3.3 Pharmacogenomics Flashcards
The mechanisms through which genetics may affect drug action:
- Pharmacokinetics, to do with drug absorption, distribution, metabolism and excretion
Transport Proteins
- Move drugs and other substances in and out of cells
- Divided into two types: ____________________
Metabolism
- Phase I (e.g. cytochrome P450)
- Phase II enzymes (e.g. thiopurine methyltransferase)
solute carriers (SLC) & ATP-binding cassette (ABC)
Example of a transport protein and a drug-induced ADR
- ____________ is responsible for the transport of simvastatin into hepatocytes. A variant in the gene encoding ___________is associated with_________________
- A mutation at SNP ______________– (in which the base T is replaced by C) of this gene reduces the transport of simvastatin into cells and leads to higher blood concentration of the drug which likely leads to myopathy
SLCO1B1; SLCO1B1; statin-inducedmyopathy;
rs4149056
Drug metabolism
- When taken into the body, all drugs are either: Excreted (1/4) though the bile or urine Or metabolised (3/4)
- Drug metabolism is classified into phase I and phase II
- Most drugs are metabolised by the cytochrome system, a component of phase I
Cytochromes are electron-carrying mitochondrial proteins
- They are highly conserved ____________________ with a single heme group
- They play a vital role in cellular oxidations in both plants and animals
Cytochrome proteins are classified by the similarity in the gene sequences. They are assigned:
- A family number (e.g., CYP1, CYP2)
- A subfamily letter (e.g., CYP1A, CYP2D)
- Then differentiated by a number for the isoform or individual enzyme (e.g., CYP1A1, CYP2D6)
¾ of drugs are metabolised and ¾ of this is carried out by the cytochrome system Relative importance of the individual cytochromes in drug metabolism: - \_\_\_\_\_\_\_\_\_\_\_ family 46% - \_\_\_\_\_\_\_\_\_ 16% - CYP2C19 12% - CYP2D6 12% - CYP1A family 9% - CYP2B6 2% - CYP2E1 2%
~12kDa proteins consisting of a single 104 amino acid peptide;
CYP3A;
CYP2C9
How cytochrome function may be affected
Genetic variation
- The usual phenotype is referred to as the ____________________
- Genetic variants that affect the function of the gene lead to a poorly functional or non-functional protein
- The phenotype is a poor metaboliser if both copies of the gene are non-functional
- If a person has a copy of a wildtype gene and a mutated gene, he may be an ________________
- If a person has multiple copies of a wildtype gene, he may be a ______________
- Some drugs inhibit cytochrome function E.g. _________________________
Inhibition can occur rapidly - Some drugs induce or potentiate cytochrome function E.g. ______________
Induction takes hours or days
rapid metaboliser;
intermediate metaboliser;
ultrarapid metaboliser
cimetidine, fluconazole, fluoxetine, clarithromycin, (grapefruit juice);
rifampicin, carbamazepine, (chargrilled meat)
Warfarin is metabolised by ____________
- People with the minor allele metabolise the drug more slowly and need a ____________ to reach the therapeutic effect
CYP2C9;
lower dose
Clopidogrel, an anti-platelet agent, is an inactive pro-drug that is converted to the active form by _____________
- It is found that slow metabolisers do worse than rapid metabolisers in the secondary prevention of ischaemic heart disease
CYP2C19
Codeine is also a pro-drug that is converted to ___________ by __________
- Slow metabolisers require a ___________ dose of the drug to achieve the same analgesic effects as rapid metabolisers
morphine;
CYP2D6;
larger
Tamoxifen is an inactive drug that is converted both by ________________ to the active form _______________
- Poor metabolisers may not benefit from the protection against breast cancer recurrence
CYP2D6 and CYP3A4/5;
endoxifen
Phase II metabolising proteins
Phase II proteins conjugate the phase I metabolites, other intermediates, or the parent compound, for ______________
Phase II proteins include:
_____________________
renal or biliary excretion;
- Glutathione S transferases (GSTs)
- Thiopurine methyltransferase (TMPT)
- UDP glycurosyltransferases (UGT)
- N-acetyltransferases (NAT)
- Glutathione S transferases (GSTs)
- NADH quinone oxidases.
Thiopurine methyltransferase
- Some patients develop _____________ when prescribed azathioprine
- Polymorphism in the gene that encodes the enzyme thiopurine methyltransferase (TPMT) is an important determinant of this adverse effect
- Azathioprine is used in the treatment of ________________________
- Patients with normal TPMT activity or normal genotype may receive the normal dose of azathioprine (such as 50 mg daily)
- Intermediate metabolisers should receive ____________________
- Poor metabolisers should use an alternate drug; if none are available, azathioprine must be used very cautiously (_______________) with close monitoring
life-threatening leucopaenia;
cancers, inflammatory bowel disease and systemic lupus erythematosus;
30-80% of the usual dose;
10% of the dose and thrice weekly dosing
Hardy-Weinberg equilibrium
- The Hardy–Weinberg principle states that _____________________________
- It was first proposed to dispel the thinking that the dominant allele will gradually displace the recessive one in succeeding generations
- Therefore the proportion of people with certain conditions, such as thalassaemia and colour blindness, will remain fairly stable in subsequent generations provided there is no mutation, migration, or selection
allele and genotype frequencies in a population will remain constant from generation to generation in the absence of other evolutionary influences
Azathioprine and NUDT15
- Asian populations have a lower frequency of poor TPMT activity but also experience leucopaenia with thiopurine treatment
- The_______________ is more important in determining the adverse effect of thiopurine in Asians
- The NUDT15 gene product is also responsible for the metabolism of the thiopurines
NUDT15 gene
Warfarin pharmacogenetics
- Warfarin is an anticoagulant that is still widely used
- Warfarin is a chiral drug that is marketed as a racemate consisting of equal amounts of R- and S-enantiomers
- The _______________ is more potent
- Warfarin accounted for more drug-related emergency room visits in the US than any others
- Warfarin is metabolised by ________
- Its target is ____________
- Variants in the genes encoding these proteins affect the therapeutic dose of warfarin
- _____________ metabolises vitamin K
- Variants of its gene have a small effect on the warfarin dose
S-enantiomer;
CYP2C9;
VKORC1;
CYP4F2
Pharmacogenetics of warfarin in Singapore
- Patients carrying the minor allele in
_______________ genes require lower doses of warfarin
- Proportionately more Chinese and Malay carry the CC allele of VKORC1 gene than Indians. Which explains why patients of these two ethnicities require lower doses of warfarin
CYP2C9 and VKORC1 ;
Human leucocyte antigen and severe cutaneous allergic reactions
In the mid 2000’s, HLA subtypes were discovered to be associated with severe cutaneous reactions to drugs, namely:
- HLA-B*1502 and _____________
- HLA-B*5701 and ____________
- HLA-B*5801 and ______________
The mechanism behind the association has been worked out only for one.
- Abacavir interferes with _____________ to the cleft of HLA-B*5701
- So that self-antigens could be presented
- Leading to immune response against many of the patient’s own tissues
carbamazepine;
abacavir;
allopurinol;
binding of peptides
Severe Cutaneous Allergy Reactions
- Severe Cutaneous Allergy Reactions (SCARs) include: Stevens-Johnson syndrome (SJS) (mortality 15%), __________________ (mortality 35%), Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS), also known as ____________________
- They continue to be important causes of morbidity and mortality in medical practice worldwide
- Common causes of SCARs are __________________
toxic epidermal necrolysis (TEN)
Drug Hypersensitivity Syndrome (DHS);
allopurinol, anticonvulsants (carbamazepine, phenytoin), penicillins, and sulfonamides
Human leucocyte antigen and severe cutaneous allergic reactions
- Because of the differences in prevalence and HLA associations, each country has to decide its own practice about how the tests should be used
- In Singapore, from April 2013, the Ministry of Health and the Health Sciences Authority announced that testing for ________________ is considered standard of care before prescribing carbamazepine. Genotyping is not required for prescribing abacavir and allopurinol
- In countries such as Australia and USA, it is mandatory to test ____________ before prescribing abacavir
HLA-B*1502;
HLA-B*5701
Tools in molecular biology relevant to genotyping in pharmacogenetics
1) To begin, the patient’s blood is drawn and the DNA extracted:
- This is the ______________ (not somatic DNA)
- ________________ is used to make multiple copies of a segment of the gene we want to study
- Because it is not always possible to have sufficient genomic DNA for analysis
2) To test for specific genetic mutation:
- endonuclease digestion gel electrophoresis (also known as _______________)
- Real-time PCR and Taqman technology
- High-resolution melt curve analysis.
