Cancer genes and detecting mutations

Question 1

Define cancer

Answer 1

A condition where abnormal cells grow and reproduce uncontrollably. The cancerous cells can invade and destroy surrounding healthy tissue, including organs.

Question 2

Evidence that cancer is a genetic disease

Answer 2

Most carcinogens are also mutagens

not contagious (usually)

incidence increases with age, as does damage to DNA

Some cancers segregate in families

Chromosomal instability: a common feature and specific chromosomal changes are found in some cancers

Defects in DNA repair increase the probability of cancer

Question 3

What did Bert Vogelstein say in 1988 about cancer

Answer 3

“the tremendous progress made in understanding tumorigenesis in large part is owing to the discovery of the genes, that when mutated, lead to cancer”

Question 4

What percentage of cancer is inherited (germ-line mutations)

Answer 4

5-10%

Question 5

Features of germline mutations

Answer 5

Every cell of the body, including the reproductive cells (egg and sperm)

Passed directly from a parent to a child

Less common cause of cancer

Cancer caused by germline mutations is called inherited cancer

Question 6

Features of somatic mutations

Answer 6

Also known as ‘acquired mutations’

Occur from damage to genes during a person’s lifetime [e.g. tobacco, ultraviolet (UV) radiation and age]

They are not passed from parent to child

The most common cause of cancer

Cancer caused by somatic mutations is called sporadic cancer

Question 7

What are the different types of substitution (point) mutations?

Answer 7

Silent
Nonsense (STOP)
Missense (both conservative and non-conservative)

These can also be classed into coding (occurring in the coding region) and non-coding (Non-coding mutations can still have some effects e.g. transcription)

Question 8

Different types of mutations

Answer 8

Substitution (point)

Insertions – may cause transcription of a functional protein, frame shift ect

Deletions – may cause a protein to therefor not be transcribed

Duplication – can lead to over-expression

Inversions

Translocations (e.g. Philadelphia chr, BCR-ABL gene fusion in CML/AML (fusion protein))

Question 9

Explain simply the inheritance of mutations based on cell type

Answer 9

Mutation in germ-line cells passed onto offspring

Mutation in somatic cells not passed onto offspring

Question 10

Two main groups of cancer genes

Answer 10

Oncogenes and Tumour suppressor genes

Question 11

what are proto-oncogenes

Answer 11

The normal versions of oncogenes (without mutations)

Question 12

Features of oncogenes

Answer 12

Tend to be dominantly acting (gain of function/switched on)

Mutations in these genes are not usually inherited (exceptions = MEN2 (RET oncogene), HPRCC (MET oncogene))

Activated oncogene in the germ line normally affects embryonic development so severely that it causes embryonic lethality

Genes which normally function to PROMOTE cell growth/division

Question 13

How can oncogenes be activated? give examples for each

Answer 13

Amplification (e.g. Myc oncogene)

Translocation (e.g. gene fusion BCR-ABL)

Point mutations (e.g. Ras family genes– Kras, Nras, Hras)

Question 14

Features of TSGs

Answer 14

Recessively acting (two copies mutated to have impact on function)

Mutations in these genes inherited in family cancer syndromes

Mutated tumor-suppressor gene

These genes normally function to PREVENT cell growth/division

Mutations cause loss of function (switched off)

Question 15

Examples of cancers linked to TSGs

Answer 15

Retinoblastoma - RB1 (gene)

Familial adenomatous polyposis (FAP) – APC (gene)

Li-Fraumeni syndrome – TP53 (gene)

Question 16

Explain how oncogenes and tumour suppressor genes differ in the distribution of mutations within them

Answer 16

Oncogenes:
- tend to have mutations in few codons affecting particular domains
- Bias towards missense mutations

Tumour suppressor genes:

• tend to have mutations more widely spread across the gene
• more evenly missense mutations and mutations inducing premature termination codons (nonsense) (truncated protein leading to loss of function)

Question 17

Explain how cancer is more complex than just one mutation in a gene

Answer 17

Usually several mechanisms need to fail in order for cancer to occur (several mutations need to occur)

Question 18

Explain a more recent way of classifying genes

Answer 18

classified as ‘caretaker’, ‘gatekeeper’ and ‘landscaper’ genes

Reflecting the nature of the function of the gene which causes cancer, some genes can function in different ways in different situations

Question 19

Features of gatekeeper genes

Answer 19

Act directly

Restrain cell proliferation

e.g. Classical tumour suppressors (RB1) and some oncogenes (RET)

Question 20

Features of Caretaker genes

Answer 20

Act indirectly

Maintain integrity of genome, disruption leads to genomic instability

DNA repair genes; A subgroup of tumour suppressor genes (e.g. BRCA1,BRCA2)

