Causal effects of immune cell surface antigens and functional outcome after ischemic stroke: a Mendelian randomization study.

Objective: While observational studies link immune cells with post-stroke functional outcome, the underlying immune mechanisms are not well understood. Immune cell surface antigens are actively involved in the biological behavior of immune cells, investigating immune cell surface antigens could deepen our comprehension of their role and biological processes in stroke recovery. Therefore, we aimed to investigate the immunological basis of stroke outcome by exploring the causal relationship between immune cell surface antigens and functional outcome after ischemic stroke in a Mendelian randomization study. Methods: Genetic variants related to immune cell surface antigens and post-stroke functional outcome were selected for two-sample Mendelian randomization (MR) analysis. 389 fluorescence intensities (MFIs) with surface antigens were included. Inverse variance weighted (IVW) modeling was used as the primary MR method to estimate the causal effect of exposure on the outcome, followed by several alternative methods and sensitivity analyses. Additional analysis of the association between immune cell surface antigens and risk of ischemic stroke for assessment of collider bias. Results: We found that suggestive associations between CD20 on switched memory B cell (OR = 1.16, 95% CI: 1.01-1.34, p = 0.036) and PDL-1 on monocyte (OR = 1.32, 95% CI: 1.04-1.66, p = 0.022) and poor post-stroke functional outcome, whereas CD25 on CD39+ resting Treg (OR = 0.77, 95% CI: 0.62-0.96, p = 0.017) was suggestively associated with good post-stroke functional outcome. Conclusion: The elevated CD20 on switched memory B cell, PDL-1 on monocyte, and CD25 on CD39+ resting Treg may be novel biomarkers and potential causal factors influencing post-stroke functional outcome.

Knockdown of lncRNA AU020206 could inhibit microglia apoptosis in ischemic stroke.

The diagnostic biomarkers associated with ischemic stroke (IS) that may have clinical utility remain elucidated. Thus, the potential functional lncRNAs in IS were explored. The Gene Expression Omnibus database provided the transcriptome profile of IS for download. WGCNA analysis and integrated bioinformatics were used to find genes that were differentially expressed (DEGs). The Starbase database created the lncRNA-based ceRNA network. In order to investigate the molecular mechanism and involved pathways of DEGs in IS, functional enrichment analysis was carried out. Using qRT-PCR, lncRNAs identified as putative IS biomarkers were confirmed to be expressed in a permanent middle cerebral artery occlusion (MCAO) model. Using the annexin V/PI apoptosis test, the amount of apoptosis in oxygen-glucose deprivation (OGD) cells was measured. A total of 1600 common differentially expressed - protein-coding RNA (DE-pcRNAs) and 26 DE-lncRNAs were identified. The results of enrichment analysis indicate that the cytokine may be regulated by common DE-pcRNAs and are vital in the progress of IS. A lncRNAs-mediated ceRNA network including lncRNAs AU020206, Brip1os, F630028O10Rik and 9530082P21Rik was constructed. The expression of these lncRNAs was significantly increased in MCAO model. Knockdown of lncRNA AU020206 inhibited microglia apoptosis in OGD cell model. We constructed a lncRNAs-mediated ceRNA network and found that lncRNA AU020206 inhibited microglia apoptosis in OGD cell model. These findings provided further evidence for the diagnosis and a novel avenue for targeted therapy of IS.

Molecular subtype identification of cerebral ischemic stroke based on ferroptosis-related genes.

Cerebral ischemic stroke (CIS) has the characteristics of a high incidence, disability, and mortality rate. Here, we aimed to explore the potential pathogenic mechanisms of ferroptosis-related genes (FRGs) in CIS. Three microarray datasets from the Gene Expression Omnibus (GEO) database were utilized to analyze differentially expressed genes (DEGs) between CIS and normal controls. FRGs were obtained from a literature report and the FerrDb database. Weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) network were used to screen hub genes. The receiver operating characteristic (ROC) curve was adopted to evaluate the diagnostic value of key genes in CIS, followed by analysis of immune microenvironment, transcription factor (TF) regulatory network, drug prediction, and molecular docking. In total, 128 CIS samples were divided into 2 subgroups after clustering analysis. Compared with cluster A, 1560 DEGs were identified in cluster B. After the construction of the WGCNA and PPI network, 5 hub genes, including MAPK3, WAS, DNAJC5, PRKCD, and GRB2, were identified for CIS. Interestingly, MAPK3 was a FRG that differentially expressed between cluster A and cluster B. The expression levels of 5 hub genes were all specifically highly in cluster A subtype. It is noted that neutrophils were the most positively correlated with all 5 real hub genes. PRKCD was one of the target genes of FASUDIL. In conclusion, five real hub genes were identified as potential diagnostic markers, which can distinguish the two subtypes well.

