Association of CD28 IVS3 +17T/C Polymorphism with Soluble CD28 in Rheumatoid Arthritis

25 Disease Markers 30 (2011) 25–29 DOI 10.3233/DMA-2011-0760 IOS Press Association of CD28 IVS3 +17T/C polymorphism with soluble CD28 in rheumatoid arthritis I.Y. Ledezma-Lozanoa, J.J. Padilla-Martı́neza, S.D. Leyva-Torresb, I. Parra-Rojasc , M.G. Ramı́rez-Dueñasd, Ana Laura Pereira-Suárezd , H. Rangel-Villalobose, S.L. Ruiz-Quezadaf , P.E. Sánchez-Hernándezd and J.F. Muñoz-Vallea,∗ a Departamento de Biologı́a Molecular y Genómica. Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, Jalisco, México b Department of Rheumatology. Hospital “Valentı́n Gómez-Farı́as ISSSTE”, Zapopan, Jalisco, México c Unidad Académica de Ciencias Quı́mico-Biológicas, Universidad Autónoma de Guerrero, Chilpancingo, Guerrero, México d Laboratorio de Inmunologı́a. Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, Jalisco, México e Instituto de Investigación en Genética Molecular. Centro Universitario de la Ciénega, Universidad de Guadalajara; Ocotlán, Jalisco, México f Laboratorio de Biologı́a Molecular, Centro Universitario de Ciencias Exactas e Ingenierı́as, Universidad de Guadalajara; Guadalalajara, Jalisco, México Abstract. Objective: Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology in which inflammatory pathology involves T cell activation and the CD28 costimulatory molecule involved in T cell presentation. The gene includes the CD28 IVS3 +17T/C polymorphism that could be associated with susceptibility to RA whereas the soluble concentrations of CD28 (sCD28) could be related to clinical activity. Methods: We investigated the CD28 IVS3 +17T/C polymorphism in 200 RA patients and 200 healthy subjects (HS). Furthermore, we quantified the sCD28 concentrations in 77 samples of each group. We applied indexes focused to determine the activity and disability (DAS28 and Spanish HAQ-DI, respectively) in RA patients. Results: RA patients had significantly higher frequencies of the CD28 T allele compared to HS (p = 0.032 OR = 1.59, C.I. 1.02–2.49). In addition, the IVS3 +17 T/T genotype frequency was also increased in RA vs. HS (p = 0.026). The RA patients showed higher sCD28 serum levels than HS (p = 0.001). Carriers of the T/T genotype in RA patients showed higher sCD28 levels than C/C carriers (p = 0.047). In addition, a correlation between sCD28 and Spanish HAQ-DI (correlation, 0.272; p = 0.016), was found. Conclusion: The T allele in CD28 IVS3 +17T/C polymorphism is associated with a susceptibility to RA in Western Mexico. In addition, increased sCD28 levels are related to T/T genotype in RA patients. Keywords: CD28, IVS3 +17T/C, polymorphism, rheumatoid arthritis, sCD28 1. Introduction ∗ Corresponding author: José Francisco Muñoz-Valle, PhD, Departamento de Biologı́a Molecular y Genómica. Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara. Insurgentes 244-1, Colonia Lomas de Atemajac, Zapopan, Jalisco, C.P. 45178, México. Tel.: +52 (33) 10585309, ext. 3635; E-mail: . Rheumatoid arthritis (RA) is an autoimmune disease characterized by progressive sinovitis and the formation of pannus, which can lead to articular damage and the loss of function in the diarthrodial joints. This process involves T cell activation and hyperactivity which ISSN 0278-0240/11/$27.50  2011 – IOS Press and the authors. All rights reserved 26 I.Y. Ledezma-Lozano et al. / Association of CD28 IVS3 +17T/C polymorphism with soluble CD28 have been related to an irregular function of the costimulatory molecules in autoimmune diseases [1]. CD28 (mCD28) in membranes is an important costimulator of T lymphocytes, their principal events are increasing the response and promotion of T cells survival through positive signalization [2]. Moreover, soluble CD28 (sCD28) could regulate T cell proliferation and contribute to the loss of their own tolerance [3], in competition with their ligands B7.1 (CD80) and B7.2 (CD86). The IVS3 +17T/C single nucleotide polymorphism (SNP) of CD28 gene, with a T/C substitution at position +17 in the third intron, is located in the 2q33 region [4]. This polymorphism has been related with Behçet disease [5], but has not been considered in other inflammatory diseases like RA. Based on the importance of this molecule our purpose was to determine if CD28 IVS3 +17T/C polymorphism and sCD28 could be related to susceptibility and clinical activity to RA in Western Mexico. 2. Subjects, materials and methods This study included 200 RA patients (190 female, 10 male; mean age 48 ± 14) classified according to the American College of Rheumatology criteria [6] from the Rheumatology Departments of the O.P.D. Hospital Civil Fray Antonio Alcalde and Hospital Valentı́n Gómez Farias, Jalisco, Mexico. As a control group, we recruited 200 healthy subjects (HS) (123 female, 77 male; mean age 35 ± 11.8). The RA patients and HS were born in Western Mexico with similar genetic background and a family history of Mexican ancestors, at least back to the third generation. Disease activity and disability in RA patients were evaluated through the Disease Activity Score using 28 joint counts (DAS28) and Health Assessment Questionnaire (Spanish HAQDI), respectively. The internal Committee of Ethics of both Institutions mentioned above, approved the present study in compliance with the Helsinki declaration (CE3/CI-03 and No. 1934/07). Informed consent was obtained from both study groups. 2.1. Polymerase Chain Reaction (PCR) – Restriction fragment length polymorphism (RFLP) analysis of CD28 IVS3 +17T/C Genomic DNA was isolated from peripheral blood leucocytes using standard procedures. In order to amplify the 147 bp region that includes the CD28 IVS3 +17T/C polymorphism, we used the following primers for the PCR reaction: forward 5’TTTTC TGGGTAAGAGAAGCAGCGC-3’ and reverse 5’-GA ACCTACTCAAGCATGGGG-3’. Each of the PCR reactions included 5 µL of DNA, 3 µM of each primer, 2.5 µL of 1X reaction buffer (Invitrogen ), 1.5 mM magnesium chloride (Invitrogen ), 2.5 mM dNTPs (Invitrogen ), and 0.125 µL of Taq DNA polymerase (Invitrogen ) in a total volume of 18 µL. The CD28 gene amplification conditions were as follows: initial denaturation at 95◦ C for 2 min, followed by 30 cycles of 94◦ C for 30 s, 62◦ C for 30 s, and 72◦ C for 30 s and a final extension at 72◦ C for 2 min. 2.2. CD28 IVS3+17T/C restriction pattern polymorphism The T to C transition was identified by digestion with 5 U of AfeI restriction enzyme (New England BioLabs ) for 3 h at 37◦ C. Digested products were electrophoresed on a 3% agarose gel (Invitrogen Life Technologies ) and then stained with ethidium bromide. Digestion fragments of 125 and 22 bp represent the wild type genotype (T/T); fragments of 147, 125 and 22 bp represent the heterozygote (T/C) whereas 147 bp represents the polymorphic genotype (C/C). To confirm this technique, random samples were taken of each genotype and sequenced using an ABIPRISM 310 Sequencer (Applie (...truncated)