Downloaded from ard.bmj.com on August 13, 2013 - Published by group.bmj.com ARD Online First, published on February 26, 2013 as 10.1136/annrheumdis-2012-202742 Basic and translational research EXTENDED REPORT New insight on the Xq28 association with systemic sclerosis F David Carmona,1 M Carmen Cénit,1 Lina-Marcela Diaz-Gallo,1 Jasper C A Broen,2 Carmen P Simeón,3 Patricia E Carreira,4 José-Luis Callejas-Rubio,5 Vicente Fonollosa,3 Francisco J López-Longo,6 Miguel A González-Gay,7 Nicolas Hunzelmann,8 Gabriela Riemekasten,9 Torsten Witte,10 Alexander Kreuter,11 Jörg H W Distler,12 Rajan Madhok,13 Paul Shiels,13 Jacob M van Laar,14 Annemie J Schuerwegh,15 Madelon C Vonk,16 Alexandre E Voskuyl,17 Carmen Fonseca,18 Christopher P Denton,18 Ariane Herrick,19 Jane Worthington,19 Frank C Arnett,20 Filemon K Tan,20 Shervin Assassi,20 Timothy R D J Radstake,2,16 Maureen D Mayes,20 Javier Martín,1 Spanish Scleroderma Group Handling editor Tore K Kvien ABSTRACT In some cases, part of the genetic component is Objective To evaluate whether the systemic sclerosis shared among different immune disorders, sug- ▸ Additional material is fl published online only. To view (SSc)-associated IRAK1 non-synonymous single- gesting that these pathologies may be in uenced please visit the journal online nucleotide polymorphism rs1059702 is responsible for by disease-specific and common molecular path- – (http://dx.doi.org/10.1136/ the Xq28 association with SSc or whether there are ways.1 4 For instance, most of the genetic associa- annrheumdis-2012-202742). other independent signals in the nearby methyl-CpG- tions described for systemic sclerosis (SSc), a For numbered affiliations see binding protein 2 gene (MECP2). fibrotic autoimmune disease of skin and internal end of article. Methods We analysed a total of 3065 women with organs, have also been reported to play a role in SSc and 2630 unaffected controls from five independent the susceptibility to systemic lupus erythematosus Correspondence to 56 Dr F David Carmona, Caucasian cohorts. Four tag single-nucleotide (SLE). Instituto de Parasitología polymorphisms of MECP2 (rs3027935, rs17435, Remarkably, a wide spectrum of autoimmune y Biomedicina López-Neyra. rs5987201 and rs5945175) and the IRAK1 variant diseases shows a significant female preponderance. Consejo Superior de rs1059702 were genotyped using TaqMan predesigned SSc represents a clear example of a sex biased fi Investigaciones Cientí cas, assays. A meta-analysis including all cohorts was immune disorder, with women reaching almost Parque Tecnológico Ciencias de la Salud. Avenida del performed to test the overall effect of these Xq28 90% of total affected individuals in some popula- Conocimiento s/n, Armilla, polymorphisms on SSc. tions. Different factors have been proposed to Granada 18100, Spain; Results IRAK1 rs1059702 and MECP2 rs17435 were explain this marked sexual dimorphism, including [email protected] associated specifically with diffuse cutaneous SSc reproductive and sex hormones, fetal microchimer- −3 FDC, MCC and LMDG (PFDR=4.12×10 , OR=1.27, 95% CI 1.09 to 1.47, and ism, and gender differences in the immune system −4 fi contributed equally. PFDR=5.26×10 , OR=1.30, 95% CI 1.14 to 1.48, and lifestyle. Nevertheless, de nitive evidences are TRDJR, MDM and JM share respectively), but conditional logistic regression analysis still lacking and cumulative knowledge points to a senior authorship showed that the association of IRAK1 rs1059702 with major role of sex chromosomes in the immune this subtype was explained by that of MECP2 rs17435. system homeostasis. Due to the statistical complex- Received 27 September 2012 Revised 3 January 2013 On the other hand, IRAK1 rs1059702 was consistently ity of testing for association between phenotype Accepted 3 February 2013 associated with presence of pulmonary fibrosis (PF), and genetic markers on the sex chromosomes, because statistical significance was observed when very few associations of sex-linked genes with the comparing SSc patients PF+ versus controls development of autoimmunity have been reported 7–9 (PFDR=0.039, OR=1.30, 95% CI 1.07 to 1.58) and SSc to date. patients PF+ versus SSc patients PF− (p=0.025, One of the shared risk loci between SSc and SLE OR=1.26, 95% CI 1.03 to 1.55). within the non-homologous region of the X chromo- Conclusions Our data clearly suggest the existence of some is the interleukin-1 receptor-associated kinase 1 two independent signals within the Xq28 region, one gene (IRAK1),10 11 that encodes a serine/threonine located in IRAK1 related to PF and another in MECP2 protein kinase with a special relevance in the signal- related to diffuse cutaneous SSc, indicating that both ling pathways of Toll-like receptors (TLRs)/ genes may have an impact on the clinical outcome of IL-1R.12 13 However, IRAK1 is in the same haploty- the disease. pic block as the methyl-CpG-binding protein 2 gene (MECP2) on Xq28, which is also associated with