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High Frequency of CYP3A4*1B among Opiate Dependent Patients in
Nasir Mohamad1,2*, Nurfadhlina M2, Nazila T2, Ahmad A2, Nor Hidayah Abu Bakar3, Hussein H4, Khafidz I5 and Ismail R2,6
1Department of Emergency Medicine, School of Medical Sciences, Universiti Sains Malaysia, Kelantan, Malaysia
2Pharmacogenetics Research Group, Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Kelantan, Malaysia
3Department of Pathology, Hospital Raja Perempuan Zainab II, Kelantan, Malaysia
4Department of Psychological Medicine, Universiti of Malaya, Kuala Lumpur, Malaysia
5Klinik Dr Khafidz, 43000 Semenyih Bandar Teknologi Kajang, Selangor, Malaysia
6Pharmacogenetics Research Group, Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Kelantan, Malaysia
Department of Emergency
Medicine/Institute for Research in Molecular Medicine (INFORMM), School of
Health Campus, Universiti Sains Malaysia
Kerian, Kelantan, Malaysia Tel: 609-7672407 Fax: 609-7657267 E-mail:
Received July 14, 2012; Accepted July 24, 2012; Published July 27, 2012
Citation: Mohamad N, Nurfadhlina M, Nazila T, Ahmad A, Abu Bakar NH, et al.
(2012) High Frequency of CYP3A4*1B among Opiate Dependent Patients in
Malaysia. J Addict Res Ther 3:130.
The sharing of injection needles among drug user is a leading cause for the spread of HIV/AIDS. Malaysia introduced
methadone as a management of heroin dependents to reduce HIV spread. Methadone has variable pharmacokinetics
and CYP3A4 has been implicated in its metabolism. The objective of this study therefore was to determine if
polymorphisms exist with CYP3A4 among opiate users in Malaysia. This study was approved by Ethics Committees at
University of Malaya and Universiti Sains Malaysia. Control subjects comprised blood donors, students and residents
of a village. Opiate-dependents were from methadone clinics and drop-in centers. They signed a written-informed
consent to participate and gave blood for DNA CYP3A4 genotyping. DNA was extracted using QIAgen DNA mini kit. A
nested two-step allele specific PCR method was developed to detect CYP3A4*1B, CYP3A4*3, CYP3A4*4, CYP3A4*5,
CYP3A4*6, CYP3A4*7, CYP3A4*8, CYP3A4*9, CYP3A4*10, CYP3A4*11, CYP3A4*12, CYP3A4*13, CYP3A4*14,
CYP3A4*15 and CYP3A4*16. Normal controls comprised Malays, Chinese and Indians but opiate-dependent subjects
were majority Malay males. Control subjects all carried the wild-type gene. Mutant CYP3A4*1B allele was found in
2.17% of opiate-dependent subjects. Our results revealed that CYP3A4 was not polymorphic among Malaysian Malays,
Chinese and Indians who were not opiate-dependent. To date, we are not aware of any study to associate CYP3A4
polymorphism and heroin addiction. It is conceivable that altered CYP3A4 function may contribute towards addiction
liabilities in subsets of individuals. We conclude that CYP3A4 is polymorphic among heroin-dependent individuals.
The mutation, CYP3A4*1B is not silent. This may have implications on heroin addiction liability as well as on dose
requirements for MMT and HAART.
Heroin use and the sharing of injection needles among drug
users is a leading cause for the spread of HIV/AIDS in Malaysia and
several other South East Asian countries. In 2006, Malaysia introduced
methadone as a management of heroin dependents in an effort to break
the viscous cycles of heroin addiction and HIV/AIDS. Methadone is
taken orally and it prevents withdrawal and reduces illicit drug use.
It is a vital public health strategy for HIV/AIDS risk reduction .
