L15 - mutations Flashcards Preview

LS1 MGD > L15 - mutations > Flashcards

Flashcards in L15 - mutations Deck (72):

Why are mutation in most of the genome of little consequence?

Only 1-2% of the human genome encodes proteins - it is mutations close to or within these genes that are most likely to cause disease


What is the most common form of sequence variation?

single base substitution/ SNPs (single nucleotide polymorphisms)


Which is the most common SNP?

C to T changes


Which is a transition?

purine to purine SNP


What is a transversion?

purine to pyrimidine (or vice versa) SNP


What are silent substitutions?

Base substitutions that have no effect on the amino acid sequence


Which codon position is most likely to produce a silent substitution?

Third codon position


Which codon position(s) are most likely to produce amino acid changes?

Codon positions 1 and 2


Define a missense mutation

A mutation that causes an amino acid substitution: normally by a single base change


Define a silent mutation

A single base substitution which does not substitute the amino acid


Why can "silent" substitutions sometimes still cause heritable diseases?

They can interupt RNA spicing - introduce or disrupt spice sites


Define nonsense mutations

When an amino acid codon is mutated to a stop codon


Define a frameshift mutation

A mutation which causes the reading frame of mRNA to be altered in some way


What could cause a frameshift mutation?

splice-site mutations


Why do frameshift mutations often result in ribosomes terminating translation prematurely?

They often introduce stop codons


What is a conservative missense mutation?

An amino acid substitution that substitutes in an amino acid with similar properties that is tolerated in the protein and does not cause a functional change.

(e.g. not in active site but in a structural, non-functional part of the protein like the outside of a protein)


How has evolution maximised the chance of conservative missense rather than missense mutations?

The genetic code means that often amino acids with similar properties, have similar codons


Which type of indel or duplication will preserve the reading frame?

One that causes a gain or loss of a multiple of 3


What is the name of the protective mechanism in which mRNAs with premature termination codons (PTCs) are degraded?

Nonsense mediated decay (NMD) - mRNA which have stop codons that are not near the end are degraded. Evolutionary favoured process - better to have less product than a product which has been altered


What is often the result of mutations at intron splice sites?

Skipping of the adjacent exon - may lead to frameshift


What internal factors can cause base changes?

Tautomeric shift - altered base-pairing
DNA strand slippage during replication


What external factors can cause base changes?

1. Chemicals - direct alteration of bases or altered stacking
2. Radiation - UV light or radioactive substances


What is a tautomeric shift?

When a proton in a DNA base briefly changes position


Why can a tautomeric shift result in altered base pairing?

Rare forms of tautomers have altered base pairing and so behave as an altered template base during replication


Describe the two ways that slippage can occur during replication (fairly common replication error)

1. Newly synthesised strand can loop out-> insertion of one base
2. Template strand loops out -> omission of one base


What does the mutagen nitrous acid (oxide) do to DNA?

Replaces amino groups with keto groups which alters their base pairing


What does the mutagen Ethyl methane sulphonate (EMS) do to DNA?

Causes removal of purine rings which can then be filled with ANY base during replication


The mutagen 2-amino-3-methylimidazo[4,5-f] quinoline (IQ) is found in cooked meats and cigarette smoke condensates. What effect does its disruption of DNA base pair packing have?

Intercalation of IQ forces the base pairs of one strand further apart which leads to misreading by DNA pol and deletion of a single base pair


What mutagen used to stain DNA in electrophoresis, also disrupts DNA packing by intercalation?

Ethidium bromide


Why is radiation with short wavelengths called ionising radiation?

The shorter the wavelength the more energy they have and the more damage they cause.
Ionising radiation produce ions during interaction with cellular molecules.


List some environmental sources of ionising radiation

Solar radiation (UV)
Nuclear power accidents
Environmental sources such as radon gas and granite rocks


What are the only practicable ways of avoiding exposure to radiation

Avoid flying and X-rays!


What type of UV light induces the production of Vitamin D in the skin?



Which type of UV light damage collagen fibres in the skin which leads to skin ageing?



Which vitamin in the skin does UVA and UVB destroy?

Vitamin A


What can over-exposure to UVB cause?

Some forms of skin cancer


What effect can UV light photons have on DNA?

Thymine dimer formation


What is thymine dimer formation?

When UV light photons cause adjacent thymine bases on the same DNA strand to base pair with one another


What is the name of the spontaneous process which often resolves thymine dimers?



How are most DNA errors corrected?

DNA polymerase's proofreading ability


What is meant by 'proofreading'?

When the DNA polymerase detects the mis-paired 3' base in the newly synthesised strand and corrects it 99% of the time.


After replication, what type of DNA repair occurs?

Nucleotide mismatch repair


Explain the process of nucleotide mismatch repair

Enzymes detect mismatch. 3 stages:
1. Mismatch recognition
2. Strand discrimination - recognises newly synthesised strand (somehow)
3. Strand excision and resynthesis


How are damaged bases (oxidised, alkylated, de-aminated, uracil) that accumulate repaired?

Base excision repair


What can the failure of DNA repair cause?



