Association of a Single Nucleotide Variant in TERT with Airway Disease in Japanese Rheumatoid Arthritis Patients

Interstitial lung disease and airway disease (AD) are often complicated with rheumatoid arthritis (RA) and have a poor prognosis. Several studies reported genetic associations with interstitial lung disease in RA. However, few genetic studies have examined the susceptibility to AD in RA patients. Here, we investigated whether single nucleotide variants susceptible to idiopathic pulmonary fibrosis might be associated with interstitial lung disease or AD in Japanese RA patients. Genotyping of rs2736100 [C/A] in TERT and rs1278769 [G/A] in ATP11A was conducted in 98 RA patients with usual interstitial pneumonia, 120 with nonspecific interstitial pneumonia (NSIP), 227 with AD, and 422 without chronic lung disease using TaqMan assays. An association with AD in RA was found for rs2736100 (p = 0.0043, Pc = 0.0129, odds ratio [OR] 1.40, 95% confidence interval [CI] 1.11–1.77). ATP11A rs1278769 was significantly associated with NSIP in older RA patients (>65 years, p = 0.0010, OR 2.15, 95% CI 1.35–3.40). This study first reported an association of rs2736100 with AD in RA patients and ATP11A rs1278769 with NSIP in older RA patients.


Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory disease that destroys synovial joints [1].RA is often associated with extra-articular manifestations that include interstitial lung disease, airway disease (AD), vasculitis, pleuritis, pericarditis, and Felty's syndrome [2].Interstitial lung disease is characterized by interstitial inflammation of the lung and is found in about 10% of RA patients [3].It confers poor outcomes in RA patients [4].Interstitial lung disease in RA includes usual interstitial pneumonia (UIP) and nonspecific interstitial pneumonia (NSIP); RA patients with UIP are associated with a poor prognosis [5].AD is also frequently detected in RA [6][7][8][9] and worsens its prognosis [10,11].Thus, it is necessary to clarify the pathogenesis of interstitial lung disease and AD in RA.
The pathogenesis of RA is still unknown, but the susceptibility is thought to be influenced by genetic and environmental factors.In our previous study, the MUC5B single nucleotide variant (SNV) was revealed to be associated with RA-associated interstitial lung disease [12].The MUC5B gene encodes mucin 5B, a secretory mucin expressed in the lung.The risk allele of the SNV upregulates mucin 5B expression and might interfere alveolar repair.In a study by another group, an SNV in RPA3-UMAD1 was also associated with RA-associated interstitial lung disease [13], but this was not confirmed in our replication study [14].Associations of TOLLIP, MUC5B, TERT, and FAM13A SNVs with interstitial lung disease were reported in RA patients from European populations [15].TOLLIP encodes an adaptor protein that regulates the degradation of the TGF-β type 1 receptor.The SNV in FAM13A was confirmed to be associated with interstitial lung disease in our study [16].The FAM13A protein plays some roles in the Wnt signaling pathway involved in the pathogenesis of idiopathic pulmonary fibrosis.Taken together, these studies have demonstrated genetic associations with interstitial lung disease in RA.In contrast, few genetic studies have investigated the susceptibility to AD in RA [7,[17][18][19][20][21].
Associations of TERT or ATP11A with idiopathic pulmonary fibrosis were reported in previous studies [22][23][24].TERT was also associated with interstitial lung disease in RA [15] and combined pulmonary fibrosis and emphysema [25].TERT encodes a component of the telomerase complex that regulates the telomere length and cell survival.ATP11A is expressed in the lung and the methylation of ATP11A correlated with the severity of cystic fibrosis [26].TERT and ATP11A might be candidate causative genes for interstitial lung disease or AD in RA.This study was performed to clarify whether SNVs in TERT and ATP11A are associated with interstitial lung disease or AD in Japanese RA patients.

Patients
RA patients fulfilled the Rheumatoid Arthritis Classification Criteria [27] or American College of Rheumatology Criteria for RA [28] and were native Japanese and living in Japan.RA patients with chest conventional or high-resolution computed tomography (CT) images as clinical information were recruited from 2010 to 2023 at the hospitals of the research group organized by NHO Tokyo National Hospital.Based on the predominant findings of CT images interpreted by two rheumatologists, specialists of lung diseases in RA [21], RA patients were diagnosed with UIP, NSIP, AD, or no chronic lung disease (CLD).Patients with other predominant findings of CT were excluded.
This study was conducted in accordance with the Declaration of Helsinki and was approved by the Tokyo National Hospital Research Ethics Committee (190010) and Research Ethics Committees of all other institutes involved in this study.In this study, written informed consent was obtained from each patient.

Genotyping
Genomic DNA was extracted from peripheral blood of the RA patients.Genotyping of rs2736100 [C/A] in TERT and rs1278769 [G/A] in ATP11A was conducted from the genomic DNA of RA patients with TaqMan assays (Assay ID: C___1844009_10 and C___8701049_10, respectively, Thermo Fisher Scientific Inc., Waltham, MA, USA) using Real-Time PCR System.

