Published online before print June 28, 2007, doi: 10.1161/STROKEAHA.107.483115
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(Stroke. 2007;38:2241.)
© 2007 American Heart Association, Inc.
Original Contributions |
Confirmation of an Association Between the TNF(–308) Promoter Polymorphism and Stroke Risk in Children With Sickle Cell Anemia
From the Department of Hematology/Oncology (C.H., K.D., J.N., L. Styles), Children’s Hospital & Research Center Oakland, the Public Health Institute (W.K.), Oakland, and the Department of Human Genetics (S.C., M.G., L. Steiner), Roche Molecular Systems Inc, Alameda, Calif; and the Department of Neurology (R.A.), Medical College of Georgia, Augusta.
Correspondence to Carolyn Hoppe, MD, Department of Hematology/Oncology, Children’s Hospital & Research Center Oakland, 747 52nd St, Oakland, CA 94609. E-mail choppe{at}mail.cho.org
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionAppendix 1Appendix 2References |
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Background and Purpose— The etiology of stroke in children with sickle cell anemia (SCA) is complex and poorly understood. Growing evidence suggests that genetic factors beyond the sickle cell mutation influence stroke risk in SCA. We previously reported risk associations with polymorphisms in several proinflammatory genes in SCA children with ischemic stroke. The aim of this replication study was to confirm our previous findings of associations between the TNF(–308) G/A, IL4R 503 S/P, and ADRB2 27 Q/E polymorphisms and large vessel stroke risk.
Methods— Using previously collected MRA data, we assessed an independent population of SCA children from the multicenter Stroke Prevention Trial in Sickle Cell Anemia (STOP) for the presence or absence of large vessel stenosis. Samples were genotyped for 104 polymorphisms among 65 candidate vascular disease genes. Genotypic associations with risk of large vessel stroke were screened using univariable analysis and compared with results from our original study. Joint analysis of the 2 study populations combined was performed using multivariable logistic regression.
Results— A total of 96 children (49 MRA-positive, 47 MRA-negative) were included in this study. Of the SNP associations previously identified in the original study, the TNF(–308) G/A association with large vessel stroke remained significant and the IL4R 503 S/P variant approached significance in the joint analysis of the combined study populations. Consistent with our original findings, the TNF(–308) GG genotype was associated with a >3-fold increased risk of large vessel disease (OR=3.27; 95% CI=1.6, 6.9; P=0.006). Unadjusted analyses also revealed a previously unidentified association between the LTC4S(–444) A/C variant and large vessel stroke risk.
Conclusions— Similar findings in 2 independent study populations strongly suggest that the TNF(–308) G/A promoter polymorphism is a clinically important risk factor for large vessel stroke in children with SCA. The previously observed association with the IL4R 503 S/P variant and the novel association with the LTC4S(–444) A/C variant suggest that these loci may also contribute to large vessel stroke risk in children with SCA.
Key Words: genetics • pediatric stroke • risk factors • sickle cell anemia
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionAppendix 1Appendix 2References |
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Children with sickle cell anemia (SCA) carry a 300-fold increased risk for stroke, making SCA the most common cause of childhood stroke. In children with SCA, ischemic stroke is usually a result of stenosis or obstruction of the large intracranial vessels, including the distal internal carotid (dICA), proximal middle cerebral (MCA), and anterior cerebral (ACA) arteries.1,2 Although the etiology of stroke in children with SCA is not fully understood, twin and sibling studies in SCA support a genetic component beyond the sickle mutation.3,4 Attractive candidates for investigation of stroke predisposition in SCA include genes known to be involved in endothelial injury, thrombosis, and inflammation in the general stroke population. Although several studies have reported stroke associations with particular candidate gene polymorphisms in SCA, replication studies to confirm these associations are lacking.