Machine learning identifies novel coagulation genes as diagnostic and immunological biomarkers in ischemic stroke.

BACKGROUND: Coagulation system is currently known associated with the development of ischemic stroke (IS). Thus, the current study is designed to identify diagnostic value of coagulation genes (CGs) in IS and to explore their role in the immune microenvironment of IS. METHODS: Aberrant expressed CGs in IS were input into unsupervised consensus clustering to classify IS subtypes. Meanwhile, key CGs involved in IS were further selected by weighted gene co-expression network analysis (WGCNA) and machine learning methods, including random forest (RF), support vector machine (SVM), generalized linear model (GLM) and extreme-gradient boosting (XGB). The diagnostic performance of key CGs were evaluated by receiver operating characteristic (ROC) curves. At last, quantitative PCR (qPCR) was performed to validate the expressions of key CGs in IS. RESULTS: IS patients were classified into two subtypes with different immune microenvironments by aberrant expressed CGs. Further WGCNA, machine learning methods and ROC curves identified ACTN1, F5, TLN1, JMJD1C and WAS as potential diagnostic biomarkers of IS. In addition, their expressions were significantly correlated with macrophages, neutrophils and/or T cells. GSEA also revealed that those biomarkers may regulate IS via immune and inflammation. Moreover, qPCR verified the expressions of ACTN1, F5 and JMJD1C in IS. CONCLUSIONS: The current study identified ACTN1, F5 and JMJD1C as novel coagulation-related biomarkers associated with IS immune microenvironment, which enriches our knowledge of coagulation-mediated pathogenesis of IS and sheds light on next-step in vivo and in vitro experiments to elucidate the relevant molecular mechanisms.

Inflammatory factors mediated the effect of air pollution on ischemic stroke: a two-step, mediation Mendelian randomization study.

OBJECTIVE: Numerous investigations have indicated a correlation between air pollution (AP) and an elevated ischemic stroke (IS) likelihood. The existing literature does not provide a consensus about the possible link between AP and IS. A two-sample Mendelian randomization (MR) analysis was utilized to systematically measure the causal link between AP and ischemic stroke. Furthermore, the mediating impact of inflammatory factors was also performed by a two-step MR. MATERIALS AND METHODS: A two-sample MR analysis was utilized to examine the AP impact on the incidence of IS. Additionally, a two-step MR approach was carried out to account for possible mediating variables. The indirect impact was determined by employing the product approach, which included multiplying the AP impact on inflammatory factors by the inflammatory factors' impacts on IS. The MR effect was identified through inverse variance-weighted (IVW) meta-analysis of each Wald Ratio. Additionally, complementary studies were conducted using the weighted median and MR-egger approaches. RESULTS: The IVW method with random effects showed that the per unit increase in genetically predicted PM2.5 was linked to the 0.362-fold elevated ischemic stroke risk (OR: 1.362, 95% CI: 1.032-1.796, p=0.029). Furthermore, the IVM technique, incorporating random effects, demonstrated that the per unit increase in genetically predicted PM2.5 was related to an elevated Interleukin (IL)-1ß risk (OR: 1.529, 95% CI: 1.191-1.963, p=0.001), IL-6 (OR: 1.498, 95% CI: 1.094-2.052, p=0.012) and IL-17 (OR: 1.478, 95% CI: 1.021-2.139, p=0.038). IL-1ß, IL-6, and IL-17 modulated the PM2.5 impact on ischemic stroke, while the proportion mediated by them was 59.5%. CONCLUSIONS: A positive correlation between genetically predicted PM2.5 levels and elevated ischemic stroke risk is mediated by IL-1ß, IL-6, and IL-17.

Association of single nucleotide polymorphisms in ITLN1 gene with ischemic stroke risk in Xi'an population, Shaanxi province.

