Mutations Flashcards
How many protein coding genes are there in the human genome?
~22,000
What % of the human genome do protein coding genes constitute?
~1-2% of all DNA
What is the result of only a small percentage of the human genome coding for proteins?
Mutations in much of the genome are of little/no consequence
What happens if a mutation is within/close to gene?
They are likely to cause disease
What determines the consequences of a mutation?
The type of mutation and its location within a gene
What is the most common form of sequence variation?
Single-base substitutions
How often do single base substitutions occur?
About every 300bp
What is a single-base substitution also known as?
A single nucleotide polymorphism (SNP)
What are ~2/3 SNPs?
C→ T changes
What are more common, transitions or transversions?
Transitions
How many amino acids are specified by the genetic code?
20
How many codon triplets are there per amino acid?
Can be 1, 2, 3, 4 or 6
How many stop codons are there?
3
What normally happens where there is a single base change at the 3rd codon position?
Does not normally result in amino acid change
What is it called when a single base change doesn’t result in an amino acid change?
A silent substitution
Are silent substitutions always truly silent?
No
What do most amino acid changes result fmor?
Changes at codon positions 1 and 2
What are the possible mutations that changes amino acids?
- Missense mutations
- Silent mutations
- Nonsense mutations
- Frameshift mutations
- Promoter, start codon and stop codon mutations
- Intron splice site mutations
What happens in a missense mutation?
One amino acid is substituted by another, normally by a single base change
What happens in a silent mutation?
Single base substitution that does not change the amino acid
Why can some silent mutations affect amino acids?
Can disrupt RNA splicing, and therefore still cause heritable disease
What happens in a nonsense mutation?
An amino acid codon is changed to a stop codon by change of a single base
What happens in a frameshift mutation?
The reading frame of mRNA is altered in some way
How can the reading frame of mRNA be altered?
- Insertions
- Deletions
- Splice-site mutations
What are often found in the alternate reading frame?
Stop codons
How many alternate reading frames are there following a frameshift mutation?
2
What is the result in a stop codon being in a alternative reading frame?
Causes the ribosome to terminate translation prematurely
What is meant by a conservative missense mutation?
Some amino acid substitutions are better tolerated that others
Give an example of a fairly well tolerated missense mutation?
Substitution of valine by alanine might be well tolerated in non-critical regions of proteins
Why may a valine to alanine substitution be well tolerated?
They have similar properties
What has resulted from the tolerance of valine to alanine substitutions?
They are adjacent in the genetic code, by evolution
What is the effect of small insertions/duplications?
Either maintain or disrupt the reading frame
What results in the maintenance of the reading frame?
Gain or loss of multiples of 3bp
Why does the gain/loss of multiples of 3 bp result in the maintenance of the reading frame?
As this is the gain/loss of a whole amino acid(s)
What do gains/losses of non-multiple of 3’s cause?
Frameshifts
What can frameshifts produce?
Premature termination codons (PTCs)
What happens to mRNAs that contain PTCs?
They are degraded by ‘nonsenses mediated decay’ (NMD), so little/no protein produced
Why is NMD essential?
To mop up mutated mRNA
What is NMD?
An evolutionary protective mechanism
Why is NMD advantageous?
It’s better to have produced reduced amounts of protein than mutated proteins
What do mutations at intron splice sites usually result in?
Skipping of the exon immediately adjacent to the mutation
What happens in the skipped exon is a multiple of 3bp?
The mRNA is shortened, but remains in frame
What happens if the skipped exon is not a multiple of 3bp?
The mRNA is shortened, and will contain a frameshift and a PTC, leading to NMD
Why may it better if the mutation is not a multiple of 3?
Because the protein will be broken down, so no mutated protein made
What causes base changes?
- Sequence changes during DNA replication
- Chemicals can induce mutation
- Exposure to different types of radiation
How can sequence changes during DNA replication cause mutation?
NAME?
How can chemicals induce mutation?
NAME?
What kinds of radiation can cause mutation?
NAME?
What happens in tautomeric shift?
A proton briefly changes position, and then jumps back to where it should be
Where does tautomeric shift occur?
In the 4 bases of DNA
What are the rare forms?
The forms of the bases where the proton is in the alternative position
What is different about the rare forms?
They have altered base pairing properties
How do the rare forms behave?
As an altered template base during DNA replication
What do the rare forms cause?
Bonding to another base
What anomalous base pairing is caused by tautomeric forms?
- A - C ( with 2 H bonds)
- T - G (with 3 H bonds)
What happens if the C base is in it’s rare tautomeric form?
