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FREQUENCY OF COMMON ABCB1 GENE VARIANT C3435T POLYMORPHISM IN PAKISTANI POPULATION.

Byline: Kulsoom Farhat, Akbar Waheed, Nusrat Nazir, Muhammad Ismail, Qaisar Mansoor and Anwar Kamal Pasha

ABSTRACT

Objective: To determine the frequency of the C3435T; a single nucleotide polymorphism of the ABCB1 gene for the first time in Pakistani population and compare it with the data available from other populations.

Study Design: A cross-sectional study.

Place and Duration of Study: Sampling was carried out at Combined Military Hospital, Rawalpindi from August 2012 to May 2013 and institute of Biomedical and Genetic Engineering (IBGE).

Material and Methods: The genotype frequency of C3435T polymorphism of ABCB1 gene was investigated in 491 Pakistani subjects. This frequency observed in our population was also compared with the published data on Asians and Caucasians.

Results: The distribution of frequencies of C3435T genotypes in our study population were; CC 7.1%, CT 59.1% and TT 33.8%. The Pakistanis differed significantly from Asian populations in the distribution of the TT genotype of C3435T ABCB1 (pless than 0.05). The study population also differ significantly in the distribution of CC genotype from rest of the populations compared (pless than 0.05).The data obtained may give the basis for predicting effects of drugs that are substrates for ABCB1 in Pakistani population and may be useful for individualized therapy of some diseases.

Keywords: ABCB1, Genotypes, Pakistani population, P-glycoprotein, Polymerase chain reaction, Polymorphism.

INTRODUCTION

A trans-membrane transporter-P- glycoprotein (P-gp) plays an important role in the efflux of drugs and thus influences the drug treatment outcome. Primarily being over expressed in tumor cells, it was found to be associated with resistance to chemotherapeutic agents1. Consequently, the localization of P-gp was identified at many sites in the body and it included blood-brain barrier, liver, intestine, kidneys, placenta, testes, salivary glands and other blood tissue barriers. Moreover P-gp has also been found in stem cells, mononuclear cells and macrophages, suggesting its physiological function over there too2. P-gp transports a wide range of substrates and thus plays a significant role in bioavailability, distribution and excretion of many drugs3. A difference in expression and function of P-gp has been identified responsible for inter- individual differences, and one reason for this difference is said to the genetic variability in the form of polymorphism.

Up till now over 60 single nuc leotide polymorphisms (SNPs) and 3 insertion/deletion polymorphisms naturally occurring in different populations have been reported in ABCB14.

In most of the studies done so far the functional impact of these SNPs on the P-gp remains unclear however some among these do have proved their functional relevance. And to be more specific, the C3435T- a silent but simultaneously a functional polymorphism has been studied repeatedly in different populations and various disease conditions5-8.

The frequencies of the homozygous and heterozygous genotypes of C3435T have been studied in various populations including Caucasians and Asians where a large difference has been revealed among these populations. We in our study have analyzed an existing silent but functional ABCB1 C3435T polymorphism in a Pakistani population.This is the first ever study from this area to estimate C3435T genotype frequencies in a Pakistani population.These frequencies were then also compared with those in other populations.

MATERIAL AND METHODS

It was a cross-sectional study that was conducted at Combined Military Hospital, Rawalpindi from August 2012 to May 2013. analysis was performed in accordance with the principles of the Declaration of Helsinki.The genomic DNA from whole blood was isolated using the standard organic methods9.

Table-1: Comparison of C3435T of Pakistani population with other populations.

###C3435T###References

Countries###CC###CT###TT

Pakistani (n= 491)###0.07###0.59###0.34###(This study)

Serbian (n = 158)###0.19###0.54###0.27###Milojkovic et al, 2011

German (n = 461)###0.21###0.50###0.29###Cascorbi et al, 2001

Russian (n = 290)###0.21###0.49###0.30###Gaikovitch et al, 2003

Portuguese (n =###0.12###0.47###0.41###Cavaco et al, 2003

100)

Turkish (n = 150)###0.20###0.53###0.27###Gumus-Akay et al,

###2008

Polish (n = 204)###0.22###0.51###0.27###Kurzawski et al, 2006

Czech (n = 189)###0.21###0.45###0.34###Pechandova et al,

###2006

UK (n = 190)###0.24###0.48###0.28###Ameyaw et al, 2001

Spanish (n = 204)###0.27###0.51###0.22###Vicente et al, 2008

Japanese (n = 154)###0.36###0.47###0.17###Komoto et al, 2006

Chinese (n = 200)###0.30###0.53###0.17###Zhang et al, 2008

Indian (n = 87)###0.18###0.37###0.45###(Chowbay et al, 2003)

Unrelated healthy volunteers both males (n=220) and females (n=271)between the ages of 18 to 65 years were included in the study through non-probability consecutive sampling. Subjects having any history of chronic diseases like cancer, hepatitis, HIV, cardiac and neural diseases or those receiving continuous medical treatment (substrates for P-gp) were excluded from the study. Consent was taken from all the subjects in writing. All the subjects were recruited in a way that they provided representation from all regions of Pakistan including Punjab, Sindh, Balochistan, Khyber Pakhtunkhwa, Hazara/Baltistan and Azad Jammu and Kashmir. This study was approved by Ethical Committee of Centre for Research in Experimental and Applied Medicine (CREAM), Army Medical College, Rawalpindi, Pakistan.A blood sample measuring to 5 ml was taken from all the subjects included in the study.

