Transcript Diapositive 1

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
Breast cancer risk factors
Linked to:
-Environment
-Diet
-Hormones and reproductive life
-Familial history
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
Chr17
Miki et al, Science, 1994
BRCA1: 17q21
BRCA1
Genomic region
Exons
Coding sequence
81,092 bp
23
5,592 bp
Identification of the BRCA2 gene
Chr13
Wooster et al, Nature, 1995 BRCA2: 13q12-13
BRCA2
Genomic region
Exons
Coding sequence
84,190 bp
27
11,385 bp
Mendelien genetic diseases
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.
Frequency of BRCA germline mutations
BRCA1 & 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
Breast Cancer
Ovarian Cancer
70
Cumulative Risk (%)
60
50
40
30
20
10
0
30
40
50
60
70
Age (years)
Antoniou et al, Am J Hum Genet, 2003
Breast and ovarian cancer risk
conferred by BRCA2 mutations
Breast Cancer
Ovarian Cancer
70
Cumulative Risk (%)
60
50
40
30
20
10
0
30
40
50
60
70
Age (years)
Antoniou et al, Am J Hum Genet, 2003
Point mutations linked to diseases
5’ UnTranslated
Region (5’ UTR)
UAG
UGA
UAA
AUG
Coding sequence
3’ UnTranslated
Region (3’ UTR)
mRNA
CGG ACG AUG AAA UGC GUA GUA
Arg
Thr
Met
Lys
Cys
Val
Val
CGC ACG AUG AAG UGC GUA GUA
Arg
Thr
Met
Lys
Cys
Val
Val
CGC ACG AUG CAA UGC GUA GUA
Arg
Thr
Met
Gln
Cys
Val
Thr
Met
stop
Cys
Val
Thr
Met
Asn
Ala
stop
Nonsense mutation
Val
CGC ACG AUG AAU GCG UAG UAC
Arg
Missense mutation
Val
CGC ACG AUG UAA UGC GUA GUA
Arg
Silent mutation
Tyr
Frameshift mutation
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.Faivre
CHU de Dijon
S.Giraud
M.-A.Collonge-Rame
CHU de Besançon
C.Lasset
V.Bonadona
Centre Léon Bérard
S.Giraud
Hospices Civils de Lyon
J.Lespinasse
S.Fert-Ferrer
CHU de Chambéry
D.Leroux
H.Dreyfus
C.Rebichung
CHU de Grenoble
F.Prieur
CHU de St-Etienne
Molecular diagnosis in France
GENETIQUE CONSTITUTIONNELLE
DES CANCERS FREQUENTS
Hospices Civils de Lyon – Centre Régional Léon Bérard
Diagnostic génétique des formes héréditaires des cancers du sein et du côlon
Olga SINILNIKOVA
Responsable of the molecular diagnosis for familial breast
cancer in the Rhône-Alpes and Burgundy area.
Molecular diagnosis of breast
cancer susceptibility
Main difficulties:
- Identification of the mutations
- Interpretation of the results
Frameshift and nonsense
BRCA1/2 mutations
BRCA1
11
1 kb
BRCA2
11
1 kb
Frameshift, nonsense, missense and
splice site BRCA1/2 mutations
Screening of one index case :
122 amplicons (PCR fragments)
(analysis of ~17 kb of coding sequence, 96 exon/intron junctions)
Non causal nonsense mutation
Ser3326ter
Ser3291
BRCA2 protein
BRC Repeats
TR2
HTH
1
1
2
3 4 5
OB1
OB2
OB3
3418
6 78
NLS
Mazoyer et al, Nature Genet, 1996
- 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
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 BRCA2
is 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)
BRCA1 partners
RING finger
BRCT
Non exhaustive list
BARD1
BAP1
Rb
p53
c-myc
STAT1
ZBRK1
RAD50
Mre11
ATM
RAD51
Importin a
g tubulin
RNApol/RHA
Ctip
BACH1
HDAC1/2
CBP/p300
BRCA2
MSH2
MSH6
Cell cycle proteins
Ubiquitinases
DNA repair proteins
Transcription proteins
Transport proteins
Cytoskeleton protein
Biological interpretation of BRCA
missense variants
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
Large rearrangements (1)
Puget et al, Am J Hum Genet, 1999
Mazoyer et al, Am J Hum Genet, 2000
Founder duplication in BRCA1 in the anglo-saxon population
(7th most frequent mutation, identified in >100 families)
Large rearrangements (2)
homologous region (11.4 kb)
25 kb
y BRCA1 (17 kb)
NBR1 (41 kb)
4c
4b 4a 3
2
14.5 kb
NBR2 (19 kb)
1 1 2
5
4 3
2
BRCA1 (81 kb)
11 2
3
37 kb deletion
4c
5 kb
4b 4a 3
NBR1
2
1 1 2
3
y BRCA1/BRCA1
Puget et al, Am J Hum Genet, 2002
Hot-spot for recombination in the BRCA1 promoter
(identified in more than 20 independant families world-wide)
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
Clinical follow-up
High variability in cancer risks in carriers of BRCA mutations
Acknowledgements
Olga SINILNIKOVA
Monique BUISSON
Almoutassem ZETOUNE
Amandine GARCIA
Past members:
Nadine PUGET
Laure PERRIN-VIDOZ
Mark WARE
Olga ANCZUKÓW
Marc BILLAUD, Director of the CNRS unit UMR5201
Faculté de Médecine, LYON, FRANCE