Variability in methadone clearance, susceptibility to drug interactions,
and a long elimination half-life can however be major impediments
to optimal methadone use [2,3]. In in vitro drug metabolism studies,
CYP3A4 has been implicated in methadone metabolism . Authors
have suggested dosing guidelines for methadone and warned about the
potential for CYP3A4-mediated interactions and suggested the need to
adjust doses accordingly [2,3,5-11].
CYP3A4 is the most abundant CYPs in human. Substrates of
CYP3A4 include methadone, anti-depressants, immunosuppressive
agents, macrolide antibiotics, benzodiazepines, calcium channel
blockers , and several antiretroviral [4,13,14] used in HIV/AIDS.
Also importantly, CYP3A4 is involved in the metabolism of endogenous
substances that include testosterone , progesterone , cortisol
, and 17β-estradiol , and this may be important in the patho
physiology of diseases including drug dependence. CYP3A4 exhibits
genetic polymorphism and as with other polymorphic enzymes, the
identification of molecular variants in the CYP3A4 gene is a major
focus of pharmaco genetic studies.
Malaysia is a multiethnic country where genetic polymorphisms of several drug metabolizing enzymes have been previously described
[19-26]. Although our earlier studies failed to detect polymorphism
at the CY3A4 locus, mutant alleles have recently been described in
Malaysia . The objective of this study therefore was to determine
if polymorphisms exist with CYP3A4 among opiate users in Malaysia
given its role in endogenous metabolism and in the metabolism of
Recruitment of subjects
The protocols for this study received the approval of the Ethics
Committees at the University of Malaya in Kuala Lumpur and Universiti
Sains Malaysia in Kelantan. For the normal controls, subjects comprised
blood donors at Universiti Malaysia Medical Centre, Universiti Sains
Malaysia Hospital, students at the two universities and residents of a
Malaysian Indian community in Kuala Krai, Kelantan. For opiate-dependent individuals, subjects were enrolled from several methadone
clinics and drop-in centres for drug users in Kuala Lumpur and in
Kota Bharu, Kelantan. They were given an explanation about the study
and were invited to participate if they were willing to sign a writteninformed
consent. They were administered standard questionnaires
to obtain demographic data and to establish opiate dependence. Five
milliliter of blood was then obtained from them for DNA extraction
and CYP3A4 genotyping.
Isolation of DNA and PCR genotyping
Genomic DNA was extracted from subjects’ blood by using
QIAgen DNA mini kit (QIAGEN, Hilden) according to the protocols
recommended by the manufacturer. The quantity and quality of the extracted DNA was determined on the spectrophotometer with
measurements done at 260 and 280 nm.
A nested two-step allele specific PCR method was developed to
detect CYP3A4*1B, CYP3A4*3, CYP3A4*4, CYP3A4*5, CYP3A4*6,
CYP3A4*7, CYP3A4*8, CYP3A4*9, CYP3A4*10, CYP3A4*11,
CYP3A4*12, CYP3A4*13, CYP3A4*14, CYP3A4*15 and CYP3A4*16.
The first PCR used the primers listed in Table 1 to isolate the CYP3A4
gene to improve on specificity. The products were used as templates for
three allele specific second PCR using primers also listed in Table 1 to
detect the alleles of interest. All PCR reactions were performed using
Bio-Rad MyCycler™ Thermal Cycler.
Table 1:Primer sequences used in nested two-step allele specific PCR of CYP3A4
In the first PCR, the reaction mixture consisted of 1X Biotools
Reaction Buffer, 3.0 mM Biotools MgCl2, 0.2 mM of each Biotools dNTP
Mix, 0.2 pmol of each primer which were divided into two groups;
Set A and Set B as listed in Table 4, 1.0 U Biotools DNA Polymerase,
and 20-100 ng of DNA in a total volume of 25 μL. The cycle condition
consisted of pre-denaturation for 5 min at 94°C, followed by 10 cycles of
denaturation at 94°C for 45 s, annealing at 65°C for 45 s with touchdown
from 65°C to 60.5°C (reduce by 0.5°C every cyle) and extension at 72°C
for 30 s. This was followed by another 25 cycles of denaturation at 94°C
for 45 s, annealing at 60°C for 45 s and extension at 72°C for 30 s. A final
extension 72°C for 5 min was also included.
