Novel germline mutations in BRCA2 gene among 96 ... - Springer Link

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We analyzed 102 Breast/Ovarian cancer patients from 96 breast and/ovarian cancer families for BRCA2 gene mutations using Conformation-Sensitive Gel ...
J Cancer Res Clin Oncol (2007) 133:867–874 DOI 10.1007/s00432-007-0229-6

ORIGINAL PAPER

Novel germline mutations in BRCA2 gene among 96 hereditary breast and breast–ovarian cancer families from Kerala, South India Vani Syamala · Leelakumari Sreeja · Volga S. Syamala · B. Vinodkumar · Praveenkumar B. Raveendran · Hariharan Sreedharan · Ratheesan Kuttappan · Lekshmi Balakrishnan · Ravindran Ankathil

Received: 14 December 2006 / Accepted: 23 March 2007 / Published online: 15 May 2007 © Springer-Verlag 2007

Abstract Purpose Aim of the present study was to identify the genetic heterogeneity, prevalence and frequency of germline mutations of BRCA2 gene in Hereditary Breast/Ovarian cancer patients from Kerala, South India. Methods We analyzed 102 Breast/Ovarian cancer patients from 96 breast and/ovarian cancer families for BRCA2 gene mutations using Conformation-Sensitive Gel Electrophoresis (CSGE) followed by sequencing. Results Sequence variations in BRCA2 gene were detected in 27 (26.4%) patients. Sixteen distinct sequence variants were detected of which 11 were (69%) in exon 11. We have identiWed two novel disease-causing frameshift mutations (c.4642delAA and c.4926insGACC) in two unrelated patients. Apart from this, fourteen distinct sequence variants were detected in 25 breast/ovarian cancer patients of which 8 (57%) were also novel. These include nine mis-

V. Syamala · L. Sreeja · V. S. Syamala · P. B. Raveendran · H. Sreedharan · R. Ankathil (&) Division of Cancer Research, Regional Cancer Centre, Thiruvananthapuram, Kerala 695011, India e-mail: [email protected] V. Syamala e-mail: [email protected] L. Sreeja e-mail: [email protected] B. Vinodkumar Department of Microbiology and Immunology, Emory Vaccine Center and Yerkes National Primate Research Center, Emory University School of Medicine, Atlanta, GA, USA R. Kuttappan · L. Balakrishnan Division of Radiation Oncology, Regional Cancer Centre, Thiruvananthapuram, Kerala 695011, India

sense mutations, one silent mutation, one-nonsense mutation and three intronic variants. Conclusions The results of this study suggest that germline mutations of BRCA2 gene account for rather small proportion of Hereditary Breast/Ovarian cancer in Kerala, South India. Keywords BRCA2 · Breast cancer · Ovarian cancer · Germline mutations · CSGE · HBOC Families

Introduction Breast cancer is the most common malignancy aVecting women world wide (Parkin 2004), accounting for 25% of all new cases of cancer. Inheritance of germline mutation(s) in cancer susceptibility gene(s) accounts for 5–10% of all breast and ovarian cancer, and a higher proportion of cancers associated with a strong family history of the disease. In the context of high-risk families, one important gene is the BRCA2 gene which was localized to chromosome 13q by linkage analysis in 1994 and cloned in 1995 (Wooster et al. 1994, 1995). Germline mutations in BRCA2 gene (GI: 4502450, OMIM# 600185) account for the genetic predisposition and increased risk for breast cancer in almost 35% of the families with inherited predisposition to this cancer (Gauthier-Villars et al. 1999). To date, more than 500 germline mutations have been identiWed within the BRCA2 gene (BIC database 1996). The majority of these mutations are nonsense mutations or frameshift mutations that generate premature termination codons (Rahman and Stratton 1998). Studies on highly selected families with at least four cases of breast cancer suggest that BRCA2 accounts for the majority of breast cancer families where males as well as females are aVected, for