The analytical procedures were carried out at Institute of Biomedical and Genetic Engineering (IBGE), Islamabad. The genetic The genotyping for C3435T was made by PCR- RFLP. The amplification of DNA was carried out using forward and reverse primers for the region harboring the C3435T. The forward primers were: 5'- TGC AGG CTA TAG GTT CCA GG 3' and the reverse primers were: R5'- TTT AGT TTG ACT CAC CTT CCC G 3'. The PCR was then carried out. The digestion of PCR products were carried out with Mbo1 restriction enzyme10. There were three fragments of 172 bp, 60 bp,16bpin individuals homozygous for major allele C. There were four fragments of 248bp, 172 bp, 60 bp, 16b pin individuals heterozygous for both the major and minor allele T and C. There was a single fragment of 248 bp in individuals homozygous for minor allele T (Fig-1).

Microsoft excel was used for data analysis. Mean and standard deviation were calculated for age. Frequency and percentage were calculated for gender and genotype. Allele and genotype frequencies were calculated by direct counting. Chi square test was used to compare the observed genotype frequencies to published data for other populations. A p value of less than 0.05 was considered significant.

RESULTS

Genotyping of C3435T SNP of ABCB1 gene was assessed by PCR-RFLP in 491 Pakistani subjects with an average age of 42.67 years (SD=8.74). Two hundred and seventy one (55.2%) were females and 220 were males (44.8%). Figure 1 was the genotype results for ABCB1 C3435T polymorphisms. After digestion with MboI restriction enzyme the genotypes CC, CT and TT was classified into different band sizes.

Out of 491 subjects, 35 (7.1%) had CC genotype, 290 (59.1%) had CT and 166 (33.8%) had TT genotype. The comparison of C3435T of Pakistani population with other populations is given in Table-1 and Fig-2.

DISCUSSION

How one responds to the drug is said to be largely influenced by the genetic makeup and it is here that the role of polymorphism in human genome arises. These polymorphisms can alter the pharmacokinetics as well as dynamics of the drugs and thus can modify the response of the individuals to the drugs. The pharmacogenetic testing in this way can be quite useful in adjusting the therapy with the drugs that are the substrates of P-gp which is encoded by ABCB1. This dose adjustment will in turn increase the efficacy and safety of drugs.

Table-1 shows the comparison between the distribution of frequencies of CC, CT and TT genotypes in the Pakistani population and other populations; data being retrieved from previous studies. The frequencies of genotypes of C3435T polymorphism of ABCB1 vary among different population. The frequency of TT polymorphism reported in this study (0.34) is close to that found in the Serbian11 (0.27), German12 (0.29), Russian13 (0.30), Portuguese14 (0.41), Polish15 (0.27), Czech16 (0.34) and Caucasians of UK17 (0.28). The frequency distribution of TT genotype of 3435 in the present study is significantly different from that documented in the Spanish18 (0.22), Japanese19 (0.17), Chinese20 (0.17) and Indian21 (0.45) populations (pless than 0.05). This study has depicted that the frequency distributions of CT heterozygous variant were similar between the Pakistani (0.59), Serbian11 (0.54), Turkish22 (0.53), Polish15 (0.51), Spanish18 (0.51) and Chinese20 (0.53) populations.

The frequency of this genotype was statistically different from German12 (0.50), Russian13 (0.49), Portuguese14 (0.47), Czech16 (0.45), Caucasians of UK17 (0.48), Japanese19 (0.47) and Indian21 (0.37) populations (pless than 0.05). We (0.07) are much closer to Portuguese14 (0.12) in the distribution of the frequency of homozygous CC genotype. However this frequency in our population is significantly different from that reported in Serbian11 (0.19), German12 (0.21), Russian13 (0.21), Portuguese14 (0.12), Turkish22 (0.20), Polish15 (0.22), Czech16 (0.21), Caucasians of UK17 (0.24), Spanish18 (0.27).Japanese19 (0.36), Chinese20 (0.30) and Indian21 (0.18) population (pless than 0.05). As can be seen in Table-1, the frequency of TT genotype in comparison to Asian populations is statistically different. The wild-type CC genotype is significantly different from the rest of the populations compared.

To conclude we have put forward the frequency distribution of C3435Tgenotypes in a Pakistani population. We have observed significant difference in the genotype frequency compared to other populations. This study has provided a framework for future pharmacogenetic and pharmacokinetic studies on this polymorphic variant of ABCB1 gene in the Pakistani population. This will promote validation of more and more studies besides decreasing the economic costs and ethical risks for the individuals included in such studies. This knowledge of high frequency of functional polymorphisms in our population is also of prime importance to the practicing physicians, more specifically when they are prescribing drugs having a narrow therapeutic window. They will thus prevent the development of therapeutic failure and serious adverse drug reactions.

We can foresee that in the near future this routine testing for the SNPs will become a useful tool-adjusting the dose of the drugs that are substrates for P-gp.

CONFLICT OF INTEREST

This study has no conflict of interest to declare by any author.

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Publication:Pakistan Armed Forces Medical Journal
Geographic Code:9PAKI
Date:Jun 30, 2015
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