Brief Report
Arthritis & Rheumatism DOI 10.1002/art.38520
RUNNING HEAD: TLR2 Polymorphism and Thrombosis in Lupus A Polymorphism in TLR2 is Associated with Arterial Thrombosis in a Multi-Ethnic SLE Population Rachel Kaiser1, Ling Fung (Paul) Tang2, Kimberly E. Taylor1, Kirsten Sterba1, Joanne Nititham1, Elizabeth E. Brown3, Jeffrey C. Edberg3, Gerald McGwin, Jr.3, Graciela S. Alarcón3, Rosalind Ramsey-Goldman4, John D. Reveille5, Luis M. Vilá6, Michelle Petri7, Joyce Rauch8, Emily Miller9, Kara Mesznik9, Pui-Yan Kwok2, Robert P. Kimberly3, Jane E. Salmon9 and Lindsey A. Criswell1,2 1
Rosalind Russell Medical Research Center for Arthritis, Department of Medicine, UCSF, San Francisco, CA 2 UCSF Institute for Human Genetics, San Francisco, CA 3 Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 4 Department of Medicine, Northwestern University, Chicago, IL 5 Department of Medicine, University of Texas Health Science Center at Houston, Houston, TX 6 Department of Medicine, University of Puerto Rico Medical Sciences Campus, San Juan, PR 7 Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 8 Department of Medicine, McGill University, Research Institute of the McGill University Health Centre, Montreal, QC, 9 Department of Medicine, Hospital for Special Surgery, Weill Cornell Medical College, New York, NY SUPPORT This publication was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through UCSF-CTSI Grant Number UL1 TR000004. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. This research was also supported by an Arthritis Foundation Postdoctoral Fellow Award, an American College of Rheumatology Investigator Award, an Alliance for Lupus Research Target Identification in Lupus award, a Kirkland Scholar Award, the UCSF Rosalind Russell Medical Research Center for Arthritis, operating grant MOP-97916 from the Canadian Institutes of Health Research, and the following NIH grants: P60 AR053308, R01 AR44804 and K24 AR02175. For the PROFILE collaborators – Dr. Kimberly: P60 AR048095, P01 AR049084, UL1TR000165, the Hopkins population is supported by NIH AR 43727, the Northwestern population is supported by K24 AR 002138, P60 2 AR 30692, PO1 AR 49084, and UL 1 RR 025741. The Hospital for Special Surgery Registry and Repository is supported by the Mary Kirkland Center for Lupus Research. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/art.38520 © 2014 American College of Rheumatology Received: Aug 06, 2013; Revised: Dec 29, 2013; Accepted: Feb 18, 2014
Arthritis & Rheumatology
The University of Puerto Rico population is supported by 8U54MD007587 from the National Institute on Minority Health and Health Disparities. CORRESPONDING AUTHOR Lindsey A. Criswell MD MPH UCSF Division of Rheumatology 513 Parnassus Ave, room 857 Medical Sciences Building San Francisco, CA 94143-0500 tel: (415) 476-9026 fax: (415) 476-9370 email: [email protected]
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Arthritis & Rheumatology
ABSTRACT
Objectives: Thrombosis is a serious complication of systemic lupus erythematosus (SLE). Limited prior work has investigated the genetics of thrombosis in SLE. We focus on candidate genes, including toll-like receptor (TLR) genes, which have been implicated in thrombosis pathogenesis.
Methods: SLE patients (n=3587) from 3 multi-ethnic populations were genotyped for 77 single nucleotide polymorphisms (SNPs) in 10 genes, primarily in TLR-2, 4, 7, and 9, and a set of 64 ancestry informative markers (AIMs). Association with arterial and venous thrombosis was first analyzed in the combined population via logistic regression, adjusting for top principal components of the AIMs and other covariates. An associated SNP, rs893629, was also meta-analyzed (after stratification by ethnicity and population) to confirm the association and test for population or ethnicity effects.