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about one third of the families with female breast cancer alone, and only for a few families containing multiple cases of breast and ovarian cancer (Ford et al. 1998). Populationbased studies have deWned high- and low-risk subsets for developing breast cancer based on ethnic origin and have shown speciWc mutations associated with speciWc populations and also variable number of novel mutations in diVerent populations (Gayther et al. 1997). Despite the large number of BRCA2 gene mutations reported worldwide, only few reports on the prevalence of BRCA2 mutation in India are available which has come from northern states (Saxena et al. 2002, 2006; Valarmathi et al. 2004). But the contribution of these genes to breast/ ovarian cancer remains relatively unexplored among the South Indian populations and the only data available is from a very small series (only 22 cases) (Rajkumar et al. 2003). The present study was undertaken to determine the genetic heterogeneity and prevalence of germline mutations of BRCA2 gene in 102 breast/ovarian cancer patients from 96 breast and breast–ovarian cancer families from Kerala, South India.

Materials and methods

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collected from each proband after her/his written informed consent was obtained. This study has been approved by the Ethical Review Committee of Regional Cancer Centre, Thiruvananthapuram, Kerala, South India. DNA isolation and PCR ampliWcation Genomic DNA was extracted from peripheral blood lymphocytes with phenol–chloroform following digestion with Proteinase K (100 mg/ml) in the presence of 0.1 M EDTA and 0.5% SDS at 55°C for 12 h, and recovered by ethanol precipitation (Sambrook et al. 1989). The entire coding region (exons 2–27) and exon–intron junctions of BRCA2 were PCR ampliWed. Coverage of this region was accomplished using 44 PCR primer sets, which generated products ranging from 164 to 591 bp in length (Table 1). Generally, the PCR assay was carried out in a volume of 25 l containing 100–200 ng of genomic DNA, 100 M dNTPs (Bioenzyme), 1£ PCR buVer (Bangalore Genei), 1.5–3 mM MgCl2, 0.5 U of Taq DNA Polymerase (Bangalore Genei) and 10 pmol of each primer (Microsynth). AmpliWcation was for 35–40 cycles in an Applied Biosystems 2700 thermal cycler, with each cycle consisting of 1 min at 94°C, 45 s at TA, and 30 s at 72°C, with a 10 min extension at 72°C following the last cycle.

IdentiWcation of breast and breast–ovarian cancer families Conformation sensitive gel electrophoresis (CSGE) Breast and/or ovarian cancer patients referred to the outpatient clinic of Regional Cancer Centre, Thiruvananthapuram, Kerala were selected for mutation analysis of the BRCA2 gene if they fulWlled any of the following inclusion criteria: (a) Families with two cases of breast and/or ovarian cancer diagnosed under age 50 years in Wrst-degree relatives, (b) families with one case of breast cancer diagnosed under age 50 years and one case of ovarian cancer diagnosed at any age in Wrst degree relatives, (c) families with four cases of breast or ovarian cancer diagnosed at age under 60 years among Wrst and second degree relatives, (d) sporadic patients diagnosed with breast or ovarian cancer the age of 38 years, (e) sporadic patients diagnosed with multiple primary breast cancers or concomitant breast and ovarian cancer before age of 60 years, and (f) Sporadic patients with male breast cancer diagnosed at any age. Among the 102 patients meeting these criteria, four probands were diagnosed with ovarian cancer at the ages of 36, 46, and 46 and 49 years, four were male breast cancer cases and the rest were all female breast cancer patients. For all families, a detailed pedigree was constructed through an index case and available related family members. All information concerning the family history of cancers, age at onset, either ages at death or current ages and other clinical parameters were obtained by personal interview of the index case and most relatives. Blood samples were

