Variants in 3p24.3 predicts the risk of early neurological deterioration in large artery atherosclerotic stroke.

The rate of early neurological deterioration (END) differs in different subtypes of ischaemic stroke. Previous studies showed PLCL2 gene is a novel susceptibility locus for the occurrence of atherosclerosis and thrombotic events. The objective of this research is to examine the efficacy that PLCL2 may have on the risk of END in large artery atherosclerotic (LAA) stroke. Tagged single nucleotide polymorphisms (SNPs) were identified by a strategy of fine-mapping. The genotyping of the selected SNPs was performed by SNPscan. The impact of PLCL2 on indicating the susceptibility of END in LAA patients was evaluated by binary logistic regression. The SNP-SNP interactions of PLCL2 for END was assessed by generalized multifactor dimensionality reduction (GMDR). A total of 1527 LAA stroke patients were recruited, 582 patients (38 %) experienced END. Compared to participants without END, participants experienced END were much older (P = 0.018), more likely to suffer pre-existing diabetes mellitus (P = 0.036), higher frequent in active tobacco users (P = 0.022) and had much higher median NIHSS on admission (P < 0.001). Rs4685423 was identified to be a predictor to the risk of END: the frequency of END in AA genotype patients is lower than that in AC or CC genotype patients (multivariate-adjusted, OR 0.63; 95 % CI 0.49-0.80; P < 0.001). The SNP-SNP interactions analysis indicates rs4685423 has the greatest impacton the risk of END for LAA patients. The time from admission diagnosis to END onset in AA genotype patients is much later than that in CA or CC genotype patients (log-rank, P = 0.005). In summary, the PLCL2 rs4685423 SNP is probably associated with the END risk in LAA stroke patients.

Apolipoprotein E genotypes were not associated with intracranial atherosclerosis: a population-based autopsy study.

BACKGROUND: Apolipoprotein E gene (APOE) ε4 allele is associated with a higher risk of carotid atherosclerosis, but less is known about the association of APOE with intracranial atherosclerotic disease (IAD). We aimed to investigate the association of APOE alleles with IAD in a cross-sectional autopsy study. METHODS: We measured the stenosis in the 12 arteries of the Circle of Willis using postmortem morphometric measurements. The APOE polymorphism was determined by real-time polymerase chain reaction. We assessed the association between APOE polymorphism and IAD using regression models adjusted for sociodemographic and clinical variables. We also verified the modifier effect of age, sex, and race on this association. We stratified the analysis by age group to investigate the possibility of attrition bias. RESULTS: In 400 participants (mean age=73.2±12.3 years old, 51% female, and 64% White), IAD was evaluated in 4,504 artery segments. APOE-ε4 was not associated with IAD nor with the number of artery stenosis compared to non-APOE-ε4 carriers. Sociodemographic variables did not modify this relationship. Among participants older than 70 years, there was a trend towards an association between APOE allele ε4 and a lower stenosis index in the middle cerebral artery, suggesting attrition bias related to the APOE-ε4 effect on mortality. CONCLUSIONS: APOE alleles were not associated with IAD in this population-based autopsy study. Lower stenosis in older participants suggests the possibility of attrition bias.

Circulating miR-130a-3p is elevated in patients with cerebral atherosclerosis and predicts 2-year risk of cerebrovascular events.

BACKGROUND: Cerebral atherosclerosis (AS) leads to high risk of cerebrovascular events. This study aims to evaluate the diagnostic performance of serum microRNA-130a-3p (miR-130a-3p) in cerebral AS patients, and construct a logistic risk model for 2-year cerebrovascular events on the basis of the prognostic potential of miR-130a-3p. METHODS: Serum samples were collected from 74 cerebral AS patients and 62 control individuals, and miR-130a-3p expression was investigated using reverse transcription quantitative PCR. Risk factors related with cerebral AS were assessed using a logistic regression analysis, and the receiver operating characteristic analysis was performed to evaluate the diagnostic value of miR-130a-3p. The relationship between miR-130a-3p and cerebrovascular events was analyzed using a Kaplan-Meier method, and a logistic risk model was constructed for 2-year cerebrovascular events. RESULTS: Cerebral AS patients had elevated serum miR-130a-3p compared with controls (P < 0.001). Serum miR-130a-3p had diagnostic value (AUC = 0.899), and could significantly improve the diagnostic accuracy of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in cerebral AS patients (AUC = 0.992). High serum miR-130a-3p was independently related with high probability of cerebrovascular events (HR = 1.993, 95% CI = 1.205-2.897, P = 0.006), and a logistic risk model was constructed based on serum miR-130a-3p, hs-CRP, TC and LDL-C. CONCLUSION: All the findings indicated that high serum miR-130a-3p had diagnostic potential to screen cerebral AS, and predicted the probability of cerebrovascular events after AS. The logistic risk model based on miR-130a-3p may provide an efficient method to predict 2-year cerebrovascular events in AS patients.

Single-Cell RNA Sequencing Reveals the Pathogenic Relevance of Intracranial Atherosclerosis in Blood Blister-Like Aneurysms.

Background: Intracranial non-branching site blood blister-like aneurysms (BBA) are extremely rare and vicious. Their etiology remains elusive, and no molecular study has been carried out to reveal its pathogenic relevance to intracranial atherosclerosis. To investigate its transcriptomic landscape and underlying potential pathogenesis, we performed single-cell RNA sequencing with extensive pathological validation. Methods: In total, 12,245 cells were recovered for single-cell RNA sequencing analysis from 1 BBA and 2 saccular intracranial aneurysms (IAs). Unbiased clustering using Seurat-based pipeline was used for cellular landscape profiling. Cellchat was used to understand intracellular communications. Furthermore, 10 BBAs and 30 IAs were retrospectively collected for pathological validations like scanning electron microscopy, H&E stain, Masson stain, Verhoeff Van Gielson stain, and immunofluorescence. Results: Single-cell transcriptome profiled 14 total subclusters in 6 major groups, namely, 6 monocyte/macrophage clusters, 2 T&NK clusters, 3 vascular smooth muscle cell (VSMC) clusters, 1 dendritic cell, 1 B cell, and 1 endothelial cell cluster. The only mural cell identified in BBAs was VSMC-2 cluster, while mural cells in IAs comprise most clusters of VSMCs and endothelial cells. Upregulated genes in BBA-derived VSMCs are related to arterial mineralization and atherosclerosis, such as PTX3, SPP1, LOX, etc., whereas vasodilation and physiological regulatory genes such as MGP, ACTA2, and MYL9 were conversely enriched in conventional IA-derived VSMCs. Immune cells in the BBA were predominantly macrophages, with a low fraction of T&NK cells, while conventional IAs had a higher percentage of T&NK. Gene enrichment analysis suggested that macrophages in BBA were highly enriched in lipid metabolism as well as atherosclerosis. Ligand-receptor interaction suggested that secretory phosphoprotein 1 (also known as osteopontin) played a major role in mediating the intracellular communication between VSMC and macrophages, especially in BBA. Pathological experiments corroborate with the bioinformatic findings and further characterized BBAs as a thin-walled thrombotic aneurysm with severe atherosclerotic lesions, where ApoE+ macrophages and OPN+ mural cells are intimately involved in the inflammation process. Conclusions: The preexisting intracranial atherosclerosis might predispose the parent artery to the pathogenic occurrence of BBAs. These data shed light on the pathophysiology of intracranial aneurysms and might assist in the further resolution of the complexity in aneurysm pathogenesis.

Association of plaque enhancement on vessel wall MRI and the phosphodiesterase 4D variant with stroke recurrence in patients with symptomatic intracranial atherosclerosis.

PURPOSE: Vessel wall MRI (VW-MRI) can be used to evaluate the nature of intracranial atherosclerosis (ICAS) plaque in vivo. Phosphodiesterase 4D (PDE4D) participates in stroke development. This study aims to explore the value of VW-MRI findings and the PDE4D gene variant in predicting stroke recurrence in patients with ICAS. METHODS: We prospectively recruited 324 symptomatic ICAS patients. VW-MRI was performed to determine luminal and wall changes. PDE4D gene single-nucleotide polymorphisms (SNPs)-namely, SNP32, SNP83, and SNP87-were determined by direct sequencing. The risk factors of stroke recurrence were analyzed using the multivariate Cox proportional hazards model. RESULTS: Of the 324 subjects, 97 (29.9%) experienced recurrent ischemic stroke during the follow-up period. A total of 254 patients (78.4%) showed plaque enhancement; 87 of these patients experienced stroke recurrence. The CT/CC genotype frequencies of PDE4D83 were significantly higher in participants with recurrent stroke than in patients without stroke recurrence (p = 0.019 and p < 0.001, respectively). However, the PDE4D32 and PDE4D87 variants were not correlated with recurrent stroke. Multivariate analysis showed that plaque enhancement from VW-MRI (HR 4.52, 95% CI 2.35-8.73, p < 0.001) and the PDE4D83 variant (HR 7.43, 95% CI 1.75-31.87, p = 0.005) were independently correlated with stroke recurrence. Kaplan-Meier curves showed significant differences in stroke recurrence rates between the plaque-enhanced group and the non-enhanced group (p < 0.001) and between the PDE4D83 variant carriers and noncarriers (p = 0.002). CONCLUSION: Plaque enhancement on VW-MRI and the presence of the PDE4D83 variant are associated with ischemic stroke recurrence in subjects with symptomatic ICAS.