DNA polymerase will recognise it as a T base, and an A will be inserted into the new strand.
What happens in slippage during replication?
The newly synthesised strand loops out, or the template strand loops out
What is the result of the newly synthesised strand looping out?
The addition of one nucleotide to the new stand, to maintain base pairing
What is the result of the template stand looping out?
The omission of one of the nucleotides on the new strand, so the newly synthesised strand is one short of what it should be
Is slippage during replication common?
Fairly
When does slippage during replication occur?
When several of the same bases occur one after the other
Give two chemical mutagens that cause base changes
- Nitrous acid
- Ethyl methane sulphonate (EMS)
What does nitrous acid do?
Replace amino groups with keto groups
What is the result of the replacement of amino groups with keto groups?
Changes the properties of the bases
What are the bases changed to when amino groups are substituted for keto groups?
- C → U; base pairs with A
- A → H; base pairs with C
- G → X; base pairs with C
What does EMS cause?
Removal of purine rings
What happens when a purine ring is removed?
The purine backbone stays the same, but the ring is removed, so no template base there
What can apurinic sites be paired with during replication?
Any base
What is the result of apurinic sites pairing with any base during replication?
3/4 times, it pairs with the wrong base
What is2-amino-3-methylimidazo[4,5-f]quinoline(IQ)?
A heterocyclic aromatic amine food mutagen?
Where is IQ identified?
Cooked meats and cigarette smoke condensates
What does IQ do?
Disrupts the packing of bases
How doesIQ disrupt the packing of bases?
The planar molecule slips into the DNA double helix
What does IQ cause?
Mostly single base deletions at G-C base pairs, as it interferes with separation of bases in the 2 strands
How does IQ act?
- Intercalation of a molecule of IQ forces the bases further part on one DNA strand
- This leads to misreading by the DNA polymerase, and deletion of a single base
Other that IQ, what else intercalates into DNA?
Ethidium bromide
Where is ethidium bromide used?
In electrophoresis to obtain DNA
What does ionising radiation produce?
Ions during interactions with cellular molecules
Where does ionising radiation come from?
- Solar radiation (UV)
- X-rays
- Nuclear power plant accidents
- Environmental sources
- Radon gas
- Rocks
How can exposure to radiation be avoided?
You can’t- it’s in the environment, food and air
How can radiation exposure be reduced?
Avoiding x-rays and flying
What is the wavelength of UV-B light?
280-315nm
What does UV-B exposure induce?
Production of vitamin D in skin
What does overexposure to UV-B cause?
Sunburn, and some forms of skin cancer
What is the wavelength of UV-A?
315-400nm
What is the wavelength of UV-C?
100-280nm
What can UV-A, B and C all damage?
Collagen fibres
What does collagen fibre damage cause?
Skin ageing
What do UV-A and B destroy?
Vit A in skin
What do UV light photons cause?
Adjacent thymine bases to base pair with another
What happens to thymine dimers?
They often resolve spontaneously
What is the spontaneous resolution of thymine dimers called?
Photo-reactivation
What happens if photo-reactivation doesn’t occur?
It’s the basis of damage from UV light
What is the incorporation error rate for DNA polymerase?
~1 in 100,000 nucleotides
What does the incorporation error rate of DNA polymerase account for in a single cell replication?
~120,000 mistakes per DNA molecule
What is the problem with the number of mistakes caused by DNA polymerase incorporation errors?
Intolerable genetic load
How are most error caused by DNA polymerase corrected?
‘Proof-reading
What happens in proof-reading?
The polymerase detects the mispaired 3’ base in the newly synthesised stand and corrects these errors
What % of errors are corrected by proof reading?
99%
What happens to repair remaining mistakes after replication?
Mismatch repair
What happens in mismatch repair?
Enzymes detect mismatched bases in newly synthesised strand and replace a patch of DNA sequence around the mutation (rather than just the mutation)
What damaged bases does DNA accumulate?
NAME?
How can the damaged bases that DNA accumulates be repaired?
Base excision repair
What can failure of DNA repair mechanisms result in?
Cancer
What encodes mismatch repair enzymes?
The human genes MLH1, MSH2, MSH6
What do mismatch repair enzymes do?
Fix where DNA polymerase have mis-incorporated the bases
Give an example of where are the mismatch repair enzyme genes commonly mutated
In cases of hereditary non-polyposis colorectal cancer
What can virtually all cases of non-polyposis colorectal cancer be accounted for by?
On of the three genes involved in mismatch repair being mutated
When does natural selection in cancer occur?
When changes in a cell in which a mutation has occurred gives a growth advantage over other genes
What kind of mutations give rise to cancer?
