Human Polymorphism
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Transcript of Human Polymorphism
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Types of polymorphism
SNPs
Indel (insertion, deletion)
VNTR (Variable Number Tandem Repeat)*
Haplotype
Overview
Populations
Types of populations
HapMap Geographic project
Significance
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Single-Nucleotide Polymorphisms
Genetic polymorphism: A variation in DNA sequence
occurring with a frequency of at least 1% in thepopulation.
Majority of genetic variations aresingle-nucleotide
polymorphisms (SNPs; snips).
- SNPs are common DNA sequence variations amongindividuals.
- Many SNPs that do not themselves change proteinexpression and cause disease. But they may be closeon the chromosome to deleterious mutations.
- SNPs are used asmarkersto unearth mutations andaccelerate efforts to find therapeutic drugs.
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SNPs = single nucleotide polymorphisms
Estimated 1 every 1000 bases
Approx 10 million common SNPs in
human genome Most examples show 2 alleles (I.e. 2
bases observed)
A/G A/C G/T
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How many SNPs are there in
Humans today?
Human Mutation rate is ~2.5 x 10-8
mutations/site/gen
~150 mutations/diploid genome/generation
6.3 billion people in the world =
945,000,000,000 mutations in the world
today. With 3 billion nucleotides = each nucleotide in
the world today is mutated 315 times.
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The most common sources of variation
between humans are SNPs: single base
differences between genome sequences.
Fragments of two sequences, with eight
SNPs, are shown.
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SNPs
Critical SNPs can be clinically relevant andimpact drug response or lead to expression orsuppression of inheritable disease phenotype.
SNPs have important role in PGx due to theirhigh prevalence rate and ease of their analysis.
Once specific SNPs are discovered, the nextstep is to determine whether these SNPs have
phenotypic or physiological consequences.
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How do SNPs arise?
DNA polymerase is the enzyme thatreplicates DNA one base at a time
Polymerase has base substitution error
rate of 1/10,000-100,000 Most of these are fixed by proofreading
and then DNA mismatch repair
Still leaves errors but less than
1/1,000,000,000 bases
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Indel
Insertion - one or more extra bases at aspecific location relative to the reference
sequence
Deletion - one or more bases less than thereference sequence at a specific location
Reference sequence - can be hard to define
what the most common allele is (due to
population differences) so better to usecommon standard E.g. Golden Path whole
genome sequence at UCSC
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Indel in ACE
ACE is involved in the pathways that controlblood pressure
Indel in intron 16
I allele has 289bp alu type repeat
D allele is deletion
D allele associated with higher expression ofACE
Implicated in hypertension, response toantihypertensives, physical endurance,cardiovascular disease.
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G Protein Beta 3 Gene (GNB3) Case:
There is a polymorphism (C825T) in the gene
encoding the G protein b3 subunit (GNB3)
This SNP is associated with some diseases
such as hypertension and obesity.
The G-protein 825T allele is associated with
altered drug responses while the underlying
mechanism is not fully understood. Differential expression of transcripts from the
C and T alleles could contribute to this
process.
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Conclusion: The GNB3 825 CC genotype isassociated with non-response in HCV-1-
infected patients.
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NAT2
N
OHO
N
N N HO
H
H
N
N NO
H
H
CH3
O
minor
Isoniazid N-Acetylisoniazid
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N-acetylation (NAT2) Polymorphism
Peripheral Neuropathy was noticed inpatients treated for tuberculosis withisoniazid, an antitubercular drug (Late
1940s).
Genetic factors influencing isoniazid
blood levels in humans wasdocumented (1959)
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Plasma isoniazid concentrations were measured in
267 subjects six hours after an oral dose. Bimodal
distribution in the rate of acetylation is due to genetic
polymorphisms within NAT2 gene.(N Engl J Med. 348: 529, 2003)
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Examples of drugs whose metabolism
is affected by acetylation
Isoniazid: peripheral neuropathy
Sulfasalazine: hemolytic anemia
Benzidine: urinary bladder cancer
Problem: Slow acetylation may lead to
exaggerated responses and toxicity !!!
Solution: May need to lower the dose !!!
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EXAMPLE 2: DIFFERENCE IN METABOLIC
ACTIVITY OF ENZYME DUE TO MUTATIONS
OR DUPLICATIONS
The superfamily of cytochrome P450 (CYP)
enzymes is the most important oxidative
enzymatic system involved in drug metabolism.
There may be a defect in drug oxidation by
CYP2D6, a member of CYP super family,
resulting in a major problem.
