Genetic Polymorphism

What is Genetic Polymorphism?

(1) The existence together of many forms of DNA
sequences at a locus within the population.

(2) A discontinuous genetic variation that results in

different forms or types of individuals among the
members of a single species.
Genetic Polymorphism & Drug
Metabolism
• inter-individual variation of drug effects
• Genetic polymorphisms of drug-metabolizing enzymes give
rise to distinct subgroups in the population that differ in their
ability to perform certain drug biotransformation reactions.
• Polymorphisms are generated by mutations in the genes for
these enzymes, which cause decreased, increased, or absent
enzyme expression or activity by multiple molecular
mechanisms.
 Polymorphism
• polymorphism= the inheritance of genes in different forms
termed alleles
alleles have different DNA sequences

• polymorphic locus: the frequency of the most common allele is

less than 99%.
1 allele in 100 alleles
100 alleles =50 people
1 person in 50 (2%) is heterozygous
Important points

• every conceivable type of variation in DNA has been

identified

• some genes demonstrate many alleles (>50). Some of

these alleles may have a high population frequency
 Examples of polymorphic variation
gene allele change/ phenotype
GSTM1 GSTM1*0 gene deleted: no enzyme
GSTM1*A G519: active
GSTM1*B C519: active
GSTM1*1X2 gene duplicated:high activity

GSTM3 GSTM3*A wild type: active

GSTM3*B 3bp deletion in intron 6: linkage dysequilibrium with M1*A

CYP2D6 CYP2D6*4 G/A intron 3/exon 4: splice site defect, inactive enzyme

TNF-alpha TNF*A substitution in the promotor region at -308

 Polymorphism:
• is common (probably all genes show allelic variation)

• BUT does it matter?

(outside celebrated examples such as cystic fibrosis,
haemoglobin)
Individual variation in response to drugs is a
substantial clinical problem

1 in 15 British hospital admissions is due to adverse drug

reactions

In the US, 106,000 patients die and 2.2 million are injured
each year by adverse reactions to prescribed drugs.
Polymorphisms
• Genetic differences in drug metabolism are the result of
genetically based variation in alleles for genes that code
for enzymes responsible for the metabolism of drugs.
• In polymorphisms, the genes contain abnormal pairs or
multiples or abnormal alleles leading to altered enzyme
function.
• Differences in enzyme activity occur at different rates
according to racial group.
Single Nucleotide Polymorphisms (SNPs)
• Single changes in one allele of a gene responsible for a variety of
metabolic processes including enzymatic metabolism.
• The combination of alleles encoding the gene determines the activity
and effectiveness of the enzyme.
• The overall function of the enzyme is the phenotype of enzyme
function.
• Phenotype: the observable physical or biochemical characteristics
determined by both genetic makeup and environmental influences
• Poor metabolizers
• two defective alleles (ex: CYP2D6*4/*5 and CYP2D6*4/*4)
• Combination of alleles including one resulting in no enzyme
(ex: CYP2D6*5 and CYP2D6*4 deletion)
• Intermediate metabolizers
• Heterozygous – having only one wild type allele and one
defective allele
• Normal metabolizers
• Carry wild type alleles (ex: CYP2D6*1/*3).
• Wild type alleles encode genes for normal enzyme function
• Extensive metabolizers
• Carry one wild type and one amplified gene
• ex: CYP2D6*1/*2, CYP2D6*A/*1a, and CYP2D6*1A/*5

• Ultra-rapid metabolizers
• Carry two or more copies of amplified gene
• ex: CYP2D6*2/*3
• Genetic changes may inactivate or reduce enzyme
activity leading to increase in the substrate drug.

• Genetic duplication may increase enzyme activity

resulting in lower levels of substrate drug.
Inhibitors & Inducers
• Polymorphisms affect drug interactions by altering the effect of
inhibitors and inducers on the enzyme.
•  results in an exaggerated effect or minimal effect on the substrate

• Inhibitor: An enzyme inhibitor is a molecule, which binds to enzymes

and decreases their activity.

