The Contribution of the BRCA1 and BRCA2 Genes to Hereditary Breast Cancer and Ovarian Cancer

1. The Contribution of the BRCA1 and BRCA2 Genes to Hereditary Breast Cancer and Ovarian Cancer in Israel Tieling Wang Department of Human Genetics Hadassah Hebrew University Hospital, Israel

2. New cases of cancer in women in Israel (1993), adjusted per age (x10-5) % 30 Others Breast cancer 25 80.9 20 Colon cancer 15 Brain tumors Ovarian cancer 30.2 Uterus cancer 10 Lung cancer Lymphoma Melanoma 5 13.4 11.1 11.0 10.8 9.5 9.1 S2: The incidence of BC is the most highest one and the incidence of OC is the third one among all the cancers in Israel.

4. New Breast Cancer Cases in Israel (1993), According to Country of Birth Place of birth Israel America and Europe Asia Africa non-Jewish (Arabs) No. 442 1166 262 206 70 Rate* 89.9 89.8 70.7 55.6 24 * adjusted per age (x10-5)

5. The lifetime risk of a Jewish woman living in Israel to develop breast cancer is 1 in 9

6. Risk Factors for Breast Cancer Excess of estrogen: early menarche (<14) late birth (>30) or no birth late menopause (>55) hormone therapy Relative risk 1.3 1.9 1.5 1.4 Family history: one 1st degree relative with BC <50 2.0 two 1st degree relative with BC <50 4.0-6.0 5

7. Familial Breast & Ovarian Cancer 3 50y 50y 35y 58y 43y 2 Breast cancer Thyroid 20y Ovarian cancer

8. Breast and Ovarian Cancer Susceptibility Gene BRCA1 • Linkage analysis with early onset breast cancer • families, BRCA1was localized to chromosome 17q • and the sequence was identified in 1994. • Mutations in BRCA1 gene responsible for breast • cancer, ovarian cancer, prostate cancer and colon • cancer.

9. Breast and Ovarian Cancer Susceptibility Gene BRCA2 • Some families with early onset breast cancer showing • linkage evidence against BRCA1, lead to second breast • cancer susceptibility gene BRCA2 was identified and • was localized to chromosome 13q, the sequence was • completely identified in 1996. • Except for breast cancer and ovarian cancer, mutations • in BRCA2 gene responsible for male BC and pancreas • cancer.

10. Function domains of BRCA1 and BRCA2

11. Suggested role for BRCA1 and BRCA2 1. Transcriptional regulation 2. DNA repair associated with Rad51 and maintenance of genome integrity 3. BRCA1&2 are essential for a normal cell proliferation in early embryogenesis.

13. Mutation types and distribution • Several hundreds mutations were identified in • BRCA1&BRCA2 genes. • The majority of the cancer-predisposing mutations • result in protein truncation. These include small • deletions or insertions, nonsense, splicing sites and • gross rearrangement. • The mutations in both BRCA1 and BRCA2 are scattered • throughout the genes with no evidence of hot spots.

14. Some founder mutations were found in different • populations. In Iceland, a founder mutation 999del5 in BRCA2, accounts for more than 75% of BC families with more than 4 cases. In Finland, 6 founder mutations in BRCA1 and 5 in BRCA2 were identified. In Israel, 3 founder mutations were identified in Ashkenazi Jews.

15. PENETRANCE Cancer risk at age 70 associated with germ-line mutations in BRCA1 and BRCA2

16. BRCA1/2 founder mutations in Ashkenazi BC/OC patients BRCA1 (17q) 1. 185delAG, exon 2 2. 5382insC, exon 20 BRCA2 (13q) 3. 6174delT, exon 11

17. Frequencies of BRCA Carriers in the general Ashkenazi Population BRCA1 185delAG 1.05% 5382insC 0.11% BRCA2 6174delT 1.36% Total: 2.5% (1 in 40)

18. Carrier frequency 1 in 40 No. of Ashkenazi females in Israel ~1 000 000 Female carriers n=25 000

19. The Contribution of the BRCA Mutations (185delAG, 5382insC, 6174delT) to Breast & Ovarian Cancer Morbidity in Ashkenazi Patients BC&OC OC BBC BC <40y BC Healthy individuals % 83 53 33 30 11 2.5 17

20. The Aims of This Study To identify cancer predisposing mutations in BC/OC patients in BRCA1 & BRCA2 genes.

21. The study group

22. The study group

23. BRCA1 and BRCA2 Genome organization BRCA1 BRCA2 Methods for mutation analysis DNA Sequencing Screening methods Single Strand Confirmation Polymorphism (SSCP ) Restriction Endonuclease Fingerprinting (REF) 1863 Amino acids 3418 Amino acids

24. SSCP: Single Strand Conformation Polymorphism SSCA: Single Strand Conformation Analysis normal allele mutant allele denaturation (heat & formamide) dilution separation on MDE gel

25. REF: Exons 10 & 11 in the BRCA2 Gene: Ex10 Ex11A-F Ex11AF-J Ex11J-P Ex11P-U Ex11U-Z 1221bp 1160bp 1123bp 1193bp 1324bp 1327bp

26. Restriction Endonuclease Fingerprinting (REF) Principle of the Method Mbo II + Hinf I BRCA2: Exon 11 U-Z (1327bp)

27. Identification of a Mutation (4093delAAAT ) in BRCA2 Exon 11K by the REF method Normal Mutant SSCP of Ex11K The REF of fragment Ex 11J-P

