Original Article

Genes and Immunity (2009) 10, 248–253; doi:10.1038/gene.2008.95; published online 18 December 2008

Replication of the TNFSF4 (OX40L) promoter region association with systemic lupus erythematosus

A M Delgado-Vega1, A-K Abelson1, E Sánchez2, T Witte3,9, S D'Alfonso4,9, M Galeazzi5, J Jiménez-Alonso6,9, B A Pons-Estel7,9,10, J Martin2,11 and M E Alarcón-Riquelme1,8,11

  1. 1Unit of Medical Genetics, Department of Genetics and Pathology, Rudbeck Laboratory, University of Uppsala, Uppsala, Sweden
  2. 2Instituto de Biomedicina ‘López-Neyra’, CSIC, Granada, Spain
  3. 3Department of Medicine, Medical School Hannover, Hannover, Germany
  4. 4Department of Medical Sciences and IRCAD, University of Eastern Piedmont, Novara, Italy
  5. 5Rheumatology Unit, Department of Clinical Medicine, Siena University, Siena, Italy
  6. 6Servicio Medicina Interna, Hospital Virgen de las Nieves, Granada, Spain
  7. 7Sanatorio Parque, Rosario, Argentina
  8. 8Arthritis and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA

Correspondence: Professor ME Alarcón-Riquelme, The Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Dag Hammarskjölds väg 20, Uppsala SE-751 85, Sweden. E-mail: marta.alarcon@genpat.uu.se

9Other clinical collaborators who provided samples are listed in the acknowledgements.

10Bernardo Pons-Estel is the coordinator of the Argentine Collaborative Group

11These authors contributed equally to this work.

Received 3 September 2008; Accepted 17 November 2008; Published online 18 December 2008.

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Abstract

The tumor necrosis factor ligand superfamily member 4 gene (TNFSF4) encodes the OX40 ligand (OX40L), a costimulatory molecule involved in T-cell activation. A recent study demonstrated the association of TNFSF4 haplotypes located in the upstream region with risk for or protection from systemic lupus erythematosus (SLE). To replicate this association, five single nucleotide polymorphisms (SNPs) tagging the previously associated haplotypes and passing the proper quality-control filters were tested in 1312 cases and 1801 controls from Germany, Italy, Spain and Argentina. The association of TNFSF4 with SLE was replicated in all the sets except Spain. There was a unique risk haplotype tagged by the minor alleles of the SNPs rs1234317 (pooled odds ratio (OR)=1.39, P=0.0009) and rs12039904 (pooled OR=1.38, P=0.0012). We did not observe association to a single protective marker (rs844644) or haplotype as the first study reported; instead, we observed different protective haplotypes, all carrying the major alleles of both SNPs rs1234317 and rs12039904. Association analysis conditioning on the haplotypic background confirmed that these two SNPs explain the entire haplotype effect. This first replication study confirms the association of genetic variation in the upstream region of TNFSF4 with susceptibility to SLE.

Keywords:

systemic lupus erythematosus, TNFSF4, OX40L, genetic association study

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Introduction

The tumor necrosis factor ligand superfamily member 4 gene (TNFSF4) gene is a recent acquisition to the soon long list of non-major histocompatibility complex susceptibility genes associated with systemic lupus erythematosus (SLE),1 such as PDCD1,2 FCGRIIA,3 ITGAM,4, 5 IRF5,6, 7, 8 BANK1,9 STAT4,10 PTPN2211, 12 and TNFAIP3.13, 14 However, putative genetic associations need to be verified using sufficiently large independent cohorts of patients and controls to confirm true susceptibility genes. TNFSF4 belongs to the tumor necrosis factor ligand superfamily of genes and encodes the ligand for OX40 (OX40L, CD252). This receptor–ligand pair is involved in various important regulatory functions associated with lymphocyte activation. OX40L is expressed primarily on antigen-presenting cells, including plasmacytoid dendritic cells, whereas OX40 is expressed on CD4+ T cells.15 OX40L has been found to inhibit the generation of interleukin-10-producing CD4+ type 1 regulatory T cells (Tr1) from naive and memory T cells, and to strongly inhibit interleukin-10 production and suppressive function of differentiated Tr1 cells.16