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For mutation analysis of BRCA2 coding sequence, we adopted CSGE (Ganguly et al. 1993) method as described below. PCR products were denatured by heating at 98°C for 5 min and reannealed at 68°C for 1 h to generate heteroduplexes. These samples were loaded on 1 mm thick 10% polyacrylamide gel containing 1, 4 bis acryloyl piperazine (Fluka) as cross linker and ethylene glycol (Sigma) and formamide (Sigma) as denaturants. The gel was run in Tris Taurine EDTA buVer for 16 h at 400 V. The gels were stained with ethidium bromide (1 g/ml) for 10 min and destained for 10 min in double distilled water. PCR products were visualized by ultra violet light with the aid of gel documentation system (Bio-Rad). Samples displaying abnormal CSGE proWles compared to that of controls were identiWed. Direct sequencing of PCR products Potential sequence variants identiWed by altered electrophoretic mobility in CSGE analyses were reampliWed from genomic DNA; Amplicons were puriWed using QIA quick PCR puriWcation kit (Qiagen) and subjected to sequence analysis using an ABI BigDye terminator kit and a Prism 377 automated DNA sequencer (Applied Biosystems). All mutations were conWrmed by sense and antisense strand

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Table 1 Primer sequences used for PCR ampliWcation of BRCA2 gene sequence Exon

Primer sequences (5⬘-3⬘)

Base Pair

2

F CTC AGT CAC ATA ATA AGG AAT R ACA CTG TGA CGT ACT GGG TTT T

256

3

F TCT GGG TCA CAA ATT TGT CTG TCA R TTC CTA GTT TGT AGT TCT CCC CAG TC

356

4

F AGA ATG CAA ATT TAT AAT CCA GAG TA R AAT CAG ATT CAT CTT TAT AGA ACA AA

249

5

F AAC AAT TTA TAT GAA TGA GAA TC R AAT TGT TAA GTT TTA TTT TTA TTA

220

6

F CCA CAA AGA GAT AAG TCA GGT A R TGT AAA TCT CAG GGC AAAGGT A

234

7

F TAA GTG AAA TAA AGA GTG AA R AAC AGA AGT ATT AGA GAT GAC

275

8

F AAT AGT AGA TGT GCT TTT TGA R ACA TAT AGG ACC AGG TTT AGA GAC

285

9

F CTA GTG ATT TTA AAC TAT AAT TTT G R GTT CAA CTA AAC AGA GGA CT

164

10.1

F TGC CAA GTA CTC AGA ATA ACC C R ATC ATT TGG TTC CAC TTC AG

440

10.2

F CCA CAT TGG AAA GTC AAT GC R TGG TAG GCT AGA AAT ACG TGG C

517

10.3

F TCA GGT CAT ATG ACT GAT CCA A R AAA CAC AGA AGG AAT CGT CAT C

496

10.4

F TCA GGT CAT ATG ACT GAT CCA A R AAA CAC AGA AGG AAT CGT CAT C

416

11.1

F ATT TAG TGA ATG TGA TTG ATG G R TGA TTC TTT GCC TCT AGA AA

521

11.2

F CAA AAG TGG AAT ACA GTG ATA C R TCT GTT TCA TGA AGT TCC TT

507

11.3

F TTC AAA AAT AAC TGT CAA TCC R TCT TTG AAG AAC ATT TTG CT

488

11.4

F ACA AAT GGG CAG GAC TCT TAG G R TCT GCA TTC CTC AGA AGT GG

508

11.5

F GAA TCA GGA AGT CAG TTT GA R TAT CAG TTG GCA TTT ATT ATT TTT

521

11.6

F TGA GGA AAC TTC TGC AGA GG R ACA TGC TTC TTG AGC TTT CG

453

11.7

F AAG ATG AAA CGG ACT TGC TA R TGT ATG AAA ACC CAA CAG AGT AGG

526

11.8

F GAA AGA ACA AAA TGG ACA TTC T R TGG CAC CAC AGT CTC AAT AG

424

11.9

F CCC TAA AGT ACA GAG AGG CC R CGG AGA GAT GAT TTT TGT CA

503

11.10 F ACT TGA AGC AAA AAA ATG GC R CCA CTG GCT ATC CTA AAT GC

488

11.11 F AAA TGA AGA TAT TTG CGT TGA R GAC TGA CTT ATG AAG CTT CCC

495

11.12 F GTT TTT GCT GAC ATT CAG AG R CAA ATT CCT CTA ACA CTC CC

492

11.13 F ATA TCC CAA AAA GGC TTT TC R TCA CAG GAA CAT CAG AAA AA

498

11.14 F AAC AAG ACA AAC AAC AGT TGG R AGC ATA CCA AGT CTA CTG AAT AAA C

438

Table 1 continued Exon Primer sequences (5⬘-3⬘)

