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Table of Contents    
ORIGINAL ARTICLE
Year : 2022  |  Volume : 70  |  Issue : 4  |  Page : 1575-1579

Association of Paraoxonase-2 (C1053G) Gene Polymorphism with the Expression of Paraoxonase-2 Gene in Patients of Ischemic Stroke – A Pilot Study in Indian Population


1 Department of Biochemistry, Lady Hardinge Medical College, New Delhi, India
2 Department of Biochemistry, University College of Medical Sciences, New Delhi, India

Date of Submission22-Nov-2018
Date of Decision24-Oct-2019
Date of Acceptance05-Jul-2022
Date of Web Publication30-Aug-2022

Correspondence Address:
Sudhir Chandra
Department of Biochemistry, Lady Hardinge Medical College, Shaheed Bhagat Singh Road, Connaught Place, New Delhi - 110 001
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0028-3886.355082

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 » Abstract 


Background and Objective: Oxidative stress plays an important role in atherosclerosis and ischemic stroke. Due to antioxidant properties of Paraoxonase-2, we studied the implication of Paraoxonase-2 gene polymorphism (C1053G) on expression of Paraoxonase-2 gene at mRNA level in ischemic stroke patients.
Material and Methods: 40 patients of ischemic stroke and 40 age and sex-matched controls were included. Paraoxonase-2 genotypes were evaluated by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism and expression of Paraoxonase-2 gene at mRNA level was determined by quantitative real time Polymerase Chain Reaction analysed as delta-CT (△CT).
Result and Discussion: The observed allele frequencies in patients for C and G allele were 0.61 and 0.39 respectively, and were 0.72 and 0.28 in control group. No significant association was found in C allele of C1053G polymorphism and ischemic stroke. The average △ CT value is significantly (p = 0.0001) higher in patients group (7.68 ± 2.0) as compared to controls (5.70 ± 1.8). We found a significant difference in the average delta-CT value (p = 0.0001), wherein down-regulated paraoxonase-2 gene expression (approximately 0.25 fold) was observed in case of patients as compared to controls. Down-regulated expression of paraoxonase-2 gene was observed in patients with GG genotype as compared to CG and CC genotypes in patients with ischemic stroke (p = 0.0001).
Conclusion: Down-regulated Paraoxonase-2 gene expression, as evidenced by low mRNA levels in GG genotype may be one of the contributory factors in the progression of ischemic stroke.


Keywords: Atherosclerosis, ischemic stroke, oxidative stress, paraoxonase-2 gene expression
Key Message: Association of Paraoxonase-2 gene in patients with ischemic stroke.


How to cite this article:
Kumari S, Singh R, Chandra S, Mehndiratta M, Debnath E, Dhamija RK. Association of Paraoxonase-2 (C1053G) Gene Polymorphism with the Expression of Paraoxonase-2 Gene in Patients of Ischemic Stroke – A Pilot Study in Indian Population. Neurol India 2022;70:1575-9

How to cite this URL:
Kumari S, Singh R, Chandra S, Mehndiratta M, Debnath E, Dhamija RK. Association of Paraoxonase-2 (C1053G) Gene Polymorphism with the Expression of Paraoxonase-2 Gene in Patients of Ischemic Stroke – A Pilot Study in Indian Population. Neurol India [serial online] 2022 [cited 2022 Oct 2];70:1575-9. Available from: https://www.neurologyindia.com/text.asp?2022/70/4/1575/355082




Stroke remains the second most important cause of death globally, with approximately 5.5 million deaths attributed to this disease in 2016.[1] Ischemic stroke is a multifaceted trait, with genetic factors expected to work in the course of various risk factors for cerebrovascular disease.[2] Timely detection and modification of risk factors are important in reducing the risk of stroke and decreasing morbidity as well as mortality due to stroke. Paraoxonase, a 45-kDa glycoprotein, is well known for being antiatherogenic, subsequently preventing stroke.[3] The paraoxonase gene maps to chromosome 7 (q21–23) and exists in three isoforms: paraoxonase-1, paraoxonase-2, and paraoxonase-3.[4] It has been demonstrated that paraoxonase-2 decreases the intracellular oxidative stress by reducing the levels of reactive oxygen species (ROS), inhibiting low-density lipoprotein (LDL) lipid peroxidation, and reversing the oxidation of mildly oxidized LDL, and it also inhibits the ability of minimally modified LDL to induce monocyte chemotaxis.[5] Paraoxonase-2 has antioxidative and antiatherosclerotic potential; therefore, researchers have shown interest to investigate the association of paraoxonase-2 with ischemic stroke. A number of studies have revealed association of paraoxonase polymorphisms with ischemic stroke and atherosclerosis,[6] while other studies are less conclusive.[7],[8],[9] Two common polymorphisms have been studied in paraoxonase-2 gene in relation to stroke and atherosclerosis: the first one is either cysteine or serine at codon 311 (C1053G) and the other one is alanine or glycine at codon 148.[10],[11]

The upregulated mRNA expression of paraoxonase-2 has also been shown to reduce intracellular oxidative stress and the cell's capacity to oxidize LDL, and thus prevents atherosclerosis.[12] Paraoxonase-2 polymorphism[13],[14] and its mRNA expression[15] have been evaluated separately in many studies; however, correlation between these two parameters has not been studied in ischemic stroke patients. This study was undertaken to determine the correlation between paraoxonase-2 polymorphism and its mRNA expression in ischemic stroke in Indian population.


 » Materials and Methods Top


Study population

We conducted a case–control study in the Department of Biochemistry and the Department of Neurology, Lady Hardinge Medical College and Associated Hospitals, New Delhi. In group I, 40 patients with ischemic stroke (diagnosed by history and clinical examination and confirmed by neuroimaging), aged between 50 and 80 years, were enrolled in the study. Patients with history of diabetes, hypertension, previous history of immunologic or neoplastic disease, ongoing infection, impaired renal function, surgery, traumatic brain injury, and subjects on vitamin supplements within 2 weeks before the start of the study were excluded from the study. Group II included 40 unrelated, healthy age- and sex-matched controls.

A detailed history and physical examination, routine hematology, biochemistry including lipid profile, and neuroimaging was done in all cases. Written informed consent was obtained from all the participants recruited for the study. An approval from the ethics committee for human research, Lady Hardinge Medical College, Delhi, India was obtained before the study.

Sample collection and processing

Six milliliters of venous blood was collected in an ethylenediaminetetraacetic acid (EDTA) vial from the subjects under sterile conditions. One milliliter of whole blood was used for RNA extraction and the remaining volume of blood was used for DNA extraction and analysis of other biochemical parameters.

DNA extraction and genotyping

Genomic DNA was extracted using QIAamp DNA mini kit (QIAGEN®, USA) according to manufacturer's instructions and quantified by using basic Biospectrometer® (Eppendorf, Germany). Genotype and allele frequencies were calculated using the Hardy–Weinberg equation. Genotyping for paraoxonase-2 C1053G gene polymorphism was carried out by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) technique with slight modifications. The amplified 256-base pair (bp) PCR product was digested with HpyF3I (DdeI) Thermo Scientific FastDigest restriction enzyme (Thermo Fisher Scientific, USA) by incubating at 37°C for 5 min followed by separation of the fragments on 2% agarose gel. RFLP of the PCR products yielded a band of 256 bp in homozygous wild type (CC), two bands of 159 and 67 bp corresponding to the presence of the homozygous mutant type (GG), and three bands (256, 159, 67 bp) in the heterozygous (CG) genotype [Figure 1].
Figure 1: RFLP gel picture of paraoxonase-2 C1053G gene polymorphism. The figure represents the RFLP of C1053G variant of paraoxonase-2 gene on 2% agarose gel electrophoresis. Lane M represents the DNA ladder, lane 1 = sample no. P13, lane 2 = sample no. P16, lane 3 = sample no. P17, lane 4 = sample no. P21, lane 5 = sample no. P22, lane 6 = sample no. P29, lane 7 = sample no. P30, and lane 8 = sample no. P39. Band pattern: homozygous (CC)- 256 bp; heterozygous (CG)- 256, 159, and 67 bp; homozygous (GG)- 159 and 67 bp. RFLP = restriction fragment length polymorphism

