast cancer).The family histories of these women are summarisedin Table I.Breast cancer patients from the three main ethnicgroups seen in Kuala Lumpur Hospital between1994 and 1998 in average were 45% Malays, 35%Chinese and 18% Indians. Individuals from thedifferent ethnic groups in Malaysia were representedin our series of patients - i.e. 16 (53%) Malays, 9 (30%)Chinese and 5 (17%) Indian.Patients identified were attending the Breast Clinic,Kuala Lumpur Hospital, Malaysia from 1994 to 1998and blood was collected from consenting individuals.Polymerase chain reaction was carried out withprimers(16,17) on leukocyte DNA for the entire codingregion of the BRCA1 gene in 37 separate reactions,followed by direct sequencing with the ABI PRISMTMdRhodamine Terminator Cycle Sequencing ReadyReaction Kit on ABI PRISMTM 310 Genetic Analyser.Sequences obtained were compared with the publishedsequence (GenBank Acc. No. L78833).RESULTSBetween 1994 and 1996 approximately 10% of thepatients diagnosed at the Kuala Lumpur Breast Clinicwere at or below the age of 35 while 2 - 5% of patientshad two or more relatives with breast and/or ovariancancer. Among the patients aged 35 years or below,none of them had any family history of cancer andonly two of the patients had bilateral breast cancer.Patients studied comprised of 23 patients who wereaged 35 or below of which two of them had a familyhistory of breast cancer, and seven patients agedbetween 40 to 70 with a history of breast and/orovarian cancer. An insertion was found in exon 21in nucleotide 5447 codon 1776 in a 32-year-old patientof the Malay origin (Fig. 1). This mutation is predictedto result in a premature stop codon at position 1829.The eight polymorphisms identified and the numberof cases in which they were identified are listed inTable II. All polymorphisms identified in patientswere from the three ethnic groups studied in exceptionof one.DISCUSSIONIn our study, 4.3% (1/23) of our breast cancer patientsaged 35 years or below had BRCA1 mutations. Thedetection of one frameshift mutation in this groupTable I. Age and family history of patients.Patient no. Age (years) Family history1 40 Mother, sister and maternal aunt (breast cancer)2 55 Sister and 3 maternal aunts (breast cancer);Father (stomach cancer)3 57 2 sisters (breast cancer)4 25 Paternal grandmother and 1 maternal aunt(breast cancer);1 maternal aunt (ovarian cancer)5 70 2 maternal aunt (breast cancer)6 45 Mother (breast cancer);Sister (ovarian cancer)7 50 2 sisters (breast cancer)8 49 2 maternal aunts and 1 maternal aunt’s daughter(breast cancer)Fig. 1 Electropherogram of the frameshift mutation, c.5447-5448insC of exon 21. The mutant sequence (mut) with the insertionof C underlined and the wild type sequence (wt) are shown.196 : 2002 Vol 43(4) Singapore Med Jcould explain the moderate role of BRCA1 in thepathogenesis of breast cancer in this age group.BRCA1 mutational studies conducted amongCaucasian patients from the same age group andwithout family history as in this study identifiedBRCA1 mutations in 6 (5.7%) of the 105 patients(18)and 12 (6.2%) of the 193 patients(6). These are quitesimilar as seen in our study.The frameshift mutation, c.5447-5448insC(insC5447) (codon 1776 of exon 21) was found in apatient aged 32 with non-cancer family history. Thepatient had a grade 3 cancer with negative oestrogenreceptor. Johnasson et al(19) found BRCA1 relatedtumours to be oestrogen receptor negative. Anotherstudy which analysed tumours of patients agedbelow 40 years observed that younger patients arelinked with adverse pathological features such asgrade 3 tumour, oestrogen receptor negative andlymphatic vessel involvement(20). In our patient therewere histologically positive nodes. The same mutationidentified in our patient had been identified in aDutch Caucasian study among familial breast cancerpatients(21). This truncating mutation would disrupt