Background: Ischemic stroke (IS) is the main cause of death and adult disability. However, the pathogenesis of this complicated disease is unknown. The present study aimed to assess the relationship between ITLN1 single nucleotide polymorphisms (SNPs) and the susceptibility to IS in Xi'an population, Shaanxi province. Methods: In this study, we designed polymerase chain reaction (PCR) primers located at -3,308 bp upstream of the transcription initiation site within promoter region of the ITLN1 gene. The target fragment was amplified by PCR and identified by agarose gel electrophoresis. Sanger sequencing was then performed in the samples extracted from a cohort comprising 1,272 participants (636 controls and 636 cases), and the obtained sequences were compared with the reference sequences available on the National Center for Biotechnology Information (NCBI) website to detect SNPs in the ITLN1 gene promoter region. Logistic regression analysis was employed to assess the relationship between ITLN1 polymorphisms and IS risk, with adjustments for age and gender. Significant positive results were tested by false-positive report probability (FPRP) and false discovery rate (FDR). The interaction among noteworthy SNPs and their predictive relationship with IS risk were explored using the Multi-Factor Dimensionality Reduction (MDR) software. Results: The results of Sanger sequencing were compared with the reference sequences on the NCBI website, and we found 14 SNPs in ITLN1 gene promoter satisfied Hardy-Weinberg equilibrium (HWE). Logistic regression analysis showed that ITLN1 was associated with a decreased risk of IS (rs6427553: Homozygous C/C: adjusted OR: 0.69, 95% CI [0.48-0.97]; Log-additive: adjusted OR: 0.83, 95% CI [0.70-0.98]; rs7411035: Homozygous G/G: adjusted OR: 0.66, 95% CI [0.47-0.94]; Dominant G/T-G/G: adjusted OR: 0.78, 95% CI [0.62-0.98]; Log-additive: adjusted OR: 0.81, 95% CI [0.69-0.96]; rs4656958: Heterozygous G/A: adjusted OR: 0.74, 95% CI [0.59-0.94]; Homozygous A/A: adjusted OR: 0.51, 95% CI [0.31-0.84]; Dominant G/A-A/A: adjusted OR: 0.71, 95% CI [0.57-0.89]; Recessive A/A: adjusted OR: 0.59, 95% CI [0.36-0.96]; Log-additive: adjusted OR: 0.73, 95% CI [0.61-0.88]), especially in people aged less than 60 years and males. Conclusions: In short, our study revealed a correlation between ITLN1 variants (rs6427553, rs7411035 and rs4656958) and IS risk in Xi'an population, Shaanxi province, laying a foundation for ITLN1 gene as a potential biomarker for predicting susceptibility to IS.

Upregulation of miR-499a Targets Beclin1 to Modulate Astrocyte Autophagy and Promote Ischemic Stroke.

OBJECTIVE: To investigate the clinical significance of miR-499a expression in the serum of ischemic stroke patients and its potential mechanism in regulating astrocytes to promote ischemic stroke. METHODS: Serum samples from 99 ischemic stroke patients and 99 healthy individuals were collected and analyzed for miR-499a expression through RT-PCR. Statistical analysis was performed to compare the expression differences between the two groups, and correlation between miR-499a expression and clinical pathological indices in stroke patients was analyzed. MiR-499a mimic, inhibitor, and negative control vectors were constructed and transfected into astrocyte SVGp12 cells. Afterward, miR-499a expression was validated by RT-PCR, cell viability was assessed by CCK8 assay, and apoptosis was detected using flow cytometry. The binding sites of miR-499a and Beclin1 were predicted by the Target-scan database and confirmed by dual luciferase assay. After overexpressing Beclin1, co-transfection with miR-499a mimic or negative control was conducted to observe the reverse effect of miR-499a mimic on Beclin1 overexpression. RESULTS: MiR-499a was significantly upregulated in the stroke group (p<0.001), it was positively correlated with TC (Total Cholesterol), LDL-C (Low-density lipoprotein cholesterol), and APO-A1 (Apolipoprotein A1) (R2>0.3, p<0.001). MiR-499a mimics promoted cell viability while inhibiting apoptosis of astrocytes. MiR-499a targeted Beclin 1 and inhibited its mRNA and protein expression, as well as the expression of autophagy-related proteins LC-3 and p62. MiR-499a could reverse the impact of Beclin1 overexpression on SVGp12 astrocyte proliferation and apoptosis. CONCLUSION: Serum miR-499a in stroke patients may serve as a potential diagnostic indicator. MiR-499a-mediated inhibition of Beclin 1, subsequently leading to suppression of astrocytic autophagy and viability, may represent a pivotal mechanism underlying its promotion of IS.

Stroke genetics and how it Informs novel drug discovery.