The recently identified association of TNFSF4 with SLE1 described a haplotype block in the upstream region of the gene with four single nucleotide polymorphisms (SNPs) (rs10912580, rs12039904, rs2205960 and rs1234317) tagging a unique risk haplotype, and one SNP (rs844644) tagging a protective haplotype. Although no functional variants were identified, correlation with expression of OX40L and CD86 was observed.1 This study replicates the genetic association between TNFSF4 polymorphisms and SLE in four independent European SLE sets from Germany, Italy, Spain and Argentina.

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Results

Single marker analysis

Six SNPs were genotyped in the upstream region of TNFSF4. After frequency and genotyping quality-control filters, five markers were tested for differences in allelic frequencies between cases and controls (Table 1). All the markers were strongly associated in the Argentineans and Italians, whereas three markers (rs1234317, rs12039904 and rs844648) displayed only weak association in the Germans, and none reached statistical significance in the Spanish set. In terms of statistical significance, the SNPs with the strongest effect were rs1234317 in the Argentine set (odds ratio (OR)=1.73 (1.36–2.20), P-value=6.23 × 10−6), rs12039904 in Germans (OR=1.36 (1.05–1.77), P-value=2.05 × 10−2) and rs844644 in the Italian set, which was the strongest marker with a protective effect (OR=0.64 (0.49–0.82), P-value=5.68 × 10−4) (Table 1). Owing to the observed heterogeneity for some markers, the pooled OR estimates were considered under a random effects model (DerSimonian–Laird method) in the meta-analysis.


In general, the minor alleles of the SNPs rs1234317 (T) and rs120399904 (T) displayed the most consistent association, as they were associated with an increased risk to develop SLE in three of the four populations studied and showed the strongest effect size (odds ratio) in the meta-analysis (rs1234317: ORDL=1.39 (1.15–1.69), P-value=9 × 10−4; rs12039904: ORDL=1.38 (1.14–1.68), P-value=1.2 × 10−3). The Breslow–Day test indicated homogeneity of the OR for rs12039904 (P>0.05) and borderline heterogeneity for rs1234317 (P=0.045) (Table 1). The rare A allele of rs844644 previously shown to tag a protective haplotype1 was associated only in the Italian (P=1.59 × 10−3) and Argentine sets (5.68 × 10−4) (Table 1). The Breslow–Day test for this marker was significant (P=0.0014), suggesting heterogeneity in the distribution of protective haplotypes.

Proxy association

To refine the SNP's association signals, we reanalyzed the data in PLINK by the proxy association function on imputation mode.17 The proxy association test enables the extension of the SNP coverage by imputing nongenotyped SNPs on the basis of patterns of haplotype structure from HapMap,17, 18 and may reveal markers with higher association values not detectable otherwise.17 We successfully imputed five SNPs that were associated in the previous TNFSF4 fine mapping study1 (Supplementary Table S1). We observed a similar magnitude of association between the SNPs rs1234317 (genotyped), rs12039904 (genotyped), rs2205960 (imputed) and rs10912580 (imputed) in Argentineans, Germans and Italians (Supplementary Table S1), which was expected because rs12039904 is highly correlated with rs2205960 (r2=0.90) and rs10912580 (r2=0.95) according to the HapMap-CEU data. The linkage disequilibrium (LD) between rs1234317 and rs12039904 is high but not enough to be complete proxies of each other (r2=0.64 in HapMap, r2=0.65-0.72 in our cohorts). We did not detect any imputed SNP with an effect stronger than the genotyped markers.

Haplotype association

After determining the LD structure of the region, and given the overall high correlation between SNPs, we considered a single five-markers block for subsequent haplotype analyses (rs1234314–rs1234317–rs844644–rs12039904–rs844648) (Table 2). The haplotype omnibus test results showed a similar association pattern as the single marker results: higher effects in the Argentine (P=8 × 10−5) and Italian (P=5.8 × 10−6) cohorts, weak association in the Germans (P=1.86 × 10−2) and no association in the Spanish group. A unique haplotype (GTCTA) was associated with increased risk of disease but, unlike the first association report,1 different protective haplotypes were observed (Table 2).