Genome sequencing reveals the role of rare genomic variants in Chinese patients with symptomatic intracranial atherosclerotic disease.

OBJECTIVES: The predisposition of intracranial atherosclerotic disease (ICAD) to East Asians over Caucasians infers a genetic basis which, however, remains largely unknown. Higher prevalence of vascular risk factors (VRFs) in Chinese over Caucasian patients who had a stroke, and shared risk factors of ICAD with other stroke subtypes indicate genes related to VRFs and/or other stroke subtypes may also contribute to ICAD. METHODS: Unrelated symptomatic patients with ICAD were recruited for genome sequencing (GS, 60-fold). Rare and potentially deleterious single-nucleotide variants (SNVs) and small insertions/deletions (InDels) were detected in genome-wide and correlated to genes related to VRFs and/or other stroke subtypes. Rare aneuploidies, copy number variants (CNVs) and chromosomal structural rearrangements were also investigated. Lastly, candidate genes were used for pathway and gene ontology enrichment analysis. RESULTS: Among 92 patients (mean age at stroke onset 61.0±9.3 years), GS identified likely ICAD-associated rare genomic variants in 54.3% (50/92) of patients. Forty-eight patients (52.2%, 48/92) had 59 rare SNVs/InDels reported or predicted to be deleterious in genes related to VRFs and/or other stroke subtypes. None of the 59 rare variants were identified in local subjects without ICAD (n=126). 31 SNVs/InDels were related to conventional VRFs, and 28 were discovered in genes related to other stroke subtypes. Our study also showed that rare CNVs (n=7) and structural rearrangement (a balanced translocation) were potentially related to ICAD in 8.7% (8/92) of patients. Lastly, candidate genes were significantly enriched in pathways related to lipoprotein metabolism and cellular lipid catabolic process. CONCLUSIONS: Our GS study suggests a role of rare genomic variants with various variant types contributing to the development of ICAD in Chinese patients.

Association of PI3K-Akt Pathway-Related Gene Polymorphisms with Symptomatic Intracranial Atherosclerotic Stenosis with Hypertension in a Chinese Han Population.

BACKGROUND: PI3K-Akt signaling has been proved to be closely related to atherosclerosis, and hypertension has been shown to be an important risk factor for atherosclerosis. Studies have shown that genetic susceptibility is important in the etiology of symptomatic intracranial atherosclerotic stenosis (sICAS). However, few candidate genes have been identified. In the present study, we explored latent connections between single nucleotide polymorphisms (SNPs) of PI3K-Akt-related genes and sICAS with hypertension in Han Chinese subjects. METHODS: Eight genes related to the PI3K-Akt pathway in 400 patients with sICAS and 1007 healthy controls of Han nationality were sequenced, and further subgroup analysis stratified by the presence of hypertension was performed. The χ2 test and multiple logistic regression in dominant, recessive, and additive models were used to evaluate the association between the SNPs and the risk of sICAS with hypertension. When linkage disequilibrium was found in different loci of the same gene, tagSNP represents the SNP in the haplotype block. RESULTS: We found 4 common variants of 1 candidate gene differently distributed between those with sICAS with and without hypertension. Among these 4 common variations, INSR (insulin receptor) rs3745551 was significantly related to the risk of sICAS with hypertension after multiple regression analysis, with the T allele more prevalent in sICAS with hypertension. CONCLUSIONS: The variant of the INSR rs3745551 loci might be crucial to the pathogenesis of sICAS with hypertension in Chinese Han populations. Furthermore, the C allele at this locus might be a potentially harmful variant in sICAS with hypertension.

A Genetic Study of Cerebral Atherosclerosis Reveals Novel Associations with NTNG1 and CNOT3.