• Inducer: An enzyme inducer is a type of drug that increases the

metabolic activity of an enzyme either by binding to the enzyme and
activating it, or by increasing the expression of the gene coding for the
enzyme.
Extensive Metabolizers - Inhibitors
• Extensive metabolizer ----- level of substrate drug is normally
low due to rapid metabolism by the enzyme.
• An inhibitor to the enzyme will inhibit the extensive
metabolism and cause significant elevations in the
substrate drug.
• Effect of inhibitors is much greater in an EM  inc. level of
substrate levels
Poor Metabolizers - Inhibitors
• In a poor metabolizer, the level of substrate drug remains
high because the metabolism of the substrate is much less
than normal.
• When an inhibitor is added, the additional inhibition of
metabolism is not much greater than is already occurring
in the PM.
• The effect of inhibitor is less in a PM than in normal
metabolizers.
• The drug interaction might not occur.
Extensive Metabolizers - Inducers
• Level of substrate drug is lower than in a normal
metabolizer due to rapid metabolism.
• The addition of an inducer does not cause a greater
difference in the level of substrate because the
metabolism is already increased greatly.
• The drug interaction might not occur.
Poor Metabolizers - Inducers
• Level of substrate drug is higher than expected in normal
metabolizer because of the lower metabolism of substrate.
• The addition of inducer will cause a signification increase in
the metabolism of the substrate  much lower level of
substrate than expected in a normal metabolizer.
• Drug interaction may occur to a greater extent.
• Drug interaction may result in substrate levels similar to
those of normal metabolizers.
**NOTE**
• The effect of inhibitor is great in EMs than in PMs.

• The effect of inducer is greater in PMs than in EMs.

 Genetic Polymorphisms in
Genes that Can Influence Drug
Metabolism – CYP450 Isoforms
Phase I Enzymes
Enzyme Substrate Clinical Consequence
CYP1A1 Benzopyrine, phenacetin Inc. or dec. cancer risk
CYP1A2 Acetaminophen, amonafide, caffeine, paraxanthine, ethoxyresorufin, Decreased theophylline metabolism
propranolol, fluvoxamine
CYP1B1 Estrogen metabolites Possible inc. cancer risk
CYP2A6 Coumarin, nicotine, halothane Dec. nicotine metabolism and
cigarette addiction
CYP2B6 Cyclophosphamide, aflatozin, mephenytoin Significance unknown
CYP2C8 Retinoic acid, paclitaxel Significance uknown
CYP2C9 Tolbutamide, warfarin, phenytoin, NSAIDS Anticoagulant effect on warfarin
CYP2C19 Mephenytoin, omeprazole, hexobarbital, mephobartibal, Peptic ulcer response to omeprazole
propranolol, proquanil, phenytoin
CYP2D6 Betablockers, antidepressants, antipsychotics, codeine, debrisoquin, Tardive dyskinesia from
dextromethorphan, encainide, flecanide, fluoxetine, guanoxan, antipsychotics; narcotic side effects,
methxyamphetamine, phenacetin, propafenone, sparteine efficacy and dependency, imipramine
dose requirement; beta blocker
effects
CYP2E1 Acetaminophen, ethanol Possible effects on alc consumption
Possible inc cancer risk
CYP3A4/3A7/3A7 Macrolides, cyclosporine, tacrolimus, calcium channel Tacrolimus dose requirement in
blockers, midazolam, tefrenadie, lidocaine, dapsone, pediatric cancer patients
quinidine, triazolam, etoposide, teniposide, loastatian,
alfentanil, tamoxifen, steroids, benzo(a)pyrene
Aldehyde Cyclophosphamide, vinyl chloride SCE frequency in lymphocytes
dehydrogenase
Alcohol Ethanol Inc. alc consumption and
dehydrogenase dependence
Dihydrodyrimidine 5-fluorouracil Inc. 5-flurorouracil toxicity
dehydrogenase
(DPD)
NQO1 Ubiquinone, menadione, mitomycin C Menadione-associated orlithiasis, dec
tumor sensitivity to mitomycin-C;
possible inc. cancer risk
P450 Enzymes in Drug Metabolism
• The polymorphic P450 (CYP) enzyme superfamily is the most
important system involved in the biotransformation of many
endogenous and exogenous substances including drugs, toxins, and
carcinogens.
• Genotyping for CYP polymorphisms provides important genetic
information that help to understand the effects of xenobiotics on
human body.
• For drug metabolism, the most important polymorphisms are those of
the genes coding for CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5, which
can result in therapeutic failure or severe adverse reactions.
CYTOCHROME P4502D6
• Most extensively studied polymorphic drug metabolizing enzyme
• Debrisoquin --- marked hypotension
• Impaired ability to hydroxylate, and therefore, inactivate debrisoquin
• 5-10% of white subjects have relative deficiency in ability to oxidize
debrisoquin
• Also have impaired ability to metabolize the antiarrhythmic and oxytocic drug
sparteine
• PM  lower urinary concentration, higher plasma concentrations
• Subjects inherited two copies of a gene or genes that encoded an enzyme
with either decreased CYP2D6 activity or no activity at all
• Prominent in East African population – frequency as high as 29%
CYTOCHROME P4502C SUBFAMILY
• Accounts for approximately 18% of the CYP content in the liver
• Catalyzes roughly 20% of the CYP-mediated metabolism of drugs

