Genetic polymorphism: Genetic polymorphism: (i) individual handling of drugs (ii) inherited...
-
Upload
harold-barker -
Category
Documents
-
view
217 -
download
2
Transcript of Genetic polymorphism: Genetic polymorphism: (i) individual handling of drugs (ii) inherited...
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
Menu
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.