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Total RNA extraction and cDNA synthesis

Total RNA was extracted using QIAamp RNA blood mini kit (QIAGEN, USA) according to manufacturer's instructions and quantified by using basic Biospectrometer (Eppendorf, Germany). Five hundred nanograms of total RNA was used for cDNA preparation using the iScript cDNA synthesis kit (Bio-Rad Laboratories®, USA), according to the manufacturer's instructions.

Determination of expression of paraoxonase-2 gene by quantitative PCR

Expression of paraoxonase-2 gene was determined by quantifying mRNA using real-time PCR method. The real-time PCR was performed by using CFX96 real-time system (Bio-Rad Laboratories, USA) with iTaq Universal SYBR® Green Supermix (Bio-Rad Laboratories, USA) PCR core reagent. Amplification was performed in triplicate in 10 μl volume by using the following primers for paraoxonase-2 gene and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as reported earlier:[16]

Paraoxonase-2 gene.

Forward: 5′-TGATTCAGCAAATGGGATCA-3′.

Reverse: 5′-GCTTCTGGCCATTAGGATGA-3′.

GAPDH (housekeeping gene).

Forward: 5′-AGAAGGCTGGGGCTCATT-3′.

Reverse: 5′-GGGCCATCCACAGTCTTCT-3′.

Delta CT (△CT) value was then calculated as

△CT of group I (patients) = CT of paraoxonase2 − CT of housekeeping gene and

△CT of group II (controls) = CT of paraoxonase 2 – CT of housekeeping gene.

Further, the delta-delta-CT (△△CT) value was calculated between both groups as

△△CT = △CT of group I – △CT of group II.

The paraoxonase-2 gene expression was compared between both groups in terms of fold change using △△CT equation, and specificity of real-time assay was determined by analyzing the melting curve either for paraoxonase-2 gene or for the housekeeping gene.

Statistical analysis

All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) program, version 16. Chi-square goodness of fit was used to verify the agreement of Hardy–Weinberg equilibrium. The analysis of variance (ANOVA) using Bonferroni's method was used to calculate the difference between genotype groups. An odds ratio at (95% confidence interval [CI]) was calculated as an index of the association of the gene with the disease. Results for gene expression analysis are expressed as mean ± standard deviation (SD). Statistical significance was defined as a P value < 0.05.


 » Results Top


Demographic profile

Demographic profile of stroke patients in this study showed the mean age as 60.2 years in group I and 58.8 years in group II. Also, 35% of the stroke cases (group I) were women, 30% of the patients were vegetarian by diet, and 62.5% of them were illiterate. Mean plasma glucose level was 86.8 mg/dl in group I [Table 1].
Table 1: Demographic profile of the study subjects

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Distribution of genotype and allele frequencies of paraoxonase-2 C1053G

The distribution of genotype in the patient and control groups was in accordance with the Hardy–Weinberg equilibrium. The homozygous genotypic pattern (CC) was more frequent in group I as well as in group II. The observed allele frequencies for C and G alleles in group I were 0.61 and 0.39, respectively, and in group II were 0.72 and 0.28, respectively. No significant association was found in C allele of C1053G polymorphism and ischemic stroke. The adjusted odds ratio for C versus G allele frequencies was found to be 1.66 ([0.85–3.24], χ2 = 2.28; P = 0.13) [Table 2].
Table 2: Genotype and allele frequencies of C1053G variant of the PON-2 gene in the study subjects