CYP2C19
• Study using mephenytoin as probe drug determined that individuals
can be segregated into EMs and PMs.
• PM trait is autosomal recessive – present in 3-5% of Caucasians & 12-23% of
Asian populations
CYP2C19 (cont.)
• Also catalyzes the metabolism of several
proton pump inhibitors (i.e. omeprazole),
diazepam, thalidomide, and some
barbiturates.
• Responsible for inactivation or propranolol
and metabolic activation of antimalarial drug
proquanil.
CYP2C19 & Diazepam
• Diazepam is demethylated by CYP2C19
• Plasma diazepam half-life is longer in individuals who are homozygous
for the defective CYP2C19*2 allele compared to those who are
homozygous for the wild type allele.
• Half-life of the desmethyldiazepam metabolite is also longer in
CYP2C19 poor metabolizers.
• High frequency in Asian population.
• Diazepam induced toxicity may occur as a result of slower metabolism
– careful dosing in Asian population.
CYP2C9
• Major CYP2C subfamily member in the liver
• Primarily responsible for the oxidative metabolism of important
compounds – warfarin, phenytoin, tolbutamide, glipizide, losartan,
etc.
• 6 different polymorphisms – CYP2C9*1, *2, *3, *4, *5, *6
• CYP2C9*1 – wild type allele, CYP2C9*2-*6 – variants
• Variants *2 and *3 alleles are common in Caucasians (≈35%)
• CYP2C9*2 and *3 alleles associated with significant reduction in the
metabolism and clearance of selected CYP2C9 substrates
CYP2C9 & Warfarin
• Polymorphisms linked to both toxicity and dosage
requirements for optimal anticoagulation with warfarin.
• *2 and *3 variants – higher risk of acute bleeding
complications than patients with *1 wild type genotype.
• Require 15-30% lower maintenance dose of warfarin to
achieve target INR
• Patients with variant CYP2C9 genotype take a median of
95 days longer to achieve stable dosing compared to wild-
type group
Dihydropyrimide Dehydrogenase
• Metabolism of antineoplastic agent fluorouracil.
• In the 1980s, fatal CNS toxicity developed in several patients after
treatment with standard doses fluorouracil.
• Patients had inherited deficiency of dihyropyrimidine dehydrogenase.
• DPD metabolizes fluorouracil and endogenous pyrimidines.
• Severe fluorouracil toxicity occurs when DPD activity < 100 pmol/min/mg
protein.
• 3% of population carries heterozygous mutations that inactivate DPD and 1%
are homozygous for the inactivating mutations.
• Heterozygous individuals do not exhibit no phenotype until challenged with
fluorouracil.
CYTOCHROME P4503A SUBFAMILY
• CYP3A subfamily plays a critical role in the metabolism of more drugs
than any other phase I enzyme.
• Expressed in liver and small intestine
• Contribute to oral absorption, first-pass, and systemic metabolism
• Expression is highly inducible – enzyme activity influence by factors
such as variable homeostatic control mechanisms, up- or down-
regulation by environment factors, and polymorphisms.
CYP3A4
• More than 30 SNPs have been identified for CYP3A4 gene
• Unlike other P450s, there is no evidence for deleted or null allele for
CYP3A4.
• The most common variant in CYP3A4, CYP3A4*1B is an A392G
transition in the promoter region referred to as the nifedipine
response element.
• One study shows that this variant may be associated with a slower clearance
of cyclosporine.
• This is a rather controversial finding.
CYP3A5
• Polymorphically expressed in adults in about 10-20% in Caucasians,
