Effects of 469 E/K polymorphism of ICAM1 gene in ischemic stroke and its association with stroke severity and outcome

Pethusamy, et al.: Polymorphism of ICAM1 Gene in Ischemic Stroke Asian Journal of Medical Sciences | Jan 2021 | Vol 12 | Issue 1 3 Adhesion Molecule 1) also known as CD54 (Cluster of Differentiation 54) is a cell adhesion molecule and a surface glycoprotein of immunoglobulin superfamily,4 expressed in endothelial cells and cells of immune system. Ligand for ICAM-1 is an integrin superfamily member namely leukocyte function-associated antigen-1 (LFA-1), also known as CD11a/CD18. ICAM-1/LFA-1 interaction which is essential for leukocyte infiltration into tissue, cell proliferation and local inflammatory response; it is an important factor for thrombosis in ischemic stroke. SNP in the sixth exon of ICAM1 gene (rs5498) is also associated with stroke risk.5,6 Soluble intercellular adhesion molecule-1 (sICAM-1) is the circulating form of ICAM-1.7 Plasma sICAM-1 levels have been correlated with the risk of ischemic stroke,8 peripheral arterial disease9 and type 2 diabetes mellitus.10 Studying the effect of the ICAMI gene polymorphism can help to identify genetic profiles that are associated with high risk of stroke, along with the severity and outcome in acute ischemic stroke. Studies relating ICAM1 469 E/K polymorphism in ischemic stroke studies on Indian population have been sparse. Therefore, we have devised this study to understand the role of ICAM1 genetic polymorphism in ischemic stroke in north Indian population. MATERIALS AND METHODS This retrospective case control study was conducted in the Department of Biochemistry, Maulana Azad Medical College and the Department of Neurology, Govind Ballabh Pant Institute of Postgraduate Medical Education & Research, New Delhi from September 2012 to December 2014, after obtaining approval from the Institutional Ethical Committee (F.11/IEC/ MAMC/2012/130). The investigation conforms to the principles outlined in the Declaration of Helsinki. 50 adult patients (>18 years) of either sex, with history of ischemic stroke diagnosed by history and MRI brain were selected as cases. Patients presenting with hemorrhagic stroke, transient ischemic attack (TIA), cardio embolic stroke, history of fever in the recent past (1 week prior to stroke), rheumatological, autoimmune disease, or any kind of acute or chronic infection were excluded. Patients with impaired hepatic function or renal function or those patients on immunosuppressive therapy or regular analgesic intake were excluded. 100 age and sex matched healthy controls were also selected for the study. Written informed consent was obtained from all the study subjects. The study group was subjected to structured questionnaire (regarding demographic, medical, lifestyle and reproductive information). A detailed history and baseline National Institutes of Health Stroke Scale/Score (NIHSS)11 were used for assessment of clinical severity. Radiological severity was assessed by calculation of infarct volume by diffusion weighted magnetic resonance imaging (DWI). The etiological classification of acute ischemic stroke was done by TOAST (Trial of ORG 10172 in Acute Stroke Treatment) criteria.12 The outcome was measured at 3 months and 6 months in terms of stroke recurrence, death, disability, functional dependence and cognitive functioning. Disability was assessed by Modified Rankin scale13 and functional dependence was assessed by Barthel index.14 Cognitive functioning was assessed by Mini-Mental State Exam (MMSE). 15 Blood sampling for genetic analysis was done within three months after the onset of stroke. 5 mL of peripheral blood sample was collected in EDTA vial for DNA analysis. It was stored at -80 degree Celsius till further analysis. DNA from peripheral blood samples was extracted by genomic DNA Mini Kit (Genaid, Taiwan) according to the manufacturer’s instructions and was stored at -20 0C. ICAM1 469 E/K polymorphism was detected by Allele Specific PCR (GeneQTM Thermal Cycler, Bulldog Bio, Portsmouth, USA)using the following allele specific primers: G-allele specific forward primer: 5’-CACTCAAGGGGAGGTCAC CCGCG-3’ A-allele specific forward primer: 5 ’ -CACTCAAGGGGAGGTCA CCCGCA-3’ Common Reverse primer: 5 ’ -TTGTAGTCTGTATTTCTT GATCTT-3’ PCR conditions were: initial denaturation at 94oC for 10 minutes for 1 cycle, 35 cycles of denaturation at 94oC for 40 seconds, annealing at 55oC for 1 minute and extension at 72oC for 1 minute followed by final extension 72oC for 10 minutes 1 cycle. The amplified products were resolved using electrophoresis in 1.5% agarose gel. Product length obtained was of 249 bp (Figure 1). Statistical analysis The data entered in excel sheet was analysed using Statistical Package for the Social Sciences (SPSS) version 18 and VassarStats.net programs.Shapiro-Wilk test was employed to test for the normality of data; at p-value< 0.05, the null hypothesis that data is normally distributed was rejected. Parametric data were expressed in mean and standard deviation. Nonparametric data were expressed as median and quartiles. The continuous data was subjected to unpaired t-test. Dichotomous variables were analysed by Pethusamy, et al.: Polymorphism of ICAM1 Gene in Ischemic Stroke 4 Asian Journal of Medical Sciences | Jan 2021 | Vol 12 | Issue 1 Chi-square test. E allele was taken as a reference to compare the frequency of K allele.p-value< 0.05 was considered statistically significant.