theinvolvement of BRCA1 in DNA binding and inprotein-protein interactions(22).Ethnicity differences and the small sample sizeamong our patients may contribute to the low numberof mutations identified in the study. The potentialfounder mutation, 589delCT reported among Chinesewas not found in our series of nine Chinese patients(12).In the Malay ethnic group, we studied 16 patients butdid not find the 2846insA and 2885delA mutationsfound among Malay patients in Singapore(13).Studies have shown that BRCA1 mutations arenot common among breast cancer patients with amodest history of breast cancer. A study of 169 casesof women with a history of one to 11 cases of breastcancer per family only reported 27 BRCA1 mutations(7).Malone et al(6) found BRCA1 mutations in 1.2% ofpatients (14 out of 312) with families having fewerthan four cases of breast cancer only, with no historyof ovarian cancer. In our study, it is possible that breastcancer among the four patients with at least threerelatives was not due to BRCA1 mutations. The lackof BRCA1 mutations among the breast cancerpatients with family history also may be due tomutations in the noncoding regions of BRCA1 andthe existence of other breast cancer susceptibilitygenes such as BRCA2 and PTEN. The presence ofmutations in the intron and noncoding regions ofthe gene would affect RNA transcription, splicing andstability. Swensen et al(23) identified a 14kb deletionthat removed both of the BRCA1 transcription startsites. In addition, mutational analysis using directsequencing could miss large deletions. Puget et al(24)identified large germline rearrangements amongAmerican and French breast cancer families thatdid not indicate BRCA1 mutation using classicaldetection techniques.Polymorphisms previously found in Caucasians(16,25)and also identified in Chinese(12): 2201C>T, 2430T>C,2731C>T, 3232A>G, 4227T>C and 4956A>G werefound in our series of patients. The IVS8-57delTpolymorphism found in Hispanics was detected inthe three ethnic groups among our patients(26). Ourfinding of at least one heterozygous polymorphism in20 of our 30 patients indicate that the deletion of theentire BRCA1 gene did not occur in these patients.ACKNOWLEDGMENTThe authors thank the Director of the Institute forMedical Research for permission to publish the findingsand Kaur B, Lim GCC, Chia KP, Lily M, Mak JW,Duraisamy G, Tan SK, Lim PKC as well as otherstaff of the Institute for Medical Research andKuala Lumpur Hospital for their support.REFERENCES1. Ministry of Health. Cancer. Ministry of Health Annual Report 1996.2. World Health Statistics. World Health Organisation 1998.Table II. Distribution of BRCA1 polymorphisms among the Malays, Chinese and Indians.Polymorphism Number of patients with Amino acid change Ethnic group with polymorphismpolymorphism/total tested2201C>T 4/30 silent Malay, Chinese2430T>C 18/30 silent Malay, Chinese, Indian2731C>T 19/30 P871L Malay, Chinese, Indian3232A>G 14/30 E1038G Malay, Chinese, Indian3667A>G 19/30 K1183R Malay, Chinese, Indian4427T>C 18/30 silent Malay, Chinese, Indian4956A>G 20/30 S1613G Malay, Chinese, IndianIVS8-57delT 16/30 – Malay, Chinese, IndianSingapore Med J 2002 Vol 43(4) : 1973. Hall JM, Lee MK, Newman B, Morrow JE, Anderson LA, Huey B, et al.Linkage of early-onset familial breast cancer to chromosome 17q21.Science 1990; 1684-9.4. Ford D, Easton DF, Stratton M, Narod S, Goldgar D, Devilee P, et al.Genetic heterogeneity and penetrance analysis of the BRCA1 andBRCA2 genes in breast cancer families. Am J Hum Genet 1998;62:676-89.5. Greenman J, Mohammed S, Ellis D, Watts S, Scott G, Izatt L, et al.Identification of missence and truncation mutations in the BRCA1gene in sporadic and familial breast and ovarian cancer. Genes,Chromosomes & Cancer 1998; 21:244-9.6. Malone KE, Daling JR, Thompson JD, O Brien GA, Franciso LV,Ostrander EA. BRCA1 mutations and breast cancer in the generalpopulation. Analysis