Dr. dr. Mgs. Irsan Saleh, M.

Biomed
Dept of Pharmacology
Faculty of Medicine Sriwijaya University
irsan_saleh_hasani@yahoo.com
 Genetic variation occuring with a frequency
of 1% or more in the population
 SNP (Single Nucleotide Polymorphism):
▪ most frequent type
▪ difference in a single base of the
genomic sequence
▪ usually 1/1000 base
▪ most does not influence the structure or
function of proteins
 SNPs occur :
 In exons (may alter the structure of proteins
and may lead to functional consequences)
 In introns (may influence splicing)
 In the regulatory regions (may influence
expression of the gene)
 Insertion/deletion polymorphism:
insertion or deletion of a few nucleotides
 Variable number tandem repeats:
variation in the number of times a sequence
of several hundred base pairs is repeated
 Simple tandem repeats (microsatellites):
2-4 nucleotides repeated a variable number
of times
 CYTOPLASM

NUCLEUS

DNA EXON INTRON EXON INTRON EXON

GENE Transcription
Primary RNA transcript

Add 5’ Cap and

RNA Cap Poly (A) tail
 AAAA
RNA SPLICING
mRNA  AAAA
EXPORT

mRNA  AAAA
TRANSLATION

PROTEIN
 Genotype: genetic structure encoding for the given
characteristics
 Phenotype : the manifestation of the genotype,
which can be observed and can be influenced by
other factors:
 Other gene products
 Environment
 Acquired characteristics
 Frequency of genetic polymorphisms differs greatly
among ethnic groups
 The functional relevance of a given polymorphism
can vary across ethnic groups
 Genetic traits are mainly
 polygenic (influenced by several genes, the
effect may be additive or interactive)
 multifactorial (both genetic and environmental
factors contribute)
 Some genetic traits are
 monogenic
The most common genetic diseases
an autosomal recessive disorders
affecting hemoglobin synthesis
homozygous patients:
 severe anemia
 require regular blood transfusion
Heterozygous individuals (carriers):
 Clinically normal
 Small size and pale of red blood cells
Thalassemia belt ~ Malarial belt
 Adult hemoglobin

  Heme




HbA (22) 98%

HbA2 (22) ~ 2.5%
HbF (22) <1%
 -90 -70 -30 * CAP SITE

CACCC CCAAT ATAAAA ACATT

*
Exon1 Intron1 Exon2 Intron2 Exon3

Promotor
elements Polyadenilation
Splice sites
Initiator signal
5’ GT………AG 3’
codon (ATG)

• RNA Polymerase binding Terminator

• RNA transcription codon (TAA)
 Mutations affecting initiation of transcription located at the promoter regions
(-28, -101, - )  + or ++
Mutation affecting of RNA from intron located at intron (IVS1-nt1, IVS1-nt5) or
exon (HbE, HbMalay, Cd 30)  o, +, or ++
Polyadenilation signal mutation +, ++
Mutation affecting initiation of translation (ATG to ACG, ATG to AGG)  o+
Nonsense mutations  o
 Frameshift mutations  
o
BETA THALASSEMIA MUTATIONS CAUSE:
• Shortened amino acids  mostly degraded in the nucleus
• Long amino acids can be transferred to cytoplasm
• Lengthened amino acids and burdened proteolysis
recessive and dominant heterozygous thalassemia

Hb structure:
• Globin chain folding
• Heme binding
• 11, 22, 12, 21 contact

Functional and stable Hb require:

• correct amino acids sequence
• corrects number of amino acids
 Molecular defects in
-globin gene -globin gene
(-thalassemia) (-thalassemia)

Reduced or absence
-globin chain -globin chain

Excess of
-globin -globin chain (fetal)
-globin chain (adult)
 Molecular basis of -thalassemia
excess of -chains

4 


precipitate 

marrow circulation

red cells precursor mature red cells

• physical damage
• metabolic changes

• ineffective erythropoiesis
• intramedullary destruction haemolysis in the
microcirculation (spleen)
HETEROZYGOTE HOMOZYGOTE o

 



 


Asymptomatic Severe anemia

Mild anemia to normal Liver & spleen enlargement
MCV < 80 fl MCV < 70 fl
HbA2 > 3.5% only consists of HbA2 and HbF
HbF > 1% HbF > 90% or less
HOMOZYGOTE + HbE