Base Pair

12

F AGG TCA CTA TTT GTT GTA AG R AGT GGC TCA TGT CTG TAA T

358

13

F TAA AGC CTA TAA TTG TCT CA R CTT CTT AAC GTT AGT GTC ATT

271

14

F TGC AAC AAA GGC ATA TTC CT R CAA AGG GGG AAA ACC ATC AG

591

15

F GGC CAG GGG TTG TGC TTT TT R AGG ATA CTA GTT AAT GAA ATA

314

16

F TTT GGT AAA TTC AGT TTT GGT TT R AAC ACA CAA TCT TTT TGC ATA GA

330

17

F CAG AGA ATA GTT GTA GTT GTT GAA R AGA AAC CTT AAC CCA TAC TGC

306

18

F GTG ACT TGT TTA AAC AGT GGA A R ATT GAG CAT CCT TAG TAA GCA

500

19

F AAG TGA ATA TTT TTA AGG CAG TT R TAT ATG GTA AGT TTC AAG AAT

296

20

F CAC TGT GCC TGG CCT GAT AC R ATG TTA AAT TCA AAG TCT CTA

296

21

F GGG TGT TTT ATG CTT GGT TCT R CAT TTC AAC ATA TTC CTT CCT G

304

22

F TTT TGT TCT GAT TGC TTT TTA TTC R AAT CAT TTT GTT AGT AAG GTC AT

314

23

F CCA CTA CTA ATG CCC ACA AA R AAA ACA AAA CAA AAA TTC AAC ATA

362

24

F CAG TTT TGA TAA GTG CTT GTT R AGC TCC AAC TAA TCA TAA GA

290

25

F TTA GAG TTT CCT TTC TTG CAT C R AAG CTA TTT CCT TGA TAC TGG A

470

26

F AAG GAA ATA CTTTTG GAA ACA TAA R TTT ACT AGG TAT ACA ACA GAA

299

27.1

F TAG GAG TTA GGG GAG GGA GAC TGT GT R CAA GGC TCT TCT CTT TTT GC

300

27.2

F CTG TCTC AGC CCA GAT GAC T R TGT TGA ACC AGA CAA AAG AGC

350

27.3

F TCA ATG AAA TTT CTC TTT TGG A R TGT GTG GTT TGA AAT TAT AAT C

300

sequencing. Sequence variants were designated according to recommendations of the HUGO Nomenclature Working Group, using the sequence listed in GenBank accession #U43746 as a reference.

Results Out of the 102 patients selected for germline BRCA2 mutation analysis, 94 women presented Breast cancer, 4 women presented ovarian cancer and 4 were male breast cancer patients. The breast/ovarian cancer cases ranged from ages 22 to 79 years with a mean age of 42.88 years (Median: 41.50 years). Mutation analysis of the coding region and

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intron–exon boundaries of BRCA2 gene in 102 hereditary breast/ovarian cancer patients from 96 independently ascertained breast/ovarian cancer families showed sequence variations in 27 patients (26.4%). A total of 16 distinct germline BRCA2 sequence variants were detected in these patients, including 10 novel variants (Tables 2, 3). Among the 16 sequence variants, 11 (69%) were detected in exon 11 of the BRCA2 gene.

