PGx Lecture 10 Flashcards
Four distinct genetic tests
paternity/maternity
DNA forensics
Disease predisposition
Pharmacogenomics
Steps in PGx testing
- DNA sample collection
- DNA isolation
- DNA target sequence amplification via polymerase chain reaction (PCR)
- Genotyping
- Data Interpretation
Types of DNA samples
Human Genomic DNA
Nonhuman DNA
examples of Human DNA
holds complete set of genetic data obtained from any cell blood lymphocytes (non-nucleated) (peripheral whole blood) = gold standard buccal cells saliva sample
examples of nonhuman DNA
tumor, bacteria, virus
why is blood the gold standard for DNA samples
pure, high quality, large quantity, easy to amplify target sequence. DNA obtained from WBC
To determine a person’s TPMT genotype, which of the following DNA sample needs to be used?
human genomic DNA
Purposes for Nonhuman DNA collection
somatic mutations in tumors
infectious disease - to sequence viral DNA (HIV)
To determine the genotype of codon 389 in beta1-adrenergic receptor gene (ADRB1), which of the following DNA samples needs to be used
human
Most pharmacogenomic markers require what type of DNA
human
Only tumor biomarkers related to what type of mutation use tumor DNA
somatic mutations
Gene-specific analysis
to identify specific SNPs of interest - eg, to determine a person’s genotype for CYP2C19 or a panel of drug metabolizing enzyme genes
PGx test categories
Gene-specific analysis
genome-wide analysis
Types of PGx tests
cytogenetic
DNA-based genotyping
matabolic assay
cytogenetic
FISH
Flourescent in situ hybridization
A technique used to identify the presence of specific chromosomes or chromosomal regions through hybridization (attachment) of flourescently-labeled DNA probes to denatured chromosomal DNA
Steps in FISH/cytogenetic test
- preparation of probe. A probe is a florescent-labeled segment of DNA complementary to a chromosomal region of interest
- Hybridization. Denatured chromosomes fixed on a microscope slide are exposed to the florescent-labeled probe. Hybridization (attachment) occurs between the probe and complementary (matching) chromosomal DNA
- slide examined under a fluorescent microscope. Fluorescent signals indicate the presence of complementary chromosomal DNA’ absence signals absence of complementary chromosomal DNA
FISH Green signal
normal control
two green signals
two pink signals
FISH Pink signal
chromosomal region of interest
Patient with deletion:
two green signals
one pink signal
What does FISH detect
deletions
duplications
Common DNA-based genotyping methods
- restriction fragment length polymorphisms (RFLP) analysis
- TaqMan SNP
- mass spectrometry
- Microarray (gene clip)
- DNA sequencing
often a combo of methods is used
Why is whole-genome so expensive
lots of redundancy
can only sequence once b/c of cost and time –> mistakes easy to make
Metabolic test
measure MR to identify extensive and poor metabolizers
do metabolic tests identify genotype or phenotype
phenotype
problems with metabolic tests
phenotype not always stable, metabolic capacity affected by various factors
Enzyme assay
metabolic test
measurement of enzyme activity with a particular substrate (ex TPMT)
Two examples of FDA-approved genotyping tests
- Roche AmpliChip CYP450 test
- to identify a pts CYP2D6 and CYP2C19 genotype - Invader UGT1A1 Molecular Assay
- genotyping of alleles in the UGT1A1 gene
- specifically *1 and *28
First approved test
Roche AmpliChip CYP450 test extracted from whole blood sample DNA microarray using Affymetrix gene chip technology assay of 27 alleles of 2D6 get genotype results
Invader UGT1A1 molecular assay
for the detection and genotyping of the *1 (TA6) and *28 (TA7) alleles in the UGT1A1 gene in genomic DNA obtained from whole blood
What does UGT1A1 determine
genotype. homozygous –> reduce dose of irinotecan therapy
PGx testing in key therapeutic areas
infectious diseases oncology cardiovascular disease psychiatry neurology organ transplantation
Tamoxifen test
Tamoxifen -> CYP2D6
Trastuzumab test
HER2IHC
HER2/CEP17, FISH
Mercaptopurine test
PredictRx, TPMT
Irinotecan test
Invader
UGT1A1
Warfarin test
CYP2C9 and VKORC1
Abacavir test
HLA-B*5701