We previously found distinct allelic associations with stroke in children who were enrolled in the national multicenter Cooperative Study of Sickle Cell Disease (CSSCD).5 In this representative population of children with sickle cell anemia, the TNF(–308)A, IL4R 503P, and ADRB2 27E polymorphisms were specifically associated with the large vessel (LV) subtype of stroke. To confirm these findings, we carried out the present study in an independent population of children drawn from the Stroke Prevention in Sickle Cell Anemia (STOP) trial and from our local institution. We applied the same multiplexed genotyping assay previously developed to screen for genes involved in pathways of inflammation, thrombosis, lipid metabolism, and blood pressure regulation. This panel included several functional polymorphisms, including the TNF(–308) promoter polymorphism, that have been associated with ischemic stroke risk in other populations.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionAppendix 1Appendix 2References |
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Study Population
A total of 96 unrelated subjects with SCA (homozygous Hb S) were included in this study. Of these, 77 eligible subjects were drawn from the multicenter STOP study and 19 local subjects who had not previously participated in the STOP study were included as cases. Cerebral MRI/MRA scanning was previously performed and DNA archived on all patients. To determine whether regular red cell transfusions could prevent primary stroke, the STOP study enrolled children who were at increased risk of stroke based on an abnormally elevated flow velocities by transcranial Doppler ultrasound screening. As children with a history of completed LV stroke were not included in the STOP study, we classified cases and controls based on a "prestroke" phenotype of LV disease using MRA data. Cases (n=49) were defined as having MRA-documented stenosis or obstruction of the middle cerebral artery (MCA) or the internal carotid artery (ICA) indicative of LV disease. Children who had a normal MRA and were not assigned to treatment with chronic transfusions as part of the STOP study were included as controls (n=47).
Participants in the STOP study previously consented to the use of stored DNA and neuroimaging data for future research. Personal identifiers were removed and patient samples were collected and stored with a study number previously assigned by STOP. Study subjects from our local institution gave written informed consent for this study. The present study was reviewed and approved by the Institutional Review Board of Children’s Hospital & Research Center Oakland (CHRCO).
MRA Scanning
Cerebral MRA data used to ascertain cases and controls in this study were based on predefined protocols to classify intracranial vascular lesions. Briefly, MRAs were performed according to standard protocol of image acquisition using a 3-dimensional time of flight technique as previously described.6,7 Criteria for acceptable MRA images were established by the STOP study neuroradiologists and included minimization of TE to 5 ms, the smallest feasible voxel size, field of view (FOV) 15 to 20 cm, a 256x512 matrix, and shortest obtainable echo time to minimize loss of flow-related signal. Uniform procedures were used for MIP image generation and filming.
All MRA images performed as part of the STOP study were previously reviewed by a central panel of experts. Each MRA was read independently by 2 neuroradiologists. If the interpretations differed, consensus was reached in discussion with a third neuroradiologist. All films underwent quality review by a member of the expert panel. Data on the presence, location, and severity of vascular lesions were recorded. MRAs were defined as normal or abnormal with reference to the ACA, MCA, and ICA segments. Arterial segments were defined as normal, mildly (25%), moderately (50%), or severely (75%) stenosed or occluded. For the 19 case subjects included from our local institution, MRA imaging was performed using a similar protocol and read by a CHRCO neuroradiologist. The results and interpretation for each MRA were reported in a standardized fashion, using criteria similar to those used by the STOP study panel.
Multilocus Genotyping
A previously developed panel comprising 104 single nucleotide polymorphisms among 65 candidate "vascular disease" genes was used for this study.5 Genotyping was performed using multiplex PCR and immobilized probe-based assays developed for multi-locus variant detection (Roche Molecular Systems) as previously described.5,8,9 Briefly, each sample was amplified using 3 mixes of biotinylated primer pairs, each targeting between 24 and 50 genomic fragments. Amplified fragments within each PCR product pool were then detected colorimetrically with sequence-specific oligonucleotide probes immobilized on nylon membranes in a linear array.10 Probe specificities were previously confirmed by sequencing, use of DNAs genotyped independently by other methods such as restriction fragment length polymorphism analysis, or by confirming observed frequencies against published values. Interpretation of alleles represented by positive probe signals was carried out by 2 independent investigators blinded to case-control status. Discordant interpretations (<1%) were resolved with repeat genotyping.
Statistical Methods
Hardy-Weinberg equilibrium was assessed by X2 analysis. Genotype frequencies were compared between MRA-positive case and MRA-negative control subjects with the X2 goodness-of-fit test using 2x2 contingency tables. For each of the diallelic markers examined at a particular gene locus, the 2 genotypes with the less common allele were compared with the homozygous genotype of the more common allele. Initial testing was performed to identify markers nominally associated with LV disease. To eliminate testing with low statistical power, the minimum allele frequency required for testing a particular gene locus was set at 10%. For those markers known to be in linkage disequilibrium, we chose a single marker for the analysis based on allelic frequency distributions (ie, those that were closest to an allelic frequency of 0.5). To reduce type 2 statistical error, the maximum associated probability value for reporting individual marker effects was set at P<0.10.