The A allele of rs844644, previously reported to tag a protective haplotype,1 was not a consistent finding among the protective haplotypes observed in this study. Instead, the common feature of the protective haplotypes was the background xCxCx, in other words, the presence of the major alleles for the markers rs1234317 (C) and rs12039904 (C), suggesting that they hold the main effects (Figure 1). We examined whether the haplotype block was still associated after controlling for the SNPs rs1234317 and rs12039904 by means of conditional haplotype association test. The omnibus association did not remain significant and therefore we concluded that these two markers explain the complete haplotype effect in our material (Supplementary Table S2).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Risk and protective haplotypes of TNFSF4 associated with SLE. This schematic representation illustrates how the risk and protective effects of the TNFSF4 haplotypes might be well summarized by the effects of the SNPs rs1234317 and rs12039904. Whereas the risk haplotype carries the major alleles of both SNPs, all the protective haplotypes have in common the major alleles for these SNPs.

Full figure and legend (117K)

Conditional tests controlling for each haplotype at a time (Supplementary Table S3) indicated that only GTCTA and CCCCG are true disease haplotypes, the former associated with risk and the latter with protection.

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Discussion

This study confirms the genetic association of SLE with a risk haplotype in the TNFSF4 upstream region, in three independent sets of European cases and controls. The lack of association in the Spanish cohort may reflect locus heterogeneity between populations, but could also be affected by the relatively small effect (OR ~1.35) of the TNFSF4 variants on the risk for SLE. A certain degree of heterogeneity between sets might be expected and, even though the Spanish set was the largest in this study, the strength of the genetic association may vary between populations. Genetic associations are more difficult to determine unambiguously at effects with small ORs and with larger number of individuals needed for confirmation. For example, PDCD1,19, 20 PTPN2221, 22 or CTLA423, 24, 25, 26 all with odds ratios below 1.40 have displayed inconsistent results in various independent multiethnic sets. The original study describing TNFSF4 also showed inconsistency between the sets used, from the United Kingdom and the United States of America.1

We observe a risk haplotype including two SNPs in strong LD, although not complete proxies of each other. The SNP contributing to the main effect at TNFSF4 is not known, and by conditional haplotype analysis we showed that rs1234317 and rs12039904 explain the complete haplotypic effect in our data set. Owing to the high degree of correlation between these SNPs, it cannot be determined if they constitute independent effects. Neither rs1234317 nor rs12039904 remained significant when conditioning on the other due to colinearity between them (data not shown). Thus, additional functional studies are needed to shed light on the true functional variants of TNFSF4.

In contrast to the previous study, we do not identify an independent protective haplotype for TNFSF4. Instead, the protection is explained by the major alleles of the two most important SNPs contributing to the risk effect.

TNFSF4 is located in a previously identified linkage region for SLE in multicase families (1q25).27, 28 Although no fine mapping across the region has been performed yet, and other candidates cannot be excluded, it is a plausible candidate gene. In the work by Graham et al.,1 this gene was selected as a biologically feasible candidate and we hereby replicated the association. The haplotypes associated with SLE susceptibility were correlated with an increase in surface expression of OX40L and CD86 and mRNA levels of TNFSF4.1 The SNP rs1234317 was found to potentially disrupt a putative binding site for the transcriptional repressor E4BP4,1 a transcription factor with a role in the interleukin-13-mediated survival of B-cell progenitors.1, 29, 30 Our genetic data support a role for this SNP. To date, no mobility shift assay have been performed to corroborate this bioinformatic finding, and no antibodies are available for E4BP4.

Taking all results into account, it is very likely that the region upstream of the TNFSF4 gene presents genomic elements involved in susceptibility to SLE by a mechanism, as yet, completely unknown.

