|Year : 2021 | Volume
| Issue : 3 | Page : 80-84
Clopidogrel resistance in cardiovascular disease patients and its association with gene polymorphisms: A pilot study
S Parameshwara Assistant Professor 1, B Manjula Research Scientist-I 2, Geetha Bhaktha Research Scientist-II 2, Gurupadappa Kallaganad Professor 3, GK Ranjith Kumar Assistant Professor 4
1 Department of Cardiology, Super-Speciality Hospital, SIMS, Shimoga, Karnataka, India
2 Multidisciplinary Research Unit (MRU), Shimoga Institute of Medical Sciences, Shimoga, Karnataka, India
3 Department of Biochemistry, Shimoga Institute of Medical Sciences, Shimoga, Karnataka, India
4 General Medicine, McGann Teaching District Hospital, SIMS-Shimoga, Karnataka, India
|Date of Submission||22-Dec-2020|
|Date of Decision||02-Feb-2021|
|Date of Acceptance||13-Feb-2021|
|Date of Web Publication||23-Sep-2021|
Dr. S Parameshwara
Department of Cardiology, Super-Speciality Hospital, SIMS, Shimoga, Karnataka
Source of Support: None, Conflict of Interest: None
Aims: To determine the genotypic and allelic frequencies of multidrug resistance protein 1 (MDR1), CYP2C19, and P2Y12 gene and their polymorphisms in the Shimoga population who admitted for treatment in McGann teaching district hospital-Shimoga Institute of Medical Sciences (SIMS), Shimoga. Materials and Methods: It is a cross-sectional study that was conducted in McGann teaching district hospital, SIMS, Shimoga. Patients who were admitted to the Intensive coronary care unit due to cardiovascular disease (CVD) were recruited for the study. Sociodemographic data with venous blood samples (5 ml) were collected along with informed consent. Genotyping of the MDR1, CYP2C19, and P2Y12 polymorphisms were done using the polymerase chain reaction (PCR) restriction fragment length polymorphism method. Descriptive and inferential statistics were done using the Statistical Package for the Social Sciences software. The genotypic and allelic frequency was calculated using the Hardy-Weinberg equilibrium. Results: Out of 40 CVD cases, 60% were male and 40% were female and the mean age was found to be 57.13 ± 11.57 years. The percentage of the obese group was more compared to other groups of body mass index. Risk factors such as diabetes, hypertension, food custom, smoking, and tobacco consumption were not significant while alcohol consumption was seen to be significant among the study population at a 5% level of significance. The genotypic frequencies for a heterozygous and mutant type of MDR1 (C3435T) and CYP2C19*2 (G681A) were found to be 35%, 2.5%, 57.5%, and 5%, respectively. Whereas the gene polymorphism of CYP2C19*3 and P2Y12 was not observed in the present study population. Conclusions: This is the first gene polymorphism study with respect to clopidogrel resistance in the Shimoga population. We have demonstrated the presence of polymorphism in the MDR1 and CYP2C19 genes in this study population. A further elaborate study should be conducted for a better understanding of genetic with nongenetic factors involved in poor response toward clopidogrel drug using a large population.
Keywords: Blood pressure, body mass index, cardiovascular disease, intensive coronary care unit, polymerase chain reaction, restriction fragment length polymorphism, single nucleotide polymorphism
|How to cite this article:|
Parameshwara S, Manjula B, Bhaktha G, Kallaganad G, Ranjith Kumar G K. Clopidogrel resistance in cardiovascular disease patients and its association with gene polymorphisms: A pilot study. J Clin Prev Cardiol 2021;10:80-4
|How to cite this URL:|
Parameshwara S, Manjula B, Bhaktha G, Kallaganad G, Ranjith Kumar G K. Clopidogrel resistance in cardiovascular disease patients and its association with gene polymorphisms: A pilot study. J Clin Prev Cardiol [serial online] 2021 [cited 2021 Dec 3];10:80-4. Available from: https://www.jcpconline.org/text.asp?2021/10/3/80/326475
| Introduction|| |
Cardiovascular disease (CVD) is an important cause of mortality and morbidity in the world. Nowadays, these diseases are epidemic in both urban and rural areas. To treat CVD many antiplatelet drugs such as clopidogrel, prasugrel, or ticagrelor, were used in different parts of India. Compared to other antiplatelet drugs, clopidogrel was generally used to treat CVD, due to its efficiency, cost, and limited side effects. These events are associated with poor response to the clopidogrel known as clopidogrel resistance. The precise mechanism for the resistance of clopidogrel is not very well understood/established but is likely multifactorial such as poor absorption, drug–drug interaction, platelet function, single-nucleotide polymorphism (SNP), and many more.
