Indian Journal of Rheumatology 2012 September Volume 7, Number 3; pp.

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Original Article

Study of PTPN22 1858C/T polymorphism in rheumatoid arthritis patients from Western India
Vandana D. Pradhana,*, Heba Dalvib, Devraj Parsannavarc, Anjali Rajadhyakshad, Manisha Patwardhane, Kanjaksha Ghoshf

ABSTRACT
Background: Rheumatoid arthritis (RA) is an inammatory autoimmune disorder with etiologies including genetic and environmental factors. Protein tyrosine phosphatase non-receptor type22 (PTPN22) 1858C/T polymorphism is widely suspected to be a susceptibility gene for RA in the non-HLA genes group. Aim: This study aimed at determining whether PTPN22 1858C/T polymorphism is associated with RA patients from Western India and to evaluate its possible association with rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) autoantibodies. Methods: A total of 130 Indian RA patients and 100 age and sex matched normal controls were genotyped by polymerase chain reaction restriction fragment length polymorphism (PCReRFLP) for the PTPN22 1858C/T polymorphism. Results: RF positivity was seen among 73.8% RA patients studied and the overall incidence of anti-CCP antibodies was 86.2%. The homozygous genotype (T/T) was absent in both groups. Among RF positives, C/C homozygosity was 90.6% whereas 9.4% patients were C/T heterozygous. Among anti-CCP positives, 89.1% had C/C genotype while the remaining 10.9% have the C/T genotype. Statistically signicant association was obtained between the polymorphism and anti-CCP positivity in RA patients (OR: 2.939, p value 0.0595). Conclusion: Our study suggested that a positive autoantibody status may predispose an individual to RA. PTPN22 may act as a susceptibility gene only in certain ethnic groups and there is no direct association between PTPN22 C1858 polymorphism and RA patients from Western India. Still a larger study is needed to understand whether this polymorphism predisposes individual to disease-associated antibodies among Indian RA patients. Copyright 2012, Indian Rheumatology Association. All rights reserved. Keywords: Rheumatoid factor, Anti-CCP antibodies, Non-HLA gene polymorphism

INTRODUCTION
Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disorder, characterized by synovitis e an inammation of synovial membranes, which enclose joints. The aficted

joints become warm, swollen, tender, stiff, and in the nal stage, deformed. The disease is two to three times more prevalent in women than in men.1 Early diagnosis, coupled with aggressive use of disease-modifying anti rheumatic drugs (DMARDs), has been shown to have a favorable effect on

Scientist, bMSc Student, eResearch Assistant, Department of Clinical & Experimental Immunology, fDirector, National Institute of Immunohaematology, Indian Council of Medical Research, 13th oor, dProfessor and Head, Department of Medicine, King Edward Memorial Hospital, Parel, Mumbai, cScientist National Institute of Nutrition, Hyderabad, India. * Corresponding author. Tel.: 91 22 24138518; fax: 91 22 24138521, email: pradhanv69@rediffmail.com Received: 25.4.2012; Accepted: 28.6.2012; Available online: 7.7.2012 Copyright 2012, Indian Rheumatology Association. All rights reserved.
http://dx.doi.org/10.1016/j.injr.2012.06.003

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the course of disease. RA is the commonest inammatory joint disease affecting nearly 0.75% of adult Indian population.2 RA is characterized by the presence of autoantibodies rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibodies in a signicant majority of patients. RF, which is an immunoglobulin directed against the Fc portion of IgG, is found in over 70% of patients with RA and is used as serological markers of RA; high RF may confer a worse prognosis.3 Anti-CCP antibodies have emerged as sensitive and specic serological markers of RA, being present before the onset of RA symptoms, hence providing superior alternative of the RF test and proving to be a valuable diagnostic test early in the course of the disease.4 RA is believed to be a heterogeneous disease because the manifestations are greatly varied across patients in terms of severity, progression, and response to therapy. Although the etiology of RA is unknown, both genetic and environmental factors have been shown to play a role in the development of RA. Genetic factors have a substantial role and are likely to account for 50e60% of disease susceptibility.5 The HLA-DRB1 alleles encoding the shared epitope (SE) have been proved to be the major RA susceptibility locus accounting for 30% of the genetic contribution.6,7 RA genetic studies have reported polymorphisms in various non-HLA genes to be associated with the disease. Of these, PTPN22 has been most strongly replicated across multiple independent studies. The PTPN22 gene is located on chromosome 1p13 and encodes a lymphoid protein tyrosine phosphatase (LYP) that is important in negative control of T-cell activation and in T-cell development. PTPN22 belongs to a family of protein tyrosine phosphatases involved in preventing spontaneous T-cell activation by dephosphorylation and inactivation of T-cell receptor-associated kinases and their substrates.8,9 The C1858T substitution resulting in an arginine to tryptophan change in codon 620 (R620W) of the protein product has been shown to affect the proteineprotein interaction with tyrosine kinase Csk in T-cell activation. It is suggested that reduced T-cell signaling may occur as a result and, in turn, this may enable auto-reactive T cells to escape deletion during thymic selection and thus persist in the circulation to become activated at a later stage.10 Association between a functional single-nucleotide polymorphism (SNP) in the coding region of the gene PTPN22 and RA in Caucasians was rst described by Begovich et al and had been replicated by several other groups in the US, UK, Spanish and Dutch RA patient populations.3,11e15 However, the association between the polymorphism and RA was not found in Indian populations.3,11,16,17 This study was aimed at achieving two objectives: rstly, to determine whether the PTPN22 1858C/T polymorphism is associated with RA and secondly, to determine whether this polymorphism is associated with positive autoantibody (RF and anti-CCP) status.

