Mutations in the BRCA1 and BRCA2 breast cancer susceptibility genes

Breast cancer in Europe

1 in 10 women will develop breast cancer during their life

430,000 new cases and 132,000 deaths in 2006(number 1 killer in women)

27.4% of cancer cases and 17.4% of cancer deaths in women

Linked to:

-Environment

-Diet

-Hormones and reproductive life

-Familial history

Breast cancer risk factors

Familial breast cancer

5 to 10% of breast cancer cases have a family history of the disease

Represents at least 22,000 cases in Europe each year

Link with ovarian cancer

Identification of the BRCA1 gene

Genomic region 81,092 bpExons 23

Coding sequence 5,592 bp

BRCA1

Miki et al, Science, 1994

Chr17

BRCA1: 17q21

Identification of the BRCA2 gene

BRCA2

Genomic region 84,190 bpExons 27

Coding sequence 11,385 bp

Wooster et al, Nature, 1995

Chr13

BRCA2: 13q12-13

Mendelien genetic disease are classically characterized by :

√ the type of inheritance (dominant versus recessive)

√ the frequency of the mutations in the population

√ the penetrance of the mutations

√ the risks associated with the mutations.

Mendelien genetic diseases

Frequency of BRCA germline mutationsBRCA1 & BRCA2 breast and ovarian cancer susceptibility: one of the most prevalent high-risk hereditary disorders• 1/500 individuals in the general population of

Western

European descent carry a BRCA1 or BRCA2

mutation

Antoniou et al, 2002; Whittemore et al, 2004; Antoniou et al, 2008

• 1/40 individuals in the Ashkenazi Jewish population

carry

one of three ancestral BRCA1 and BRCA2

mutations

Streuwing et al, 1995; Neuhausen et al, 1996; Roa et al, 1996

Breast and ovarian cancer risk conferred by BRCA1 mutations

Antoniou et al, Am J Hum Genet, 2003

0

10

20

30

40

50

60

70

30 40 50 60 70

Breast Cancer

Ovarian Cancer

Cum

ula

tive R

isk

(%)

Age (years)

Antoniou et al, Am J Hum Genet, 2003

0

10

20

30

40

50

60

70

30 40 50 60 70

Breast Cancer

Ovarian Cancer

Cum

ula

tive R

isk

(%)

Age (years)

Breast and ovarian cancer risk conferred by BRCA2 mutations

Point mutations linked to diseases

AUG

UAGUGAUAA

5’ UnTranslatedRegion (5’ UTR)

3’ UnTranslatedRegion (3’ UTR)

Coding sequence

CGG ACG AAAAUG UGC GUA GUA

CGC ACG AAGAUG UGC GUA GUA

CGC ACG CAAAUG UGC GUA GUA

CGC ACG UAAAUG UGC GUA GUA

CGC ACG AAUAUG GCG UAG UAC

Silent mutation

Missense mutation

Nonsense mutation

Frameshift mutation

Arg Thr Met Lys Cys Val Val

Arg Thr Met Lys Cys Val Val

Arg Thr Met Gln Cys Val Val

Arg Thr Met stop Cys Val Val

Arg Thr Met Asn Ala stop Tyr

mRNA

BRCA mutation spectrum

> 90% of mutations introduce a premature termination codon in the coding sequence (PTC) = truncating mutations

- small insertions/deletions that create a frameshift- nonsense mutations - splice sites mutations - deletion/duplication of one or several exons

Very few missense mutations

60% of the mutations are unique

Molecular diagnosis in France

L.FaivreCHU de Dijon

S.GiraudM.-A.Collonge-RameCHU de Besançon

C.LassetV.BonadonaCentre Léon Bérard

S.GiraudHospices Civils de Lyon

D.LerouxH.DreyfusC.RebichungCHU de Grenoble

F.PrieurCHU de St-Etienne

J.LespinasseS.Fert-FerrerCHU de Chambéry

Olga SINILNIKOVAResponsable of the molecular diagnosis for familial breast cancer in the Rhône-Alpes and Burgundy area.

