the Departments of Anesthesia and Perioperative Care and Center for Cerebrovascular Research (A.S.A., K.Y.P., C.L., W.L.Y.), Cardiovascular Research Institute (L.P., C.H., P.Y.K.), Departments of Epidemiology and Biostatistics (C.E.M., S.C.J.), Neurology (S.C.J., W.L.Y.), Medicine (J.G.Z.), Neurological Surgery (M.T.L., W.L.Y.), University of California, San Francisco, California
Division of Research, Kaiser-Permanente Medical Care Program, Oakland, Calif (S.S.)
the Department of Genetics, Duke University Medical Center, Durham, NC (D.A.M.).
Abstract
Background and Purpose— Identification of single-nucleotide polymorphisms (SNPs) associated with increased risk of new intracranial hemorrhage (ICH) after brain arteriovenous malformation (BAVM) diagnosis would facilitate risk stratification and identify potential targets for therapeutic intervention.
Methods— Patients with BAVM were longitudinally followed. Primary outcome was new ICH after diagnosis; censoring events were last follow-up or any BAVM treatment. We genotyped 4 promoter SNPs in 2 inflammatory cytokine genes: interleukin-6 (IL-6–174G>C; IL-6–572G>C) and tumor necrosis factor- (TNF-–238G>A; TNF-–308G>A). Association of genotype with risk of new ICH was screened using 2; SNPs associated with new ICH were further characterized using Cox proportional hazards.
Results— We genotyped 280 patients (50% female; 59% white, mean±SD age at diagnosis 37±17 years; 40% presenting with ICH). TNF-–238G>A was associated with increased risk of new ICH after diagnosis (2; P=0.003). After adjusting for age, race/ethnicity, and clinical presentation, the risk of new ICH was increased for patients with TNF-–238 AG genotype (hazard ratio, 4.01; P=0.015). No other SNP was found to be associated with new ICH.
Conclusion— A TNF- SNP was associated with increased risk of new ICH in the natural course of BAVMs. The role of inflammatory cytokines in the pathogenesis of BAVM hemorrhage merits further study.
Key Words: cerebral hemorrhage genetics vascular malformations
Introduction
We previously reported on genes implicated in inflammatory and angiogenic pathways and found an interleukin-6 (IL-6) single-nucleotide polymorphism (SNP), IL-6–174G>C, was associated with presenting intracranial hemorrhage (ICH) at brain arteriovenous malformation (BAVM) diagnosis; a tumor necrosis factor- (TNF-) SNP, TNF-–308G>A, showed a similar trend.1
In this study, we sought to examine whether promoter polymorphisms in these 2 genes (IL-6: -174G>C, -572G>C; TNF-: -238G>A, -308G>A) were associated with risk of new ICH after diagnosis in the natural course of BAVMs.
Materials and Methods
With informed consent, we recruited BAVM patients at the University of California San Francisco (UCSF)1 or at Kaiser Permanente Northern California (KPNC)2 and classified them using standardized guidelines.3 We genotyped 4 polymorphisms by template-directed dye-terminator incorporation assay with fluorescence polarization detection.1 The primary outcome was occurrence of new ICH (symptomatic new hemorrhage with intracranial blood on computed tomography or MRI) after initial presentation, before any treatment.
Association of genotype with risk of new ICH after diagnosis was screened (2 tests; t tests). SNPs found to have association with new ICH that survived Bonferroni correction (P=0.0125) were selected for survival analyses (Cox regression; Kaplan–Meier).
The period at risk was defined from date of BAVM diagnosis to date of an event (ie, onset of new [first subsequent] ICH) or censoring attributable either to initiation of first BAVM treatment (surgery, embolization, or radiosurgery) or loss to follow-up (using date of last available follow-up), whichever occurred first.
The predictors in our multivariate model were chosen based on their clinical/statistical importance to the outcome in an attempt to achieve maximum parsimony. The predictors included genotype, age at diagnosis (years), race/ethnicity (white versus nonwhite) and initial presentation (presenting ICH versus nonhemorrhagic presentation at diagnosis); their effects were reported as hazard ratios (HRs) and CIs.
Results
We genotyped 280 patients (Table 1); 189 were evaluated at UCSF and 91 at KPNC. As reported previously,1 patients homozygous for IL-6–174G had a greater risk of presenting with ICH than IL-6–174C carriers (univariate logistic regression analysis; HR, 2.00 [CI, 1.20 to 3.35]; P=0.008). Neither the more distal IL-6–572G>C SNP nor the 2 TNF- SNPs were associated with ICH presentation.
In contrast, only the TNF- promoter SNP TNF-–238G>A was associated with increased risk of new ICH after diagnosis (mean±SD follow-up 2.7±6.7 years). Overall, 224 (80.0%) cases were followed until censoring treatment (74 surgery, 107 embolization, and 43 radiosurgery), and 38 (13.6%) were untreated at last follow-up. New ICH in the natural course after diagnosis occurred in 18 (6.4%) patients and was associated with the AG genotype of TNF-–238G>A (2; P=0.003); this association survived correction for multiple testing.
TNF-–238G>A was selected for further analysis to estimate prediction of BAVM hemorrhage risk (Table 2). In Cox regression analysis, risk of new ICH was increased for patients with TNF-–238 AG genotype, adjusting for race/ethnicity, age, and presenting ICH (HR, 4.01 [CI, 1.31 to 12.29]; P=0.015; Table 2).
Kaplan–Meier analysis exploring association of TNF-–238 AG genotype with new ICH supported this trend (Figure; P=0.06).
Kaplan–Meier statistics.
