Screening for CYP 2 C 19 Gene variants in a healthy Jordanian population

Purpose: To genotype healthy Jordanian population from three different provinces (Amman, Zarqa and Irbid) for cytochrome P4502C19 and to identify the allelic distribution of CYP2C19 variants in comparison with other findings around the world. Methods: Healthy Jordanian volunteers were recruited from government hospitals. Two hundred and sixty volunteers were included in the study regardless of sex and age. CYP2C19*2, *3,*4, *5, and *6 alleles were studied using polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) technique. Results: The results show that the Jordanian population tested exhibited 9 genotypes out of the 21 expected CYP2C19 genotypes. CYP2C19*1/*1 and *2/*2 genotypes were the most prominent in the sample population, while CYP2C19*2/*5 was the least prevalent genotype. The frequencies of the CYP2C19 variants did not deviate from Hardy-Weinberg equilibrium. Allele frequency of CYP2C19*2 in the Jordanian population was statistically different from that found in most Europeans, North and South Americans, Africans, and some Asian ethnic communities but not with South-East Asian populations (China, Chinese-Taiwanese, and Philippines) and Australian Aborigines. Conclusion: The findings of this study confirm the importance of CYP2C19 genotyping prior to drug therapy administration to achieve optimal dosage and cost-effective therapy.


INTRODUCTION
Individual variation in response to drugs is a substantial clinical problem that can be inherited.The genetically determined variability in drug response defines the research area known as pharmacogenetics [1].
One of the main directions in development of pharmacogenetics is identifying genes and allelic variants of genes that affect human's response to drugs [2].Polymorphisms of genes encoding drug metabolism enzymes, drug transporters, and drug receptors, which are involved in drug responses, have been reported [3,4].Genetic variation can account for as much as 95 % of variability in drug disposition and effect [5].As the main cause of the variation in drug response, attention has focused on genetic polymorphisms.Single nucleotide polymorphism (SNP) of DNA produces variation in drug response and has become a representative research target in pharmacogenetics [6].
It is known that many polymorphisms that influence drug response and which probably contribute significantly to phenotypic variation in drug response have significant allele frequency differences among racial or ethnic groups [3,7,8].
Of all genetic factor that affect drug treatment, those affecting the activity of the drug metabolizing enzyme cytochrome P450 are currently considered the quantitatively most important [6].Humans have been estimated to have at least 57 different CYP genes, but the major drug metabolizing human P450 genes are CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A [9,10].The CYP2C19 gene is located within a cluster of CYP genes on the 4th band of region 2 of the long arm of chromosome 10 (10q24) [11].
CYP2C19 exhibits high genetic polymorphism, including common defective variants.Singlebase substitutions in the coding sequence lead to a splicing defect and premature stop codon, and therefore to null function of the enzyme.These variants together (CYP2C19*2 and CYP2C19*3) are responsible for the majority of the CYP2C19related poor metabolism (PM) phenotypes in different populations [12].
Pharmacogenetics is a rapidly evolving field and a lot of information regarding genetic polymorphism is being generated for many ethnic groups, particularly Caucasians and Orientals.However, data on Arab populations is still sparse.There is a need to fill the gaps in pharmacogenetics knowledge pertaining to the Jordanian population.Thus, the aim of this study was to genotype healthy Jordanian people for cytochrome P4502C19, to identify the variant alleles of CYP2C19 and to compare the results with findings in other countries.

Study group
Healthy Jordanian volunteers were randomly recruited from governmental hospitals of three provinces (Amman, Zarqa and Irbid).Two hundred and sixty volunteers were included in the study regardless of sex and age.All volunteers gave their written, informed consent.The study was approved by the Institutional Review Board (IRB) [AM/16/13/10/1200085] of the Hashemite University, Zarqa, Jordan which conforms to the World Medical Association Declaration of Helsinki [13].

CYP2C19 genotyping
Three milliliters of peripheral blood were withdrawn on ethylenediaminetetraacetic acid EDTA tube from each participant by venous puncture.DNA was extracted from 300 µL blood using a commercially available kit (Wizard Genomic DNA Purification Kit, Promega, Madison, USA).The CYP2C19*2, *3,*4, *5, and *6 alleles were studied using the PCR-RFLP technique.The PCR reactions were carried out in a BioRad thermocycler (Mycycler, BioRad, USA).All PCR primers are indicated from 5' to 3' end and are provided in Table 1.The primers sequences were based on published literature [14][15][16][17].
The amplification products were then digested overnight with the appropriate restriction endonuclease (Table 1).The digested PCR products were resolved by electrophoresis in 3 % agarose gels stained with ethidium bromide.The result of the electrophoresis was detected and documented using the CSL-MicroDoc and Canon digital camera (Cleaver-Scientific, UK).

Statistical analysis
The observed genotypes and allele frequencies were compared with those expected, in order to verify the Hardy-Weinberg equilibrium.Allelic frequencies and prevalence were compared between Jordanians and other populations using the test for differences between proportions.The statistical analysis has been performed using STATISITCA 7 analysis program (StatSoft Inc., Ok, USA).A value of p < 0.05 was considered statistically significant.

