3.3 Pharmacogenomics

Question 1

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)

Answer 1

solute carriers (SLC) & ATP-binding cassette (ABC)

Question 2

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

Answer 2

SLCO1B1; SLCO1B1; statin-inducedmyopathy;

rs4149056

Question 3

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%

Answer 3

~12kDa proteins consisting of a single 104 amino acid peptide;

CYP3A;

CYP2C9

Question 4

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

Answer 4

rapid metaboliser;

intermediate metaboliser;

ultrarapid metaboliser

cimetidine, fluconazole, fluoxetine, clarithromycin, (grapefruit juice);

rifampicin, carbamazepine, (chargrilled meat)

Question 5

Warfarin is metabolised by ____________

- People with the minor allele metabolise the drug more slowly and need a ____________ to reach the therapeutic effect

Answer 5

CYP2C9;

lower dose

Question 6

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

Answer 6

CYP2C19

Question 7

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

Answer 7

morphine;

CYP2D6;

larger

Question 8

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

Answer 8

CYP2D6 and CYP3A4/5;

endoxifen

Question 9

Phase II metabolising proteins

Phase II proteins conjugate the phase I metabolites, other intermediates, or the parent compound, for ______________

Phase II proteins include:
_____________________

Answer 9

renal or biliary excretion;

• Glutathione S transferases (GSTs)
• Thiopurine methyltransferase (TMPT)
• UDP glycurosyltransferases (UGT)
• N-acetyltransferases (NAT)
• Glutathione S transferases (GSTs)
• NADH quinone oxidases.

Question 10

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

Answer 10

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

Question 11

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

Answer 11

allele and genotype frequencies in a population will remain constant from generation to generation in the absence of other evolutionary influences

Question 12

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

Answer 12

NUDT15 gene

Question 13

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

Answer 13

S-enantiomer;

CYP2C9;

VKORC1;

CYP4F2

Question 14

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

Answer 14

CYP2C9 and VKORC1 ;

Question 15

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

Answer 15

carbamazepine;
abacavir;
allopurinol;

binding of peptides

Question 16

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 __________________

Answer 16

toxic epidermal necrolysis (TEN)

Drug Hypersensitivity Syndrome (DHS);

allopurinol, anticonvulsants (carbamazepine, phenytoin), penicillins, and sulfonamides

Question 17

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

Answer 17

HLA-B*1502;

HLA-B*5701

Question 18

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

Answer 18

germline DNA;
Polymerase chain reaction (PCR)

restriction fragment length polymorphism, RFLP;

Question 19

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

Answer 19

DNA to RNA to protein;

RNA polymerases;

ribosome

Question 20

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

Answer 20

94–98;

50–65;

heat-resistant DNA polymerase

Question 21

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

Answer 21

specific site;

endonucleases

Question 22

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

Answer 22

report dye and a quencher;

hybridises;

polymerase

Question 23

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

Answer 23

single strands (ss-DNA);

SYBR Green;

Sanger sequencing

Question 24

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

Answer 24

many genes (up to hundreds);

Dideoxy chain-terminating bases;

mass

Question 25

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

Answer 25

length;

hydroxy moiety

dye of a different colour

Question 26

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

Answer 26

HLA-B*1502 genotyping

Question 27

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.

Answer 27

Human Epidermal growth factor Receptor 2-positive (HER2+)