This study enrolled 200 subjects divided into 2 groups. Group 1 included 100 patients (64 males and 36 females) presented with atherosclerotic ischemic stroke to Stroke Unit, Kasr El Aini Hospital, Cairo University, Egypt. Group 2 included 100 age and sex-matched individuals (61 males and 39 females) have more than one risk factor for atherosclerotic ischemic stroke with no evidence of cerebrovascular disease, ischemic heart diseases, or peripheral arterial diseases, recruited from Kasr El Aini outpatient clinic. The enrolment period extended from January 2017 to April 2019.
Hypertension was defined as systolic pressure > 140 mm Hg and diastolic pressure > 90 mm Hg on > 1 occasion, included patients currently taking hypertensive medications. Diabetes mellitus was defined as a fasting plasma glucose level > 126 mg/dL, included patients taking diabetic medications. Smoking refers to patients who currently smoke. Hyperlipidemia was defined as serum total cholesterol level ≥ 240 mg/d or an antihyperlipidemic agent treatment history.
Inclusion criteria: (1) Age ranging from 20 to 80 years. (2) Adult patients with atherosclerotic ischemic stroke whether males or females. Exclusion criteria: (1) Age less than 20 years. (2) Patients with cerebral hemorrhage, transient ischemic attack, cerebral embolism, cerebral trauma, cerebrovascular malformations. (3) Non-Egyptians.
All participants were subjected to full history: especially for risk factors for ischemic heart disease, peripheral artery disease, or cerebrovascular events as transient ischemic attack (TIA).
Clinical assessment: Eligible patients were defined as those who were diagnosed as acute ischemic stroke according to neurological examination and radiological imaging, including a sudden onset of focal neurological deficit for more than 24 h with corresponding infarction on brain imaging (computed tomography and magnetic resonance imaging). Patients were also subjected to the National Institutes of Health Stroke Scale (NIHSS)  scale on admission to quantifying the impairment and degree of severity caused by stroke. NIHSS is composed of 11 elements: level of consciousness, best gaze, visual, facial palsy, motor arm, motor leg, limb ataxia, sensory, best language, dysarthria, extinction, and inattention. For each item, a score of 0 typically indicates normal function, while a higher score indicates the level of impairment. These 11 components are summed then the score was correlated with stroke severity as follow: 0 = no stroke symptoms, 1-4 = minor stroke, 5-15 = moderate stroke, 16-20 = moderate to severe stroke, 21-42 = severe stroke.
Grading of large artery atherosclerosis ultrasound of the extra-cranial carotid system and intra-cranial vasculature was done to assess large vessel atherosclerosis of the cerebrovascular circulation. The machine used was the Philips iU22 (Philips iU22–series made in the USA) with a linear high frequency probe (5-12 MHz) for extra-cranial carotid system and sector low frequency probe (1-5 MHz) for transcranial examination.
Extra-cranial stenosis: Using the Von Reutern and colleagues criteria , low grade stenosis was 0-40%, moderate stenosis was 50-60%, and severe stenosis was ≥ 70%.
Intracranial stenosis: Based on a large multicenter study , we considered moderate stenosis of intracranial vessels if it was from 50-70% and severe if the stenosis was > 70%. The stenosis was based on mean flow velocity of large intracranial arteries. Presence of turbulence (in absence of anemia or hyperdynamic circulation) with no major hemodynamic affection was considered the mildest degree of stenosis.
Laboratory investigations: Random blood sugar and lipid profile were recruited from patients’ files.
Sample collection and DNA Extraction: Two milliliters of venous blood was withdrawn from all subjects and collected in a sterile vacutainer EDTA, for DNA extraction. GeneJET Genomic Purification Mini Kit (#K0781), Thermo Fisher, Life Technologies (Carlsbad, California 92008) was used for DNA extraction. Real-time PCR TaqMan probes allelic discrimination assays for Genotyping assay C > T variant of of miR-196a2 (rs11614913) and T > C variant of miR-149 (rs2292832) were determined using Custom TaqMan® SNP Genotyping Assays from Applied Biosystems and consequent analysis was performed on DNA-Technology DTlite Real-Time PCR Thermal Cycler (LLC Varshavskoe shosse, Moscow, Russia).
The context sequence for rs11614913 of miR-196a2 was
Forward 5′-TTTTGAACTCGGCAACAAGAAACTG-3′ [C/T]
Reverse 5′-CTGAGTTACATCAGTCGGTTTTCGT-3′. Variant: C/T, transition substitution.
The context sequence for rs2292832 of miR-149 was
Forward 5′-GGGACGGGGGCTGTGCTGGGGCAGC- 3′ [T/C]
Reverse 5′-GGAACAACGCAGGTCGCCGGGCCGG-3′ Variant: T/C, transition substitution.
All reactions were performed in a total volume of 20 μL containing 10 μL of master mix, 0.5 μL of SNP-readymade assay, 1-5 μL purified DNA solution according to DNA concentration, to be completed to 20 μL of nuclease-free water. (c) Allelic Discrimination Plate Read and Analysis: After PCR amplification, an endpoint plate read was performed using The DTlite master Software to plot fluorescence values based on the signals from each well which indicated allele’s type in sample. Automatic allele calls were made then converted to genotypes.
Data were entered and analyzed using the IBM-SPSS software (IBM Corp. Released 2017.IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.). Data was summarized using mean and standard deviation for quantitative variables, while frequencies (number of cases) and relative frequencies (percentages) for categorical variables. Comparisons between quantitative variables were done using unpaired T test. For comparing categorical data, chi-square test was performed. Exact test was used instead when the expected frequency is less than 5. Odds ratio (OR) with 95% confidence intervals was calculated . P values less than 0.05 were considered as statistically significant.