Clopidogrel is an oral thienopyridine that acts as a potent antiplatelet drug. It irreversibly inhibits platelet aggregation induced by adenosine diphosphate (ADP), low concentrations of thrombin, or by collagen. For inhibition of platelet aggregation, the clopidogrel first absorbs and transported through the small intestine by multidrug resistance protein 1 (MDR1) and was converted into its active form in the liver by the Cytochrome P450 (CYP2C19) enzymes system and then acts via irreversible antagonism of the platelet purinergic (P2Y12) ADP receptor. By blocking this receptor, Clopidogrel prevents platelet degranulation and inhibits the action of the glycoprotein IIb/IIIa (GP IIb/IIIa) receptor which is responsible for the binding of fibrinogen and platelet aggregation. Therefore, clopidogrel can be considered as a precursor of an active metabolite. Clopidogrel resistance has been documented in the range of 5%–44% worldwide including India. The percentage varies from state to state and place to place due to regional variation in the Indian population. More work was conducted in the north region of India,,, whereas in the south region of India only diminutive work was done with a small sample size in Kerala, Hyderabad, and Tamil Nadu, but we do not have any type of information/data about clopidogrel resistance related to gene polymorphism in Karnataka (Shimoga).
For clopidogrel resistance, only one gene polymorphism was not a reason, so in this study, we are going to detect/screen for SNPs of MDR1, CYP2C19, and P2Y12 genes are proposed which influence the response to clopidogrel.
| Materials and Methods|| |
This is a cross-sectional study conducted in the McGann teaching district hospital-SIMS Shimoga, India. Patients with CVDs were enrolled prospectively for genotyping of the MDR1 (C3435T), CYP2C19*2 (G681A), CYP2C19*3 (G636A), and P2Y12 (i-T744C) polymorphisms. The patients were enrolled according to the inclusion criteria which include patients admitted to the intensive coronary care unit and on the clopidogrel therapy with the age of >18 years and exclusion criteria which includes active neoplasm or history of neoplasm, severe renal and hepatic insufficiency, hemorrhagic diathesis, hematocrit <35% or >50%, pregnant women, cancer patients with treatment and concomitant GPIIb/IIIa inhibitor administration such as abciximab and eptifibatide. Informed written consent was obtained from all the subjects before enrolment.
Patient information was documented in a study pro forma which included demographic data of individuals such as age, gender, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), smoking, alcohol, and tobacco consumption, hypertension, type of occupation, diabetes mellitus, and family history of CVD.
Venous blood samples (5 ml) were collected in ethylene-diamine tetra-acetic acid vacutainer from each patient for genetic analysis.
DNA was extracted from peripheral blood using the spin column kit method (HimediaHiPurA™ Blood Genomic DNA Mini Purification) and DNA yield was estimated by measuring absorbance at 260 nm using Nanodrop (Eppendorf BioSpectrometer® basic). Primers were designed using Primer 3 and BLAST from the National Center for Biotechnology Information and purchased from bioengineering (India). Amplification of the gene was carried out using polymerase chain reaction (PCR) using ×2 PCR Taq Mixture (Himedia) in a thermal cycler (Biorad). The primers used for PCR of each polymorphism are given in [Table 1] and PCR conditions are depicted in [Table 2]. Restriction fragment length polymorphism (RFLP) analysis was done using Sau3A1, SmaI, BamHI, and HpyCH4IV restriction enzymes (New England Biolabs) to identifying the genotypes of MDR1 (C3435T), CYP2C19*2 (G681A), CYP2C19*3 (G636A), and P2Y12 (i-T744C), respectively and bands were analyzed by 1.5% Agarose gel electrophoresis which was stained with ethidium bromide and the banding pattern was documented using Bio-Rad gel doc instrument.