- Mass-array spectrometry
- It is also possible to sequence the PCR product to determine to exact sequence of the DNA (Sanger sequencing)
- Real-time PCR is the most common technique used in clinical pharmacogenetic tests
germline DNA;
Polymerase chain reaction (PCR)
restriction fragment length polymorphism, RFLP;
The central dogma of molecular biology
1) First expounded in 1958 by Francis Crick:
- Sequence information flows from ______________
- Information cannot be recovered from protein
2) The sequence of three bases in the DNA (a codon) determines the amino acid to be added to the polypeptide
3) Transcription is the copying of a gene’s DNA sequence to make an RNA molecule (the messenger RNA or mRNA)
- Performed by enzymes called _________________
4) Intranslation, mRNA is decoded in the ____________ to produce a specific polypeptidechain, orprotein
DNA to RNA to protein;
RNA polymerases;
ribosome
Polymerase chain reaction
- This laboratory technique make many faithful copies (millions) of a specific DNA segment. It was developed in the early 1980s by Kary Mullis
1) The double-stranded DNA separates when heated (denaturing, _________°C)
2) Differently designed primers attach to the strands when cooled (annealing, ______°C)
3) And enzymatic elongation (with ___________________) takes place at 75–80°C
- The cycles are repeated
94–98;
50–65;
heat-resistant DNA polymerase
Restriction fragment length polymorphism
- RFLP is a method to detect mutation at a ______________
- It depends on the fact the _____________ (also known as restriction enzymes) cut DNA at precise regions depending on the sequence
Let’s say a segment of DNA from PCR has one site of attack (restriction site) for the endonuclease
- The end result will be two smaller pieces of DNA that can be detected in a gel analysis
specific site;
endonucleases
Real time or quantitative PC R
- Using an ingenious reporter system, it is possible to detect if a particular allele is present in genomic DNA
As in PCR, the genomic DNA is denatured - to separate the strands
- The Taqman probe contains a _________________, which when placed together suppresses the reporter fluorescence
The Taqman probe is allowed to interact with the DNA
- If the appropriate complementary DNA sequence is present, the probe ____________ to it
- It is possible to have more than one probe with a different coloured dyes
Now the DNA is allowed to elongate as in the usual PCR, with forward and reverse primers and DNA polymerase
- When the _____________ reaches the Taqman probe that is hybridised, it cleaves the reporter dye from the quencher dye, releasing fluorescence
- Thus, the fluorescence signal generated by PCR amplification indicates which alleles are in the sample
report dye and a quencher;
hybridises;
polymerase
High-resolution melt curve analysis
- Mutations that are difficult to detect with RFLP or Taqman can be analysed with high-resolution melt curve analysis
- When double-stranded DNA (ds-DNA) is heated, it separates into _________
- If there is a mutation, even at a single position, the temperature at which this separation occurs (‘melting point’) may differ
- Some dyes such as ____________ bind ds-DNA much better than ss-DNA
- As the strands separate, the fluorescence falls
- Using modern sensitive equipment, it is possible to detect if there is a difference between two PCR products
- Of course, this method reports a difference but does not identify where and what the mutation is
- When necessary, _______________ may be carried out
single strands (ss-DNA);
SYBR Green;
Sanger sequencing
Mass array spectrometry
- To test for mutations in ______________ at one time, mass-array spectrometry may be used
- PCR product containing the gene of interest is used
A primer is created just one base proximal to the allele of interest
- _________________, of differing molecular weights are added to the reaction well
- Therefore, only a single base complementary to the DNA will be added
- The identity of the dideoxy base that is added can be determine by its ______. This method lends itself to high throughput workload
many genes (up to hundreds);
Dideoxy chain-terminating bases;
mass
This clever technique allows us to work out the sequence of the bases in DNA
- It uses DNA polymerase to extend the DNA strands
- Besides the usual bases A, T, C and G, dideoxy bases are added to the mixture
- These special bases are analogous to A, T, C and G, but they do not allow the next base to be added after them
These chain-terminating bases are proportionately lower in number than the bases
- So that they are incorporated randomly and the DNA chains are terminated at different lengths
- By arranging these strands according to ________ and reading off the final bases that is added, we can work out the sequence of the DNA
- The only difference between a dideoxy base and the naturally occurring one is the absence of a _________________
- This difference means that the next base cannot be added after a dideoxy one
- The strands of DNA of various lengths can be separated in a gel
- Each of the 4 dideoxy bases can be attached to a _____________, simplifying the reading of the sequence
length;
hydroxy moiety
dye of a different colour
Applications of pharmacogenomics in personalised medicine
- Mandatory _______________ before prescribing carbamazepine in Singapore
- Computerised clinical decision support system in the electronic medical record (EMR)
Pre-emptive pharmacogenomics
- With the lowering costs of next-generation sequencing (of the whole exome or whole genome), it is possible that people may be sequenced ahead of time, with the data stored in the cloud or any medium
- When the clinician prescribes a medication with known drug-gene rule, the relevant gene may be interrogated
HLA-B*1502 genotyping
Applications of pharmacogenomics in drug development
Drugs may be specially designed for patients who carry certain mutations. For example, Herceptin (trastuzumab) is used to treat patients only if the breast tumour is __________________. This type of testing is also known as companion diagnostics.
Human Epidermal growth factor Receptor 2-positive (HER2+)
