Mutations in the BRCA 1 and BRCA 2
Mutations in the BRCA 1 and BRCA 2 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 BRCA 1 gene Chr 17 Miki et al, Science, 1994 BRCA 1: 17 q 21 BRCA 1 Genomic region Exons Coding sequence 81, 092 bp 23 5, 592 bp
Identification of the BRCA 2 gene Chr 13 Wooster et al, Nature, 1995 BRCA 2: 13 q 12 -13 BRCA 2 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 BRCA 1 & BRCA 2 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 BRCA 1 or BRCA 2 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 BRCA 1 and BRCA 2 mutations Streuwing et al, 1995; Neuhausen et al, 1996; Roa et al, 1996
Cumulative Risk (%) Breast and ovarian cancer risk conferred by BRCA 1 mutations Age (years) Antoniou et al, Am J Hum Genet, 2003
Cumulative Risk (%) Breast and ovarian cancer risk conferred by BRCA 2 mutations Age (years) Antoniou et al, Am J Hum Genet, 2003
Point mutations linked to diseases 5’ Un. Translated Region (5’ UTR) AUG UAG UGA UAA Coding sequence 3’ Un. Translated Region (3’ UTR) m. RNA CGG ACG AUG AAA UGC GUA Arg Thr Met Lys Cys Val CGC ACG AUG AAG UGC GUA Arg Thr Met Lys Cys Val CGC ACG AUG CAA UGC 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 UAC Arg Missense mutation Val CGC ACG AUG UAA UGC 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 BRCA 1/2 mutations BRCA 1 11 1 kb BRCA 2 11 1 kb
Frameshift, nonsense, missense and splice site BRCA 1/2 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 Ser 3326 ter Ser 3291 BRCA 2 protein BRC Repeats TR 2 HTH 1 1 2 3 4 5 OB 1 OB 2 OB 3 3418 6 78 NLS Mazoyer et al, Nature Genet, 1996 - Ser 3326 ter 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 Glu 1694 ter in BRCA 1 exon 18 Mazoyer et al, Am J Hum Genet, 1998
Missense mutations The amino acid sequence of BRCA 1 and BRCA 2 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 BRCA 1 and BRCA 2 (1) BRCA 1 is involved in a number of cellular processes, the main one being DNA damage signaling and DNA repair. BRCA 2 is mainly involved in DNA repair.
Function of BRCA 1 and BRCA 2 (2)
BRCA 1 partners RING finger BRCT Non exhaustive list BARD 1 BAP 1 Rb p 53 c-myc STAT 1 ZBRK 1 RAD 50 Mre 11 ATM RAD 51 Importin a g tubulin RNApol/RHA Ctip BACH 1 HDAC 1/2 CBP/p 300 BRCA 2 MSH 6 Cell cycle proteins Ubiquitinases DNA repair proteins Transcription proteins Transport proteins Cytoskeleton protein
Biological interpretation of BRCA missense variants üAmino-acid modification (Grantham matrix) ü Phylogenic conservation of the modified amino-acid ü Functionnal evaluation : related to the disease? ü Co-segregation of the variant with the disease in the family : samples from affected relatives are needed ü Presence of a deleterious mutation on the other allele of the same gene: in trans or in cis ? ü 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 BRCA 1 in the anglo-saxon population (7 th most frequent mutation, identified in >100 families)
Large rearrangements (2) homologous region (11. 4 kb) 25 kb y BRCA 1 (17 kb) NBR 1 (41 kb) 4 c 4 b 4 a 3 2 14. 5 kb NBR 2 (19 kb) 1 1 2 5 4 3 2 BRCA 1 (81 kb) 11 2 3 37 kb deletion 5 kb 4 c 4 b 4 a 3 NBR 1 2 1 1 2 3 y BRCA 1/BRCA 1 Puget et al, Am J Hum Genet, 2002 Hot-spot for recombination in the BRCA 1 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 UMR 5201 Faculté de Médecine, LYON, FRANCE
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