NAME?
What does formation of a tumour result from?
Growth advantage conferred on cells which acquire 6 new characteristics
What 6 characteristics result in tumour formation?
- Divide independently of external growth signals
- Ignore external anti-growth signals
- Ignore apoptosis
- Divide indefinitely wth senescence
- Stimulate sustained angiogenesis
- Invade tissues and establish secondary tumours
What is overcome with cancer?
Any barrieres that were made to stop synthesis of mutated base sequences
What is angiogenesis?
Synthesise of new components that make blood capillaries
What is the importance of angiogenesis in tumour cells?
Provides energy
What is the change of picking up the 6 successive mutations required for the development of cancer?
Assuming a mutation rate of 10 -6 per generation , 10 -36
How is the improbability of picking up the 6 successive mutations causing cancer overcome?
Early mutations affect functions which raise the probability of successive mutations occurring, so the successive mutations aren’t truly independent, with early mutations making it more likely that the later mutations will happen, so the probability is actually much higher
What do all chromosomes exhibit?
Chromosomal instability and microsatellite instability, leading to deletions and duplications of chromosomes
How many people in the UK will develop breast cancer?
~1 in 8
How many cases of breast cancer are familial?
~5-10%
What is known to be true of familial breast cancer?
It maps the genes BRCA1 and BRCA2
What are BRCA1 and BRCA2 involved in?
Detecting DNA damage, and signally to cell-cycle checkpoints
What is the purpose of cell-cycle checkpoints?
It’s an opportunity for cell mechanisms to say it’s safe for replication to go ahead, and if not, programmed cell death
Do sporadic tumours have BRCA1 or BRCA2 mutations?
Rarely
Are affected males common in families with inherited breast cancer?
Fairly
How many mutations in BRCA1 and BRCA2 been found?
Hundreds, but some more common than others in specific populations
What is the advantage of BRCA1 and BRCA2 varying in commonality between different populations?
It makes screening more effective, as the population can influence the choice of genes that are screened for to make it more precise
What are some cancers known to be associated with?
The presence of specific retro-viruses in humans and animals
Give an example of a retro-virus associated with cancers?
Infection with human papilloma virus, especially HPV16 and 1B is associated with development of cervical, anal and penile cancer
What does study of retro-viruses show?
That they main contain genes able to transform cells to a cancerous phenotype
What has been identified to show remarkable sequence similarity to viral oncogenes?
Several human genes
What are the human genes that show sequence similarity to viral oncogenes called?
Protooncogenes
What do human proto-oncogenes do?
Perform a range of cell-cycle control functions
What can happen to proto-oncogenes?
They can be activated into dominantly acting cancer causing oncogenes
What causes the activation of proto-oncogenes?
Key amino acid substitutions
What do inherited cancer genes tends to harbour?
Recessive mutation
What does the development of cancer display?
A dominant pattern of inheritance
What does initiation of tumour formation require?
Both copies must be mutated, or the functional wild-type copy must be deleted
What explains the apparent contradiction in inheritance patterns of cancer?
Alfred Knudson’s two hit theory
What are the mechanisms of homozygosity?
- Loss of wild type chromosome
- Deletion
- Point mutation
- Mitotic recombination
How common is the loss of a wild type chromosome?
Incredibly rare
What happens in the case of the loss of the wild type chromosome?
Leaves only one chromosome carrying the mutant gene, setting of a chain of events resulting in tumour formation
How common is deletion causing homozygosity?
Rare
What happens when a deletion causes homozygosity?
Portion of wild type chromosome possessing the normal gene is deleted, so the cell effectively only has the mutant copy of the gene
What happens when a point mutation cause homozygosity?
Acquisition of another point mutation
Does the second point mutation have to be the same as the first to cause homozygosity?
No, just has to inactive protein
How does mitotic recombination cause homozygosity?
Individual becomes homozygous for region of chromosome where the mutant gene
How does mitotic recombination differ from the other mechanisms of homozygosity?
- Pathway by which there is no loss of genetic material
- Happens further along the pathway than others- after the chromosome has divided once
Why is the probability of cancer higher in inherited cases?
- In inherited cases, all cells carry the mutation, so probability of the second mutation is nµ
- In sporadic cases, the probability somatic mutation in one cell is nµ, so the probability of a second mutation is µ- much lower
How many exons are there in the CFTR gene?
27
How many bases in the CFTR gene?
189kb
What is the chromosomal position of the CFTR gene?
7q31.2
What must happen for a person to have CF?
Both copies of the CFTR gene must be mutated
Where can mutations in the CFTR gene occur to cause CF?