HbE/HbE HbE/ o or +

 E
 
E

  
  

Mild to severe anemia - mild anemia - moderate to

Liver & spleen may/not >> - MCV < 70 fl severe anemia
Still has HbA - only consists of HbE,
HbF range from 10 to > 90% HbA2 and HbF
 The distribution and the frequency (%) of
beta-thalassemia carriers in Indonesia

P’baru

Dayak
Batak

Banjar
1 9 5

M’hasa
0

Kaili
5 2

M’kasar
Minang

9
P’bang

7
15
Bangka

Java

Flores
9
Bali

8 Sumba
Sasak

7 6
Bima

7 36
12

A.S. Sofro & F. Lanni, University of Gajah Mada (1995)

 Spectrum of beta thalassemia mutations &
hemoglobinopathies in Indonesia
ACEH BATAK SOUTH CELEBES
IVS1-nt1 HbMalay HbE
HbD Los-Angeles IVS1-nt5
Poly-A (Black’s) Hb Lepore
Codon 35
PADANG Filipino deletion
IVS1-nt5 Poly-A (Malay)
Codon 35 Initiation Codon
Codon 41-42 Codon 17
HbS
MALAY
IVS1-nt5 JAVANESE
HbMalay BETAWI SUNDANESE
IVS1-nt5 IVS1-nt5 HbE Codon 30
HbE IVS1-nt5 Codon 41-42
Codon 26 IVS1-nt1 IVS1-nt1
Codon 17 HbE IVS1-nt1 IVS2-nt654
Codon 41-42 Codon 35 IVS1-nt2
Codon 15 Codon 15
25bp deletion Codon 17 Codon 8-9
IVS1-nt1 Codon 26 HbG Makassar
HbS HbG Sirriraj
 2 2 1

2 2 1

Normal -globin gene cluster

Common mutations Less common mutations

2 2 1
Usually misense mutations or
2 2 1
mutations at codon termination
which produce:
one -globin gene deletion - unstable Hb
2 2 1 - unstable -globin chains

2 2 1

two -globin genes deletion

World’ Distribution of -Thalassemia

From: Hill, A.V.S. (1992) Bailliere’s Clinical Hematology 5:209-238

 Molecular basis of -thalassemia

 Excess of:
-globin chain (fetal) 
 -globin chain (adult)
  

4 (Hb Bart)
4 (HbH)

Precipitate in mature red cells

hemolysis in spleen & microcirculation

Normal -thalassemia

--/-- - -/-



  4=HbBart 4=HbH



  
 

• death during fetal life • HbH disease

• Hydrops fetalis • mild to severe anemia
o-thalassemia deletions homozygote (--/--)
 Normal -thalassemia
- -/  - / 

   


  
  

MCH : < 26-32 pg < 25 pg 25-27pg

HbA2 : 2.5-3.5% normal or low normal or low
HbF : <1% low or absent low or absent
 Global Malaria Problem

Malaria cases (UN common database, prevalence in 100,000 people)

919.8 (Indonesia, 26th), 75385.8 (Guinea 1st), 1688.3 (PNG, 22nd)
EIJKMAN INSTITUTE
Epidemiology of antimalarial drug resistance

EIJKMAN INSTITUTE
 Available antimalarial drugs
cinchona alkaloids quinine
4-aminoquinolines chloroquine, amodiaquine
8-aminoquinolines primaquine, tafenoquine
4-quinoline methanols mefloquine
9-phenanthrene methanols halofantrine

antifolic drugs pyrimethamine, proguanil

sulfa drugs a) sulphones; dapsone

b) sulphonamides; sulfadoxine

sesquiterpene lactones a) artemisinin

b) artemisinin derivatives, artemether
antibiotics tetracycline, chloramphenicol, azithromycin,
clindamycin, rifampicin
drug combinations pyrimethamine + sulfadoxine ('Fansidar'),
atovaquone + proguanil ('Malarone'), ACT

EIJKMAN INSTITUTE
Antimalarial drug targets in the malarial parasite

FV Ct

FV

EIJKMAN INSTITUTE
 Mechanisms of Antimalarial drug resistance

Up to 1980, little is known about:

How does the parasite resist treatment?