Which genes encode the three enzymes involved in mismatch repair that are commonly mutated in cases of hereditary non-polyposis colorectal cancer?

MSH6 genes


List the 6 new characteristics that cancer cells have acquired that give them a growth advantage?

1. Divide independently of external growth signals
2. Ignore external anti-growth signals
3. Avoid apoptosis
4. Divide indefinitely without senescence
5. Stimulate sustained angiogenesis
6. Invade tissues and establish secondary tumours


Why when mutation rate is so low is the probability of getting cancer not improbable?

Early mutations affect functions which increase the probability or successive mutations occurring.
All cancer cells exhibit chromosomal instability and microsatelite instability.


Which two genes (found in familial cases) are known to cause 5-10% of cases of breast cancer?

Sporadic tumours rarely have BRAC1 or BRAC2 mutations


What is the role of BRAC1 and BRAC2 genes?

Detecting DNA damage and signalling in cell-cycle checkpoints


What are oncogenes?

Oncogenes are genes that have the potential to cause cancer. In tumor cells, they are often mutated or expressed at high levels.


What are proto-oncogenes?

A normal gene which, when altered by mutation or by increased expression, becomes an oncogene that can contribute to cancer.


The cancers: cervical, anal, penile and human papilloma viruses (esp HPV 16 and 18) are associated with what?

The presence of specific retro-viruses


How can retro viruses cause cancer?

They have genes which are able to transform cells to a cancerous phenotype (viral oncogenes)


What is the role of human equivalents to viral oncogenes?

They perform a range of cell-cycle control functions


How can proto-oncogenes be converted into dominantly acting cancer causing oncogenes?

Key amino acid substitutions.


How come inherited cancer genes tend to be recessive but the development of cancer often displays a dominant pattern of inheritance?

Two Hit Theory
In inherited cases every cell already contains one mutated form of a protooncogene. The chance of a mutation in the same gene occuring is nx. This is the same chance of a mutation occurring in that same gene for a sporadic case. However for a sporadic case this does not cause cancer, they require a second mutation (chance of occurring now only x), whereas in inherited cases it does.


How can an inherited recessive cancer gene become homozygous after meiosis?

1. mitotic recombination (occurs very occasionally)
2. loss of wildtype chromosome
3. deletion of wildtype gene
4. point mutation of wildtype gene (doesn't need to be the same mutation just in the same gene)


What is a multiplex PCR-based test?

When PCR is used to amplify several different DNA sequences simultaneously using multiple primers that have been optimised to work at the same annealing temperature. Each primer set produces amplicons (products) of different lengths which are specific to different sequences.


How does SSCP mutation scanning work?

Heterozygotes for a mutation will yield a mix of normal and mutant sequences after PCR amplification. If this DNA is heated to denature it and then cooled rapidly, the individual strands will adopt sequence-specific partly double-stranded forms (withing the stand NOT between strands). This can then be electrophoresed in a polyactylamide gel and detected by staining with silver. Each strand has different 3D shapes and therefore different motility on the gel. These can be run on gels comparing with other patients with the same disease.


Why do you have to be careful not to jump to conclusion when your analysis finds a sequence variant?

It is not necessarily disease-causing but may just be a harmless variant. One way to decide this is by looking at the variants that the general population have at this position. If it is a common variant you can assume it is not disease-causing


How can foetal DNA be isolated?

Amniotic fluid cells
Chorion villus biopsy (placenta)
Foetal DNA in mother's blood (area still under development).


When is amniocentesis performed?

At 15-20 weeks of gestation


How is amniocentesis performed?

Amniotic fluid is drawn up with a syring that has penetrated the mother's abdomen, under ultrasound guidance


What is the risk of causing miscarriage with amniocentesis?



When is chorion villus biopsy performed?

10-13 weeks of gestation (earlier than amniocentesis)


How is chorion villus biopsy performed?

With ultrasound guidance, either:
trans-cervical or


What is the potential issue of chorion villus biopsies?

Foetal villi must be separated from maternal tissue


What is the risk of miscarriage with chorion villus biopsy?

2% risk (higher risk than amniocentesis)


Name two inherited diseases which are caused by the deletion or duplication of whole exons

Osteogenesis imperfecta (collagen gene) - AD
Duchenne muscular dystrophy (dystrophin gene) - XR


How could you analyse whether a whole exon had been duplicated or deleted?

Using multiplex ligation-dependent probe amplification to count the number of many probes in parallel (in a single test)


How does multiplex ligation-dependent probe amplification work (MPLA)?

Two probes for each exon of interest. Both probes bind to an adjacent sequence. Each probe has a stuffer region which makes each ligation product a different length for analysis by electrophoresis and a primer binding site (for two primer (for all the exons) X and Y). The forward primer used for PCR amplification is also labelled. DNA ligase is used to ligate the gap between adjacent bound probes. The DNA is then amplified. If the probes have bound to each exon, they will be ligated and have a primer on each end and therefore be amplified. By comparing the PCR amplified DNA by analysis of the fluorescent signal by capillary electrophoresis gel to a reference sample you can see if half the amount of an exon is present.