Abstract
Background:
Rheumatoid arthritis (RA) is occasionally complicated with interstitial lung disease (ILD). A recent genome-wide association study of ILD in RA reported an association with the polymorphism rs12702634 in RPA3-UMAD1. We conducted an association study of this variant with ILD in Japanese RA patients to replicate this association.
Methods:
Genotyping of rs12702634 was performed in 175 RA with ILD and 411 RA without chronic lung disease.
Results:
No association was detected for rs12702634 with ILD in RA (P = .6369, odds ratio [OR] 1.13, 95% confidence interval [CI] 0.72-1.78). Meta-analysis of these data combined with the data from the recent report showed no significant association (P = .0996, OR 1.52, 95% CI 0.92–2.49).
Conclusions:
The present study demonstrated no association of RPA3-UMAD1 rs12702634 with ILD in RA, suggesting the heterogeneity of the disease.
Keywords: Rheumatoid arthritis, interstitial lung diseases, polymorphism, genetic association, extraarticular manifestation
Introduction
Rheumatoid arthritis (RA) is characterized by the distraction of the synovial joints and is occasionally complicated with the development of interstitial lung disease (ILD). ILD is detected in about 10% of RA cases 1 and precedes RA diagnosis in about 10% of RA cases with ILD. 2 The prognosis of RA patients with ILD is quite poor. 3 Although the etiology of RA is vague, it is thought that the disease susceptibility of RA is associated with genetic factors. Many genetic factors for RA or idiopathic interstitial pneumonia were reported; a few genetic analyses had been conducted for ILD in RA. A recent genome-wide association study (GWAS) of ILD in RA in a Japanese population identified a significant association with a single nucleotide polymorphism (SNP), rs12702634, in the RPA3-UMAD1 gene. 4 An association of this SNP with ILD in Japanese RA patients was analyzed in the present study to replicate this association.
Materials and Methods
Patients
Japanese RA patients with available chest computed tomography images were recruited at outpatient departments or hospital wards of the rheumatology centers for this case control study; all the RA patents fulfilled American College of Rheumatology criteria for RA 5 or Rheumatoid Arthritis Classification Criteria. 6 Usual interstitial pneumonia (UIP), nonspecific interstitial pneumonia, (NSIP), or no chronic lung diseases (CLDs) complications in RA patients were diagnosed using chest conventional or high-resolution computed tomography images. 7 RA patients with other computed tomography findings, bronchiolitic airway disease, bronchiectatic airway disease, cryptogenic organizing pneumonia, mosaic perfusion, pleural effusion, pneumonia, or cancer were excluded.
175 RA with ILD (76 UIP and 99 NSIP) and 411 RA without CLD were enrolled in the present study. This study was reviewed and approved by Yokohama Minami Kyosai Hospital Research Ethics Committee (22-6-2), Tokyo National Hospital Ethics Committee (190010), Tama Medical Center Research Ethics Committee (H23-30), Sagamihara National Hospital Research Ethics Committee (2009061621), Niigata Rheumatic Center Research Ethics Committee (2017-018), Nagoya Medical Center Research Ethics Committee (2012-526), Nagasaki Medical Center Research Ethics Committee (22081), Miyakonojo Medical Center Research Ethics Committee, Kumamoto Center for Arthritis and Rheumatology Research Ethics Committee, Hyogo College of Medicine Research Ethics Committee (178), and all the institutes involved in the recruitment of the subjects. Written informed consent was obtained from all subjects. This study was conducted in accordance with the principles expressed in the Declaration of Helsinki.
Genotyping
Genotyping of rs12702634 [C/G] in RPA3-UMAD1 was conducted with TaqMan genotyping assay (Assay ID: C__31572862_10, Thermo Fisher Scientific Inc., Waltham, MA) and 7500 Fast Real-Time PCR System (Thermo Fisher Scientific Inc.). Thermal cycling conditions comprised denaturation at 95°C for 20 seconds, followed by 40 cycles at 95°C for 3 seconds then at 60°C for 30 seconds.