Question 21

Features of landscaper genes

Answer 21

Act indirectly

Control the environment in which cells grow, creating a microenvironment aiding cancer cell growth (e.g. extracellular matrix genes)

Question 22

Describe DNA repair genes

Answer 22

Often thought of as a subclass of tumor suppressor genes:

Targeted by loss of function mutations, similar to classical tumour suppressor genes

However;

Classical tumour suppressor genes are directly involved in growth inhibition or differentiation (gatekeeper function)

DNA repair genes are indirectly involved in growth inhibition or differentiation (caretaker function)

Question 23

Explain the impact of inactivation of DNA repair genes by mutations

Answer 23

results in DNA damage going unrepaired

leads to accumulation of mutations in the other cellular genes

Increasing the likelihood of damaging mutations in other critical genes (i.e. other tumour suppressors or proto-oncogenes)

Question 24

Source for mutations within DNA repair henes can lead to cancer

Answer 24

JH Hoeijmakers 2001

Question 25

Features of Retinoblastoma

Answer 25

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)

Question 26

Out of unilateral and bilateral RB, which has an earlier age of diagnosis

Answer 26

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

Question 27

Who came up with the two hit hypothesis

Answer 27

Alfred Knudson 1971

RB was the prototype for this hypothesis

Question 28

Why do inherited forms of RB occur earlier?

Answer 28

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

Question 29

Explain the genetics of inherited RB

Answer 29

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

Question 30

Explain the 2 hit hypothesis in terms of RB

Answer 30

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

Question 31

Explain loss of heterozygosity (LOH) using RB as an example

Answer 31

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)

Question 32

Explain haploinsufficiency

Answer 32

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

Question 33

What gene encodes for the p53 (a tumour surpressor)

Answer 33

TP53

Question 34

features of p53

Answer 34

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

Question 35

Explain % survival of mice with different p53 zygosity

Answer 35

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

Question 36

Explain the ‘Dominant-negative’ effect, using p53 as an example

Answer 36

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

Question 37

Explain the multistep model

Answer 37

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

Question 38

Explain multstep tumourigenesis in terms of colorectal cancer

Answer 38

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

Question 39

Most sporadic cancers follow which model? give an example

Answer 39

Multi-step model e.g. colorectal cancer

Question 40

What is the cancer gene census?

Answer 40

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

Question 41

Examples of oncogenes and functions

Answer 41

MDM4 - p53 inhibator (breast, colon, lung)

Cyclin E - cyclin (gastric cancers)

N-ras - small G protein (head and neck cancers)

Question 42

Examples of TSGs and function of gene product

Answer 42

p16 (INK4A) - CDK inhibator (familial melanoma)

VHL - ubiquitylation of HIF (von Hippel-Lindau syndrome)

Question 43

Examples of technologies that detect larger scale changes to genetics involved in cancer

Answer 43

larger scale changes = chromosomal abnormalities, deletions, duplications

Cytogenetics, FISH, array-CGH (lower res – megabase lvl)

Question 44

What are SNP arrays?

Answer 44

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

Question 45

What method can be used for detecting known mutations?

Answer 45

PCR based methods

Question 46

What methods are used to sequence genes, exons and mutation hotspots

Answer 46

Sanger sequencing and next gen

Question 47

Explain sanger sequencing

Answer 47

▪ 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

Question 48

What does deciding to sequence the genome, exome and gene pannel depend on?

Answer 48

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

Question 49

Explain normal variation

Answer 49

• 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

Question 50

How are germline and somatic mutations detected?

Answer 50

▪ 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

Question 51

What are the two types of somatic mutations in cancer

Answer 51

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)

Question 52

Explain driver mutations

Answer 52

▪ 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

Question 53

Explain passenger mutations:

Answer 53

▪ 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.

Question 54

Explain tumour heterogeneity/clonality

Answer 54

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

Question 55

What may understanding which mutations occurred when help us understand?

Answer 55

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

Question 56

Chemotherapy acts as..

Answer 56

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

Question 57

What are the challenges of tumour sequencing?

Answer 57

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)

Question 58

What can be used to detect aneuploidy

Answer 58

array GCH

Question 59

explain the challenged when it comes to samples for tumour sequencing

Answer 59

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

Question 60

Explain why mutation frequency is a challenge when sequencing tumours

Answer 60

Particular somatic mutations may occur in only a few cells

Need to sequence more of the tumour cells to detect mutations in all subclones

Question 61

Explain the challenge of mutations evolving when it comes to tumour sequencing

Answer 61

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

Question 62

What are the aims of large NGS projects into the sequencing of cancer

Answer 62

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

Question 63

What are the challenges of genome sequencing with relation to cancer?

Answer 63

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

Question 64

Explain single cell technologies and some technical hurdles

Answer 64

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