Advance in Molecular Parasitology:

• Drug transport alteration
Quinoline antimalarials, sesquiterpene lactones

• Binding affinity alteration

Antifolate, sulfa drugs, Coenzyme Q analogues etc.

EIJKMAN INSTITUTE
 Molecular basis of parasite resistance to quinoline
antimalarials

¤ Resistant-parasites accumulate less chloroquine

¤ Resistance is reversed by Ca-channel blockers (similar to multi-

drug resistance in cancer cells, mdr gene: ATP-dependent drug effluxer)

¤ Identification of P-glycoprotein homologue (PfPgh1) in the

malaria parasite that is encoded pfmdr1 gene (Foote et al, 1989)

¤ Identification of pfcrt gene and its product, pfCRT, a transporter

protein localized to food vacuole (Fidock et al, 2000)

¤ Multifactorial mechanisms?

EIJKMAN INSTITUTE
Polymorphisms in pfmdr1 gene associated
with chloroquine resistance

Chromosome 5, Pfmdr1 codes for P-glycoprotein homologues 1 (Pgh1)

Pfmdr1 gene

Restriction enzymes D1246Y

AflIII and ApoI for N86T S1034C
N86Y Y184F
DdeI for 1034C N1042D
AseI for 1042D
EcoRV for D1246Y

• Allelic replacement experiment (Reed et al, 2000)

184F, 1034C, 1042D and 1246Y confers chloroquine resistance
and modulate sensitivity to Quinine, Mefloquine, Halofantrin,
and Artemisinin

EIJKMAN INSTITUTE
Polymorphisms in the pfcrt gene associated
with chloroquine resistance

Chromosome 7; pfcrt gene codes for Chloroquine Transporter Protein (CRT)

pfCRT pfcg9 pfcg1

pfcg4 pfcg3

2kb

M74I, N75I, K76T, A220S, Q271E, N326S, I356T and R371I

The roles of pfcrt in chloroquine resistance were not proven

in P. vivax and P. chabaudi

EIJKMAN INSTITUTE
 Mechanisms of actions of Antifolate
and sulpha drugs

• Pyrimethamine and cycloguanil inhibit dihydrofolate

reductase (DHFR) enzyme through competitive binding
(Proven by genetic crossing and has been used as selectable genetic marker
in transfection)

• Sulfadoxine and sulfone act through antagonistic effect

with PABA, to inhibit dihydropteroate synthase (DHPS)
enzyme

EIJKMAN INSTITUTE
Folate Biosynthesis in the Malaria parasites
GTP
2 H2O
GTP cyclohydrolase
formate
Dihydroneopterin trophosphate
Dihydroneopterin triphosphate Pyrophospate
pyrophosphohydrolase
Dihydropterin phosphate
Dihydroneopterin monophosphate dephosphorylase
Phosphate

2-amino-4-hydroxy-6-(D-erythro trihydroxypropyl dihydropteridine

Dihydroneopterin aldolase
hydroxyacetaldehyde

2-amino-4-hydroxy-6-hydroxymethyl- 7,8-dihydropteridin diphosphate

ATP 6-hydroxymethyl- pterin-6-aldehyde
7,8-dihydropterin pyrophosphokinase
AMP PABG Pteridine-6-
2-amino-4-hydroxy-6-hydroxymethyl- 7,8-dihydropteridin diphosphate methylhydrolase

PABA Dihydropteroate SULFONE /

synthase SULFONAMIDE folate
pyrophosphate
7,8-dihydropteroate
ATP
L-glutamate Dihydrofolate synthase

ADP-Phosphate
7,8-dihydrofolate
NADPH Dihydrofolate PYRIMETHAMINE
Reductase CYCLOGUANIL
NADP
tetrahydrofolate
 PCR-RFLP method to detect mutant alleles of dhps gene

Chromosome 8, bifunctional pppk-dhps gene

nt1784
pppk

dhps
PfF3717 PfR199 PfR186

PfJR82 PfRJR83

PfFJR84 PfRJR85

PfF153 Pf165R

L L’
RFLP analysis
MspA1I for S436F/A
Ava II for A437G
Fok I for K540E
BstU1I and Bsl1 for A581G
437G is the most common allele
MwoI for A613S/T