33% in Japanese, and 55% in African Americans.
• The variable CYP3A5*3 is a result of improper mRNA splicing and
reduced translation of functional protein.
• CYP3A5 is the primary extra-hepatic CYP3A isoform, its polymorphic
expression has been implicated in disease risk and the metabolism of
endogenous steroids or drug in tissues other than liver.
• CYP3A5 has been linked to tacrolimus dose requirements to maintain
adequate immunosuppression in solid organ transplant patients.
CYP3A7
• Expressed in fetal liver during development
• Hepatic expression is generally down-regulated after birth, but the
CYP3A7 protein has been detected in some adults
• Increased CYP3A7 expression has been associated with the
replacement of 60 nucleotide fragment of the CYP3A7 promoter with
the corresponding region form of the CYP3A4 promoter (CYP3A7*1C
allele.)
• This promoter swap results in increased gene expression of the
pregnane X receptor response element.
• PXR signaling serves as a central regulator of inducible CYP3A4
expression as well as several other genes involved in drug
detoxification.
• Polymorphisms in PXR suggest observed variability in CYP3A4
enzymatic activity may be due to, in part, inherited differences in the
upstream signaling proteins that control induction of gene expression.
Phase 2 Enzymes
Enzyme
N-acetyltransferase (NAT1)
N-acetyltransferase (NAT2)
Glutathione transferase (GSTM1, M3,
T1)
Glutathione transferase (GSTP1)
Sulfotransferases
Catechol-O-methyltransferases
Substrate
Aminosalicylic acids, aminobenzoic
acid, sulfamethoxazole
Isoniazid, hydralazine, sulfonamides,
amonifidide, procainamine, dapsone,
caffeine
Busulfan, aminochrome, dopachrome,
adrenochrome, noradrenochrome
13-cis retinoic acid, busulfan,
ethacrynic acid, epirubicin
Steroids, acetaminophen, tamoxifen,
estrogens, dopamine
Estrogens, levodopa, ascorbic acid
Clinical Consequence
Possible increased cancer risk
Hypersensitivity to sulfonamides;
amonafide toxicity; hydralazine-
induced lupus, isoniazid neurotoxicity
and hepatitis
Possible inc cancer risk; cisplatin
induced ototoxicity
Possible inc cancer risk
Possible inc or dec cancer risk; clinical
outcomes in women receiving
tamoxifen for breast cancer
Decreased response to amphetamine,
substance abuse, levodopa response
Thiopurine methyltransferase Mercatopurine, thioguanine, Thiopurine toxicity and efficacy, risk of
azathioprine second cancers
UDP-glucuronosyl-transferase Irinotecan, troglitazone, bilirubin Irinotecan glucuronidation and
(UGT1A1) toxicity, hyperbilirubinemia (Crigler-
Najjar syndrome, Gilbert’s syndrome)
UDP-glucuronosyl-transferase (UGT2B) Opioids, morphine, naproxen, Significance unknown
ibuprofen, epirubicin
N-ACETYLTRANSFERASE
• N-acetylation of isoniazid to acetylisoniazid
• Individuals are slow or rapid acetylators
• Ethnic variation is seen
• Slow acetylation: Japanese (10%), Chinese (20%), Caucasians (60%)
• NAT2 protein is the specific protein isoform that acetylates isoniazid.
• 27 unique NAT2 alleles identified
• NAT2*4 is the wild type allele
• NAT2 alleles containing the G191A, T341C, A434C, G590A, and/or G857A
missense associated substitutions are associated with slow acetylator
phenotype.
Cytochrome P450 CYP2D6: debrisoquine
hydroxylase
• Strongly expressed in liver
• gene located on chromosome 22
• many drugs are substrates for CYP2D6:
amitriptyline, clozapine, haloperidol
propanolol, amiodarone, flecainide