Cerebral atherosclerosis is a leading cause of stroke and an important contributor to dementia. Yet little is known about its genetic basis. To examine the association of common single nucleotide polymorphisms with cerebral atherosclerosis severity, we conducted a genomewide association study (GWAS) using data collected as part of two community-based cohort studies in the United States, the Religious Orders Study (ROS) and Rush Memory and Aging Project (MAP). Both studies enroll older individuals and exclude participants with signs of dementia at baseline. From our analysis of 1325 participants of European ancestry who had genotype and neuropathologically assessed cerebral atherosclerosis measures available, we found a novel locus for cerebral atherosclerosis in NTNG1. The locus comprises eight SNPs, including two independent significant SNPs: rs6664221 (ß = -0.27, 95% CI = (-0.35, -0.19), p = 1.29 × 10-10) and rs10881463 (ß = -0.20, 95% CI = (-0.27, -0.13), p = 3.40 × 10-8). We further found that the SNPs may influence cerebral atherosclerosis by regulating brain protein expression of CNOT3. CNOT3 is a subunit of CCR4-NOT, which has been shown to be a master regulator of mRNA stability and translation and an important complex for cholesterol homeostasis. In summary, we identify a novel genetic locus for cerebral atherosclerosis and a potential mechanism linking this variation to cerebral atherosclerosis progression. These findings offer insights into the genetic effects on cerebral atherosclerosis.

Serum MicroRNA-137 Serves as a Novel Biomarker for Cerebral Atherosclerosis Diagnosis and Cerebrovascular Event Prediction.

ABSTRACT: MicroRNAs have been reported as biomarkers for various diseases, including cerebral atherosclerosis (AS). In this study, whether serum microRNA-137 (miR-137) could be used as a biomarker for diagnosing cerebral AS and predicting cerebrovascular event was investigated. Quantitative real-time PCR was used to measure the expression of miR-137 in serum. Logistic analysis was used to evaluate the risk factors for the occurrence of cerebral AS, and receiver operating characteristic curves were used to estimate the diagnostic value of miR-137 and other risk factors for AS occurrence. Furthermore, the prognostic value of miR-137 for patients with AS was estimated using Kaplan-Meier survival analysis and Cox regression analysis. The results indicated that serum miR-137 levels were decreased in patients with cerebral AS. The expression of miR-137 was negatively correlated with total cholesterol and low-density lipoprotein cholesterol levels in patients with cerebral AS. The levels of miR-137, total cholesterol, low-density lipoprotein cholesterol, and hypersensitivity C response protein may serve as risk factors for the occurrence of cerebral AS, and miR-137 had diagnostic value for AS screening. Cerebral AS patients with positive cerebrovascular events have low miR-137 expression. Patients with high miR-137 expression had a lower incidence of cerebrovascular adverse events (log-rank P = 0.013), and miR-137 was an independent prognostic marker for the prediction of cerebrovascular event occurrence in patients with cerebral AS. In conclusions, our findings indicate that serum miR-137 levels are decreased in patients with cerebral AS and may be a new biomarker for diagnosing cerebral AS and predicting cerebrovascular events.

Genetic polymorphisms in lncRNAs predict recurrence of ischemic stroke.

Genetic polymorphisms in long non-coding RNAs (lncRNAs) are considered as potential genetic biomarkers for the prediction of human complex diseases such as ischemic stroke (IS). However, so far, no reports have focused on the relationship of lncRNA polymorphisms with IS onset and prognosis. In our study, eight potential functional polymorphisms of four well-known lncRNAs (H19 rs2107425 and rs2251375, MALAT1 rs4102217 and rs3200401, MEG3 rs11160608 and rs4081134, SENCR rs4526784 and rs555172) were genotyped in 657 ischemic stroke patients. Then, the association between lncRNA polymorphisms and IS onset and recurrence were investigated. These lncRNA variants were not associated with age onset of IS. However, we observed that MEG3 rs4081134 AA genotype was statistically related with a reduced risk of stroke recurrence, particularly for patients with large-artery atherosclerotic stroke. Also, the decreased risk was more prominent in elders, non-smokers, non-drinkers and hypertensive patients. Furthermore, the variant genotype AA of rs4081134 was an independent predictor for IS recurrence using the multivariate Cox regression model. Our findings indicated that MEG3 rs4081134 can serve as a useful biomarker and potential therapeutic target in IS recurrence. More researches are needed to verify our results and explore the underlying molecular mechanisms.

