Stroke is a common acquired disorder with the highest mortality rate globally. It is of ischemic or hemorrhagic type . An ischemic type accounts for 85% of the affected stroke cases and is caused by thrombosis, embolism, or hypo-perfusion . Atherosclerosis is the leading cause of IS with multiple environmental and genetic interacting predisposing factors classified as modifiable and non-modifiable risk factors. The modifiable risk factors for atherosclerotic IS include smoking, HTN, hyperlipidemia, and DM .
Several susceptibility genes, such as non-modifiable risk factors, are linked to the risk and prognosis of stroke . miRNAs can involve in cell proliferation, apoptosis, and cellular hemostasis . SNVs of pre-miRNAs affect synthesis processing of miRNAs that may involve the action of Drosha and Dicer enzymes, with subsequent function disruption of the mature form of miRNA and emergence of various disorders . Genetic variants in miRNAs or their precursors, pre-miRNAs, play an essential role in IS pathogenesis and its risk factors susceptibility, including HTN, triglyceride disturbance, and DM . The rs2910164 C > G of pre-miRNA-146a and rs3746444 A > G of pre-miRNA-499 are related to several disorders like congenital heart disorder, coronary heart diseases, and malignancies . Both variants play a pivotal role in inflammatory stimulation proposing their effect on stroke risk [10, 30]. Our study confirmed their role in the risk of atherosclerotic IS in the Egyptian population. Both variants were investigated by real-time PCR. As well, CRP levels, as one of the genetic inducing mechanisms to IS, were analyzed by immunoturbidimetry.
Regarding rs3746444 A > G, our study revealed that the G allele and its involving genotypes demonstrated a higher IS risk. Combining the G allele of rs3746444 A > G with either C or G allele of rs2910164 C > G was associated with increased IS susceptibility.
There were increased frequencies of HTN in the GG genotype of rs3746444 A > G than in AG and AA genotypes (p = 0.028). No statistically significant difference was found regarding the genotypic frequencies to age, gender, smoking, DM, and hyperlipidemia. Also, no difference was detected regarding NIHH score and duplex atherosclerotic scoring, p > 0.05. There was a significant association of the genotypic variants GG, AG of rs3746444 A > G compared to the AA genotype with increased levels of CRP at a cut value of 7.5 mg/L.
The rs3746444 A > G of pre-miRNA-499 is present in 20q11.22 in the 3p strand of the mature form of miRNA. Its association with IS is linked to multiple inflammatory pathways with subsequent thrombosis susceptibility. Several inflammatory mediators are regulated by rs3746444, including IL-2, IL-6, IL-17 receptors, and B, T lymphocytes immune regulatory receptors . The rs3746444 A > G variant affects the expression of the mature form of miR-499a and the control of mRNA functions to influence IS susceptibility . The miR-499a disruption is found to be related to cellular hypoxic state and apoptosis .
CRP levels enhance one of the pathways of rs3746444 A > G, inducing inflammation. CRP was found to be raised in G-involved genotypes and could cause hypertension, hyperlipidemia, and insulin resistance with induction of cerebral anoxia . Another possible mechanism of rs3746444 A > G in IS maybe its linkage disequilibrium with other genetic variants involved in stroke pathogenesis .
Compatible to our findings, Luo et al.  studied both variants in IS versus normal controls with analysis of CRP in the Chinese population. They found that rs3746444 A > G, GG + AG genotypes and G allele demonstrated increased IS risk [(p = 0.027, OR = 1.621 and 95% CI = 1.079–2.516) and (p = 0.039, OR = 1.455 and 95% CI = 1.019–2.381) respectively]. Also, they detected higher levels of CRP in AG + GG genotypes than found in AA genotypes, p < 0.001.
In agreement with our results, Liu et al.  investigated both variants in IS compared to normal controls by PCR- Restriction Fragment Length Polymorphism (PCR-RFLP), and 10% of the results were confirmed by direct sequencing. For rs3746444 A > G, they reported that the G allele showed a significant linkage to IS risk (p = 0.003, OR = 1.509, 95% CI = 1.151–1.978). The GG genotype and AG + GG genotypes had an increased risk to IS than the AA genotype (p = 0.007, OR = 1.563, 95% CI = 1.135–2.153). However, they did not find an association between the genotypic frequencies with NIHH scores.