|Table 2: Details of the polymerase chain reaction protocol for genotyping polymorphisms|
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The data obtained were coded and entered into Microsoft Excel and analyzed using the Statistical Package for the Social Sciences IBM SPSS software version 22, Armonk, NY: IBM Corp. Descriptive statistics such as frequencies, mean, and standard deviation were calculated. Inferential statistics like analysis of variance and Chi-square test were applied. The statistical significance was evaluated at a 5% level of significance. Allelic frequencies were calculated from genotype frequencies. Genotypes were tested for deviations from the Hardy-Weinberg equilibrium.
| Results|| |
In this study, there were 24 males and 16 females with a mean age of 57.13 ± 11.57 years. The mean and standard deviation of SBP, DBP, and BMI of the study population was found to be 131.0 ± 23.18 mmHg, 81.65 ± 12.64 mmHg, and 25.2 ± 3.65 Kg/m2.
In our study population, the percentage of nonvegetarians, nontobacco users, nonalcoholic and nonsmokers, nondiabetic were more when compared to vegetarians, tobacco users, alcoholics, smokers, and diabetic. Risk factors such as diabetes, hypertension, food habit, smoking, and tobacco consumption were not significant, while alcohol consumption was seen to be significant among the study population at a 5% level of significance [Table 3].
|Table 3: Cardiovascular disease risk factors in the study population (n=40)|
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The percentage of CVD patients was more in ≥61 years age group when compared to other groups [Figure 1]a.
|Figure 1: Percentage of cardiovascular disease v/s age (a), Type of occupations (b) and body mass index classification (c)|
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The percentage of CVD patients in underweight, normal weight, overweight, obese I, and obese II was found to be 5%, 22.5%, 10%, 57.5%, and 5% [Figure 1]c. Another risk factor of CVD is the lifestyle/type of work of the individuals. In this study, heavy type (52.5%) lifestyle was significantly higher compared to moderate (7.5%) and sedentary (40%) type of occupation [Figure 1]b.
PCR-RFLP for the MDR1 (C3435T), CYP2C19*2 (G681A), CYP2C19*3 (G636A), and P2Y12 (i-T744C) was done using Sau3A1, SmaI, BamHI, and HpyCH4IV restriction enzymes, and the banding pattern is presented in [Figure 2] and [Supplementary Figure 1].
|Figure 2: The restriction digestion pattern of MDR1-C3435T (a), CYP2C19*2-G681A (b), CYP2C19*3-G636A (c) and P2Y12-i-T744C (d) on 1.5% Agarose gel electrophoresis: (a) represents the restriction digestion pattern of MDR1 (C3435T) with Sau3A1.Lane 1: Wild type genotype showing one band of 248bp. Lane 2: Homozygous mutant genotype showing two bands of 208bp and 40bp. Lane 3: Heterozygous genotype showing three bands of 248bp, 208bp and 40bp.(b) represents the restriction digestion pattern of CYP2C19*2 (G681A) with SmaI. Lane 1: Homozygous mutant genotype showing one band of 220bp. Lane 2: Wild type genotype showing two bands of 117bp and 103bp. Lane 3: Heterozygous genotype showing three bands of 220bp, 117bp and 103bp. (c) represents the restriction digestion pattern of CYP2C19*3 (G636A) with BamHI. Lane 1: Homozygous mutant genotype showing one band of 288bp. Lane 2: Wild type genotype showing two bands of 208bp and 122bp. (d) represents the restriction digestion pattern of P2Y12 (i-T744C) with HpyCH4IV.Lane 1: Homozygous mutant genotype showing one band of 266bp. Lane 2: Wild type genotype showing two bands of 175bp and 91bp. Lane M: molecular markers (50 -1500 base pairs)|
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MDR1 (C3435T) polymorphism was found to be in 37.5% which included 35% heterozygous (Loss of function allele) and 2.5% homozygous mutant (total loss of function). CYP2C19*2 (G681A) polymorphism was found to be in 62.5% which included 57.5% heterozygous and 5% homozygous mutant. Whereas in the case of CYP2C19*3 (G636A) and P2Y12 (i-T744C) gene polymorphism was not observed in the present study population [Table 4].