Table 2 BRCA2 disease associated germline mutations identiWed in South Indian breast and breast–ovarian cancer families Sl Family Age Exon no:

Nucleotide

Predicted eVect

BIC entry

1

BF15

43

11

c.4642delAA

STOP 1480

Nil

2

BF11

55

11

c.4926 insGACC STOP 1575

Nil

Disease associated BRCA2 mutations Out of the 16 sequence variants detected, two were distinct deleterious germline mutations, identiWed in 2/102 (1.9%) patients (Table 2). These two pathogenic mutations included two fraeshift mutations, c.4642delAA (Fig. 1) and c.4926insGACC. The frame shift deletion mutation [c.4642delAA] was detected in the BRCA2 exon 11 of a proband who developed breast cancer at the age of 43 and was from a breast cancer only family in which the proband’s mother was deceased with breast cancer at age 59. The second pathogenic mutation was a frame shift insertion mutation, also in exon 11 [c.4926insGACC]. It was detected in a proband who developed breast cancer at the age of 55, and was also from a Breast cancer only family in which the proband’s sister (age 38) was aVected with breast

BF Breast cancer only Family, Bold face novel mutation

Table 3 BRCA2 sequence variants of unclassiWed signiWcance identiWed in South Indian breast and breast–ovarian cancer families Sl Family Age Exon/ No: intron

Nucleotide

Predicted EVect

BIC Entry

1

BF45

33

11

c.3257G > A

R1010K

Nil

2

BF57

28

3

BOF61 45

4

BF62

39

5

BF87

39

11

c.3447G > A

Q1073Q

Nil

6

BF45

33

11

c.3578T > C

I 1117 T

Nil

7

BOF03 37

11

c.4486G > T

D1420Y

Yes

8

BF16

38

9

BF67

36

10

BF02

40

11

c.4501G > A

D1425N

Nil

11

BOF30 68

11

c.5007A > C

E1593D

Yes

12

BF29

30

11

c.5206C > T

P1660S

Nil

13

BOF32 41

11

c.5299A > C

K1691Q

Nil

14

BF74

60

11

c.5332C > T

P1702S

Yes

15

BF54

40

18

c.8415G > T

K2729N

Yes Yes

16

45

17

BF82

52

27

c.10204A > T

K3326X

18

BF52

54

Intr8

c.909 + 56C > T

Non coding Yes

19

BF53

44

20

BF55

38

21

BF56

79

22

BF45

33

Intr10

c.2137 + 22delT Non coding Nil

23

BF65

67

24

BF82

52

25

MBF

61

Intr 24 c.9729¡17T > C Non coding Nil

BOF Breast ovarian cancer family, MBF male breast cancer family, BF breast cancer only family, Bold face novel mutation

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Fig. 1 A representative gel picture and electropherogram of a BRCA2 Pathogenic mutation. a An ethidium bromide stained gel picture of CSGE showing BRCA2 pathogenic mutation c. 4642delAA. Abnormal migration pattern can be seen in index case P15. b Sequence analysis of sample showing altered migration conWrmed the presence of c. 4642delAA. c Sequence analysis of the corresponding wild genotype

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cancer and her mother was deceased due to breast cancer at age 65. Sequence variants in HBC/HBOC families Fourteen sequence variants were detected in 25 breast/ovarian cancer patients: 9 missense mutations, one silent mutation, one nonsense mutation and 3 sequence variations in introns 8,10 and 24 (Table 3). Out of the nine nucleotide substitutions that encoded missense aminoacid changes, Wve BRCA2 missense mutations [c.3257G > A (p.R1010K), c.3578T > C (p.I1117T) (Fig. 2), c.4501G > A (p.D1425N), c.5206C > T (p.P1660S) and c.5299A > C (p.K1691Q)] have not been previously reported either in BIC database or published elsewhere, and hence are considered to be novel. Three missense alterations were recurrently found among our study subjects. Two missense variants in exon 11, c.3257G > A (p.R1010K), c.4486G > T (p.D1420Y) were found in four and three patients, respectively. Another nucleotide substitution in exon 18, c.8415G > T (p. K2729N) was observed in a breast cancer only family (BF 54) where two sisters aVected with breast cancer appeared to harbor the mutation. Also we detected a silent mutation p.Q1073Q (c.3447G > A) in one patient and two intronic variants [c.2137 + 22delT, c.9729-17T > C] that have not been previously reported. The intronic variant c.9729-17T > C was

Fig. 2 A representative gel picture and electropherogram of a BRCA2 Sequence variant. a An ethidium bromide stained gel picture of CSGE showing BRCA2 sequence variant c. 3578T > C. Abnormal migration pattern can be seen in index case P45. b Sequence analysis of sample showing altered migration conWrmed the presence of c.3578T > C. c Sequence analysis of the corresponding wild genotype

871

detected in a male breast cancer patient and also in a female breast cancer patient in intron 24. A nonsense mutation K3326X was detected in exon 27 of 1 patient, and an intronic variant c.909 + 56C > T was detected in the intron 8 of 4 patients.

