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| ORIGINAL ARTICLE |
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| Year : 2019 | Volume
: 6
| Issue : 2 | Page : 53-57 |
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Impact of CD40 gene polymorphism on coronary artery disease in an Indian population: A pilot study
Sapna Singh, Manisha Naithani, Sarama Saha
Department of Biochemistry, AIIMS, Rishikesh, Uttarakhand, India
| Date of Web Publication | 18-Nov-2019 |
Correspondence Address:
Dr. Sarama Saha
Department of Biochemistry, AIIMS, Rishikesh, Uttarakhand
India
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/ami.ami_54_19
Context: Coronary artery disease (CAD) is number one killer in India. CD40/CD40 L may have crucial contribution in the development of CAD because of its dual prothrombotic and proinflammatory role. However, no study has been conducted to observe the effect of CD40 gene polymorphism on CAD in Indian populations. Aims: To determine the allelic frequency of CD40 gene and its influence on Indian individuals having unstable chest pain and cardiac muscle infarction which are variants of CAD. Settings and Design: A clinic-based observational study was conducted in Maulana Azad Medical College. Eighty angiographically proven patients of CAD and fifty healthy individuals registered for this study. Materials and Methods: Polymerase chain reaction and restriction fragment length polymorphism were used for studying CD40 gene polymorphism. Statistical Analysis Used: Frequency distribution of genotype was analyzed by standard Chi-square test using SPSS software version 21. Results: The C and T allele frequencies were 71.25% and 28.75%, respectively, in diseased individuals. Significant difference was observed among patients with CAD and healthy references regarding distribution of genotypes (χ2 = 10, P = 0.007) although allele frequencies (χ2 = 2.94, P = 0.09) of CD40 gene did not show any significance. The presence of C allele augmented the chance of having CADs compared to the wild type (odds ratio: 1.13; 95% confidence interval: 0.687–1.887). Conclusions: Important correlation was noted between rs1883832 C/T polymorphism of CD40 gene and risk of development of CADs among Indian populations. Keywords: CD40 L, coronary artery disease, restriction fragment length polymorphism, single-nucleotide polymorphism
How to cite this article:
Singh S, Naithani M, Saha S. Impact of CD40 gene polymorphism on coronary artery disease in an Indian population: A pilot study. Acta Med Int 2019;6:53-7 |
| Introduction | |  |
Coronary artery disease (CAD) is considered to be a serious health issue worldwide and primary killer in all over India. Approximately one-fourth of deaths due to CAD occur in the age group of 25–69 years, indicating its premature appearance and its severity.[1] High CAD rates in Indians are not fully explained by conventional risk factors.[2],[3] Diverse proinflammatory markers become evident as risk factors for CAD.[4] CD40/CD40 L may have crucial contribution in the development of CAD because of its dual prothrombotic and proinflammatory role.[5] Moreover, a strong association was documented between CD40 gene polymorphism and various diseases.[6],[7],[8] There was an evidence of a strong association of single-nucleotide polymorphism (SNP) in CD40 (-1C/T) gene with acute coronary syndrome in the Chinese population.[9] Moreover, the risk of disruption of unstable coronary atherosclerotic plaque is augmented by the presence of CC genotype of CD40 (-1C/T) gene polymorphism.[10] However, no such study has been documented in Indian populations. Furthermore, polymorphism varies as a function of ethnic backgrounds.[11],[12] Therefore, this study has been aimed to determine the allelic frequency of SNPs in human CD40 gene and its influence on Indian patients with unstable angina and myocardial infarction which are two variants of clinical presentation of CAD.
| Materials and Methods | | |
The present collaborative study was conducted in the Department of Biochemistry, Maulana Azad Medical College, New Delhi, India. Patients were enrolled from the Department of Cardiology of G. B. Pant Hospital, New Delhi. The study group included a total of 80 angiographically proven patients of CAD, of which 64 had unstable angina and 16 had acute MI. The patients presented with manifestations suggestive of unstable angina, including those with ischemic discomfort occurring at rest or with minimal exertion and usually lasting >20 min, ischemic discomfort of severe intensity described as commencement of frank pain within 1 month of presentation and it appears frequently, more severe in nature and persisting for longer time than earlier. Patients suffering from other heart diseases such as valvular heart disorder and cardiomyopathy and patients diagnosed with other diseases such as tumors, liver, lung, and renal diseases; acute infections; and chronic inflammatory diseases such as arthritis were excluded from the study. The study was performed following the rules and regulations stated in the Declaration of Helsinki and accepted by the Institutional Ethics Committee, and informed consent was taken from all participants recruited in this study.
After taking consent from all participants, the blood pressure of the participant was taken by a trained nurse after a rest period of 5 min. Following this, fasting venous blood was drawn from participants for the measurement of total cholesterol, high-density lipoprotein, low-density lipoprotein, urea, creatinine, and blood glucose using cholesterol oxidase–peroxidase, immunoinhibition method, enzymatic method, urease, Jaffe's reaction, and hexokinase method, respectively.
