Lecture 15- Epidemiology Flashcards
Why Identify cancer genes??
- Understanding of disease mechanism
- High risk
- Identify targets for therapy
- Pharmacogenomics
- Potential for bad reactions to drugs
Familial cancer caused by Tumour suppressor genes
- Familial adenomatous polyposis – APC gene
- Retinoblastoma – RB1
Knudsons Hypothesis
2 hit hypothesis
- 2 this required to turn a normal cell into a cancer cell
Rare syndromes above – one hit causes the mutation – to completely KO other copy become mutated- no longer any working gene version
Knudsons Hypothesis- explained
1st hit often a point mutation
2nd hit usually a larger region e.g big deletion – measure throughheterozygosity
Using microsatellite markers
Rb was the first Ts gene to be cloned
How have genes for familial cancers been identified ?
- By positioning cloning
- Identification of deletion and LOH in tumours
- Testing for mutation know it be involved in cell cycle regulation
- Sequencing within in families
RB, APC Genotype risk
> 1000
Breast/colon Genotype risk
10-100 genotype risk
Antitypical Analysis
Positional cloning – genotype their DNA using microsateelile markers that span the whole chromosome
Look to see whether the disease co-segregates with markers on a particular chromosome
Breast Cancer
4-5% familial Early age of onset Breast and ovarian in the same family Frequent bilateral In males also
Segregation Analysis in Breast Cancer
Looking for dominant or recessive
Frequency’s
Risk
Use this info to carry out linkage analysis to work out where the allele is
Linkage Analysis
Localise a disease gene in a chromosome
Statistical
Genotype markers and observe whether or not particular marker allele segregate with disease in families with multiple cases
Looking at family tree
Calculate a Lod score if greater than 3 significant result
»> Chromosome 17 is linked to breast cancer
BRCA1 study
- BRCA1 gene
- Positional cloning of BRCA1 in 1994
- Chromosome 17
- Other families shown not to be linked to chromosome 17»_space; BRCA2 on chromosome 13
-BRCA1
24 exons 1863a,a
BRCA2
BRCA2
27 exons
Chromsome 13
Similarity between BRCA1 and 2
Both got Nuclear localisation signals / multifunctional domains
What has linkage analysis been applied to ??
Rare recessvie syndromes
e.g AT a mutation in ATM
Positional Cloning
- assemble some families with many affected individuals
- genotype their DNA using microsatellite markers that span the whole genome
- look to see whether the disease co-segregates with markers on a particular chromosome - this is done by linkage analysis
- fine-map the region of linkage using more markers
- identify likely candidate genes in the region of interest and look for mutations carried by people with the disease but not by healthy family members
lessons learnt from rare recessive syndromes: Signalling DNA Damage
- ATM phosphorylates MRE11 and NBS1 in response to DNA damage.
- The RAD50, NRE11, NBS1 complex binds at sites of DNA double strand breaks and initiates DNA repair.
- Mutation in these genes causes familial syndromes with genomic instability
NBS and AT have several common features
- Cells show increased sensitivity to ionising radiation
- Fail to induce p53 at G1/S check point > fail to stop DNA synthesise in response to ionising radiation
Ataxia telangiectasia (AT; caused by mutation in ATM)
- autosomal recessive
- cerebellar dysfunction, chromosomal instability, predisposition to cancer
Nijmegen Breakage syndrome (NBS; caused by mutation in NBS1)
- autosomal recessive
- microcephaly, immunodeficiency, predisposition to haematopoietic malignancies
BRCA1
associated with the MRE11/RAD50/NBS1 complex
Cortez et al (1999) showed that ATM also phosphorylates BRCA1.
They took wild-type and AT-mutant fibroblasts and g-irradiated them, then showed by Western blotting using anti-Brca1 antibodies that Brca1 was phosphorylated in the wild-type and not in the mutant cells.
A mutated Brca1 lacking the 2 phosphorylation sites failed to rescue the radiation hypersensitivity of a Brca1 deficient cell line; thus this pathway is important to protect cells against DNA damage.
ATM and Breast Cancer
ATM mutations have been identified in breast cancer families
variant of cell cycle arrest gene > CHEK2 also contributes to familial breast cancer
Non-familial or sporadic cases of cancer are caused by what ?
Non-familial or sporadic cases of cancer are caused by a combination of genes and environment
In this case the genes have weaker effects and act in combination with each other and environmental influences
The genetic variation in this case is polymorphisms rather than mutations; i.e. common variation such as single nucleotide changes (SNPs)
We can identify SNPs affecting breast cancer risk in genome-wide association studies
Genome Wide Association Studies
- SNP bead-chip Array
-24 DNA samples per chip
Assay thousands of SNPs
GWAS
- 400 patients vs 400 control
- 300,00 tag SNPs
- Compared frequencies between the two groups
- Take top 5% and genotype this set in stage 2
- Compare genotype frequencies
- Stage 3 – 30 most significant SNPs from stage 2
- Identify 5 regions significantly associated with breast cancer
FGFR2 - Chr10
TNCR9 -Chr 16
MAP3K1-Chr 5
LSP1- Chr 11
?? – Chromosome 8 is a gene desert snps may be in enhancer region
Oncoarray study –
Huge analysis by multiple groups
Formed the Oncoarray chip
Allowed fine mapping
Combined result of GWAS, iCOGS and oncorarray
Manhattan plot further up the smaller the P value
123000 cases
All SNPS
Idenfitied 65 new regions significantly associated with breast cancer
Conclusion of
Combined result of GWAS, iCOGS and oncorarray
Strong overlap between candidate target gene identified in the oncoarray and somatic driver genes identified by sequencing in of cancer tumours.
20 genes identified with both inherited and somatic genetic roles in breast cancer
Few of them coding most regulators of expression
