Exome Array Analysis of Early-Onset Ischemic Stroke.

BACKGROUND AND PURPOSE: The genetic contribution to ischemic stroke may include rare- or low-frequency variants of high-penetrance and large-effect sizes. Analyses focusing on early-onset disease, an extreme-phenotype, and on the exome, the protein-coding portion of genes, may increase the likelihood of identifying such rare functional variants. To evaluate this hypothesis, we implemented a 2-stage discovery and replication design, and then addressed whether the identified variants also associated with older-onset disease. METHODS: Discovery was performed in UMD-GEOS Study (University of Maryland-Genetics of Early-Onset Stroke), a biracial population-based study of first-ever ischemic stroke cases 15 to 49 years of age (n=723) and nonstroke controls (n=726). All participants had prior GWAS (Genome Wide Association Study) and underwent Illumina exome-chip genotyping. Logistic-regression was performed to test single-variant associations with all-ischemic stroke and TOAST (Trial of ORG 10172 in Acute Stroke Treatment) subtypes in Whites and Blacks. Population level results were combined using meta-analysis. Gene-based aggregation testing and meta-analysis were performed using seqMeta. Covariates included age and gender, and principal-components for population structure. Pathway analyses were performed across all nominally associated genes for each stroke outcome. Replication was attempted through lookups in a previously reported meta-analysis of early-onset stroke and a large-scale stroke genetics study consisting of primarily older-onset cases. RESULTS: Gene burden tests identified a significant association with NAT10 in small-vessel stroke (P=3.79×10-6). Pathway analysis of the top 517 genes (P<0.05) from the gene-based analysis of small-vessel stroke identified several signaling and metabolism-related pathways related to neurotransmitter, neurodevelopmental notch-signaling, and lipid/glucose metabolism. While no individual SNPs reached chip-wide significance (P<2.05×10-7), several were near, including an intronic variant in LEXM (rs7549251; P=4.08×10-7) and an exonic variant in TRAPPC11 (rs67383011; P=5.19×10-6). CONCLUSIONS: Exome-based analysis in the setting of early-onset stroke is a promising strategy for identifying novel genetic risk variants, loci, and pathways.

Detection of Uncommon Enteric Bacterial Pathogens from Acute Diarrheal Specimens Using SYBR-Green Real Time PCR.

Acute diarrheal disease is a major health problem, and the second most common cause of death in children under 5 years of age. Conventional diagnostic methods are laborious, time consuming, and occasionally inaccurate. We used SYBR-Green real-time PCR for the detection of 10 uncommon bacterial pathogens using fecal specimens from acute diarrheal patients. In the SYBR-Green real-time PCR assay, the products formed were identified based on a melting point temperature curve analysis, and the assay was validated with the respective reference strain. In a retrospective study, we tested 1,184 stool specimens previously examined using conventional culture methods. Enterotoxigenic Bacteriodes fragilis was detected in 6.7% of the samples followed by enterotoxigenic Bacillus cereus (5.1%), Clostridium perfringens (3.9%), and Aeromonas hydrophila (3.8%). In the prospective study, A. hydrophila, Staphylococcus aureus, and C. perfringens were predominantly detected in 11 > 5 years of age, using real-time PCR. The real-time PCR assay is comprehensive, rapid, accurate, and well suited for surveillance or diagnostic purposes to detect uncommon bacterial pathogens, and should be useful in initiating appropriate care and thereby reducing patient risk.

Factor V leiden and ischemic stroke risk: the Genetics of Early Onset Stroke (GEOS) study.

BACKGROUND: Factor V Leiden (FVL) has been associated with ischemic stroke in children but not in adults. Although the FVL mutation is associated with increased risk for venous thrombosis, its association with ischemic stroke in young adults remains uncertain. Therefore, we examined the association between FVL and ischemic stroke in participants of the Genetics of Early Onset Stroke (GEOS) study. METHODS: A population-based case control study identified 354 women and 476 men 15 to 49 years of age with first-ever ischemic stroke and 907 controls. Participant-specific data included vascular risk factors, FVL genotype and, for cases, the ischemic stroke subtype by modified Trial of ORG 10172 in Acute Stroke criteria. Logistic regression was used to calculate odds ratios for the entire population and for subgroups stratified by risk factors and ischemic stroke subtype. RESULTS: The frequency of the FVL mutation was similar between ischemic stroke patients (3.6%; 95% confidence interval [CI] 2.5%-5.1%) and nonstroke controls (3.8%; 95% CI 2.7%-5.2%). This frequency did not change significantly when cases were restricted to patients with stroke of undetermined etiology (4.1%; 95% CI 2.6%-6.4%). CONCLUSIONS: Among young adults, we found no evidence for an association between FVL and either all ischemic stroke or the subgroup with stroke of undetermined etiology.