INTRODUCTION: Stroke is one of the main causes of death and disability worldwide. Nevertheless, despite the global burden of this disease, our understanding is limited and there is still a lack of highly efficient etiopathology-based treatment. It is partly due to the complexity and heterogenicity of the disease. It is estimated that around one-third of ischemic stroke is heritable, emphasizing the importance of genetic factors identification and targeting for therapeutic purposes. AREAS COVERED: In this review, the authors provide an overview of the current knowledge of stroke genetics and its value in diagnostics, personalized treatment, and prognostication. EXPERT OPINION: As the scale of genetic testing increases and the cost decreases, integration of genetic data into clinical practice is inevitable, enabling assessing individual risk, providing personalized prognostic models and identifying new therapeutic targets and biomarkers. Although expanding stroke genetics data provides different diagnostics and treatment perspectives, there are some limitations and challenges to face. One of them is the threat of health disparities as non-European populations are underrepresented in genetic datasets. Finally, a deeper understanding of underlying mechanisms of potential targets is still lacking, delaying the application of novel therapies into routine clinical practice.

The Impact of PCSK9 Gene Polymorphisms on Ischemic Stroke: A Systematic Review and Meta-Analysis.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene are known to be associated with susceptibility to several cerebrovascular diseases, including ischemic stroke (IS). The aims of this study was to evaluate associations between PCSK9 gene polymorphisms and the risk of IS. Based on previous reports linking PCSK9 SNPs to plasma lipid levels and to atherosclerosis, and to inconsistencies in the reported associations between the SNPs, plasma lipid levels and IS risk, we choose the PCSK9 rs505151, rs529787, and rs17111503 to performe the association analysis. METHODS: Using multiple databases, all relevant case-control and cohort studies that matched our search criteria were collected. Quality assessment of included studies was performed using the Newcastle-Ottawa Scale. Demographic and genotype data were extracted from each study, and meta-analysis was performed using Stata/MP 17.0. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using fixed and random effects models. RESULTS: A critical evaluation was conducted on ten case-control studies, involving a total of 2426 cases and 2424 controls. Pooled results from the allelic models indicated the PCSK9 rs505151 G allele (OR: 1.41, 95% CI: 1.06-1.87, p = 0.019, I2 = 53.9%) and the PCSK9 rs17111503 A allele (OR: 1.38, 95% CI: 1.22-1.55, p < 0.001, I2 = 43.5%) were significantly associated with IS. Study qualities ranged from moderate (n = 4) to good (n = 6). Begg's and Egger's tests results indicated there was no evidence of publication bias in the findings (p > 0.05). CONCLUSIONS: This meta-analysis demonstrated that G allele variant of PCSK9 rs505151 and A allele variant of PCSK9 rs17111503 were associated with an increased risk of IS. Based on our findings, these SNPs could serve as potential targets for the diagnosis and treatment of IS. The integration of information on genetic polymorphism into IS risk prediction model may be beneficial in routine clinical practice.

The lncRNA lnc_AABR07044470.1 promotes the mitochondrial-damaged inflammatory response to neuronal injury via miR-214-3p/PERM1 axis in acute ischemic stroke.

PURPOSE: We investigated the role of lnc_AABR07044470.1 on the occurrence and development of acute ischemic stroke (AIS) and neuronal injury by targeting the miR-214-3p/PERM1 axis to find a novel clinical drug target and prediction and treatment of AIS. METHODS: The mouse AIS animal model was used in vivo experiments and hypoxia/reoxygenation cell model in vitro was established. Firstly, infarction volume and pathological changes of mouse hippocampal neurons were detected using HE staining. Secondly, rat primary neuron apoptosis was detected by flow cytometry assay. The numbers of neuron, microglia and astrocytes were detected using immunofluorescence (IF). Furthermore, binding detection was performed by bioinformatics database and double luciferase reporter assay. Lnc_AABR07044470.1 localization was performed using fluorescence in situ hybridization (FISH).Lnc_AABR07044470.1, miR-214-3pand PERM1mRNA expression was performed using RT-qPCR. NLRP3, ASC, Caspase-1 and PERM1 protein expression was performed using Western blotting. IL-1ß was detected by ELISA assay. RESULTS: Mouse four-vessel occlusion could easily establish the animal model, and AIS animal model had an obvious time-dependence. HE staining showed that, compared with the sham group, infarction volume and pathological changes of mouse hippocampal neurons were deteriorated in the model group. Furthermore, compared with the sham group, neurons were significantly reduced, while microglia and astrocytes were significantly activated. Moreover, the bioinformatics prediction and detection of double luciferase reporter confirmed the binding site of lnc_AABR07044470.1 to miR-214-3p and miR-214-3p to Perm1. lnc_AABR07044470.1 and PERM1 expression was significantly down-regulated and miR-214-3pexpression was significantly up-regulated in AIS animal model in vivo. At the same time, the expression of inflammasome NLRP3, ASC, Caspase-1 and pro-inflammatory factor IL-1ß was significantly up-regulated in vivo and in vitro. The over-expression of lnc_AABR07044470.1 and miR-214-3p inhibitor could inhibit the neuron apoptosis and the expression of inflammasome NLRP3, ASC, Caspase-1 and pro-inflammatory factor IL-1ß and up-regulate the expression of PERM1 in vitro. Finally, over-expression of lnc_AABR07044470.1 and miR-214-3p inhibitor transfected cell model was significant in relieving the AIS and neuronal injury. CONCLUSION: Lnc_AABR07044470.1 promotes inflammatory response to neuronal injury via miR-214-3p/PERM1 axis in AIS.