EIJKMAN INSTITUTE
 PCR-RFLP method to detect mutant alleles of dhfr gene

Chromosome 4, bifunctional dhfr-ts gene

1875 bp

DHFR Junction TS

JR76 JR77
JR78 JR79 Restriction enzymes
NlaIII for 16Val
Tai I and Mae II for 50Arg
Tsp5091 for 51Ile
XmnI for 59Arg
BsrI for 108Asn, ScrfI for 108Thr
Alu I for Ser108
Allele 108N is the most common allele
Alleles 108T+16V confers resistance to Cycloguanil
but retains sensitivity to Pyrimethamin
Molecular Analyses of Plasmodium falciparum isolates

Minahasa
(North Sulawesi)

Kutai (East Kalimantan)

Nias (North Sumatera)
Armopa (West Papua)

Mamuju (South Sulawesi) Genyem

Hanura (Lampung)
Purworejo Flores (East Nusa Tenggara)
Kokap (Central Java)

EIJKMAN INSTITUTE
 Frequency distribution of mutant alleles of the genes
associated with chloroquine resistance among
P. falciparum isolates in Indonesia
Number % of mutant alleles
Area of
pfmdr1 pfCRT
samples
86Y 1034C 1042D 1246Y 76T
Kokap 35 100 (32) 0(32) 0 (32) 0 (24) 94.7 (19)
Purworejo 111 92 (111) 0 (111) 4.5 (111) 0 (111) 99.1 (111)
Hanura 48 100 (33) 0 (33) 0 (33) 0 (33) 100 (25)
Nias 20 100 (19) 0(19) 0 (20) 0 (19) 100 (19)
Kutai 28 100 (28) 0 (19) 5.2 (19) 0 (19) 100 (19)
Mamuju 25 62.5 (16) 0 (22) 13.6 (22) 0 (24) 100 (16)
Minahasa 41 94.7 (19) 0 (24) 13 (23) 0 (24) 94.7 (19)
Armopa 21 26.6 (15) 0 (17) 0 (15) 0 (17) 88.2 (17)
Genyem 93 27.3 (93) 0 (93) 19.3 (93) 0 (93) 89.1 (93)
Flores 21 16.6 (19) 0 (19) 89.4 (19) 0 (19) 100 (19)
Numbers in parentheses indicate number of samples that yield PCR products for analysis

EIJKMAN INSTITUTE
 Frequency distribution of mutations in dhfr gene of
P. falciparum isolates in Indonesia

Area Number % of mutant alleles

of 16V 50R 51I 59R 108N 108T 164L
samples
Kokap 35 33.3 (27) 0 (27) 0 (27) 33.3 (27) 40.7 (27) 66.6 (27) 0 (27)
Purwore jo 111 29.3 (111) 0 (111) 0 (111) 46.2 (111) 51 (111) 27.4 (111) 0 (111)
Hanura 48 0 (32) 0 (32) 0 (32) 62.5 (32) 78.1 (32) 34.3 (32) 0 (32)
Nias 20 0 (17) 0 (17) 0 (17) 75 (16) 94.1 (17) 0 (17) 0 (17)
Kutai 28 0 (18) 0 (18) 0 (18) 0 (18) 55.5 (18) 5 (18) 0 (18)
Mamuju 25 9 (22) 0 (22) 0 (22) 27.2 (22) 27.2 (22) 72.7 (22) 0 (22)
Minahasa 41 0 (20) 0 (20) 0 (20) 10 (20) 55 (20) 75 (20) 0 (20)
Armopa 21 0 (19) 0 (19) 0 (19) 68.4 (19) 68.4 (19) 0 (19) 0 (19)
Genyem 93 0 (93) 0 (93) 0 (93) 58.2 (93) 78.7 (93) 0 (93) 0 (93)
Flores 21 10 (20) 0 (21) 0 (21) 35.2 (17) 0 (20) 90 (20) 0 (20)
Numbers in parentheses indicate number of samples that yielded PCR products for analysis

EIJKMAN INSTITUTE
 Frequency distribution of mutations in dhps gene
among P. falciparum isolates in Indonesia

