CYP 3 A Genotypes in Bangladeshi Tuberculosis Patients

The purpose of this study is to investigate the genotype and allelic frequencies of CYP3A in Bangladeshi Tuberculosis (TB) patients which may help for individualized drug dosing and improved therapeutics. Genotyping was done using the extracted genomic DNA from 90 TB patients followed by amplification of target alleles by Polymerase Chain Reaction (PCR). Amplified alleles were then digested by restriction enzymes followed by gel electrophoresis & sequencing to identify the targeted alleles namely CYP3A4*1B, CYP3A4*2, CYP3A4*4, CY3A4*5, CYP3A4*6, CYP3A4*10, CYP3A4*18, and CYP3A5*3. In TB patients, no samples were positive for CYP3A4*2, CYP3A4*4, CYP3A4*5, CYP3A4*6, CYP3A4*10, and CYP3A4*18 alleles. One sample was found to be heterozygous for CYP3A4*1B (1.11%). The wild homozygous (CYP3A5*1/*1) genotype frequency was 7.78%, the heterozygous (CYP3A5*1/*3) frequency was 42.22% and the homozygous mutant (CYP3A5*3/*3) frequency was 50% in Bangladeshi TB patients. The absence of the common polymorphic gene suggests that there will be no impact of CYP3A drug metabolizing enzymes on antituberculosis drugs.


Introduction
Genetic polymorphisms in the drug metabolizing enzymes (DMEs) can result in varied interindividual and interethnic pharmacological and toxicological responses upon exposure to therapeutics and environmental pollutants 1 .Interindividual variation in drug metabolism is caused by many factors including environmental factors, concurrent drug therapy as well as genetic factors 2 .Much of this variation, however, has shown to be caused by genetic polymorphisms of the human cytochrome P450 enzymes (CYP) 3 .The cytochrome P450 (CYP) 3A subfamily plays a major role in the oxidative, peroxidative, and reductive biotransformation reactions of 50-60% of all currently used drugs 4,5 , exogenous carcinogens, and endogenous substrates such as steroids 6,7 .The CYP3A subfamily composed of CYP3A4, CYP3A5, CYP3A7, and CYP3A43 in humans is of special importance because it accounts for as much as 30% of total liver cytochrome P450 content 8 .The most abundant CYP3A isoform in liver and intestine is CYP3A4.Its interindividual hepatic expression varies 60-fold 9 , and the in vivo function as assessed by clearance displays at least a 20-fold difference 10 .Induction by xenobiotics (e.g.rifampin) and endogenous compounds (e.g.steroid hormones) further modulates the variability of CYP3A4 expression among individuals 11 .Although the substrate specificity of CYP3A5 is similar to that of CYP3A4, CYP3A5 has been regarded to be less important for drug elimination because it is expressed at much lower levels than CYP3A4 12 .This variability in CYP3A expression and function explains why the intensity and duration of drug action and the occurrence of side effects show large patient-to-patient variability 11 .
To date, several polymorphic CYP3A isoforms have been described in different populations in particular the CYP3A4 and 3A5 isoforms [13][14][15] .The most common CYP3A4 variant reported so far, CYP3A4*1B, is an A-392G transition in the promoter region 16,17 .A number of rarer variants, mainly nonsynonymous polymorphisms, have also been reported 18 .
CYP3A5 expression is polymorphic with a variant allele of CYP3A5, CYP3A5*3, conferring low or undetectable CYP3A5 expression as a result of a single point mutation (6986A>G) 12,16 .However, carriers of at least one wild type CYP3A5*1 allele expresses CYP3A5 in the small intestine, liver and the kidneys 12,16,19 .
Tuberculosis (TB) is a common and often deadly infectious disease usually caused by Mycobacterium tuberculosis in humans.Tuberculosis is a major public health problem in Bangladesh 20 .Different drugs, namely isoniazid, rifampin, rifapentine, pyrazinamide, ethambutol (as first-line drugs), cycloserin, ethionamide, p-amino salicylic acid, streptomycin, capreomycin (as second-line drugs), are used for the treatment and management of tuberculosis 21 .Rifabutin (as firstline drugs) and levofloxacin, moxifloxacin, gatifloxacin, amikacin/kanamycin (as first-line drugs) are also used for the treatment of TB which are not approved by the United States Food and Drug Administration (FDA) 21 .
Most antituberculosis drugs are liposoluble and their elimination requires biotransformation into more water-soluble compounds.This is mostly performed by hepatic phase I and phase II biotransformation enzymes.In the phase I reaction, oxidation or demethylation occurs, usually performed by cytochrome P450 (CYP450) enzymes.The compound is usually still not very water soluble, and requires further metabolism.Phase I reactions often produce toxic intermediates.In a typical phase II reaction, a large water-soluble compound is attached by glucuronidation or sulfation, resulting in non-toxic metabolites which can easily be eliminated 22 .To date, allelic frequencies and genotypes of CYP3A4 and CYP3A5 variant alleles have been reported in different ethnic groups 2,18,[23][24][25][26] .In Bangladeshi healthy subjects, no sample (n200) was positive for CYP3A4*2, *4, *5, *6, *10 & *18 alleles 25 .Two samples heterozygous for CYP3A4*1B (1.0%) and twenty six samples with the genotype CYP3A5*1/*1 (13.0%) were found in Bangladeshi healthy subjects 25 .In North Indians, two heterozygotes with genotype CYP3A4*1/1B were found in the high enzyme activity group whereas CYP3A4*2, *4, *5, *6 & *10 were absent (n200) 18 .Hence, the present study has been designed to investigate the genotypes and allelic frequencies of CYP3A in Bangladeshi TB patients.SNPs, which are known to have genetic variation and can affect CYP3A activity among Asian subjects, are selected based on the findings of different published papers 2,18,[23][24][25][26] .