Bioinformatics identification of potential biomarkers and therapeutic targets for ischemic stroke and vascular dementia.

Ischemic stroke and vascular dementia, as common cerebrovascular diseases, with the former causing irreversible neurological damage and the latter causing cognitive and memory impairment, are closely related and have long received widespread attention. Currently, the potential causative genes of these two diseases have yet to be investigated, and effective early diagnostic tools for the diseases have not yet emerged. In this study, we screened new potential biomarkers and analyzed new therapeutic targets for both diseases from the perspective of immune infiltration. Two gene expression profiles on ischemic stroke and vascular dementia were obtained from the NCBI GEO database, and key genes were identified by LASSO regression and SVM-RFE algorithms, and key genes were analyzed by GO and KEGG enrichment. The CIBERSORT algorithm was applied to the gene expression profile species of the two diseases to quantify the 24 subpopulations of immune cells. Moreover, logistic regression modeling analysis was applied to illustrate the stability of the key genes in the diagnosis. Finally, the key genes were validated using RT-PCR assay. A total of 105 intersecting DEGs genes were obtained in the 2 sets of GEO datasets, and bioinformatics functional analysis of the intersecting DEGs genes showed that GO was mainly involved in the purine ribonucleoside triphosphate metabolic process，respiratory chain complex，DNA-binding transcription factor binding and active transmembrane transporter activity. KEGG is mainly involved in the Oxidative phosphorylation, cAMP signaling pathway. The LASSO regression algorithm and SVM-RFE algorithm finally obtained three genes, GAS2L1, ARHGEF40 and PFKFB3, and the logistic regression prediction model determined that the three genes, GAS2L1 (AUC: 0.882), ARHGEF40 (AUC: 0.867) and PFKFB3 (AUC: 0.869), had good diagnostic performance. Meanwhile, the two disease core genes and immune infiltration were closely related, GAS2L1 and PFKFB3 had the highest positive correlation with macrophage M1 (p < 0.001) and the highest negative correlation with mast cell activation (p = 0.0017); ARHGEF40 had the highest positive correlation with macrophage M1 and B cells naive (p < 0.001), the highest negative correlation with B cell memory highest correlation (p = 0.0047). RT-PCR results showed that the relative mRNA expression levels of GAS2L1, ARHGEF40, and PFKFB3 were significantly elevated in the populations of both disease groups (p < 0.05). Immune infiltration-based models can be used to predict the diagnosis of patients with ischemic stroke and vascular dementia and provide a new perspective on the early diagnosis and treatment of both diseases.

Identification of significant m6A regulators and immune microenvironment characterization in ischemic stroke.

The role of m6A modification in the regulation of the immune microenvironment (IME) of ischemic stroke (IS) is barely known. Thus, we aim to investigate the impact of m6A modification on the IME of IS and its diagnostic value in IS. We comprehensively assessed the m6A modification patterns, the relationship between these modification patterns and the characteristics of the IME. The m6A modification patterns of individual IS sample were quantified by m6Ascore. The performance of m6A phenotype-related genes as potential biomarkers was evaluated by the area under the receiver operating characteristic curve. Experimental validation was also performed by qRT-PCR. Six dysregulated m6A regulators were identified and a classification model consisting of four key m6A regulators (METLL3, RBMX, RBM15B, YTDHF3) could distinguish IS and healthy control samples well. METTL3 and YTHDF3 are closely related to circulating neutrophil abundance. Two distinct m6A modification patterns were determined which differed in immunocyte abundance. We also identified six m6A phenotype-related genes (APOBEC3A, PTMA, FCGR3A, LOC440926, LOC649946, and FTH1L11), and further explored their biological function. Among them, APOBEC3A, FCGR3A, and FTH1L11 were positively associated with neutrophil abundance. APOBEC3A and FCGR3A were stable diagnostic m6A-associated genes in both the discovery and validation cohorts. This study reveals that m6A modification plays a non-negligible role in the formation of a diversified and complex IME in IS. The m6A phenotype-related genes could be diagnostic biomarkers of IS.