Population characteristics
The total number of subjects enrolled in the current study was 260.One hundred and fifty nine of them were females (61.2 %) and the other 101 (38.8 %) were males.The age of the subjects ranged from 18 to 59 years with a mean of 24.1 years.There was no significant difference (data not shown) between the mean age of females (24.3 years) and males (23.7 years) (p > 0.05).

CYP2C19 genotyping results
DNA extraction, PCR amplification and restricted PCR products were performed and recorded for all samples.Restriction products of the genomic DNA for the CYP2C19 variants (*2, *3, *4, *5 and *6) and their respective restriction enzymes were as shown in Table 1.

Genotype characteristics
The size of PCR amplicons and restriction digestion products were similar to what have been previously reported (Table 1 and Figure1).Genotypic and allelic frequencies for the six CYP2C19 variants were determined according to the count method (Table 2).In addition, Table 2 shows that the Jordanian population tested exhibited nine genotypes out of the 21 expected CYP2C19 genotypes.CYP2C19*1/*1 and *2/*2 genotypes were the most prominent genotypes in the Jordanian sample population with a frequency of 0.5731 and 0.3038, respectively.CYP2C19*2/*5 was the least prevalent genotype with a frequency of 0.0038.Furthermore, Table 2 shows the allelic frequencies of the six CYP2C19 variants.CYP2C19*1 and *2 were the most dominant variants with allelic frequencies of 0.5904 and 0.3365, respectively.CYP2C19*5 was the least dominant variant with an allelic frequency of 0.0019.The frequencies of the CYP2C19 variants in the Jordanian population were found not to deviate from the Hardy-Weinberg equilibrium.

Comparison
between Jordanian allelic frequencies of CYP2C19*2 and *3 alleles and that of other ethnic populations worldwide is shown in Table 3 [18,19].Allele frequency of CYP2C19*2 in the Jordanian population was statistically different (p < 0.05) from that found in most of the European, North and South Americans, Africans, and some Asian ethnic communities.No significant differences regarding the CYP2C19*2 allele frequency were found when comparing Jordanians to South-East Asian populations (China, Chinese-Taiwanese, and Philippines) and Australian Aborigines.In the case of CYP2C19*3 allele, a lower distribution as in Jordanians have been found in European, North and South America, Africa, and some South-East Asian ethnic groups.Compared to the Jordanian population, a significantly higher allelic frequency of CYP2C19*3 allele was found in East-South Asian ethnic groups (China, Chinese-Taiwanese, Philippines, Japan and Korea), Australian Aborigines and Oceanians (p < 0.05).In this study, no statistical analysis was carried out for CYP2C19*4, *5, and *6 variants due to the absence of data for most of the populations tested [18,19].

DISCUSSION
Various studies indicated the genetic basis for interethnic and inter-individual variability in the metabolism of CYP2C19 substrates [20,21].This leads to dividing individuals according to their CYP2C19 genotype and the associated CYP2C19 enzyme activity into four categories: Extensive, Intermediate, Poor and Ultrarapid metabolizers, according to individuals' response to drug therapy [6].
The distribution of CYP2C19*2 allele in the Jordanian population is consistent with the relatively high frequency of CYP2C19*2 allele worldwide [22], suggesting that CYP2C19*2 was present in humans way before the separation of humans into different distinctive populations [23].However, the higher frequency of such variant in the Jordanian population compared to some regional countries such as Saudi Arabia, Egypt, Gaza Strip (Palestine), and Turkey [19] may be hypothesized to be the result of emigration and admixture between the native Jordanians and different populations such as the Circassians, Palestinians, Syrian, Iraqi, Lebanese, Egyptians and other ethnic Arab populations.On the other hand, the distribution of CYP2C19*3 allele in the Jordanian population was found to be similar to its distribution within the international populations except the south-East Asians, Australian Aborigines and Oceanians [19], suggesting that this allele may be specific to these populations and that it occurred quite recently, after the separation of Caucasian and Oriental groups [23,24].

Limitations of the study
One major limitation of the current study is the scarce of information regarding CYP2C19 allele frequencies in Arab populations in order to make any kind of comparison.In addition, allele frequencies of CYP2C19*4, *5 and *6 in other populations are also lacking which made it impossible to draw any conclusion regarding these genotypes.

CONCLUSION
Earlier studies indicate that CYP2C19 polymorphic nature plays a crucial role in the pharmacokinetics of drug therapy, thus suggesting the importance of CYP2C19 genotyping prior to drug administration to achieve optimal dosage adjustment and costeffective therapy.The findings of this study confirm the importance of CYP2C19 genotyping prior to drug therapy administration to achieve optimal dosage and cost-effective therapy.

Table 1 :
CYP2C19 variants, PCR primers, PCR amplicons and their respective restriction digestion products

Table 2 :
CYP2C19 genotypic and allelic frequencies in the Jordanian population