Results: In combined analysis, SNP rs893629 in the KIAA0922/TLR2 region was significantly associated with arterial thrombosis (logistic p=6e-5, false discovery rate p=0.0044). Two additional SNPs in TLR2 were also suggestive: rs1816702 (p=0.002) and rs4235232 (p=0.009). In meta-analysis, the OR for arterial thrombosis with rs893629 was 2.44 (95% CI 1.58–3.76) without evidence for heterogeneity (p=0.51). By ethnicity, the effect was most significant among African-Americans (OR=2.4, p=4e-4) and European Americans (OR 3.5, p=0.024).
Conclusion: TLR2 gene variation is associated with thrombosis in SLE, particularly among African-Americans and European Americans. There was no evidence of association among Hispanics, and results in Asian Americans were limited due to insufficient sample size. These results may help elucidate the pathogenesis of this important clinical manifestation.
John Wiley & Sons
Arthritis & Rheumatology
BACKGROUND Thrombosis is a complication that occurs in up to 37% of SLE patients (1). It occurs more frequently and at younger ages than in the general population. Prior research has elucidated clinical risk factors associated with thrombosis such as smoking and the presence of anti-phospholipid antibodies (aPL) (2). Work to identify the role of genes in this outcome, however, has been limited, especially among patients of non-European ancestry. Toll like receptors (TLR) are a component of the innate immune system. TLRs recognize pathogen-associated molecular patterns (PAMPs) such as microbial peptides and viral RNA as well as endogenous ligands released by cells undergoing apoptosis. TLR signaling leads to innate immune activation and an inflammatory response. This inflammatory response can activate the local endothelium (via TNFα, for example) leading to increased levels of tissue factor and activation of the coagulation cascade (3,4). Recent work has implicated innate immune activation in the pathogenesis of the anti-phospholipid syndrome (APS). Innate immune activation, particularly via TLR2 and TLR4, may be necessary for initiating aPL production and provides one mechanism for endothelial activation that leads to thrombosis (reviewed in (3,4)). In addition to their importance in the pathogenesis of thrombosis, several TLRs - including 4, 7 and 9 have been implicated in SLE pathogenesis (5). We investigated single nucleotide polymorphisms (SNPs) in TLRs 2, 4, 7 and 9 and an additional 6 genes associated with thrombosis risk in the general population in
John Wiley & Sons
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Arthritis & Rheumatology
the literature to assess their association with venous and arterial thrombosis in a multiethnic SLE population.
PATIENTS AND METHODS Our population consisted of 3,587 SLE patients enrolled in one of three North American study populations. Characteristics of these patients are shown in Table 1. All subjects met American College of Rheumatology criteria for SLE and all sites had Institutional Review Board approval. A total of 1,748 patients were derived from the Lupus Genetics Project at the University of California, San Francisco (UCSF) (6). The UCSF patients completed an extensive questionnaire, gave permission for medical record review, and provided a DNA sample. Recruitment sources included tertiary care and community hospitals and clinics as well as lupus support groups in northern California and nationwide. Baseline questionnaire data included demographic (e.g. self-report ethnicity based on four grandparental origin, sex), clinical (e.g. disease duration) and behavioral (e.g. smoking) factors. Thrombotic events were documented in the questionnaire then confirmed by medical record review (where thromboses not reported were also noted). Events included deep venous thrombosis (DVT), pulmonary embolism (PE), myocardial infarction (MI), cerebral vascular accident (CVA), recurrent miscarriages (at least 3 in the first trimester or one in the 2nd or 3rd trimester), and retinal vein thrombosis. 435 patients were recruited as part of the SLE Registry and Repository at the Hospital for Special Surgery in New York, NY. For this collection, patients who meet ACR criteria for SLE are invited to participate at the time of their visit with their HSS
John Wiley & Sons
Arthritis & Rheumatology
rheumatologist and follow-up evaluations and sample collections take place at subsequent physician visits. Over 670 data points are collected per visit, including detailed demographics (including self-reported ethnicity), family history, SLE manifestations, activity (SLEDAI) and damage (SLICC), comorbidities (including arterial and venous thrombotic events and pregnancy outcomes), laboratory tests and standard lupus and aPL serologies. Finally, 1404 SLE patients were obtained from the PROFILE population, a multicenter prospective cohort based at the University of Alabama at Birmingham (7). Patients meet ACR criteria, are at least 16 years of age, and have disease duration of ≤ 10 years at enrollment. Phenotype data obtained for these SLE patients includes age, sex, ethnicity, smoking, medication use, renal involvement, aPL positivity and thrombosis outcomes of deep venous thrombosis (DVT), cerebral vascular accident (CVA), and myocardial infarction (MI). Explanatory variables investigated for association with thrombosis risk included ethnicity (European American, Hispanic, African-American and Asian-American), age at diagnosis, and SLE disease duration. Other variables available for a majority of patients included smoking (ever vs. never exposure), immunomodulating medications (including cyclophosphamide, azathioprine, methotrexate, and mycophenalate mofetil, ever vs. never use), other important medications (hydroxychloroquine, prednisone) and the presence of aPL antibodies. aPL positivity was defined as the presence of at least one positive laboratory test (lupus anticoagulant [LAC] measured by Russell Viper Venom Time [RVVT] (including confirmatory studies)), anticardiolipin [aCL] IgG and
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Arthritis & Rheumatology
IgM, or beta-2 glycoprotein-1 antibodies (B2GP1)). Our outcome measure was at least one arterial or venous thrombosis.
Genotyping and Quality Control Primary predictors included 77 SNPs in ten genes including genes involved in innate immunity [toll-like receptors (TLRs) 2, 4, 7, and 9], platelet glycoproteins, and the coagulation cascade. Seventy tagging SNPs for TLR 2, 4, 7, and 9 with 1000bp upstream and downstream were included. Tagging SNPs were selected with the web tool SNPinfo (http://snpinfo.niehs.nih.gov/snpinfo/snptag.htm). In order to adjust for confounding by population substructure in our analyses, 64 ancestry informative markers (AIMs) (8) as well as a 3-marker set as a proxy for north-south European ancestry (9) were also genotyped. Supplemental Table 1 contains the complete list of 144 genotyped SNPs. Genotyping was performed using a custom 144-SNP Illumina GoldenGate Assay in the Genomics Core Facility at the University of California, San Francisco. Four SNPs were dropped because of low genotyping (>10% missing) and 6 SNPs were dropped because of minor allele frequency (MAF)
Arthritis & Rheumatism DOI 10.1002/art.38520
RUNNING HEAD: TLR2 Polymorphism and Thrombosis in Lupus A Polymorphism in TLR2 is Associated with Arterial Thrombosis in a Multi-Ethnic SLE Population Rachel Kaiser1, Ling Fung (Paul) Tang2, Kimberly E. Taylor1, Kirsten Sterba1, Joanne Nititham1, Elizabeth E. Brown3, Jeffrey C. Edberg3, Gerald McGwin, Jr.3, Graciela S. Alarcón3, Rosalind Ramsey-Goldman4, John D. Reveille5, Luis M. Vilá6, Michelle Petri7, Joyce Rauch8, Emily Miller9, Kara Mesznik9, Pui-Yan Kwok2, Robert P. Kimberly3, Jane E. Salmon9 and Lindsey A. Criswell1,2 1
Rosalind Russell Medical Research Center for Arthritis, Department of Medicine, UCSF, San Francisco, CA 2 UCSF Institute for Human Genetics, San Francisco, CA 3 Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 4 Department of Medicine, Northwestern University, Chicago, IL 5 Department of Medicine, University of Texas Health Science Center at Houston, Houston, TX 6 Department of Medicine, University of Puerto Rico Medical Sciences Campus, San Juan, PR 7 Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 8 Department of Medicine, McGill University, Research Institute of the McGill University Health Centre, Montreal, QC, 9 Department of Medicine, Hospital for Special Surgery, Weill Cornell Medical College, New York, NY SUPPORT This publication was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through UCSF-CTSI Grant Number UL1 TR000004. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. This research was also supported by an Arthritis Foundation Postdoctoral Fellow Award, an American College of Rheumatology Investigator Award, an Alliance for Lupus Research Target Identification in Lupus award, a Kirkland Scholar Award, the UCSF Rosalind Russell Medical Research Center for Arthritis, operating grant MOP-97916 from the Canadian Institutes of Health Research, and the following NIH grants: P60 AR053308, R01 AR44804 and K24 AR02175. For the PROFILE collaborators – Dr. Kimberly: P60 AR048095, P01 AR049084, UL1TR000165, the Hopkins population is supported by NIH AR 43727, the Northwestern population is supported by K24 AR 002138, P60 2 AR 30692, PO1 AR 49084, and UL 1 RR 025741. The Hospital for Special Surgery Registry and Repository is supported by the Mary Kirkland Center for Lupus Research. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/art.38520 © 2014 American College of Rheumatology Received: Aug 06, 2013; Revised: Dec 29, 2013; Accepted: Feb 18, 2014
Arthritis & Rheumatology
The University of Puerto Rico population is supported by 8U54MD007587 from the National Institute on Minority Health and Health Disparities. CORRESPONDING AUTHOR Lindsey A. Criswell MD MPH UCSF Division of Rheumatology 513 Parnassus Ave, room 857 Medical Sciences Building San Francisco, CA 94143-0500 tel: (415) 476-9026 fax: (415) 476-9370 email: [email protected]
John Wiley & Sons
Page 2 of 15
Page 3 of 15
Arthritis & Rheumatology
ABSTRACT
Objectives: Thrombosis is a serious complication of systemic lupus erythematosus (SLE). Limited prior work has investigated the genetics of thrombosis in SLE. We focus on candidate genes, including toll-like receptor (TLR) genes, which have been implicated in thrombosis pathogenesis.
