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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

The complication of interstitial lung disease (ILD) in polymyositis/dermatomyositis (PM/DM) is associated with anti–aminoacyl–transfer RNA synthetase (anti-aaRS) antibody or anti–melanoma differentiation–associated gene 5 (anti–MDA-5) antibody positivity. Anti–MDA-5 antibody is associated with clinically amyopathic DM and fatal outcome due to rapidly progressive ILD in Asian populations. The association between genetic factors and anti–MDA-5 antibody–positive DM is unclear. This study was undertaken to investigate the HLA–DRB1 genotype in patients with anti–MDA-5 antibody–positive DM.

Methods

We examined genetic differences among 17 patients with anti–MDA-5 antibody–positive DM, 33 patients with anti-aaRS antibody–positive PM/DM, 33 patients with PM/DM without anti-aaRS antibody or ILD, and 265 healthy controls.

Results

The frequencies of HLA–DRB1*0101 and DRB1*0405 were 29% and 71%, respectively, in patients with anti–MDA-5 antibody–positive DM, which were higher than the frequencies in healthy controls (10% and 25%, respectively). Among the 17 patients with anti–MDA-5 antibody–positive DM, 16 (94%) harbored either the DRB1*0101 or DRB1*0405 allele. The combined frequency of the DRB1*0101 allele and the DRB1*0405 allele was significantly higher in patients with anti–MDA-5 antibody–positive DM than in patients with PM/DM without anti-aaRS antibody or ILD, with an odds ratio (OR) of 42.7 (95% confidence interval [95% CI] 4.9–370.2) (P = 1.1 × 10−5), or in patients with anti-aaRS antibody–positive PM/DM (OR 13.3 [95% CI 1.6–112.6], P = 4.5 × 10−3).

Conclusion

Our findings indicate that HLA–DRB1*0101/*0405 is associated with susceptibility to anti–MDA-5 antibody–positive DM in the Japanese population.

Dermatomyositis (DM) is characterized by inflammation of the skin and muscle (1) and is occasionally complicated by interstitial lung disease (ILD). In particular, rapidly progressive ILD is an intractable and life-threatening complication. Clinically amyopathic DM (CADM) includes typical skin lesions with amyopathy or hypomyopathy (2). It has recently been reported that patients with CADM who are positive for the anti–melanoma differentiation–associated gene 5 (MDA-5) antibody frequently have complications with rapidly progressive ILD, especially in the Japanese population (3–5). In general, anti–MDA-5 antibody is specific for rapidly progressive ILD associated with CADM and is not detected in patients with CADM or DM without ILD or in patients with polymyositis (PM). The MDA-5 protein plays a role in the innate immune system. MDA-5 initially recognizes picornaviruses, such as coxsackievirus, and induces antiviral responses by producing type I interferons and tumor necrosis factor α (6). Hyperferritinemia is complicated by rapidly progressive ILD in anti–MDA-5 antibody–positive DM (4, 5). Although the pathogenesis of rapidly progressive ILD associated with anti–MDA-5 antibody–positive DM has been tentatively attributed to a cytokine storm triggered by viral infection, especially in the skin and lungs, its exact mechanism is unknown.

In PM/DM, complication with ILD is associated with the anti–aminoacyl–transfer RNA synthetase (anti-aaRS) antibody or anti–MDA-5 antibody. It has been reported that 90% of Caucasian patients with the anti-aaRS antibody are carriers of HLA–DRB1*03 (7). In the Japanese population, HLA–DRB1*0405 is associated with susceptibility to anti-aaRS antibody–positive PM/DM (8). However, associations between genetic factors and anti–MDA-5 antibody–positive DM have remained unclear.

Therefore, we investigated the HLA–DRB1 gene in patients with anti–MDA-5 antibody–positive DM. In addition, we compared genetic differences in HLA among patients with anti–MDA-5 antibody–positive DM, patients with anti-aaRS antibody–positive PM/DM, and patients with PM/DM without anti-aaRS antibody or ILD.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Patients.

