7 - Diagnostic Tests

Question 1

Compare and contrast genotype vs. phenotype regarding pharmacogenetic testing and recommendations

Answer 1

• Genotype
• • Generally easier to obtain and interpret
• • Results have less ambiguity (b/c you’re either homozygous or heterozygous, no in-between)
• • Cheap and less expertise needed
• • Potentially all genotyping can be done on a single instrument and for multiple genes (for multiple drugs simultaneously)
• Phenotype
• • More expensive and more expertise needed
• • Results can be ambiguous
• • Each phenotype assay is unique and needs to be developed and validated by a highly trained professional

Question 2

How can you determine TPMT phenotype and genotype by measuring TPMT activity?

Answer 2

• If we can measure TPMT activity, we can identify the phenotype of the individual
• • We can infer the genotype from the phenotype; we have to define all reduced or no activity alleles as defective
• We could use a genetic test to determine the genotype for TPMT and make dosing recommendations based on that

Question 3

Describe phenotyping and the TPMT assay

Answer 3

• Measure the actual enzymatic activity of TPMT
• Use whole blood from a pt prior to giving AZA
• Collect whole blood, spin down RBC, lyse RBC
• Add a known amount of 6-MP to the lysed RBC
• Initiate reaction by addition of S-adenosyl-methionine (SAM) (methyl donor to convert 6-MP -> 6-methylmercaptopurine)
• Use HPLC to measure the amount of 6-methylmercaptopurine
• Those who have low TPMT activity will produce less 6-methyl-mercaptopurine and therefore may have toxicity if given AZA at normal doses

Question 4

Issues w/ TPMT phenotyping

Answer 4

• Results of phenotyping will not be accurate in px who have received recent blood transfusions
• TPMT activity is affected by any change in hematopoiesis (ex: results will be falsely low in px w/ leukemia)
• Rather than phenotyping, can also genotype for TPMT*2, *3A, and *3C b/c they account for more than 90% of defective alleles

Question 5

Why genotype?

Answer 5

• All clinical recommendations are based on an individual px genotype info
• We can’t tell what a px genotype is w/o a test
• It is getting increasingly easy, rapid, and cheap to genotype individuals (so why not?)

Question 6

Genotyping using qPCR

Answer 6

• qPCR = quantitative polymerase chain reaction AKA real-time PCR
• Differs from normal PCR b/c the amount of product (amplicon) can be quantified
• • In some cases, we actually quantify the # of amplicons or amount of DNA amplified
• • In most cases, we measure the amount of DNA relative to a gene that has a constant background expression (house-keeping gene)

Question 7

Describe PCR – what is needed and what it does

Answer 7

• Use an enzyme to synthesize a copy of a user defined sequence of dsDNA
• Requires:
• • 2 oligonucleotide primers directing the sequence to be synthesized
• • The thermo-stable DNA polymerase (Taq polymerase) -> has to be able to survive being heated and cooled multiple times during multiple cycles
• • A mixture of all 4 dNTPs, Mg2+, sterile water, buffer
• • A device to cycle temp. accurately and precisely over an accurate and precise time (a thermocycler)

Question 8

How do we detect the accumulation of DNA copiens in qPCR?

Answer 8

• Use a reporter dye
• Dye is actually a fluorescent molecule, and somehow the fluorescent dye must be a reporter for the synthesis of DNA
• TaqMan method:
• • In order to understand the TaqMan method, have to understand some concepts
• • DNA polymerase (ie: like Taq) have 5’-3’ exonuclease activity; this means that they digest any DNA that is already annealed to the template sequence as it is replicating DNA
• • Primers can be made w/ a reporter dye and a quencher on the same primer; when the reporter and quencher are in close proximity, the reporter will not be fluorescent

Question 9

Describe the TaqMan assay

Answer 9

• Probe of a reporter and quencher binds to a specific region on template DNA where primers are amplifying DNA
• • Only binds if there is an exact match w/ the template DNA
• Taq polymerase will start making DNA and when it comes into contact w/ probe, will use 5’-3’ exonuclease to break this up, releasing the reporter dye

Question 10

Genotyping w/ multiple dyes

Answer 10

• One allele uses a fluorescence dye and the other allele uses a different fluorescence dye; each dye is attached to a primer that is specific for a particular allele
• Getting only 1 colour means that the individual is homozygous based on the colour
• Getting a mixture of 2 colours (ex: green is a mixture of yellow and blue) means that the individual is heterozygous
• This test is only qualitative