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Paraoxonase-2 gene expression

Expression of paraoxonase-2 gene was compared between both groups in terms of average △ CT values [Figure 2]. The average △ CT value was significantly (P = 0.0001) higher (required a higher number of cycles to cross the threshold levels) in group I (7.68 ± 2.0) compared to group II (5.70 ± 1.8), which implies that the gene expression was less in group I compared to that in group II. The ΔΔCT value for group I (1.97) showed the downregulated expression of paraoxonase-2 gene. The relative expression of mRNA for paraoxonase-2 gene to GAPDH in group I was decreased by approximately 0.25-fold, compared to group II.
Figure 2: △CT values and expression of paraoxonase-2 gene in the study subjects. The figure represents the expression of paraoxonase-2 gene in terms of average △ CT values for the patient and control groups. Higher △ CT value represents lower expression of the gene at the mRNA level. *P < 0.05 is significant

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Intergenotypic (C1053G variant of the paraoxonase-2 gene) variations in gene expression (△CT values) in the study subjects

The intergenotypic variation in the △ CT values in group I with CC, CG, and GG genotypes was found to be 6.11 ± 1.65, 8.5 ± 1.15, and 11.1 ± 1.06, respectively, and was statistically significant (P = 0.0001). The intergenotypic variation in the △ CT values in group II with CC, CG, and GG genotypes was found to be 5.29 ± 1.97, 5.97 ± 1.71, and 7.50 ± 1.15, respectively (P = 0.20) [Table 3]. The GG genotype exhibited minimal level of expression of paraoxonase-2 gene compared to CG and CC genotypes in group I (P = 0.0001) [Figure 3].
Figure 3: Intergenotypic (C1053G variant of the paraoxonase-2 gene) variations in gene expression (△CT values) in the study subjects. The figure represents the intergenotypic variations in gene expression (average △ CT values) in the study subjects. Results are expressed as mean ± SD. *P < 0.05 is considered to be significant. SD = standard deviation

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Table 3: Intergenotypic (C1053G variant of the PON-2 gene) variations in the gene expression of PON-2 (△CT values) in the study subjects

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 » Discussion Top


This was a hospital-based case–control study conducted to determine paraoxonase-2 polymorphism and the expression of paraoxonase-2 gene, and also to look for the correlation between these two parameters in patients of ischemic stroke.

In our study of paraoxonase-2 C1053G gene polymorphism, we observed that CC genotype was more frequent than CG and GG genotypes in both the groups. However, the frequency of GG genotype was higher in group I (20%) compared to group II (5%). The odds ratio observed in our study for GG genotype was 1.3, indicating that the subjects with GG genotype may be at an increased risk of development of ischemic stroke compared to other genotypes, although it was statistically nonsignificant (P = 0.5). These findings are similar to those of the study carried out by Pasdar et al.,[3] where the authors observed that there was no significant genotypic association of paraoxonase-2 with ischemic stroke in Caucasians. However, a previous study in Asian Indians reported a significant (P = 0.01) association between paraoxonase-2 C1053G polymorphism and development of ischemic stroke.[6] The authors suggested that there is a significant positive association between the “G” allele and ischemic stroke, and that GG genotype is an independent risk factor for development of the disease.