Allelic effects identified by univariate analysis were then tested in the combined population using the Cochran-Mantel-Haenszel method of stratified analysis.11,12 Before calculating the common odds ratio the Breslow-Day test for homogeneity of the odds ratio was performed. Odds ratios (OR) are presented with corresponding 95% confidence intervals. A Bonferroni correction for multiple comparisons was applied to significant associations when appropriate, with a correction factor derived from the markers previously identified in our original study. The R statistical software package was used for the stratified Mantel-Haenszel analysis; all other data were analyzed using Stata 8.0.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionAppendix 1Appendix 2References |
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The study sample comprised 53 females and 43 males with a mean age of 9.5±4.2 years (median, 10.2 years; range, 1.8 to 17.7 years). All subjects were African-American and were homozygous for the Hb S mutation (Hb SS). Although there was greater number of female than male subjects in the MRA(–) control group, this difference did not reach statistical significance (P=0.11). The case and control groups were otherwise similar to those described in our original study (Table 1).
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Brain MRA Results
Of the 96 SCA patients included in this study, 47 had normal brain MRA scans with a mean age of 8.75±3.16 years at the time of the last follow-up MRA. The 49 case children (51%) demonstrated stenosis/obstruction of at least 1 of the large intracranial arteries (ICA, MCA, ACA) on MRA and had a mean age of 9.51±4.16 years at the time of a first positive MRA. Of the 47 MRA-negative control subjects, 20 (43%) also had a normal brain MRI and 27 (57%) demonstrated subclinical white matter hyperintensities attributable to small vessel disease (SV) on MRI. For the purposes of this study, subjects were placed into 2 groups: those with LV disease and those without LV. Controls included patients with SV disease on MRI, because our original study did not show any overlapping associations. Forty-three (88%) of the 49 MRA-positive case subjects had coexisting MRI abnormalities consistent with infarction attributable to LV or SV disease. A clinical history of stroke was documented in 28 (57%) of the MRA-positive cases.
Genotypic Associations
Of the 104 total variant sites examined, 61 were sufficiently polymorphic to permit statistical testing, using 2x2 exact tests. The observed genotype distributions were in Hardy-Weinberg equilibrium and are shown for the SNPs with nominally significant results in the individual study populations (STOP/CHO and CSSCD), as well as combined study populations (Table 2
). Genotype distributions for the remaining SNPs are provided separately (Supplemental Table I, available online at http://stroke.ahajournals.org).
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Genotyping results were first examined for replication of the previously reported TNF(–308), IL4R, and ADRB2 27 associations with LV disease in the CSSCD population. Comparison of MRA-positive case subjects and MRA-negative control subjects revealed nominally significant differences (P<0.10) in 1 of these 3 SNPs, TNF(–308), with 18% carriers of the minor A allele in the MRA(+) group versus 38% in the control group (P=0.04).
Joint analysis of data from the combined study populations was performed to increase the power of this study to detect significant associations.13 Testing for homogeneity of the odds ratios was not significant, indicating that none of the pairs of OR values calculated for each of the variants differed between the 2 study populations. When analyzed jointly, the TNF(–308)A association persisted and the previously reported IL4R 503P association with stroke risk now approached significance (OR=2.1; 95% CI=1.1 to 3.8; P=0.03; Pcorr=0.09). In addition to the TNF(–308)A and IL4R 503P associations, the LTC4S (–444)A association identified by univariable analysis was still significant on joint analysis (OR=0.35; 95% CI=0.1 to 0.9; P=0.03).
