L1 - Genomic Instability

Question 1

Describe the principle of cancer arising from mutation

Answer 1

Heritable variation leads to selection which then leads to a change in allelic frequency

Question 2

Describe how allelic frequency changes in canccer

Answer 2

Initially the cell has a single mutation - over time cell proliferation leads to even more mutations arising and these have increased allelic frequency

Question 3

What is the first hallmark of cancer

How was it discovered

Answer 3

Genomic instability

Systematic sequencing of cancer cell genomes

Question 4

What can instability of genomes be represented on

Answer 4

Cytogeneitic maps - circos plots

Question 5

Describe how tumour cells harbour enormous genomic instability

Answer 5

There is a huge mutational burden #24000 mutations
Mutations within the whole tumour
Individual cells vary in their genomic composition

Question 6

Describe the hierarchy of alterations that occur in cancer cell genomes

Answer 6

Interchromosomal strucutural varsiations
Intrachromosomal strucutural variations
Loss of heterozygosity/allelic imbalance
Copy number variation
Single nucleotide variation

Question 7

How are cancer cell genomes sequenced?

Answer 7

Massive parallel sequencing

Question 8

Describe the process of massive parallel sequencing

Answer 8

Shear genomic DNA to 100bp ss fragments
Ligate chemically at the end to a known adaptor sequence
Chemically attach chimeric DNA to solid chip surface
Introduce an anchor which anneals to the adaptor sequence
Introduce probes each labelled with a different flurophore
Only the probe perfetly complementary to the genomic DNA will perfectly anneal in presence of DNA ligase
Raise temp - if not ligated probe will fall off
Spot on chip will be the same colour as that of the flurophore
Specificity of DNA ligand enablees multiple related probes

Question 9

Describe how the specificity of DNA enables configuration of multiple related probes

Answer 9

Start with using position -1 probes - then can use position -2 and -5 probes

Question 10

How is the problem of sequence depth addressed

Answer 10

Usiig offset anchors with variable length offsets

Question 11

What is mean by HUGE parallel seqeucning

Answer 11

Huge arrays are used - many many chips used

Question 12

Describe how the issue of signal strength is addressed

Answer 12

MORE than one molecule is used

DNA polymersase is used to amplify circular lengths which produces DNA nanoballs

Question 13

What does CIN stand for

Answer 13

Chromosomal instability

Question 14

What is CIN

Answer 14

The inability to maintain the correct number and gross composition of chromosomes

Question 15

What is numerical CIN

Answer 15

Aneuploidy

The wrong number of chromosomes

Question 16

What is strucutural CIN

Answer 16

Gross compositional failure in chromosomes

Question 17

What can CIN be visualised with

Answer 17

Chromosomal painting

Question 18

Describe the process of chromosome painting

Answer 18

Using probes that stain each chromosome a different colour for each chromosome
Can see through colour rearrangement if chromosomal rearrangement has occured

Question 19

The philadelphia chromosome leads to what clinical condition

Answer 19

Chronic myeloid leukaemia

Question 20

What is the fusion protein formed in the philadelphia chromosome

Answer 20

BCR-ABL fusion protein

Question 21

What is the function of the BCR-ABL fusion protein

Answer 21

It encodes a consituitively active tyrosine kinase

Question 22

How can the Philadelphia chromosome be visualised

What is seen

Answer 22

Using FISH
Flourescent in-situ hybridisation

Can see part of chromsomes 9 fused to 22 and part of 22 fused to 9

Question 23

What is copy number variation an example of

Answer 23

Chromsomsal instability

Question 24

Describe an instance where copy number variation is observed in cancer

Answer 24

N-myc is amplified in neuroblastoma

Question 25

How can copu number variation be visualised

Answer 25

Use probes to the gene that you think has be amplified - would see many spots Compare to a control

Question 26

What is FAp

Answer 26

Familial adenomatous poplyposis

Question 27

What is the gene that is mutated in FAP

Answer 27

The APC gene | This is a recessive mutation

Question 28

Where does FAP occur

Answer 28

In the intestinal crypt

Question 29

Describe NORMAL signalling in the intestinal crypt

Answer 29

Stem cells at the bottom of the cryp B catenin signalling causes the proliferation and upwards movement Towards the top of the crypt B-catenin is turned OFF leading the cell cycle arrest and the differentiation of these cells

Question 30

Describe how FAP may occur

Answer 30

Failure to turn OFF B-catenin signalling at the top of the crypt leads to all of the cells at the top having a progenitor like phenotype and the formation of a polyp

Question 31

Describe the Wnt signalling cascade - in the presence of Wnt

Answer 31

Wnt binds frizzled which activates dishevelled Dsh inhibits GSK3B which releases B catenin from the Axin-APC-GSK3B complex B catenin translocates into the nucleus binding Tcf/Lef causing transcription of Wnt target genes

Question 32

APC is a What its normal function What happens if this is not present

Answer 32

Tumour suppressor gene Normally part of the complex that in the absence of Wnt, binds and prevents the nuclear translocation of B-catenin When mutant there is no way of stopping B-catenin entering the nucleus and thus this pathway is constituitively on

