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RCPath Session 5: 22/01/2009 Stephanie Batey

Describe the clinical features, the genes involved and briefly the testing strategies for familial breast and ovarian cancer. RCPath Session 5: 22/01/2009 Stephanie Batey. Introduction.

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RCPath Session 5: 22/01/2009 Stephanie Batey

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  1. Describe the clinical features, the genes involved and briefly the testing strategies for familial breast and ovarian cancer. RCPath Session 5: 22/01/2009 Stephanie Batey

  2. Introduction • Germline mutations in known and unknown susceptibility genes account for approximately 5-10% of breast and/or ovarian cancer cases . • Two genes, BRCA1 and BRCA2, are the most common known cause of familial breast and ovarian cancer. • Predisposition due to BRCA1 or BRCA2 mutation shows an autosomal dominant mode of inheritance, with variable penetrance. • Other genetic factors are likely to contribute to the etiology of breast and ovarian cancer, including BRCA3, p53, PTEN and CHEK2 mutations.

  3. Clinical features • A hereditary predisposition to breast and ovarian cancer may be indicated by: - Multiple blood relatives with the same, or related, cancers on the same side of the family. - Unusually young age of onset - Individuals with bilateral of multifocal/ multiple cancers. - Personal family history of male breast cancer. • NICE guidelines provide criteria for classifying individuals or families into appropriate risk categories, based on family history.

  4. Breast cancer risk assessment High risk group referral to Clinical Genetics • 4 or more relatives with breast cancer at any age • 3 relatives with breast cancer: average age of diagnosis <50yrs • 2 relatives with breast cancer: average age of diagnosis <40 yrs • A first degree relative diagnosed with BrCa <30yrs • 3 or more relatives with breast and ovarian cancer • A relative diagnosed with BrCa <50 yrs and a relative diagnosed with OvCa at any age • A first degree relative with both breast and ovarian cancer • A male first degree relative with breast cancer • Rare syndromes eg. Li-Fraumeni, Cowden syndrome • Known BRCA1 or BRCA2 family.

  5. BRCA1 gene • 17q21 • 24 exons (22 coding) • Exons range in size from 100 -500bp. • Exon 11 largest: accounts for ~60% of the coding region. • 7.8kb mRNA encoding BRCA1 protein: nuclear location • Genomic sequence rich in Alu repeats: promotes instability

  6. BRCA2 gene • 13q12 • 27 exons (26 coding) • Exons 10 and 11 large (account for ~60% coding region) • 11-12kb mRNA encoding BRCA2 protein: normally located in nucleus Very little sequence homology between BRCA1 and BRCA2

  7. Gene functions • BRCA1 and BRCA2 genes are essential for cellular development • BRCA1 and BRCA2 proteins required for maintenance of genome integrity. Specific roles in: - DNA damage repair - cell cycle regulation - transcriptional regulation • Both have characteristics of tumour suppressor genes: 1) Autosomal dominant inheritance 2) Complies with Knudson’s two-hit hypothesis: loss of heterozygosity at relevant gene locus in familial tumours with retention of disease-predisposition allele. • Ubiquitous expression: highest levels in testes, thyroid and ovaries.

  8. DNA damage repair (1) • Distinct complexes formed by BRCA1 and BRCA2 are involved in damage signalling, protein degradation, homologous recombination and DNA repair. • BRCA1 is part of a genome surveillance complex- BASC. • BASC contains other proteins important for efficient DNA repair including the Rad50/MRE11/NBS1 complex which acts in response to dsDNA breaks • Wild-type BRCA1 alleles can restore DSB repair to BRCA1 mutated cell line. • BRCA2 interacts with the essential homologous recombination/ repair protein RAD51: - Mediates DSB repair in response to DNA damage.

  9. DNA damage repair (2) • BRCA1 has a conserved RING finger motif, which has ubiquitin ligase activity. Stimulated by binding to BARD1 protein: - mutations in RING finger motif show increased sensitivity to DNA-damaging agents. - role in targeting proteins for ubiquitin-mediated degradation eg. FANCD2 protein • Ubiquitin ligase activity may downregulate proteins involved in cell cycle progression.

