Genetic polymorphism:Genetic polymorphism:

(i) individual handling of drugs(ii) inherited susceptibility to disease

Much individuality in drug response is inherited (polymorphism)

PHARMACOGENETICS

Identify specific genes associated with specific diseases and that may be targets for new drugs

Identify polymorphic genesthat mediate response to drugs

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Metabolism of drugs- phase 1 and 2 reactions

what is polymorphism? Examples of polymorphic genes relevant

in drug metabolism polymorphism and disease susceptibility

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.

Drug metabolism

Drugs are usually lipophilic. This allows them to cross membranes and enter cells.

Lipophilic compounds are difficult to remove from the body.

Enzymatic transformation of drugs into hydrophilic, inactive metabolites is usually necessary.

Detoxication reactions often involve phase 1 and phase 2 steps

Phase 1 reactions add or expose a functional group through oxidative reactions:

N-dealkylation

O-dealkylation

hydroxylation

N-oxidation

S-oxidation

deamidation

The cytochrome P-450 mono-oxygenase system is largely responsible for catalysing phase 1 reactions.

Complex supergene family: at least 40 enzymes expressed in human tissues.

CYP1A2, 3A4, 2C9, 2C19, 2D6, 2E1 exert a major role in drug metabolism.

Enzymes located on smooth endoplasmic reticulum

ARE RESPONSIBLE FOR THE METABOLIC ELIMINATION

OF MOST DRUGS CURRENTLY USED IN MEDICINE

Examples of phase 1 reactions

N-dealkylation RNHCH2 RNH2 +CHO2 codeine

theophylline

aliphatic RCH2CH2 RCHCH2 cyclosporine

hydroxylation tolbutamide

OH

Aromatic hydroxylation

OHO

R R R

phenytoin

Detoxication reactions often involve phase 1 and phase 2 steps

Phase 2 reactions are conjugations with:

glucuronic acid

sulphate

acetate

amino acids

reduced glutathione

that result in water-soluble molecules that are inactive and easily excreted.

Phase 2 reactions involve an extensive enzymology

Glucuronidation: uridine 5’-diphosphate glucuronyltransferase

Methylation: catechol O-methyltransferase

histamine N-methyltransferase

thiopurine methyltransferase

Sulphation: sulphotransferases

Glutathione: glutathione S-transferases

Example of phase 2 reaction

sulphation

ROH+3’-phosphoadenosine 5’phosphosulphate

R O S OH

O

O+3’-phosphoadenosine 5’phosphate

Eg: acetaminophen

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 (GA 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

environment

susceptibilitygenesSET2

susceptibilitygenesSET3

susceptibilitygenesSET4

susceptibility genes SET1

high environment low genetic risk

low environment high genetic risk

less environment more genetic risk

even less environmenteven more genetic risk

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.

susceptibilitygenesSET2

susceptibilitygenesSET3

susceptibilitygenesSET4

susceptibility genes SET1

Assume SET1 comprises 3 genesgenes 1 2 3alleles a, b x, y m,nrisk genotypes: aa xx mm

? effects of the aa/xx/mm combination influenced by other genes

? completely different genes and arrangement of genes

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

GOOD

BAD

OU

TC

OM

E

Generalpopulation

SusceptibilitygenesSET 1

SusceptibilitygenesSET 2

SusceptibilitygenesSET 4

SusceptibilitygenesSET 3

environment

Diseasegroup

Modifier genesSET G1SET B1

Modifier genesSET G4SET B4

Modifier genesSET G3SET B3

Modifier genesSET G2SET B2

Susceptibility genes: compare gene frequencies in affected and unaffected subjects.case-control and/or family studies

Modifier genes:compare gene frequencies in cases withdifferent outcomes; young/old, good/bad outcomestudies 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

Biochemically interesting

?clinically significant

Useful for MedicalScreening/Diagnosis

p Odds ratio Change n=

<0.05 1.1 55% 57% >7100

<0.05 2.5 55% 75% >94

<0.05 5.0 55% 86% >39

<0.05 15 55% 95% >22

The glutathione S-transferase supergene family

alpha mu theta pi zeta

ancestral GST gene

Chrom 6p 1p 22q 11q 14q 4q ND10q

Genes A1-A4 M1-M5 T1,T2 P1 Z1 S1 K1O1

Allelic yes yes yes yes yes ? ?? Gene products expressed in cytosol

kappasigma omega

O2-

H2O2

H2O

Hydroxyl

radical (OH.)

Lipidhydroperoxides

classGlutathione

S-Transferases

Superoxide dismutase(Cu,Zn-SOD,

Mn-SOD)

Catalase,Glutathione peroxidase

DNA

DNAhydroperoxides

Lipid

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

41.7%

33.3%

25.0%

54.2%

39.6%

6.3%

0.0%

30.0%

60.0%

Ile/Ile Ile/Val Val/Val

Non-asthmaticAsthmatic

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.