Diapositive 1

1
Mutations in the BRCA1 and BRCA2 breast cancer
susceptibility genes
2
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
3
Breast cancer risk factors

• Linked to
• Environment
• Diet
• Hormones and reproductive life
• -Familial history

4
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
5
Identification of the BRCA1 gene
Miki et al, Science, 1994
BRCA1 17q21
BRCA1
Genomic region 81,092 bp Exons 23 Coding
sequence 5,592 bp
6
Identification of the BRCA2 gene
Wooster et al, Nature, 1995
BRCA2 13q12-13
BRCA2
Genomic region 84,190 bp Exons 27 Coding
sequence 11,385 bp
7
Mendelien genetic diseases
Mendelien genetic disease are classically
characterized by v the type of inheritance
(dominant versus recessive) v the frequency of
the mutations in the population v the
penetrance of the mutations v the risks
associated with the mutations.
8
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

9
Breast and ovarian cancer risk conferred by
BRCA1 mutations
Cumulative Risk ()
Age (years)
Antoniou et al, Am J Hum Genet, 2003
10
Breast and ovarian cancer risk conferred by
BRCA2 mutations
Cumulative Risk ()
Age (years)
Antoniou et al, Am J Hum Genet, 2003
11
Point mutations linked to diseases
3 UnTranslated Region (3 UTR)
5 UnTranslated Region (5 UTR)
UAG UGA UAA
AUG
Coding sequence
mRNA
Arg Thr Met Lys Cys
Val Val
Silent mutation
Arg Thr Met Lys Cys
Val Val
Missense mutation
Arg Thr Met Gln Cys
Val Val
Nonsense mutation
Arg Thr Met stop Cys
Val Val
Frameshift mutation
Arg Thr Met Asn Ala
stop Tyr
12
BRCA mutation spectrum

• gt 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

13
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
14
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.
15
Molecular diagnosis of breast cancer
susceptibility

• Main difficulties
• Identification of the mutations
• Interpretation of the results

16
Frameshift and nonsense BRCA1/2 mutations
BRCA1
1 kb
BRCA2
1 kb
17
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)
18
Non causal nonsense mutation
Ser3326ter
Ser3291
BRCA2 protein
BRC Repeats
TR2
OB1
OB2
OB3
HTH
3418
1 2 3 4 5 6 7 8
1
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
19
Splice mutations
Mutations in canonical sites
Mutations in exonic splicing enhancers and
silencers
20
Exonic splicing mutation
Glu1694ter in BRCA1 exon 18
Mazoyer et al, Am J Hum Genet, 1998
21
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.
22
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.
23
Function of BRCA1 and BRCA2 (2)
24
BRCA1 partners
RING finger
BRCT
Non exhaustive list
BARD1 BAP1
Rb p53 c-myc STAT1 ZBRK1 RAD50 Mre11 ATM RAD51 Imp
ortin 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
25
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

26
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 gt100 families)
27
Large rearrangements (2)
homologous region (11.4 kb)
25 kb
14.5 kb
y
(17 kb)
BRCA1
(41 kb)
BRCA1
(81 kb)
NBR2
(19 kb)
NBR1
1
2
3
1
2
3
4
5
2
1
2
1
3
4a
4b
4c
37 kb deletion
2
1
2
1
3
4a
4b
4c
3
5 kb
y
BRCA1/BRCA1
NBR1
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)
28
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

29
Clinical follow-up
High variability in cancer risks in carriers of
BRCA mutations
30
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