Causal relationship between lactate dehydrogenase and risk of developing ischemic stroke: A Mendelian randomized study.

BACKGROUND AND OBJECTIVE: Ischemic stroke (IS) is one of the major global health problems. It is not clear whether there is a causal relationship between lactate dehydrogenase (LDH) and the risk of IS attacks. The purpose of this study was to investigate whether LDH has a causal relationship with the development of IS. METHODS: The genome-wide association data of LDH and IS were obtained through a Mendelian randomization-based platform. Single nucleotide polymorphisms (SNP) that were significantly associated with LDH were identified and used as instrumental variables, and a two-sample Mendelian randomization study was used to examine the causal relationship between LDH and IS. The statistical methods included Inverse-variance weighted approach, MR-Egger regression, and weighted median estimator. RESULTS: We selected 15 SNPs of genome-wide significance from Genome-wide association study database with LDH as instrumental variables. A consistent causal association between LDH and IS was observed by different assessment methods. The results of the inverse-variance weighted method suggested an inverse association between LDH and higher genetic predictability of IS risk (OR, 0.997; 95%CI 0.995-0.999). The weighted median estimate showed consistent results with the MR-Egger method (weighted median estimate: OR, 0.995; 95%CI 0.992-0.999; MR-Egger method: OR, 0.996; 95%CI 0.992-0.999). The inverse-variance weighted method indicates a causal association between LDH and IS (ß = -0.002563, SE = 0.00128, p = .0453). MR-Egger analysis (ß = -0.004498, SE = 0.001877, p = .03) and the weighted median method suggested that LDH and IS also existed causal relationship (ß = -0.004861, SE = 0.001801, p = .00695). CONCLUSIONS: Our Mendelian randomization results suggest that LDH is inversely associated with the risk of developing IS, and are contrary to the results of previous observational studies.

lncRNA-MIAT rs9625066 polymorphism could be a potential biomarker for ischemic stroke.

BACKGROUND: Ischemic stroke (IS) is a common and serious neurological condition that is highly fatal but so far no early diagnostic markers are available. Myocardial infarction-associated transcript (MIAT) is a long non-coding RNA (lncRNA) that could lead to IS by inducing autophagy and apoptosis in neuronal cells. However, there has been no report on the link between susceptibility to IS and the single-nucleotide polymorphisms (SNPs) of MIAT. This study aimed to investigate the association between MIAT gene polymorphisms and IS risk. METHODS: A total of 320 IS patients and 310 age-, sex- and race-matched controls were included in this study. Four polymorphisms (rs2157598, rs5761664, rs1894720, and rs9625066) were genotyped by using SNPscan technique. RESULTS: Among the 4 polymorphisms of MIAT, only rs9625066 was associated with IS risk (CA vs. CC: adjusted OR = 0.55, 95% CI, 0.37-0.85, P = 0.006; AA vs. CC: adjusted OR = 0.39, 95% CI, 0.16-0.94, P = 0.036; (AA + CA vs. CC: adjusted OR = 0.53, 95% CI, 0.35-0.80, P = 0.002; A vs. C adjusted OR = 0.59, 95% CI, 0.42-0.82, P = 0.002). Haplotype analysis showed a 1.32-fold increase (95% CI, 1.05-1.67, P = 0.017) in IS risk for rs2157598-rs5761664-rs1894720-rs9625066 (A-C-G-C). Logistic regression analysis identified some independent impact factors for IS including rs9625066 AA/AC, TC, TG, HDL-C (P < 0.05). CONCLUSION: The rs9625066 polymorphism of MIAT might be associated with IS susceptibility in Chinese population, in which AA/CA plays a protective role in IS, whereas the CC genotype increases the risk of developing IS, suggesting it might be a marker predictive of IS risk.

Prevalence of Fabry disease and GLA variants in young patients with acute stroke: The challenge to widen the screening. The Fabry-Stroke Italian Registry.

