Article Text

Extended report
Positive association between STAT4 polymorphisms and polymyositis/dermatomyositis in a Japanese population
  1. Tomoko Sugiura1,
  2. Yasushi Kawaguchi1,
  3. Kanako Goto2,
  4. Yukiko Hayashi2,
  5. Rie Tsuburaya2,
  6. Takefumi Furuya1,
  7. Takahisa Gono1,
  8. Ichizo Nishino2,
  9. Hisashi Yamanaka1
  1. 1Department of Rheumatology, Tokyo Women's Medical University, Tokyo, Japan
  2. 2Department of Neuromuscular Research, Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan
  1. Correspondence to Yasushi Kawaguchi, Institute of Rheumatology, Tokyo Women's Medical University, 10-22 Kawada-cho, Shinjuku-ku, Tokyo 162-0054, Japan; y-kawa{at}ior.twmu.ac.jp

Abstract

Objectives To investigate associations between signal transducer and activator of transcription 4 (STAT4), one of the most commonly acknowledged genes for the risk of multiple autoimmune diseases, with susceptibility to adult-onset polymyositis/dermatomyositis among Japanese individuals.

Methods A single nucleotide polymorphism of STAT4, rs7574865, was genotyped using TaqMan assay in 1143 Japanese individuals. The first set comprised 138 polymyositis/dermatomyositis patients and 289 controls and the second set comprised 322 patients and 394 controls. 460 patients (273 polymyositis and 187 dermatomyositis patients) and 683 controls were genotyped.

Results rs7574865T conferred a risk of polymyositis/dermatomyositis with an OR of 1.37 (95% CI 1.16 to 1.64; p=4x10−4; pcorr=0.0012). Both polymyositis and dermatomyositis exhibited high associations with the rs7574865T allele (polymyositis: OR=1.36, 95% CI 1.11 to 1.67; p=0.0039; pcorr=0.012; dermatomyositis: OR=1.40, 95% CI 1.10 to 1.78; p=0.0054; pcorr=0.016). The association between this STAT4 polymorphism and interstitial lung disease (ILD) was also investigated in the first set of polymyositis/dermatomyositis patients (n=138); those with ILD (n=79) bore rs7574865T more frequently compared with controls (OR 1.59, 95% CI 1.10 to 2.28; p=0.013; pcorr=0.039).

Conclusion This is the first study to show a positive association between a STAT4 polymorphism and polymyositis/dermatomyositis, suggesting that polymyositis/dermatomyositis shares a gene commonly associated with the risk of other autoimmune diseases.

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Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of diseases that affect skeletal muscles. Their common clinical feature is muscle weakness, and muscle biopsies typically show inflammatory cell infiltrates. IIM are clinically subdivided into several subgroups, including polymyositis, dermatomyositis, inclusion body myositis, myositis overlapping with another connective tissue disease (CTD), and cancer-associated myositis. Although the pathogenesis of IIM remains unclear, some environmental factors, such as viral infections, might trigger disease onset in genetically susceptible individuals, as is often the case with other autoimmune diseases.

Several studies have attempted to clarify the contributions of genetic factors for IIM susceptibility. Among possible candidate genes, major histocompatibility complex (human leucocyte antigen (HLA)) genes have been investigated most frequently.1 In North American Caucasian patients, HLA alleles of the 8.1 ancestral haplotype (particularly HLA-B*0801 and DRB1*0301) are the principal HLA risk loci.2 Among Japanese, HLA-DRB1*0803 was found to be associated with IIM and anti-aminoacyl-tRNA synthetase antibody.3 Several genes outside the HLA regions, including the proinflammatory cytokines tumour necrosis factor alpha, interleukin (IL)-1α, IL-1β and interferon (IFN) γ, and an immunoglobulin gene4,,7 were found to be associated with specific IIM subgroups, particularly juvenile dermatomyositis.

However, because these diseases are rare and there is a broad spectrum of disease entities, genetic risk factors for IIM have not been thoroughly investigated. A functional variant of the protein tyrosine phosphatase N22 gene (PTPN22), an R620W polymorphism, was recently found to be associated with adult and juvenile IIM in British Caucasian patients.8 This suggested that IIM might share a common genetic background with other autoimmune diseases.

Most susceptibility genes common to autoimmune diseases were originally identified in systemic lupus erythematosus (SLE) patients using genome-wide association studies. Among these, the following genes contributed most prominently: IFN regulatory factor 5 (IRF5), signal transducer and activator of transcription 4 (STAT4), PTPN22, B-lymphoid tyrosine kinase (BLK), B-cell scaffold protein with ankyrin repeats (BANK1) and tumour necrosis factor alpha-induced protein 3 (TNFAIP3). Their involvement has been replicated in different ethnic groups.9 Furthermore, these genes were also found to be associated with the risk of several other autoimmune diseases including rheumatoid arthritis (RA),10 systemic sclerosis (SSc),11 ,12 and type I diabetes mellitus.13

The present study is the first to investigate the possible involvement of STAT4, the best established gene for susceptibility to autoimmune diseases across different ethnic groups, in the susceptibility to adult-onset polymyositis/dermatomyositis among Japanese individuals.