This retrospective study included patients admitted to Tokyo Women's Medical University Aoyama Hospital or Keio University Hospital from August 1992 to February 2010. Medical records were obtained for 142 and 57 patients diagnosed as having DM and CADM, respectively. The anti–MDA-5 antibody was detected in 31 patients. DNA samples were available for 17 patients with the anti–MDA-5 antibody, and all of these patients were enrolled in the study. All of the enrolled patients had skin rashes, myopathy, or respiratory symptoms (or a combination thereof) at admission. The patients were diagnosed as having DM or CADM based on the criteria of Bohan and Peter (9) or Sontheimer (10), respectively. Specific rashes, including heliotrope rash, Gottron's sign, or Gottron's papules, were used to define DM or CADM. In general, CADM patients present with typical skin lesions and amyopathy or hypomyopathy with a duration of >6 months. A subset of the CADM group included patients who developed fatal ILD within the first 6 months of this study. Clinical data were obtained from hospital admission records.

To investigate the characteristics of the HLA–DRB1 genotype in anti–MDA-5 antibody–positive DM, HLA data were obtained in patients with anti-aaRS antibody–positive PM/DM, patients without anti-aaRS antibody or ILD, and healthy controls. These HLA genotype databases have been described previously (8). All of the subjects in the present study were Japanese. None of the subjects had rheumatoid arthritis (RA) or other connective tissue diseases. This study was approved by the ethics committee of Tokyo Women's Medical University and was performed in accordance with the Declaration of Helsinki.

Evaluation of autoantibodies.

Anti–MDA-5 antibody was detected by immunoprecipitation (IP) assay and enzyme-linked immunosorbent assay using recombinant MDA-5 as an antigen, as previously described (3). Anti-aaRS antibodies, including Jo-1, EJ, PL-7, PL-12, and OJ; anti–signal recognition particle (anti-SRP) antibody; anti-Ku antibody; and anti–U1 small nuclear RNP (anti-U1 snRNP) antibody were assessed by RNA IP assays.

Classification of ILD.

Patients were evaluated for ILD by chest radiography and computed tomography (CT) or high-resolution CT of the chest. Rapidly progressive ILD was defined as a progressive ILD within 3 months of the onset of respiratory symptoms. Chronic ILD was defined as ILD that was asymptomatic and non–rapidly progressive or slowly progressive over 3 months (11).

HLA–DRB1 genotyping.

HLA–DRB1 genotyping was performed using polymerase chain reaction–reverse sequence-specific oligonucleotide techniques and standard methods. The DNA for the HLA–DRB1 genotyping of the patients was extracted from peripheral blood mononuclear cells using standard methods.

Statistical analysis.

The chi-square test was used for the comparison of frequencies, and Fisher's exact test was used when appropriate. Data were analyzed using JMP software (SAS Institute). P values were adjusted by Bonferroni correction when appropriate.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Clinical characteristics and HLA–DRB1 genotype of patients with anti–MDA-5 antibody–positive DM.

As shown in Table 1, 17 patients with anti–MDA-5 antibody–positive DM were enrolled in the study. Their mean ± SD age was 46 ± 16 years. Seventy-one percent were women. The HLA–DRB1*0101 and DRB1*0405 alleles were identified in 5 patients (29%) and 12 patients (71%), respectively. The HLA–DRB1*0101 or *0405 allele was identified in 16 (94%) of the 17 patients. No patients had homoalleles of HLA–DRB1*0101 or DRB1*0405. One patient had both DRB1*0101 and DRB1*0405. With respect to the clinical phenotype, 16 patients had CADM. ILD complication was observed in all of the patients. Moreover, the frequency of rapidly progressive ILD was high (65%). No patients had RA or other connective tissue diseases as complications.