Area Number % of mutant alleles

of 436F/A 437G 540E 581G 613S/T
samples
Kokap 35 0 (28) 3.57 (28) 3.57 (28) 0 (28) 0(28)
Purworejo 111 0 (111) 35.3 (111) 26.5 (111) 0 (111) 0 (111)
Hanura 48 0 (48) 20.8 (48) 0 (48) 0(48) 0(48)
Nias 20 0 (20) 0(20) 0 (20) 0(20) 0(20)
Kutai 28 0 (28) 17.8 (28) 0 (28) 0(28) 0(28)
Mamuju 25 0 (25) 4 (25) 0 (25) 0(25) 0(25)
Minahasa 41 0 (40) 22.5 (40) 2.5 (40) 0(40) 0(40)
Armopa 21 0(21) 23.8 (21) 4.7 (21) 0(21) 0(21)
Genyem 93 0 (93) 25 (93) 21.7 (93) 0 (93) 0 (93)
Flores 21 0 (20) 0 (20) 0 (20) 0 (20) 0 (20)
Numbers in parentheses indicate number of samples that yielded PCR products for analysis

EIJKMAN INSTITUTE
Results of Molecular Analysis (2004)
• The pfmdr1 86Y and the pfcrt 76T have been fixed in all
of the P. falciparum isolates examined

• The frequency distribution of isolates carrying double

mutants 108N+59R increases significantly (52% to 90%)

• The frequency distribution of allele 437G of the dhps gene

increases significantly

• Isolates carrying atovaquone-resistant alleles were not

found in Indonesia

EIJKMAN INSTITUTE
Collaborating Institutions
Eijkman Institute for Molecular Biology, Jakarta

Din Syafruddin
Josephine E Siregar
Puji BS Asih
Irsan Saleh
Sangkot Marzuki
National Institute of Health, Research and Development,
The Ministry of Health (LITBANGKES)/United States Naval Medical
Research Unit II (NAMRU-II), Jakarta
Sekartuti
Rita Marleta
Emiliana Tjitra
Jason Maguire
J. Kevin Baird

The Walter and Eliza Hall Institute of Medical Research,

Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia

Nagesha Hadya, Gerard Casey, Michael Duffy, John Reeder and Alan F Cowman
EIJKMAN INSTITUTE
Pharmacogenomics:
an overview

Dr. dr. Mgs. Irsan Saleh, M.Biomed

Dept of Pharmacology
Faculty of Medicine Sriwijaya University
irsan_saleh_hasani@yahoo.com
 Interindividual variability in drug response
Disease Drug Class Rate response (%)
Asthma β-agonists, others 25-60
Solid cancer various 0-30
Depression SSRIs, tryciclic, others 60-80
Diabetes Sulfonylurea, others 25-50
Arthritis NSAIDs, COX-2 50-80
inhibitors, others
Migraine Triptan, NSAIDs, ergot 40-70
Schizoprenia Various 25-75
Major drug Various 2 million hospitalized
toxicity patients/y, 4th-6th leading
cause of death in US
1994
 Factors contributing to interindividual
variability in drug response
• Age
• Environmental
• Weight
• Gender
• Concomitant Diseases
• Concomitant Drugs
• Social factors
• Genetics
 Definition

Pharmacogenomics

Pharmacon Genome

The chemical substance that The entire DNA content of a

influence biological function at cell, including all of genes and
molecular, cell or organs of all of the intergenic regions.
organism.

The study of genome-derived data, including human genetic

variation, RNA and protein expression differences, to predict drug
response in individual patients or groups of patients.
Other definition

• The application of genome science

(genomics) to the study of human
variability in drug response.
• The study of genetic content (DNA
sequence) of humans for drug discovery &
optimization.
• Refer to the general study of all many
different genes that determine drug
behavior.
 History

1. Vision and some predictive observations

• Garrod (1902) : alcaptonuria and phenylketonuria due to biochemical
individuality.
• Snyder (1932) : a heritable disability to taste phenylthiocarbamide.
• Savin & Glick (1943) : a genetic lack atropine esterase in some rabbits.
 History (cont. …)