Statistical Analysis
BellCurve for Excel (Social Survey Research Information, Tokyo, Japan) was employed for statistical analyses.Clinical manifestations of RA patients were analyzed using Mann-Whitney U test or Fisher's exact test using 2 × 2 contingency tables.Associations of the SNVs were analyzed using Fisher's exact test or Cochran-Armitage test.For the adjustment of multiple comparisons, the Bonferroni method was used: p values were multiplied by the number of tests to generate corrected p values (Pc).p values less than 0.05 were considered to indicate statistical significance.Statistical power of 80% was obtained when the odds ratio (OR) was 1.40 (AD vs. CLD(-)) or higher for rs2736100.It was calculated to be 1.63 (NSIP vs. CLD(-)) for rs1278769 (http://biostat.mc.vanderbilt.edu/wiki/Main/PowerSampleSize,accessed on 19 April 2022) [30].

Results
Association of TERT rs2736100 with AD in RA and ATP11A rs1278769 with NSIP in RA A total of 98 RA patients with UIP, 120 with NSIP, 227 with AD, and 422 without CLD were recruited to the study (Table 1).TERT rs2736100 and ATP11A rs1278769 were genotyped in the RA patients (Table 2).A deviation from the Hardy-Weinberg equilibrium was not observed for TERT rs2736100 (p = 0.3019), but it was observed in ATP11A rs1278769 (p = 0.0110).We investigated whether TERT rs2736100 was associated with AD, UIP, or NSIP in Japanese RA patients (Table 2, Supplementary Table S1).TERT rs2736100 was associated with AD under the allele (p = 0.0043, Pc = 0.0129, OR 1.40, 95% confidence interval [CI] 1.11-1.77),recessive (p = 0.0074, Pc = 0.0222, OR 1.84, 95% CI 1.18-2.86),and codominant models (p = 0.0042, Pc = 0.0127).No association with UIP or NSIP in RA was detected for TERT rs2736100.No association with UIP or AD in RA was detected for ATP11A rs1278769.To exclude the effects of age differences between the AD and CLD(-) groups, the association of TERT rs2736100 was investigated in older RA patients (>65 years, Table 3).TERT rs2736100 was still significantly associated with AD in older RA patients (p = 0.0438, OR 1.40, 95% CI 1.02-1.92).Analogically, the association of ATP11A rs1278769 was also investigated in older RA patients because age differences were detected between the NSIP and CLD(-) groups.An association of ATP11A rs1278769 with NSIP in older RA patients was found (p = 0.0010, OR 2.15, 95% CI 1.35-3.40).Thus, the association of TERT rs2736100 with AD was found in Japanese RA patients and ATP11A rs1278769 was significantly associated with NSIP in older RA patients.We also investigated whether TERT rs2736100 or ATP11A rs1278769 were associated with RA per se (Table 4).However, these variants were not associated with RA.

Discussion
We detected that TERT rs2736100C is predisposing to AD in RA patients in Japan.An association of TERT rs2736100 with interstitial lung disease was known in European RA patients [15], but this is the first study, to the best of our knowledge, to reveal an association of the SNV with AD.ATP11A rs1278769 was also found to be associated with NSIP in older RA patients.ATP11A was previously found to be associated with idiopathic pulmonary fibrosis [22,23], but the association of this SNV with NSIP in RA has not been reported.
TERT rs2736100A was reported to be a risk allele for idiopathic pulmonary fibrosis [22][23][24] and interstitial lung disease in European RA patients [15].TERT rs7726159 was previously investigated in Mexican and European populations [12], but it was not associated with interstitial lung disease in RA.This allele was associated with lower levels of TERT gene expression [31] and with a shorter leukocyte telomere length [32], which contributed to the development of idiopathic pulmonary fibrosis [33].However, in this study, TERT rs2736100C, a protective allele for idiopathic pulmonary fibrosis, was found to be a risk allele for AD in RA.These results suggested that a high TERT gene expression and long leukocyte telomere length might contribute to the development of AD in RA, although dysfunction of the telomere was involved in the development of AD [34].TERT rs2736100C was also associated with combined pulmonary fibrosis and emphysema [25].TERT rs7726159A was a risk allele for systemic lupus erythematosus in Asian populations [35] and was in moderate linkage disequilibrium with rs2736100C in Japanese populations (D = 0.932, r2 = 0.768, http://www.ensembl.org/,accessed on 16 April 2022).Moreover, TERT rs2736100A was a risk allele for microscopic polyangiitis [36].These data suggest that SNVs in TERT might be involved in the development of autoimmune features in a protective or predisposing manner.
It was reported that ATP11A was associated with idiopathic pulmonary fibrosis [22,23].ATP11A rs1278769 was previously tested in Mexican and European and populations [12], but it was not associated with interstitial lung disease in RA.The ATP11A gene was reported to be expressed in the lung and its methylation correlated to the severity of cystic fibrosis [26].In the present study, ATP11A rs1278769G was revealed to be associated with NSIP in older RA patients.The Genotype-Tissue Expression (GTEx) database showed an association of rs1278769G with the down-regulation of ATP11A in the lung (p = 5.0 × 10 −11 , Supplementary Figure S1, https://gtexportal.org/home/snp/rs1278769,accessed on 17 April 2022) [37].Low expression levels of ATP11A were associated with the severity of Coronavirus Disease 2019 [38].In our study, ATP11A rs1278769 was associated with NSIP in older RA patients, but it was not associated with UIP in RA patients, suggesting the heterogeneity of the pathogenesis of RA chronic lung disease.
To the best of our knowledge, the present study is the first report on an association of TERT rs2736100 with AD in RA and ATP11A rs1278769 with NSIP in older RA patients.This study included several limitations: the sample size was modest, and it was based on the results of Japanese populations.Multi-ethnic and larger-scale studies should be conducted to evaluate the etiology of AD or NSIP in RA.

