Lecture 6-7 - Testing and Tech Flashcards
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
PCR Substrates and Products
Substrates:
-DNA template
-dNTPs (dATP, dGTP, dCTP, dTTP)
-Primers: 2 short sequences specific to the region of interest
-Buffer: pH, Mg2+
-Enzyme: Taq DNA polymerase
Products:
-DNA molecules (fragments start and end w/primers)
PCR - chain reaction
From 2 copies to 2^n+1 copies (n= # of thermal cycles)
Starts from very small amount of template DNA
-5-20 ng
Enzyme (Taq polymerase) is the key
-Thermal stable
important point on PCR
PCR amplifies DNA from both DNA molecules of homologous chromosomes
This is why you can tell a genotype
The PCR reaction products (amplicon) are a mixture of double-strand DNA products generated from both homologous chromosomes (the primers equally bind to each chromosome)
-We need additional specific technique to distinguish each allele
Major PGx testing techs
The process to determine a genotype using certain techniques
Detect Known alleles
-DNA chip
Detecting both known and unknown alleles
-Sequencing
DNA Chip
Detecting known SNPs or targeted SNPs
High throughput
-Up to 5M SNPs can be genotyped simultaneously
Medium cost
-Low per SNP cost
Large-scale used for research
-Genome-wide based studies
Mid-throughput use for PGx testing
Chip-based PGx Testing Platform
Amplichip CYP450 Array
-Roche Molecular diagnostics
-Covers CYP2D6 and CYP2c19
DNA (Sanger) Sequencing
Several methods have been developed for DNA sequencing
conventional sequencing (Sanger sequencing)
Low throughput
Targeted sequencing: sequencing one specific DNA fragment
Next generation sequencing
high-throughput sequencing
parallel sequencing
massive sequencing - sequencing multiple DNA fragments simultaneously
Sanger Sequencing explained
A method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication
Sanger Sequencing - what makes it different?
Can detect both known and unknown alleles: SNPs, indel, small CN
low throughput
-96 samples per overnight for one DNA fragment ~700bp
Relatively higher cost per base pair
High per SNP cost
Widely used in PGx testing
Next generation sequencing (NGS)
Sequencing by the synthesis in parallel
Data processing based on overlap sequences
Common NGS produces very short reads (~100bp)
Long-read sequencing was recently introduced (20-100kb)
NGS explained
high throughput, can simultaneously sequence DNA of multiple individuals
Customizable
Whole genome/whole exome vs/ targeted genes
higher total cost
very low cost per SNP
detect all known or unknown alleles
detect almost all kinds of polymorphisms
Sequencing depth and coverage
The NGS coverage level often determines whether variant discovery can be made with a certain degree of confidence at particular base positions
for detecting human genome mutations, SNPs, and rearrangements:
10x to 30X depth of coverage recommended
Reads are not distributed evenly over an entire genome because
The reads will sample the genome in a random and independent manner
You need multiple observations per base to come to a
reliable base call
Germline
Sequence of germ cells that may be passed to a child
Exists in the somatic genome
Exists since the individual was born
Somatic
Sequence of nongermline cells that is NOT passed to a child
-Does not exist in the germline genome
-Acquired (e.g. in cancer, sunshine induced, etc.)
Does de novo mutation refer to somatic?
Yes
Not a germline - new mutation not from parents
Detection methods for somatic mutations
DNA chips are usually not used for somatic mutation detection
Sanger: point mutations, small indels
NGS: almost all kinds of mutations
Other methods: karyotyping, IHC
HIPPA
Health Insurance Portability and Accountability Act
Data sharing (what should be the default? Opt-in or opt-out? evolving concept
GINA
Genetic information Non-Discrimination Act
