Summary Introduction The discovery of disease-associated loci through genome-wide association studies (GWAS) is the leading approach to the identification of novel biological pathways for human disease. To date, GWAS have had been limited by relatively small sample sizes and yielded relatively few loci associated with ischemic stroke The National Institute of Neurological Disorders Stroke Genetics Network (NINDS-SiGN) is an international consortium that has taken a systematic approach to phenotyping and produced the largest ischemic stroke GWAS to date. Methods In order to identify genetic loci associated with ischemic stroke, we performed a two-stage genome-wide association study. The first stage consisted of 16,851 cases with state-of-the-art phenotyping and 32,473 stroke-free controls. Cases were aged 16 to 104 years, recruited between 1989 and 2012, and subtyped by centrally trained and certified investigators using the web-based protocol, Causative Classification of Stroke (CCS). We constructed case-control strata by identify samples genotyped on (nearly) identical arrays and of similar genetic ancestral background. Data was cleaned and imputed using dense imputation reference panels generated from whole-genome sequence data. Genome-wide testing was performed within each stratum for each available phenotype, and summary level results were combined using inverse variance-weighted fixed effects meta-analysis. The second stage consisted of in silico look-ups of 1,372 SNPs in 20,941 cases and 364,736 stroke-free controls, with cases previously subtyped using the TOAST classification system according to local standards. The two stages were then jointly analyzed in a final meta-analysis. Findings We identified a novel locus at 1p13.2 near TSPAN2 associated with large artery atherosclerosis (LAA)-related stroke (stage I OR for the G allele at rs12122341 = 1·21, p = 4.50 × 10−8; stage II OR = 1·19, p = 1·30 × 10−9). We also confirmed four loci robustly associated with ischemic stroke and reported in prior studies, including PITX2 and ZFHX3 for cardioembolic stroke, and HDAC9 for LAA stroke. The 12q24 locus near ALDH2, originally associated with all ischemic stroke but not with any specific subtype, exceeded genome-wide significance in the meta-analysis of small artery stroke. Other loci, including NINJ2, were not confirmed. Interpretation Our results identify a novel LAA-stroke susceptibility gene and now indicate that all loci implicated by GWAS to date are subtype specific. Follow-up studies will be necessary to determine whether the locus near TSPAN2 yields a novel therapeutic approach to stroke prevention. Given the subtype-specificity of these associations, the rich phenotyping available in SiGN is likely to prove vital for further genetic discovery in ischemic stroke. Funding National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH).

Background and Purpose-The aim of our study was to identify factors associated with stroke recurrence after an initial minor stroke or transient ischemic attack (TIA) in a prospective hospital-series. Methods-Included in the series were 689 patients with NIHSS lower than 4 at hospital admission. The end point was a new neurological event (worsening Ն4 points in the initial NIHSS was considered as recurrence) at 90 days (and additionally at 7 days). Factors based on two previous reported scores (ABCD and SPI-II) were analyzed in relation with stroke recurrence: age, duration of symptoms Ͼ1 hour, weakness, speech impairment, initial hypertension, hypertension, diabetes, coronary disease, minor stroke versus TIA, prior stroke, and heart failure. We also analyzed: gender, hyperlipidemia, severe alcohol intake (Ͼ60gr/d), current smoking habits, peripheral arterial disease, atrial fibrillation, acute lesion in initial head computed tomography, severe symptomatic extra or intracranial arterial disease (SSAD; arterial stenosis Ն70%), previous TIA, and vertebrobasilar event. Patients were also analyzed separately according to diagnosis of TIA or minor stroke. Results-90-day recurrence occurred in 111 patients (16.1%), whereas 62 patients had 7-day recurrence (9%). The independent variables associated with 90-day recurrence were: SSAD (ORϭ4.97), weakness (ORϭ3.25), speech impairment (ORϭ1.96), severe alcohol intake (ORϭ4.18), heart failure (ORϭ2.41), previous TIA (ORϭ4.62), and vertebrobasilar events (ORϭ2.87). SSAD was independently associated with 7-day recurrence (ORϭ7.73) and also for TIA (ORϭ3.45) and minor stroke (ORϭ5.15) patients. Conclusions-An arterial study to discard SSAD would be necessary, in combination with clinical factors, to improve the identification of patients with a higher risk of 90-day recurrence after an initial minor stroke or TIA.