Community-associated methicillin-resistant Staphylococcus aureus bacteremia and endocarditis among HIV patients: a cohort study.

BACKGROUND: HIV patients are at increased risk of development of infections and infection-associated poor health outcomes. We aimed to 1) assess the prevalence of USA300 community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) among HIV-infected patients with S. aureus bloodstream infections and. 2) determine risk factors for infective endocarditis and in-hospital mortality among patients in this population. METHODS: All adult HIV-infected patients with documented S. aureus bacteremia admitted to the University of Maryland Medical Center between January 1, 2003 and December 31, 2005 were included. CA-MRSA was defined as a USA 300 MRSA isolate with the MBQBLO spa-type motif and positive for both the arginine catabolic mobile element and Panton-Valentin Leukocidin. Risk factors for S. aureus-associated infective endocarditis and mortality were determined using logistic regression to calculate odds ratios (OR) and 95% confidence intervals (CI). Potential risk factors included demographic variables, comorbid illnesses, and intravenous drug use. RESULTS: Among 131 episodes of S. aureus bacteremia, 85 (66%) were MRSA of which 47 (54%) were CA-MRSA. Sixty-three patients (48%) developed endocarditis and 10 patients (8%) died in the hospital on the index admission Patients with CA-MRSA were significantly more likely to develop endocarditis (OR = 2.73, 95% CI = 1.30, 5.71). No other variables including comorbid conditions, current receipt of antiretroviral therapy, pre-culture severity of illness, or CD4 count were significantly associated with endocarditis and none were associated with in-hospital mortality. CONCLUSIONS: CA-MRSA was significantly associated with an increased incidence of endocarditis in this cohort of HIV patients with MRSA bacteremia. In populations such as these, in which the prevalence of intravenous drug use and probability of endocarditis are both high, efforts must be made for early detection, which may improve treatment outcomes.

Evaluation of self-reported ethnicity in a case-control population: the stroke prevention in young women study.

BACKGROUND: Population-based association studies are used to identify common susceptibility variants for complex genetic traits. These studies are susceptible to confounding from unknown population substructure. Here we apply a model-based clustering approach to our case-control study of stroke among young women to examine if self-reported ethnicity can serve as a proxy for genetic ancestry. FINDINGS: A population-based case-control study of stroke among women aged 15-49 identified 361 cases of first ischemic stroke and 401 age-comparable control subjects. Thirty single nucleotide polymorphisms (SNPs) throughout the genome unrelated to stroke risk and with established ancestry-based allele frequency differences were genotyped in all participants. The Structure program was used to iteratively evaluate for K = 1 to 5 potential genetic-based subpopulations. Evaluating the population as a whole, the Structure output plateaued at K = 2 clusters. 98% of self-reported Caucasians had an estimated probability >/=50% of belonging to Cluster 1, while 94% of self-reported African-Americans had an estimated probability >/=50% of belonging to Cluster 2. Stratifying the participants by self-reported ethnicity and repeating the analyses revealed the presence of two clusters among Caucasians, suggesting that potential substructure may exist. CONCLUSIONS: Among our combined sample of African-American and Caucasian participants there is no large unknown subpopulation and self-reported ethnicity can serve as a proxy for genetic ancestry. Ethnicity-specific analyses indicate that population substructure may exist among the Caucasian participants indicating that further studies are warranted.

Ischemic stroke risk, smoking, and the genetics of inflammation in a biracial population: the stroke prevention in young women study.

