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| RESEARCH ARTICLE |
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| Year : 2023 | Volume
: 55
| Issue : 1 | Page : 27-33 |
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Prevalence of CYP2C9 and CYP2C19 variants and the impact on clopidogrel efficacy in patients having CYPC19*2 variant
Meha P Mehta1, Normi D Gajjar1, Rutvi J Patel1, Lipi P Joshi1, Gaurang B Shah2
1 Department of Pharm.D, L. M. College of Pharmacy, Ahmedabad, Gujarat, India
2 Department of Pharmacology, L. M. College of Pharmacy, Ahmedabad, Gujarat, India
| Date of Submission | 04-Oct-2022 |
| Date of Decision | 07-Feb-2023 |
| Date of Acceptance | 08-Feb-2023 |
| Date of Web Publication | 20-Mar-2023 |
Correspondence Address:
Gaurang B Shah
Department of Pharmacology, L. M. College of Pharmacy, Navrangpura, Ahmedabad - 380 009, Gujarat
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/ijp.ijp_706_22
OBJECTIVE: Human cytochrome p450 enzymes play an important role in the metabolism of various substances. The CYP2C subfamily consists of various important drug-metabolizing enzymes such as CYP2C9 and CYP2C19. The objectives of the study include the determination of the frequency of genetic variants (CYP2C9*2, CYP2C9*3, and CYP2C19*2) of selected enzymes using allele-specific polymerase chain reaction (ASPCR) and its comparison with Indian as well as global past frequencies. We also aimed to study the impact of genetic mutation on clopidogrel efficacy and compare the efficacies between patients with and without CYP2C19*2 genetic variant.
METHODOLOGY: In this study, the prevalence of variants CYP2C19*2, CYP2C9*2, and CYP2C9*3, the most popular variants of the respective enzymes, was determined using the ASPCR method. The correlation between the CYP2C19*2 variant and the antiplatelet activity of clopidogrel was studied using platelet aggregation assay (PAA).
RESULTS: The determined frequencies of CYP2C19*2, CYP2C9*2, and CYP2C9*3 are 46%, 9%, and 12%. These frequencies are indicative of homozygous as well as heterozygous mutations. Reduced clopidogrel efficacy was observed in patients with a heterozygous mutation of CYP2C19*2 variant.
CONCLUSIONS: The observed frequencies are not significantly different from that observed in earlier reported studies conducted across India and the world. Antiplatelet activity, as measured using the PAA method, was significantly lesser in patients having the CYP2C19*2 variant. The therapy failure in these patients can lead to serious cardiovascular consequences, and we propose determining the presence of the CYP2C19*2 variant before initiation of clopidogrel therapy.
Keywords: Clopidogrel, CYP2C19, CYP2C9, cytochrome p450, pharmacogenetics
How to cite this article:
Mehta MP, Gajjar ND, Patel RJ, Joshi LP, Shah GB. Prevalence of CYP2C9 and CYP2C19 variants and the impact on clopidogrel efficacy in patients having CYPC19*2 variant. Indian J Pharmacol 2023;55:27-33 |
How to cite this URL:
Mehta MP, Gajjar ND, Patel RJ, Joshi LP, Shah GB. Prevalence of CYP2C9 and CYP2C19 variants and the impact on clopidogrel efficacy in patients having CYPC19*2 variant. Indian J Pharmacol [serial online] 2023 [cited 2023 May 31];55:27-33. Available from: https://www.ijp-online.com/text.asp?2023/55/1/27/372168 |
| » Introduction | |  |
Pharmacogenomics and pharmacogenetics, often used interchangeably, have distinct definitions. Pharmacogenomics takes into account the individual's entire genome and its impact on the drug response, while pharmacogenetics involves studying the impact of a single gene on the individual's drug response. The human genome is made up of around 3 million bases which form around 40,000 genes in different combinations, which ultimately code for around 1,00,000 proteins. The pharmacogenomic studies began when differences were observed in the drug response between individuals. The initial and primary application of pharmacogenomics was studying and justifying adverse drug reactions (ADRs).
