Association of CYP2C19*2 and *17 genetic variants with hypertension in Pakistani population

Purpose: To investigate the association of *2 and *17 single nucleotide polymorphisms (SNPs) of CYP2C19 gene with hypertension in Pakistani population. Methods: The study was conducted on 527 hypertensive patients and 530 unrelated healthy controls from selected regions of Pakistan. DNA was extracted from leukocytes and all patients and controls were genotyped for two SNPs (rs4244285 and rs12248560) of CYP2C19 gene by allele specific polymerase chain reaction (AS-PCR). Results: Multi-allelic polymorphism in CYP2C19 identified four distinct phenotypes known as ultra-rapid metabolizer (UM), extensive metabolizer (EM), intermediate metabolizer (IM) and poor metabolizer (PM) in hypertensive patients and controls. For CYP2C19*2 polymorphisms, overall wild type and mutant allele frequency were 75 and 25 % in hypertensive patients, and 64.2 and 35.8 % in controls. For CYP2C19*17 polymorphisms, the overall wild type and mutant allele frequency were 66.6 and 33.4 % in hypertensive patients and 75.6 % and 24.4 % in controls. Significant difference in allele frequencies for CYP2C19*2 and *17 was demonstrated between hypertensive and non-hypertensive subjects. Conclusion: To the best of our knowledge, this is the first report on CYP2C19 frequencies in hypertensive Pakistani patients. The finds should help clinicians to determine a suitable optimal dosage of some drugs in order to reduce side effects.


INTRODUCTION
Cytochrome P450 2C19 (CYP2C19), an important enzyme of the cytochrome P450 family, is involved in the metabolism of several drugs. Inter-individual variation in treatment response is due to genetic polymorphisms in CYP2C19 [1].
PM of CYP2C19*2 sustain omeprazole for a longer time period which helps in stronger inhibition of gastric acid secretion for longer interval [2]. Psychiatric

EXPERIMENTAL Subjects
The study group included 1057 subjects, consisting of 527 hypertensive patients and 530 controls from representative regions of Pakistan. The study was conducted in compliance with the Declaration of Helsinki [9] and was approved by the Ethical Committee and Institutional Review Board (IRB) of Quaid-i-Azam University, Islamabad (#IRB-QAU-97). A written informed consent was obtained from all subjects before participation in the study.
PCR amplification of CYP2C19*2 region was carried out by initial denaturation at 95 o C for 5 min, followed by 30 cycles of denaturation at 94 o C for 1 min, annealing at 62 o C for the amplification of "A" allele-specific DNA fragment and 60 o C for "G" allele-specific DNA fragment for 40 seconds, extension at 72 o C for 1 min followed by final extension at 72 o C for 5 min. The thermal profile for the amplification of CYP2C19*17 was similar to CYP2C19*2 amplification except annealing at 58 o C for both alleles. Once DNA amplification was done, the PCR products were run on 2 % agarose gel in order to confirm the genotype of patients. The phenotype was inferred theoretically from the genotype.

Statistical analysis
Association between the CYP2C19*2 and CYP2C19*17 variants and HTN was determined using SPSS version 20.0 [10]. The level of significance for all the results was set at p < 0.05. Chi-square test was used to test the significance of the Hardy-Weinberg equilibrium of the polymorphism of the two genes in the entire data of the patient and the control groups.
HTN is a major public health issue affecting approximately 1 billion people worldwide [21]. Among hypertensive drugs, most commonly prescribed were CCB and BB [22]. Previous studies show that in case of severe kidney patients, ACE inhibitor or ARB alone or in combination should be the first choice of therapy regardless of ethnic background [23]. On the other hand, patients receiving ACE inhibitor as initial treatment instead of CCB are on 51 % increase threat of having stroke [24]. Previous studies in a large trial show that compared to an ACE inhibitor, thiazide-type diuretic plays vital role in improving cerebrovascular heart failure and combined cardiovascular outcomes [25].

CONCLUSION
Assessment of CYP2C19*2 and *17 variants and HTN association has been conducted for the first time among Pakistani population. This findings indicate that the frequency of CYP2C19*2 and *17 allele are high. The findings should be helpful for physicians to determine the correct drug dosage according to individual's metabolic capacity, lead to decreasing adverse drug reactions and improve therapeutic outcomes.

Acknowledgement
Thanks are due to Mr. Amjad Farooq for his technical assistance, and BioMES, Alpha Genomics Private Limited, Islamabad -Pakistan for editing and reviewing the manuscript. Thanks are also due to the patients who participated in the study.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
We declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors.