Lecture 15- Epidemiology

Question 1

Why Identify cancer genes??

Answer 1

• Understanding of disease mechanism
• High risk
• Identify targets for therapy
• Pharmacogenomics
• Potential for bad reactions to drugs

Question 2

Familial cancer caused by Tumour suppressor genes

Answer 2

• Familial adenomatous polyposis – APC gene

- Retinoblastoma – RB1

Question 3

Knudsons Hypothesis

Answer 3

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

Question 4

Knudsons Hypothesis- explained

Answer 4

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

Question 5

How have genes for familial cancers been identified ?

Answer 5

• 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

Question 6

RB, APC Genotype risk

Answer 6

> 1000

Question 7

Breast/colon Genotype risk

Answer 7

10-100 genotype risk

Question 8

Antitypical Analysis

Answer 8

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

Question 9

Breast Cancer

Answer 9

	4-5% familial 
	Early age of onset 
	Breast and ovarian in the same family 
	Frequent bilateral 
	In males also

Question 10

Segregation Analysis in Breast Cancer

Answer 10

Looking for dominant or recessive
Frequency’s
Risk
Use this info to carry out linkage analysis to work out where the allele is

Question 11

Linkage Analysis

Answer 11

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

Question 12

BRCA1 study

Answer 12

• BRCA1 gene
• Positional cloning of BRCA1 in 1994
• Chromosome 17
• Other families shown not to be linked to chromosome 17&raquo_space; BRCA2 on chromosome 13
  -BRCA1
  24 exons 1863a,a

Question 13

BRCA2

Answer 13

BRCA2
27 exons
Chromsome 13

Question 14

Similarity between BRCA1 and 2

Answer 14

Both got Nuclear localisation signals / multifunctional domains

Question 15

What has linkage analysis been applied to ??

Answer 15

Rare recessvie syndromes

e.g AT a mutation in ATM

Question 16

Positional Cloning

Answer 16

• 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

Question 17

lessons learnt from rare recessive syndromes: Signalling DNA Damage

Answer 17

• 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

Question 18

NBS and AT have several common features

Answer 18

• 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

Question 19

Ataxia telangiectasia (AT; caused by mutation in ATM)

Answer 19

• autosomal recessive

- cerebellar dysfunction, chromosomal instability, predisposition to cancer

Question 20

Nijmegen Breakage syndrome (NBS; caused by mutation in NBS1)

Answer 20

• autosomal recessive

- microcephaly, immunodeficiency, predisposition to haematopoietic malignancies

Question 21

BRCA1

Answer 21

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.

Question 22

ATM and Breast Cancer

Answer 22

ATM mutations have been identified in breast cancer families
variant of cell cycle arrest gene > CHEK2 also contributes to familial breast cancer

Question 23

Non-familial or sporadic cases of cancer are caused by what ?

Answer 23

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

Question 24

Genome Wide Association Studies

Answer 24

• SNP bead-chip Array
  -24 DNA samples per chip
  Assay thousands of SNPs

Question 25

GWAS

Answer 25

• 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

Question 26

Oncoarray study –

Answer 26

Huge analysis by multiple groups
Formed the Oncoarray chip
Allowed fine mapping

Question 27

Combined result of GWAS, iCOGS and oncorarray

Answer 27

Manhattan plot further up the smaller the P value
123000 cases
All SNPS
Idenfitied 65 new regions significantly associated with breast cancer

Question 28

Conclusion of

Combined result of GWAS, iCOGS and oncorarray

Answer 28

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