Ryan, Kathleen A.

Introduction: Even though neuroinflammation is increasingly recognized as an essential contributor to ischemic brain injury, the exact underlying mechanisms remain unclear. Higher expression of the vascular cell adhesion molecule 1 (VCAM-1) increases leukocyte-brain endothelium interaction and has been associated with larger infarct size following acute ischemic stroke (AIS). The activation of the TGF-β signaling pathways can down-regulate VCAM-1 expression and ameliorate deleterious tissue outcome during neuroinflammation. Hypothesis: We sought to investigate whether genetic variation in the TGF-beta pathway and adhesion molecule genes is associated with acute stroke lesion size. Methods: We completed genome-wide association (GWA) testing and diffusion-weighted imaging lesion volume (DWIv) analysis in a discovery cohort of 532 AIS patients of European ancestry enrolled within 48 hours of symptom onset. An independent European ancestry cohort of 724 AIS patients with GWA data and automated DWIv served as replication cohort. GWA testing per SNP was performed using linear regression modeling of natural log-transformed DWIv adjusted for age, sex and relevant principal components. We selected 42 inflammatory genes in the TGF-beta pathway for a gene-based analysis using VEGAS (Versatile Gene-based -Association Study) software. A pre-specified discovery phase Bonferroni-corrected threshold was set at p<0.001. In the replication phase, 14 SNPs overlapping were tested at the Bonferroni-corrected p-value threshold of p<0.004. Results: Of all genes in the TGF-beta pathway, VCAM1 (p=0.0006) was significantly associated with DWIv in the discovery AIS cohort (age: 66 ± 14.9 years, sex: 63.4% male, DWIv: 2.2cm 3 (IQR: 0.6-11.7cm 3 )). A single SNP within the VCAM1 gene boundaries (rs3176876 (BETA=0.2341, p=0.003)) was significantly associated with DWIv in the replication AIS cohort (age: 65 ± 14.1 years, sex: 68.6% male, DWIv: 4cm 3 (IQR: 1.3-17.2cm 3 )). Conclusion: The genetic variant rs3176876 in the VCAM1 gene is associated with larger infarct lesion size measured on brain MRI of AIS patients. These findings suggest genetic contribution to the pro-inflammatory mechanisms in acute cerebral ischemia and warrant further investigation.

Introduction: The MRI-Genetics Interface Exploration (MRI-GENIE) study is the first international collaboration that aims to facilitate genetic discoveries in clinical cohorts of patients with acute ischemic stroke (AIS). We have amassed the largest-to-date collection of AIS cases with brain MRI scans and genome-wide genotyping to test the role of genetic susceptibility in MRI-based cerebrovascular traits. Objective/Hypothesis: To elucidate the genetic architecture of white matter hyperintensity (WMH) burden in AIS patients. Methods: Using a novel automated algorithm, we extracted WMH volume (WMHv) from clinical MRI scans of 2704 AIS patients (age 63.1 ± 14.7 years, 60.6% male) of European ancestry. Quality control (QC) measures were undertaken per subject and per SNP, excluding subjects with non-European ancestry and poor genotyping, as well as SNPs deviating from Hardy-Weinberg equilibrium and high levels of missingness. Imputation to the Haplotype Reference Consortium (HRC version r1.1) was conducted for 1712 remaining subjects with 2.8 million SNPs on the Michigan Imputation Server. After exclusion of poorly imputed SNPs (R 2 <0.5) and SNPs with minor allele frequency < 1%, 7.7 million SNPs remained for further analysis. Genome-wide association testing of natural log-transformed WMHv on the allelic dosage per SNP was adjusted for age, sex and principal components 1-10. Results: Genome-wide association testing has identified a novel locus on chromosome 2 (T allele at rs72856504) near the LDL Receptor related Protein 1B gene (LRP1B) that was significantly associated with WMHv burden in AIS (β=0.54, SE=0.098, p=3.65*10 -8 ). Conclusion: We have identified a novel locus (T allele rs72856504) on chromosome 2 near the LRP1B gene, which is specific for WMH in AIS and has not been previosuly described in stroke-free WMH cohorts. A replication effort involving additional independent cohorts of AIS patients with brain MRI and genome-wide genotyping is ongoing.

Background: Current ischemic stroke (IS) subtyping systems are very labor-intensive and costly to implement in large samples. There is a need to develop phenotyping approaches for genetic and other studies that can be implemented using generally available items captured in electronic medical records. Objective: In this report, we seek to validate a simplified approach using results from a genetic association study. Specifically, the strength of association for known genetic signals in the NINDS Stroke Genetics Network study (SiGN) is compared between the established IS subtyping systems (TOAST and Causative Classification System (CCS) subtypes) and phenotyping based on a single item within the CCS. Methods: Non-mutually exclusive “Simple” IS subtypes were defined from single CCS items as follows: 1) Small vessel stroke- lacunar infarct on imaging (with no other imaging or other requirements); 2) large artery stroke - vascular imaging showing > 50% stenosis or <50% stenosis with plaque rupture or thrombosis judged to be due to atherosclerosis in clinically relevant artery; and 3) cardioembolic stroke - persistent or paroxysmal atrial fibrillation. We compared the GWAS signals using these definitions to the published SiGN GWAS results for TOAST and CCS, among those of European ancestry only. Results: The Table shows that, for loci significant in SiGN GWAS study, the “Simple” IS subtypes had similar strengths of association and p-values compared to results from TOAST and the causative and phenotypic CCS subtypes. Conclusions: “Simple” IS subtypes perform as well in GWAS analyses as more complex phenotyping systems and could potentially be derived through automated or semi-automated methods applied to electronic medical record systems. Since the ‘Simple” phenotypes were based on high quality CCS data, the current findings reflect optimal performance. Additional validation studies will be required for implementation in other settings.

Background Bone-marrow derived progenitor cells (PCs) may play a role in maintaining vascular health by actively repairing damaged endothelium. The purpose of this study in asymptomatic Old Order Amish men (n = 90) without hypertension or diabetes was to determine if PC count, as determined by CD34+ cell count in peripheral blood, was associated with 10-year risk of cardiovascular disease (CVD) and measures of subclinical atherosclerosis. Methods and Results CD34+ cell count by fluorescence-activated cell sorting, coronary artery calcification (CAC) by electron beam computed tomography, and CVD risk factors were obtained. Carotid intimal-medial thickness (CIMT) also was obtained in a subset of 57 men. After adjusting for 10-year CVD risk, CD34+ cell count was significantly associated with CAC quantity ( p =0.03) and CIMT ( p < 0.0001). A 1-unit increase in natural-log transformed CD34+ cell count was associated with an estimated 55.2% decrease (95% CI: −77.8% to −9.3%) in CAC quantity and an estimated 14.3% decrease (95% CI: −20.1% to −8.1%) in CIMT. Conclusions Increased CD34+ cell count was associated with a decrease in extent of subclinical atherosclerosis in multiple arterial beds, independent of 10-year CVD risk. Further investigations of associations of CD34+ cell count with subclinical atherosclerosis in asymptomatic individuals could provide mechanistic insights into the atherosclerotic process.