PGx Lecture 10

Question 1

Four distinct genetic tests

Answer 1

paternity/maternity
DNA forensics
Disease predisposition
Pharmacogenomics

Question 2

Steps in PGx testing

Answer 2

1. DNA sample collection
2. DNA isolation
3. DNA target sequence amplification via polymerase chain reaction (PCR)
4. Genotyping
5. Data Interpretation

Question 3

Types of DNA samples

Answer 3

Human Genomic DNA

Nonhuman DNA

Question 4

examples of Human DNA

Answer 4

holds complete set of genetic data 
obtained from any cell
blood lymphocytes (non-nucleated) (peripheral whole blood) = gold standard
buccal cells
saliva sample

Question 5

examples of nonhuman DNA

Answer 5

tumor, bacteria, virus

Question 6

why is blood the gold standard for DNA samples

Answer 6

pure, high quality, large quantity, easy to amplify target sequence. DNA obtained from WBC

Question 7

To determine a person’s TPMT genotype, which of the following DNA sample needs to be used?

Answer 7

human genomic DNA

Question 8

Purposes for Nonhuman DNA collection

Answer 8

somatic mutations in tumors

infectious disease - to sequence viral DNA (HIV)

Question 9

To determine the genotype of codon 389 in beta1-adrenergic receptor gene (ADRB1), which of the following DNA samples needs to be used

Answer 9

human

Question 10

Most pharmacogenomic markers require what type of DNA

Answer 10

human

Question 11

Only tumor biomarkers related to what type of mutation use tumor DNA

Answer 11

somatic mutations

Question 12

Gene-specific analysis

Answer 12

to identify specific SNPs of interest - eg, to determine a person’s genotype for CYP2C19 or a panel of drug metabolizing enzyme genes

Question 13

PGx test categories

Answer 13

Gene-specific analysis

genome-wide analysis

Question 14

Types of PGx tests

Answer 14

cytogenetic
DNA-based genotyping
matabolic assay

Question 15

cytogenetic

Answer 15

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

Question 16

Steps in FISH/cytogenetic test

Answer 16

1. preparation of probe. A probe is a florescent-labeled segment of DNA complementary to a chromosomal region of interest
2. 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
3. slide examined under a fluorescent microscope. Fluorescent signals indicate the presence of complementary chromosomal DNA’ absence signals absence of complementary chromosomal DNA

Question 17

FISH Green signal

Answer 17

normal control
two green signals
two pink signals

Question 18

FISH Pink signal

Answer 18

chromosomal region of interest
Patient with deletion:
two green signals
one pink signal

Question 19

What does FISH detect

Answer 19

deletions

duplications

Question 20

Common DNA-based genotyping methods

Answer 20

• restriction fragment length polymorphisms (RFLP) analysis
• TaqMan SNP
• mass spectrometry
• Microarray (gene clip)
• DNA sequencing
  often a combo of methods is used

Question 21

Why is whole-genome so expensive

Answer 21

lots of redundancy

can only sequence once b/c of cost and time –> mistakes easy to make

Question 22

Metabolic test

Answer 22

measure MR to identify extensive and poor metabolizers

Question 23

do metabolic tests identify genotype or phenotype

Answer 23

phenotype

Question 24

problems with metabolic tests

Answer 24

phenotype not always stable, metabolic capacity affected by various factors

Question 25

Enzyme assay

Answer 25

metabolic test | measurement of enzyme activity with a particular substrate (ex TPMT)

Question 26

Two examples of FDA-approved genotyping tests

Answer 26

1. Roche AmpliChip CYP450 test - to identify a pts CYP2D6 and CYP2C19 genotype 2. Invader UGT1A1 Molecular Assay - genotyping of alleles in the UGT1A1 gene - specifically *1 and *28

Question 27

First approved test

Answer 27

``` 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 ```

Question 28

Invader UGT1A1 molecular assay

Answer 28

for the detection and genotyping of the *1 (TA6) and *28 (TA7) alleles in the UGT1A1 gene in genomic DNA obtained from whole blood

Question 29

What does UGT1A1 determine

Answer 29

genotype. homozygous --> reduce dose of irinotecan therapy

Question 30

PGx testing in key therapeutic areas

Answer 30

``` infectious diseases oncology cardiovascular disease psychiatry neurology organ transplantation ```

Question 31

Tamoxifen test

Answer 31

Tamoxifen -> CYP2D6

Question 32

Trastuzumab test

Answer 32

HER2IHC | HER2/CEP17, FISH

Question 33

Mercaptopurine test

Answer 33

PredictRx, TPMT

Question 34

Irinotecan test

Answer 34

Invader | UGT1A1

Question 35

Warfarin test

Answer 35

CYP2C9 and VKORC1

Question 36

Abacavir test

Answer 36

HLA-B*5701