Lecture 1/2 - the complexity of cancer

Question 1

Circos plot

Answer 1

represent data sequence of whole cancer cell genomes

all lines, dots and colours show extent of change

Question 2

Inter-chromosomal structural variations

Answer 2

parts of the chromosome stuck to different chromosomes - swapped around

Question 3

Intra-chromosomal structure variations

Answer 3

normally separated parts in chromosomes come together or parts are swapped around within a single chromosome

Question 4

Loss of heterozygosity/allelic imbalance

Answer 4

loss of heterozygous during proliferation/mitotic recombination
one is non-functional so loss of the functional allele is particularly important for tumour suppressors

Question 5

Copy number variation

Answer 5

in porto-oncogenes, changes in expression

Question 6

Name the progressive hierarchy in order in cancer genomes

Answer 6

Interchromosomal
intrachromosomal structural variations
loss of heterozygosity/allelic imbalance
copy number variation
single nucleotide variation

Question 7

Cancer genome sequencing process

Answer 7

1. Shear DNA to 100bps fragments
2. chemically ligate to a known adaptor sequence
3. chemically attached chimeric DNA to solid chip surface
4. Introduce probes and anchor which are colour coded with fluorescent fluorophores
5. DNA ligase forms bonds between probes to anchor if complementary - shape must match in order for them to catalyse the reaction
6. raise temperature as temp it takes for probe to be removed determines length
7. Using DNA ligase and melting temp to ensure only correct probe in place

Question 8

Cancer genome sequencing can also…

Answer 8

can also investigate positions 1-5 in any probe set
can also use offset anchor in order to extend the sequencing so build up with smaller parts
only take one signal therefore must amplify it

Question 9

Cancer genome sequencing must amplify the signals by…

Answer 9

take two anchors to form a circle
add DNA polymerase and many nucleotides
DNA polymerase continuously goes around circle repeatedly to make many copies to increase signal strength

Question 10

Nanomoles

Answer 10

attached to amplifying circle array and use probes to determine sequences

Question 11

Raising temperature in cancer genome sequencing means…

Answer 11

the temperature it takes to be removed determines length. If the wrong one binds the ligase will not catalyse the reaction so temperature increase will just get it to fall off.

Question 12

Chromosomal instability (CIN)

Answer 12

the ability to maintain the correct number and gross composition of chromosomes, two types

Question 13

Numerical CIN

Answer 13

aneuploidy, wrong number of chromosomes

Question 14

Structural CIN

Answer 14

gross compositional failure in chromosomes

Question 15

Philadelphia chromosome

Answer 15

structural CIN underpinning myeloblastic leukaemia

reciprocal translocation between ends of chromosomes 9 and 22

Question 16

Philadelphia chromosome is…

Answer 16

the fusion of the BCR-ABL proteins to produce a non-natural protein which is unregulated and highly expressed

Question 17

ABL

Answer 17

protein kinase involved in proliferation and cell cycle progression
drives WBC progenitor proliferation

Question 18

Myc CIN

Answer 18

Myc is a porto-oncogene that is amplified in neuroblastomas

Question 19

Colorectal cancer occurs when…

Answer 19

loss of the second APC allele in familial adenomatous polyposis
Loss of heterozygosity

Question 20

Wnt signalling and APC loss

Answer 20

Wnt down regulation doesn’t occur, so B-catenin is always released, so cells migrating up the crypt doesn’t stop proliferating
GSK cannot induce B-catenin degradation

Question 21

Microsatellite instabilità (MIN)

Answer 21

all single nucleotide variations occurred due to unpaired DNA damage

Question 22

microsatellite

Answer 22

variable length dinucleotide repeats

Question 23

Microsatellite mutations

Answer 23

these are hypermutable with mismatches occur. DNA repair cuts out the loop but does not fix properly so require realigning instead

Question 24

Deamination

Answer 24

changes cysteine to uracil, so when DNA is replicated one stranded results in U-A pairing rather than C-G

Question 25

Depurination

Answer 25

Adenine is removed so when DNA is replicated, an A-T nucleotide pair is deleted

Question 26

Mutations in mismatch repair proteins precipitate MIN

Answer 26

MSH creates ring around mismatch site, nucleoside creates nick in mismatch vicinity, ring finds nick and chooses way to create ssDNA to replace mismatch, polymerase replicates this properly and follows template to fix it

Question 27

HNPCC

Answer 27

aka Lynch syndrome, a MIN example

Loss of function alleles for MSH and loop

Question 28

Driver mutations

Answer 28

viral oncogenes, drivers of carcinogenesis

Question 29

Driver mutation experiments

Answer 29

take fragmented DNA and transfect normal mice fibroblasts, inject these into mice
if oncogenes are present then tumours are formed