MATERIALS AND METHODS

A total of 130 RA patients from Western India were included in the study who fullled American College of Rheumatology criteria for RA.18 One hundred age and sex matched healthy individuals of the same ethnicity were included as controls. Genomic DNA samples were extracted from peripheral blood leukocytes from EDTAtreated whole blood using the phenol-chloroform method. Genomic DNA samples were stored at 20  C until required. For the determination of the PTPN22 alleles, PCR-based restriction fragment length polymorphism (RFLP) analysis was performed modifying protocol used by Cantn et al19 A 400 bp fragment of the PTPN22 gene was amplied by PCR using a mixture containing 25 mM MgCl2, 10 mM dNTP, and 10 pM of each of the forward and reverse primers in a total reaction mixture of 25 ml. The sequences of the forward and reverse primers were: forward primer, 50 -GCCTCAATGAACTCCTCAA30 ; reverse primer 50 -CCTCCTGGGTTTGTACCTTA-30 . The PCR conditions were as follows: denaturation at 94  C for 5 min, followed by 35 cycles of denaturation at 95  C for 45 s, annealing at 62.7  C for 40 s, elongation at 72  C for 45 s and a nal elongation at 72  C for 7 min Fig. 1 shows the specic and control bands obtained after amplication. The amplied product was then subjected to restriction digestion using RsaI restriction endonuclease for 16 h at 37  C. Digested products were electrophoresed on a 2% agarose gel and visualized using ethidium bromide. RsaI recognizes its target sequence only when the PTPN22 1858C allele is present. Digestion of the amplied product gives two fragments of 238 bp and 120 bp for the C/C genotype and three fragments of 300 bp, 238 bp and 120 bp for the C/T genotype (Fig. 2).

Fig. 1 Gel photograph of the PTPN22 gene fragment (specic band) and control band. Lanes 1, 2, 3 and 4 show amplied RA samples with control band (434 bp) and the specic band (400 bp). Lane M contains marker 8/pUC8 mix 100 bp ladder.

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RESULTS
Among 130 RA patients studied, 117 were females (90%) and 13 were males (10%). The ages ranged between 21 and 65 years. Among these, 30 patients had an early disease onset (age <30 years), with mean age 26.3 3.8 years and 100 patients showed the late onset of disease (!30 years of age) with mean age 46.5 12.4 years. Clinical manifestations revealed that 70% had multiple joint swelling, 62% patients had hand joint involvement, 64% patients had more than three joints involvement, 57% had symmetrical joint involvement, 59% patients had morning stiffness, 31% had tender joints whereas 14% had erosive joints. RF positivity was seen among 73.8% RA patients studied where titer values ranged between 61 RU/ml and 325 RU/ml. The overall incidence of anti-CCP antibodies was 86.2%. The genotype frequencies of PTPN22 C1858T polymorphism in RA patients and healthy controls are shown in Table 1. The frequency of heterozygous genotype (C/T) was 9.2% in RA patients and 4% in healthy controls. The homozygous genotype (T/T) was absent in both RA patients as well as healthy controls. Statistical analysis revealed no statistically signicant difference between RA patients and healthy controls with respect to the frequency of heterozygous genotype (OR: 2.441, 95% CI: 0.7625e7.813, p value 1.222). Among RF positives 87/96 patients, C/C (90.6%) was C/C homozygous where as remaining 9 patients (9.4%) were C/T heterozygous. Among anti-CCP positives, 89.1% had C/C genotype while the remaining 10.9% had the C/T genotype. Statistically signicant association was obtained between the polymorphism and anti-CCP positivity in RA patients (OR: 2.939, p value 0.0595).