Subsequent to successful first PCR, the products were subjected
to two parallel allele-specific second PCR, one with wild-type specific
primers and the other with mutation-specific primers, were carried
out in separate PCR reactions. In this second PCR, five set of reaction
mixtures and cycle condition were required to detect all the alleles as
listed in Table 2 and Table 3. The predicted PCR products as listed in
Table 4 were analyzed on 2.5 % agarose gel in 1X TBE buffer run at 90
V for 75 min.
Table 2:Reaction mixtures and cycle condition for allele-specific second PCR.
Table 3:Final concentration of each primers used for each Set in allele-specific
Table 4:Primer combinations for amplification of first and allele-specific second
PCR of CYP3A4.
Upon successful PCR, three heterozygous samples were sent for
sequencing. The PCR products were purified using QIAquick PCR
Purification kit before being sent for DNA sequencing by standard kit
of ABI PRISM Big Dye Terminator. The sequencing results were verified against the published sequence for CYP3A4 (Gene bank accession
Control subjects comprised 270 Malays, 172 Chinese and 174
Indians and their average age are 29.4, 30.2, and 26.1 respectively. The
control subject consist of 174 males and 96 females for Malays, 132
males and 40 females for Chinese and 123 males and 51 females Indian.
Opiate dependent subjects comprised 114 Malays with average age of
39.57. Out of the 114 subjects, 112 are males and 2 are females.
Figure 1.1 shows a gel picture from a successful Set 1 for second
PCR for two representative samples from two different subjects.
CYP3A4*1B was detected in five subjects who carried heterozygous
CYP3A4*1A/CYP3A4*1B genotype. The remaining subjects did not
carry any mutant allele. The heterozygous subjects then were validated
using DNA sequencing and the chromatogram is shown in Figure
1.2. Double peaks was produced at position 262 which indicates the presence of heterozygous CYP3A4*1A/CYP3A4*1B genotype.
Figure 1:PCR products for amplification using primers in second PCR
(1.1) and sequencing chromatogram (1.2). In Figure 1.1, Lane 1is DNA
marker (100 bp) and Lane 2-3 are amplification of CYP3A4*8, CYP3A4*1B and CYP3A4*15 alleles respectively and (a) for wild type and (b) for mutant
type. Lane 2 had shown heterozygous which produce specific band for wild
and mutant type for SNP CYP3A4*1B. This corresponds with sequencing
chromatogram in Figure 1.2. The chromatogram had shown two peaks at
position 262 which indicates heterozygous for sample in lane 2.
The most common allele carried by subjects was CYP3A4*1A that
occurred at a frequency of 97.83%. CYP3A4*1B was found in 2.17%
of subjects and another fourteen alleles were not detected. In terms
of observed genotypes, 95.65 % were CYP3A4*1A/*1A and 4.35%
CYP3A4*1A/*1B. However in terms of predicted genotype frequency
by Hardy-Weinberg Law, 95.70 % were CYP3A4*1A/*1A, 4.25%
CYP3A4*1A/*1B and 0.05% CYP3A4*1B/*1B. Both observed and
predicted genotype frequency is shown in Table 5 with 95% confidence
Table 5:Genotype frequency according to Hardy-Weinberg equilibrium in opiatedependent
Malaysia is facing a double challenge of illicit drug use and a rapid
spread of HIV/AIDS. As a “harm-reducing” measure, the Malaysian
Government introduced Methadone Maintenance Therapy (MMT)
for heroin users in Malaysia. In our previous study in 52 Malay male
subjects given a single 8 mg dose of methadone, we found a wide
variability in its clearance, consistent with other studies previously
reported . The variability in our 52 subjects could not be explained
by polymorphisms at the CYP2D6 locus, a very polymorphic gene
in our population [19-21], another enzyme implicated in methadone
metabolism . This led us to investigate the genetic polymorphism
at the CYP3A4 locus among heroin drug users. Apart from being
involved in the metabolism of methadone, CYP3A4 is also involved
in the metabolism of several anti-retrovirals used in the treatment of
AIDS , thus our interest. We were also led to study this because
of the role of CYP3A4 in endogenous metabolism, especially involving
testosterone, as a possible contributing factor to heroin addiction.
