The Contribution of the BRCA1 and BRCA2 Genes to Hereditary

Breast Cancer and Ovarian Cancerin Israel

Tieling Wang

Department of Human Genetics

Hadassah Hebrew University Hospital, Israel

New cases of cancer in New cases of cancer in women in Israel (1993), women in Israel (1993),

adjusted per age (x10adjusted per age (x10-5-5))

Breast cancerBreast cancerC

olon

can

cer

Col

on c

ance

r

Ova

rian

can

cer

Ova

rian

can

cer

Mel

anom

aM

elan

oma

Bra

in t

um

ors

Bra

in t

um

ors

Lym

ph

oma

Lym

ph

oma

Lu

ng

can

cer

Lu

ng

can

cer

Ute

rus

can

cer

Ute

rus

can

cer

80.980.9

30.230.2

13.413.411.111.1 11.011.0 10.810.8 9.59.5 9.19.1

OthersOthers

5

10

15

20

25

30

%

New Breast Cancer Cases in Israel (1993), According to Country of Birth

Place of birthPlace of birth

IsraelIsraelAmerica and EuropeAmerica and EuropeAsiaAsiaAfricaAfricanon-Jewish (Arabs)non-Jewish (Arabs)

No.No.

44244211661166

2622622062067070

Rate*Rate*

89.989.989.889.870.770.755.655.62424

* adjusted per age (x10adjusted per age (x10-5-5) )

The lifetime risk of a Jewish woman

living in Israel to develop

breast cancer is

1 in 9

Risk Factors for Breast CancerRisk Factors for Breast Cancer

Excess of estrogen:Excess of estrogen:early menarche (<14)early menarche (<14)late birth (>30) or no birthlate birth (>30) or no birthlate menopause (>55)late menopause (>55)hormone therapyhormone therapy

Relative riskRelative risk1.31.31.91.91.51.51.41.4

Family history:Family history:one 1st degree relative with BC <50one 1st degree relative with BC <50 2.02.0two 1st degree relative with BC <50two 1st degree relative with BC <50 4.0-6.04.0-6.0

5858yy

Breast cancerBreast cancer

Ovarian cancerOvarian cancer

4343yy

2

ThyroidThyroid20y20y

5050yy

5050yy

33535yy

Familial Breast & Ovarian Familial Breast & Ovarian CancerCancer

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.

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.

Function domains of BRCA1 and BRCA2

Suggested role for BRCA1 and BRCA2Suggested role for BRCA1 and BRCA2

1. 1. Transcriptional regulationTranscriptional regulation

2. DNA repair associated with Rad51 and2. DNA repair associated with Rad51 and maintenance of genome integritymaintenance of genome integrity

3. BRCA1&2 are essential for a normal cell3. 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

BRCA1 BRCA2

Data from family studies:BC risk in BC or BC &OC families 40-85% 40-85%OC risk in BC and OC families 10-40% 10-25%

Data from population-based studies:BC risk in Jewish founder mutation 55-60% 30-55%OC risk in Jewish founder mutation 15% 15%BC risk in Icelandic founder mutation 35%

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

BRCA1/2 founder mutations BRCA1/2 founder mutations in Ashkenazi BC/OC patientsin Ashkenazi BC/OC patients

BRCA1 (17q)BRCA1 (17q)1. 185delAG, exon 21. 185delAG, exon 22. 5382insC, exon 202. 5382insC, exon 20

BRCA2 (13q)BRCA2 (13q)3. 6174delT, exon 113. 6174delT, exon 11

Frequencies of BRCA Carriers Frequencies of BRCA Carriers in the general Ashkenazi in the general Ashkenazi

PopulationPopulation

BRCA1BRCA1

185delAG 1.05%185delAG 1.05%

5382insC 0.11%5382insC 0.11%

BRCA2BRCA2

6174delT 1.36%6174delT 1.36%

Total: 2.5% (1 in 40)Total: 2.5% (1 in 40)

