The Association of the 4q25 Susceptibility Variant for Atrial Fibrillation With Stroke Is Limited to Stroke of Cardioembolic Etiology
Background and Purpose—Genome-wide association studies recently identified 2 variants on chromosome 4q25 as susceptibility factors for atrial fibrillation. Interestingly, these variants were subsequently also shown to be associated with stroke. However, it remains unclear whether 4q25 associates with all the stroke subtypes or with cardioembolic stroke in particular, which is often attributable to atrial fibrillation.
Methods—We performed a large case-control association study in 4199 ischemic stroke patients, all subtyped according to Trial of Org 10172 in Acute Stroke Treatment criteria, and 3750 controls derived from 6 studies conducted in Australia, Austria, Belgium, Poland, Spain, and Sweden. Two variants on chromosome 4q25, rs1906591 and rs10033464, were genotyped.
Results—Within cases, the A-allele of rs1906591 was associated with atrial fibrillation (odds ratio, 1.64 [95% CI, 1.43 to 1.90]; P=9.2 · 10−12), whereas rs10033464 was only marginally associated. There was an association between overall ischemic stroke and rs1906591 (odds ratio, 1.20 [95% CI, 1.09 to 1.32]; P=1.2 · 10−4). However, this was probably caused by the large effect of stroke of cardioembolic etiology because no relation was obtained in any other subgroup of stroke. The rs10033464 variant failed to show any relationship with ischemic stroke.
Conclusions—We replicated the association of the rs1906591 variant on chromosome 4q25 with atrial fibrillation and ischemic stroke of cardioembolic etiology. The 4q25 locus failed to associate with noncardiac subtypes of ischemic stroke.
In recent years, genome-wide association studies have been performed in several complex diseases to identify genetic risk factors.1 On average, these studies have led to the unbiased identification of genetic susceptibility factors that modestly elevate risk of disease. Stroke is a complex disease, in which several genetic variants, together with environmental factors, influence the risk of developing a cerebrovascular event. Several genome-wide studies have also been published on patients with stroke or MRI-defined brain infarction.2–6 One of the first sufficiently powered genetic association studies in stroke identified 2 variants on chromosome 4q25 as susceptibility factors for ischemic stroke.3 A small replication study in the Chinese Han population failed to show association for ischemic stroke.7 The 4q25 variants had been shown previously to confer risk for atrial fibrillation (AF).8 The 4q25 haplotype block consists of ≈1.5 million bp without any known genes. The closest gene, PITX2, is >50 000 bp away from these variants. PITX2 is a transcription factor critical for determining left–right asymmetry and for the differentiation of the left atrium.9 So far, it is unknown whether they also relate to other stroke-specific mechanisms beyond AF risk. Studies focusing on these 4q25 variants in AF were replicated extensively, and a meta-analysis revealed that the largest risk effect could be attributed to the rs2200733 variant (odds ratio [OR], 1.90), which is in full linkage with the rs1906591 variant, and a smaller risk effect to the rs10033464 variant (OR, 1.36).10 Unlike coronary artery disease and peripheral artery disease, ischemic stroke is characterized by heterogeneity. There are several subtypes of stroke, such as cardioembolic (CE) stroke, large-artery atherosclerosis, small vessel disease, undetermined (cryptogenic, more causes, or incomplete evaluation), or other causes.11 It has been suggested that risk factors could be subtype specific.12 The association of the variants on 4q25 with stroke was most striking in the CE subgroup.3 Because most patients with CE stroke have been diagnosed with AF, the most straightforward explanation might be that the 4q25 locus associates with AF and therefore indirectly also with CE stroke. However, because the association remained after excluding the patients with CE stroke in the analysis, the intriguing possibility could be proposed that AF represents a common and more important risk factor for non-CE stroke than generally has been anticipated. Other explanations are that the association with other subtypes is driven by the phenotypic misclassification or underdiagnosis of AF, or that 4q25 increases the risk for stroke through AF-independent mechanisms.
To carefully assess these hypotheses, we genotyped 2 genetic variants on chromosome 4q25 (ie, rs1906591 and rs10033464) in a large combined cohort of ischemic stroke patients and assessed their association with each etiologic subtype.
We assembled study cohorts from Belgium (principal investigator [PI] V.T., Leuven), Spain (PI J.M., Barcelona), Austria (PI R.S., Graz), Australia (PI C.L., New Lambton Heights), Poland (PI A.S., Krakow), and Sweden (PI C.J., Göteborg) through a collaboration with investigators from the International Stroke Genetics Consortium. Informed written consent was obtained from all subjects or their next of kin, and each local ethics committee approved the study.