BACKGROUND: Fabry disease (FD) is a treatable X-linked lysosomal storage disorder caused by GLA gene variants leading to alpha-galactosidase A deficiency. FD is a rare cause of stroke, and it is still controversial whether in stroke patients FD should be searched from the beginning or at the end of the diagnostic workup (in cryptogenic strokes). METHODS: Fabry-Stroke Italian Registry is a prospective, multicentric screening involving 33 stroke units. FD was sought by measuring α-galactosidase A activity (males) and by genetic tests (males with reduced enzyme activity and females) in patients aged 18-60 years hospitalized for TIA, ischemic stroke, or intracerebral hemorrhage. We diagnosed FD in patients with 1) already known pathogenic GLA variants; 2) novel GLA variants if additional clinical, laboratory, or family-derived criteria were present. RESULTS: Out of 1906 patients, we found a GLA variant in 15 (0.79%; 95%CI 0.44-1.29) with a certain FD diagnosis in 3 (0.16%; 95%CI 0.03-0.46) patients, none of whom had hemorrhage. We identified 1 novel pathogenic GLA variant. Ischemic stroke etiologies in carriers of GLA variants were: cardioaortic embolism (33%), small artery occlusion (27%), other causes (20%), and undetermined (20%). Mild severity, recurrence, previous TIA, acroparesthesias, hearing loss, and small artery occlusion were predictors of GLA variant. CONCLUSION: In this large multicenter cohort the frequency of FD and GLA variants was consistent with previous reports. Limiting the screening for GLA variants to patients with cryptogenic stroke may miss up to 80% of diagnoses. Some easily recognizable clinical features could help select patients for FD screening.

CircSKA3 is Associated With the Risk of Extracranial Artery Stenosis and Plaque Instability Among Ischemic Stroke Patients.

Circular RNA circSKA3 (spindle and kinetochore-related complex subunit 3) has been identified as a prognostic factor in ischemic stroke. The objective of this study was to investigate the association of circSKA3 with the risk of extracranial artery stenosis (ECAS) and plaque instability in patients with ischemic stroke. We constructed a competing endogenous RNA (ceRNA) network regulated by circSKA3 based on differentially expressed circRNAs and mRNAs between five patients and five controls. Gene Ontology (GO) analysis was performed on the 65 mRNAs within the network, revealing their primary involvement in inflammatory biological processes. A total of 284 ischemic stroke patients who underwent various imaging examinations were included for further analyses. Each 1 standard deviation increase in the log-transformed blood circSKA3 level was associated with a 56.3% increased risk of ECAS (P = 0.005) and a 142.1% increased risk of plaque instability (P = 0.005). Patients in the top tertile of circSKA3 had a 2.418-fold (P < 0.05) risk of ECAS compared to the reference group (P for trend = 0.02). CircSKA3 demonstrated a significant but limited ability to discriminate the presence of ECAS (AUC = 0.594, P = 0.015) and unstable carotid plaques (AUC = 0.647, P = 0.034). CircSKA3 improved the reclassification power for ECAS (NRI: 9.86%, P = 0.012; IDI: 2.97%, P = 0.007) and plaque instability (NRI: 36.73%, P = 0.008; IDI: 7.05%, P = 0.04) beyond conventional risk factors. CircSKA3 played an important role in the pathogenesis of ischemic stroke by influencing inflammatory biological processes. Increased circSKA3 was positively associated with the risk of ECAS and plaque instability among ischemic stroke patients.

Expression, Functional Polymorphism, and Diagnostic Values of MIAT rs2331291 and H19 rs217727 Long Non-Coding RNAs in Cerebral Ischemic Stroke Egyptian Patients.

Cerebral ischemic stroke (CIS) is a severe cerebral vascular event. This research aimed to evaluate the role of single-nucleotide polymorphisms (SNPs) of the lncRNAs MIAT rs2331291 and H19 rs217727 and epigenetic methylation in the expression patterns of serum lncRNA H19 in CIS Egyptian patients. It included 80 CIS cases and 40 healthy subjects. Serum MIAT expression levels decreased, whereas serum H19 expression levels increased among CIS compared to controls. For MIAT rs2331291, there were significant differences in the genotypic and allelic frequencies between the CIS and healthy subjects at p = 0.02 and p = 0.0001, respectively. Our findings illustrated a significantly increased MIAT T/T genotype frequency in hypertensive CIS compared to non-hypertensive CIS at p = 0.004. However, H19 rs217727 gene frequency C/C was not significantly higher in non-hypertensive CIS than in hypertensive CIS. The methylation of the H19 gene promoter was significantly higher in CIS patients compared to healthy subjects. The level of MIAT was positively correlated with serum H19 in CIS. Receiver operating characteristics (ROC) analysis revealed that serum MIAT and H19 have a high diagnostic potential for distinguishing CIS subjects from healthy ones. In conclusion, the MIAT-rs2331291 polymorphism might serve as a novel potential indicator of CIS.

Novel diagnostic biomarkers of oxidative stress, ferroptosis, immune infiltration characteristics and experimental validation in ischemic stroke.