Results were unaffected by including center of ascertainment (UCSF versus KPNC) as a factor in the models. TNF-–238 AG genotype conferred similar magnitude of risk in subset analysis within the major ethnic group (whites) and in stratified analysis irrespective of presenting ICH versus non-ICH presentation (data not shown). Haplotype analysis was performed for TNF- and IL-6 genes; results were consistent with single-SNP results but did not contribute additional information (data not shown).
Discussion
This study demonstrates the first evidence of an association between genotype and risk of new ICH in the natural course of BAVMs. The AG genotype of the TNF-–238G>A promoter polymorphism was associated with a 4-fold increase in risk of new ICH. This finding further implicates inflammatory processes in the pathogenesis of vessel rupture. TNF- is a proinflammatory and immunomodulatory cytokine implicated in inflammatory conditions that involve proteolytic processes.4 TNF-–238 AG genotype has been associated with a high cytokine expressor phenotype.5
As an upstream modulator for many inflammatory cytokines and proteolytic enzymes, TNF- induces IL-6 and matrix metalloproteinases, a family of proteolytic enzymes that degrade extracellular matrix around blood vessels and damage endothelial cells, which could result in destabilization and potential weakening of the vessel wall, passive dilation, and rupture.6 Surgical BAVM specimens display elevated matrix metalloproteinase expression.6 IL-6 expression appears to be increased as well and is associated with IL-6–174 GG genotype.7
Although these observations suggest a common pathway whereby genetic variation in IL-6 and TNF- influences hemorrhagic risk, it remains unclear why only the IL-6–174 GG genotype was associated with hemorrhagic presentation at diagnosis, whereas the current study only implicates TNF-–238 AG genotype in increased risk of new ICH in the natural course of disease after presentation.
A limitation of the current study is that the results depend on events in a small subset of our patients, which makes it difficult to assess more complicated models and fully explore possible confounders. Another consequence is a wide CI for the HR for TNF- in Table 2 (1.31–12.29), although this does bound the HR to be 1.31. Nevertheless, we believe these findings indicate that the role of inflammatory cytokines in the pathogenesis of BAVM hemorrhage merits further study.
Acknowledgments
This work was supported in part by Public Health Service grants R01 NS34949 & NS41877, P01 NS44155. The following members of the UCSF BAVM Study Project contributed to portions of this work: Nancy Quinnine, Esteban Burchard, Guo-Yuan Yang, Yongmei Chen, Frankye Pang, Tomoki Hashimoto, Eric Theise, Brad Dispensa, Van Halbach, Randall T. Higashida, and Christopher Dowd. The authors thank Henry Matallana and the UCSF DNA Bank.
References
Pawlikowska L, Tran MN, Achrol AS, McCulloch CE, Ha C, Lind DL, Hashimoto T, Zaroff J, Lawton MT, Marchuk DA, Kwok P-Y, Young WL. Polymorphisms in genes involved in inflammatory and angiogenic pathways and the risk of hemorrhagic presentation of brain arteriovenous malformations. Stroke. 2004; 35: 2294–2300.
Halim AX, Johnston SC, Singh V, McCulloch CE, Bennett JP, Achrol AS, Sidney S, Young WL. Longitudinal risk of intracranial hemorrhage in patients with arteriovenous malformation of the brain within a defined population. Stroke. 2004; 35: 1697–1702.
Joint Writing Group of the Technology Assessment Committee ASoIaTN, Joint Section on Cerebrovascular Neurosurgery, a section of American Association of Neurological Surgeons and Congress of Neurological Surgeons, and Section of Stroke and the Section of Interventional Neurology of the American Academy of Neurology. Reporting terminology for brain arteriovenous malformation clinical and radiographic features for use in clinical trials. Stroke. 2001; 32: 1430–1442.
Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med. 2005; 352: 1685–1695.
Reich K, Mossner R, Konig IR, Westphal G, Ziegler A, Neumann C. Promoter polymorphisms of the genes encoding tumor necrosis factor-alpha and interleukin-1beta are associated with different subtypes of psoriasis characterized by early and late disease onset. J Invest Dermatol. 2002; 118: 155–163.
Hashimoto T, Wen G, Lawton MT, Boudreau NJ, Bollen AW, Yang GY, Barbaro NM, Higashida RT, Dowd CF, Halbach VV, Young WL. Abnormal expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in brain arteriovenous malformations. Stroke. 2003; 34: 925–931.
Chen Y, Shen F, Yao JS, Zhai W, Chopra M, Yang G-Y, Young WL. Interleukin-6 expression is increased in BAVM patients carrying a high-risk interleukin-6 gene polymorphism. Ann Neurol. 2005; November 8 [Epub ahead of print].
Cardiac Troponin Elevation, Cardiovascular Morbidity, and Outcome After Subarachnoid Hemorrhage
Presence of Intraplaque Hemorrhage Stimulates Progression of Carotid Atherosclerotic Plaques
Randomized Controlled Trial of Acetylsalicylic Acid in Aneurysmal Subarachnoid Hemorrhage
Lack of Evidence for an Association Between Hemodynamic Variables and Hematoma Growth in Spontaneous Intracerebral Hemorrhage
Akt/GSK3 Survival Signaling Is Involved in Acute Brain Injury After Subarachnoid Hemorrhage in Rats
Pretreatment With Antiplatelet Agents Is Not Independently Associated With Unfavorable Outcome in Intracerebral Hemorrhage
Adrenoceptor Polymorphisms and the Risk of Cardiac Injury and Dysfunction After Subarachnoid Hemorrhage
Vasospasm and p53-Induced Apoptosis in an Experimental Model of Subarachnoid Hemorrhage
Intracerebral Hemorrhage Elicits Aberration in Cardiomyocyte Contractile Function and Intracellular Ca2+ Transients
D-Dimer as an Independent Predictor for Poor Outcome After Aneurysmal Subarachnoid Hemorrhage