Genetic epidemiology of cancer

Question 1

What are examples of rare familial cancers and the mutated tumour suppressor genes that cause them?

Answer 1

Familial adenomatous polyposis- APC
Breast cancer- BRCA1/2
Retinoblastoma- RB1
Familial melanoma- CDKN2A

Question 2

What are the mutations associated with the first and second hits in cancer?

Answer 2

1st hit- point mutation- premature termination of the protein
2nd hit- loss of a larger region on the homologous chromosome- eliminates the WT allele on the other chromosome

Question 3

How do you look for the loss of a region of a chromosome?

Answer 3

Look at LoH- by comparing polymorphic microsatellite markers between the tumour and blood
blood= heterozygosity
tumour= homozygosity

Question 4

How have genes for familial cancers been identified?

Answer 4

1. By positional cloning- linkeage analysis studies
2. Identification of deletions and LoH in tumours
3. Testing tumours for mutations in candidate genes
4. Sequencing of candidate genes in those with a family history

Question 5

What are the main steps in positional cloning?

Answer 5

1. Assemble families and genotype their DNA
2. Look to see if disease cosegregates with a marker on a particular chromosome
3. If you identify a chromosomal region linked to cancer- use more families and markers to narrow down the region

Question 6

What are the features of familial breast cancer?

Answer 6

Familial breast cancer has a set of characteristics:
early age of onset
breast and ovarian cancer seen in the family
bilateral disease
males affected

Question 7

What is the genetic model of familial breast cancer? What did this help do?

Answer 7

The identification that the inheritance familial breast cancer is dominant
1% frequency in population
In people carrying a risk allele- risk of almost 40% before the age of 40
Use this info to carry out linkage analysis

Question 8

What is the basis of linkeage analysis?

Answer 8

Genotype markers across the family
observe whether or not a particular marker allele cosegregates with disease in the family
can use this data to work out the likelihood that our marker is linked to the disease gene
generates a lod score- measure of how likely it is that the disease gene and marker is linked
lod score of greater than 3= statistically significant

Question 9

What was the lod score of a marker at chromosome 17? which studies followed suit?

Answer 9

Lod score of 5.98
identified that chromosome 17 was linked to breast cancer
further linkage studies: failed to show linkage to marker in chromosome 17 in 20 families
Another study: 5 families showed linkage, 2 didnt
genetic heterogeneity= some families are linked to 17, but in other families a different region is relevant

Question 10

What did the gene on chromosome 17 turn out to be?

Answer 10

BRCA1
Narrowed down the region to 600kb
Cloned the gene- identified BRCA1- candidate gene causing breast cancer in these families

Question 11

What gene was identified in the families not linked to chromosome 17?

Answer 11

BRCA2
15 families analysed by linkage analysis
the gene was localised on chromosome 13

Question 12

What sort of proteins do BRCA1 and BRCA2 encode?

Answer 12

Multifunctional proteins

they have structural features in common, but share no sequence similarity

Question 13

What similarities do the BRCA1 and BRCA2 proteins share?

Answer 13

They both have a region that binds RAD5 (DNA repair protein)
they both have nuclear localisation signals
both have transcriptional activation domains
The proteins are performing lots of different functions in the cell

Question 14

How does ATM interact with RAD50, MRE11 and NBS1?

Answer 14

ATM phosphorylates MRE11 and NBS1 in response to DNA damage

The RAD50, MRE11 and NBS1 complex binds at sites of DNA double-strand breaks and initiates DNA repair

Question 15

What does a mutation in the genes in this complex cause?

Answer 15

Familial syndromes with genomic instability
Ataxia telangiectasia (AT)- ATM mutation
Nijmegan breakage syndrome (NBS)- NBS1 mutation
AT like syndrome- MRE11 mutation

Question 16

What common features does NBS and AT share?

Answer 16

Increased sensitivity to ionising radiation

fail to induce p53 at the G1/S checkpoint- fail to stop DNA synthesis when exposed to ionising radiation

Question 17

Why are people with AT/NBS so prone to cancers?

Answer 17

When DNA damage is too severe the cells dont activate their checkpoint and thus continue with cell division regardless
mutations accumulate in DNA as a result- oncogenes and tumour suppressor gene mutations

Question 18

How does ATM interact with BRCA1? Which technique proves this?

Answer 18

ATM phosphorylates BRCA1-
irradiating WT and ATM mutant fibroblasts- did a western blot using anti-BRCA1 antibodies
BRCA1 was phosphorylated in the WT but not mutant cells

Question 19

What signified the importance of phosphorylation of BRCA1?

Answer 19

A mutated BRCA1 lacking the phosphorylation sites- failed to rescue the radiation hypersensitivity of the BRCA1 deficient cell line

Question 20

What other gene variants have been associated with breast cancer?

Answer 20

ATM mutations
CHEK2 mutations
PALB2
all identified through sequencing of candidate genes in families who had breast cancer- but didn’t have BRCA1 or BRCA2 mutations

Question 21

What underlies sporadic breast cancer?

Answer 21

A combination of genes and the environment

genetic variation in this case is polymorphisms rather than mutations

Question 22

What happens in a GWAS?

Answer 22

Assemble lots of cases and matched controls-
genotype them for their SNPs
count up the alleles in the cases and compare to the controls
if a SNP is at a higher frequency in the cases- likely that there is a disease gene close

Question 23

What is a SNP bead chip assay?

Answer 23

SNPs are genotyped using the chip arrays
can assay 1000s of SNPs at the same time
need very large samples

Question 24

Why are there many stages needed in a GWAS?

Answer 24

1. Get lots of SNPs that dont work well- have to run replicates to check everything is working correctly- reduces the amount of usable data
2. Likely to have a large number of false-positive results- to make sure results are believable and can be replicated- need multiple steps

Question 25

What was the summary from the GWAS of breast cancer?

Answer 25

Top hit= FGFR2
MAP3K1 kinase
lots of cancer association in region of chromosome 8-but theres no genes here?
on another region of chromo 8- CMIC gene- they think the SNPS associated with the cancer- affecting the transcription of this gene

Question 26

What is the oncoarray network?

Answer 26

International cancer genotyping effort- to try and identify as many genes as possible
5 cancers studied- created a SNP chip with half a million SNP markers

Question 27

What did the oncoarray study find in terms of breast cancer? How did these results overlap with previous driver genes identified in breast cancer?

Answer 27

65 new regions associated with breast cancer- with quite significant p values
Strong overlap between the target genes (600+) identified in the oncoarray study and somatic driver genes (147) identified by sequencing of breast cancer tumours
20 overlapped- statistically significant enrichment