Type 2 diabetes mellitus (DM) is an established risk factor for a wide range of vascular diseases, including ischemic stroke (IS). Glycated hemoglobin A1c (HbA1c), a marker for average blood glucose levels over the previous 12 weeks, is used as a measure of glycemic control and also as a diagnostic criterion for diabetes (HbA1c levels ≥ 6.5%). Epigenetic mechanisms, such as DNA methylation, may be associated with aging processes and with modulation of the risk of various pathologies, such as DM. Specifically, DNA methylation could be one of the mechanisms mediating the relation between DM and environmental exposures. Our goal was to identify new CpG methylation sites associated with DM. We performed a genome-wide methylation study in whole-blood DNA from an IS patient cohorts. Illumina HumanMethylation450 BeadChip array was used to measure DNA methylation in CpG sites. All statistical analyses were adjusted for sex, age, hyperlipidemia, body mass index (BMI), smoking habit and cell count. Findings were replicated in two independent cohorts, an IS cohort and a population-based cohort, using the same array. In the discovery phase (N = 355), we identified a CpG site, cg19693031 (located in the TXNIP gene) that was associated with DM (P = 1.17 × 10(-12)); this CpG was replicated in two independent cohorts (N = 167 and N = 645). Methylation of TXNIP was inversely and intensely associated with HbA1c levels (P = 7.3 × 10(-16)), specifically related to diabetic patients with poor control of glucose levels. We identified an association between the TXNIP gene and DM through epigenetic mechanisms, related to sustained hyperglycemia levels (HbA1c ≥ 7%).

Background White matter hyperintensity (WMH), or leukoaraiosis, is a radiological finding generally assumed to reflect diseased small cerebral vasculature. WMH has significant functional impact through its relationship to cognitive decline and risk of ischemic and hemorrhagic stroke. Accumulating evidence suggests that some manifestations of small vessel disease such as intracerebral hemorrhage (ICH) are associated with low levels of cholesterol. We sought to determine the relationship between hyperlipidemia (HL) and WMH severity in patients with acute ischemic stroke (AIS). Methods We analyzed two independent hospital-based AIS cohorts. Demographic and clinical data were collected prospectively. WMH was measured using semi-automated volumetric image analysis and a semi-quantitative visual grading scale. Univariate and multivariable regression analyses were used to assess the relationship between WMH severity and study variables. Results A total of 631 and 504 subjects in the first and second cohorts, respectively, were included. In univariate analyses, advancing age and hypertension were associated with severity of WMH (p<0.001) in both cohorts. In the multivariable analysis, after controlling for age, gender, and those significant risk factors in the univariate and age-adjusted analyses, patients with a history of HL had less severe WMH in both cohorts (p<0.01). Conclusions Results from two independent cohorts demonstrate that AIS patients with a history of HL have less severe WMH at the time of stroke. These data support the hypothesis that HL may play a relatively protective role on cerebral small vessel disease.

Background and Purpose— Few data are available on very early stroke recurrence evaluated within the first hours after onset of symptoms and outcome for unselected patients with first-ever mild stroke or TIA and symptomatic carotid stenosis ≥50%. Methods— One hundred sixty-three patients with symptomatic carotid stenosis and initial mild stroke (121) or TIA (42) were evaluated within 6 hours from onset of symptoms in a single tertiary hospital. Neurological recurrence (NR) was defined as a clearly defined new neurological event (TIA or stroke) or an increase of 4 points in the initial NIHSS. The NR rate was determined at 72 hours, 7 days, and 14 days. Disability was defined as a score of 3 to 6 on the modified Rankin scale at 14 days. Results— Forty-five patients (27.6%) had NR, including 6 patients with 2 episodes in different time periods: 34 (20.9%) within the first 72 hours; 11 (6.7%) between 72 hours and 7 days; and 6 (3.7%) at 14 days. Only carotid stenosis ≥70% was associated with NR; diabetes was marginally associated. At 2 weeks, 19 patients (11.7%) had disability; 14 of them experienced NR in the first 72 hours. Conclusions— Patients with first-ever mild stroke or TIA and symptomatic carotid stenosis are at high risk for NR, especially within the first 72 hours. Our results suggest the necessity of testing pharmacological or interventional strategies for use during the hyperacute stroke phase in these patients.

Apart from the classical outcome predictors, the previous use of antiplatelet agents and the glucose value at admission are independent predictors of 30-day mortality in patients suffering a supratentorial ICH.

Both forms of HF (with or without decreased systolic function) are associated with poor outcome in AIS.

Microvascular endothelial cells at the blood-brain barrier exhibit a protective phenotype, which is highly induced by biochemical and biomechanical stimuli. Amongst them, shear stress enhances junctional tightness and limits transport at capillary-like levels. Abnormal flow patterns can reduce functional features of macrovascular endothelium. We now examine if this is true in brain microvascular endothelial cells. We suggest in this paper a complex response of endothelial cells to aberrant forces under different flow domains. Human brain microvascular endothelial cells were exposed to physiological or abnormal flow patterns. Physiologic shear (10-20 dyn/cm) upregulates expression of tight junction markers Zona Occludens 1 (1.7-fold) and Claudin-5 (more than 2-fold). High shear stress (40 dyn/cm) and/or pulsatility decreased their expression to basal levels and altered junctional morphology. We exposed cells to pathological shear stress patterns followed by capillary-like conditions. Results showed reversible recovery on the expression of tight junction markers. Flow protection of barrier phenotype commensurate with junctional signaling pathways decrease (Src, 0.25-fold, ERK, 0.77-fold) when compared to static conditions. This decrease was lost under high shear and pulsatile flow. In conclusion, abnormal shear stress inherent to systemic vascular disease leads to barrier impairment, which could be reverted by hemodynamic interventions.