Discussion Mutation analysis of BRCA2 gene in 102 breast/ovarian cancer patients from 96 diVerent breast ovarian cancer families has identiWed 16 distinct germline alterations in 27 individuals (26.47%). To the best of our knowledge, these results represent the largest number of BRCA2 sequence variants published so far in Indian HBOC families. The two pathogenic frame shift BRCA2 mutations detected in our cases were c.4642delAA and c.4926insGACC. The c.4642delAA is a frame shift deletion mutation, which changes the reading frame of mRNA and causes a premature termination codon at position 1,480. The c.4926insGACC is a frame shift insertion mutation, which results in the ceasing of the translation of the BRCA2 protein at codon 1575, leading to the production of a truncated BRCA2 protein. These two disease causing germline mutations were identiWed in 2.08% [2/96] of breast/breast ovarian cancer families analyzed. This mutation frequency is in agreement with previous reports in other populations (Chen et al. 1998; Yazici et al. 2000; Salazar et al. 2006). But studies on several other populations had reported comparatively higher pathogenic mutation frequencies for BRCA2 gene (Austria 8% by Wagner et al. 1999; Dutch 12% by Peelen et al. 2000; Germany 12% by Hamann et al. 2002; Portuguese 7% by Peixoto et al. 2006). However, contradictory results among these studies might probably be because of diVerences in the characteristics of the patient population analyzed, and criteria used for the selection of these families. Variation in the sensitivity of the screening techniques might also have accounted for the diVerences in the reported results. Additional variability in the results of population-based studies might also be attributed to the variable contribution of founder mutations in diVerent ethnic populations (Thorlacius et al. 1998; Syrjakoski et al. 2000; Loman et al. 2001; Shih et al. 2002). Besides true pathogenic mutations, we identiWed fourteen (nine missense mutations, one silent mutation, one nonsense mutation and three intronic sequence variants) genetic variants of uncertain signiWcance. Although truncating mutations may be assumed to cause disease, pathogenicity involving missense/unclassiWed variants is more equivocal because of insuYcient information concerning both protein function and genetic variation. A sequence variant that leads to a stop codon was identiWed in exon 27. The very same missense mutation

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10204A > T[K3326X] was identiWed previously with a frequency of 0.02% in a US control population, and was not associated with an increase of susceptibility to Breast cancer and ovarian cancer (Mazoyer et el. 1996). Another sequence variant in exon 18 (c.8415G > T) resulting in a substitution of Lysine to Asparagine at codon 2729 (K2729N) was identiWed in two patients in the present study. This same type of missense alteration has been reported earlier in one young breast cancer case and six patients belonging to Wve families (Zhi et al. 2002). Further, this substitution is non-conservative and occurs in an amino acid residue located in the highly conserved BRCA2 COOH domain. In our study, the two breast cancer patients identiWed to harbor this mutation belonged to the same family (BF53). These observations are suggestive of disease association which is further supported by the absence of this variant in the previously studied Indian control samples (Saxena et al. 2002, 2006; Valarmathi et al. 2004). The missense variant c. 5332 C > T (p.P1702S) obtained in our study was a rare alteration, reported only once at the BIC database and the ethnicity has not been speciWed. The c.4501G > A missense change in exon 11 which results in the substitution of asparagine for aspartic acid at codon 1425 (D1425N) may aVect the conformation of the protein, since an acidic residue is replaced by a polar uncharged residue. Similarly, the c.5299A > C missense change in the same exon which results in the substitution of lysine for glutamine at codon 1691 (K1691Q), may also aVect the conformation of the protein, since a polar uncharged residue replaces a residue that is basic. However, the missense variant c.5007A > C obtained in the present study has previously been reported in two breast cancer patients from North India (Saxena et al. 2002). Although Wagner et al. (1999) identiWed this variant as a neutral polymorphism, the fact that it was found only in one breast cancer patient (age: 68 years) in the present study suggest the possibility of local variation in ethnic-speciWc alteration of the BRCA2 gene (Khoo et al. 2002). However, further studies of this variant are warranted to establish whether it is disease associated. Interestingly, 57% (8/14) BRCA2 sequence variants identiWed were novel, all (except intronic variants) localized in exon 11. Five of the novel BRCA2 germline alterations (including the two truncation mutations) described in this study are located in the short BRC repeat sequences in exon 11. Using in vitro studies, it has been shown that BRCA2 binds directly to RAD51 through 6 of the 8 BRC repeats (BRC 1, 2, 3, 4, 7, and 8 motifs) (Wong et al. 1997) that are highly conserved between species (Bignell et al. 1997). Therefore, inactivating mutations in the RAD51 binding sites may be responsible for breast and ovarian cancer predisposition in these families. However, biological signiWcance of these missense mutations has been proven