|Table 4: Genotype and allele frequencies of cardiovascular disease patients (n=40)|
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Among 15 patients with MDR1 (C3435T) polymorphisms, eight (20%) were female and 7 (17.5%) were male, whereas in CYP2C19*2 (G681A) polymorphism, eight (20%) were female and 17 (42.5%) were male among 25 patients. Females had a significantly higher incidence of MDR1 (C3435T) polymorphisms whereas males had a significantly higher incidence of CYP2C19*2 (G681A) polymorphisms [Table 5].
| Discussion|| |
This is a pilot study that demonstrated the polymorphism of drug absorption (MDR1), and clopidogrel metabolizing enzyme (CYP2C19*2) genes in our study population.
The human MDR1 gene is positioned in chromosome 7 which consists of 28 exons and core promoter regions. C3435T SNP is a silent mutation in exon 26 of the MDR1 gene. It affects the expression and function of P-glycoprotein which is associated with drug absorption in the intestine.,, In our study population, the frequency of variant genotype of MDR1 (C3435T) which affects the intestinal absorption of clopidogrel was found to be 37.5% (35% heterozygous and 2.5% mutant) which was similar to the other studies conducted in different ethnic groups.,,
The human CYP2C19 gene plays a significant role in clopidogrel metabolism by converting into its active pro-drug form. There will be variability in the concentration of active metabolites due to polymorphism in the CYP2C19 gene. CYP2C19*1(wild type), CYP2C19*2-G681A (with splicing defect) and CYP2C19*3-G636A (with stop codon) were some genetic variants of the CYP2C19 gene. Many studies show that polymorphism of the CYP2C19*2 is the key marker of low responsiveness to clopidogrel when compared to other alleles of the CYP2C19 gene. Similar to CYP2C19*2 other alleles such as CYP2C19*3, CYP2C19*4, CYP2C19*5, CYP2C19*6, CYP2C19*7, and CYP2C19*8 will also diminish the clopidogrel metabolism, but their frequency is less when compared to CYP2C19*2., The relative risk for major cardiac events among patients who were treated with clopidogrel was found to be 1.53 to 3.69 times higher for carriers of CYP2C19*2 and CYP2C19*3 when compared with noncarriers. In our study population, the frequency of variant genotype of CYP2C19*2 (G681A) was found to be 62.5% (57.5% heterozygous and 5% mutant) which was similar to the other studies in the Indian population.,,, In the present study population, CYP2C19*3 (G636A) and P2Y12 (i-T744C) polymorphisms were absent as in other studies.,
- It is a pilot study with a small sample size
- It was a single-center study and hence the results cannot be generalized to the total population.
| Conclusions|| |
Compared to other studies worldwide our present study also supports the importance of MDR1 and CYP2C19*2 gene variants as a potential marker of clopidogrel resistance. Further, an elaborate study should be conducted in a larger population with follow-up cases and healthy controls for a better understanding of the phenomenon of clopidogrel heterogeneity of response toward clopidogrel drug and also we should look into the effect of nongenetic components on clopidogrel resistance along with genetic components.
Authors acknowledge the Department of Health Research, New Delhi, India for providing the facilities at Multidisciplinary Research Unit for the study. Also thank Mrs. Revathy, Biostatician for helping through the statistics.
Financial support and sponsorship
Multidisciplinary Research Unit, Department of Health Research, New Delhi, India.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]