Statistical analysis
The distribution of allele frequencies was compared between RA patients with ILD and those without CLD by Fisher’s exact test using 2 × 2 contingency tables under the allele model. The 80% statistical power was estimated to be provided when the odds ratio (OR) was 1.84 or higher under the allele model (http://biostat.mc.vanderbilt.edu/wiki/Main/PowerSampleSize) 8 ; this threshold was lower than the previously reported OR. 4 Meta-analysis was conducted using EZR (http://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmed.html) 9 with the DerSimonian-Laird method under the random effects model. 10
Results
Demographic features of the RA patients
Demographic features of RA with ILD, UIP, or NSIP were compared with RA without CLD (Table 1). Age at onset, male percentage, and mean age at enrollment in RA patients with ILD, UIP, or NSIP were higher compared with RA patients without CLD. Rheumatoid factor was increased in RA with ILD or UIP. Surfactant protein-D and Krebs von den lungen-6 in RA with ILD, UIP, or NSIP were also increased. No difference was detected in anti-citrullinated peptide antibody, smoking status, and Steinbrocker stage. 11
Table 1.
Characteristics of RA patients.
| ILD | UIP | NSIP | CLD(-) | ||||
|---|---|---|---|---|---|---|---|
| Number | 175 | 76 | 99 | 411 | |||
| Mean age, years (SD) | 68.8 (10.5) | 1.61 × 10−12 | 69.7 (10.2) | 1.48 × 10−8 | 68.1 (10.7) | 1.34 × 10−7 | 61.2 (12.7) |
| Male, n (%) | 67 (38.3) | *4.74 × 10−9 | 34 (44.7) | *6.84 × 10−8 | 33 (33.3) | *0.0002 | 63 (15.4) |
| Age at onset, years (SD) | 58.7 (13.6) | 0.0002 | 63.0 (14.0) | 0.0026 | 56.9 (13.1) | 0.0083 | 50.2 (14.5) |
| Steinbrocker stage III and IV, n (%) | 21 (39.6) | *0.8706 | 6 (42.9) | *0.7772 | 15 (38.5) | *1.0000 | 59 (37.8) |
| Smoker or past smoker, n (%) | 24 (58.5) | *0.1544 | 3 (42.9) | *1.0000 | 21 (61.8) | *0.0882 | 60 (45.1) |
| RF, IU/ml (SD) | 489.8 (1141.4) | 0.0347 | 442.2 (489.3) | 0.0146 | 527.5 (1469.1) | 0.1445 | 233.7 (661.6) |
| ACPA, IU/ml (SD) | 248.4 (275.9) | 0.8009 | 268.8 (289.5) | 0.8487 | 234.0 (267.6) | 0.5916 | 258.5 (433.7) |
| KL-6, U/ml (SD) | 1112.7 (1395.8) | 3.90 × 10−7 | 1268.3 (1563.4) | 0.0003 | 985.0 (1241.4) | 0.0004 | 353.1 (324.1) |
| SP-D, ng/ml (SD) | 139.1 (122.4) | 2.19 × 10−5 | 148.7 (125.8) | 0.0024 | 131.4 (121.6) | 0.0029 | 49.3 (46.5) |
Abbreviations: RA, rheumatoid arthritis; ILD, interstitial lung disease; UIP, usual interstitial pneumonia; NSIP, nonspecific interstitial pneumonia; CLD, chronic lung disease; RF, rheumatoid factor; ACPA, anti-citrullinated peptide antibody; KL-6, Krebs von den lungen-6, SP-D: surfactant protein-D. Number or average value of each group are shown. Standard deviations or percentages are shown in parentheses. Difference was tested in the comparison with the CLD(-) population by Fisher’s exact test using 2 × 2 contingency tables or Student’s t-test. *Fisher’s exact test was employed.
Lack of association of rs12702634 with ILD in RA patients
Genotyping of rs12702634 was conducted; deviation from Hardy-Weinberg equilibrium was not detected in RA with ILD (P = .7829) or RA without CLD (P = .3243). There was no significant association of rs12702634 with ILD in RA (P = .6369, OR 1.13, 95% confidence interval [CI] 0.72-1.78, Table 2). There was no significant association of rs12702634 with UIP or NSIP.
Table 2.