SNP rs2043211 (p.C10X) in CARD8 Is Associated with Large-Artery Atherosclerosis Stroke in a Chinese Population.

SNP rs2043211 in CARD8 was found to have significant association with ischemic stroke. This study aimed to explore the possible association between rs2043211 and large-artery atherosclerosis stroke in Chinese and explain the possible mechanism. In total, 716 large-artery atherosclerosis stroke patients and 1088 controls were included in the study. Co-dominant, dominant, and recessive genetic models were constructed to evaluate the relationship between rs2043211 and large-artery atherosclerosis stroke risk by odds ratios with 95% confidence intervals. Stratified and interaction analyses were also done. We selected another 111 large-artery atherosclerosis stroke patients and measured the CARD8 levels in their plasma samples by enzyme-linked immunosorbent assay. Participants who carry T/T genotype have a higher risk of large-artery atherosclerosis stroke compared with those carry A/T or A/A genotypes (odds ratio = 1.35, 95% confidence intervals 1.03-1.77, P = 0.029). The higher risk for the T/T genotype is still notable in female, people with hypertension, and people without diabetes. In the interaction analysis, compared to the non-hypertensive participants with the wild homozygote type A/A, the hypertensive participants with the A/T+T/T homozygote had 3.27-fold increased risk (odds ratio = 3.27, 95% confidence intervals 2.33-4.60). The A/A group had lower CARD8 levels in plasma than the A/T and T/T group (P < 0.001). Further bioinformatics prediction indicated that the rs2043211 could significantly influence the mRNA secondary structure and protein expression of CARD8 (eQTL P = 9.8 × 10-198). The rs2043211 is probably a novel biomarker for large-artery atherosclerosis stroke in Chinese.

Missense Variants in Hypoxia-Induced VEGFA/VEGFR2 Signaling Predict the Outcome of Large Artery Atherosclerotic Stroke.

Collateral density variations are a major determinant of stroke outcome. Here, we explored the association of missense variants in hypoxia-induced VEGFA/VEGFR2 signaling and stroke outcome. We recruited 683 large artery atherosclerotic (LAA) stroke patients as the training set from Nanjing Stroke Registry Program between August 2013 and January 2016. To validate the findings from the training set, we recruited an additional 333 LAA stroke patients between February 2016 and January 2017 as the validation set. Genotyping of target SNPs (rs11549465 [HIF-1α], rs11549467 [HIF-1α], rs1870377 [VEGFR2], and rs2305948 [VEGFR2]) was conducted using a SNPscan method. Unfavorable outcome was defined as a modified Rankin Scale (mRS) score > 2 at three months after index event. In the training set, the AA genotype of rs1870377 led to a decreased risk of unfavorable outcomes in the recessive model (AA vs. TA + TT, OR 0.60, 95% CI 0.38-0.95, P = 0.031). This was confirmed in the validation set (OR 0.43, 95% CI 0.21-0.86, P = 0.017) and the combined set (OR 0.54, 95% CI 0.36-0.79, P = 0.002). We also found that A allele was a protective factor for stroke outcome in both validation set and combined set (OR 0.70, 95% CI 0.49-0.99, P = 0.044 and OR 0.77, 95% CI 0.63-0.94, P = 0.012, respectively). In silico analysis indicated that the rs1870377 variant led to structural alterations in VEGFR2 that may influence its activity. Our findings demonstrate that the rs1870377 in the hypoxia-induced VEGFA/VEGFR2 axis predicts the 3-month outcome of patients with LAA stroke.

MMP9 rs17576 Is Simultaneously Correlated with Symptomatic Intracranial Atherosclerotic Stenosis and White Matter Hyperintensities in Chinese Population.

