W. Mark Brown

A genome-wide genotyping study in patients with ischaemic stroke: initial analysis and data release

BACKGROUND Despite evidence of a genetic role in stroke, the identification of common genetic risk factors for this devastating disorder remains problematic. We aimed to identify any common genetic variability exerting a moderate to large effect on risk of ischaemic stroke, and to generate publicly available genome-wide genotype data to facilitate others doing the same. METHODS We applied a genome-wide high-density single-nucleotide-polymorphism (SNP) genotyping approach to a cohort of samples with and without ischaemic stroke (n=278 and 275, respectively), and did an association analysis adjusted for known confounders in a final cohort of 249 cases and 268 controls. More than 400,000 unique SNPs were assayed. FINDINGS We produced more than 200 million genotypes in 553 unique participants. The raw genotypes of all the controls have been posted publicly in a previous study of Parkinsons disease. From this effort, results of genotype and allele association tests have been publicly posted for 88% of stroke patients who provided proper consent for public release. Preliminary analysis of these data did not reveal any single locus conferring a large effect on risk for ischaemic stroke. INTERPRETATION The data generated here comprise the first phase of a genome-wide association analysis in patients with stroke. Release of phase I results generated in these publicly available samples from each consenting individual makes this dataset a valuable resource for data-mining and augmentation.

Whole Genome Analyses Suggest Ischemic Stroke and Heart Disease Share an Association With Polymorphisms on Chromosome 9p21

Background and Purpose— Recently independent studies reported an association between coronary heart disease and single-nucleotide polymorphisms (SNPs) located at chromosome 9p21, near CDKN2A and CDKN2B genes. Given that stroke is a common complication after myocardial infarction, we investigated if the same SNPs were associated with ischemic stroke in our population. Methods— We recently initiated a whole genome analysis of ischemic stroke and published the first stage of a case control study using >400 000 SNPs from Illumina Infinium Human-1 and HumanHap300 assays. We focused on SNPs recently associated with heart disease by Helgadottir and colleagues and SNPs from the same haplotype block. Results— In analyses both unadjusted and adjusted for stroke risk factors, significant associations with ischemic stroke were observed for SNPs from the same haplotype block previously associated with myocardial infarction. Significant association was also seen between disease and haplotypes involving these SNPs, both with and without adjustment for stroke risk factors (odd ratios: 1.01 to 2.65). Conclusions— These data are important for 3 reasons: first, they suggest a genetic association for stroke; second, they suggest that this association shares pathogenic mechanisms with heart disease and diabetes; and third, they illustrate, that public release of data can facilitate rapid risk locus discovery.

Phosphodiesterase 4D and 5-lipoxygenase activating protein in ischemic stroke.

Risk for ischemic stroke is mediated by both environmental and genetic factors. Although several environmental exposures have been implicated, relatively little is known about the genetic basis of predisposition to this disease. Recent studies in Iceland identified risk polymorphisms in two putative candidate genes for ischemic stroke: phosphodiesterase 4D (PDE4D) and 5‐lipoxygenase activating protein (ALOX5AP). A collection of North American sibling pairs concordant for ischemic stroke and two cohorts of prospectively ascertained North American ischemic stroke cases and control subjects were used for evaluation of PDE4D and ALOX5AP. Although no evidence supported linkage of ischemic stroke with either of the two candidate genes, single‐nucleotide polymorphisms and haplotypic associations were observed between PDE4D and ischemic stroke. There was no evidence of association between variants of ALOX5AP and ischemic stroke. These data suggest that common variants in PDE4D may contribute to the genetic risk for ischemic stroke in multiple populations. Ann Neurol 2005;58:351–361

Chronic and Recurrent Otitis Media: A Genome Scan for Susceptibility Loci

Otitis media (OM) is the most common childhood disease. Almost all children experience at least one episode, but morbidity is greatest in children who experience chronic/recurrent OM (COME/ROM). There is mounting evidence that COME/ROM clusters in families and exhibits substantial heritability. Subjects who had tympanostomy tube surgery for COME/ROM (probands) and their families were recruited for the present study, and an ear examination was performed, without knowledge of the subjects history, to determine presence of OM sequelae. In addition, tympanometric testing was performed at three frequencies (226, 630 or 710, and 1,400 Hz) to detect abnormal middle-ear mechanics, and hearing was screened at 20 dB for the speech frequencies. Of these families, 121 had at least two individuals who had received the diagnosis of COME/ROM (364 affected and genotyped individuals), of whom 238 affected and informative relative pairs were used for analyses. Single-point nonparametric linkage analysis provided evidence of linkage of COME/ROM to chromosome 10q at marker D10S212 (LOD 3.78; P=3.0 x 10(-5)) and to chromosome 19q at marker D19S254 (LOD 2.61; P=5.3 x 10(-4)). Analyses conditional on support for linkage at chromosomes 10q and 19q resulted in a significant increase in LOD score support on chromosome 3p (between markers D3S4545 and D3S1259). These results suggest that risk of COME/ROM is determined by interactions between genes that reside in several candidate regions of the genome and are probably modulated by other environmental risk factors.