highly polymorphic with more than 50 allelic variants

identified
CYP2D6 polymorphic variants
Relatively common gene inactivating mutations:
CYP2D6*4 splice site variants (GA transition
at intron 3/exon 4)
CYP2D6*3 base pair deletion in exon 5 CYP2D6*5 gene
deletion

homozygotes for these (and other rarer) mutant alleles are

PM (poor metabolisers) and comprise about 10% of
European populations
Further CYP2D6 polymorphic variants

• Amplification of the entire CYP2D6 gene with up to 13 copies is

found: ultra rapid individual.
• Found in 1.5% Scandinavians, 7% Spaniards, 20% Ethiopians
• Affected people metabolise CYP2D6 substrates so quickly that a
therapeutic effect cannot be obtained at conventional doses.

Nortriptyline:
CYP2D6 PM individual requires 10-20 mg/day
CYP2D6 ultra rapid individual requires 500mg/day
 Thiopurine methyltransferase

• potentially important polymorphism

• responsible for the metabolism of anti-tumour agents, 6-mercaptopurine,

6-thioguanine

• polymorphism associated with difficulty in achieving effective dose of

these drugs in children with leukaemia

• children with TPMT deficiency show severe haematopoietic toxicity when

exposed to drugs like 6-mercaptopurine.
Should patients be tested for specific
polymorphisms?

• Not generally available at present.

• likely to become more available since:
(a) particularly sensitive individuals may avoid serious adverse
reactions.
(b) can avoid giving drugs to patients who cannot benefit from them.

• Trials in psychiatric patients are underway.

• Technology may allow a detoxication DNA chip that screens for all
relevant polymorphisms.
What is the true function of drug metabolising
enzymes?
• Cytochrome P450 gene family is believed to be the product of
an ancestral gene formed about 3 billion years ago.

• Possible that P450s are the result of evolution of plants

producing toxins and animals evolving enzymes to detoxify
these chemicals.
 Genetic susceptibility to most
diseases appears to be due to
multiple genes that interact with
each other and the environment.
 general population

susceptibility high environment low genetic risk

genes
SET1
susceptibility
genes less environment more genetic risk
SET2
environment
susceptibility even less environment
genes even more genetic risk
SET3

susceptibility
genes low environment high genetic risk
SET4
Problems
unknown:
• number of patient subgroups

• number of susceptibility sets

• number of genes in a susceptibility set

• how genes interact within a susceptibility set- perhaps

two/three genes critical and a variable number of others
modify their effect.
 Assume SET1 comprises 3 genes
susceptibility genes 1 2 3
genes alleles a, b x, y m,n
SET1 risk genotypes: aa xx mm
susceptibility
genes ? effects of the aa/xx/mm combination
SET2 influenced by other genes

susceptibility
genes
SET3

susceptibility
genes ? completely different genes and
arrangement of genes
SET4
 Why have we identified so few
genuine epistatic effects?
• there is no basis for predicting epistatic effects- need to examine all
possible 2-way, 3-way etc genotype combinations.
problems for statistical analysis (multiple testing)

• many genotypes are found in low frequency:

genotype aa found in 20% of cases and genotype xx found in 30% of cases
aa/xx interaction present in only 6% of cases
 General Disease
population group
GOOD
Susceptibility Modifier genes
genes SET G1
SET 1 SET B1

Susceptibility Modifier genes

genes SET G2

OUTCOME
SET 2 SET B2
environment
Susceptibility Modifier genes
genes SET G3
SET 3 SET B3

Susceptibility Modifier genes

genes SET G4
SET 4 SET B4
BAD
Susceptibility genes:
compare gene frequencies in affected and unaffected subjects.
case-control and/or family studies

Modifier genes:
compare gene frequencies in cases with
different outcomes; young/old, good/bad outcome
studies in cases
Selection of candidate genes