PURPOSE: The aim of this study was screening for single nucleotide polymorphisms (SNPs) associated with white matter hyperintensities (WMHs) in symptomatic intracranial atherosclerotic stenosis (sICAS) patients and exploring a possible connection in the genetic background between macrovascular disease and small vessel disease. METHODS: There were 400 sICAS patients enrolled in the study. Fazekas scores were applied to WMH classification. Healthy controls were referred to 1,000 Genome Project and GeneSky company who provided 1,007 Chinese healthy controls. Fast target sequencing technology was used to select the SNPs of 102 genes related to the pathogenesis of sICAS in the sICAS patients. RESULTS: The allele frequencies of 88 SNPs were significantly different between the sICAS group and the healthy controls (p < 0.05). The allele frequencies of 53 SNPs were significantly different between the sICAS patients with and without WMHs (p < 0.05). Further analysis found that matrix metalloproteinase 9 (MMP9) rs17576 was simultaneously related to sICAS and WMHs. The frequency of the rs17576 A allele was significantly lower in sICAS patients when compared to the normal controls (p = 0.03, OR [95% CI] = 0.75 [0.625-0.91]). Also, the frequency of the rs17576 genotypes was significantly different under codominant (p = 0.009), dominant (p = 0.014), and recessive (p= 0.023) models. The frequency of the rs17576 A allele was significantly higher in sICAS with WMH patients, compared to those without WMHs (p = 0.022, OR [95% CI] = 1.54 [1.06-2.22]); the frequency of the rs17576 genotypes was significantly different under codominant (p = 0.019) and recessive (p = 0.032) models. Logistic regression analysis showed that age, hypertension, and MMP9 rs17576 AA genotype were independent risk factors for sICAS with WMHs. CONCLUSION: MMP9 rs17576 may be simultaneously associated with the risk of sICAS and WMHs.

Role of the RNF213 Variant in Vascular Outcomes in Patients With Intracranial Atherosclerosis.

Background The RNF213 (ring finger protein 213 gene) variant R4810K is a susceptibility allele not only for Moyamoya disease (MMD) but also for intracranial atherosclerosis (ICAS) in East Asian populations. We hypothesized that this variant would affect the distribution of ICAS and recurrence of cerebrovascular events. Methods and Results We conducted a prospective study of patients with ICAS and MMD using high-resolution magnetic resonance imaging and RNF213 R4810K genotyping. Patients were included in the ICAS group when relevant plaques existed on high-resolution magnetic resonance imagingand in the MMD group when they carried the variant and high-resolution magnetic resonance imaging showed no plaques but characteristic features of MMD. We compared clinical and neuroimaging features of patients with ICAS-RNF213+ with patients with ICAS-RNF213- and of patients with MMD. Of 477 patients, 238 patients were in the ICAS group and 239 were in the MMD group. Among patients with ICAS, 79 patients (33.2%) were in the ICAS-RNF213+ group and 159 (66.8%) in the ICAS-RNF213- group. Tandem lesions were significantly more common in the ICAS-RNF213+ group than in the ICAS-RNF213- group (40.3% versus 72.2%, P<0.001), and their distributions were similar between the ICAS-RNF213+ and MMD groups. The presence of the R4810K variant (hazard ratio [HR], 3.203; 95% CI, 1.149-9.459; P=0.026) and tandem lesions (≥3) (HR, 8.315; 95% CI, 1.930-39.607; P=0.005) were independently associated with recurrent cerebrovascular events. Conclusions Patients with ICAS carrying the RNF213 R4810K variant showed clinical and imaging features distinct from patients with ICAS without the variant, suggesting that the R4810K variant plays a role in intracranial atherosclerosis in East Asian patients.

Are Genetic Variants Associated with the Location of Cerebral Arterial Lesions in Stroke Patients?

BACKGROUND: Genetic variants may play a role in determining the location of cerebral atherosclerosis. We aimed to investigate the association between RNF213, MMP2, and genetic polymorphisms linked to vascular tortuosity with the location of cerebral arterial atherosclerosis. METHODS: A prospective case-control study was conducted on patients with ischemic stroke and age- and sex-matched stroke-free controls. The stroke patients were categorized into those with intracranial artery atherosclerosis (ICAS), extracranial artery atherosclerosis (ECAS), and small vessel occlusion (SVO). Six single nucleotide polymorphisms (SNPs) including rs2118181 (FBN1), rs2179357 (SLC2A10), rs1036095 (TGFBR2), rs243865 (MMP2), rs1800470 (TGFB1), and rs112735431 (RNF213) were analyzed with the TaqMan Genotyping Assay, and the distribution of genotypes across groups was compared. RESULTS: None of the 6 SNPs were associated with stroke on comparing the 449 stroke patients (71 with ECAS, 169 with ICAS, and 209 with SVO) to the 447 controls. In the subgroup analysis, the adjusted odds ratios (aORs) for age and sex indicated a significant association between rs112735431 and ICAS in the allele comparison analysis and in the additive and dominant model analyses. rs112735431 was associated with anterior circulation involvement and increased burden of cerebral atherosclerosis. rs2179357 was significantly associated with ICAS in the recessive model analysis, and rs1800470 was significantly associated with ECAS in the recessive model analysis when compared to controls. CONCLUSION: rs112735431 was associated with ICAS and increased atherosclerosis burden in Korean stroke patients. Further studies are needed to elucidate the role of rs112735431 and to confirm the association of rs2179357 and rs1800470 with cerebral atherosclerosis.

