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The R620W C/T polymorphism of the gene PTPN22 is associated with SLE independently of the association of PDCD1

Abstract

The gene PTPN22 is located on chromosome 1p13 and encodes a protein tyrosine phosphatase called the lymphoid-specific phosphatase (Lyp). Lyp is expressed in lymphocytes, where it physically associates through its proline-rich motif (called P1) with the SH3 domain of the protein tyrosine kinase Csk, an important suppressor of the Src family of kinases Lck and Fyn, which mediate TCR signaling. Therefore, it is said that interaction between Lyp and Csk enables these effectors to inhibit T-cell activation synergistically. It was reported that a missense single nucleotide polymorphism , R620W (rs2476601), 1858C—>T encodes an amino-acid change in the P1 proline-rich motif of the gene PTPN22 and is associated with SLE in North American white individuals. PTPN22 gene polymorphisms were genotyped in 571 Swedish SLE patients and 1042 healthy controls using TaqMan SNP Genotyping Assay. Differences were observed between cases and control subjects at both the allele (χ2=11.2895;P=0.0007,1df) and genotype (χ2=10.2243;P=0.0013, 1df) levels. We also found evidence of a genetic association between PTPN22 and renal disorder (χ2=9.5660;P=0.0019). We then analyzed if in patients with renal disorder associations with PDCD1 and PTPN22 were independent. Our data suggest that this appears to be the case although we observed some degree of interaction.

Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease in which a consistent attack of the body's own immune system is directed against self-tissue through the production of autoantibodies. There is immune complex formation and deposition in tissues often but not always associated with inflammatory damage. Immune complexes are found in glomeruli, skin, lungs, or synovium.1

SLE is a disorder of the immune system, where the result of the combination of multiple genetic factors, interacting with one another and with the environment, leads to pathology. This means that multiple genes and their combinations contribute to SLE. Not every individual with SLE will have the same set of genes, and different combinations of genes are likely to influence the severity of the disease. It has been hypothesized that each gene will contribute a relatively small individual risk to disease; however, once a sufficient number of genes are inherited then an individual will become susceptible to the development of SLE. This is known as the gene threshold liability hypothesis.2

The gene protein tyrosine phosphatase nonreceptor type 22 (PTPN22) encodes the lymphoid protein tyrosine phosphatase (Lyp) that is known to be involved in the control of T-cell activation. Under normal conditions, this enzyme (Lyp) works as a ‘negative regulator’ and keeps immune cells from becoming overactive.

Lyp interacts through one of its proline-rich motifs (referred to as P1) with the SH3 domain of the Csk kinase.3 The ability of Csk and Lyp to inhibit T cell receptor signaling requires their physical association. As a result, Csk acts as a suppressor of the Src family of kinases Lck and Fyn, which mediate TCR signaling. Therefore, it is said that interaction between Lyp and Csk enables these effectors to synergistically inhibit T-cell activation.4, 5

A single nucleotide polymorphism (SNP), a C to T transition at position 1858, recently identified, leads to a crucial amino acid substitution of a highly conserved arginine to tryptophane in Lyp. Unlike the variant encoded by the more common allele 1858C, the tryptophane (1858T) does not allow binding of Lyp to Csk.6, 7

This variant was firstly associated with type I diabetes6 and later with SLE8 and rheumatoid arthritis.9 This same variation has also been associated with Graves’ disease although not with multiple sclerosis10, 11 suggesting that it may be a common risk factor for many autoimmune diseases. Indeed, multiplex families segregating various autoimmune diseases also show genetic association with PTPN22.12

In cases where the SNP is present in one or both copies of an individual's genes, it was found that the negative regulation by this enzyme appears to be inefficient, so that T cells and other immune cells are hyper-responsive causing increased inflammation and tissue damage.13

PDCD1 codes for PD-1 an immunoreceptor tyrosine-inhibitory motif-containing protein that, similarly to PTPN22, regulates T and B-cell activation through the transmission of inhibitory signals via its interaction with SHP-2 and SHP-1.

We had previously identified an association of the PD1.3A polymorphism of PDCD1 in European individuals and SLE, in particular those individuals with nephritis.14, 15 Although this polymorphism does not disrupt the coding structure of the PDCD1 gene, it does disrupt the repressive effect of the regulator RUNX1 on an enhancer in the fourth intron of PDCD1.16

Whether the products of PTPN22 and PDCD1 interact biologically in some way is unknown; however the function of both genes suggests that their mode of action in the pathophysiology of SLE is similar, that is, to inhibit lymphocyte cell activation. There is a possibility that when T cells are not controlled by PTPN22 or by PD-1, the effect of the other would take over. Therefore, having polymorphisms of both that would disrupt their function or expression would imply a higher risk to develop SLE and would follow the threshold liability model. We therefore tested if the genetic effects of PDCD1 and PTPN22 were independent or not .

