No Association of PTPN22 Polymorphisms with Susceptibility to Ocular Behcet's Disease in Two Chinese Han Populations

Background Behcet's disease is known as a recurrent, multisystem inflammation and immune-related disease. Protein tyrosine phosphatase non-receptor 22 (PTPN22) is a key negative regulator of T lymphocytes and polymorphisms of the PTPN22 gene have been shown to be associated with various immune-related diseases. The present study was performed to assess the association between PTPN22 polymorphisms and Behcet's disease in two Chinese Han populations. Methodology/Principal Findings A total of 516 patients with ocular Behcet's disease and 690 healthy controls from two Chinese Han populations were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for three single nucleotide polymorphisms (SNPs). Hardy-Weinberg equilibrium was tested using the χ2 test. Genotype frequencies were estimated through direct counting. Allele and genotype frequencies were compared between patients and controls using logistic regression analysis. The results revealed that there was no association between the tested three PTPN22 SNPs (rs2488457, rs1310182 and rs3789604) and ocular Behcet's disease (p>0.05). Categorization analysis according to the clinical features did not show any association of these three polymorphisms with these parameters (p>0.05). Conclusions/Significance The investigated PTPN22 gene polymorphisms (rs2488457, rs1310182 and rs3789604) were not associated with ocular Behcet's disease in two Chinese Han populations, and showed that it may be different from other classical autoimmune diseases. More studies are needed to confirm these findings for Behcet's disease in other ethnic backgrounds.


Introduction
Behcet's disease (BD) is known as a multisystem inflammatory disorder of unknown etiology. It is characterized by recurrent attacks of uveitis, oral ulceration, genital ulceration, skin lesions, arthritis, and vascular inflammation [1]. It is curently recognized as an immune-related disease presenting with signs of immune hyper-reactivity mainly affecting elements of the innate immune system. Although the precise triggering factors for BD are still unclear, complex mechanisms including genetic, environmental and immunologic, are presumed to be involved [2][3][4][5]. Genetic susceptibility to BD has been investigated in different populations throughout the world. Human leukocyte antigen (HLA)-B51 is thought to be the most strongly associated genetic factor predisposing to BD. The contribution of HLA-B51 to the genetic risk of BD has however been estimated to be less than 20%. Therefore study on non-HLA genes, especially related to the immune response and inflammation is of great importance. Several immune relevant genes, such as the IL-23 receptor gene, IL-10 gene and SUMO4 gene, have been revealed to be risk factors for BD [6][7][8][9].
The protein tyrosine phosphatase non-receptor 22 (PTPN22) gene maps to chromosome 1p13.3-p13.1, and encodes the lymphoid-specific phosphatase known as Lyp, which contains a catalytic N-terminal domain and a non-catalytic C-terminus composed of four proline-rich domains. Lyp is an important down-regulator of T-cell activation through interacting with protein tyrosine kinase (Csk) and inhibiting signaling pathways mediated by the T-cell receptor (TCR) [10]. Several studies have revealed that the nucleotide diversity of PTPN22 could trigger the immune and inflammation response by decreasing the affinity between Lyp and Csk, thereby inhibiting the TCR signaling pathway [11][12][13]. PTPN22 has been shown to be associated with susceptibility to a number of immune related-diseases, such as type 1 diabetes (T1D), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) [14][15][16].
Earlier studies in a Turkish population with BD analyzed the rs2476601 genotype of the PTPN22 gene and concluded that this polymorphism had a limited (protective) role in disease pathogenesis [17]. A similar study including UK and Middle-East patients with BD showed a protective effect of the T1858 allele in the Middle East patients but not in the UK group [18]. As yet, in Chinese Han populations, no data are available concerning a possible association of PTPN22 gene with BD, and this was therefore the purpose of the study presented here. The frequencies of some alleles display great variation in different regions and ethnic groups worldwide. The International HapMap data and recent reports showed the SNP rs2476601 (1858C/T) are not polymorphic in Chinese Han populations, although they have been shown to be associated with susceptibility for immune diseases in other ethnic groups [19][20][21]. In Asian populations, other candidate SNPs of this gene, namely rs2488457, rs1310182 and rs3789604, were confirmed to be associated with several immune diseases, such as T1D, RA and Graves disease (GD) [22][23][24][25][26]. In view of these earlier findings we limited our analysis to these three SNPs (rs2488457, rs1310182 and rs3789604). The results failed to show an association of PTPN22 gene polymorphisms with risk to ocular BD.

