Cancer genes and detecting mutations Flashcards

Question

Features of Retinoblastoma

Answer 1

Most common eye tumor in children Tumour of retinal stem cell Affects 1 in 20,000 children Males and Females equally affected Signs and symptoms of retinoblastoma include "white pupil" and eye pain or redness Treatments include surgery, chemotherapy, radiation therapy Identifying at-risk infants substantially reduces morbidity and mortality Diagnosed in the first few years of life Two forms; inherited and sporadic (non-inherited)

Answer 2

Bilateral (also a higher incidence of other cancers in lifetime)

Answer 3

Alfred Knudson 1971 RB was the prototype for this hypothesis

Answer 4

Mathematically showed that with a certain mutation rate you could explain this discrepancy by assuming that the sporadic cases needed two somatic mutations and the inherited (unilateral) cases only one This assumes; Recessively acting alleles The inherited cases had already inherited 1 mutation in the germline

Answer 5

Autosomal dominant transmission (around 50% offspring will Inherit the mutated copy) (gatekeeper function) Gene localized to chromosome 13 in 1978 RB1 gene cloned in 1986 First tumor suppressor gene discovered Gene encodes Rb protein which is a negative regulator the of cell cycle Large gene spanning 27 exons, with more than 100 known mutations – spread across the gene

Answer 6

Similar to explanation of recessive inheritance, when a child inherits one mutated copy of a gene from mum and one mutated copy from dad i.e. 2 mutated copies are required But not both inherited - instead 2 separate events are required One germline and one somatic mutation –> Inherited form Two somatic mutations -> Sporadic form Retinal cells are growing very rapidly in early life, likelihood of a second somatic mutation is very high when one gene is already out of action – average of 5 cells gain a second mutation i.e. 5 tumours/eye This disease is therefore inherited in a dominant fashion at the individual level and acts recessively at the cellular level 2-hit model explains RB and some other inherited cancers very well, but most cancers not due to single genes

Answer 7

Sporadic retinoblastoma - following single mutation, cell is left heterozygous (Rb +/-) and would exhibit wild-type phenotype, therefore loss of 2nd allele is required for cancer How could 2 separate mutational events occur in one cell disrupting both alleles? These types of mutational events in a single cell are highly unlikely second allele is not hit by a mutation – instead a recombination event occurred leading to loss of wild-type allele (allelic deletion or LOH)

Answer 8

about half a dozen tumour suppressor genes have abnormal cellular phenotypes when a single wild-type gene copy is present (not recessive acting at cellular level) [e.g. PTEN and NF1] Haplo-insufficient WT gene, therefore the mutated gene acts dominantly

Answer 9

Known as the “guardian of the genome” In the presence of DNA damage, p53 induces either cell-cycle arrest to allow for DNA repair, or apoptosis. May also be involved directly in DNA repair. Somatic mutations in TP53 commonly found in human tumour cells

Answer 10

p53 -/- mice develop normally so homozygous p53 not affecting embryonic growth, although do develop tumours and die young Does not fit the recessive action of tumours suppressors – p53+/- also get cancer Is only one mutation required? 'Dominant-negative’ effect

Answer 11

p53 subunits form a functional tetramer Mutated subunit still forms tetramers but 15/16 lack normal function The mutated alleles ‘cancel out’ the effects of the normal alleles

Answer 12

Most cancers involve multiple acquired mutations leading towards cancer Known as ‘Multi-Step Tumourigenesis’ Formation of tumours is complex and usually progresses over decades Epidemiological studies show age is a large factor in the incidence of cancer Cells acquire increasing cancer-like qualities Pathology provides evidence of a multistep process Pre-cancerous cells in cervical cancer screening

Answer 13

Cells of the intestinal wall have a high turnover, each day ~15-20% of the epithelial cells of the colon die and are replaced Changes occur in mucosal surface of the intestinal wall during a person's lifetime ~10% of all adenomas become cancerous can take ≥10 years to develop Estimated that mutations in ~5 critical genes are required Involves both tumour suppressor genes and oncogenes

Answer 14

Multi-step model e.g. colorectal cancer

Answer 15

an ongoing effort to catalogue those genes for which mutations have been causally implicated in cancer More than 1% of all human genes are implicated via mutation in cancer Of these, approximately 90% have somatic mutations in cancer, 20% bear germline mutations that predispose to cancer and 10% show both somatic and germline mutations

Answer 16

MDM4 - p53 inhibator (breast, colon, lung) Cyclin E - cyclin (gastric cancers) N-ras - small G protein (head and neck cancers)

Answer 17

p16 (INK4A) - CDK inhibator (familial melanoma) VHL - ubiquitylation of HIF (von Hippel-Lindau syndrome)

Answer 18

larger scale changes = chromosomal abnormalities, deletions, duplications Cytogenetics, FISH, array-CGH (lower res – megabase lvl)