Question 33

What is MIN

Answer 33

Microsatellite instability | Refers to variation of a single nucleotide

Question 34

What are microsatellites

Answer 34

Variable length dinucleotide repeats

Question 35

Why are microsatellites vulnerable to DNA damage

Answer 35

Since mismatches are able to occur easily during replication

Question 36

MIN is a consequence of

Answer 36

Unrepaired DNA damage

Question 37

What are two ways in which unrepaired DNA damage can cause MIN

Answer 37

Deamination of C | Depurination of A

Question 38

Deamination of C

Answer 38

Loss of the amino group C-->U When replicated pairs with A Introduces a point mutation C-G ==> U-A

Question 39

Depurination of A

Answer 39

Loss of the (big) purine group When replicated one of the strands in a base short (A-T nucelotide has been deleted)

Question 40

In both the deamination of C and depurination of A what does MIN rely on

Answer 40

The replicaition before DNA damage can be recognised

Question 41

What sort of mutations precipate MIN

Answer 41

Mutations in the mismatch repair proteins

Question 42

Describe a clinical condition underpinned by a mutation in a mismatch repair pathway

Answer 42

HNPCC (Lynch Sundrome) Hereditary non-polyposis colon cancer Loss of function in MLH1 PMS2 MSH2 MSH6

Question 43

What are MLH1 PMS2 MSH2 MSH6 involved in

Answer 43

Mismatch repair pathways

Question 44

If the geneomic instability is a feature of carcinogenesis then what does this imply regarding the importance of mutations

Answer 44

Many of the mutations will be irrelevant

Question 45

What are the two classed of mutations

Answer 45

Driver | passenger

Question 46

Describe how driver mutations were identified

Answer 46

Compare viral oncogene sequences with human homologues | Then use assays of transformation to identify oncogenes in mouse modesl

Question 47

Describe how transformed cells can be identified in the dish

Answer 47

Display both anchorage and growth factor independent growth

Question 48

Describe how you would test if a gene is an oncogene in mice

Answer 48

Inject cellls that have been trasnformed into a mouse model | Should see tumour growth if it is an oncogene

Question 49

Many forms of inherited cancer involves single gene mutations. What gene is linked to retinoblastoma what does this encode

Answer 49

Rb Transcriptional repressor

Question 50

Many forms of inherited cancer involves single gene mutations. What gene is linked to Li Fraumeni what does this encode

Answer 50

P53 DNA damage response protein

Question 51

Many forms of inherited cancer involves single gene mutations. What gene is linked to Wilm's tumour what does this encode

Answer 51

Wt-1 Transcriptional regulator

Question 52

Many forms of inherited cancer involves single gene mutations. What gene is linked to Gorlins syndrome what does this encode

Answer 52

Pyc Membrane receptor signalling

Question 53

Many forms of inherited cancer involves single gene mutations. What gene is linked to Breast cancer what does this encode

Answer 53

BRCA-1 DNA repair and recombination

Question 54

Many forms of inherited cancer involves single gene mutations. What gene is linked to FAP what does this encode

Answer 54

APC B catenin signalling transducer/regulator

Question 55

The early attempts to identify driver mutations favoured what type of genes

Answer 55

Genes promoting proliferation and clonal expnsion

Question 56

What techniques were used to identify driver mutations

Answer 56

Homology to viral oncogenes Cytogenetics Differential gene expression ALL COMBINED WITH TESTING IN A MODEL SYSTEM

Question 57

What is progressive lymphoma associated with How can this be visualised

Answer 57

Increased Myc expression See using Western blot

Question 58

What is NGS | What does it allow

Answer 58

Next generation sequencing technologies | Allows us to identify mutations

Question 59

When using NGS to identify driver mutations what was the hypothesis

Answer 59

Driver mutations are likely to occur frequently while passenger mutations are likely to occur randomly

Question 60

What were some of the genes commonly seen to be mutated in the 131 colon cancers that were sequenced

Answer 60

``` APC K-Ras P53 PIK3CA FBXW7 ```

Question 61

Genes commonly mutated are seen as what on the chromosome 1-X, gene position, frequency (x,y,z) plot

Answer 61

Mouthains

Question 62

Hills and mountains ...

Answer 62

Are genes that are frequently mutated and thus these are likely to be dirver mutatiosn

Question 63

Hillocks (invisible)

Answer 63

Occur randomly with no pattern and thus are likely to be passenger mutations

Question 64

In any given cancer around how many driver mutations are there

Answer 64

around 15

Question 65

What can be seen in terms of mountains, hills and hillocks if we compare two colorectal cancer side by side

Answer 65

Some common mountains Very few common hills Many disparate hullocks

Question 66

Describe what is seen when plotting cancer against age

Answer 66

That the probability is the number of genetic changes that are required to cause cancer (lots of changes would mean that cancer would be very rare)

Question 67

Describe the mathematical model related probability of getting cancer with the number of genetic changes that need to occur

Answer 67

LogP(T) = nLogA + constant

Question 68

LogP(T) = nLogA + constant What is the slope

Answer 68

Slop is the indication of what needs to occur Steep slope means that many changes have to occur and the probability is low

Question 69

What does bioinformatics allows us to look at with regards pathways

Answer 69

Looks at the links between various genes | Each line = some link and suggests that genes are here involved in the same pathway

Question 70

In different types of cancers ______

Answer 70

Different parts of the pathway are targetted e.g. some mutations more common in colon cancer and others more common in breast cancer