Allele frequencies of RPA3-UMAD1 rs12702634 in the RA patients with ILD or without CLD.
| n | Genotype | [C/G] | [G/G] | Allele | P | OR (95%CI) | |
|---|---|---|---|---|---|---|---|
| [C/C] | [C] | ||||||
| ILD(+)RA, n (%) | 175 | 1 (0.6) | 28 (16.0) | 146 (83.4) | 30 (8.6) | 0.6369 | 1.13 (0.72-1.78) |
| UIP(+)RA, n (%) | 76 | 1 (1.3) | 13 (17.1) | 62 (81.6) | 15 (9.9) | 0.3332 | 1.32 (0.73-2.38) |
| NSIP(+)RA, n (%) | 99 | 0 (0.0) | 15 (15.2) | 84 (84.8) | 15 (7.6) | 1.0000 | 0.99 (0.55-1.77) |
| CLD(-)RA, n (%) | 411 | 1 (0.2) | 61 (14.8) | 349 (84.9) | 63 (7.7) |
Abbreviations: RA, rheumatoid arthritis; ILD, interstitial lung disease; UIP, usual interstitial pneumonia; NSIP, nonspecific interstitial pneumonia; CLD, chronic lung disease; OR, odds ratio; CI, confidence interval. Genotype and allele frequencies are shown in parentheses (%). Association was tested by Fisher’s exact test using 2 × 2 contingency tables under the allele model.
Meta-analysis of our data combined with the data from the previous report 4 was performed under the random effects model and did not reach the genome-wide significance threshold (Supplemental Figure 1; P = .0996, OR 1.52, 95% CI 0.92-2.49; the weight of this study in the random effects model was 42.2%). The lack of heterogeneity was not confirmed between our data and the previously reported data on rs12702634 (I2 = 74.6%, τ2 = 0.0980, H = 1.99, Q = 3.94, P = .0470); these data supported the analysis under the random effects model.
Discussion
Although the association of the MUC5B promoter variant rs35705950 with ILD was reported in RA, the allele frequency of the susceptible variant was low (0.2%) in Japanese populations 12 , suggesting that this variant could not explain the predominant pathogenesis of ILD in Japanese RA patients. The disease-susceptible genes are occasionally different between different ethnic populations; PTPN22, a RA-susceptible gene in European populations, is not the susceptible gene for RA in Japanese populations. 13
It was suspected that other genetic factors would be associated with ILD in Japanese RA. Hence, a genome-wide association study of ILD in RA was conducted, 4 and the association of rs12702634 in RPA3-UMAD1 was reported. However, the results from the previous study were not reproduced in the present study and the results of meta-analysis did not indicate the association, suggesting the heterogeneity of the disease. The pathogenesis of ILD in RA would be heterogeneous, since male is dominant in RA with UIP but not in RA with NSIP. 7 Thus, different results of genetic analyses between different populations in the same ethnic group may provide the explanation for the heterogeneity of ILD in RA.
To the best of our knowledge, this is the first replication study on the association of rs12702634 with ILD in RA, but it failed to confirm the association. This study has some limitations. This study revealed a strong heterogeneity of ILD in RA patients, although our sample size was modest. Moreover, multi-ethnic studies with other populations should be conducted, since this study was performed only in Japanese. The genetic analyses should be continued in future larger scale studies to reveal the true etiology of ILD in RA.
Conclusions
The present study demonstrated no association of RPA3-UMAD1 rs12702634 with ILD in RA. The results of meta-analysis could not confirm the association, suggesting the heterogeneity of the disease.
Supplemental Material
Supplemental material, sj-JPG-1-bmi-10.1177_11772719221091758 for Lack of Association of rs12702634 in RPA3-UMAD1 With Interstitial Lung Diseases in Japanese Rheumatoid Arthritis Patients by Takashi Higuchi, Shomi Oka, Hiroshi Furukawa, Kota Shimada and Shigeto Tohma in Biomarker Insights
Acknowledgments