Meta-analysis of genome-wide association studies identifies 1q22 as a susceptibility locus for intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is the stroke subtype with the worst prognosis and has no established acute treatment. ICH is classified as lobar or nonlobar based on the location of ruptured blood vessels within the brain. These different locations also signal different underlying vascular pathologies. Heritability estimates indicate a substantial genetic contribution to risk of ICH in both locations. We report a genome-wide association study of this condition that meta-analyzed data from six studies that enrolled individuals of European ancestry. Case subjects were ascertained by neurologists blinded to genotype data and classified as lobar or nonlobar based on brain computed tomography. ICH-free control subjects were sampled from ambulatory clinics or random digit dialing. Replication of signals identified in the discovery cohort with p < 1 × 10(-6) was pursued in an independent multiethnic sample utilizing both direct and genome-wide genotyping. The discovery phase included a case cohort of 1,545 individuals (664 lobar and 881 nonlobar cases) and a control cohort of 1,481 individuals and identified two susceptibility loci: for lobar ICH, chromosomal region 12q21.1 (rs11179580, odds ratio [OR] = 1.56, p = 7.0 × 10(-8)); and for nonlobar ICH, chromosomal region 1q22 (rs2984613, OR = 1.44, p = 1.6 × 10(-8)). The replication included a case cohort of 1,681 individuals (484 lobar and 1,194 nonlobar cases) and a control cohort of 2,261 individuals and corroborated the association for 1q22 (p = 6.5 × 10(-4); meta-analysis p = 2.2 × 10(-10)) but not for 12q21.1 (p = 0.55; meta-analysis p = 2.6 × 10(-5)). These results demonstrate biological heterogeneity across ICH subtypes and highlight the importance of ascertaining ICH cases accordingly.

A Genome Scan for ESRD in Black Families Enriched for Nondiabetic Nephropathy

Nephropathy is a complex disorder, with predisposition influenced by the interplay of both genetic and environmental factors. As part of an effort to map genes that predispose to ESRD, a genome scan was performed in 264 black pedigrees that contained 296 ESRD-affected sibling pairs using multipoint nonparametric linkage analysis methods. The cause of ESRD in index cases was consistent with hypertension-associated ESRD. Nonparametric linkage (NPL) regression provided modest evidence of linkage to 9p21.3 near D9S1121 (logarithm of odds [LOD] = 2.03), 1q25.1 near D1S1589 (LOD = 1.62), and 13q33.3 near D13S796 (LOD = 1.02). Adjusting for the evidence of linkage at the other loci through the NPL regression analysis provided evidence for linkage to 1q25.1, 6p23, and 9p21.3. The NPL regression and ordered subset analyses suggest that the evidence for linkage significantly increased with early onset of ESRD (2q32.1 LOD = 3.89, 13q13.1 LOD = 3.90), increased BMI (8p22 LOD = 3.37, 13q33.3 LOD = 5.20, 18p11.3 LOD = 2.38), early onset of hypertension (14q21.1 LOD = 3.19, 20q13.2 LOD = 2.32), and late onset of hypertension (4q13.1 LOD = 3.44, 5p15.33 LOD = 2.82). Multipoint single-locus linkage analysis provided modest evidence of linkage to nondiabetic ESRD on 9p21.3, 1q25.1 (in the region of the podocin gene), and 13q33.3. NPL regression and ordered subset analyses also identified loci on 13q13.1 and 13q33.3 as contributing to early-onset ESRD and ESRD in the presence of increased BMI, respectively. These regions should receive priority in the search for loci that contribute susceptibility to nondiabetic nephropathy.

Age-stratified heritability estimation in the Framingham Heart Study families.