Australia: Vascular Ischemia Study
All patients with cerebral ischemia (ischemic stroke only) who were admitted to 4 acute stroke units in NSW Australia were enrolled in the Vascular Ischemia Study from 2004 to 2008. Stroke patients from the Vascular Ischemia Study underwent brain imaging (48% had MRI, 96% had CT, and 46% had both) and a standardized protocol including laboratory examination, carotid ultrasound or CT angiography, and cardiac examination (transesophageal echocardiography or transthoracic echocardiography and Holter). Control subjects were selected randomly from the same geographical population and were healthy community-dwelling volunteers drawn from the Hunter Community Study.13 Cases and controls were predominantly of self-reported European descent.
Austria: Graz With Controls From Austrian Stroke Prevention Study
White patients with cerebral ischemia (ischemic stroke and transient ischemic attack) admitted to the stroke unit of the Medical University Graz Department of Neurology were included. All patients underwent either CT or MRI of the brain and a standardized protocol including a laboratory examination and carotid ultrasound or magnetic resonance angiography and ECG. More extensive cardiac examination, including transesophageal echocardiography or transthoracic echocardiography and Holter, was done in subjects with suspected cardiac embolism. Controls were participants of the Austrian Stroke Prevention Study14 and came from the same catchment area. All control subjects were free of stroke and dementia and underwent full risk factor assessment, brain MRI, Duplex scanning of the carotid arteries, ECG, and transthoracic echocardiography.
Belgium: Leuven Genetics Stroke Study
White patients with cerebral ischemia (ischemic stroke and transient ischemic attack) who were admitted to the stroke unit of the University Hospitals in Leuven were enrolled in the Leuven Stroke Genetics Study.15 All patients from the Leuven Stroke Genetics Study underwent brain imaging (MRI in >91% of patients and CT in the remainder) and a standardized protocol including laboratory examination, carotid ultrasound or CT angiography, and cardiac examination (transesophageal echocardiography or transthoracic echocardiography and Holter). Control subjects were selected from the same population and were either spouses of patients with multiple sclerosis, amyotrophic lateral sclerosis or stroke, or healthy community-dwelling subjects, for whom stroke-free status was ensured in 89%.
Patients were recruited in the stroke unit of the Jagiellonian University in Krakow, Poland (a single-center study). All stroke patients and controls, ≥18 years of age, were white. All stroke patients underwent CT, and MRI was performed in 10% of patients. A total of 86% underwent extracranial arterial ultrasound examination, 13% had intracranial angioimaging (digital subtraction angiography, angio-CT, or angio-MRI), and 64.5% patients had transthoracic echocardiography performed. Holter monitoring was performed when indicated. Patients <50 years of age underwent transesophageal echocardiography, Holter monitoring, angiography, or blood tests for hypercoagulability to exclude rare causes of stroke. The control group included unrelated subjects taken from the population of southern Poland. Control subjects had no apparent neurological disease based on the findings in a structured questionnaire and a neurological examination. Controls were matched to the patients with respect to the age of disease onset and gender.
Patients with ischemic stroke were recruited consecutively at the stroke unit of Vall d'Hebron Hospital, Barcelona, Spain. Only patients admitted at the stroke unit with stroke resulting from large and small vessel disease as well as CE origin were included in the genetic study. All patients underwent brain imaging by CT scan, and some had additional MRI performed. ECG and extracranial and intracranial arterial ultrasound examination were performed in all patients, and Holter monitoring and echocardiography were performed on patients with clinical suspicion of a CE source or undetermined origin.
Control participants were selected from relatives of patients (wife or husband, without any consanguinity among cases and controls) and healthy volunteers visiting the same hospital for routine testing. They were >65 years of age and classified as free of neurovascular and cardiovascular history and familial history of stroke by direct interview before recruitment.