Genetic Risk Factors of Intracranial Atherosclerosis.

PURPOSE OF REVIEW: Intracranial atherosclerosis (ICAS) is the most common cause of stroke throughout the world. It also increases the risk of recurrent stroke and dementia. As a complex and multifactorial disease, ICAS is influenced by multiple genetic, biological, and environmental factors. This review summarizes the candidate gene and genome-wide studies aimed at discovering genetic risk factors of ICAS. RECENT FINDINGS: Numerous studies have focused on the association between single-nucleotide polymorphisms (SNPs) of atherosclerosis-related genes and the risk of ICAS. Variants in adiponectin Q (ADIPOQ), ring finger protein 213 (RNF213), apolipoprotein E (APOE), phosphodiesterase 4D (PDE4D), methylenetetrahydrofolate reductase (MTHFR), lipoprotein lipase (LPL), α-adducin (ADD1) genes, angiotensin-converting enzyme (ACE), and other genes related to renin-angiotensin-aldosterone system have been associated with ICAS. We review the available evidences on the candidate genes and SNPs associated with genetic susceptibility to ICAS, and point out future developments of this field. Genetic discoveries could have clinical implications for intracranial atherosclerotic disease.

Association of AMPK Pathway-Related Gene Polymorphisms with Symptomatic Intracranial Atherosclerotic Stenosis in a Chinese Han Population.

Background and Objective: Recently, AMP-activated protein kinase (AMPK) signaling was confirmed to be intimately associated with atherosclerosis. Evidence indicates that genetic susceptibility plays an important role in the etiology of symptomatic intracranial atherosclerotic stenosis (sICAS), however few genes have been pinpointed being etiologically associated. This study investigated possible links between single nucleotide polymorphisms (SNPs) of AMPK-related genes and sICAS in Han Chinese subjects. Methods: Target gene sequencing was carried out in 400 sICAS Han Chinese patients and 1007 healthy controls for 11 AMPK pathway-related genes. Chi-squared testing and multiple logistic regression in dominant, recessive, and additive models were used to evaluate the association between SNPs and risk of sICAS. Bonferroni corrections were performed with a p < (0.05/44 = 0.0011) as statistically significant. Further subgroup data analyses was conducted using chi-squared or t-tests. Results: There were 44 common variants of 11 candidate genes distributed differently between sICAS patients and healthy controls, among which the INSR rs78312382 SNP remained significant even after a Bonferroni correction. Logistic regression analysis showed that rs78312382 was significantly associated with the risk of sICAS in both dominant and additive models (pBonferroni = 7.874e-5 and 0.000506, respectively), with the A allele being much more prevalent in the sICAS group (p = 0.000404). Conclusions: Variants of the INSR rs7831282 locus may play an important role in the development of sICAS among the Han Chinese with the A allele being a risk factor and a potential biomarker for this illness.

Two Novel SNPs in the PLCL2 Gene Associated with Large Artery Atherosclerotic Stroke Identified by Fine-Mapping.

A genome-wide association study (GWAS) reported that the single nucleotide polymorphism (SNP) rs4618210 in the PLCL2 gene is related to myocardial infarction (MI) in the Japanese population, but no study has examined the correlation of PLCL2 with ischemic stroke (IS). The present study was designed to investigate whether the genetic variation in PLCL2 is associated with large artery atherosclerotic (LAA) stroke in a Han Chinese population. Tagging SNPs (tSNPs) of the PLCL2 gene were determined by a fine-mapping strategy and were genotyped by improved multiplex ligation detection reaction (iMLDR) technology in 669 LAA stroke patients and 668 healthy controls. A logistic regression model was used to analyze the associations between genetic variation at PLCL2 and the risk of LAA stroke. Two SNPs were significantly associated with the risk of LAA stroke after adjusting for potential confounders: for rs4685423, the AA genotype and CA genotype decreased the risk of LAA stroke compared with the CC genotype (multivariate-adjusted, P = 0.001); for rs4618210, the AA genotype and GA genotype decreased the risk of LAA stroke compared with the GG genotype (multivariate-adjusted, P = 0.007). In addition, haplotype analysis indicated that compared with haplotype TTT, haplotype TAT decreased the risk of LAA stroke in block 2 (adjusted OR, 0.706; 95% CI, 0.550-0.907; P = 0.006). The analysis of SNP-SNP interactions showed that rs4685423 was the most influential contributor to LAA stroke risk. SNPs rs4685423 and rs4618210 in the PLCL2 gene may be related to the risk of LAA stroke in Han Chinese.