These 3 variants met criteria for inclusion into the logistic regression model. Protective effects were found in association with the TNF(–308)A (OR=0.39, P=0.006) and LTC4S(–444)C alleles (OR=.39, P=0.03), whereas the risk-conferring effect of the IL4R 503P variant only approached significance (OR=1.6, P=0.09; Table 3).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionAppendix 1Appendix 2References |
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Our findings of an association between the TNF(–308)G/A promoter polymorphism and LV stroke risk were replicated in this study of an independent population of children with SCA. Consistent with our previous results, the TNF(–308)A minor allele was associated with protection from LV disease and, in our analysis of the 2 study populations combined, homozygosity for the corresponding TNF(–308) G allele was associated with a >3-fold increased risk of LV disease (OR=3.27; 95% CI=1.6, 6.9). Given a prevalence of 75% for the TNF(–308) GG genotype in our combined study population of SCA patients, the proportion of LV strokes attributable to this genotype may be as high as 63%.14
The TNF gene has been linked to increased susceptibility to a variety of conditions characterized by inflammation. Several studies have documented a TNF(–308) allelic association with ischemic stroke, but the results have not been consistent across populations. Whereas the TNF(–308)A allele was found to be protective in Korean adults with ischemic stroke15 and patients with lacunar infarcts,16 it conferred an increased risk of ischemic stroke in younger Italian patients17 and patients with a preceding febrile episode.18 Another study in Turkish children found no association between the TNF(–308) SNP and stroke.19 Population-specific differences in reported TNF(–308) allele frequencies, variability in stroke classification criteria, and allelic heterogeneity may potentially limit these comparisons, emphasizing the importance of replication studies to confirm original results.20
Studies investigating the functional relevance of the TNF(–308) polymorphism have yield mixed results, showing both positive and negative allelic correlations with TNF expression.21–25 These inconsistencies may be explained by data indicating that the differential expression of the TNF(–308) alleles is dependent on both the stimulus and the cell type examined.26 Nonetheless, the proinflammatory TNF gene may have a direct impact on predisposition to stroke in children with SCA, as vascular inflammation, marked by activated monocytes and endothelium, plays a significant role in the pathophysiology of this disease. Even at baseline, SCD patients demonstrate elevations of several inflammatory markers, including TNF.27,28 In vitro gene expression studies have shown sickled red blood cells, either directly or indirectly, promote endothelial cell upregulation of the TNF gene.29 Jison et al recently demonstrated differential peripheral blood mononuclear cell expression of 112 genes, including IL-15 which induces TNF production, in steady-state SCD patients.30
The replication of our findings showing a TNF(–308)G/A association with LV stroke risk in children with SCA is compelling and suggests that this association is unlikely to spurious. Although we hypothesize that the TNF(–308) SNP itself has a true effect on LV stroke risk, we cannot rule out the possibility that another marker in LD with this SNP is causative. Further studies of the LD patterns in this region and haplotype analyses are needed to determine whether it is indeed the TNF locus or other genes in linkage disequilibrium that are responsible for this association.
In addition to the TNF(–308) SNP association, the previously reported IL4R 503P association with LV disease was also reproduced in this study. Although the increased risk of LV disease associated with the IL4R 503P variant did not reach significance after adjustment for multiple testing, the magnitude and direction of this association were similar to the results reported in our original study. Finally, we found an association between the leukotriene C4 synthase, LTC4S (–444)C, variant and protection from LV disease that was not previously observed in our original study. How the LTC4S gene might plausibly influence stroke risk in SCA is less clear, but the (–444)C variant upregulates LTC4S mRNA expression, increasing the synthesis of proinflammatory leukotrienes, and has been associated with increased mean carotid artery intimal-medial thickness.31
A major strength of this replication study was the source population from which our study population was derived. As in our original study population, the sample of children came from a demographically representative multicenter cohort of SCA patients. However, subjects in the present study were classified based on MRA findings of LV disease rather than MRI evidence of ischemic stroke. The novel risk polymorphisms identified in this study may thus reflect a genetic distinction between the intermediate phenotype of MRA-detected vasculopathy and the final phenotype of ischemic stroke, and it is possible that these variants may be involved in earlier pathways leading to stroke.
Our study may have been limited by unrecognized population stratification, but this possibility was minimized, as cases and controls were drawn equally from representative sickle cell centers across the US. Moreover, the equal distribution of ß-globin haplotypes and similar frequencies of deletional alpha thalassemia among African-American SCA patients across the US argue against population admixture in this group.6,32 We also controlled for misclassification of cases and controls by using predefined phenotyping criteria and a central rating system in both studies.
Conclusions
In summary, replication of our results in a second independent population of children with SCA provides evidence for a true association between the TNF(–308) G/A variant and LV stroke risk. Persistence of the previously reported IL4R 503P association, as well as the novel risk association found in this study with the LTC4S (–444)C variant, suggest that these genes may also influence LV stroke risk in SCA. Further mechanistic studies would augment our findings to determine whether the gene products regulated by the TNF(–308) promoter polymorphism are involved in the pathways leading to LV disease and ultimately stroke in SCA. Nonetheless, these polymorphisms may prove informative for prediction of the genetic risk for LV disease and thereby contribute to the primary prevention of stroke in this inherently susceptible population of children.