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Genetic Classification of CYPs
Several ( at least 12) CYP Families identified
Main CYP gene family members: CYP1
CYP2 CYP3
CYP4
Classification is based on amino acidsequence and is lot limited to a particularspecies
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CYP 2D6
CYP 2D6 is responsible for metabolism ofmore than 25% of drugs available in themarket including
- antiarrhytmic drugs,
- antidepressants,
- neuroleptics,
- beta-adrenoceptor blockers,
- others eg. debrisoquine, codeine, etc.(Ref: British Journal of Pharmacology, 53:111-122)
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Some drugs whose metabolism is catalyzed by CYP2D6
(Ref: British Journal of Pharmacology, 53:111-122)
Diff i t b li ti it f CYP2D6 i tt ib t d t
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Differences in metabolic activity of CYP2D6 is attributed to
mutations or duplications ofCYP2D6gene
Example: Patients taking antidepressantnortriptyline require different doses based onCYP2D6 genotype
- Normal CYP2D6 activity (one or more active alleles):
100-150 mg/day nortriptyline
- Poor metabolizer(2 inactive alleles):
10-30 mg/day nortriptyline
- Ultra-rapid metabolizer(CYPD26 duplication):
500 mg/day nortriptyline !!!
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Patients taking antidepressant nortriptyline
require different doses
based on CYP2D6 genotype
- Normal CYP 2D6 activity (one or more active alleles):
100-150 mg/day nortriptyline
- Poor metabolizers (2 inactive alleles):
10-30 mg/day nortriptyline
- Ultra-rapid metabolizers (CYP D26 duplication):
500 mg/day nortriptyline !!!
(Lancet,
356:1667,2000)
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CYP2D6 and Codeine
Codeine is metabolized by CYP2D6 tomorphine.
Since morphine is the active form havinganalgesic properties, poor CYP2D6metabolizer will not benefit relief fromcodeine !
H3CO
NCH3
HO
O
CODEINE
O-demethylation
MorphineCYP2D6
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A. PM
(poormetabolizer
B. IM(intermediatemetabolizer)
C. EM
(extensivemetabolizer)
D. UM
(UltraMetabolizer)
Pharmacogenetic Effect of Cytochrome Genotypes
(http://www.healthanddna.com/professional/pharmacogenetics.html)
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Michael died from anadverse reaction toFluoxetine hydrochloride(Prozac).
(Prozac is the most widelyprescribed antidepressantin history)
He was a slow metabolizerfor CYP2D6 gene
His Doctor would havechanged his medication ifs/he had known !!!
(Ref:http://www.hcroi.com/presentations/Coleman,%20Howard%20(session%201
1.05).ppt)
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Based on PGx data,
FDA started to include PGx-based
drug safety and efficacy drug labels !
Drug Biomarker Drug Label
Propafenone is used to treat arrhythmias and to maintain a normal heart rate
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SUCCINYLCHOLINE APNEA
Succinylcholine is a rapid acting, rapid recovery
neuromuscular blocking agent.
Often used to produce muscular relaxation during surgery usual paralysis lasts 2 to 6 min in patients
Succinylcholine is metabolized by
pseudocholinesterase
Genetic variation is one of the major factors determining theactivity of enzyme
O C CH2CH2
O
(H3C)3NH2CH2C C
O
O CH2CH2N(CH3)3
+ +
SUCCINYLCHOLINE
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SUCCINYLCHOLINE APNEA
Pseudocholinesterase deficiency
decreases succinylcholine inactivation.
There are several variants of geneaffecting metabolization of succinylcholine
Occasionally even when conventionaldoses of succinylcholine are used,prolonged paralysis of the respiratorymuscles results.
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G6PD Deficiency
G6PD is present in all human cells but is
particularly important to red blood cells (RBCs) It is required to make NADPH (Nicotinamide
adenine dinucleotide phosphate) in RBCs.
It is also required to make glutathione.
Glutathione and NADPH both help protect redblood cells against oxidative damage.
Thus, when G6PD is defective, oxidative
damage to red blood cells readily occurs, andthey break open as a result. This event iscalled hemolysis, and multiple hemolysis in ashort time span constitute an episode ofhemolytic anemia.
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The Pentose Phosphate Pathway. Note the importance ofG6PD in the production of reduced G-SH, ribose, andNADPH (adapted from: Yoshida and Beutler, 1986, pg.8).
- NADP+ = nicotinamide adenine dinucleotide phosphate
- NADPH = reduced nicotinamide adenine dinucleotide phosphate
- GS-SG = oxidized glutathione
- G-SH = reduced glutathione
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G6PD Deficiency
The most commonenzyme deficiency !
Worldwide 400 million (?) patients !!!
Patients with G6PD deficiency are at increased risk of
developing hemolytic anemia when given oxidant drugs,
such as antimalarial (e.g., Chloroquine, primaquine),
aspirin, probenecid, and vitamin K.(http://www.merck.com/mrkshared/mmanual/section22/c
hapter301/301a.jsp)
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Compounds reported in the literature that may induce hemolysis in
G6PD deficient individuals (from: Avery, 1980; Koda-Kimble, 1978).