In regard to expression of paraoxonase-2 gene at the mRNA level, we observed a significant (P = 0.0001) increase in the value of average △ CT in group I compared to group II. An increase in average △ CT values clearly shows that the paraoxonase-2 gene expression has been significantly decreased (~25-fold) in group I. While analyzing the intergenotypic variation, it was observed that paraoxonase-2 gene expression was significantly (P = 0.0001) decreased in GG genotype compared to CC and CG genotypes. This suggests that “GG” genotype might lead to decreased levels of mRNA of paraoxonase-2 gene in group I. This suggests the possible association of paraoxonase-2 gene and its reduced expression in GG genotype with pathogenesis of ischemic stroke. While the elevated levels of paraoxonase-2 mRNA in CC genotype might have a protective role against ischemic stroke, an association of the paraoxonase-2 C1053G polymorphism with decreased paraoxonase-2 activity has not been reported previously in Indian population. Stroke is a rising public health problem, since it is a foremost cause of long-term disability in industrialized countries.[17] Various researchers have postulated that risk factors for stroke, like smoking, hypertension, diabetes, and hypercholesterolemia, lead to production of ROS, resulting in increased oxidative stress.[18],[19] This promotes the modification of LDL, which is a major cause of plaque formation. This modification of LDL is prevented and reverted by paraoxonases.[20] Paraoxonase-3 and paraoxonase-1 both are associated with high-density lipoprotein (HDL) and show the capacity to delay LDL oxidation.[21] However, paraoxonase 2 is unique and different, and it is not associated with HDL.[21]


 » Conclusion Top


Thus, we conclude that GG genotype is associated with downregulated paraoxonase-2 gene expression at the mRNA level, which may be one of the major contributing factors to oxidative stress and pathophysiology of ischemic stroke. Further studies are necessary to confirm, evaluate, and replicate this study in a larger sample size with different ethnicities.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published, and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Acknowledgements

This work was supported by Lady Hardinge Medical College, Delhi, India.

Financial support and sponsorship

This work was supported by Lady Hardinge Medical College, Delhi, India.

Conflicts of interest

There are no conflicts of interest.



 
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Jamall S, Ishaq M, Ahmad S. Paraoxonase activity in patients with atherosclerosis. Pak J Biochem Mol Biol 2010;43:189-91.  Back to cited text no. 7
    
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Zhang G, Li W, Li Z, Lv H, Ren Y, Ma R, et al. Association between paraoxonase gene and stroke in the Han Chinese population. BMC Med Genet 2013;14:16. doi: 10.1186/1471-2350-14-16.  Back to cited text no. 9
    
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Levy E, Trudel K, Bendayan M, Seidman E, Delvin E, Elchebly M, et al. Biological role, protein expression, subcellular localization, and oxidative stress response of paraoxonase 2 in the intestine of humans and rats. Am J Physiol Gastrointest Liver Physiol 2007;293:G1252-61.  Back to cited text no. 10
    
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Guxens M, Tomás M, Elosua R, Aldasoro E, Segura A, Fiol M, et al. Association between paraoxonase-1 and paraoxonase-2 polymorphisms and the risk of acute myocardial infarction. Rev Esp Cardiol 2008;61:269-75.  Back to cited text no. 12
    
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Jayashree S, Arindam M, Vijay KV. Genetic epidemiology of coronary artery disease: An Asian Indian perspective. J Genet 2015;94:539-49.  Back to cited text no. 14
    
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Anand Babu K, Bharathi devi SR, Sripriya S, Sen P, Prakash VJ, Bindu A, et al. Serum paraoxonase activity in relation to lipid profile in age-related macular degeneration patients. Exp Eye Res 2016;152:100-12.  Back to cited text no. 15
    
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Précourt LP, Marcil V, Ntimbane T, Taha R, Lavoie JC, Delvin E, et al. Antioxidative properties of paraoxonase 2 in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 2012;303:G623-34.  Back to cited text no. 16
    
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Dhamija RK, Gaba P, Arora S, Kaintura A, Kumar M, Bhattacharjee J. Homocysteine and lipoprotein (a) correlation in ischemic stroke patients. J Neurol Sci 2009;281:64-8.  Back to cited text no. 17
    
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Rodríguez-Esparragón F, López-Fernández JC, Buset-Ríos N, García-Bello MA, Hernández-Velazquez E, Cappiello L, et al. Paraoxonase 1 and 2 gene variants and the ischemic stroke risk in Gran Canaria population: An association study and meta-analysis. Int J Neurosci 2017;127:191-8.  Back to cited text no. 21
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

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