Conclusions
In conclusion, this study showed associations of TERT rs2736100 with AD in RA and ATP11A rs1278769 with NSIP in older RA patients, suggesting the heterogeneous pathogenesis of chronic lung disease in RA patients.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/genes14112084/s1, Figure S1: An association of rs1278769G with low expression levels of the ATP11A gene in the lung; Table S1: Associations of rs2736100 TERT and rs1278769 ATP11A with the RA subsets.

Funding:
The study was supported by grants-in-aid for clinical research from the National Hospital Organization, research grants from the Mitsui Sumitomo Insurance Welfare Foundation, research grants from The Nakatomi Foundation, grants-in-aid from the Japan Agency for Medical Research and Development, grants-in-aid for scientific research (18K08402, 15K09543, 22591090, 26293123) from the Japan Society for the Promotion of Science, a grant from Bristol-Myers Squibb Co., research grants from Daiwa Securities Health Foundation, research grants from the Japan Research Foundation for Clinical Pharmacology, research grants from the Ministry of Health, Labour, and Welfare of Japan, research grants from the Takeda Science Foundation, and research grants from Abbott Japan Co., Ltd., Mitsubishi Tanabe Pharma Corp., Merck Sharp and Dohme Inc., Pfizer Japan Inc., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd., Chugai Pharmaceutical Co., Ltd., Astellas Pharma Inc., Eisai Co., Ltd., and Bristol-Myers K.K.

Institutional Review Board Statement:
This study was conducted in accordance with the Declaration of Helsinki and was approved by the Tokyo National Hospital Research Ethics Committee (190010) and Research Ethics Committees of all other institutes involved in this study.

Informed Consent Statement:
In this study, written informed consent was obtained from each patient.Data Availability Statement: Data supporting the findings of this study will be provided upon reasonable request to the authors.However, the clinical information and genotype data of each participant are not available based on the conditions of informed consent and the Act on the Protection of Personal Information.

Table 1 .
Characteristics of the RA patients.

Table 2 .
Genotype frequencies of TERT rs2736100 and ATP11A rs1278769 in the RA patients.: rheumatoid arthritis; UIP: usual interstitial pneumonia; NSIP: nonspecific interstitial pneumonia; AD: airway disease; CLD: chronic lung disease; OR: odds ratio; CI: confidence interval.Genotype and allele frequencies are shown in parentheses (%).Associations were tested using Fisher's exact test using 2 × 2 contingency tables under the allele model. RA

Table 3 .
Genotype frequencies of TERT rs2736100 and ATP11A rs1278769 in RA patients older than 65 years.
RA: rheumatoid arthritis; CLD: chronic lung disease; NSIP: nonspecific interstitial pneumonia; OR: odds ratio; CI: confidence interval; AD: airway disease.Allele and genotype frequencies are shown in parentheses (%).Associations were tested in comparison with the CLD(-) RA older than 65 years using Fisher's exact test.

Table 4 .
Allele frequencies of TERT rs2736100 and ATP11A rs1278769 in the RA patients and controls.
Interest: Shigeto Tohma received honoraria from AbbVie GK., Chugai Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co. Asahi Kasei Pharma Corp., Astellas Pharma Inc., and Pfizer Japan Inc. Hiroshi Furukawa received honoraria from Ayumi Pharmaceutical Corp., Pfizer Japan Inc., Luminex Japan Corp. Ltd., Ajinomoto Co. Inc.Dainippon Sumitomo Pharma Co., Ltd., Daiichi Sankyo Co., Ltd., and Takeda Pharmaceutical Co. Shigeto Tohma was supported by research grants from Abbott Japan Co., Ltd., Takeda Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corp., Merck Sharp and Dohme Inc., Eisai Co., Ltd., Chugai Pharmaceutical Co., Ltd., Pfizer Japan Inc., Teijin Pharma Ltd., and Astellas Pharma Inc. Hiroshi Furukawa was supported by research grants from Japan Research Foundation for Clinical Pharmacology supported by Daiichi Sankyo, Daiwa Securities Health Foundation established by Daiwa Securities Group Inc., Takeda Science Foundation run by Takeda Pharmaceutical Co., Mitsui Sumitomo Insurance Welfare Foundation established by Mitsui Sumitomo Insurance Co., Ltd., Bristol-Myers-Squibb Co., and the Nakatomi Foundation established by Hisamitsu Pharmaceutical Co. Inc.The other authors have no conflict of interest.