Sweden: The Sahlgrenska Academy Study on Ischemic Stroke
The design of the Sahlgrenska Academy Study on Ischemic Stroke has been reported previously.16 Briefly, 600 patients who presented with first-ever or recurrent acute ischemic stroke before reaching 70 years of age were recruited consecutively at 4 stroke units. For each subject, a healthy community control, matched for age (±1 year), sex, and geographic area of residence, was randomly selected. All participants were white. All patients underwent ECG and neuroimaging at the acute stage (all by CT and 62% also by MRI). Additional diagnostic work-up was performed when clinically indicated as described.16
Stroke subtype was assessed according to modified Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria11 by stroke neurologists blinded to genotype. The classification was performed independently for each stroke cohort but in a standardized manner. The TOAST classification was divided in 5 categories: (1) large-artery occlusive disease (large vessel disease); (2) cardioembolism, (3) lacunar stroke (small vessel disease); (4) other determined causative factor; and (5) undetermined: causative agent unknown despite diagnostic efforts or >1 cause or incomplete evaluation. In the Spain cohort, patients with strokes classified as undetermined or attributable to other causes were excluded from the study. AF was diagnosed based on ECG or Holter findings.
This project was part of a larger study that was aimed at evaluating 39 single-nucleotide polymorphisms (SNPs) that had been shown previously to be associated with cardiovascular risk factors of stroke (supplemental Table I, available online at http://stroke.ahajournals.org).
All SNPs from the Australian, Belgian, Polish, and Swedish cohorts, as well as the cases from the Austrian cohort, were genotyped in 2 assays in a blinded manner using iPLEX technology on a matrix-assisted laser desorption/ionization time-of-flight based MassARRAY Compact Analyser (Sequenom, Inc.) as described previously.17 Primers were designed using AssayDesign software (version 22.214.171.124; Sequenom, Inc.). SNP genotypes were assigned with the TyperAnalyser software (version 126.96.36.199; Sequenom, Inc.). Only samples with ≥80% call success were retained for additional analysis (6266 of 6525; 96.03%). The average SNP call rate was 99.26%, with all individual call rates >96.66%. Quality control was performed by genotyping 95 samples in duplicate with a 99.57% concordance, as well as 79 Centre Etude Polymorphism Humain (CEPH) samples obtained from the Coriell Institute for Medical Research, with a concordance of 99.60%.
Genetic analysis of the Spanish samples was performed at the Spanish National Genotyping Centre (CeGen) using SNPlex (Applied Biosystems, Inc.) with GeneMapper 3.5 as allele calling algorithm (error rates <0.5%; call rates ≈90%).
Genotyping of the controls in the Austrian Stroke Prevention Study cohort was conducted on the Illumina Human610–Quad BeadChip platform. Genotyped SNPs underwent quality control filters for call rate >98%, minor allele frequency >0.01, and Hardy–Weinberg equilibrium P>1×10−6. DNA samples were excluded in the case of sample call rate <97.5%, an excess of autosomal heterozygosity, mismatch between called and phenotypic gender, or being outliers identified by the identity-by-state (IBS) clustering analysis. After quality control (QC), the database contained data on 550 635 markers for 856 subjects. The Markov Chain Haplotyping package (version 1.0.15 or 1.0.16 software) for imputation to plus strand of NCBI build 36, HapMap release No. 22 was used (only samples in the Austrian controls). The number of SNPs after imputation was 2 543 887. SNPs not present in the imputed set were genotyped using the TaqMan assay (Applied Biosystems) and the calling algorithm SDS Software v2.3 on the Applied Biosystems 7900HT Fast 384-well real-time polymerase chain reaction system.
Here we report the results on rs1906591 (r2=1 with rs2200733) and rs10033464 on chromosome 4.
Allele frequencies of the risk variants were compared within each study population within cases and between cases and controls using the Fisher exact test. Meta-analyses of the individual studies using a random-effects model were performed. For the subgroup analyses, allele frequencies in the different subgroups in each individual study were compared with the controls from the same study. Similarly, meta-analysis of the different subgroup results was performed. Logistic regression analysis was used to correct for age and sex differences between cases and controls for each individual study. Adjusted ORs and 95% CIs from the logistic regression were then combined in a random-effects meta-analysis. An interaction test was performed to compare the OR obtained for the cardiac embolism subgroup versus the OR for the subgroup without cardiac embolism.18 Statistical analysis was performed using PLINK (http://pngu.mgh.harvard.edu/∼purcell/plink/), SPSS 16.0 for Windows and the Comprehensive Meta-analysis Package 2.0. All tests were 2-tailed, and a P value of 0.05 was considered significant. A Bonferroni correction was applied where indicated because 39 SNPs were tested in this experiment.