28. Nonsense mutation Missense mutation Nucleotide change in introns The mutations and sequence variants in BRCA1&2 genes 3053 T G BRCA1 IVS18+66 CA 4965AG 3232AG 3667AG 2430 TC IVS 8-57 7T 6T IVS16-68 AG 2731 CT IVS16-92 AG 5553CG 8558delA 8765delAG 4093delAAAT BRCA2 IVS16-14 T C IVS8+56 C T 5972 CT 3624 AG 1593 AG 3326 A T IVS21+1470 new EcoR I site 5426 CT IVS2-7 T A 3199 AG IVS14+53 C T 1342 AC Silent mutation

29. RESULTS The mutations were identified in study group

30. The 8765delAG Mutation in Exon20 in BRCA2 gene SSCP

31. Mutation carriers in breast cancer family 10 Prostate Ca 60yr. * * * * * * BC 46yr. BC 31yr. BBC 42yr. BC BC 36yr. BsmA I restriction analysis

32. 8765delAG in Exon 20 of BRCA2 * Themutation was found in 3 unrelated Jewish families of Yemenite origin The mutation was found in 1: 140 Jewish healthy individuals of Yemenite origin The mutation was not found in 68 Jewish breast cancer patients with positive family history or early age at diagnosis (<30yr.) of Ashkenazi or Sephardic origin * * 8765delAG is a founder mutation in Jews from Yemen

33. Haplotype analysis of BRCA2 8765delAG mutation carriers in French Canadian and Yemenite Jewish hereditary breast cancer families Diseased associated haplotypes in BRCA2 8765delAG mutation carriers The haplotype of French Canadian families 4-3-11-2-C-C-4-9 The haplotype of Yemenite Jewish families 7-3- 2- 2-T-C-4-6 8765delAG mutation arose independently in French Canadian and Yemenite Jewish populations. Conclusion

34. Are missense mutations can be disease-causing? The frequency of the mutation in the general population. 2. Co-segregation of the mutation with the disease. 3. Gene expression.

35. The effect of the missense mutation T1915M on the expression of BRCA2 Ex12 Ex13 Ex14 Ex11 U T1915M Ex11UF Ex14R

36. The RT-PCR of T1915M mutation carrier in BRCA2 gene

37. Normal GA Mutant The sequence variants T1915M in BRCA2 didn`t affect the transcription.

38. Detection of gross rearrangements Deletions and duplications • PCR-based methods such as SSCP or REF are likely • to miss Gross rearrangements. • Gross rearrangements can be detected by Southern • hybridization.

39. BRCA1 1026 bp Ex11 Ex1-11 1653 bp 3032 bp Ex11-24 Hind III / Ex11 Hind III / Ex11-24 Southern Hybridization in BRCA1 Hind III / Ex1-11

40. BRCA2 Southern Hybridization in BRCA2 NS Ex23-27 Ex10 Ex18-22 Ex12-17 Ex2-9 Ex11

41. Kb N N N M Kb N N M M 9.5 9.0 Southern Hybridization EcoRI/exon11 (BRCA2) 7.3 6.1 5.6 4.3 3.9 3.6 3.6 2.9 2.3 NS (L+R) NS-R 5‘ 3‘ 3362bp 2175bp 4931bp 11 12 13 EcoR I EcoR I EcoR I L R E x 1 1 N S 6.1kb 9.5kb

42. Identification of the deletion position deletion 5’ 3’ 4931bp 3362 bp 2175 bp 7967 bp 13 14 15 16 11 12 Ban II Ban II Ban II Ban II NS Ex11 (F)79951 IVS13 (R)87771

43. Identification of the deletion mutation: Long PCR Long PCR: Ex11 (F)79951 IVS13 (R)87771 Normal: 7828 bp deletion Del mutant: ~1700 bp Deletion of ~ 6Kb Including exons 12 & 13

44. PolyT/PolyA Identification of the deletion breakpoints 81181-agactccgtctcaaaaaaaaaaaaaaa-81207------------- (50a)---------- 87421-gaaattaaaaatatgat insertion deletion (81207) (87421) Nucleotide No. 6213bp 3’ 5’ 4931bp 13 14 11 12 Ban II Ban II Ban II The size of the deletion is 6.2 kb which include of exons 12 &13, there is an insertion of about (A/T)50 at the break point junction.

45. Pedigree of the deletion/insertion mutation carrier family Esophgeal Ca 129-3 BC (60y) 129-1 BC (43y) 129-2 BC (54y)

46. The Study of Promoter regions in BRCA1&BRCA2 genes • BRCA1&BRCA2 promoter regions were screened • for mutations by REF and Southern hybridization and • no mutations were identified. • Three polymorphisms were identified in BRCA2 • promoter area.

47. SUMMARY 50% 11% 6.3% 10%

48. The mutation frequency in the study group Jewish BC&OC • patients is 8.6% (6/70). • The mutation frequency of non-Ashkenazi Jewish BC&OC • patients is 16.7%(4/24). • The mutations frequency of Ashkenazi Jewish BC&OC • patients is 4.3% (2/46). • In Ashkenazi Jew the impact of BRCA1&2 genes on BC&OC • patients is mainly due to the “ 3 founder mutations”, other • mutations that were identified are mainly “ private mutations”.

49. No mutations were identified in 35 BC/OC patients • with a definite family history which indicated other • BRCA gene exist or they were affected by other factors.

50. Acknowledgements