Table 1. Clinical characteristics and HLA–DRB1 genotype of the patients with anti–MDA-5 antibody–positive DM*
Patient/age/sexGenotypePhenotypeILD type
  • *

    Anti–MDA-5 = anti–melanoma differentiation–associated gene 5; DM = dermatomyositis; ILD = interstitial lung disease; CADM = clinically amyopathic DM.

1/48/MDRB1*0101/1602CADMRapidly progressive
2/25/FDRB1*0101/1501CADMChronic
3/53/FDRB1*0101/0803CADMRapidly progressive
4/18/MDRB1*0101/1502CADMRapidly progressive
5/47/FDRB1*0101/0405DMRapidly progressive
6/58/MDRB1*0405/1406CADMRapidly progressive
7/16/FDRB1*0405/0401CADMRapidly progressive
8/53/FDRB1*0405/1501CADMRapidly progressive
9/53/FDRB1*0405/0410CADMRapidly progressive
10/44/FDRB1*0405/1406CADMChronic
11/45/FDRB1*0405/1202CADMChronic
12/39/MDRB1*0405/0401CADMChronic
13/47/FDRB1*0405/1201CADMChronic
14/76/FDRB1*0405/0802CADMRapidly progressive
15/56/FDRB1*0405/1502CADMRapidly progressive
16/43/MDRB1*0405/0901CADMChronic
17/66/FDRB1*0901/1502CADMRapidly progressive

Comparison of the HLA–DRB1 genotype in patients with anti–MDA-5 antibody–positive DM, patients with anti-aaRS antibody–positive PM/DM, and patients with PM/DM without anti-aaRS antibody or ILD.

To investigate the characteristics of the HLA–DRB1 genotype in anti–MDA-5 antibody–positive DM, the frequency of the HLA–DRB1 genotype was compared among patients with anti–MDA-5 antibody–positive DM, patients with anti-aaRS antibody–positive PM/DM, patients with PM/DM without anti-aaRS antibody or ILD, and healthy controls (Table 2).

Table 2. Comparison of HLA–DRB1 genotypes among patients with anti–MDA-5 antibody–positive DM, patients with anti-aaRS antibody–positive PM/DM, and patients with PM/DM without anti-aaRS antibody or ILD*
GenotypePatients with anti–MDA-5 antibody–positive DM (n = 17)Patients with anti-aaRS antibody–positive PM/DMPatients with PM/DM without anti-aaRS antibody or ILDHealthy controls (n = 265)
PM/DM (n = 33)DM (n = 19)PM/DM (n = 33)DM (n = 21)
  • *

    Values are the percent of subjects. Anti-aaRS = anti–aminoacyl–transfer RNA synthetase; PM = polymyositis (see Table 1 for other definitions).

  • P = 0.0003 versus patients with PM/DM without anti-aaRS antibody or ILD; P = 0.00018 versus healthy controls.

DRB1*0101291211121410
DRB1*04011200352
DRB1*0403095655
DRB1*0405714253182425
DRB1*0406065357
DRB1*0407000002
DRB1*0410635952
DRB1*0802618219107
DRB1*080362421271914
DRB1*0901122421181930
DRB1*1101030002
DRB1*12016916357
DRB1*1202660004
DRB1*1301000300
DRB1*1302065212919
DRB1*14010359105
DRB1*14030306104
DRB1*1405035356
DRB1*14061200353
DRB1*15011212169511
DRB1*150218611212420
DRB1*1602600603
Other000005

Data previously obtained at our institution indicated that 33 PM/DM patients (14 patients with PM and 19 with DM) exhibited anti-aaRS antibody, as follows: 8 PM patients and 8 DM patients had anti–Jo-1; 4 PM patients and 6 DM patients had anti-EJ; 2 PM patients and 2 DM patients had anti–PL-7; 0 PM patients and 3 DM patients had anti–PL-12; and none of the patients had anti-OJ. Of the 33 patients with anti-aaRS antibody–positive PM/DM, 24 (73%) had ILD. Moreover, 33 PM/DM patients (12 PM patients and 21 DM patients) had neither anti-aaRS antibody nor ILD, and in all 21 of these DM patients, the clinical phenotype was classic DM, not CADM. In patients with PM/DM without anti-aaRS antibody or ILD, anti-SRP antibody, anti-U1 snRNP antibody, and anti-Ku antibody were detected in 3 PM patients, 1 DM patient, and 0 patients, respectively.