Angiographic vessel scoring was also done for all patients admitted in the Coronary Care Unit of G. B. Pant Hospital suspected of having unstable angina or myocardial ischemia. This score estimates the degree of stenosis, which correlates with severity of disease. The score was computed by assigning a severity score to each coronary stenosis according to the degree of luminal narrowing and number of arteries involved. Absent stenosis, mild stenosis in single artery having more than 50% stenosis, moderate stenosis with two arteries having more than 50% stenosis with no coronary lesions, and severe stenosis with three arteries having more than 50% stenosis were assigned vessel scores of 0, 1, 2, and 3, respectively.
Blood collection and genetic analysis
Peripheral venous blood (5 ml) was collected within 24 h of admission before any intervention or angioplasty in a tube containing ethylenediaminetetraacetic acid as an anticoagulant. Two milliliters of whole blood was used for total genomic DNA extraction using DNA extraction kit (Geneaid Biotech Ltd., Taiwan). A region of DNA having 5' untranslated region of CD40 gene was amplified by standard polymerase chain reaction (PCR). The primers used were as per the method described earlier.[13] PCR was conducted with reaction mixture including 4 μL isolated DNA, 10 μL master mix, 0.3 μL of each primer (forward: 5'-CCTCTTCCCCGAAGTCTTCC-3'; reverse, 5'-GAAACTCCTGCGCGGTGAAT-3'), and 12.4 μL nuclease free water. Thermocycling was carried out using initial denaturation at 94°C for the duration of 7 min, followed by 40 cycles at 94°C for 35 s; this was succeeded by 60°C for 45 s and 72°C for 45 s, and the final extension was done at 72°C for 10 min. The expanded product was restricted with 1 μL of the digestion enzyme NcoI (Fermentas Life sciences, India) in 31 μl volume for 15–20 min, according to the manufacturer's instructions. Restriction fragments were separated on 2.5% agarose gel and visualized by ethidium bromide ultraviolet illumination.
Statistical analysis
Statistical analysis was carried out using SPSS software version 21 (IBM, India). The data were presented as mean ± standard deviation. The frequency distribution of genotype was analyzed using classic Chi-square test. Continuous variables in [Table 1], [Table 2], [Table 3] were compared by ANOVA. Odds ratios (ORs: with 95% confidence intervals [CI]) were estimated to investigate the relative risk of developing CAD.P < 0.05 was accepted as statistically significant. | Table 1: Demographic profile and biochemical parameters of different genotypes among study participants
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 | Table 3: Blood levels of emerging parameters related to coronary artery disease among different genotypes
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| Results | | |
Identification of three distinct CD40 genotypes
Identification of genotypes was carried out by restriction fragment length polymorphism method. This was performed based on the digestion of PCR products by NcoI restriction endonuclease and separation of digested and undigested fragments by 2.5% agarose gel preparation. The uncut product of 302 base pairs represents that the T allele and the C allele have two fragments (169 and 133 bp) following digestion [Figure 1]. This resulted in three genotypes (CC, CT, and TT). Of 130 study participants, 44 (55.5%), 26 (35%), and 10 (12.5%) individuals having features of CAD belonged to CC, CT, and TT genotypes, respectively [Table 4]. The C allele and T allele frequencies were 71.25% and 28.75%, respectively, in diseased individuals. The distribution of alleles for cases is consistent with the Hardy–Weinberg equilibrium (χ2 = 3.42;P>.05). Significant difference was observed among patients with CAD and healthy references regarding distribution of genotypes (χ2 = 10;P= 0.007), whereas allele frequencies (χ2 = 2.94;P= 0.09) of CD40 gene were not significantly different. Interestingly, CT genotype was observed significantly higher in patients with CAD (35%) as compared to the control (14%). The C allele frequency of the patients with disease (CAD) was higher than that in the control (71.25% vs. 61%). The presence of C allele enhanced the chance of having CAD compared to the wild type (OR: 1.13; 95% CI: 0.687–1.887). | Figure 1: Agarose gel electrophoresis of PCR products from restriction fragment length polymorphism (Lane M = 100 bp DNA ladder, Lane 1 = PCR product, Lane 2, 5 = CC genotype, Lane 1,3,6 = CT genotype, Lane 4 = TT genotype). T = 302 bp, C = 169 and 133 bp. CC = 55.5%, CT = 35%, TT = 12.5% in cases. The C allele frequency = 71.25% and T allele frequency = 28.75% in diseased individuals
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 | Table 4: Genotype and allele frequencies of CD40 polymorphism in coronary artery disease patients and controls
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Clinical characteristics of the participants
Demographic profiles of these three genotypes (CC, CT, and TT) are presented in [Table 1]. There was no significant difference among these three genotypes. However, the CC genotype had slightly higher level of blood pressure, pulse rate, urea, creatinine, and random blood sugar.
Risk factors for coronary artery disease and genotype distribution
This study demonstrated that the CC genotype has the highest frequency of exposure to the risk factors such as smoking (54.4%), alcohol intake (52%), hypertension (67.7%), diabetes mellitus (82.4%), and family history of CAD (61.1%) as compared to other genotypes CT and TT [Table 2]. However, there was no significant difference among these three groups with respect to the risk factors except in case of exposure to hyperglycemia (P = 0.029). Moreover, thrombolysis in myocardial infarction score > 4 which is an important indicator of CAD has the highest frequency in CC genotype (88.6%) in comparison with CT (80.8%) and TT (70%) genotypes, respectively.