Methods

Subjects

We enrolled polymyositis or dermatomyositis patients who were 18 years or older at disease onset and who had probable or definite myositis based on the criteria of Bohan and Peter.14 All patients underwent muscle biopsy. For our study group, dermatomyositis patients included those with clinically defined amyopathic dermatomyositis who fulfilled the traditional criteria of Sontheimer.15 We excluded patients with myositis overlapping with other CTD, who met either the following published criteria (American College of Rheumatology (ACR) criteria for systemic sclerosis,16 ACR criteria for systemic lupus erythematosus,17 ACR criteria for rheumatoid arthritis18 and American and European consensus criteria for Sjögren's syndrome)19 or the criteria for mixed CTD by Sharp et al.20 Inclusion body myositis is much less prevalent among Japanese than among European individuals, and was excluded on the basis of careful pathological examinations, clinical features, age of onset and response to immunosuppressive therapy. Patients with inherited, metabolic, or infectious myopathies, or muscle diseases caused by other factors were systematically excluded.

Polymyositis/dermatomyositis patients were recruited from two different institutions. For the first set for analysis, 138 patients (46 polymyositis patients and 92 dermatomyositis patients) were recruited from the Institute of Rheumatology, Tokyo Women's Medical University (TWMU), Tokyo, Japan, along with 289 healthy unrelated Japanese subjects as controls (60.4% women; mean age 42.3±12.3 years). For the second set for analysis, 322 patients (227 polymyositis patients and 95 dermatomyositis patients) were recruited from the National Center of Neurology and Psychiatry (NCNP), Kodaira, Tokyo, Japan, along with 331 control subjects (54.9% women; mean age 36.0±10.9 years).

Finally, a total of 460 adult-onset polymyositis/dermatomyositis patients (69.8% women) was enrolled, including 273 polymyositis patients (68.8% women) and 187 dermatomyositis patients (71.1% women). These included five polymyositis patients and 13 dermatomyositis patients with malignancies. The mean ages for polymyositis patients and dermatomyositis patients were 51.2±16.9 and 52.1±16.7 years, respectively. The combined control group included 683 subjects (57.1% women; mean age 38.6±11.9 years). All patients and controls were Japanese individuals.

For a subanalysis regarding the association between STAT4 polymorphisms and the presence or absence of interstitial lung disease (ILD), 138 polymyositis/dermatomyositis patients recruited from TWMU were evaluated. Of these patients, data on ILD for six patients were missing; therefore, 132 polymyositis/dermatomyositis patients (43 polymyositis patients and 89 dermatomyositis patients) were investigated and their allele and genotype frequencies were compared with those of the 289 control subjects who were included in the first study set. The presence of ILD was confirmed or excluded by CT, high-resolution CT, if available and spirometry.

This study was reviewed and approved by the research ethics committees of TWMU and NCNP.

Selection of single nucleotide polymorphisms

To date, rs7574865 of STAT4 and related single nucleotide polymorphisms (SNP) have shown the strongest associations with autoimmune disease susceptibility. Therefore, we investigated rs7574865 and rs11889341, which are in strong linkage disequilibrium.

Genotyping

Genotyping for each SNP site was performed using the TaqMan fluorogenic 5′ nuclease assay according to the manufacturer's instructions (Applied Biosystems, Tokyo, Japan). Endpoint fluorescence readings were made with an ABI Prism 7900 HT sequence detection system (Applied Biosystems).

Statistical analysis

Association analysis used χ2 tests for 2×2 contingency tables. For the association analysis between STAT4 polymorphisms and the three clinical subsets (all polymyositis/dermatomyositis patients, polymyositis patients and dermatomyositis patients vs control subjects); Bonferroni's correction was applied. Corrected p values (pcorr) were calculated by multiplying the p values by the results of these three comparisons. OR and 95% CI were also determined. For the subanalysis regarding the association study for STAT4 polymorphisms and the presence of ILD, pcorr values were also calculated by multiplying p values by the results of the following three comparisons: all polymyositis/dermatomyositis patients, those with ILD and those without ILD versus control subjects.

Results

STAT4 polymorphisms and polymyositis/dermatomyositis susceptibility

Because the patients in the first set for analysis were recruited from the Department of Rheumatology, whereas those in the second set for analysis were from neurology, the patient compositions in these two groups were different: the first set included predominantly dermatomyositis patients (polymyositis:dermatomyositis, 1:2), whereas the second set included predominantly polymyositis patients (polymyositis:dermatomyositis, 2.4:1). Therefore, the polymyositis and dermatomyositis patients in each set were separately compared with their corresponding control groups. Finally, all patients and control subjects were combined; the risk allele frequencies among polymyositis/dermatomyositis, polymyositis and dermatomyositis patients were compared with those among the combined control subjects (table 1), from which corrected p values were calculated.