Differential expression of circulating exosomal microRNAs in refractory intracranial atherosclerosis associated with antiangiogenesis.

Intracranial atherosclerotic disease (ICAD) is a common cause of stroke with high rates of ischemic recurrence. We aimed to investigate the role of circulating exosomal microRNAs (e-miRNAs) in recurrent ischemic events in ICAD. Consecutive patients with severe ICAD undergoing intensive medical management (IMM) were prospectively enrolled. Those with recurrent ischemic events despite IMM during 6-month follow up were algorithmically matched to IMM responders. Baseline blood e-miRNA expression levels of the matched patients were measured using next generation sequencing. A total of 122 e-miRNAs were isolated from blood samples of 10 non-responders and 11 responders. Thirteen e-miRNAs predicted IMM failure with 90% sensitivity and 100% specificity. Ingenuity pathway analysis (IPA) determined 10 of the 13 e-miRNAs were significantly associated with angiogenesis-related biological functions (p < 0.025) and angiogenic factors that have been associated with recurrent ischemic events in ICAD. These e-miRNAs included miR-122-5p, miR-192-5p, miR-27b-3p, miR-16-5p, miR-486-5p, miR-30c-5p, miR-10b-5p, miR-10a-5p, miR-101-3p, and miR-24-3p. As predicted by IPA, the specific expression profiles of these 10 e-miRNAs in non-responders had a net result of inhibition of the angiogenesis-related functions and up expression of the antiangiogenic factors. This study revealed distinct expression profiles of circulating e-miRNAs in refractory ICAD, suggesting an antiangiogenic mechanism underlying IMM failure.

Ring Finger Protein 213 Variant and Plaque Characteristics, Vascular Remodeling, and Hemodynamics in Patients With Intracranial Atherosclerotic Stroke: A High-Resolution Magnetic Resonance Imaging and Hemodynamic Study.

Background Intracranial atherosclerotic stroke is prevalent in Asians. We hypothesized that patients with the ring finger protein 213 (RNF213) variant, a susceptibility locus for moyamoya disease in Asians, have different neuroimaging characteristics in terms of the vessel wall and hemodynamics. Methods and Results We analyzed consecutive patients with ischemic events in middle cerebral artery distribution and relevant plaques of the distal internal carotid artery or proximal middle cerebral artery on high-resolution magnetic resonance imaging. Patients with carotid/cardiac sources of embolism or moyamoya disease were excluded. High-resolution magnetic resonance imaging features (eg, outer vessel diameters and plaque characteristics) and fractional flow (as measured by adjusted signal intensity ratio on time-of-flight magnetic resonance angiography) were compared between RNF213 p.Arg4810Lys variant carriers and noncarriers. Among 144 patients included, 44 (29.9%) had the RNF213 variant. Clinical characteristics, including age, sex, body mass index, and vascular risk factors, were not significantly different between RNF213 variant carriers and noncarriers. However, the outer vessel diameter was smaller in RNF213 variant carriers than in noncarriers (P<0.0001 for middle cerebral artery of relevant stenosis [2.05-mm analysis of RNF213 gene for moyamoya disease in the Chinese HAN population 2.75 mm]; P<0.0001 for contralateral side [2.42  versus 3.00 mm] and P<0.001 for basilar artery [3.19 versus 3.53 mm]). Other high-resolution magnetic resonance imaging features, including plaque morphology and eccentricity, were not significantly different. Fractional flow was diminished in patients with smaller-diameter intracranial arteries with a similar degree of stenosis. Conclusions The RNF213 variant may be associated with vasculogenesis, but not with atherogenesis. Patients with this variant had small intracranial arteries predisposing hemodynamic compromise in the presence of intracranial atherosclerosis. In addition to antiatherosclerotic strategies, further studies are warranted to develop novel therapeutic strategies against RNF213 vasculopathy in Asians.