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

New cases of cancer in women in Israel (1993), adjusted per age (x10 -5 ) Breast cancer Colon cancer Ovarian cancer Melanoma Brain tumors Lymphoma Lung cancer Uterus cancer Others % 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.

New Breast Cancer Cases in Israel (1993), According to Country of Birth Place of birth Israel America and Europe AsiaAfrica non-Jewish (Arabs) No Rate* adjusted per age (x10 -5 ) * adjusted per age (x10 -5 )

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

Risk Factors for Breast Cancer Excess of estrogen: early menarche (<14) late birth (>30) or no birth late menopause (>55) hormone therapy Relative risk Family history: one 1st degree relative with BC <502.0 two 1st degree relative with BC <

58y Breast cancer Ovarian cancer 43y 2 Thyroid20y 50y 50y 50y 3 35y Familial Breast & Ovarian Cancer

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 Mutations in BRCA1 gene responsible for breast cancer, ovarian cancer, prostate cancer and colon cancer.

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 Except for breast cancer and ovarian cancer, mutations in BRCA2 gene responsible for male BC and pancreas cancer.

Function domains of BRCA1 and BRCA2

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

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.

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.

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

BRCA1/2 founder mutations in Ashkenazi BC/OC patients BRCA1 (17q) delAG, exon insC, exon 20 BRCA2 (13q) delT, exon 11

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)

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

Healthyindividuals % BC BC<40y BBC BC&OC OC The Contribution of the BRCA Mutations (185delAG, 5382insC, 6174delT) to Breast & (185delAG, 5382insC, 6174delT) to Breast & Ovarian Cancer Morbidity in Ashkenazi Patients 17

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

The study group

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

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

1221bp 1160bp 1123bp 1193bp 1324bp 1327bp Ex10Ex11A-F Ex11AF-J Ex11J-P Ex11P-U Ex11U-Z REF: Exons 10 & 11 in the BRCA2 Gene:

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

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

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

The mutations were identified in study group RESULTS

The 8765delAG Mutation in Exon20 in BRCA2 gene SSCP

BsmA I restriction analysis Mutation carriers in breast cancer family 10 ***** * Prostate Ca 60yr. BC46yr.BC31yr.BCBBC42yr.BC36yr.

8765delAG in Exon 20 of BRCA2 The mutation 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

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

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

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

The RT-PCR of T1915M mutation carrier in BRCA2 gene

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

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.

BRCA bp Ex bp Ex bp Ex11-24 Hind III / Ex11 Hind III / Ex11-24 Hind III / Ex1-11 Southern Hybridization in BRCA1

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

Southern Hybridization EcoRI/exon11 (BRCA2) 3‘ Ex11NS RL 5‘ 6.1kb9.5kb 3362bp2175bp bp EcoR I NS (L+R) N N N M Kb NS-R N N M M 3.6 Kb

’5’ Ban II 4931bp3362 bp 2175 bp7967 bp deletion Ex11 (F)79951 IVS13 (R)87771 NS Identification of the deletion position

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

Identification of the deletion breakpoints agactccgtctcaaaaaaaaaaaaaaa (50a) gaaattaaaaatatgat insertion 1314 Ban II PolyT/PolyA 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. deletion ’ Ban II 4931bp (81207) (87421) 6213bp Nucleotide No. 3’

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

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.

11% 50% 6.3% 10% SUMMARY

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”.

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.