Pre-implantation Genetic
Diagnosis (PGD)
Genetic analysis of a single cell from an eight-cell embryo done in
conjunction with in vitro fertilization (IVF) to improve the chances
of a “normal” pregnancy.
• Polar body
• Blastomere
• Trophectoderm
• Remove a single cell from the 6-8-cell embryo using a fine glass
needle to puncture the zona pellucida and aspirate the cell
FISH
using fluorescent probes specific for each chromosome. These allow number and size of
each chromosome to be checked.
• useful for identifying aneuploidies (incorrect chromosome numbers) and
translocations
• procedure destroys the tested cell
• limited number of chromosomes can be checked simultaneously; some abnormalites
undetectable
• Aneuploidy is the most frequent cause of spontaneous abortions.
genetic testing (PCR or gene chips)
• Array CGH (aCGH)
• PGD of monogenic diseases
based on indirect diagnostics using STR markers
direct diagnostics - sequencing
Limitations of PCR-based tests:
• Both alleles may not amplify equally, leading to misdiagnosis or
inconclusive results
ADO (alelic drop out) – amplification of one alelle under the detection
limit. Reasons unknown but influenced by cell lysis, PCR conditions,
target DNA for sequencing, PCR product size
• PCR-based tests only detect disorders at target loci; other
mutations may exist elsewhere
Cell free fetal DNA
• Short segments (<200 base pairs) of fetal DNA circulates in maternal
plasma
• Origin is primarily placenta
• Placental cells undergoing apoptosis
• Reliably detected >7 weeks
• Increases throughout pregnancy (10-22 weeks constant)
• Cleared within hours of birth
• Short half life (16 min), undetectable by 2 hours postpartum
• Both cell-free fetal and cell-free maternal DNA circulate in
maternal plasma.
• Cell-free fetal and maternal DNA circulate in maternal plasma as
relatively short fragments (150-200 base pairs) and represent
the entire genome.
• Fetal DNA comes primarily from the placenta.
• Maternal DNA comes primarily from maternal blood cells.
• Fetal DNA is 5-25% of the total cell-free DNA (~10% on
average).
Cell free DNA Clinical Applications
• Sex Determination
• Single gene disorders – paternal origin
• Isoimmunization: noninvasively determine fetal Rh type
• Aneuploidy: detect abnormal ratio of a particular
chromosome;
• Does not detect nontargeted aneuploidies
• Next generation sequencing
• Real-time PCR
• Many platforms and methods
• Why cell free fetal DNA fails?
• Confined placental mosaicism
Pharmacogenetics:
From DNA to Drug Treatment
• Pharmacogenomics
• The science of how genes affect the way people people respond to drugs
• How genes affect…
…the way our body processes drugs (pharmacokinetics)
…the interaction of drugs with receptors (pharmacodynamics)
…the treatment efficacy and adverse side effects
• Pharmacogenetics
• A subset of ‘pharmacogenomics’
• The study of how inherited variation affects drug response and metabolism
Why is this a good approach?
• Drugs can be dangerous
• Many people have severe adverse reactions to drugs
• Many people respond to drugs at different doses
• Many drug treatments are horribly unpleasant, painful
• Drugs are expensive (to take and to make)
• Ineffective drugs are a waste of money to take
• Drug development needs to account for response variability
• Genetics provide a priori information
• Genetics don’t change (except in cancer)
• Genetics can point to the cause not just the symptom
The Goal of Personalized Medicine
• The Right Dose of
• The Right Drug for
• The Right Indication for
• The Right Patient at
• The Right Time.
Courtesy Felix W. Frueh
Pharmacogenetics
The study of variations in genes that determine an
individual’s response to drug therapy.
Common variation in DNA sequence (i.e. in >1% of
population)
Genetic Polymorphism:
SNPs; INDEL; VNTRs
Potential Target Genes are those that encode:
Drug-metabolizing enzymes
Transporters
Drug targets
Sequencer
A T C G A A A T G C A T G A C C T T T G A T A T G A T C G G C T G C A G T C A G C
T T C G A A G T G C A T G A C T T T T G A C A T G A G C G G C G G C C C A C A G C
Common Variation Rare Variation No Recorded Variation
The Technology: Deep Sequencing
• Captures every base pair in the
genome (3,000,000,000)
• (Currently) low throughput (slow)
• (Currently) Very expensive (> 10k)
• Captures common, rare, and
personal variation
• New and hard to analyze
Back to the drugs…
• The utility of pharmacogenetics:
• Determining appropriate dosing
• Avoiding unnecessary toxic treatments
• Ensuring maximal efficacy
• Reducing adverse side effects
• Developing or choosing novel treatments
• Can also explain variable response to illicit drugs
Warfarin: A dosage story
• Most widely used
anticoagulant in the world
• A “blood thinner”
• Prescribed doses vary widely
(1-40mg / daily)
• Therapuetic index is very low
• High risk of bleeding early in
treatment
• Two genes involved in
metabolism: CYP2C9 and
VKORC1
Homozygous wild-type CYP2C9 and VKORC1
Carrier of CYP2C9 mutant allele
Carrier of VKORC1 mutant allele
Purine Analogs:
A Case Study in
Pharmacogenetics• 6-mercaptopurine, 6-thioguanine, azathioprine
• Used to treat lymphoblastic leukemia, autoimmune disease,
inflammatory bowel disease, after transplant
• Interferes with nucleic acid synthesis
• Therapeutic index limited by myelosuppression
(treatment limited by immune suppression side effect)
6-thioguanine azathioprine6-mercaptopurine
Metabolism of 6-MP
L Wang and R Weinshilboum, Oncogene 25, 1629-1638 (2006)
Pharmacogenetics: A Case Study
Courtesy of Michelle Whirl-Carillo
Pharmacogenetics: A Case Study
Courtesy of Michelle Whirl-Carillo
Pharmacogenetics: A Case Study
Courtesy of Michelle Whirl-Carillo
Second Example: Codeine and
Cytochrome P450 CYP2D6
• Codeine is a commonly used opioid
• Codeine is a prodrug
• It must be metabolized into morphine for activity
• Cytochrome P450 allele CYP2D6 is the
metabolizing enzyme in the liver
• 7% of Caucasians are missing one copy of
the Cytochrome P450 CYP2D6 gene
• codeine does not work effectively in these
individuals
Courtesy of Michelle Whirl-Carillo
Cytochrome Oxidase P450 Enzymes
• 57 Different active genes
• 17 Different families
• CYP1, CYP2 and CYP3 are primarily involved in drug
metabolism.
• CYP2A6, CYP2B6, CYP2C9 ,CYP2C19, CYP2D6,
CYP2E1 and CYP3A4 are responsible for
metabolizing most clinically important drugs
Effect of Metabolic Rate on Drug Dosage
© 2006 American Medical Association. All rights reserved
FDA Requires Genetic Tests
for Certain Therapies