SLE,14 15 and functional genetic variants of the latter To cite: Carmona FD, Cénit MC, Diaz-Gallo L-M, INTRODUCTION locus may explain the association signals with SLE et al. Ann Rheum Dis Autoimmune diseases are complex polygenic con- observed in IRAK1.16 Published Online First: ditions in which multiple susceptibility genes In this study we aimed to evaluate whether the [please include Day Month interact with epigenetic and environmental SSc-associated IRAK1 polymorphism rs1059702 Year] doi:10.1136/ factors for their predisposition and progression. et al10 annrheumdis-2012-202742 (Phe196Ser) described by Dieudé is the CopyrightCarmona FD,Articleet al. Ann author Rheum Dis (or2013; their0:1– 7.employer) doi:10.1136/annrheumdis-2012-202742 2013. Produced by BMJ Publishing Group Ltd (& EULAR) under licence.1 Downloaded from ard.bmj.com on August 13, 2013 - Published by group.bmj.com Basic and translational research causal variant of the Xq28 association or whether it reflects Single-nucleotide polymorphism selection another association signal from the nearby MECP2. We followed a single-nucleotide polymorphism (SNP) tagging strategy using Haploview V.4.2 (http://www.broad.mit.edu/mpg/ haploview)18 to identify taggers that may cover all the common genetic variation within MECP2 (r2≥0.8) in the Utah residents PATIENTS AND METHODS with ancestry from northern and western Europe (CEU) popula- Study population tion of the HapMap database (http://hapmap.ncbi.nlm.nih.gov/). MECP2 Since the IRAK1/MECP2 genes are located in a sex-linked Four polymorphisms were selected with this method: region, only women were included in the study. Informed rs3027935, rs17435, rs5987201 and rs5945175 (see online fi written consent from all participants and approval from the supplementary gure S1). Additionally, we also included in the IRAK1 local ethical committees were obtained in accordance with the study the non-synonymous genetic variant rs1059702 tenets of the Declaration of Helsinki. (Phe196Ser), which was described as the SNP that best explains IRAK1 10 We analysed a total of 3065 female SSc patients and 2630 the SSc susceptibility haplotype. The location of the fi IRAK1/MECP2 female unaffected controls of Caucasian ancestry, from an initial ve genetic variants analysed within the region fi discovery cohort of Spain (1016 SSc and 1520 controls) and is shown in online supplementary gure S2. four additional replication cohorts from USA (965 SSc and 489 controls), Germany (490 SSc and 180 controls), The Genotyping methods Netherlands (235 SSc and 278 controls) and UK (359 SSc and DNA samples were obtained from peripheral white blood cells of 163 controls). Since most samples have not been subjected to participants following standard procedures. Predesigned TaqMan 0 genome-wide association study platforms, population substruc- 5 SNP genotyping assays were used to genotype the selected ture analysis could not been performed and this may represent a Xq28 genetic variants (assay IDs: rs3027935: C__15765567_10, potential limitation. In all cases, SSc patients were classified rs17435: C___2597094_20, rs5987201: C__30089704_10, based on their skin involvement into limited cutaneous SSc rs5945175: C__30485633_20, rs1059702: C___8966367_30) (lcSSc) or diffuse cutaneous SSc (dcSSc) according to the criteria in a 7900HT Fast Real-Time PCR System (Applied Biosystems, by Leroy et al17 A subset of the German cohort (43.73%) over- Foster City, California, USA). laps with that included in the study of IRAK1 by Dieudé et al10 To perform the subphenotype analyses, they were also pheno- Statistical analyses typically characterised accordingly with the presence or absence The overall statistical power of our study, according to Power of anticentromere antibodies (ACA), antitopoisomerase anti- Calculator for Genetic Studies 2006,19 is shown in online bodies (ATA) and pulmonary fibrosis (PF). ACA were deter- supplementary table S1. mined by their characteristic distinctive pattern on HEP-2 cells, The Linux software Plink V.1.7 (http://pngu.mgh.harvard.edu/ and ATAs were detected by passive immunodiffusion against calf purcell/plink/)20 was used to perform 2×2 contingency tables, thymus extract. PF was diagnosed by high resolution CT in all χ2 and/or Fisher’s exact tests, when appropriate. p Values lower the European cohorts. However, the PF status of our US cohort than 0.05 were considered as statistically significant. ORs, and was obtained by pulmonary function test (patients were consid- 95% CI, were calculated according to Woolf’s method. The ered to have PF if they showed a forced vital capacity (FVC) less Breslow-Day (BD) test was used to estimate the OR heterogen- than 70%). Since two different methodologies were used for the eity among the different cohorts. Combined data were analysed diagnosis, the meta-analyses on PF data were performed only in by Mantel-Haenszel tests under fixed effect, or random effect the European cohorts.