Cytochrome P450 (CYP) is a superfamily of drug-metabolizing enzymes (DMEs) that are involved majorly in the Phase I Biotransformation of drugs. Their role is quite important in the metabolism of both endogenous and exogenous compounds. The CYP2 family contains several DMEs encoded by polymorphic genes. Molecular variation at loci coding for CYP2C9, CYP2C19, and CYP2D6 has a major role in producing the most important clinical consequences.[1] CYP2C9 and CYP2C19 exhibit frequent genetic polymorphisms linked with the altered activity of the respective enzymes. Regional variations in the frequency of these genetic variants can cause population-specific altered DME activity.[2] CYP2C9 and CYP2C19 are involved in the biotransformation of about 70% of commonly prescribed drugs.
Among these variants, the most common variants *2 (NM_000771.4:c. 430C>T, rs1799853) and *3 (NM_000771.4:c. 1075A>C, NM_000771.4:c. 1075A>G rs1057910) of CYP2C9 and *2 (NM_000769.4:c.681G>A, NM_000769.4:c.681G>C, NM_000769.4:c. 681G>T rs4244285) of CYP2C19, are responsible for affecting the metabolism of their respective substrates. Various studies have reported a significant inter-ethnic variation in the prevalence of CYP2C9 and CYP2C19 polymorphic alleles.[3] Variants CYP2C9*2 and *3 cause replacement in the amino acids of protein R144C and I359 L, respectively, whereas CYP2C19*2 generates splicing defects in the protein.[2] CYP2C9*2 and *3 affect the metabolism of warfarin, nonsteroidal antiinflammatory drugs, tolbutamide, glipizide, etc., CYP2C19*2 affects the metabolism of clopidogrel, diazepam, omeprazole, and lansoprazole.[4] Pharmacogenetics is a widely accepted scientific approach to studying genetic variation in individuals and applying it to make diagnostic and therapeutic decisions.
Clopidogrel is an antiplatelet medication used in the management of acute coronary syndrome (ACS), refractory stent thrombosis, refractory unstable angina, and other ischemic disorders. Clopidogrel is activated in the liver by cytochrome p450 enzymes. The active metabolite irreversibly inhibits the purinergic adenosine diphosphate (ADP) platelet receptor P2Y12 causing the prevention of platelet aggregation against ADP stimulation.[5]
In certain patients, limited response to clopidogrel is observed. Various studies attribute this reduced effect of clopidogrel to the presence of one nonfunctional copy of CYP2C19 (CYP2C19*2).[6],[7],[8] The most cost-effective approach is the implementation of genotype-guided antiplatelet therapy based on genotyping. Instead of common practice of using clopidogrel in all patients with ACS, ticagrelor or prasugrel can be prescribed in CYP2C19*2 variant carriers, whereas clopidogrel can be used in noncarriers.[9]
This research contributes establishment of the correlation between the presence of the CYP2C19*2 variant and the antiplatelet activity in patients that receive clopidogrel. In addition, the prevalence of variants of these enzymes in the community was determined.[10]
| » Methodology | | |
The present cross-sectional study involved the determination of the prevalence of CYP2C19*2, CYP2C9*2, and CYP2C9*3 genetic variants in the community and to study the influence of CYP2C19*2 variant on the efficacy of clopidogrel in the patients visiting the hospitals for their regular follow-up.
Inclusion and exclusion criteria
Based on the following inclusion criteria were selected for the study:
- Age: ≥18
- Willing to provide written informed consent.
For comparing the therapeutic efficacy of clopidogrel:
- Patients following all the above inclusion criteria and
- Patients taking clopidogrel 75 mg OD (once daily) monotherapy for more than 6 months.
Individuals, who met either one of the following exclusion criteria, were excluded from the study.
- Pregnant and lactating women
- Individuals who are unable to respond to the questions asked
- Patients taking any other antiplatelet agent or anticoagulants other than clopidogrel
- Patients with a known history of hepatic disorders or abnormal liver function tests.
Based on the inclusion and exclusion criteria, 100 individuals were included in this research after obtaining Ethics committee approval from the Institutional Ethics Committee, L M College of Pharmacy (LMIEC). The reference number for the approved protocol is LMIEC/2021-22/PD/001.