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Appendix 1 |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionAppendix 1Appendix 2References |
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The following individuals were senior investigators in the Stroke Prevention Trial in Sickle Cell Anemia (STOP): R.J. Adams, F. Nichols, V. McKie, A. Kutlar (Medical College of Georgia, Augusta); L. Hsu (Emory University, Atlanta, Ga); B. Files (East Carolina University School of Medicine, Greenville, NC); E. Vichinsky (Children’s Hospital Oakland, Calif); C. Pegelow (University of Miami School of Medicine, Miami, Fla); M. Abboud (Medical University of South Carolina, Charleston, SC); D. Gallagher, D. Brambilla (New England Research Institutes, Watertown, Mass); D. Bonds (National Heart, Lung and Blood Institute, Bethesda, Md); G. Woods (Children’s Mercy Hospital, Kansas City, Mo); N. Olivieri (Hospital for Sick Children, Toronto, Canada); C. Driscoll (Children’s National Medical Center, Washington, DC); S. Miller (State University of New York Health Science Center, Brooklyn, NY); W. Wang (St. Jude Children’s Research Hospital, Memphis, Tenn); A. Hurlett (Columbia-Presbyterian Medical Center, NY); C. Scher (Tulane University Medical School, New Orleans, La); B. Berman (Rainbow Babies and Children’s Hospital, Cleveland, Ohio); E. Carl, A. Jones (Medical College of Georgia, Augusta, Ga); E.S. Roach (University of Texas Southwestern Medical Center, Dallas); E. Wright (New England Research Institutes, Watertown, Mass); R. Zimmerman (Children’s Hospital of Philadelphia, Philadelphia, Pa); and M. Waclawiw (National Heart, Lung, and Blood Institute, Bethesda, Md).
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Appendix 2 |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionAppendix 1Appendix 2References |
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Abbreviations for candidate gene polymorphisms assessed in Table 1: ADD1, adducin
; ADRB, ß adrenergic receptor; AGT, angiotensinogen; AGTR1, angiotensin receptor 1; APO, apolipoprotein; CBS, cystathionine ß-synthase; CCR, chemokine receptor; CETP, cholesteryl ester transfer protein; CD14, monocyte differentiation antigen; CSF2, colony-stimulating factor 2; CTLA4, cytotoxic T-lymphocyte antigen 4; DCP1, dipeptidyl carboxypeptidase 1/angiotensin converting enzyme; F2, coagulation factor II; F5, factor V; F7, factor VII; FCER1B, immunoglobulin E receptor 1 ß; FGB, fibrinogen beta polypeptide; GC, human vitamin D–binding protein gene; GNB3, guanine nucleotide binding protein ß 3; ICAM1, intracellular adhesion molecule 1; IL, interleukin; IL1A, interleukin 1 ; IL1B, interleukin 1 ß; IL4R, interleukin 4 receptor; IL5RA, interleukin 5 receptor ; ITGA2, platelet glycoprotein 1a; ITGB3, platelet glycoprotein IIIa; LDLR, low-density lipoprotein receptor; LIPC, hepatic lipase; LPA, apolipoprotein(a); LPL, lipoprotein lipase; LTA, tumor necrosis factor ß; LTC4S, leukotriene C4 synthase; MMP3, matrix metalloproteinase 3; MTHFR-5, 10, methylenetetrahydrofolate reductase; NOS2A, nitric oxide synthase 2A; NOS3, nitric oxide synthase 3 endothelial; NPPA, atrial natriuretic peptide; PAI1, plasminogen activator inhibitor type 1; PON, paraoxonase; PPARG, peroxisome proliferator activated receptor gamma; SCNN1A, sodium channel epithelial alpha subunit; SCYA11, eotaxin; SDF1, stromal-derived factor 1; SELE, E-selectin; SELP, P-selectin; TCF7, T-cell transcription factor 7; TGFB1, transforming growth factor-beta 1; TNF, tumor necrosis factor; UGB, uteroglobin; VCAM, vascular cell adhesion molecule; VDR, vitamin D receptor.
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Acknowledgments |
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Sources of Funding
This work was supported in part by the Doris Duke Charitable Foundation, Clinical Scientist Development Award, and by National Institutes of Health grants NS40292, HL64556-01, and M01RR01271.
Disclosures
S.C., M.G., and L.S. are employed by a company (RMS Inc) whose research assays were used in the present work.The remaining authors report no conflicts.
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Footnotes |
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*The Stroke Prevention Trial in Sickle Cell Anemia (STOP) Investigators are listed in Appendix 1.
Received January 22, 2007; accepted February 14, 2007.
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