Methods: SLE patients (n=3587) from 3 multi-ethnic populations were genotyped for 77 single nucleotide polymorphisms (SNPs) in 10 genes, primarily in TLR-2, 4, 7, and 9, and a set of 64 ancestry informative markers (AIMs). Association with arterial and venous thrombosis was first analyzed in the combined population via logistic regression, adjusting for top principal components of the AIMs and other covariates. An associated SNP, rs893629, was also meta-analyzed (after stratification by ethnicity and population) to confirm the association and test for population or ethnicity effects.
Results: In combined analysis, SNP rs893629 in the KIAA0922/TLR2 region was significantly associated with arterial thrombosis (logistic p=6e-5, false discovery rate p=0.0044). Two additional SNPs in TLR2 were also suggestive: rs1816702 (p=0.002) and rs4235232 (p=0.009). In meta-analysis, the OR for arterial thrombosis with rs893629 was 2.44 (95% CI 1.58–3.76) without evidence for heterogeneity (p=0.51). By ethnicity, the effect was most significant among African-Americans (OR=2.4, p=4e-4) and European Americans (OR 3.5, p=0.024).
Conclusion: TLR2 gene variation is associated with thrombosis in SLE, particularly among African-Americans and European Americans. There was no evidence of association among Hispanics, and results in Asian Americans were limited due to insufficient sample size. These results may help elucidate the pathogenesis of this important clinical manifestation.
John Wiley & Sons
Arthritis & Rheumatology
BACKGROUND Thrombosis is a complication that occurs in up to 37% of SLE patients (1). It occurs more frequently and at younger ages than in the general population. Prior research has elucidated clinical risk factors associated with thrombosis such as smoking and the presence of anti-phospholipid antibodies (aPL) (2). Work to identify the role of genes in this outcome, however, has been limited, especially among patients of non-European ancestry. Toll like receptors (TLR) are a component of the innate immune system. TLRs recognize pathogen-associated molecular patterns (PAMPs) such as microbial peptides and viral RNA as well as endogenous ligands released by cells undergoing apoptosis. TLR signaling leads to innate immune activation and an inflammatory response. This inflammatory response can activate the local endothelium (via TNFα, for example) leading to increased levels of tissue factor and activation of the coagulation cascade (3,4). Recent work has implicated innate immune activation in the pathogenesis of the anti-phospholipid syndrome (APS). Innate immune activation, particularly via TLR2 and TLR4, may be necessary for initiating aPL production and provides one mechanism for endothelial activation that leads to thrombosis (reviewed in (3,4)). In addition to their importance in the pathogenesis of thrombosis, several TLRs - including 4, 7 and 9 have been implicated in SLE pathogenesis (5). We investigated single nucleotide polymorphisms (SNPs) in TLRs 2, 4, 7 and 9 and an additional 6 genes associated with thrombosis risk in the general population in
John Wiley & Sons
Page 4 of 15
Page 5 of 15
Arthritis & Rheumatology
the literature to assess their association with venous and arterial thrombosis in a multiethnic SLE population.