The Framingham Heart Study provides a unique source of longitudinal family data related to CVD risk factors. Age-stratified heritability estimates were obtained over three age groups (31–49 years, 50–60 years, and 61–79 years), reflecting the longitudinal nature of the data, for four quantitative traits. Age-adjusted heritability estimates were obtained at a single common time point for the same four quantitative traits. The importance of these groups is that they consist of the same individuals. The highest age-stratified heritability estimate (h2 = 0.88 (± 0.06)) was for height in the model adjusting for gender over all three age groups. SBP gave the lowest heritability estimate (h2 = 0.15 (± 0.11)) for the 70 age group in the model adjusting for gender, height, BMI, smoker, and drinker. BMI had slightly higher estimates (h2 = 0.64 (± 0.11)) in the 40 age group than previously published. The highest age-adjusted heritability estimate (h2 = 0.90 (± 0.06)) was for height in the model adjusting for gender. SBP gave the lowest heritability estimate (h2 = 0.38 (± 0.09)) for unadjusted model. These results indicate that some common, complex traits may vary little in their genetic architecture over time and suggest that a common set of genes may be contributing to observed variation for these longitudinally collected phenotypes.

Risk factors for intracerebral hemorrhage differ according to hemorrhage location

ABSTRACT Objectives: Risk factors have been described for spontaneous intracerebral hemorrhage (ICH); their relative contribution to lobar vs nonlobar hemorrhage location is less clear. Our purpose here was to investigate risk factors by hemorrhage location. Methods: This case-control study prospectively enrolled subjects with first-ever spontaneous ICH and matched each with up to 3 controls by age, race, and gender. Conditional stepwise logistic regression modeling was used to determine significant independent risk factors for lobar and nonlobar ICH. Results: From December 1997 through December 2006, 597 cases and 1,548 controls qualified for the analysis. Hypertension, warfarin use, first-degree relative with ICH, personal history of ischemic stroke, less than a high school education, and APOE ε2 or ε4 genotype were more common in ICH cases. Hypercholesterolemia and moderate alcohol consumption (≤2 drinks per day) were less common in ICH cases. The associations of hypertension and hypercholesterolemia were specific for nonlobar ICH. Conversely, the association of APOE ε2 or ε4 genotype was specific for lobar ICH. Conclusions: APOE ε2 or ε4 genotype was associated specifically with lobar ICH. Hypertension was associated specifically with nonlobar ICH. A protective association was seen between hypercholesterolemia and nonlobar ICH; no such association was identified for lobar ICH.

Candidate Gene Polymorphisms for Ischemic Stroke

Background and Purpose— Ischemic stroke (IS) is a multifactorial disorder with strong evidence from twin, family, and animal model studies suggesting a genetic influence on risk and prognosis. Several candidate genes for IS have been proposed, but few have been replicated. We investigated the contribution of 67 candidate genes (369 single nucleotide polymorphisms [SNPs]) on the risk of IS in a North American population of European descent. Methods— Two independent studies were performed. In the first, 342 SNPs from 52 candidate genes were genotyped in 307 IS cases and 324 control subjects. The SNPs significantly associated with IS were tested for replication in another cohort of 583 IS cases and 270 control subjects. In the second study, 212 SNPs from 62 candidate genes were analyzed in 710 IS cases with subtyping available and 3751 control subjects. Results— None of the candidate genes (SNPs) were significantly associated with IS risk independent of known stroke risk factors after correction for multiple hypotheses testing. Conclusion— These results are consistent with previous meta-analyses that demonstrate an absence of genetic association of variants in plausible candidate genes with IS risk. Our study suggests that the effect of the investigated SNPs may be weak or restricted to specific populations or IS subtypes.

IL1RN VNTR Polymorphism in Ischemic Stroke: Analysis in 3 Populations

Background and Purpose— Genetic factors influence risk for ischemic stroke and likely do so at multiple steps in the pathogenic process. Variants in genes related to inflammation contribute to risk of stroke. The purpose of this study was to confirm our earlier finding of an association between allele 2 of a variable number tandem repeat of the IL-1 receptor antagonist gene (IL1RN) and cerebrovascular disease. Methods— An association study of the variable number tandem repeat genotype with ischemic stroke and stroke subtypes was performed on samples from a North American study of affected sibling pairs concordant for ischemic stroke and 2 North American cohorts of prospectively ascertained ischemic stroke cases and unrelated controls. DNA analysis was performed on cases and controls, stratified by race. Results— After adjustment for age, sex, and stroke risk factors, the odds ratio for association of allele 2 and ischemic stroke was 2.80 (95% confidence interval, 1.29 to 6.11; P=0.03) for the white participants. The effect of allele 2 of IL1RN on stroke risk most closely fits a recessive genetic model (P=0.009). For the smaller sample of nonwhite participants, the results were not significant. Conclusions— Allele 2 of IL1RN, present in nearly one-quarter of stroke patients, may contribute to genetic risk for ischemic stroke and confirm the previously identified association with cerebrovascular disease. These results are driven by the association in the white participants. Further exploration in a larger nonwhite sample is warranted.