Cancer genes and detecting mutations Flashcards
Define cancer
A condition where abnormal cells grow and reproduce uncontrollably. The cancerous cells can invade and destroy surrounding healthy tissue, including organs.
Evidence that cancer is a genetic disease
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
What did Bert Vogelstein say in 1988 about cancer
“the tremendous progress made in understanding tumorigenesis in large part is owing to the discovery of the genes, that when mutated, lead to cancer”
What percentage of cancer is inherited (germ-line mutations)
5-10%
Features of germline mutations
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
Features of somatic mutations
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
What are the different types of substitution (point) mutations?
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)
Different types of mutations
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))
Explain simply the inheritance of mutations based on cell type
Mutation in germ-line cells passed onto offspring
Mutation in somatic cells not passed onto offspring
Two main groups of cancer genes
Oncogenes and Tumour suppressor genes
what are proto-oncogenes
The normal versions of oncogenes (without mutations)
Features of oncogenes
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
How can oncogenes be activated? give examples for each
Amplification (e.g. Myc oncogene)
Translocation (e.g. gene fusion BCR-ABL)
Point mutations (e.g. Ras family genes– Kras, Nras, Hras)
Features of TSGs
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)
Examples of cancers linked to TSGs
Retinoblastoma - RB1 (gene)
Familial adenomatous polyposis (FAP) – APC (gene)
Li-Fraumeni syndrome – TP53 (gene)
Explain how oncogenes and tumour suppressor genes differ in the distribution of mutations within them
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)
Explain how cancer is more complex than just one mutation in a gene
Usually several mechanisms need to fail in order for cancer to occur (several mutations need to occur)
Explain a more recent way of classifying genes
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
Features of gatekeeper genes
Act directly
Restrain cell proliferation
e.g. Classical tumour suppressors (RB1) and some oncogenes (RET)
Features of Caretaker genes
Act indirectly
Maintain integrity of genome, disruption leads to genomic instability
DNA repair genes; A subgroup of tumour suppressor genes (e.g. BRCA1,BRCA2)
Features of landscaper genes
Act indirectly
Control the environment in which cells grow, creating a microenvironment aiding cancer cell growth (e.g. extracellular matrix genes)
Describe DNA repair genes
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)
Explain the impact of inactivation of DNA repair genes by mutations
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)
Source for mutations within DNA repair henes can lead to cancer
JH Hoeijmakers 2001
Features of Retinoblastoma
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)
Out of unilateral and bilateral RB, which has an earlier age of diagnosis
Bilateral (also a higher incidence of other cancers in lifetime)
Who came up with the two hit hypothesis
Alfred Knudson 1971
RB was the prototype for this hypothesis
Why do inherited forms of RB occur earlier?
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
Explain the genetics of inherited RB
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
Explain the 2 hit hypothesis in terms of RB
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
Explain loss of heterozygosity (LOH) using RB as an example
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)
Explain haploinsufficiency
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
What gene encodes for the p53 (a tumour surpressor)
TP53
features of p53
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
Explain % survival of mice with different p53 zygosity
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
Explain the ‘Dominant-negative’ effect, using p53 as an example
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
Explain the multistep model
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
Explain multstep tumourigenesis in terms of colorectal cancer
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
Most sporadic cancers follow which model? give an example
Multi-step model e.g. colorectal cancer
What is the cancer gene census?
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
Examples of oncogenes and functions
MDM4 - p53 inhibator (breast, colon, lung)
Cyclin E - cyclin (gastric cancers)
N-ras - small G protein (head and neck cancers)
Examples of TSGs and function of gene product
p16 (INK4A) - CDK inhibator (familial melanoma)
VHL - ubiquitylation of HIF (von Hippel-Lindau syndrome)
Examples of technologies that detect larger scale changes to genetics involved in cancer
larger scale changes = chromosomal abnormalities, deletions, duplications
Cytogenetics, FISH, array-CGH (lower res – megabase lvl)
What are SNP arrays?
assess millions of SNPs (single nucleotide polymorphisms) of known variation
What method can be used for detecting known mutations?
PCR based methods
What methods are used to sequence genes, exons and mutation hotspots
Sanger sequencing and next gen
Explain sanger sequencing
▪ 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
What does deciding to sequence the genome, exome and gene pannel depend on?
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
Explain normal variation
- 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
How are germline and somatic mutations detected?
▪ 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
What are the two types of somatic mutations in cancer
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)
Explain driver mutations
▪ 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
Explain passenger mutations:
▪ 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.
Explain tumour heterogeneity/clonality
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
What may understanding which mutations occurred when help us understand?
why some cancers are resistant to specofoc treatments, and how to prevent this
Chemotherapy acts as..
a selective bottleneck BUT the fittest subclones survive and dominate the tumour
What are the challenges of tumour sequencing?
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)
What can be used to detect aneuploidy
array GCH
explain the challenged when it comes to samples for tumour sequencing
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
Explain why mutation frequency is a challenge when sequencing tumours
Particular somatic mutations may occur in only a few cells
Need to sequence more of the tumour cells to detect mutations in all subclones
Explain the challenge of mutations evolving when it comes to tumour sequencing
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
What are the aims of large NGS projects into the sequencing of cancer
Aims to understand genetic basis of cancer progression, prognosis, metastasis and to guide cancer treatment
What are the challenges of genome sequencing with relation to cancer?
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
Explain single cell technologies and some technical hurdles
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