BACKGROUND: Although cigarette smoking is a well-established risk factor for vascular disease, the genetic mechanisms that link cigarette smoking to an increased incidence of stroke are not well understood. Genetic variations within the genes of the inflammatory pathways are thought to partially mediate this risk. Here we evaluate the association of several inflammatory gene single nucleotide polymorphisms (SNPs) with ischemic stroke risk among young women, further stratified by current cigarette smoking status. METHODS: A population-based case-control study of stroke among women aged 15-49 identified 224 cases of first ischemic stroke (47.3% African-American) and 211 age-comparable control subjects (43.1% African-American). Several inflammatory candidate gene SNPs chosen through literature review were genotyped in the study population and assessed for association with stroke and interaction with smoking status. RESULTS: Of the 8 SNPs (across 6 genes) analyzed, only IL6 SNP rs2069832 (allele C, African-American frequency = 92%, Caucasian frequency = 55%) was found to be significantly associated with stroke using an additive model, and this was only among African-Americans (age-adjusted: OR = 2.2, 95% CI = 1.0-5.0, p = 0.049; risk factor adjusted: OR = 2.5, 95% CI = 1.0-6.5, p = 0.05). When stratified by smoking status, two SNPs demonstrated statistically significant gene-environment interactions. First, the T allele (frequency = 5%) of IL6 SNP rs2069830 was found to be protective among non-smokers (OR = 0.30, 95% CI = 0.11-.082, p = 0.02), but not among smokers (OR = 1.63, 95% CI = 0.48-5.58, p = 0.43); genotype by smoking interaction (p = 0.036). Second, the C allele (frequency = 39%) of CD14 SNP rs2569190 was found to increase risk among smokers (OR = 2.05, 95% CI = 1.09-3.86, p = 0.03), but not among non-smokers (OR = 0.93, 95% CI = 0.62-1.39, p = 0.72); genotype by smoking interaction (p = 0.039). CONCLUSION: This study demonstrates that inflammatory gene SNPs are associated with early-onset ischemic stroke among African-American women (IL6) and that cigarette smoking may modulate stroke risk through a gene-environment interaction (IL6 and CD14). Our finding replicates a prior study showing an interaction with smoking and the C allele of CD14 SNP rs2569190.

Neuroserpin polymorphisms and stroke risk in a biracial population: the stroke prevention in young women study.

BACKGROUND: Neuroserpin, primarily localized to CNS neurons, inhibits the adverse effects of tissue-type plasminogen activator (tPA) on the neurovascular unit and has neuroprotective effects in animal models of ischemic stroke. We sought to evaluate the association of neuroserpin polymorphisms with risk for ischemic stroke among young women. METHODS: A population-based case-control study of stroke among women aged 15-49 identified 224 cases of first ischemic stroke (47.3% African-American) and 211 age-matched control subjects (43.1% African-American). Neuroserpin single nucleotide polymorphisms (SNPs) chosen through HapMap were genotyped in the study population and assessed for association with stroke. RESULTS: Of the five SNPs analyzed, the A allele (frequency; Caucasian = 0.56, African-American = 0.42) of SNP rs6797312 located in intron 1 was associated with stroke in an age-adjusted dominant model (AA and AT vs. TT) among Caucasians (OR = 2.05, p = 0.023) but not African-Americans (OR = 0.71, p = 0.387). Models adjusting for other risk factors strengthened the association. Race-specific haplotype analyses, inclusive of SNP rs6797312, again demonstrated significant associations with stroke among Caucasians only. CONCLUSION: This study provides the first evidence that neuroserpin is associated with early-onset ischemic stroke among Caucasian women.

Phosphodiesterase 4D polymorphisms and the risk of cerebral infarction in a biracial population: the Stroke Prevention in Young Women Study.

An association between polymorphisms within the phosphodiesterase 4D gene (PDE4D) and ischemic stroke was initially reported in older adults from Iceland and has been supported by studies in several other primarily elderly populations. In the present study, we examined the association between PDE4D polymorphisms and early-onset ischemic stroke in a biracial female population. A systematic search for polymorphisms in the highly evolutionary conserved regions of PDE4D was performed on 48 African-American and 48 Caucasian participants. Novel and known polymorphisms were then prioritized and genotyped in the entire study population of 224 cases of first ischemic stroke among women aged 15-49 and 211 age- and ethnicity-balanced control subjects. Forty novel and previously reported polymorphisms with a minor allele frequency greater than 0.05 were determined, with 23 polymorphisms selected for analysis in the full case-control sample. Single nucleotide polymorphism (SNP), linkage disequilibrium and haplotype analyses were performed. SNP rs918592, found in an intron near the 5' end of the gene, was significantly associated with stroke (age- and race-adjusted odds ratio (OR=1.5, P=0.007), with four other SNPs showing significant, albeit less strong, associations. The magnitude of association was similar across African-Americans and Caucasians and across multiple stroke subtypes (e.g. atherosclerotic, lacunar and non-lacunar of undetermined etiology). The association of rs918592 with stroke was confined exclusively to current smokers (OR=3.2, P=0.00014), with no association observed among never-smokers (OR=0.9, P=0.75) or former smokers (OR=1.2, P=0.66), demonstrating a gene-environment interaction (P=0.03). A strong dose-response relationship was also seen among current smokers. No specific risk haplotypes were identified.