human polymorphism

7/29/2019 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):


- 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):


- Poor metabolizers (2 inactive alleles):


- 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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human polymorphism

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