Results and Discussion
No statistical differences were observed in the distribution of age for the Chinese Han case-control cohorts (p.0.05). There were also no significant differences in the distribution of gender for the case-control study in groups (p.0.05). The age and gender distribution of the ocular BD patients and healthy controls are shown in Table 1. The clinical features of the ocular BD patients included in our study are presented in Table 2.
A total of 516 ocular BD patients and 690 healthy controls were genotyped for three SNPs of PTPN22 (rs2488457, rs1310182 and rs3789604). The RFLP genotyping matched results obtained by direct sequencing. The genotype and allele frequencies of the three SNPs of PTPN22 examined in BD patients and healthy controls are summarized in Tables 3, 4, 5. The results showed that the distribution of genotypes and alleles did not deviate from the Hardy-Weinberg equilibrium (HWE). There were no differences concerning the genotype or allele frequencies of the three SNPs between ocular BD patients and healthy controls respectively in the two cohorts (p.0.05), and similar results were observed when comparing the combined patient cohorts with the controls (p.0.05). Under the null hypothesis (absence of association between disease and genotype/allele), OR (odd ratio) is expected to be 1.000.
In addition, we investigated whether the PTPN22 SNPs were associated with the various clinical features of ocular BD, such as oral ulceration, genital ulceration, hypopyon, skin lesions, positive pathergy test and arthritis. The analysis did not indicate any significant association of these parameters with the tested three SNPs of PTPN22 (p.0.05) (data not shown).
Numerous factors have been reported to influence the results of the study on the association of gene polymorphisms with disease. We made the following efforts to ensure correctness of the results. The ocular BD patients were selected strictly according to the criteria of the International Study Group or the revised criteria from Behcet's Disease Research Committee of Japan if oral ulceration was not present, and two cohorts of the samples came from two independent uveitis centers in China. The healthy individual controls were recruited from the same geographical regions as the patients. No statistical differences were observed in the distribution of age and gender for the collected Chinese Han case-control cohorts. In order to validate the results of genotyping by PCR-restriction fragment length polymorphism, 10% of the samples were randomly chosen and confirmed by direct sequencing.
During recent years the PTPN22 gene has emerged as an important candidate susceptibility factor for a number of immune diseases [27]. A role of PTPN22 in clinical uveitis had been addressed in British, Turkish and Middle-East BD patients and described a limited (protective) effect of the T1858 allele in the  Middle East patients but not in the UK group [17,18]. Similarly, Martin et al showed no association between the R620W (rs2476601) of PTPN22, 1858C/T genotype and acute anterior uveitis [28]. Wagenleiter et al revealed that the above polymorphism of PTPN22 gene had no influence on Crohn's disease (CD) in Germans [29]. Matesanz et al also failed to demonstrate any correlation of the above SNP with Multiple sclerosis (MS) in Spanish patients [30]. On the other hand, an association with PTPN22 polymorphisms has consistently been reported as a susceptibility gene for a number of autoimmune diseases characterized by specific autoantibodies [20]. These studies suggest that BD, CD and MS may have a different genetic background and pathogenesis as compared with classical antibody mediated autoimmune diseases such as T1D, RA and SLE. On the other hand, autoimmunity in BD is still a controversial issue.
Numerous studies suggest that BD may be mainly prone to  autoinflammatory processes as opposed to autoimmunity [17,18,[31][32][33]. Since PTPN22 has always been presumed to be a genetic switch for autoimmunity, the lack of an association with the common SNPs of this gene may provide additional evidence to the hypothesis that BD is autoinflammatory rather than an autoimmune disease. Like other candidate gene research, there are some limitations in our study. The size of patient samples seems to be relatively small if the pathogenic role of the PTPN22 gene is not strong enough. The patients were only recruited from the Chinese Han population. Therefore, the association of PTPN22 polymorphisms with BD should be studied using a larger sample size and other ethnic populations. On the other hand, BD is a complex disease that affects multiple systems, and the tested patients from an ophthalmology department represent a subpopulation of this disease. The association of PTPN22 polymorphisms with BD should therefore also be investigated in BD patients from other medical departments, such as dermatology and stomatology. Finally, the present study only examined three of the PTPN22 polymorphisms, therefore it does not exclude the possibility that other SNPs of PTPN22 are associated with BD. More studies are needed to clarify these issues.
In conclusion, our study failed to find any association between the three polymorphisms of PTPN22 gene (rs2488457, rs1310182 and rs3789604) and susceptibility for ocular BD in Chinese Han patients.

Clinical Samples
Two independent Chinese Han population case-control cohorts were included in this study.

Ethics statement
The protocol was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, Chongqing, China (Permit Number: 2009-201004), and written informed consent was obtained from all the study subjects. All procedures followed the tenets of the Declaration of Helsinki.

Genomic DNA extraction and genotyping
Blood samples were collected in EDTA tubes and kept at 270uC until used. Genomic DNA was extracted by the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Amplification of the target DNA in the PTPN22 gene was analyzed by the polymerase chain reaction (PCR) using primers presented in Table 6. Each PCR reaction was performed in 10 ml containing 5 ml Premix Taq (Promega, Madison, USA), 20 pmoles primers and 0.2 mg of genomic DNA. The PCR conditions were as follow: initial denaturation at 95uC for 5 minutes followed by 37 cycles of denaturation at 94uC for 30 seconds, annealing at different temperatures (58uC for rs2488457, 58uC for rs1310182, and 60uC for rs3789604) for 30 seconds, extension at 72uC for 30 seconds, and a final extension at 72uC for 5 minutes. These SNPs were genotyped by PCR-restriction fragment length polymorphism (RFLP) analysis. PCR products of rs2488457,

Statistical methods
Hardy-Weinberg equilibrium was tested using the x 2 test. The variable age, described as mean 6 standard deviation (SD), median and range, between cases and controls was compared using the student's t-test. The variable gender was compared with Chi-square tests between cases and controls. Genotype frequencies were estimated by direct counting. Allele and genotype frequencies were compared between patients and controls by the unconditional logistic regression analysis (SAS, 9.13), which was used to compute the odds ratios (ORs) and their 95% confidence intervals (CIs). This statistical method was also performed to analyze the association between the SNPs and various clinical features of ocular BD. All statistical tests were two-sided. P,0.05 was considered significant.