According to the current study, Li et al.  meta-analysis results for rs3746444 A > G in Chinese individuals demonstrated that the G allele and AG + GG genotypes had increased risk for IS with OR = 1.24 and 1.36, respectively.
In contrary to our findings, Liu et al.  did not found a reciprocal action between the genetic variants and environmental elements as the AG + GG genotypes increased IS risk more in the young, non-smoker, non-diabetic, non-hyperlipidemic, and non-hypertensive patients with [(OR = 1.89, 95% CI = 1.22–2.93), (OR = 1.88, 95%CI = 1.31–2.69), (OR = 1.60, 95%CI = 1.13–2.25), OR = 1.61, 95% CI = 1.09–2.38), (OR = 2.38, 95% CI = 1.44–3.91)] respectively.
Jeon et al.  investigated both variants in the Korean population by PCR-RFLP and confirmed their results by direct sequencing. In contrary to our findings, they reported that rs3746444 A > G polymorphism was not distinct between stroke cases and control subjects, besides its avoidance in association to HTN. However, in agreement with us, they detected no association between its genotypic variants and age, sex, DM, and hyperlipidemia with p > 0.05. Zou et al.  also reported no association of genotypic and allelic variants of rs3746444 A > G to IS risk.
For rs2910164 C > G, our finding revealed that the G allele carried a higher risk to IS. The combined GG + GC genotypic frequencies showed a borderline statistical significance to CC genotype frequency. No statistically significant difference was detected regarding demographic data, confounding factors to IS, stroke severity assessment scores, and CRP levels in the different genotypic variants. The combination of A allele of rs3746444 A > G with C allele of rs2910164 C > G was of a protective value to IS incidence.
The rs2910164 C > G of pre-miRNA 146a is in chromosome 5 in 3p mature miRNA regions . This variant leads to precursor hairpin mispairing that changes processing and reduces the production of the mature form . It is related to various human diseases, including vascular damage . Moreover, it regulates TNF-α through the TLR signaling pathway . TNF-α is linked to increased levels of plasminogen activator inhibitor-1 that disrupt the hemostatic balance . It affects atherosclerosis and IS risk by disrupting various inflammatory cytokines release, including IL-6, IL1, monocyte chemotactic protein 1, IL 8, and nuclear factor-Kappa B (NF-kB) .
Concomitant to our findings regarding rs2910164 C > G variant, Jeon et al.  reported the association of GG + GC and G allele to IS risk [(p = 0.051, OR = 1.275 and 95% CI = 0.999–1.628) and (p = 0.011, OR = 1.244 and 95% CI = 1.051–1.472)] independently. Unlikely, they revealed high demonstration of CC genotype in females, non-hypertensive, and non-diabetic patients [(AOR = 1.431 and 95% CI = 1.022–2.002), (AOR = 1.632 and 95% CI = 1.133–2.350) and (AOR = 1.355 and 95% CI 1.028–1.785)] respectively.
Consistent with the current study, Zou et al.  meta-analysis in Asian populations revealed that the GG variants of rs2910164 C > G associated with IS risk (OR = 1.20, 95% CI = 1.02–1.42, p = 0.03). Young et al.  demonstrated no association of CRP levels to different genetic variants of rs2910164 C > G, p = 0.436.
In contrast, Luo et al.  did not find any association between genotypic and allelic frequencies of rs2910164 C > G and IS with p > 0.05. However, in agreement with our results, they reported no association between the frequency of hyperlipidemia in the different genotypic types of both variants.
Contrary to our results, Qu et al.  stated that rs2910164 C > G variants were not associated with IS incidence. However, in the follow-up of their patients, they found that the GG genotype was associated with IS recurrence risk, with OR = 1.56 and p = 0.016.
Inconsistent with our finding, Liu et al.  and Zhu et al.  found no significant difference for rs2910164 C > G genotypic and allelic frequencies between the cases and controls, p ≥ 0.05 and 0.869 independently. Li et al.  meta-analysis study in Chinese ethnic showed that the genotypic and allelic variants of rs2910164 C > G were not associated with IS risk.
The disparity in results between prior studies and our research could be attributable to differences in study method, sample size, patient selection criteria, geographical characteristics, racial factors, and ethnicity.
The limitations of our study were due to foundation absence. It represents in the lacking of confirmatory method to our results that compensated by its verification by sequencing in the previous researches. Larger sample size is recommended in future research for validation of our results.