Carrier frequency 1 in 40Carrier frequency 1 in 40

No. of Ashkenazi females in No. of Ashkenazi females in IsraelIsrael

~1 000 000~1 000 000

Female carriersFemale carriersn=25 000n=25 000

HealthyHealthyindividualsindividuals

8383 5353 3333 3030 1111 2.52.5%%

BCBC

BC BC <40y<40y

BBCBBC

BC&OCBC&OC

OCOC

The Contribution of the BRCA MutationsThe Contribution of the BRCA Mutations (185delAG, 5382insC, 6174delT) to Breast & (185delAG, 5382insC, 6174delT) to Breast &

Ovarian Cancer Morbidity in Ashkenazi PatientsOvarian Cancer Morbidity in Ashkenazi Patients

The Aims of This Study

To identify cancer predisposing mutations in BC/OC patients in BRCA1 & BRCA2 genes.

70 unrelated Jewish BC& OC patients met at least one ofthe three criteria of hereditary cancer:

Definite or positive family history of BC or OC. Two primary cancers (BBC or BC and OC). Young age at diagnosis (<30 years).

17 patients belong to more than one category.

None of the patient was a carrier of the Ashkenazifounder mutations.

The study group

The study group

Family history 2nd primarytumors

Age

Patients Definite Positive BBC BC+OC 30

AshkenaziJews (n = 46)

28 11 14 1 5

Non-AshkenaziJews (n = 24)

11 7 4 1 5

Total (n = 70) 39 18 18 2 10

DNA Sequencing

Screening methods Single Strand Confirmation Polymorphism (SSCP )Restriction Endonuclease Fingerprinting (REF)

Methods for mutation analysis

BRCA1 and BRCA2 Genome organizationBRCA1

BRCA2 3418Amino acids

1863Amino acids

SSCASSCA : : SSingle ingle SStrand trand CConformation onformation AAnalysisnalysisSSCPSSCP : : SSingle ingle SStrand trand CConformation onformation PPolymorphismolymorphism

normal allelenormal allele

mutant allelemutant allele

denaturationdenaturation(heat & formamide)(heat & formamide)

separation onseparation onMDE gelMDE gel

dilutiondilution

1221bp1221bp 1160bp1160bp1123bp1123bp

1193bp1193bp1324bp1324bp

1327bp1327bp

Ex10Ex10 Ex11A-FEx11A-F Ex11AF-JEx11AF-J Ex11J-PEx11J-P Ex11P-UEx11P-U Ex11U-ZEx11U-Z

REF: Exons 10 & 11 in the BRCA2 Gene:REF: Exons 10 & 11 in the BRCA2 Gene:

The comparison of conventional SSCP and REFmethods by PCR

Exon 10(1221 bp )

Exon 11(6127 bp )

Conventional SSCP 5 26

REF 1 5

Restriction Endonuclease Fingerprinting (REF) Restriction Endonuclease Fingerprinting (REF) Principle of the MethodPrinciple of the Method

Mbo Mbo II + II + Hinf Hinf II

BRCA2: Exon 11 U-Z (1327bp)BRCA2: Exon 11 U-Z (1327bp)

415942664272

45934362 4665 4836 4987

52235289

52955398

5486

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 The mutations and sequence variants in BRCA1&2 genesBRCA1&2 genes

IVS16-92 AG

5553CG

IVS14+53 C T

3199 AG3326 A T

IVS8+56 C T

Missense mutation

Nucleotide change in introns

8765delAG

IVS2-7 T A

IVS16-14 T C

1342 AC

1593 AG 3624 AG5426 CT

5972 CTIVS21+1470new EcoR I site

BRCA2

8558delA4093delAAAT

IVS 8-57 7T 6T

2430 TC 2731 CT3232AG 3667AG 4965AG

IVS16-68 AG

IVS18+66 CA

BRCA1

3053 T G

Nonsense mutation

Silent mutation

Genes Exons Sequence changes

Exon 20 8765 delAG stop codon 2867

Exon 11K 4093delAAAT stop codon 1291BRCA2

Exon18B 8558delA stop codon 2840

BRCA1 Exon 11K2 3053 T G stop codon 1017

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 CaProstate Ca60yr.60yr.