of women before age 35 years and in womenbefore age 45 years with first-degree family history. J Am Med Asso1998; 279:922-9.7. Couch FJ, DeShano ML, Blackwood MF, Calzone K, Stopfer J,Campeau L, et al. BRCA1 mutations in women attending clinics thatevaluate the risk of breast cancer. New Eng J Med 1997; 336:1409-515.8. Simard J, Tonin P, Durocher F, Morgan K, Rommens J, Gingras S, et al.Common origins of BRCA1 mutations in Canadian breast and ovariancancer families. Nat Genet 1994; 8:392-8.9. Shugart YY, Cour C, Renard H, Lenoir G, Goldgar D, Teare D, et al.Linkage analysis of 56 multiplex families excludes the Cowdendisease gene PTEN as a major contributor to familial breast cancer.J Med Genet 1999; 36(9):720-1.10. Chen ST, Yu SY, Tsai M, Yeh KT, Wang JC, Kao MC, et al. Mutationanalysis of the putative tumour suppression gene PTEN/MMAC1in sporadic breast cancer. Breast Cancer Res Treat 1999; 55(1):85-89.11. Li SSL, Tseng HM, Yang TP, Liu CH, Teng SJ, Huang HW, et al.Molecular characterisation of germline mutations in the BRCA1 andBRCA2 genes from breast cancer families in Taiwan. Hum Genet1999; 104:201-4.12. Tang NLS, Pang CP, Yeo W, Choy KW, Lam PK, Suen M, et al.Prevalence of mutations in the BRCA1 gene among Chinese patientswith breast cancer. J Natl Cancer Inst 1999; 91:882-5.13. Ho GH, Phang BH, NG, ISL, Law HY, Soo KC, Ng EH. Novel germlineBRCA1 mutations detected in women in Singapore who developedbreast carcinoma before the age of 36 years. Cancer 2000; 89(4):811-6.14. Inoue R, Fujutomi T, Ushijima T, Matsumoto Y, Sugimura T, Nagao.M. Germline mutation of BRCA1 in Japanese breast cancer. IntJ Cancer 1995; 68:3521-4.15. Katagiri T, Emi M, Ito I, Kobayashi K, Yoshimoto M, Iwase T, et al.Mutations in BRCA1 gene in Japanese breast cancer patients. HumMut 1996; 334-9.16. Friedman LS, Ostermeyer EA, Szabo CI, Dowd P, Lynch ED,Rowell SE, et al. Confirmation of BRCA1 by analysis of germlinemutations linked to breast and ovarian cancer in ten families. NatGenet 1994; 8:399-404.17. Castilla LH, Couch, FJ, Erdos MR, Hoskins KF, Calzone K, Garber JE,et al. Mutations in the BRCA1 gene in families with early-onsetbreast and ovarian cancer. Nat Genet 1994; 8:387-91.18. Elena GP, Blanca G, Mariano P, Jose MS, Jose MG, Pilar E, et al.Germline BRCA1 mutations in women with sporadic breast cancer:clinical correlations. J Cli Onco 1998; 16(1):115-20.19. Johannsson OT, Idvall I, Anderson C, Borg A, Barkardottir RB,Egilsson V, et al. Tumour biological features of BRCA1 inducedbreast and ovarian cancer Eur J Cancer 1997; 33:362-71.20. Jonathan AH, Delray JS, Michael EP, Lawrence JS. An analysis of ageand family history on outcome of breast conservation treatment: theUniversity of Pennsylvania Experience. Cancer J Sci Am 1998; 308-15.21. van den Ouweland, personal communication.22. Scully R, Anderson SF, Chao DM, Wei W, Ye L, Young RA, et al.BRCA1 is a component of the RNA polymerase II holoenzyme.Proc Nat Acad Sci 1997; 94(11):5605-10.23. Swensen J, Hoffman M, Skolnick MH, Neuhausen SL. Identificationof a 14kb deletion involving the promoter region of BRCA1 in abreast cancer family. Hum Mol Genet 1997; 6(9):1513-7.24. Puget N, Stoppa-Lyonnet D, Sinilnikova OM, Pages S, Lynch HT,Lenoir GM, et al. Screening for germline rearrangements andregulatory mutations in BRCA1 led to the identification of fournew deletions. Can Res 1999; 59(2):455-61.25. Miki Y, Swensen J, Shattuck-Eidens D, Furtreal PA, Hashman K,Tavtigian S, et al. A strong candidate for the breast and ovarian cancersusceptibility gene BRCA1. Science 1994; 266:66-71.26. Nunes ME, personal communication.

Mutation analysis of the BRCA1 gene in Malaysian breast cancer patients

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Mutation analysis of the BRCA1 gene in Malaysian breast cancer patients

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AIR Universita degli studi di Milano