This study was undertaken to analyze the association of polymorphism R620W of PTPN22 with SLE in the Swedish population and to analyze if there is an interaction between the genetic effects of PD1.3A in PDCD1 and the T allele of PTPN22.

Results

A total of 571 unrelated patients with SLE and 1042 controls recruited from 3 different centers in Sweden were used for association analysis of the polymorphism rs2476601 (1858C/T). Genotype frequencies of both cases (χ2=1.037; P=0.3085) and controls (χ2=2.301; P=0.1292) were found to be in Hardy–Weinberg equilibrium. The overall risk-allele frequency of the polymorphism R620W in individuals with SLE was 16.5%, compared with an allele frequency of 12.3% in control individuals (χ2=11.2895; P=0.0007; OR=1.4161, 95% CI=1.15–1.73). The risk allele was present in 29.8% of individuals with SLE, compared with 22.5% of control individuals (χ2=10.2243; P=0.0013; OR=1.4558, 95% CI=1.15–1.83). The genotype and allele data are shown in Tables 1 and 2. We show the frequency distribution among our three groups of SLE patients from North, Mid- and South Sweden. For the individual groups, only patients from North Sweden show association with PTPN22 by themselves, with a weak association in the Mid-Sweden group. Combined analysis of the data shows a genetic association (Table 2) mainly contributed by the Northern population group.

Table 1 Genotypic and allelic association to PTPN22 comparing subpopulations of Swedish patients with their respective controls
Table 2 Combined analysis of genotypic and allelic association of all Swedish SLE patients and controls

Overall 466 SLE individuals had data on renal disorder, so we analyzed whether our patients with this clinical manifestation had an increased frequency of the risk allele than those without renal disorder. Association was found between the 1858T risk allele and renal disorder (χ2=9.5660; P = 0.0019) (Table 3).

Table 3 Comparison of the frequencies of the PTPN22 R620W polymorphism in individuals having or not having renal disorder from the separate Swedish subpopulations as well as in combination

We then did a combined analysis of the risk alleles of PDCD1 and PTPN22 in patients with renal disorder (n=142) and compared to those without or to controls. Results are shown in Table 4. The strongest association was found when individuals negative for both PDCD1 and PTPN22 were compared to the control group population (χ2=19.9; P=0.000008) suggesting a strong ‘protective’ or compensatory effect of the nonrisk alleles of both genes. The test for independency was more significantly associated (χ2=8.23; P=0.004) in the PTPN22 positive and PDCD1 negative group than in the PDCD1 positive and PTPN22 negative group (χ2=3.2594; P=0.0710) when the individuals were compared to those without renal disorder, suggesting that PTPN22 1585T contributes a stronger risk than PD1-3A and may be independently associated. However, some degree of interaction, albeit weak and based on very few individuals (n=9), could be observed (χ2=5.17; P=0.02).

Table 4 PTPN22 and PD1.3A are independent genetic susceptibility factors in SLEa

Discussion

We have replicated the genetic association between the T allele of PTPN22 and SLE previously reported. It was clear to us that association was attained with two of our sets but not the third one (Southern Sweden). This suggests some substructure in our population sample, which can be overcome by increasing the numbers of individuals. Similarly, our PDCD1 data became less significant because in this paper we have not used familial cases, which contributed importantly to our previous results.14

Nephritis is known to be one of the most serious manifestations of SLE. Mainly due to selection bias of recruitment, the incidence of renal involvement in SLE varies widely between different series, ranging from 29 to 53%.17 The incidence of nephritis in our three cohorts varies, the one from Southern Sweden being the one with the lowest incidence (10%); that of renal disorder as defined by the ACR is somewhat higher.

We analyzed patients with CT and TT genotypes to see if these genotypes in particular occur frequently in individuals having renal disorder or nephritis. There was evidence of a genetic association between PTPN22 SNP R620W and renal disorder. However, the association was not stronger than that we identified for SLE in general. In contrast, we have observed a stronger association with nephritis (as defined by biopsy) when individuals are analyzed for PD1.3A.14 Note that not all patients in the present data set overlap those studied previously.

Now that we are beginning to identify the various genetic effects contributing to SLE susceptibility, the analysis of genetic interactions becomes feasible. Accordingly, we analyzed if PD.1.3 and PTPN22 SNP R620W do coexist in SLE patients. There was a weak association when we analyzed the difference of individuals positive for both risk alleles; however, this was based on a very reduced number of typed individuals. However the strongest effect was observed in patients positive for PTPN22 but negative for PDCD1 and a tendency was also observed for the opposite, suggesting independence of the effects; therefore for this particular situation the threshold liability hypothesis is not confirmed. It would be expected that individuals possessing both risk alleles would have a significantly higher risk to develop SLE, but this appears not to be the case. However, the ‘protective’ or compensatory effect of not having the risk alleles was the strongest providing support, albeit indirectly, for the importance of the genetic susceptibility of both PTPN22 and PDCD1. It is clear from these results that the relative risk of PTPN22 appears to be somewhat stronger than that for PDCD1.