PATIENTS AND METHODS Our population consisted of 3,587 SLE patients enrolled in one of three North American study populations. Characteristics of these patients are shown in Table 1. All subjects met American College of Rheumatology criteria for SLE and all sites had Institutional Review Board approval. A total of 1,748 patients were derived from the Lupus Genetics Project at the University of California, San Francisco (UCSF) (6). The UCSF patients completed an extensive questionnaire, gave permission for medical record review, and provided a DNA sample. Recruitment sources included tertiary care and community hospitals and clinics as well as lupus support groups in northern California and nationwide. Baseline questionnaire data included demographic (e.g. self-report ethnicity based on four grandparental origin, sex), clinical (e.g. disease duration) and behavioral (e.g. smoking) factors. Thrombotic events were documented in the questionnaire then confirmed by medical record review (where thromboses not reported were also noted). Events included deep venous thrombosis (DVT), pulmonary embolism (PE), myocardial infarction (MI), cerebral vascular accident (CVA), recurrent miscarriages (at least 3 in the first trimester or one in the 2nd or 3rd trimester), and retinal vein thrombosis. 435 patients were recruited as part of the SLE Registry and Repository at the Hospital for Special Surgery in New York, NY. For this collection, patients who meet ACR criteria for SLE are invited to participate at the time of their visit with their HSS
John Wiley & Sons
Arthritis & Rheumatology
rheumatologist and follow-up evaluations and sample collections take place at subsequent physician visits. Over 670 data points are collected per visit, including detailed demographics (including self-reported ethnicity), family history, SLE manifestations, activity (SLEDAI) and damage (SLICC), comorbidities (including arterial and venous thrombotic events and pregnancy outcomes), laboratory tests and standard lupus and aPL serologies. Finally, 1404 SLE patients were obtained from the PROFILE population, a multicenter prospective cohort based at the University of Alabama at Birmingham (7). Patients meet ACR criteria, are at least 16 years of age, and have disease duration of ≤ 10 years at enrollment. Phenotype data obtained for these SLE patients includes age, sex, ethnicity, smoking, medication use, renal involvement, aPL positivity and thrombosis outcomes of deep venous thrombosis (DVT), cerebral vascular accident (CVA), and myocardial infarction (MI). Explanatory variables investigated for association with thrombosis risk included ethnicity (European American, Hispanic, African-American and Asian-American), age at diagnosis, and SLE disease duration. Other variables available for a majority of patients included smoking (ever vs. never exposure), immunomodulating medications (including cyclophosphamide, azathioprine, methotrexate, and mycophenalate mofetil, ever vs. never use), other important medications (hydroxychloroquine, prednisone) and the presence of aPL antibodies. aPL positivity was defined as the presence of at least one positive laboratory test (lupus anticoagulant [LAC] measured by Russell Viper Venom Time [RVVT] (including confirmatory studies)), anticardiolipin [aCL] IgG and
John Wiley & Sons
Page 6 of 15
Page 7 of 15
Arthritis & Rheumatology
IgM, or beta-2 glycoprotein-1 antibodies (B2GP1)). Our outcome measure was at least one arterial or venous thrombosis.
Genotyping and Quality Control Primary predictors included 77 SNPs in ten genes including genes involved in innate immunity [toll-like receptors (TLRs) 2, 4, 7, and 9], platelet glycoproteins, and the coagulation cascade. Seventy tagging SNPs for TLR 2, 4, 7, and 9 with 1000bp upstream and downstream were included. Tagging SNPs were selected with the web tool SNPinfo (http://snpinfo.niehs.nih.gov/snpinfo/snptag.htm). In order to adjust for confounding by population substructure in our analyses, 64 ancestry informative markers (AIMs) (8) as well as a 3-marker set as a proxy for north-south European ancestry (9) were also genotyped. Supplemental Table 1 contains the complete list of 144 genotyped SNPs. Genotyping was performed using a custom 144-SNP Illumina GoldenGate Assay in the Genomics Core Facility at the University of California, San Francisco. Four SNPs were dropped because of low genotyping (>10% missing) and 6 SNPs were dropped because of minor allele frequency (MAF)