ANALGESICS/ANTIPYRETICS
- acetanilid
- acetophenetidin (phenacetin)- amidopyrine (aminopyrine)
- antipyrine
- aspirin
- phenacetin
- probenicid
- pyramidone
ANTIMALARIALS
- chloroquine
- hydroxychloroquine- mepacrine (quinacrine)
- pamaquine
- pentaquine
- primaquine
- quinine
- quinocide
CARDIOVASCULAR DRUGS
- procainamide
- quinidineSULFONAMIDES/SULFONES
- dapsone
- sulfacetamide
- sulfamethoxypyrimidine
- sulfanilamide
- sulfapyridine
- sulfasalazine
- sulfisoxazole
MISCELLANEOUS
- alpha-methyldopa
- ascorbic acid
- dimercaprol (BAL)- hydralazine
- mestranol
- methylene blue
- nalidixic acid
- naphthalene
- niridazole
- phenylhydrazine
- pyridium
- quinine
- toluidine blue
- trinitrotoluene
- urate oxidase
- vitamin K (water soluble)
CYTOTOXIC/ANTIBACTERIAL
- chloramphenicol
- co-trimoxazole- furazolidone
- furmethonol
- nalidixic acid
- neoarsphenamine
- nitrofurantoin
- nitrofurazone
- PAS
- para-aminosalicylic acid
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GENETIC MODIFICATION OF RECEPTORS
Effectiveness of drugs is determined bytheir binding to receptors in addition to
serum levels.
For example, Familialhypercholesterolemia (FH) is a disease
in which the ability to synthesize
receptors for low-density lipoprotein(LDL) is impaired.
LDL receptors (LDLRs) are needed for
hepatic uptake of LDL.
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GENETIC MODIFICATION OF RECEPTORS
Patients with FH have very high levels ofcirculating LDL.
HMG-CoA Reductase inhibitors (important
class of drug for lowering circulating
cholesterol levels; Atorvastatin, Lovastatin, etc.)function largely by increasing number of
hepatic LDLRs.
These drugs are useless to FH patients
since they lack the genetic material needed
for LDLR !!!(HMG : 3-Hydroxy-3-MethylGlutaryl)
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ADVERSE DRUG REACTIONS (ADRs)
ADRs are responsible for:
- 6.7% of hospitalization (2.2 million/yr)- 0.32 % of mortality (100,000 deaths/yr)
6th leading cause of death (in the USA)
Although ADRs may normally occur as result ofhigher levels of drugs, genetic variation mayincrease an individuals risk of developing
ADR.
Severity of ADRs as well as response to agiven medication vary widely amongindividuals and are determined by genetic
make-up.
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ADR Test for CYP2C9
CYP2C9 Metabolizes warfarin Warfarin associated incidence of hemorrhages
0.8% fatal, 4.9% major, 15% minor
Poor Metabolizers at 2-4 X greater risk for
hemorrhages
5-10% of population - Poor Metabolizers
Genetic testing prior to the administration of
warfarin will reduce the incidence of adversebleeding event
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12 CYPC29 alleles identified. Patients with CYP2C9*2 and CYP2C9*3alleles have lower mean daily warfarin doses and a greater risk of bleeding.Testing for gene variants could potentially alter clinical management inpatients commencing warfarin.
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TOOLS FOR PGx
Medical Bioinformatics: tocorrelate genetic information withbiological activity
Microarray Technology (DNA chipTechnology):
- In the past, gene expression analysis
was very laborious and difficult.- Using microarray DNA chips, thousands
of genes, even whole genomes, can be
analyzed in a short time !.
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FUTURE OF PGx:
It is hoped that within the next decade:
-researchers will be able to correlate DNAvariants with individual responses to
medical treatments,
- and identify particular subgroups ofpatients, and eventually develop drugs
customized for those populations.
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Top 10 PGx Tests
1) CYP 2D6 (*)
2) TPMT
3) CYP 2C9
4) CYP 2C19 (**)
5) NAT (*)
6) CYP 3A5
7) UGT1A1
8) MDR1/P-glycoprotein
9) CYP 2B6
10) MTHFR
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September 1, 2004: First DNA Chip based test for two CYP 450 genes, CYP2D6 and
CYP2C19, is approved by European Committee.
December 27, 2004: U.S. Food and Drug Administration (FDA) has granted
regulatory clearance for the Affymetrix GeneChip(R) System 3000Dx (GCS 3000Dx),
an instrumentation system to analyze in vitro diagnostic microarrays.
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AmpliChip CYP450 Array
PGx test for irinotecan an antineoplastic drug
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PGx test for irinotecan, an antineoplastic drug.
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Do not be surprised if in
the next year or two, (thiskind of) DNA testing will beconsidered as a necessary
step before writing aprescription.
Dr. Francis Collins
The Director
National Human Genome Research Institute
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Terima kasih