2. Pharmacogenetics lives: systemic case studies

• 1950 : several observation indicated clearly the

dependence drug effects on the genetic constitution.
• Variation in isoniazid acetylation
• Variation in cholinesterase activity
• Hemolysis cused deficiency G-6-PD
• Motulsky (1957) : Drug reaction, enzymes and
biochemical genetics
• Vogel (1959) : coined the Pharmacogenetics
• Kalow (1962) : summarized all available knowledge in a
book
 History (cont. …)
3. Broadening of pharmacogenetic knowledge
• Manya centers contributed new data, but data
represented monogenic variations.
• Weber’s book (1997) : listed 15 variable metabolizing
enzymes, 11 variable drug receptors, 14 other variable
• Kalow (2001) : counted 42 variable drug-metabolizing
enzymes
• The enzyme’s variation are complex: mutations,
spicing defects, gene deletion, present or stop codon.
• Many clinical case study due to enzyme’s variation
reported and genetic failure of drug-metabolizing
enzyme’s can lead to a patient’s death.
 History (cont. …)
4. Pharmacogenetic differences between population
• Paskind (1921): atropine sulfat caused a initial slowing of
heart rate in Caucasian (20) but not African-American
subjects.
• Chen & Poth (1929): variation in pupillary size after
applying mydriatic eye drop (increase largest in
Caucasian, intermediate in Asians and smallest in African
American).
• Sunahara et al. (1961): Genetical and geographical
studies in isoniazid inactivation.
• Beutler (1993): primaquine caused hemolysis on African
soldiers due to deficiency G6PD
• Kalow (2001): 11 mutations of CYP2D6 was tested :
European carried 7, Chinese 4, Japanese 3 and Africans
2.
 History (cont. …)

5. The rise of multifactorial pharmacogenetics

• Differences between people in their response to

drugs are regular occurrences.
• The causes of most differences generally remain
uninvestigated, but the presence of both genetic and
environmental cause is common.
• It is of considerable interest to know the relative
contribution of the two causes.
• Kalow et al. (1999; 1998) : assess the genetic
component in pharmacological variability
 History (cont. …)

6. Advances in molecular biology

1967 Gilbert discovered DNA ligase

1972-73 DNA cloning techniques established by
Boyer, Cohen and Berg
1988 The first biothechnology products appear.
These were tissue plaminogen factor, α-
interferon, human insulin, human growth
factors and erythropoetin.
1990 Human genome project was initiated and
finished at 2003
 Pharmacogenomics

DRUG
DRUG DRUG
METABOLIZING
TARGETS TRANSPORTERS
ENZYMES

PHARMACODYNAMICS PHARMACOKINETICS

Variability in
Efficacy/Toxicity
Johnson JA. Trends in Genetics 2003: 660-666
Drug Transporter
 Drug Transporter (cont. …)

1. Drug transporters such as ABCB1, ABCC1, ABCC2,

ABCC3 participate in opioid transport and influence
opioid efficacy and side effects.
2. Substrates of ABCB1/MDR1 include morphine,
methadone, fentanyl, sufentanil, alfentanil, and
morphine-6-glucuronide and anticancer
3. Other transporters potentially involved in opioid
distribution are MRP1, MRP2, and MRP3 (ABC
transporter subfamily C), organic anion transporters
(OAT1 and 3), and organic anion transporter
polypeptides (OATP1 and 2, solute carrier family 21).
Drug Metabolizing Enzymes
 Examples of Drug Metabolism Pharmacogenomics

NEJM 2003; 348: 529-537

 Examples of Drug Metabolism Pharmacogenomics

NEJM 2003; 348: 529-537

Examples of Drug Target Pharmacogenomics

Evans WE. NEJM 2003; 348:538-48

 Beta-blockers and
Hypertension (HTN)
• HTN is the most prevalent chronic disease in the
US and a contributor to morbidity and mortality
• Beta-blockers are first-line agent in the treatment
of HTN
• Marked variability in response to beta-blockers
– 30-60% of patients fail to achieve adequate blood
pressure lowering with beta-blockers
• Common beta-blockers used in HTN:
– Metoprolol
– Atenolol
 Beta-1 Adrenergic Receptor

Codon 49 SerGly
Codon 389
ArgGly

Podlowski, et al. J Mol Med 2000;78:90.