Ischemic stroke (IS) is a prominent type of cerebrovascular disease leading to death and disability in an aging society and is closely related to oxidative stress. Gene expression profiling (GSE222551) was derived from Gene Expression Omnibus (GEO), and 1934 oxidative stress (OS) genes were obtained from the GeneCards database. Subsequently, we identified 149 differentially expressed genes related to OS (DEOSGs). Finally, PTGS2, FOS, and RYR1 were identified as diagnostic markers of IS. Moreover, GSE16561 was used to validate the DEOSGs. Two diagnostic genes (PTGS2 and FOS) were significantly highly expressed, while RYR1 was significantly lowly expressed in the IS group. Remarkably, immune infiltration characteristics of these three genes were analyzed, and we found that PTGS2, FOS, and RYR1 were mainly correlated with Mast cells activated, Neutrophils, and Plasma cells, respectively. Next, we intersected three DEOSGs with the ferroptosis gene set, the findings revealed that only PTGS2 was a differentially expressed gene of ferroptosis. High PTGS2 expression levels in the infarcted cortex of middle cerebral artery occlusion (MCAO) rats were confirmed by immunofluorescence (IF), western blotting (WB), and Immunohistochemistry (IHC). Inhibition of PTGS2 clearly improved the neurological outcome of rats by decreasing infarct volume, neurological problems, and modified neurological severity scores following IS compared with the controls. The protective effect of silencing PTGS2 may be related to anti-oxidative stress and ferroptosis. In conclusion, this work may provide a new perspective for the research of IS, and further research based on PTGS2 may be a breakthrough.

Identification of diagnostic signatures for ischemic stroke by machine learning algorithm.

OBJECTIVE: Ischemic stroke (IS) is one of the major diseases threatening human health and survival and a leading cause of acquired mortality and disability in adults. The aim of this study was to screen diagnostic features of IS and to explore the characteristics of immune cell infiltration in IS pathogenesis. METHODS: The microarray data of IS (GSE16561, GSE58294, GSE37587, and GSE124026) in the GEO database were merged after removing the batch effect. Then integrated bioinformatic analysis and machine-learning strategies were adopted to analyze the functional correlation and select diagnostic signatures. The WGCNA was used to identify the co-expression modules related to IS. The CIBERSORT algorithm was performed to assess the inflammatory state of IS and to investigate the correlation between diagnostic signatures and infiltrating immune cells. RESULTS: Functional analysis of dysregulated genes showed that immune response-regulating signaling pathway and pattern recognition receptor activity were enriched in the pathophysiology of IS. The turquoise module was identified as the significant module with IS. By using Lasso and SVM-RFE learning methods, we finally obtained four diagnostic genes, including LAMP2, CR1, CLEC4E, and F5. The corresponding results of AUC of ROC prediction model in training and validation cohort were 0.954 and 0.862, respectively. The immune cell infiltration analysis suggested that plasma cells, resting and activated NK cells, activated dendritic cells, memory B cells, CD8+ T cells, naïve CD4+ T cells, and resting mast cells may be involved in the development of IS. Additionally, these diagnostic signatures might be correlated with multiple immune cells in varying degrees. CONCLUSION: We identified four biologically relevant genes (LAMP2, CR1, CLEC4E, and F5) with diagnostic effects for IS, our results further provide novel insights regarding molecular mechanisms associated with various immune cells that related to IS for future investigations.

The Association Between Thymidylate Synthase Gene Polymorphisms and the Risk of Ischemic Stroke in Chinese Han Population.

Family history of hypertension, smoking, diabetes and alcohol consumption and atherosclerotic plaque were identified as common risk factors in IS. We aimed at investigating the relationship between Thymidylate Synthase (TS) gene polymorphisms and ischemic stroke (IS).This case-control research selected and genotyped three single nucleotide polymorphisms (SNPs)of TS( rs699517, rs2790, and rs151264360) with Sanger sequencing in Chinese Han population. We also adopted logistic regression analysis in genetic models for calculating odds ratios and 95% confidence intervals. Genotype-Tissue Expression(GTEx) database analyzed the tissue-specific expression and TS polymorphisms. The ischemic stroke patients showed higher low-density lipoprotein cholesterol and total homocysteine (tHcy). It was found that patients with the TT genotype of rs699517 and GG genotype of rs2790 had larger degrees of tHcy than those with CC + CT genotypes and AA + AG genotypes, respectively. The genotype distribution of the three SNPs did not deviate from Hardy-Weinberg equilibrium (HWE). Haplotype analysis showed that T-G-del was the major haplotype in IS, and C-A-ins was the major haplotype in controls. GTEx database indicated that the rs699517 and rs2790 increased the expression of TS in healthy human and associated with TS expression level in a single tissue. In conclusion: This study has shown that TS rs699517 and rs2790 were significantly related to ischemic stroke patients.