Table 1

Associations between STAT4 rs7574865 and polymyositis/dermatomyositis

In the first set for analysis, only dermatomyositis patients showed higher rs7574865T allele and T/T genotype frequencies compared with those in the first set of control subjects (rs7574865T allele: OR 1.48, 95% CI 1.06 to 2.10; p=0.025; T/T genotype: OR 2.36, 95% CI 1.23 to 4.52; p=0.015). In the second study set, increased T allele and T/T genotype frequencies were observed only for polymyositis patients compared with those in the second set of control subjects (rs7574865T allele: OR 1.37, 95% CI 1.08 to 1.74; p=0.011; T/T genotype: OR 1.71, 95% CI 1.08 to 2.71; p=0.029).

When the data for the first and second sets of patients were combined, the risk allele frequencies achieved statistically significant levels for both polymyositis patients (OR 1.36, 95% CI 1.11 to 1.67; p=0.0039; pcorr=0.012) and dermatomyositis patients (OR 1.40, 95% CI 1.10 to 1.78; p=0.0054; pcorr=0.016) compared with those of the combined control subjects. The comparison between all polymyositis/dermatomyositis patients and the control subjects gave the lowest p values (rs7574865T allele: OR 1.37, 95% CI 1.16 to 1.64; p=4 (10−4 pcorr=0.0012; T/T genotype: OR 1.87, 95% CI 1.33 to 2.62; p=3.9 (10−4; pcorr=0.0012).

Because of the strong linkage disequilibrium between rs7574865 and rs11889341 (R2=0.76 and D′=0.93), very similar results were observed for rs11885361 (see supplementary table S1, available online only). None of the SNP deviated from Hardy–Weinberg equilibrium in both the disease subgroups and the control groups.

To investigate a gene dose effect for a STAT4 risk allele, patients and controls were divided into three groups: carriers of one risk allele for STAT4, carriers of two risk alleles, and carriers of no risk alleles. OR for polymyositis/dermatomyositis susceptibility were compared using individuals with no risk alleles as reference. The OR was 1.2 (95% CI 0.9 to 1.5) for carriers of one risk allele (rs7574865) and increased to 2.0 (95% CI 1.4 to 2.9) for two risk alleles. Similar results were observed for rs11889341; OR for disease susceptibility increased to 2.2 (95% CI 1.5 to 3.2) in carriers of two risk alleles, whereas that in carriers of one risk allele was 1.1 (95% CI 0.9 to 1.5).

Association between STAT4 polymorphisms and the ILD phenotype

We next investigated whether STAT4 polymorphisms were associated with a particular disease phenotype (ILD) by evaluating the polymyositis/dermatomyositis patients recruited from TWMU (first study set). For 138 polymyositis/dermatomyositis patients, data on ILD were missing for six patients. For the remaining 132 patients whose data were available, 20 of 43 polymyositis patients (46.5%) and 59 of 89 dermatomyositis patients (66.3%) had ILD. After combining these patients, 79 patients had polymyositis/dermatomyositis complicated with ILD and 53 patients did not.

As shown in table 2, the frequencies for the rs7574865T allele and T/T genotype among the polymyositis/dermatomyositis patients from the first set group showed only borderline significant differences compared with the control subjects (T allele: OR 1.38, 95% CI 1.02 to 1.86; p=0.037; pcorr=0.11; T/T genotype: OR 2.25, 95% CI 1.26 to 4.03; p=0.0074; pcorr=0.022). These differences remained significant among the 79 ILD patients (T allele: OR 1.59, 95% CI 1.10 to 2.28; p=0.013; pcorr=0.039; T/T genotype: OR 2.46, 95% CI 1.25 to 4.84; p=0.016; pcorr=0.048). In contrast, no association was observed between patients without ILD and control subjects.

Table 2

Associations between STAT4 rs7574865 and interstitial lung disease in the first polymyositis/dermatomyositis set

Nevertheless, the results of this intrasubgroup analysis failed to identify a significant difference in the risk allele frequencies between the polymyositis/dermatomyositis patients with ILD and those without ILD. Similar results were obtained for rs11889361 (see supplementary table S2, available online only).