  10. Regulation of transcription • BRCA1 and 2 shown to form complexes with various transcription proteins eg. C-myc (BRCA1) and p53 (BRCA1 and BRCA2). • Regulate genes involved in cell growth control, cell cycle regulation and DNA replication and repair. • Interactions with chromatin remodelling proteins eg. BRCA1 interacts with the SWI/SNF remodelling complex and RNA polymerase II holoenzyme. • Carboxyl terminal of BRCA1 contains two BRCT motifs: important for protein/protein interactions: many BRCA1 mutations result in a truncated product in which the carboxyl-terminal is deleted. • Transcriptional regulation of cell division kinase inhibitor p21 (induced by p53) which acts to arrest cell cycle at G/S and G/M phase. Both BRCA1 and 2 can interact with p53.

  11. Features of BRCA1 and BRCA2 proteins

  12. Mutation spectrum • Majority of BRCA1/2 mutations are truncating (nonsense or frameshift) → loss of function of the BRCA1/2 proteins. • Missense mutations less frequent: clinical significance of these changes is often not known. • Genomic rearrangements including large deletions, duplications and inversions, mediated mainly by Alu repeats eg. common ex13 duplication • No major hotspots, but some common mutations are associated with specific populations due to genetic founder effect: Ashkenazi Jewish: BRCA1:187_188delAG & 5385insC BRCA2: 6174delT Icelandic: BRCA2: 999del5 Dutch: BRCA1 large deletions

  13. Genotype/ Phenotype correlations • Variable penetrance of BRCA1 and 2 mutations with respect to the type of cancer and age of onset. • Based on high-risk families: BrCa: >80% by age 70yrs (BRCA1 & 2) OvCa:~40% (BRCA1) & 27% (BRCA2) by 70 yrs • Phenotypic differences between patients with BRCA1 or BRCA2 germline mutation. BRCA1 • BRCA1 mutation carriers: higher risk of developing ovarian cancer although the risk is thought to depend on the specific mutation. - Mutations in 3’ third of gene are associated with a lower risk of ovarian cancer. - Possession of rare alleles at the HRAS1VNTR locus can increase the risk for OvCa • BRCA1 cases generally have earlier age of onset of BrCa. • Males with BRCA1 mutations may be at 3-fold increased risk of prostate cancer

  14. BRCA2 • BRCA2 mutation carriers are at higher risk of developing other cancer types in addition to BrCa and OvCa including cancer of the larynx, oesophagus, colon, pancreas, stomach, gall bladder and haematopoeitic system. • Male breast cancer is associated predominantly with BRCA2 mutations. • Certain class of BRCA2 mutations cause the B/D1 form of Fanconi anaemia. • BRCA2 mutation families with a high proportion of OvCa compared to BrCa, tend to have mutations in 3.3kb region in exon 11: ovarian cancer cluster region (OCCR).

  15. Testing strategy Possible referrals: • Mutation screening in affected individuals • Unaffected individuals with strong FH • Testing for 3 common mutations in patients of Jewish ancestry. • Predictive testing for at-risk relatives • Confirmation of known mutation. Diagnostic testing typically involves: • Screening for large rearrangements, using MLPA • Screening of coding sequence of BRCA1 and/or BRCA2

  16. Testing strategy (2) • Mutation scanning as a pre-screen: DHPLC, SSCP, DGGE, fluorescent CSCE, PTT, Chemical or enzymatic cleavage method. Direct sequencing for characterisation of any variants identified OR • Direct sequencing: high throughput, automated approach. • Mutation specific tests for common founder mutations or known familial mutations eg. sequencing, ARMS • In rare cases, linkage analysis can be used to exclude BRCA1 or BRCA2 locus.

  17. Testing considerations • Mutations in BRCA1 and BRCA2 reported in a single individual: worth screening both genes even after one mutation found, especially if strong FH on both sides of the pedigree. • If mutation is found in an individual, ideally ascertain which side of the family it has been inherited from, prior to predictive testing. • Breast cancer is a common disease so many phenocopies may exist within families: useful to carry out segregation analysis. • Incomplete penetrance • Interpretation of unclassified variants is problematic. • Implications for patients: indications for prophylactic surgery or changes to treatment regime. Predictive testing for at-risk relatives.

  18. References • Scully, R. (2000) Breast Cancer Research 2: 324-330 • Andrulis, I. et al (2002) Human Mutation 20:65-73 • Lose, F. et al (2008) JNCI 100(21): 1519-1529 • Montagna, . Et al (2003) Human Molecular Genetics 12(9):1055-1061 • Unger et al (2000) Am. J. Hum. Genet. 67: 841-850 • GeneCards: www.genecards.org • Welsch and King (2001) • EMQN Draft Best Practice Guidelines 2001 • Liu and West (2002) Breast Cancer Research 4:9-13 • Bertwhistle and Ashworth (1999) Breast Cancer Research 1(1): 41-7

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