BCBC46yr.46yr.

BCBC31yr.31yr.

BCBCBBCBBC42yr.42yr.

BCBC36yr.36yr.

87658765delAG in Exon 20 of BRCA2delAG in Exon 20 of BRCA2

TheThe mutation was found in 3 unrelated Jewish mutation was found in 3 unrelated Jewish families of Yemenite originfamilies of Yemenite origin

The mutation was found in The mutation was found in 1: 1401: 140 Jewish healthy Jewish healthy individuals of Yemenite originindividuals of Yemenite origin The mutation The mutation was not foundwas not found in 68 Jewish breast in 68 Jewish breast cancer patients with positive family history or cancer patients with positive family history or early age at diagnosis (<30yr.) of Ashkenazi or early age at diagnosis (<30yr.) of Ashkenazi or

Sephardic originSephardic origin

**

**

**

87658765delAG is a founder mutation in Jews from YemendelAG is a founder mutation in Jews from Yemen

Diseased associated haplotypes inBRCA2 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

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

GA

The sequence variants T1915M in BRCA2

didn`t affect the transcription.

Normal

Mutant

Detection of gross rearrangementsDeletions and duplications

• PCR-based methods such as SSCP or REF are likely to miss Gross rearrangements.

• Gross rearrangements can be detected by Southern hybridization.

BRCA1

1026 bp

Ex1-11

3032 bp

Ex11

1653 bp

Ex11-24

Hind III / Ex11

Hind III / Ex11-24Hind III / Ex1-11

Southern Hybridization in BRCA1

BRCA2

Ex23-27Ex12-17 Ex18-22Ex11 Ex10 Ex2-9

Southern Hybridization in BRCA2

NS

Southern Hybridization EcoRI/exon11 (BRCA2)

3‘

E x 1 1 N S

RL

5‘

6.1kb

9.5kb

3362bp 2175bp

12 1311

4931bp

EcoR IEcoR I EcoR I

NS (L+R)

6.1

9.5

N N N M

9.07.3

5.6

4.33.93.6

2.9

2.3

Kb

NS-R

N N M M

3.6

Kb

11 12 13 14 15 16

3’5’

Ban II Ban II Ban II Ban II

4931bp 3362 bp 2175 bp 7967 bp

deletion

Ex11 (F)79951Ex11 (F)79951 IVS13 (R)87771IVS13 (R)87771

NS

Identification of the deletion position

Identification of the deletion mutation: Long PCRIdentification of the deletion mutation: Long PCRIdentification of the deletion mutation: Long PCRIdentification of the deletion mutation: Long PCR

Ex11 (F)79951Ex11 (F)79951 IVS13 (R)87771IVS13 (R)87771Long PCR:Long PCR:

7828 7828 bpbp

~1700 ~1700 bpbp

Normal:Normal:

Del mutant:Del mutant:

Deletion of ~ 6KbDeletion of ~ 6KbIncluding exons 12 & 13Including exons 12 & 13

deletiondeletion

Identification of the deletion breakpointsIdentification of the deletion breakpoints

81181-81181-agactccgtctcaaaaaaaaaaaaaaa-81207------------- (50a)---------- 87421-gaaattaaaaatatgatagactccgtctcaaaaaaaaaaaaaaa-81207------------- (50a)---------- 87421-gaaattaaaaatatgat

insertioninsertion

13 14Ban II 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

11 12

5’

Ban II

4931bp

(81207) (87421)62136213bpbp

Nucleotide No.Nucleotide No.

3’

129-1BC (43y)

129-2BC (54y)

129-3BC (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.