The RA-ILD Study Consortium includes Shouhei Nagaoka and Akiko Suda (Yokohama Minami Kyosai Hospital), Shigeru Ohno (Yokohama City University Medical Center), Shoji Sugii (Tokyo Metropolitan Tama Medical Center, Tokyo Metropolitan Matsuzawa Hospital), Keigo Setoguchi (Tokyo Metropolitan Komagome Hospital), Satoshi Shinohara (Tochigi Rheumatology Clinic), Kazuhiro Hatta (Tenri Hospital), Hajime Kono (Teikyo University), Satoshi Ito (Niigata Rheumatic Center), Yojiro Kawabe (NHO Ureshino Medical Center), Mitsuru Motegi (NHO Takasaki General Medical Center), Makoto Sueishi (NHO Shimoshizu National Hospital), Kota Shimada (NHO Sagamihara National Hospital, Tokyo Metropolitan Tama Medical Center), Shigeto Tohma and Hiroshi Furukawa (NHO Sagamihara National Hospital, NHO Tokyo National Hospital), Toshihiro Matsui, Yuko Okazaki, Tatsuoh Ikenaka, Atsushi Hashimoto, Akiko Komiya, and Naoshi Fukui (NHO Sagamihara National Hospital), Masao Katayama (NHO Nagoya Medical Center), Kiyoshi Migita (NHO Nagasaki Medical Center, Fukushima Medical University), Noriyuki Chiba (NHO Morioka Hospital), Norie Yoshikawa and Koichiro Saisho (NHO Miyakonojo Medical Center), Eiichi Suematsu (NHO Kyushu Medical Center), Shunsuke Mori (NHO Kumamoto Saishunso Medical Center), Kenji Ichikawa (NHO Hokkaido Medical Center), Akira Okamoto (NHO Himeji Medical Center), Hayato Utsunomiya and Yasuo Suenaga (NHO Beppu Medical Center), Kunio Matsuta (Matsuta Clinic), Yasuhiko Yoshinaga (Kurashiki Medical Center), Hirokazu Takaoka (Kumamoto Shinto General Hospital), Tadashi Nakamura (Kumamoto Center for Arthritis and Rheumatology, Kumamoto Shinto General Hospital, Sakurajyuji Hospital), Tatsuo Nagai, Shunsei Hirohata, and Yoshiyuki Arinuma (Kitasato University), Kiminori Hasegawa (Kin-ikyo Chuo Hospital, Sapporo Yamanoue Hospital), Takeo Sato (Japanese Red Cross Kitami Hospital, Jichi Medical University), Hajime Sano (Hyogo College of Medicine, Kyoto Okamoto Memorial Hospital), Shinichiro Tsunoda (Hyogo College of Medicine, Sumitomo Hospital), and Norihiko Watanabe (Chibaken Saiseikai Narashino Hospital).
Footnotes
Funding: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The work was supported by research grants from the following pharmaceutical companies: Teijin Pharma Limited, Takeda Pharmaceutical Company Limited, Pfizer Japan Inc., Merck Sharp and Dohme Inc., Mitsubishi Tanabe Pharma Corporation, Eisai Co. Ltd., Chugai Pharmaceutical Co., Ltd., Astellas Pharma Inc., Abbott Japan Co. Ltd, and Bristol-Myers K.K.
Additional Grants: RA Clinical Investigation Grant from Bristol-Myers Squibb Co., Research Grants from Mitsui Sumitomo Insurance Welfare Foundation, Research Grants from Takeda Science Foundation, Research Grants from The Nakatomi Foundation, Research Grants from Daiwa Securities Health Foundation, Research Grants from Japan Research Foundation for Clinical Pharmacology, Grants-in-Aid for Clinical Research from National Hospital Organization, Grants-in-Aid of the Practical Research Project for Allergic Diseases and Immunology (Research on Allergic Diseases and Immunology) from Japan Agency for Medical Research and Development, Health and Labour Science Research Grants from the Ministry of Health, Labour, and Welfare of Japan, and Grants-in-Aid for Scientific Research (B, C) (26293123, 22591090, 15K09543, 18K08402) from the Japan Society for the Promotion of Science.
Declaration of conflicting interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: ST received honoraria from Pfizer Japan Inc., Ono Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Chugai Pharmaceutical Co., Ltd., Astellas Pharma Inc., AbbVie GK., and Asahi Kasei Pharma Corporation. ST was supported by research grants from Teijin Pharma Limited, Takeda Pharmaceutical Company Limited, Pfizer Japan Inc., Mitsubishi Tanabe Pharma Corporation, Merck Sharp and Dohme Inc., Eisai Co., Ltd., Chugai Pharmaceutical Co., Ltd., Astellas Pharma Inc., and Abbott Japan Co., Ltd.
HF received honoraria from Takeda Pharmaceutical Company, Pfizer Japan Inc., Luminex Japan Corporation Ltd., Dainippon Sumitomo Pharma Co., Ltd., Daiichi Sankyo Co., Ltd., Ayumi Pharmaceutical Corporation, and Ajinomoto Co., Inc. HF was supported by research grants from Bristol-Myers-Squibb Co., Mitsui Sumitomo Insurance Welfare Foundation established by Mitsui Sumitomo Insurance Co., Ltd., Daiwa Securities Health Foundation established by Daiwa Securities Group Inc., Nakatomi Foundation established by Hisamitsu Pharmaceutical Co., Inc., Takeda Science Foundation supported by Takeda Pharmaceutical Company, and Japan Research Foundation for Clinical Pharmacology run by Daiichi Sankyo.