Clinical characteristics of the 4199 included patients are shown in Table 1. There were 700 (16.7%) patients with large vessel disease, 754 (18.0%) patients with small vessel disease, and 1376 (32.8%) patients with CE stroke. A total of 1240 (29.5%) patients had stroke of undetermined etiology, and 129 (3.1%) patients were classified as having strokes of other determined etiologies. In the 6 different studies, a total of 3750 controls were recruited. The mean age was 67.0±14.3 years in cases and 63.4±12.2 in controls. The genotyping success rate was 98% in all groups, and no deviation from Hardy–Weinberg equilibrium was observed for any of these groups (P>0.10).
The 4q25 Locus Associates With AF in Stroke
We investigated whether the reported variants on chromosome 4q25 were associated with AF within our cohort of 4199 stroke patients. Overall, 968 stroke patients (23.1%) were diagnosed with AF. Allele frequencies are shown in Figures 1 and 2⇓. A pooled analysis of all cohorts revealed that the at-risk A allele of rs1906591 (hereafter referred to as rs1906591*A) was associated with AF (OR, 1.64 [95% CI, 1.43 to 1.90]; uncorrected P=9.2 · 10−12). Only a weak association was identified for the at-risk T-allele of rs130033464 (rs130033464*T; OR for rs10033464, 1.20 [95% CI, 1.01 to 1.44]; P=0.038 [without correction]).
Association of rs1906591*A With Ischemic Stroke
We then assessed whether 4q25 variants were also associated with ischemic stroke. A pooled analysis over all cohorts revealed that A allele for rs1906591 was more frequent in stroke patients than in controls (OR, 1.20 [95% CI, 1.09 to 1.32]; P=1.2 · 10−4; Table 2). This association remained after Bonferroni correction for 39 SNPs (P=0.0048). Correction for age and gender did not alter the results (OR, 1.18 [95% CI, 1.08 to 1.30]; P=5.7 to 10−4 ;Table 2; P=0.022 after Bonferroni correction).
Association With rs1906591*A With Various Stroke Subtypes
In analysis by subtype, rs1906591*A was associated with the CE subgroup (OR, 1.48 [95% CI, 1.28 to 1.71]; P=1.2 · 10−7, P=4.7 · 10−6 after Bonferroni correction but not with other stroke subtypes; Figure 3). In the CE subgroup, 846 patients (61%) were diagnosed with AF (in addition, 122 of patients with stroke with dual potential etiology (9.9%) experienced AF). When reanalyzing the data after exclusion of the CE stroke subtype, rs1906591*A failed to show a significant increase in risk (Table 2). Interestingly, an interaction between CE stroke and rs1906591 was identified (P=0.0014), underscoring the results for this variant in stroke to arise from the CE subtype. When analyzing true cryptogenic stroke (n=940) separately, potentially harboring patients with unidentified AF, no relationship could be established (OR 1.11 [95% CI, 0.87 to 1.42]; P=0.40).
Lack of Association With rs10033464*T and Stroke
T-allele frequencies were not different between controls and cases (9.2% versus 9.0%; OR for rs10033464*T, 0.97 [95% CI, 0.87 to 1.09]). Correction for age and gender did not alter the obtained result (OR, 1.00 [95% CI, 0.89 to 1.12]; Table 2). In addition, subgroup analysis failed to show association for any category of ischemic stroke, including CE stroke (Figure 4).
It has been reported that the genetic risk factor for AF on chromosome 4q25 was also associated with stroke. We recruited several independent study populations from European ancestry with variable stroke etiologies and aimed to replicate the association between the newly identified SNPs and stroke. We replicated the previously reported genetic variant on chromosome 4q25 with AF in our cohort of stroke patients. Our results are similar to those reported in the meta-analysis suggesting the largest effect for rs1906591.10 Overall, rs1906591 was associated with stroke, most prominently in the CE subtype. We formally confirmed this because an interaction test was positive. Therefore, we assume the risk factor to be only associated with stroke resulting from AF and not to ischemic stroke in general. In our study, there was a lack of association with rs10033464 and ischemic stroke or any subgroup.
It has been reported previously that in a subgroup analysis, the association of this genetic factor with stroke remained significant after excluding all patients who experienced CE stroke.3 Potentially, this could be because of phenotypic misclassification or ascertainment bias: patients classified as having a stroke of undetermined etiology when no cause was identified could in reality have a stroke of [unidentified] CE etiology because AF was not present during the hospital stay or because a less extensive investigation into CE sources was performed. However, the variants on 4q25 were not implicated in true cryptogenic stroke in this study, arguing against a large percentage of these patients experiencing unrevealed CE stroke. In addition, the TOAST phenotypic classification requires strokes to be classified as undetermined when >1 cause is identified (eg, large vessel disease in a patient with AF).