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since neither segregation analysis nor functional analysis was done in our study. Moreover, a very high number of properly selected control individuals would be needed to search for statistically signiWcant association of these alleles with breast/ovarian cancer. Our laboratory is currently involved in evaluating the frequency of these alterations that could contribute to the development of breast/ovarian cancer, in control population. For BRCA2, several studies (Gayther et al. 1997; Thompson and Easton 2001) revealed an increased ovarian to breast cancer ratio for ovarian cancer cluster region (OCCR) mutations. On the other hand, several other investigators (Frank et al. 1998; Ikeda et al. 2001; de la Hoya et al. 2002; Claes et al. 2004) failed to demonstrate an increased incidence of ovarian cancer in the BRCA2 OCCR. Interestingly all our identiWed BRCA2 alterations in exon 11 (16 families) were located with in the OCCR but only 4 families had cases of ovarian cancer whereas the remaining 12 families were site speciWc breast cancer families. Our result, which is in agreement with an earlier report from North India (Valarmathi et al. 2004), suggest that there exists a diVerence in susceptibility of BRCA2 mutation carriers to ovarian cancer between Indians and other populations. Male breast cancer risk in BRCA2 mutation carriers has been reported to be 6% by 70 years age (Easton 1997). Several studies suggest a correlation between Male breast cancer and BRCA2 mutations (Friedman et al. 1997; Frank et al. 1998; Ellisen and Haber 1998). Linkage studies indicate that more than 80% of families with male breast cancer are related to BRCA2 mutations (Ford et al. 1998; Thompson and Easton 2001). However, none of the four male breast cancer patients included in our study showed BRCA2 mutation (except an intronic alteration in family MBF88, Table 3). Few other investigators also failed to demonstrate such association (Hamann et al. 2002). It is presumed that some other genes (other than BRCA2) may be involved in the pathogenesis of male breast cancer in these families. In summary, our study identiWed two novel pathogenic mutations as well as fourteen distinct genetic alterations in BRCA2 gene in 96 HBOC families, indicating a somewhat smaller attributable fraction of cases in Kerala population than in other populations studied. A true lower frequency of mutations could be the result of several factors, including a lower penetrance of BRCA2 mutations due to the surrounding environmental and hormonal milieu, a lower frequency of mutations, and a diVerent spectrum of mutations, supporting the notion that other genes may be important determinants of familial risk. It is necessary to establish the simultaneous eVects of environmental factors and of other tumor-associated genes in modifying the impact of inherited cancer predisposition genes in breast cancer patients. We and others are continuing such eVorts that will lead to

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more accurate risk assessment and more eVective control initiatives among Indian women at high risk for breast and ovarian cancer. Acknowledgments We are deeply indebted to the patients who accepted to participate in this study. The Wrst author is a recipient of Senior Research Fellowship from Indian Council of Medical Research (ICMR).

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