Lecture 6-7 - Testing and Tech Flashcards
(46 cards)
Regulation of PGx Tests
FDA review will add a new drug label
Actionable PGx markers
FDA-approved PGx Drug labels
One gene-multiple drugs
One drug-multiple genes
One gene-multiple alleles
Who performs PGx testing?
CLIA certified labs that are accepting PGx Tests
(GTR: Genetic Testing Registry)
Prescription of a PGx test
Collect enough info
-work closely with the therapeutic team
-Discuss w/the patient
-Understand the FDA labeling/CPIC Guidelines
-Know the principle of technologies
Make informed decision
-Strength of the PGx information vs other factors
-Cost vs Benefit
-Selection of technologies
Understand the clinical implication of a PGx test:
The strength btw a PGx marker and a clinical consequence varies
-Nature of PGx studies for discovering the marker: “how convincing”
Sample size, design, replication, etc
-Evidence in applying the PGx in clinical practice: “how effective”
Genotyping the patients first, and test the outcome
-The overall impact of the genotype on the phenotype: “how important”
20-90% in all drugs
PGx is still in its infancy
PGx levels and meaning
Red - Genetic testing is required - must do it
Orange - Genetic testing recommended - better to do it
Green - Actionable PGx - Drug label mentioned - you decide
Blue - Informative PGx - you decide
Limitations: PD/PK issues are complex
-Many genetic and non-genetic factors involved
-Don’t rely on PGx alone ESPECIALLY when there is a sign of a serious ADR
-Don’t Forget non-genetic factors
-Age, gender, BMI, diet, supplements, intake, etc.
(Not all/all) FDA-approved PGx testing is a mandatory test for all related drugs
Not all
Limitations: Cost
Cost may not bring enough benefit (Value is hard to determine)
-many tests not covered by insurance
-severe toxicity for many drugs is very rare
-Few patients may benefit from the test
-Should consider when PGx information is already available: Warfarin
Consideration for the Technologies
Know the strength and limitations of different methods for a PGx test
A targeted test focusing on the major alleles could be cheaper and quicker, but may miss other uncommon/rare important alleles
-W/o testing rare alleles, a haplotype can be assigned to the reference allele: Cyp2C9*1 vs *17
Balance the cost and info you need
Cyp2C9*5-11 may be more important for African descendants
Important factors to be considered
Family hx
-Often indicates an involvement of genetic factors
-patient him/herself: previous ADR
-Genetically related relatives
Race and Ethnicity
-Allele frequency/mutation rate can be very diff between populations
-CYP2C9
Vulnerable populations
-Children: drug metabolism can be diff from adults
-Pts w/diminished competence and/or decision-making capacity due to medical conditions
-Sciz, bipolar, some dementias, etc.
-PGx prescription might be preferred given potentially incomplete information from the patient
Consent/assent
-Pt/parent/guardians
Sample collection and data handling
Determine the most appropriate method
Consult the CLIA lab for their requirements
Consider the situation
-Patients
-Availability of facilities
-Equipment
-Personnel
Know the right procedure for sample handling, preservation and transportation
Samples for PGx testing
DNA is the target
Any nucleated cells/tissue contains germline DNA
Principles:
-Easy to collect
-Avoid contamination
-Less invasive
-Availability of standard procedure (e.g. commercial kits)
Peripheral blood
White Blood cells: DNA
-2-6ml as standard amount
-Prefer EDTA-anticoagulant tube (purple top)
-Use sterile technique to prevent bacterial contamination
-Room temp same day/overnight deliver (1-2 days)
Advantages/Limitations of WBC (peripheral blood)
Advantages:
-Good and stable yield of DNA
-Less contamination w/other DNA sources
-Standard handling procedure
-The most commonly used medical sample
Limitations:
-Invasive
-requires more professional collection and handling
-Pay attention to special patients
-pts treated w/chemotherapy, radiotherapy: fewer cells, DNA sequence may be altered
-Bone marrow transplantation patients: different DNA
Can we get DNA from RBCs?
NO!
-RBCs do not have a nucleus
Cheek Swab/Brush
-Buccal epithelial cells
-easy to collect
-Noninvasive
-Room temp handling
-Less DNA yield than blood: 1-5 nanograms, but still enough for many types of assays
-DNA yield is variable from patient to patient
Possible contamination: food, bacteria, etc
-Non-patient DNA
-Inhibitors for downstream reaction
-Rinse your mouth!
Some studies showed a lower DNA quality
Tissue
Tumor
-Fresh biopsy
High yield of DNA
Snap Frozen in liquid N2
-80 C for long term storage - ALWAYS
Dry ice for transportation
-Formalin fixed and Paraffin Embedded (FFPE)
DNA is usually degraded
However, many detections are still doable
Acceptable samples
-Formalin-Fixed paraffin embedded (FFPE) specimens, including cut slide specimens are acceptable
-Use standard fixation methods to preserve nucleic acid integrity. 10% neutral-buffered formalin for 6-72 hours is industry standard. DO NOT use other fixatives (Bouins, B5, AZF, Holland’s)
Do not decalcify
DNA handling
DNA is very stable especially pre and dried (RNA is less stable)
Factors that affect DNA quality:
-pH (neutral), avoid oxidants, UV
-Repeated freezing and thawing
-Bacteria contamination
-4 C for short term storage (1-2 m)
--80 C for long term storage) (years)
-Aliquot into small volume if possible
PGx testing methods: Goals and Technique
Goal:
-Testing the known variants
Genotyping (DNA chip)
-Testing both known and unknown alleles
-Sequencing
Sanger sequencing
High-throughput next-gen sequence (whole exome sequencing)
A fundamental technique for DNA amplification: PCR
DNA amplification: 2 Critical Steps
Target DNA amplification
Allele discrimination (HOM vs HET)
Polymerase Chain Reaction (PCR)
-The most useful technique for DAN amplification: 50-1000bp
-Amplify a specific region from the genome for making billions of copies (~2^35): detectable
-Enzymatic reaction