Authors’ Contributions: HF, KS, and ST designed the study. TH, SO, and HF conducted the experiments. TH and HF analyzed the data. HF, KS, and ST contributed to the collection of clinical information and materials. TH, HF, and ST wrote the manuscript.
Availability of Data and Material: All data are presented in the paper.
Ethics Approval and Consent to Participate: This study was reviewed and approved by Yokohama Minami Kyosai Hospital Research Ethics Committee, Tokyo National Hospital Ethics Committee, Tama Medical Center Research Ethics Committee, Sagamihara National Hospital Research Ethics Committee, Niigata Rheumatic Center Research Ethics Committee, Nagoya Medical Center Research Ethics Committee, Nagasaki Medical Center Research Ethics Committee, Miyakonojo Medical Center Research Ethics Committee, Kumamoto Center for Arthritis and Rheumatology Research Ethics Committee, Hyogo College of Medicine Research Ethics Committee, and all the institutes involved in the recruitment of the subjects. Written informed consent was obtained from all subjects. This study was conducted in accordance with the principles expressed in the Declaration of Helsinki.
ORCID iD: Hiroshi Furukawa 
https://orcid.org/0000-0003-1353-8056
Supplemental Material: Supplemental material for this article is available online.
References
- 1. Furukawa H, Oka S, Shimada K, et al. Association of human leukocyte antigen with interstitial lung disease in rheumatoid arthritis: a protective role for shared epitope. PLoS ONE. 2012;7:e33133. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Nurmi HM, Purokivi MK, Kärkkäinen MS, Kettunen HP, Selander TA, Kaarteenaho RL. Variable course of disease of rheumatoid arthritis-associated usual interstitial pneumonia compared to other subtypes. BMC Pulm Med. 2016;16:107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Koduri G, Norton S, Young A, et al. Interstitial lung disease has a poor prognosis in rheumatoid arthritis: results from an inception cohort. Rheumatology. 2010;49:1483-9. [DOI] [PubMed] [Google Scholar]
- 4. Shirai Y, Honda S, Ikari K, et al. Association of the RPA3-UMAD1 locus with interstitial lung diseases complicated with rheumatoid arthritis in Japanese. Ann Rheum Dis. 2020;79:1305-1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Arnett FC, Edworthy SM, Bloch DA, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1988;31:315-324. [DOI] [PubMed] [Google Scholar]
- 6. Aletaha D, Neogi T, Silman AJ, et al. 2010 Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum. 2010;62(9):2569-81. [DOI] [PubMed] [Google Scholar]
- 7. Oka S, Furukawa H, Shimada K, et al. Association of human leukocyte antigen alleles with chronic lung diseases in rheumatoid arthritis. Rheumatology (Oxford). 2016;55:1301-1307. [DOI] [PubMed] [Google Scholar]
- 8. Dupont WD, Plummer WD., Jr. Power and sample size calculations. A review and computer program. Control Clin Trials. Apr 1990;11:116-28. [DOI] [PubMed] [Google Scholar]
- 9. Kanda Y. Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplant. 2013;48(3):452-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin Trials. 1986;7:177-88. [DOI] [PubMed] [Google Scholar]
- 11. Steinbrocker O, Traeger CH, Batterman RC. Therapeutic criteria in rheumatoid arthritis. J Am Med Assoc. 25 1949;140:659-662. [DOI] [PubMed] [Google Scholar]
- 12. Juge PA, Lee JS, Ebstein E, et al. MUC5B Promoter Variant and Rheumatoid Arthritis with Interstitial Lung Disease. N Engl J Med. 2018;379:2209-2219. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Okada Y, Terao C, Ikari K, et al. Meta-analysis identifies nine new loci associated with rheumatoid arthritis in the Japanese population. Nat Genet. 2012;44:511-516. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplemental material, sj-JPG-1-bmi-10.1177_11772719221091758 for Lack of Association of rs12702634 in RPA3-UMAD1 With Interstitial Lung Diseases in Japanese Rheumatoid Arthritis Patients by Takashi Higuchi, Shomi Oka, Hiroshi Furukawa, Kota Shimada and Shigeto Tohma in Biomarker Insights