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Materials and methods

In this study, we analyzed a total of 1312 SLE cases and 1801 controls described previously7, 9 comprising 259 SLE cases and 322 healthy controls from Germany, 258 cases and 223 controls from Italy, 517 cases and 901 controls from Spain and 278 cases and 355 controls from Argentina. All the study subjects had an individual genotyping rate >90%. The SLE patients fulfilled the American College of Rheumatology (ACR) revised criteria for the classification of SLE.31 The study has been approved by the local ethical committees of each participating center.

Using Haploview v4.1 on aggressive mode and r2>0.8,32 six-tag SNPs were selected from the HapMap-CEU genotype data to capture the common variants (minor allele frequency >5%) across the 80-kb haplotype block associated previously with SLE.1 Genotyping was preformed using TaqMan 5′-exonuclease predeveloped assays from ABI (Applied Biosystems, Foster City, CA, USA).

Statistical analysis was performed in Haploview v4.1,32 PLINK v1.0317 and StatsDirect v2.4.6. First, quality-control filters were applied to remove SNPs with more than 10% of missing data, differential missing rate between cases and controls (P<0.05), significant deviation from Hardy–Weinberg equilibrium in controls (P<0.001) or a minor allele frequency < 1%. Allele frequencies of the remaining SNPs (five of six) were tested for significant association by a χ2 test within each study population. Pooled ORs were estimated by DerSimonian–Laird meta-analysis, including a Breslow–Day test for homogeneity of the OR between strata. Multiple testing was corrected adjusting P-values by false discovery rate control. The PLINK's proxy association function was applied to find flanking markers in strong linkage disequilibrium with the reference SNPs (proxies of all the SNPs typed) and test these proxies for association with disease within a haplotype-based framework. The proxies not genotyped were imputed from multimarker combinations using the HapMap-CEU genotypes as the reference panel. Only imputed SNPs with an INFO value >0.8 were selected. The data were also analyzed for haplotype associations by both omnibus and haplotype-specific association statistics (t-test) as implemented in PLINK. Only haplotypes present in at least 5% of the chromosomes were tested for association. The case/control omnibus test is an H-1 degree of freedom test, for H haplotypes. Conditional tests were used to refine the SNPs and haplotype association signals.

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Acknowledgements

This work was supported by the Swedish Research Council, the Gustav-Ve-80-year Jubilee Foundation, the Torsten and Ragnar Söderbegs Foundation, the Marcus Borgströms Foundation and the Swedish Association Against Rheumatism. MEAR is supported by an award from the Knut and Alice Wallenberg Foundation through the Royal Swedish Academy of Sciences. This work was also partially supported by PRIN Project (MIUR, Rome), Compagnia di San Paolo (Turin), Regione Piemonte (Ricerca Sanitaria Finalizzata Project and Ricerca Sanitaria Applicata-CIPE Project), Fondazione Monte dei Paschi di Siena, the BMBF Kompetenznetz Rheuma C2.12 (Germany), Plan Nacional de I+D (Spain, SAF06-00398) and Grant CTS1880 (Junta de Andalucía, Spain). We thank Adriana I Scollo, Armando M Perichon and Mariano CR Tenaglia (CEDIM, Diagnóstico Molecular y Forense SRL, Rosario, Argentina), for their help in preparing the Argentinean samples.