Heroin addiction in Malaysia is a predominantly male disease. It would
therefore be interesting if an association can be found between altered
CYP3A4 testosterone metabolism and heroin addiction.
Our results revealed that CYP3A4 was not polymorphic among
Malaysian Malays, Chinese and Indians who were not opiate-dependent.
Many previous studies failed to show significant polymorphism at the
CYP3A4 locus. Rebbeck, et al. (1998) reported a 9.6% frequency for
CYP3A4*1B in healthy White subjects  but Lamba, et al. (2002)
reported a very low percentage among their healthy Caucassian, African
Americans, Mexican, Pacific Islander and Middle Eastern subjects .
Mutant alleles, with some exceptions, appeared to occur only at low
percentages. This probably implies an important role of CYP3A4 in
endogenous metabolism and therefore mutations would most likely
be selected out. It is assumed that evolutionary pressure acts against
coding polymorphisms and environment tends to wipe them out.
Heroin addiction is a complex disease. It probably results from
deleterious interactions between genes and the environment. Many
genes have been studied for potential roles in heroin addiction and to
date, we are not aware of any study to associate CYP3A4 polymorphism
and heroin addiction. It was interesting therefore to find a 2.17 % frequency for CYP3A4*1B among our opiate-dependent subjects, against
the background of an absence of polymorphism among Malaysian
non-drug dependent subjects. P450 enzymes in liver microsomes
have been reported to play important roles in the metabolism of
steroids, fatty acids, fat soluble enzymes and prostaglandins  and
CYP3A4 has been shown to be a major form that catabolised steroids
in human livers. It is conceivable that altered CYP3A4 function may
contribute towards addiction liabilities in subsets of individuals, either
directly through altered metabolism of some putative, yet unknown
endogenous substance(s) or indirectly through its effects on steroid and
CYP3A4*1B allele is also known as CYP3A4-V and has an A→G
substitution at position -392 on the 5’ promoter region. It was first
identified by Rebbeck et al. in 1998 . The functional significance
of CYP3A4*1B mutation has recently been reported. CYP3A4*1B/
CYP3A4*1B genotype has been reported to reduce the absorption of
indinavir in patients initiating Highly Active Anti-Retroviral Treatment
(HAART) naïve patients . This SNP has also been associated with
the more aggressive forms and advanced clinical stages of prostate
cancer in African-Americans but not in Portugese victims . It
also linked to increase transcriptional activity. The finding of a 2.17%
frequency for CYP3A4*1B among our heroin using population was
therefore interesting and the functional significance of this mutation
may need to be elucidated. Future studies on its association should also
probably include family data to allow for a transmission disequilibrium
test to be performed. CYP3A4 is known to be involved in endogenous
metabolism of steroid hormones, both testosterone (2β-, 6β-, or
15β-hydroxytestosterone) and estrogen (4- and 16α-hydroxylation)
[32-35]. As far as addiction is concerned, stress influences the
pathophysiology of addiction, to be linkages with drug dependence.
We conclude that CYP3A4 is polymorphic among heroindependent
individuals. The mutation, CYP3A4*1B is not silent. This
may have implications on heroin addiction liability as well as on dose
requirements for MMT and HAART.
This work was supported by a USM grant under the “Research University
Program” (Grant Number: 1001/PSK/8620014)
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