As shown in Table 2, the frequency of HLA–DRB1*0101 was ∼30% in anti–MDA-5 antibody–positive DM and ∼10% in the other subsets, although the difference was not significant (P = 0.012 versus healthy controls, adjusted P value not significant). P values with Bonferroni correction for multiple comparisons less than 0.0023 were considered significant; this was determined by dividing the P value of 0.05 by 22 (the number of HLA genotypes). The inadequate statistical power may be attributed to small sample sizes. Moreover, the frequency of HLA–DRB1*0405 was significantly higher in the patients with anti–MDA-5 antibody–positive DM than in the patients with PM/DM without anti-aaRS antibody or ILD (P = 0.0003) or in the healthy controls (P = 0.00018). The frequency of HLA–DRB1*0405 was also high in patients with anti-aaRS antibody–positive PM/DM, although it was not significantly different from that in the other subsets. No significant differences were found regarding the frequencies of the other alleles.

Frequency of HLA–DRB1*0101/*0405 in patients with anti–MDA-5 antibody–positive DM compared with other PM/DM patient subsets.

In this study, the HLA–DRB1*0101 or *0405 allele was identified in all but 1 of the 17 anti–MDA-5 antibody–positive patients. In the HLA–DRB1 alleles, residues 70–74 of the DRβ chain form the third hypervariable region, an important region for antigen presentation. This amino acid sequence is QRRAA, which is a shared epitope motif in both DRB1*0101 and DRB1*0405. We speculated that QRRAA may be a critical sequence in the pathophysiology of anti–MDA-5 antibody–positive DM. We considered the role of both DRB1*0101 and DRB1*0405 in anti–MDA-5 antibody–positive DM. Therefore, the combined frequency of the DRB1*0101 allele and the DRB1*0405 allele was compared among patients with anti–MDA-5 antibody–positive DM, patients with anti-aaRS antibody–positive PM/DM, and patients with PM/DM without anti-aaRS antibody or ILD.

As shown in Table 3, the combined frequency of DRB1*0101 and *0405 was significantly higher in patients with anti–MDA-5 antibody–positive DM than in patients with PM/DM without anti-aaRS antibody or ILD, with an odds ratio (OR) of 42.7 (95% confidence interval [95% CI] 4.9–370.2, P = 1.1 × 10−5), or in patients with DM without anti-aaRS antibody or ILD (OR 32 [95% CI 3.5–293.1], P = 2 × 10−4). The combined frequency of DRB1*0101 and *0405 was also higher in patients with anti–MDA-5 antibody–positive DM than in patients with anti-aaRS antibody–positive PM/DM (OR 13.3 [95% CI 1.6–112.6], P = 4.5 × 10−3) and patients with anti-aaRS antibody–positive DM (OR 9.3 [95% CI 1.0–86.4], P = 4.4 × 10−2). Moreover, the frequency of these alleles was higher in patients with anti-aaRS antibody–positive PM/DM than in patients with PM/DM without anti-aaRS antibody or ILD (OR 3.2 [95% CI 1.1–8.9], P = 2.4 × 10−2).

Table 3. Frequency of the HLA–DRB1*0101/0405 alleles in patients with anti–MDA-5 antibody–positive DM*
 Patients with anti–MDA-5 antibody–positive DM (n = 17)Patients with anti-aaRS antibody–positive PM/DMPatients with PM/DM without anti-aaRS antibody or ILD
PM/DM (n = 33)DM (n = 19)PM/DM (n = 33)DM (n = 21)
  • *

    Anti-aaRS = anti–aminoacyl–transfer RNA synthetase; PM = polymyositis; OR = odds ratio; 95% CI = 95% confidence interval (see Table 1 for other definitions).

