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
Chr17 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
Chr13 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
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 Cumulative Risk (%) Age (years) Antoniou et al, Am J Hum Genet, 2003
Breast and ovarian cancer risk conferred by BRCA2 mutations Cumulative Risk (%) Age (years) Antoniou et al, Am J Hum Genet, 2003
CGC CGC CGC CGC CGG ACG ACG ACG ACG ACG AUG AUG AUG AUG AUG AAA AAU UAA AAG CAA UGC UGC UGC UGC GCG GUA GUA GUA GUA UAG GUA GUA GUA UAC GUA 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
BRCA mutation spectrum • > 90% of mutations introduce a premature termination codon in the coding sequence (PTC) = truncating mutations • smallinsertions/deletions that create a frameshift • - nonsensemutations • splicesitesmutations • 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
11 11 Frameshift and nonsense BRCA1/2 mutations BRCA1 1 kb BRCA2 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 OB1 OB2 OB3 3418 1 1 2 3 4 5 6 7 8 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.
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 14.5 kb y (17 kb) (41 kb) BRCA1 NBR1 NBR2 (19 kb) BRCA1 (81 kb) 4c 4b 4a 3 2 1 1 2 5 4 3 2 1 1 2 3 37 kb deletion 4c 4b 4a 3 2 1 1 2 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)
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