Relationship between different biomarkers for coronary artery disease and CD40 gene polymorphism
The blood levels of different important indicators of CAD (TroponinT, hs-CRP, ApoA-I, ApoB-100, and sCD40 L) in three genotypes are presented in [Table 3]. There was no significant difference with respect to these indicators of CAD among three genotypes (CC, CT, and TT). However, interestingly, hs-CRP, ApoB-100, and sCD40 L are to some extent higher in CT genotype compared to the other genotypes. Moreover, the CT genotype shows a slightly nonsignificant (P = 0.555) increased risk of CAD (OR: 1.023, 95% CI: 0.480–2.18) as compared to other genotypes (CC and TT).
| Discussion | | |
The SNP (C/T) of CD40 gene located at the Kozak consensus sequence, consisting of 6–8 nucleotides surrounding the initiation codon ATG, has been analyzed in this study. It is directly related to the CD40 protein expression. Recent studies reported that CD40/CD40 L interaction enhances the atherosclerotic processes through progression of the inflammatory responses.[13],[14],[15] Elevated soluble CD40 L is considered to be a marker of unstable CADs and accelerated plaque activity.[16] Therefore, changes in the Kozak sequence may be responsible for the altered CD40 protein production, resulting in susceptibility to develop various disease conditions, for example, Graves' diseases,[17] follicular lymphoma, and atherosclerotic disease progression, leading to CADs. Hence, this study has been carried out to observe the effect of CD40 gene polymorphism on CAD such as unstable angina in Indian populations.
Our study reveals that majority of the study participants both with CAD (55.5%) and reference healthy individuals (54%) belong to the CC genotype. The elevated frequency of C allele compared to the T allele (71.25% vs. 28.75%) and the C allele frequency in individuals with CAD compared to the control (71.25% vs. 61%) is in concordant with previous studies, showing that C allele has the highest frequency of distribution and strongly associated with unstable coronary atherosclerotic plaque. Moreover, the risk of disruption of the plaque is increased about 1.5 times in the presence of C allele compared to the control group.[10] The relationship between C allele frequency and the occurrence of CAD is independent of risk factors such as age, sex, hypertension, smoking, alcohol, and family history of CAD. Our data suggest that SNP of CD40 gene has an important contribution in the development of CADs such as unstable angina and myocardial infarction in the Indian population.
CD40, type I transmembrane protein belongs to tumor necrosis factor superfamily, acting as a receptor for CD40 L is a product of gene situated on long arm of chromosome 20 (20q13.12). It is expressed primarily on B cells, but also expressed on other cells such as epithelial cells, fibroblasts, endothelial cells, and platelets. CD40/CD40 L may take part in triggering the pathogenesis of atherothrombosis.[5] The current study documented an association between sCD40 level in individuals with CAD and the genotype at rs1883832 C/T polymorphism. The plasma level of sCD40 L was higher in individuals with heterozygous CT genotypes (3.12 ± 5.21 pg/ml) compared to the homozygous genotypes (CC: 2.41 ± 2.91 and TT: 2.75 ± 3.61 pg/ml). These findings are in the same line of the previous study conducted by Chen et al. where a significant association was reported between the heterozygous CT genotype and CD40 level in diseased individuals among Chinese populations.[18] Our results indicate that the CD40 gene polymorphism (C/T) may have an important contribution to the molecular mechanism of the pathophysiology of coronary atherosclerosis and may be considered as a promising genetic marker for screening of the individuals susceptible to the development of CAD among Indian populations.
Clinical implication and limitation
CAD, if it is not detected at the early stage and remains untreated, may lead to sudden cardiac arrest, one of the preventable leading causes of catatonia and fatality in modern advancement. Strength of the study which deserves attention is that the present study results related to the CD40 gene polymorphism (C/T) may help in early identification of Indian individuals susceptible to the evolution of CAD and thus reduce the catatonia and fatality through prior initiation of preventive management.
The main drawback of this study is its small sample size which may limit its accurate illustration of the results obtained from this study. Further studies with larger cohorts are warranted for better interpretation of the results with respect to the correlation of CD40 gene polymorphism and CAD among Indian populations. Second, this study recruited only North Indian populations and therefore limits the generalizability of these results.
| Conclusions | | |
Conclusion of the study could be the presence of important correlation between rs1883832 C/T polymorphism of CD40 gene and blood sCD40 level with respect to the risk of development of CADs among Indian populations. Moreover, the presence of C allele increases this risk of CAD manifestation such as unstable angina and myocardial infarction. These results also imply the necessity of conducting further studies with larger cohorts including different ethnic groups to have a better interpretation of relationship of CD40 gene polymorphism and the pathogenesis of coronary atherosclerosis in Indian populations.
Financial support and sponsorship
Maulana Azad Medical College, New Delhi, India.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1]
[Table 1], [Table 2], [Table 3], [Table 4]
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