GENETIQUE CONSTITUTIONNELLE GENETIQUE CONSTITUTIONNELLE

DES CANCERS FREQUENTSDES CANCERS FREQUENTS

Hospices Civils de Lyon – Centre Régional Léon BérardDiagnostic génétique des formes héréditaires des cancers du sein et du côlon

Molecular diagnosis in France

Molecular diagnosis of breastcancer susceptibility

Main difficulties:

- Identification of the mutations

- Interpretation of the results

Frameshift and nonsense BRCA1/2 mutations

11

11

1 kb

1 kb

BRCA1

BRCA2

Screening of one index case :

122 amplicons (PCR fragments)

(analysis of ~17 kb of coding sequence, 96 exon/intron junctions)

Frameshift, nonsense, missense and splice site BRCA1/2 mutations

BRC Repeats

OB1 OB2 OB3

NLS

HTH

TR2

Ser3291

1 2 3 4 5 6 7 81 3418

Non causal nonsense mutationSer3326ter

- Ser3326ter causes loss of the final 93 amino acids (2.7% of

the protein)

- Present in 2% of the population

- Does not increase susceptibility to breast and ovarian cancer

BRCA2 protein

Mazoyer et al, Nature Genet, 1996

Splice mutations

Mutations in canonical sites

Mutations in exonic splicing enhancers and silencers

Exonic splicing mutation

Glu1694ter in BRCA1 exon 18

Mazoyer et al, Am J Hum Genet, 1998

Missense mutations

The amino acid sequence of BRCA1 and BRCA2is not highly constrained by natural selection and can tolerate amino acid substitutions.

As a result, the causality of rare variants is difficult to evaluate.

Function of BRCA1 and BRCA2 (1)

BRCA1 is involved in a number of cellular processes, the main one being DNA damage signaling and DNA repair.

BRCA2 is mainly involved in DNA repair.

Function of BRCA1 and BRCA2 (2)

RING finger BRCT

BARD1BAP1

Rbp53c-mycSTAT1ZBRK1RAD50Mre11ATMRAD51Importin tubulin

RNApol/RHACtipBACH1HDAC1/2CBP/p300BRCA2MSH2MSH6

Cell cycle proteins

Ubiquitinases

DNA repair proteins

Transcription proteins

Transport proteins

Cytoskeleton protein

BRCA1 partners

Non exhaustive list

Amino-acid modification (Grantham matrix)

Phylogenic conservation of the modified amino-acid

Functionnal evaluation : related to the disease?

Co-segregation of the variant with the disease in the family : samples from affected relatives are needed

Presence of a deleterious mutation on the other allele of the same gene: in trans or in cis ?

Association studies (cases-controls): a large number of samples is needed

Biological interpretation of BRCA missense variants

Large rearrangements (1)

Founder duplication in BRCA1 in the anglo-saxon population(7th most frequent mutation, identified in >100 families)

Puget et al, Am J Hum Genet, 1999Mazoyer et al, Am J Hum Genet, 2000

Large rearrangements (2)

Hot-spot for recombination in the BRCA1 promoter(identified in more than 20 independant families world-wide)

BRCA1 (81 kb)NBR2 (19 kb)

1 2 31234521

BRCA1 (17 kb)

2 134a4b4c

NBR1 (41 kb)

212 134a4b4c 3

BRCA1/BRCA1NBR15 kb

37 kb deletion

homologous region (11.4 kb)

25 kb 14.5 kb

Puget et al, Am J Hum Genet, 2002

Clinical follow-up

Breast cancer:

- Mammography every year from the age of 30 + ultrasound scan ?- Clinical exam every 6 months- Prophylactic mastectomy may be suggested (reduction of breast cancer risk by 90%)

Ovarian cancer:

- Prophylactic oophorectomy recommended

High variability in cancer risks in carriers of BRCA mutations

Clinical follow-up

Acknowledgements

Olga SINILNIKOVAMonique BUISSONAlmoutassem ZETOUNEAmandine GARCIA

Past members:Nadine PUGETLaure PERRIN-VIDOZMark WAREOlga ANCZUKÓW

Marc BILLAUD, Director of the CNRS unit UMR5201Faculté de Médecine, LYON, FRANCE