Download What are gene polymorphisms and how can we use them in

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 genes
that 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
GSTM1
allele
GSTM1*0
GSTM1*A
GSTM1*B
GSTM1*1X2
change/ phenotype
gene deleted: no enzyme
G519: active
C519: active
gene duplicated:high activity
GSTM3
GSTM3*A
GSTM3*B
wild type: active
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
OH
aliphatic
RCH2CH2
RCHCH2
cyclosporine
hydroxylation
tolbutamide
R
R
R
Aromatic
hydroxylation
phenytoin
O
OH
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
O
ROH
+
3’-phosphoadenosine
5’phosphosulphate
R
O
S
OH
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 (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, 6mercaptopurine, 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
genes
SET1
high environment low genetic risk
susceptibility
genes
SET2
less environment more genetic risk
susceptibility
genes
SET3
even less environment
even more genetic risk
susceptibility
genes
SET4
low environment high genetic risk
environment
Problems
unknown:
 number of patient subgroups

number of susceptibility sets

number of genes in a susceptibility set

how genes interact within a susceptibility setperhaps two/three genes critical and a variable
number of others modify their effect.
susceptibility
genes
SET1
Assume SET1 comprises 3 genes
genes
1
2
3
alleles
a, b
x, y
m,n
risk genotypes:
aa
xx
mm
susceptibility
genes
SET2
? effects of the aa/xx/mm combination
influenced by other genes
susceptibility
genes
SET3
susceptibility
genes
SET4
? 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
General
population
environment
Disease
group
Susceptibility
genes
SET 1
Modifier genes
SET G1
SET B1
Susceptibility
genes
SET 2
Modifier genes
SET G2
SET B2
Susceptibility
genes
SET 3
Modifier genes
SET G3
SET B3
Susceptibility
genes
SET 4
Modifier genes
SET G4
SET B4
GOOD
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
Biochemically interesting
?clinically significant
Useful for Medical
Screening/Diagnosis
p
Odds ratio Change
<0.05
1.1
55% 57%
n=
>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
ancestral GST gene
alpha
mu
theta
pi
zeta
sigma
kappa
omega
Chrom
6p
1p
22q
11q
14q
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.Hydroxyl
Superoxide dismutase
(Cu,Zn-SOD,
Mn-SOD)
.
radical (OH )
H2O2
DNA
DNA
hydroperoxides
Catalase,
Glutathione peroxidase
Lipid
Lipid
hydroperoxides
a, m, q, p class
Glutathione
S-Transferases
Detoxified products
H 2O
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 nonasthmatics with >10 years exposure
60.0%
54.2%
41.7%
39.6%
33.3%
30.0%
Non-asthmatic
Asthmatic
25.0%
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.