Answer 19

assess millions of SNPs (single nucleotide polymorphisms) of known variation

Answer 20

PCR based methods

Answer 21

Sanger sequencing and next gen

Answer 22

▪ Dideoxy sequencing ▪ Chain-termination method 1. 4 reactions each terminating at a different base 2. Small amount ddNTP (~1%) so termination will occur only occasionally 3. Results in strands of all lengths 4. Strands separated on gel 5. Sequence read 5’->3’ from the bottom up ▪ Advances; ▪ Fluorescent labelling ▪ Automated sequencing ▪ Capillary sequencing ▪ Used in the Human Genome Project

Answer 23

Use depends on which question you wish to answer and your budget, whole genome = increased coverage but more costly, gene panel = decreased coverage but decreased cost

Answer 24

* Homo sapiens a young species not much time to accumulate vast genetic variation * ~0.1% variation in DNA between any two people * Germline mutation rate low, ~70 new mutations in each diploid genome * Somatic mutation rate much higher ~20x (but don’t get passed on to offspring) * Most mutations neutral – no functional effect * Selection can increase freq. of beneficial changes, and eliminate deleterious changes * Population bottlenecks reduce diversity ~300 ‘novel’ germline variants per individual in a sequenced exome, ~1000s per genome

Answer 25

▪ Can compare to databases of known germline genetic variation in the population ▪ But rare variants that may be specific to the patient/family To determine which variants are present only in the tumour matched tumour and normal samples are required Appropriate tissue – blood easy to get but may not be appropriate if blood cancer

Answer 26

driver mutations and passenger mutations ▪ Drivers give the clone a selective advantage and contribute to oncogenesis ▪ Passengers have no effect on oncogenesis Cancer cells have few drivers (<20) but many passengers (10s to >100,000)

Answer 27

▪ Confer an advantage to the cell ▪ Contribute to oncogenesis (growth of cancer/tumour) ▪ Selected for during cancer evolution ▪ The genes in which these occur are ‘cancer genes’ (oncogenes/TSGs) ▪ Average mutation rate in human cells is low 10-6 per gene per cell ▪ Majority of cancer causing mutations are recessive ▪ Seems very improbable that any cell would accumulate several mutations in cancer genes - But cancer is common ▪ Driver mutations increase chance of more mutations ▪ Growth advantage – increased growth rate means cells with mutation divide at greater rate relative to other cells so more cells/bigger target for further mutations ▪ Destabilising the genome – mutations in DNA repair genes result in greatly elevated mutation rates

Answer 28

▪ Don't contribute to the development of cancer but have occurred during the growth of the cancer ▪ Not selected for, random across the population ‘Along for the ride’ These will be carried along in the clonal expansion that follows and therefore will be present in all cells of the final cancer.

Answer 29

Cancer is clonal: a set of cells that all descend from a common ancestor cell characterised by one or more somatic driver mutations Within tumours, mutations may be fully clonal (founder mutation present in all cells) or subclonal (secondary mutations present in a proportion of cells) Different environments effect clonal expamsion, e.g. one clone ,etastesises to 2 locations in the body, two different clones (subclones) differentiate Caldas 2012

Answer 30

why some cancers are resistant to specofoc treatments, and how to prevent this

Answer 31

a selective bottleneck BUT the fittest subclones survive and dominate the tumour

Answer 32

samples (quantity, quality and purity) Distinguishing between driver and passenger mutations Tumour heterogeneity Mutation frequencing within cells tumours evolve most cancers are aneuploid (abnormal chromosome numbers)

Answer 33

Quantity: Limited DNA from biopsies Quality: Formalin-fixed paraffin-embedded (FFPE) can fragment and alter DNA Purity: -Tumour contaminated with germline DNA - Germline contaminated with tumour DNA

Answer 34

Particular somatic mutations may occur in only a few cells Need to sequence more of the tumour cells to detect mutations in all subclones

Answer 35

One sequencing experiment a snapshot in time of that tumours development May need to sequence over time, from different parts of the tumour, before and after treatment

Answer 36

Aims to understand genetic basis of cancer progression, prognosis, metastasis and to guide cancer treatment

Answer 37

Can be difficult to interpret variants in known cancer genes - known as mutations of ‘uncertain significance’ Mutations in new genes – are they involved in cancer? Further research will be needed We don’t know what all genes do and we don’t know what much of the non-coding regions (97%) of the genome do… What if we find out something we weren’t looking for? ‘Incidental findings’ e.g. Non-paternity, Mutation for a late onset disease, Carrier for some other disease Translating genetic findings into clinically useful information

Answer 38

Single cell sequencing - Whole Genome Amplification and sequencing of DNA from a single cell Allows tracing cell lineages Technical hurdles; Isolating rare cells (<1% of population) is very difficult Amplification ➢bias resulting in uneven sequencing – false negative results ➢Single bp errors by polymerase – false positive results ➢Allelic dropout - one allele in a heterozygous mutation (AB) is not amplified, resulting in a genotype which appears homozygous (A or B) – not the same as LOH - Allelic droput is result of problems in PCR amplification

Cancer genes and detecting mutations Flashcards

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