Professional Documents
Culture Documents
Genetic Polymorphisms Dan Its Role On Clinical Application
Genetic Polymorphisms Dan Its Role On Clinical Application
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
GENE Transcription
Primary RNA transcript
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
*
Exon1 Intron1 Exon2 Intron2 Exon3
Promotor
elements Polyadenilation
Splice sites
Initiator signal
5’ GT………AG 3’
codon (ATG)
Hb structure:
• Globin chain folding
• Heme binding
• 11, 22, 12, 21 contact
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
• ineffective erythropoiesis
• intramedullary destruction haemolysis in the
microcirculation (spleen)
HETEROZYGOTE HOMOZYGOTE o
HbE/HbE HbE/ o or +
E
E
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
2 2 1
2 2 1
--/-- - -/-
4=HbBart 4=HbH
EIJKMAN INSTITUTE
Available antimalarial drugs
cinchona alkaloids quinine
4-aminoquinolines chloroquine, amodiaquine
8-aminoquinolines primaquine, tafenoquine
4-quinoline methanols mefloquine
9-phenanthrene methanols halofantrine
EIJKMAN INSTITUTE
Antimalarial drug targets in the malarial parasite
FV Ct
FV
EIJKMAN INSTITUTE
Mechanisms of Antimalarial drug resistance
EIJKMAN INSTITUTE
Molecular basis of parasite resistance to quinoline
antimalarials
¤ Multifactorial mechanisms?
EIJKMAN INSTITUTE
Polymorphisms in pfmdr1 gene associated
with chloroquine resistance
Pfmdr1 gene
EIJKMAN INSTITUTE
Polymorphisms in the pfcrt gene associated
with chloroquine resistance
2kb
EIJKMAN INSTITUTE
Mechanisms of actions of Antifolate
and sulpha drugs
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
ADP-Phosphate
7,8-dihydrofolate
NADPH Dihydrofolate PYRIMETHAMINE
Reductase CYCLOGUANIL
NADP
tetrahydrofolate
PCR-RFLP method to detect mutant alleles of 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
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)
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
EIJKMAN INSTITUTE
Frequency distribution of mutations in dhps gene
among P. falciparum isolates in Indonesia
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
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
Nagesha Hadya, Gerard Casey, Michael Duffy, John Reeder and Alan F Cowman
EIJKMAN INSTITUTE
Pharmacogenomics:
an overview
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
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. …)
Codon 49 SerGly
Codon 389
ArgGly
•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
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
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
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..."
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