Consent taking and blood sample collection
The required quantity of blood samples was collected from the patients visiting Mehta Hospital and Cardio-pulmonary research center, Drive-in, Ahmedabad, and Central United Hospital, Odhav, Ahmedabad, for their regular follow-up. Successfully screened patients were requested to fill and sign the informed consent form in the presence of the witness. Demographic data were obtained from the patient interview, and medication data from their case file were collected in the case report forms. The sample population was grossly divided into two groups – one group with 30 patients taking clopidogrel 75 mg OD and the other group with seventy patients not taking clopidogrel. Of the 70 patients, 3 patients belonging to different age groups and not taking any antiplatelets or anticoagulants were selected randomly as controls. The study was designed and carried out as described in [Figure 1]. The samples collected were then analyzed according to the sample groups. The study was performed over a period of 4 months. | Figure 1: Workflow adopted for the analysis of the samples for the patients (a) Taking clopidogrel (n = 30). (b) Not taking clopidogrel (n = 70). PCR = Polymerase chain reaction, EDTA = Ethylenediaminetetraacetic acid, PAA = Platelet aggregation assay
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DNA extraction, purification, and quantification
Extraction of DNA, using a specific isolation kit (HiPuRA SSP, Himedia Labs), from collected samples was carried out as per the manufacturer's instructions. The extracted DNA samples were stored at − 20°C until further use.
BioPhotometer was used to determine the purity of the extracted DNA samples. An elution buffer was used as the blank for the calibration. The purity was checked by taking the ratio of absorbances at 260 nm and 280 nm, respectively. DNA was quantified using the following formula in the eluted solution.
Concentration of DNA sample (μg/mL) = Abs260 nm × 50 × DF
Where, Abs260 nm = Absorbance at 260 nm
DF = Dilution factor.
Primer designing, validation, and in silico polymerase chain reaction
Primer designing
The primers were self-designed using NCBI BLAST to obtain accuracy and to ensure specific binding at the gene of interest. A trio of primers was designed for each selected variant (CYP2C9*2, CYP2C9*3, and CYP2C19*3) which consisted of two forward primers – one which amplified the wild DNA template while the other that amplified the DNA template containing variant. A common reverse primer was designed for the forward primers. A strong mismatch was added at the 4th position from the 3'end in the forward primers to ensure specific binding of primers to the input template. The self-designed primers containing the mismatch were validated using SnapGene version 6.0.
In silico polymerase chain reaction and gel simulation
Polymerase chain reaction (PCR) was simulated in the SnapGene software, which demonstrated the specific binding of primers. Gel electrophoresis was also simulated using the Gel tool of the SnapGene software.
Final polymerase chain reaction process
The final PCR protocol [Table 1] was designed after several trials and errors. The optimized protocol was followed for the analysis of all 100 samples.
Gel electrophoresis
The amplified PCR products were analyzed using Gel Electrophoresis. Agarose gel with a 3% concentration was prepared using EtBr (ethidium bromide) as the staining dye.
Platelet aggregation assay
Separation of platelet-rich plasma and platelet-poor plasma
The blood samples of 30 patients taking clopidogrel were collected in citrate tubes. The platelet aggregation assay (PAA) was performed within 4 h of blood collection. The blood was centrifuged at 200 g for 15 min. The supernatant platelet-rich plasma (PRP) was collected in an autoclaved Eppendorf and was stored at 22°C with continuous shaking. The remaining blood was centrifuged at 2000 g for 10 min to obtain platelet poor plasma (PPP) and was also stored along with PRP until further use.
Platelet aggregation assay
ADP (20 μm), used as a platelet aggregation agonist, was mixed with either PRP (as test) or PPP (as blank) in the 96-well plate. The absorbances were recorded every minute at 600 nm for 20 min with continuous background shaking in VarioSkan Flash. The recorded absorbances were utilized to calculate the % aggregation of the samples using the formula:
One-way ANOVA test followed by Tukey's test for multiple comparisons was performed in Microsoft Excel to determine significance.
| » Results | | |
DNA purification and quantification
The purity of extracted DNA was determined by taking the ratio of absorbance at 260 and 280 nm using Bio Photometer. Pure DNA has a ratio of ~1.8 and the presence of contaminants such as proteins and phenol Pure DNA has a ratio of ~1.8 and the presence of contaminants such as proteins and phenol reduces the ratio well below 1.3 The ratio of extracted DNA ranged between 1.3 and 1.7, and hence, extracted DNA was considered acceptable for the PCR process. The concentration of DNA was quantified using the formula:
Concentration of DNA (μg/ml) =50 × A260 × DF.
The concentrations of extracted DNA samples were found in the range of 5–15 μg/ml.
Virtual polymerase chain reaction and simulation of gel electrophoresis
The results of virtual PCR and simulation of gel electrophoresis stated that the actual primers are likely to result in amplified products having sizes 109 bp, 180 bp, and 422 bp for CYP2C9*2, CYP2C9*3, and CYP2C19*2, respectively.