  • Versus patients with anti–MDA-5 antibody–positive DM.

DRB1*0101 or DRB1*0405, %9455632733
P4.5 × 10−34.4 × 10−21.1 × 10−52.0 × 10−4
OR (95% CI)13.3 (1.6–112.6)9.3 (1.0–86.4)42.7 (4.9–370.2)32 (3.5–293.1)

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We have demonstrated an association between a genetic factor and anti–MDA-5 antibody–positive DM. Specifically, this study shows that HLA–DRB1*0101/*0405 is associated with susceptibility to anti–MDA-5 antibody–positive DM. HLA–DRB1*0301 is associated with susceptibility to anti-aaRS antibody–positive PM/DM in Caucasians. In contrast, the frequency of HLA–DRB1*0301 is low, but the frequency of HLA–DRB1*0405 is relatively high, at ∼20%, in the Japanese population. HLA–DRB1*0405 is associated with susceptibility to anti-aaRS antibody–positive PM/DM in the Japanese population, whereas HLA–DRB1*0101 is not (8). In the present study, the frequency of HLA–DRB1*0405 was high in both anti–MDA-5 antibody–positive DM and anti-aaRS antibody–positive PM/DM. In contrast, the frequency of HLA–DRB1*0405 among patients with PM/DM without anti-aaRS antibody or ILD was similar to that in healthy controls. Type 1 diabetes mellitus, Vogt-Koyanagi-Harada disease, and autoimmune hepatitis have also been associated with HLA–DRB1*0405 in the Japanese population (12–14). HLA–DRB1*0405 may contribute to the pathophysiology of several autoimmune diseases.

In addition, this study revealed that the frequency of HLA–DRB1*0101 was higher in patients with anti–MDA-5 antibody–positive DM than in patients with anti-aaRS antibody–positive PM/DM or patients with PM/DM without anti-aaRS antibody or ILD, although the number of enrolled patients was small. Previously, the HLA–DRB1*01 and *04 alleles were shown to play roles in the susceptibility to and progression of RA (15). Specifically, these alleles are associated with anti–citrullinated protein antibody (ACPA)–positive RA. Residues 70–74 of the DRβ chain (QRRAA) in both HLA–DRB1*0101 and DRB1*0405 constitute an important region for antigen presentation. QRRAA may indirectly influence outcome via ACPA production (15). Among PM/DM patients in the Japanese population, HLA–DRB1*0101 or *0405 can also be associated with the production of autoantibodies against MDA-5 or aaRS. QRRAA may be a critical sequence in the pathophysiology of anti–MDA-5 antibody–positive DM and anti-aaRS antibody–positive PM/DM. These antibodies are strongly associated with the development of ILD in PM/DM.

The HLA class II haplotypes are more important than individual alleles. DQB1 and DPB1 should be investigated in all of the patients and healthy donors included in this study. However, DQB1 and DPB1 alleles were not sufficiently investigated in all samples. This was a limitation of the present study. We plan to analyze the HLA class II haplotypes in patients with anti–MDA-5 antibody–positive DM in a future study.

In conclusion, HLA–DRB1*0101/*0405 is associated with susceptibility to anti–MDA-5 antibody–positive DM in the Japanese population. These alleles were also associated with ILD in patients with PM/DM.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Kawaguchi had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Gono, Kawaguchi, Yamanaka.

Acquisition of data. Gono, Kuwana, Furuya, Takagi, Ichida, Katsumata, Hanaoka, Ota.

Analysis and interpretation of data. Gono, Kawaguchi, Sugiura, Katsumata.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

The authors thank Manabu Kawamoto and Mika Fujita for excellent technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
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