L1 - Genomic Instability Flashcards
Describe the principle of cancer arising from mutation
Heritable variation leads to selection which then leads to a change in allelic frequency
Describe how allelic frequency changes in canccer
Initially the cell has a single mutation - over time cell proliferation leads to even more mutations arising and these have increased allelic frequency
What is the first hallmark of cancer
How was it discovered
Genomic instability
Systematic sequencing of cancer cell genomes
What can instability of genomes be represented on
Cytogeneitic maps - circos plots
Describe how tumour cells harbour enormous genomic instability
There is a huge mutational burden #24000 mutations
Mutations within the whole tumour
Individual cells vary in their genomic composition
Describe the hierarchy of alterations that occur in cancer cell genomes
Interchromosomal strucutural varsiations
Intrachromosomal strucutural variations
Loss of heterozygosity/allelic imbalance
Copy number variation
Single nucleotide variation
How are cancer cell genomes sequenced?
Massive parallel sequencing
Describe the process of massive parallel sequencing
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
Describe how the specificity of DNA enables configuration of multiple related probes
Start with using position -1 probes - then can use position -2 and -5 probes
How is the problem of sequence depth addressed
Usiig offset anchors with variable length offsets
What is mean by HUGE parallel seqeucning
Huge arrays are used - many many chips used
Describe how the issue of signal strength is addressed
MORE than one molecule is used
DNA polymersase is used to amplify circular lengths which produces DNA nanoballs
What does CIN stand for
Chromosomal instability
What is CIN
The inability to maintain the correct number and gross composition of chromosomes
What is numerical CIN
Aneuploidy
The wrong number of chromosomes
What is strucutural CIN
Gross compositional failure in chromosomes
What can CIN be visualised with
Chromosomal painting
Describe the process of chromosome painting
Using probes that stain each chromosome a different colour for each chromosome
Can see through colour rearrangement if chromosomal rearrangement has occured
The philadelphia chromosome leads to what clinical condition
Chronic myeloid leukaemia
What is the fusion protein formed in the philadelphia chromosome
BCR-ABL fusion protein
What is the function of the BCR-ABL fusion protein
It encodes a consituitively active tyrosine kinase
How can the Philadelphia chromosome be visualised
What is seen
Using FISH
Flourescent in-situ hybridisation
Can see part of chromsomes 9 fused to 22 and part of 22 fused to 9
What is copy number variation an example of
Chromsomsal instability
Describe an instance where copy number variation is observed in cancer
N-myc is amplified in neuroblastoma
How can copu number variation be visualised
Use probes to the gene that you think has be amplified - would see many spots
Compare to a control
What is FAp
Familial adenomatous poplyposis
What is the gene that is mutated in FAP
The APC gene
This is a recessive mutation
Where does FAP occur
In the intestinal crypt
Describe NORMAL signalling in the intestinal crypt
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
Describe how FAP may occur
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
Describe the Wnt signalling cascade - in the presence of Wnt
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
APC is a
What its normal function
What happens if this is not present
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
What is MIN
Microsatellite instability
Refers to variation of a single nucleotide
What are microsatellites
Variable length dinucleotide repeats
Why are microsatellites vulnerable to DNA damage
Since mismatches are able to occur easily during replication
MIN is a consequence of
Unrepaired DNA damage
What are two ways in which unrepaired DNA damage can cause MIN
Deamination of C
Depurination of A
Deamination of C
Loss of the amino group
C–>U
When replicated pairs with A
Introduces a point mutation C-G ==> U-A
Depurination of A
Loss of the (big) purine group
When replicated one of the strands in a base short
(A-T nucelotide has been deleted)
In both the deamination of C and depurination of A what does MIN rely on
The replicaition before DNA damage can be recognised
What sort of mutations precipate MIN
Mutations in the mismatch repair proteins
Describe a clinical condition underpinned by a mutation in a mismatch repair pathway
HNPCC (Lynch Sundrome)
Hereditary non-polyposis colon cancer
Loss of function in MLH1 PMS2 MSH2 MSH6
What are MLH1 PMS2 MSH2 MSH6 involved in
Mismatch repair pathways
If the geneomic instability is a feature of carcinogenesis then what does this imply regarding the importance of mutations
Many of the mutations will be irrelevant
What are the two classed of mutations
Driver
passenger
Describe how driver mutations were identified
Compare viral oncogene sequences with human homologues
Then use assays of transformation to identify oncogenes in mouse modesl
Describe how transformed cells can be identified in the dish
Display both anchorage and growth factor independent growth
Describe how you would test if a gene is an oncogene in mice
Inject cellls that have been trasnformed into a mouse model
Should see tumour growth if it is an oncogene
Many forms of inherited cancer involves single gene mutations.
What gene is linked to retinoblastoma what does this encode
Rb
Transcriptional repressor
Many forms of inherited cancer involves single gene mutations.
What gene is linked to Li Fraumeni what does this encode
P53
DNA damage response protein
Many forms of inherited cancer involves single gene mutations.
What gene is linked to Wilm’s tumour what does this encode
Wt-1
Transcriptional regulator
Many forms of inherited cancer involves single gene mutations.
What gene is linked to Gorlins syndrome what does this encode
Pyc
Membrane receptor signalling
Many forms of inherited cancer involves single gene mutations.
What gene is linked to Breast cancer what does this encode
BRCA-1
DNA repair and recombination
Many forms of inherited cancer involves single gene mutations.
What gene is linked to FAP what does this encode
APC
B catenin signalling transducer/regulator
The early attempts to identify driver mutations favoured what type of genes
Genes promoting proliferation and clonal expnsion
What techniques were used to identify driver mutations
Homology to viral oncogenes
Cytogenetics
Differential gene expression
ALL COMBINED WITH TESTING IN A MODEL SYSTEM
What is progressive lymphoma associated with
How can this be visualised
Increased Myc expression
See using Western blot
What is NGS
What does it allow
Next generation sequencing technologies
Allows us to identify mutations
When using NGS to identify driver mutations what was the hypothesis
Driver mutations are likely to occur frequently while passenger mutations are likely to occur randomly
What were some of the genes commonly seen to be mutated in the 131 colon cancers that were sequenced
APC K-Ras P53 PIK3CA FBXW7
Genes commonly mutated are seen as what on the chromosome 1-X, gene position, frequency (x,y,z) plot
Mouthains
Hills and mountains …
Are genes that are frequently mutated and thus these are likely to be dirver mutatiosn
Hillocks (invisible)
Occur randomly with no pattern and thus are likely to be passenger mutations
In any given cancer around how many driver mutations are there
around 15
What can be seen in terms of mountains, hills and hillocks if we compare two colorectal cancer side by side
Some common mountains
Very few common hills
Many disparate hullocks
Describe what is seen when plotting cancer against age
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)
Describe the mathematical model related probability of getting cancer with the number of genetic changes that need to occur
LogP(T) = nLogA + constant
LogP(T) = nLogA + constant
What is the slope
Slop is the indication of what needs to occur
Steep slope means that many changes have to occur and the probability is low
What does bioinformatics allows us to look at with regards pathways
Looks at the links between various genes
Each line = some link and suggests that genes are here involved in the same pathway
In different types of cancers ______
Different parts of the pathway are targetted
e.g. some mutations more common in colon cancer and others more common in breast cancer