Problematical: there are 50,000 genes

use whatever information is avalable:

functional
chromosomal location

but:
allele frequencies will determine patient numbers
Selection of candidate genes: functional
approach
• atopy
• bronchial hyper-responsiveness
• detoxication of environmental irritants
• detoxication of reactive oxygen species and their oxidised lipid and
DNA by-products
• recruitment of inflammatory cells
• cytokines determining Th1/Th2 response
• eicosanoid production
• tissue remodelling (growth factors)
Selection of candidate genes: positional
approach

• chromosome 5q: regulation of IgE, pro-inflammatory cytokines,

beta-adrenergic receptor
• chromosome 6: HLA, TNF-alpha
• chromosome 11q: high-affinity IgE receptor
• chromosome 12q: IFN-gamma, nitric oxide synthase
• chromosome 13: IgE levels
• chromosome 14: T cell antigen receptor, NFkB
 Biological vs Statistical significance

Statistical significance p Odds ratio Change n=

<0.05 1.1 55%® 57% >7100

Biochemically interesting
<0.05 2.5 55%® 75% >94

?clinically significant
<0.05 5.0 55%® 86% >39

Useful for Medical

<0.05 15 55%® 95% >22
Screening/Diagnosis
 The glutathione S-transferase supergene
family
ancestral GST gene

alpha mu theta pi zeta sigma kappa omega

Chrom 6p 1p 22 q 11q 14 q 4q ND
10q

Genes A1-A4 M1-M5 T1,T2 P1 Z1 S1 K1 O1

Allelic yes yes yes yes yes ? ? ?

Gene products expressed in cytosol

 O2- .

Superoxide dismutase
Hydroxyl
(Cu,Zn-SOD,
. Mn-SOD)
radical (OH )
H2O 2

DNA Catalase,
Lipid
Glutathione peroxidase

DNA
Lipid H2O
hydroperoxides
hydroperoxides
a, m
, ,,q,p class
Glutathione
S-Transferases

Detoxified products
 GSTP1 is associated with asthma symptoms
with an OR that indicates a strong biological
impact.

Question: What do I do now?

Answer: Confirm results in a separate patient cohort

 Occupational asthma
104 unrelated Italian Caucasians occupationally exposed to
toluene isocyanate

detailed clinical history

CE Mapp et al Dept Environmental Medicine and Public Health,

University of Padova, Italy
Italian occupational asthma cases:
GSTP1 Val/Val frequency in asthmatics and non-asthmatics
with >10 years exposure
60.0% 54.2%

41.7%
39.6%
33.3%

30.0% Non-asthmatic
25.0% Asthmatic

6.3%

0.0%
Ile/Ile Ile/Val Val/Val
GSTP1 genotype
What do we do now?

(i) identify further genes to build up susceptibility sets and

identify which biochemical pathways have the greatest
impact on phenotype.

(ii) in vitro studies to determine the mechanism of

gene/phenotype associations.
Molecular epidemiology can identify associations
between genes and disease phenotypes

GSTP1 Val/Val confers protection in allergic and occupational disease.

DOES THIS MAKE BIOLOGICAL SENSE?

GSTP1 is located on a hotspot region, chromosome 11q

Chronic inflammation is a prominent feature of both asthma types, in vitro

GSTP1 substrates include ROS by-products.
References
• Shargel, Leon. Chapter 12 – Pharmacogenetics. Applied Biopharmaceutics and Pharmacokinetics, 5th edition. E-
book.
• Shargel, Leon. Comprehensive Pharmacy Review, 7th Edition. Philadelphia: Lipincott- William & Wilkins, 2010. Print.
Pages 430-433.
• David B. Troy, Paul Beringer. Remington: The Science and Practice of Pharmacy, 21st Edition. Pages 1230 – 1239.
• Brunton, Laurence. Chabner, Bruce. Knollman, Bjorn. Goodman & Gilman’s The Pharmacological Basis of
Therapeutics, 12th edition. Pages 124-130.

• http://www.biology-online.org/dictionary/Genetic_polymorphism
• http://en.wikipedia.org/wiki/Drug_metabolism
• http://www.medscape.com/viewarticle/444804_5
• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1934960/
• http://dmd.aspetjournals.org/content/29/4/570.full