Polymerase chain reaction
Allele-specific PCR (ASPCR) was performed using an established PCR protocol [Table 1].
Gel electrophoresis and result validation
The PCR products were subjected to gel electrophoresis (3%). All the bands were observed at the appropriate position, same as in silico gel [Figure 2].
Statistics and data analysis
Frequency analysis
Among the analyzed 100 samples through AS-PCR, 91% were found wild type, 7% were found with homozygous variant, and 2% with the heterozygous variant for NM_000771.4:c. 430C>T, indicating the variant CYP2C9*2. Similarly, 88% were found wild type for variant CYP2C9*3. 5% were observed with NM_000771.4:c. 1075A>C homozygous variant and 6% with NM_000771.4:c. 1075A>C heterozygous variant. Along with this, 1% were found with NM_000771.4:c. 1075A>G homozygous variant. However, no sample was found having the heterozygous variant for NM_000771.4: c. 1075A>G conversion of variant CYP2C9*3.
For the variant CYP2C19*2, 54% of people had the wild type of CYP2C19*2 variant. The remaining 46% possessed the variant allele of the same gene. Among which 20% had homozygous NM_000769.4: c. 681G>A, 19% had heterozygous NM_000769.4: c. 681G>A conversion, 2% had homozygous NM_000769.4:c. 681G>C conversion, and 4% had heterozygous NM_000769.4:c. 681G>C conversion. 1% had the heterozygosity NM_000769.4:c. 681G>T conversion. However, no sample was found with homozygosity of NM_000769.4:c. 681G>C conversion [Figure 3]. | Figure 3: Observed frequencies of CYP2C19*2, CYP2C9*2, and CYP2C9*3 variants
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Clinical significance
The platelet aggregation was determined to check the efficacy of clopidogrel in the patients with or without the CYP2C19*2 variant. Platelet aggregation was significantly less in patients having either homozygous (3.59%) or heterozygous (5.48%) variant as compared to that observed in patients having wild-type variant (2.85%) [Figure 4] (One way ANOVA test, P = 0.0015). | Figure 4: % platelet aggregation in clopidogrel-receiving patients with different variants of CYP2C19. *Significantly different from control at P < 0.05, #Significantly different from wild-type variant
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Comparison with previous studies
The comparison between the previously reported percentage frequencies across India and World with the percentage frequencies obtained from our research study was made using the Chi-square test in the GraphPad Prism 9.3.1(Dotmatics, San Diego, California, USA). The P value is higher than 0.05, suggesting a lack of significant difference between the frequencies across India [Figure 5]. | Figure 5: Comparison between observed frequency and previously reported frequency in the representative frequency graph for (a) within India and (b) around the world
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| » Discussion | | |
CYP450 proteins are metabolize large number of endogenous and xenobiotic molecules.[11] Interindividual variability in the activity of these enzymes can in many cases be correlated to the presence of genetic polymorphisms. This variability can alter drug efficacy and safety and also predisposition to various diseases.[12]
Currently, 71 different alleles have been identified for the enzyme CYP2C9. Of these, CYP2C9*1 allele is wild-type having a normal function. The most prevalent variants of CYP2C9 are CYP2C9*2 (rs1799853) and CYP2C9*3 (rs4986893) that have lesser CYP2C9 enzymatic activity. Variants CYP2C9*2 and *3 cause replacement in the amino acids of protein R14 4C and I359 L, respectively.[10]
CYP2C19 is the most polymorphic enzyme in the CYP2C family. There are 39 reported variants of the CYP2C19 enzyme. Of all these variants, CYP2C19*2 (rs4244285), CYP2C19*3 (rs4986893), and CYP2C19*17 (rs12248560) are the most studied variants.[2],[13]
ASPCR uses single-nucleotide polymorphism (SNP) base-containing primers to amplify a DNA template to obtain the PCR product of the SNP. Allele-specific primers are a trio of primers containing one common reverse primer and two forward primers with, at 3' end, one having a wild and the other an SNP complementary base. In addition, a destabilizing mismatch is added within the 5 bases of the 3' end to avoid primer mega-priming and to allow specific SNP binding.[14] Analysis of platelet aggregation is widely carried out for patients with increased bleeding tendency as well as for evaluation of antiplatelet therapy but is much used for research purposes.[15]
Among the analyzed 100 patients' samples using the ASPCR technique, 7% had homozygous, and 2% showed heterozygous CYP2C9*2 variant.