Beta-1 Receptor Polymorphisms and Response
to Metoprolol

Johnson JA et al. Clin Pharmacol Ther 2003; 74:44-52

 Beta-2 Adrenergic Receptor Polymorphisms
and Response to Albuterol in Asthma

• Hyperreactivity of the airways is the hallmark of

asthma
• Airway smooth muscle contains beta-2 receptors
that produce broncodilation
• Albuterol is a beta-2 agonist that is used in the
treatment of asthma
– Produces smooth muscle cell relaxation and
bronchodilation
• Forced expiratory volume in 1 second (FEV1)
– Phenotypic measure of response
 Beta-2 Polymorphisms and
Response to Albuterol
•Single 8 mg albuterol dose

•Albuterol-evoked increases
in FEV1 were higher and
more rapid in Arg16
homozyotes compared with
Gly carriers

• Codon 16 polymorphism is
a determinant of
bronchodilator response to
albuterol

Lima JJ et al. Clin Pharmacol

Ther 1999; 65: 519-25
Lima JJ. Clin Pharmacol Ther 1999; 65:519-25
DISTRIBUTION AND PREVALENCE
OF CYP2C19 POLYMORPHISM
THAT UNDERLIE PMs PHENOTYPE
IN MALAY POPULATION

Dr. dr. Mgs. Irsan Saleh, M.Biomed

Dept. of Pharmacology Faculty of Medicine
Sriwijaya University

Dipresentasikan pada Kongres Nasional Ikatan Ahli Farmakologi XIII 2010 di Yogyakarta
 Introduction :
 Individual variation in response to drugs is a
major problem in clinical practice and in drug
development.
 Genetic variants determine individual drugs
response.
 Polymorphism of the genes encoded CYP 450
enzyme affecting drug response, such as
CYP2C19 polymorphism
 Genetic Background of
Drug Metabolism
 CYP2C19 is cytochrome P-450 subfamily
responsible for the metabolism of drugs such
as anticonvulsant (S)-mephenytoin,
omeprazole, certain barbiturates, diazepam,
propranolol, proguanil & imipramine.
 Genetic polymorphism study, individuals can
be characterized phenotypically as ‘extensive
metabolizers’ (EMs) and ‘poor metabolizers’
(PMs).
PHARMACEUTICAL SUBSTRATES
OF CYP2C19

Drug Reference
Amitriptyline Melstrom et al, 1988
Barbiturates Adedoyin et al, 1994
Chlorproguanil Wright et al, 1995
Citalopram Sindrup et al, 1993
Clomipramine Nielsen et al, 1994
Diazepam Bertillson et al, 1989
Imipramine Haefeli, et al, 1990
Mephenytoin de Morias et al, 1994
Omeprazole Anderson et al, 1992
Proguanil Andersson et al, 1990
Propranolol Ward et al, 1989
 The poor metabolizer phenotype has
been shown resulting from variations in
the gene encoding CYP2C19.
 Two single base pair mutations of
CYP2C19; G681A in exon 5 (CYP2C19*2
or CYP2C19m1) and G636A in exon 4
(CYP2C19*3 or CYP2C19m2) have been
identified
POLYGENIC DETERMINANTS OF DRUG EFFECTS

Drug Metabolism Genotypes

100

m/m
Drug Concentration (%)

80

60 wt/m

40

wt/wt
20

0
0 5 10 15 20 25

Time (hours)
Frequency and Distribution
 The impact of genetic background to drug
prescriptions :
- medicine
- socio-economic
 Frequency of PMs has been reported:
# 3-6 % in Caucasians and Africans
# 13-23 % in Asians
# 61 % in Vanuatu population (Akira et al.)
 Methods

 CYP2C19 genotyping has been done by PCR-

RFLP methods.
 To detect CYP2C19m1, exon 5 was amplified &
digested by SmaI. Exon 4 was amplified &
digested with BamHI, to detect CYP2C19m2.

 The strategy (see next slide)

Distribution of EM and PM in
Malay population
56 %
60

50

40

24 % 20 %
30

20

10

0
hEM hEM hEM
 INTERETHNIC VARIATIONS OF
CYP2C19 GENOTYPE in
Indonesian population
Melayu
70
Jawa-Jepara
60
Sunda
50 Dayak
Bugis
40
Kajang
30

20

10

0
hmEM htEM PM
 INTERETHNIC VARIATIONS OF
CYP2C19 GENOTYPE
Caucasian
100 African
90 China
Korea
80
Japan
70 Philippines
60 Indonesia
50 Vanuatu
40
30
20
10
0
hmEM htEM PM
 CONCLUSION
 The prevalence of hmEMs, htEMs and
hmPMs in Malay populations were
found 24%, 56% and 20%
 Interethnic variation of CYP2C19 in
Indonesian population need further
exploration.
"Here's my
sequence..."