Because the genotyping of these variants has become commercially available, one might wonder whether patients with stroke of cryptogenic etiology should be offered genetic testing. Patients carrying the risk variant could, for example, be more extensively examined for paroxysmal AF. Our results do not provide evidence for performing such genotyping because no association was identified in cryptogenic stroke patients.
The variants on chromosome 4q25 have been replicated in AF patients. Meta-analysis of all studies performed in this condition identified an OR of 1.90 for rs2200733 (r2=1 with rs1906591) and 1.36 for rs10033464.10 Surprisingly, in our study, the rs10033464 was only marginally associated with AF. The meta-analysis in AF also identified a smaller effect size for this variant compared with rs2200733. Further, the replication study also failed to show significant association in 2 of the 4 replication cohorts, of which the Rotterdam Study was comparable in sample size to our study.10 This might also explain the lack of association with the subgroup of stroke attributable to CE etiology.
The mechanism by which this locus is pathologically involved in AF or stroke is unknown. The nearest gene to the genetic variants is the PITX2 gene. Mutations in this gene have been linked to Axenfeld–Rieger syndrome.19 Further, it plays a critical role in the development of the left atrium and the pulmonary vein myocardium.9 This particular region has been implicated in the initiation and maintenance of AF.20 However, mutational analysis of this gene in AF patients failed to identify genetic variants in the entire coding sequences.21
Genetic association studies in stroke have thus far identified only variants that confer limited risk. Potentially, the heterogeneity in etiology of stroke hampers the analysis, which might explain the disappointing results. One might argue that genetic risk factors are implicated only in subgroups of stroke instead of all ischemic strokes. SNPs associated with carotid atherosclerosis22 could predispose to large vessel disease, and those associated with white matter disease could be associated with lacunar stroke.23,24 Considering the heterogeneity in stroke etiology, it seems of importance to analyze genome-wide studies in this disease in each TOAST subgroup separately. In general, genome-wide association studies require large sample sizes, and because investigation of subgroups leads to loss of power, this holds even more for ischemic stroke. Ideally, all subgroups should be evenly represented and interactions between subgroups and identified genetic risk variants investigated. If stroke etiology varies strongly between populations included in meta-analysis, associations could be obscured.
Our studies had some limitations. First, not all TOAST subgroups were equally represented. This probably reflects the clinical spectrum of stroke etiology. However, in the Spain cohort, patients with undetermined or other etiology were excluded, causing differences in stroke subtype frequencies. Further, because we did not obtain a history of AF in all controls, correction for this variant was impossible, which could potentially obscure the association. The TOAST classification has been criticized because of its poor inter-rater reliability. Other stroke classification systems could be more reliable in the context of genetic studies.25,26 We also did not genotype 2 SNPs that have been found recently to be associated with AF and stroke.4,27,28 Finally, although our sample size is relatively large, when performing subgroup analysis, an even larger sample size is preferable. We did not correct for performing subgroup analysis; however, we believe our findings are quite robust given the strong statistical associations we found.
In conclusion, we replicated the association of the previously reported genetic variant on chromosome 4q25 with AF and ischemic stroke. Our data could only confirm a role of these variants in CE stroke and not stroke attributable to other etiologies. Potentially, as a result of the heterogeneity in stroke etiology, some genetic risk variants, particularly those with small effect sizes, might only be identified when analyzing subtypes of stroke. Future results of large genetic association studies are required to confirm this hypothesis.
Sources of Funding
This work was funded by the BNS Clinical Research Grant of the Belgian Neurological Society and by a grant of the Spanish Government (Geno-tPA project-FIS PJ060586 and GRECAS project EC08/00137). The Neurovascular Research Laboratory takes part in the network for Cooperative Neurovascular Research RENEVAS and the genestroke Spanish consortium. Genetic studies of the Austrian Stroke Prevention Study are supported by the Austrian Science Fond (P20545). R.L. is supported by Research Fund K.U. Leuven. V.T. is supported by FWO Research Foundation Flanders. A.S. was supported by the grant from the POLYPHARMA foundation No. 9/VII/08.
The first 2 authors contributed equally to this work.
- Received April 20, 2010.
- Accepted June 1, 2010.
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