Fig. 2 Gel photograph of the PTPN22 gene fragment post digestion. PCReRFLP analysis of PTPN22 1858C/T SNP: 2% agarose gel electrophoresis after 16 h digestion of the PCR product with RsaI restriction endonuclease. Lanes 3 and 6 show RA samples that are heterozygous with C/T genotype (three bands e 300 bp, 238 bp and 120 bp). Lanes 1, 2, 4, 7 and 8 show RA samples that are homozygous with C/C genotype (two bands e 238 bp, 120 bp). No T/T genotype was observed for any of the samples. Lane M contains marker 8/ pUC8 mix 100 bp ladder.

Rheumatoid factor was detected by IgM-RF ELISA, Euroimmun, Lubek, Germany. Anti-CCP antibodies were detected anti-CCP ELISA assay kit (Immco Diagnostics, USA). The cut-off level for positivity for IgM-RF was dened as titers >20 IU/ml whereas that for anti-CCP antibodies was dened to be an O.D. value of more than 0.3. The laboratory was blinded to the disease status of patients and their visceral involvement and a double-blinded study was conducted on the autoantibody positive samples.

STATISTICAL ANALYSIS
Continuous variables were expressed as mean SD. Pairs of groups were compared using Students t test for normally distributed continuous distribution. Statistical significance was set at p value < 0.05 by Chi square (c2) test.

DISCUSSION
PTPN22 has been reported to be a good candidate for genetic marker for RA due to its functional relevance as

Table 1 Genotype frequencies of PTPN22 1858C/T polymorphism in RA patients. Parameters Controls (n 100) Patients (n 130) RF positives (n 96) (73.8%) RF negatives (n 34) (26.2%) Anti-CCP positives (n 110) (84.6%) Anti-CCP negatives (n 18) (13.8%) RF positive anti-CCP positive (n 78) (60%) C/T n (%) 4 12 09 03 12 0 10 (4) (9.2) (9.4) (8.8) (10.9) (0) (12.8) C/C n (%) 96 118 87 31 98 18 68 (96) (90.8) (90.6) (91.2) (89.1) (100) (87.2) OR e 2.441 2.483 1.069 2.939 4.695 3.529

c2
e 2.389 2.285 0.00911 3.552 2.167 4.705

95% CI e 0.7625e7.813 0.7379e8.353 0.2717e4.206 0.9154e9.434 0.2660e8.287 1.062e11.727

p Value e 0.1222 0.1306 0.9239 0.0595 0.1410 0.0301

Bold values are p Value < 0.05 is statistically signicant by c20 test, OR odds ratio, CI condence interval, RF positives and anti-CCP positives were compared with control individually, RF positives are compared with RF negatives and anti-CCP positives were compared with anti-CCP negatives.

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a negative regulator of T-cell activation and its positional mapping to a chromosome region showing linkage to RA.13,14,20 The association between 1858T allele at rs2476601 in PTPN22 gene and RA has been documented in several cohorts especially among populations of the North American and European ancestry.3,11,12,21 These studies showed that the frequencies of mutant (T) allele have changed from 7.4% to 10.3% in healthy controls and from 13.6% to 21.2% in RA patients in Caucasians.20 However, there is wide variation among the frequencies of PTPN22 C1858T polymorphism reported from world population. The association between the SNP and RA has not been replicated in Asian populations.3,11,16,17 These discrepancies between the studies may result from genetic or clinical heterogeneity in RA patients across different populations. Ethnic differences may play a role in the conicting results among the genetic association studies as described by Mori et al.16 Our study showed that there is no direct association between PTPN22 C1858T polymorphism and RA in patients from Western India. Our ndings on PTPN22 R620W polymorphism were similar to studies on Asian populations17,22 wherein the association could not be replicated either. However, prevalence of the C/T genotype in both RF positive and anti-CCP antibody positive patients together indicated that the gene may predispose an individual to autoimmunity by facilitating the production of autoantibodies. Our study suggested that a positive autoantibody status may predispose an individual to RA. PTPN22 may act as a susceptibility gene only in certain ethnic groups and there is no direct association between PTPN22 C1858 polymorphism and RA patients from Western India. Still a larger study is needed to understand whether this polymorphism predisposes individual to disease-associated antibodies among Indian RA patients.

CONFLICTS OF INTEREST
All authors have none to declare. ACKNOWLEDGMENT We are thankful to Director General, Indian Council of Medical Research, for graciously allowing us to carry our work.

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