We have tested a large cohort of SLE patients from Sweden. In particular the set of South Sweden showed no association at all, while the set from Mid-Sweden showed a weak association. The set from South Sweden was smaller, so the tendency effects and associative effects of the other two sets do overcome the negative result observed for this set providing a replication for association of PTPN22 in Swedish SLE patients and an independent effect from that of PD1.3A of PDCD1 in SLE.

Patients and methods

Patients

The case group consisted of three sets of sporadic SLE patients from Sweden; totally there were 571 patients, from the North (Umeå), from the Middle country area (Stockholm) and from the South (Lund). Swedish patients and controls were all considered to be of Swedish ancestry (ethnic background was determined based on the ancestries of the great-grandparents using a questionnaire). The control group comprised of healthy individuals from the same geographical area as the corresponding patient cohort in the case of Lund and Stockholm. The controls for Northern Sweden were part of the World Health Organization (WHO) study for Monitoring of Trends and Determinants in Cardiovascular Disease (MONICA) and are population based. All patients were diagnosed by a specialist and fulfilled the American college of Rheumatology (ACR) 1982 revised criteria for classification of SLE. Of the Swedish patients 25% had nephritis or renal disorder documented clinically and/or through biopsy.

Genomic DNA extraction

The method described by Miller et al18 was used. About 7–10 ml of whole blood from patients and controls was collected in EDTA containing test tubes and DNA was extracted manually by using a salt-based extraction method. The DNA from first collected 161 patients from Northern Sweden was extracted using the standard phenol/chloroform extraction method.

Genotyping

We used TaqMan SNP Genotyping Assay (Applied Biosystems), and detection was made on an ABI 7900HT sequence detection system. The primers and probes were designed by Applied Biosystems (Foster City, CA, USA) assay on demand service for allelic discrimination with the 5′ nuclease assay and fluorogenic probes. The SNP number is as described (rs2476601).

The material required were TaqMan universal PCR Master mix, 40 × assay mix and Genomic DNA diluted in dH20. The 40 × Assay mix consists of unlabeled PCR primers and TaqMan probes labeled with FAM and VIC dye.

The primers and probes were as follows: Forward primer-IndexTerm5′-CCAGCTTCCTCAACCACAATAAATG-3′; Reverse primer-IndexTerm5′-CAACTGCTCCAAGGATAGATGATGA-3′; probe VIC-IndexTerm5′-TCAGGTGTCCATAC-AGG-3′; obe FAM-IndexTerm5′ TCAGGTGTCCGTACAGG-3′.

The PCR was performed as follows: after enzyme activation for 10 min at 95°C, 40 two-step cycles were performed for 15 s denaturation at 92°C followed by 1 min annealing/elongation at 60°C.

Statistical analysis

Genotype and allele frequencies as well as odds ratios and their 95% confidence intervals (95% CIs) were calculated. Comparisons of genotype and allele frequencies were done using a χ2 test with a 2 × 2 contingency table. P values less than or equal to 0.05 were considered significant. The distribution of variant genotypes was also compared between the lupus patients who were positive vs negative for various clinical manifestations.

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Acknowledgements

We would like to thank the patients and their relatives for their collaboration in this project and Ludmila Prokunina-Olsson for help with the PD-1.3 data. This work was supported by grants from the Alliance for Lupus Research to MEAR, The Swedish Research Council for Medicine (12673 and 13489), the Clas Groschinski Memorial Foundation, the Swedish Society Against Rheumatism, the Gustaf V:80-year Jubileum Foundation, the Magnus Bergvalls Foundation, the Torsten and Ragnar Söderbergs Foundation, and by grants from ‘Visare Norr’, Samverkansnämden för Norra Regionen. Professor Göran Hallmans and the Blood Bank of Northern Sweden, and Birgitta Stegmayr, PhD, Department of Public Health and Clinical Medicine/Internal Medicine kindly provided control samples from the WHO-MONICA cohort. MEAR is a Fellow at the Royal Swedish Academy of Sciences.

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Correspondence to M E Alarcón-Riquelme.

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Reddy, M., Johansson, M., Sturfelt, G. et al. The R620W C/T polymorphism of the gene PTPN22 is associated with SLE independently of the association of PDCD1. Genes Immun 6, 658–662 (2005). https://doi.org/10.1038/sj.gene.6364252

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Keywords

  • PTPN22
  • Lyp
  • Lck
  • Fyn
  • polymorphism
  • systemic lupus erythematosus

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