Discussion

This is the first study to report that STAT4 polymorphisms are involved in susceptibility to adult-onset polymyositis/dermatomyositis among Japanese individuals, regardless of the polymyositis or dermatomyositis disease phenotype. This suggests that these disorders share a genetic background common with other autoimmune diseases. The STAT4 variant rs7574865, located in the third intron, has previously been implicated in the susceptibility to autoimmune diseases;9 ,10 ,12 ,13 our results are consistent with these observations. There was also a gene dose effect for rs7574865 for polymyositis/dermatomyositis susceptibility. Although several studies have suggested that rs7574865 or a related haplotype was associated with high transcriptional levels of STAT4,21 ,22 the functional consequence of rs7574865 remains unclear.

The STAT4 protein is activated on stimulation with IL-12, IL-23 and IL-17,23 and drives T-helper (Th)1 and Th17-type immune responses. STAT4 is also activated by type I IFN cytokine signals (ie, IFNα and IFNβ), which results in a spike in IFNγ secretion by CD4 cells and natural killer cells without leading to Th1 development. Activation of a type I IFN pathway is a shared pathological phenomenon among several autoimmune diseases, and the expression of type I IFN-inducible proteins in affected muscle tissues was reported predominantly for dermatomyositis.24 Interestingly, SLE patients who carry the risk variant of STAT4 show increased sensitivity to IFNα. 25 It is plausible that STAT4 variations, which can cause increased and/or prolonged STAT4 protein activity, may trigger autoimmune disease pathology because of its impact on the immune system.

In Asian populations, in which the STAT4 risk allele is more prevalent than in Caucasians, the contribution of STAT4 to disease susceptibility has been considered to be greater.26 This is in sharp contrast to PTPN22. Because of the extremely low frequency of this risk allele, PTPN22 polymorphisms are not involved in autoimmune diseases among Asians.27 In the present study, the observed frequency of the risk allele among polymyositis/dermatomyositis patients was 0.39 with an OR of 1.37. Kobayashi et al28 investigated a total of 3567 Japanese RA patients from three independent Japanese populations, and reported that the rs7574865 allele frequency was 0.37, with a combined OR of 1.27 for RA, whereas the frequency for SLE patients (n=591) ranged from 0.39 to 0.44 with an OR of 1.61. Given the reported OR for Japanese RA and SLE patients, the contribution of the STAT4 allele in polymyositis/dermatomyositis seems strong.

In addition to their influence on autoimmune disease susceptibility, STAT4 polymorphisms can also influence disease phenotypes. For example, rs7574865 in SLE patients was associated with severe disease manifestations, such as nephritis, high double-stranded DNA antibody production and younger age of disease onset.26 ,29 For SSc patients, this polymorphism was associated with the presence of ILD.12 Therefore, we examined possible associations between STAT4 and the clinical manifestation of ILD in polymyositis/dermatomyositis patients.

The rs7574865T allele and T/T genotype frequencies remained high when polymyositis/dermatomyositis patients were limited to those with ILD, whereas these were not significant for patients without ILD. Although a total of 460 polymyositis/dermatomyositis patients was genotyped in the present study, our subanalysis only tested 138 patients in our first set. This was because clinically equivalent data were not available between the collaborating institutions. The statistical power regarding the association between STAT4 and the predisposition to ILD in polymyositis/dermatomyositis was thus rather limited.

In addition to shared susceptibility genes, it is plausible that each autoimmune disease has disease-specific risk genes that can influence each unique disease phenotype. For example, Asian RA patients who had a certain functional haplotype for PADI4 (encoding for citrullinating enzyme of peptidyl arginie deaminase type 4) had high serum titres of an autoantibody to citrullinated proteins.30 ,31 SSc patients, regardless of ethnicity, are likely to have genetic variants of CTGF that encode for a connective tissue growth factor,32 ,33 which contributes to tissue fibrosis. A unique phenotypical feature of polymyositis/dermatomyositis is that skeletal muscle is targeted. Unfortunately, a risk gene(s) specific for polymyositis/dermatomyositis remains to be determined, and genome-wide association studies might help in its (their) discovery.

Our results established STAT4 as a new polymyositis/dermatomyositis susceptibility gene. One study limitation was an insufficient association analysis regarding clinical subsets, including serological phenotypes (autoantibody profiles). However, in spite of the rarity of these diseases, we managed to obtain a large sample size, which provided sufficient statistical power for this case–control study. Further investigations will be needed to replicate the positive association between STAT4 polymorphisms and polymyositis/dermatomyositis in Asian populations, as well as in different ethnic groups.

Acknowledgments

The authors would like to thank Mr Kazutomo Ogata and Ms Mika Fujita for their technical assistance. The authors also thank Mr Manabu Kawamoto for his useful suggestions.

References

Supplementary materials

  • Supplementary Data

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Footnotes

  • Funding This study was supported, in part, by an intramural research grant (23-4, 2-5) for neurological and psychiatric disorders from NCNP.

  • Competing interests None.

  • Patient consent Obtained.

  • Ethics approval This study was reviewed and approved by the research ethics committees of the Tokyo Women's Medical University and the National Center of Neurology and Psychiatry.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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