The New Yorker

 DNA fingerprinting was developed in 1984
by Alec. J. Jeffrey at the University of
Leicester
 He was studying the gene of myoglobin.

Alec. J. Jeffrey
 The chemical structure of everyone's DNA is the
same.
 The only difference between people (or any
animal) is the order of the base pairs.
 The information contained in DNA is determined
primarily by the sequence of letters along the
zipper.

Structure of DNA
 segments that vary
in size and
composition and
have no apparent
function are called
minisatellites

The different sequences is the same as the word "POST" has a different
meaning from "STOP" or "POTS," even though they use the same letters. i
 Using these sequences, every person could be
identified solely by the sequence of their base pairs
 there are so many millions of base pairs, the task
would be very time-consuming
 Instead, scientists are able to use a shorter
method, because of repeating patterns in DNA.
 These patterns do not, however, give an individual
"fingerprint,"
 they are able to determine whether two DNA
samples are from the same person, related people, or
non-related people.
 On some human chromosomes, a short sequence of
DNA has been repeated a number of times.
 The repeat number may vary from one to thirty
repeats
 These repeat regions are usually bounded by
specific restriction enzyme sites
 Cut out the segment of the chromosome containing
this variable number of tandem repeats (VNTR's )
 Iidentify the VNTR's for the DNA sequence of the
repeat.
1.Paternity and Maternity
 person inherits his or her VNTRs from his or her
parents
 Parent-child VNTR pattern analysis has been used
to solve standard father-identification cases

Can someone tell me who is my father?

2. Criminal Identification and Forensics DNA isolated
from blood, hair, skin cells, or other genetic
evidence left at the scene of a crime can be
compared
FBI and police labs around
the U.S. have begun to use
DNA fingerprints to link suspects
to biological evidence –
blood or semen stains, hair,
or items of clothing
3. Personal Identification.
 The notion of using DNA fingerprints as a sort of genetic
bar code to identify individuals has been discussed
4.Diagnosis of Inherited Disorders
 Diagnose inherited disorders in both prenatal and
newborn babies
 These disorders may include cystic fibrosis, hemophilia,
Huntington's disease, familial Alzheimer's, sickle cell
anemia, thalassemia, and many others.
A Method of Forensic Identification
 The primary method of assessing similarities
is by use of DNA fingerprinting or DNA
restriction analysis.
 This process makes use of special proteins
called restriction enzymes and sections of the
chromosome called tandem repeats
 A region of the chromosome that contains
multiple copies of a core DNA sequence that
are arranged in a repeating fashion
 Repeats act as fillers or spacers between
coded sections of DNA
 All humans have the same type of repeats but
there is tremendous variation in the number
of repeats that each of us has.
 Restriction enzymes cut DNA but only at a
certain combination of A, G, T, and C.
 Different restriction enzymes cut DNA at
different places—each has a unique sequence
it recognizes.
 The restriction enzyme EcoRI cuts DNA at the
sequence GAATTC and will cut only at that
sequence.
 RFLPs are different fragment lengths of base
pairs that result from cutting a DNA molecule
with a restriction enzyme
 It is the length differences associated with
DNA strands or RFLPs that allow one to
distinguish one person from another.
Recall: EcoR I cuts only at GAATTC

 EcoR I cuts a similar

section of DNA on Bob,
Larry, and Mary
 After the cut how many
fragments Bob, Larry, and
Mary have?
 Answer: 2, 3, and none
 After the DNA is cut with EcoR I, Bob’s, Larry’s and Mary’s
fragments are placed in different lanes on an agarose gel
 The fragments are then subjected to an electric field
 The smaller fragments move faster, the larger ones move
slower
 This process of separating the fragments by length is called
electrophoresis.
 The bigger
fragments are near
the top
 In general the child’s
DNA must be a
combination of Mary’s
DNA and one of the
men. Which man is the
father?
 Answer: Larry
 Merci Syukron
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