Subject selection
Ninety TB patients from Tuberculosis unit, National Institute of Diseases of Chest and Hospital (NIDCH), Dhaka, consisting of 61 men and 29 women were recruited for the study.Demographic data of all the TB patients are presented in Table I.The study was conducted between July 2009 to June 2010 in the Department of Clinical Pharmacy and Pharmacology, Faculty of Pharmacy, University of Dhaka in accordance with the International Conference of Harmonization (ICH) for Good Clinical Practice (GCP) and in compliance with the Declaration of Helsinki and its further amendments 27,28 .Volunteers were informed about the experimental procedures and the study.Each volunteer signed an informed consent document before entering into the study and was free to withdraw from the study at any time without any obligation.Ethical permission was taken to approve the protocol and consent form of the clinical investigation from the Ethical Review Committee of National Institute of Diseases of Chest and Hospital (NIDCH), Dhaka.Genomic DNA Isolation About 3 ml of venous blood was drawn into a tube containing EDTA and stored at20°C until the isolation of genomic DNA.Genomic DNA from all the blood samples was isolated 29 .The purity of the DNA and their concentrations were measured by UV-Spectrophotometer (Shimadzu, Tokyo, Japan) at 260 nm.

Statistical analysis
The SPSS software package (Version 16.0, SPSS Inc., Chicago, Illinois, USA) was used to analyze the data.Descriptive statistics were used for all variables.Values were expressed as percentage, mean, and standard deviation.Data were compiled according to the genotype and allele frequencies 25 .

Results
The purity (OD 260/OD 280) of all the genomic DNA was found to be in the range between 1.7 to 1.9 and the average concentration was 50 to 70 µgm/ml.In all reactions, correct lengths of expected PCR products were obtained.Restriction endonuclease MboII digestion fragments of CYP3A4*1B and RsaI digestion fragments of CYP3A5*3 in 10% Polyacrylamide gel were shown in Figure 1 and Figure 2, respectively.III).  25 .Our studies in TB patients have also demonstrated the absence of these alleles so it can be assumed that there will be no effect on metabolism by CYP3A4.CYP3A5 gene is highly polymorphic in the Chinese, Malay, Indian and Bangladeshi populations with the *3 allele having a frequency of 76% in the Chinese population and about 60% in Malays and Indians and 50% in Bangladeshis 10,33 .Our study results are similar to that for Indians, Malays and Bangladeshi healthy subjects.The common polymorphic genes are absent in Bangladeshi population suggesting no impact of CYP3A drug metabolizing enzymes on antituberculosis drugs.
DNA sequencing was performed to further confirm the genotyping results.Six samples from each different genotype were chosen at random and sent for sequencing to the Centre for Advanced Research in Sciences, University of Dhaka, Bangladesh.Sequencing was also done for the sample containing CYP3A4*1B allele for further confirmation.The PCR products were purified using Biobasic PCR purification kit (Biobasic, Canada) before being sent for DNA sequencing by standard Kit of ABI PRISM BigDye® Terminator (Applied Biosystems, USA).The sequencing results were then verified against the published sequences for CYP3A4 and CYP3A5.GenBank Accession no. were AF185589 (CYP3A4*1B); AF209389 (CYP3A4*2, *4, *5, *6, *10, and *18); J04813 (CYP3A5*1); AC005020 (CYP3A5*3).

Table I :
Demographic data of TB patients (n=90)

Table III :
18equencies of CYP3A4 and CYP3A5 variant alleles in the Bangladeshi TB patients (n=90) whereas K m and V max of CYP3A4*2were not significantly different from CYP3A4*1 for testosterone hydroxylation32.CYP3A4*2 occurs with a frequency of 2.7% in Caucasians and is absent in Africans and Chinese32and also in our study.Hsieh et al.18reported that, those with CYP3A4*4, CYP3A4*5 and CYP3A4*6 alleles have decreased CYP3A4 activity compared to those with no mutations shown.In 200 healthy Bangladeshi subjects, no allele was found to be positive for