Similarly, 5% homozygous and 6% heterozygous for NM-000771.4:c. 1075A>C and 1% homozygous and nil heterozygous NM-000771.4:c. 1075A>G variant of CYP2C9*3 were observed.
For the variant CYP2C19*2, 46% of patients possessed the variant allele. This variant allele included 20% homozygous and 19% heterozygous NM_000769.4:c. 681G>A variant and 2% homozygous and 4% had heterozygous NM_000769.4:c. 681G>C variant and. One percentage had the heterozygous NM_000769.4:c. 681G>T variant [Figure 3]. However, not a single patient was found with homozygosity of NM_000769.4:c. 681G>C conversion. This data indicate that a significant proportion of patients possess variant alleles and are most likely to have altered metabolism rates for drugs such as clopidogrel, diazepam, omeprazole, lansoprazole. Applying pharmacogenetics may help us in reducing the ADR and/or improve the efficacy of these drugs.
The efficacy of the clopidogrel was compared in patients with and without a variant of the CYP2C19 gene, using PAA. Thirty patients taking clopidogrel 75 mg OD for more than 6 months were enrolled, and their blood samples were analyzed for antiplatelet activity. Since 75 mg is the only available dose for clopidogrel, dose has no effect on the impact of a genetic variant on clopidogrel efficacy. The % platelet aggregation of the blood samples of the control group in the presence of ADP (20 μm) was 7.84% which was considered the maximum. Compared to this maximal value, the patients with the wild variant had % aggregation of 2.85%, suggesting a significant reduction in platelet aggregation. Patients having homogeneous and heterogenous variants showed 3.59% and 5.48% suggesting 14.83% and 52.71% lesser antiplatelet activity, respectively, than that observed in clopidogrel-receiving patients with wild type variant. We had expected a higher loss of antiplatelet activity in patients with homozygous variants. The smaller number of patients having the homozygous variant could be responsible for this unexpected result. A larger study may help in confirming the real clinical outcome of pharmacogenetic variation. It is clear from our study that the antiplatelet activity of clopidogrel is significantly less in the patients having the variant of the CYP2C19 gene, and they run the risk of reduced protection against myocardial infarction and stroke. Multiple comparison using Tukey's test revealed significantly lesser antiplatelet activity in patients having the heterozygous variant (NM_000769.4:c. 681G>A) as compared to those having the wild-type variant.
The P value of the Chi-square test performed for comparison of reported and obtained percentage frequencies was higher than 0.05, suggesting no statistically significant difference between the frequencies across India.[3],[16],[17] Similarly, the prevalence of CYP2C19*2, CYP2C9*2, and CYP2C9*3 variants in Ahmedabad is not different from that observed in studies conducted across the world.[18],[19],[20],[21],[22] This study differs from the previous studies as all the past studies have either determined the prevalence of the CYP2C19*2 variant or have determined the antiplatelet activity of antiplatelet/anticoagulant agents using PAA. We observed a significant influence of the presence of the CYP2C19*2 variant on clopidogrel's antiplatelet activity.
| » Conclusions | | |
The obtained frequencies of CYP2C19*2, CYP2C9*2, and CYP2C9*3 indicate a considerable prevalence of these genetic variants in the Indian population. The prevalence of different variants observed in our studies is not significantly different from that observed in previous studies conducted in India. Furthermore, the prevalence determined in this study is similar to that reported in various international studies. PAA results give an insight into the influence of the CYP2C19*2 genetic variant on the clopidogrel's efficacy. The presence of the CYP2C19*2 variant leads to reduced efficacy of clopidogrel in such patients. There is a need to popularize genetic profiling to promote personalized drug therapies for patients. In conclusion, determining the frequencies of these polymorphisms in varied populations and understanding the clinical impact of each of these variants will provide important information on drug response and the management of toxicity as well as disease predisposition arising from them.
Acknowledgment
The authors would like to acknowledge the subjects included in the study for their invaluable contribution. They also acknowledge the Atal Incubation Centre at L M College of Pharmacy (AIC-LMCP), Ahmedabad, for infrastructural and instrumental support for the present project. The authors would also like to acknowledge teams of Mehta Hospital and Cardiopulmonary Research Centre and Central united Hospital for their support and guidance.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1]
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