Anywhere- over 100 described
What may be true of a CF patient?
They may be a compound heterozygote for 2 different CTFR mutations
What is meant by a compound heterozygote?
They have 2 mutated genes, but may be in 2 different locations
What is the chance of parents with one CF child having another affected?
01-Apr
What would pre-natal diagnostic tests for CF depend on?
First identifying the mutation in the 1st child
What is the most common CF mutation?
p.F508del CF mutation
What has happened in the p.F508 del mutation?
The phenylalanine is deleted at amino acid 508 of CFTR-encoded protein, caused by a 3bp deletion
How many specific mutations account for 92% of all mutant CF alleles?
13
What is the second most common CF mutation?
p.Gly551Asp
How many cases doesp.Gly551Asp account for?
3.50%
What accounts for the remaining 8% of CF mutations?
- 6.5% by rarer mutations
- 1.5% by unknown mutations
What CF testing is available in the UK
- For 29 most common CF mutations, using a multiplex PCR based test
- Expanded planel of 97 tests, when 29 most common test doesn’t give definitive answer
Why is sequencing of the gene not used?
Expensive in a diagnostic setting
Is SSCP mutation scanning currently used?
Yes, but diminishing to cut costs
What is better to do that SSCP mutation scanning?
Better to first identify the mutated region of the gene, then perform targeted DNA sequencing
What will PCR yield is a person is heterozygous for a mutation?
A mix of normal and mutant sequences
What happens in SSCP?
- The amplified DNA is heated to denature the DNA, and then cooled rapidly, so the individual strands adopt sequence-specific, partly double-stranded forms
- The DNA is electrophoresed in a polyacrylamide gel, and detected by staining with silver
Why does SSCP work?
Because, when rapidly cooled, even small changes have a big effect on the 3D shape, which will have a different mobility in the gel
How is it possible that it can appear that a patient has more than 2 CFTR mutant alleles?
Only one of the additional variants will be the disease causing mutation, and other will be a natural variant found in the population that is unrelated to CF
How can parent and sibling DNA samples be obtained?
From blood or salia
What is required to carry out pre-natal diagnosis?
A sample of DNA from developing foetus
How is foetal DNA isolated?
- Amniotic fluid cells
- Chorion villus biopsy
- Foetal DNA in mothers blood
How is DNA obtained from amniotic fluid cells?
Using amniocentesis
How is amniocentesis performed?
A long needle is passed through the abdominal wall into the amniotic sac, and a sample of cells are removed
When is amniocentesis?
At 15-20 weeks gestation
What is required for amniocentesis?
Ultrasound guidance
What happens to the cells removed from the amniotic fluid?
They may need to be cultured for 2 weeks
What is the miscarriage risk of amniocentesis?
0.5-1%
When in a chorion villus biopsy performed?
10-13 weeks
What is required when performing a chorion villus biopsy?
Ultrasound guidance
How is a chorion villus biopsy performed?
NAME?
What must happen when performing a chorion villus biopsy?
Foetal villi must be separated from maternal tissue, to ensure the DNA isolated is from the foetus and not maternal tissue
What is the miscarriage risk from a chorion villus biopsy?
2%
What is isolated alongside foetal DNA obtained from the mothers blood?
The mothers DNA
How can foetal DNA be used when obtained from mothers blood?
By sequencing the isolated DNA
How do some inherited disorders commonly arise?
Through whole-exon duplications or deletions
Give 2 examples of disorders caused by whole exon duplications or deletions?
- Osteogenesis imperfecta
- Duchenne muscular dystrophy
What is important when screening for exon deletions and duplications?
Both copies of the gene are screened with relation to the exon
What can be used to count the copy number for many exons in parallel?
MLPA
What is produced for each exon of the gene produced in MLPA?
A pair of primers
What do the pair of primers for MLPA cover?
The region of the exon to be tested
Describe the MLPA process
- The two ends of the primers (opposite to the binding sites) push up against each other, and try and anneal the gene, but there is a gap between the two primers
- If they anneal to the exon of interest, DNA ligase joins together the two halves of the DNA
- If one copy of the gene is deleted, then only half the exon will be ligated, ad there is less target DNA available to anneal to, so reduction in amount of ligation, and consequently half the amount of product
- The stuffer, which is also added to the exon, is a different length for each exon
- Needs PCR to amplify them to be seen in electrophoresis
Why is the stuffer added to the exon a different length for each exon?
Allows it to be separated by gel electrophoresis, as each exon is a distinctly different size
How can MLPA detect mutants?
When the size of the peak is halved compared to control