The Argentine collaborative group participants: Hugo R Scherbarth MD, Jorge A Lopez MD, Estela L Motta MD Servicio de Reumatología, Hospital Interzonal General de Agudos ‘Dr Oscar Alende’, Mar del Plata, Argentina; Susana Gamron MD, Sandra Buliubasich MD, Emilia Menso MD Servicio de Reumatología de la UHMI 1, Hospital Nacional de Clínicas, Universidad Nacional de Córdoba, Córdoba, Argentina; Alberto Allievi MD, Jose L Presas MD Hospital General de Agudos Dr Juán A Fernandez, Buenos Aires, Argentina; Guillermo A Tate MD Organización Médica de Investigación, Buenos Aires, Argentina; Simon A Palatnik MD, Mariela Bearzotti PhD Facultad de Ciencias Médicas, Universidad Nacional de Rosario y Hospital Provincial del Centenario, Rosario, Argentina; Alejandro Alvarellos MD, Francisco Caeiro MD, Ana Bertoli MD Servicio de Reumatología, Hospital Privado, Centro Médico de Córdoba, Córdoba, Argentina; Sergio Paira MD, Susana Roverano MD, Carlos Louteiro MD Hospital José M Cullen, Santa Fe, Argentina; Cesar E Graf MD, Estela Bertero PhD Hospital San Martín, Paraná; Cesar Caprarulo MD, Griselda Buchanan PhD Hospital Felipe Heras, Concordia, Entre Ríos, Argentina; Carolina Guillerón MD, Sebastian Grimaudo PhD, Jorge Manni MD Departamento de Inmunología, Instituto de Investigaciones Médicas ‘Alfredo Lanari’, Buenos Aires, Argentina; Luis J Catoggio MD, Enrique R Soriano MD, Carlos D Santos MD Sección Reumatología, Servicio de Clínica Médica, Hospital Italiano de Buenos Aires y Fundación Dr Pedro M Catoggio para el Progreso de la Reumatología, Buenos Aires, Argentina; Cristina Prigione MD, Fernando A Ramos MD, Sandra M Navarro MD Servicio de Reumatología, Hospital Provincial de Rosario, Rosario, Argentina; Guillermo A Berbotto MD, Marisa Jorfen MD, Elisa J Romero PhD Servicio de Reumatología Hospital Escuela Eva Perón Granadero Baigorria, Rosario, Argentina; Mercedes A Garcia MD, Juan C Marcos MD, Ana I Marcos MD Servicio de Reumatología, Hospital Interzonal General de Agudos General San Martín, La Plata; Carlos E Perandones MD, Alicia Eimon MD Centro de Educación Médica e Investigaciones Clínicas (CEMIC), Buenos Aires, Argentina; Cristina G Battagliotti MD Hospital de Niños Dr Orlando Alassia, Santa Fe, Argentina.

The German collaborative group participants: K Armadi-Simab, MD, Wolfgang L Gross, MD, Abteilung Rheumatologie und Immunologie, University Hospital of Schleswig-Holstein, Campus Luebeck, Rheumaklinik Bad Bramstedt, Luebeck, Germany, Erika Gromnica-Ihle, MD, Rheumaklinik Berlin-Buch, Berlin, Germany, Hans-Hartmut Peter, MD, Medizinische Universitaetsklinik, Abteilung Rheumatologie und Klinische Immunologie, Freiburg, Germany, Karin Manger, MD, Medizinische Klinik III derFAU Erlangen-Nuernberg, Erlangen, Germany, Sebastian Schnarr, MD, Henning Zeidler, MD, Abteilung Rheumatologie, Medizinische Hochschule Hannover, Hannover, Germany, Reinhold E Schmidt, MD, Klinik fuer Immunologie und Rheumatologies, Medizinische Hochschule Hannover, Hannover, Germany.

The Spanish collaborative group participants: Norberto Ortego, Servicio Medicina Interna, Hospital Clínico San Cecilio, Granada, Enrique de Ramón, Servicio de Medicina Interna, Hospital Carlos Haya, Málaga, Juan Jiménez-Alonso, Servicio de Medicina Interna, Hospital Virgen de las Nieves, Granada, Julio Sánchez-Román, Servicio de Medicina Interna, Hospital Virgen del Rocío, Sevilla, and Miguel ángel López-Nevot, Servicio de Inmunología, Hospital Virgen de las Nieves, Granada.

The Italian collaborative group participants: Maria Giovanna Danieli and Armando Gabrielli (Clinica Medica, Dipartimento di Scienze Mediche e Chirurgiche, Università Politecnica delle Marche, Ancona, Italy), Gian Domenico Sebastiani (UOC di Reumatologia Ospedale San Camillo, Roma – Italy), Patrizia Rovere Querini (IRCCS San Raffaele Scientific Institute, Milan, Italy), Sergio Migliaresi (Rheumatology Unit Second University of Naples, Naples, Italy).

Supplementary Information accompanies the paper on Genes and Immunity website (http://www.nature.com/gene)