Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of necrotizing small vessel vasculitides characterized by ANCA production, mainly against proteinase 3 (PR3) or myeloperoxidase (MPO). AAV is classified as microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA), or eosinophilic granulomatosis with polyangiitis (EGPA) according to the European Medicines Agency (EMA) algorithm (1). In AAV, epidemiological differences between European and Asian populations are well known. In European populations, GPA and PR3-ANCA positive AAV (PR3-AAV) are predominant, whereas in the Japanese population, MPA and MPO-ANCA-positive AAV (MPO-AAV) account for the majority of AAV cases (2). Such ethnic differences in epidemiology imply that the genetic background of AAV may play a role in its development.

Although patients with AAV achieve remission with immunosuppressive therapy (3), a substantial proportion experience relapse. In European populations, relapse occurs in approximately half of the patients with GPA within 5 years after achieving complete remission (4). Among AAV patients, the risk of relapse has been shown to be higher in those with GPA and PR3-AAV compared with MPA and MPO-AAV (5). In Japanese observational studies conducted to identify risk factors for AAV relapse, the dosage and tapering speed of prednisolone were associated with relapse (6, 7).

With respect to susceptibility to MPA and GPA, three genome-wide association studies (GWAS) in European populations have been reported thus far (8–10). In GPA and PR3-AAV, the most striking association was identified in the HLA-DP region, which is consistent with a previously reported association of GPA with HLA-DPB1*04:01 in a German population (11). Additionally, PRTN3 and SERPINA1 genes, encoding PR3 and α1-antitrypsin, respectively, were identified as susceptibility genes (8, 10). With respect to MPA and MPO-AAV, the HLA-DQ region has been associated with susceptibility in the GWAS (8, 10). In agreement with this, the HLA-DRB1*09:01-DQB1*03:03 haplotype was found to be associated with susceptibility to MPA and MPO-AAV in the Japanese population (12–15). In addition, the DQA1*03:02-DQB1*03:03 haplotype was recently reported to be associated with MPO-AAV in a Chinese population (16). The HLA-DRB1*09:01-DQA1*03:02-DQB1*03:03 haplotype is common in general East Asian populations, but rare in European populations. Additionally, DRB1*13:02 has been found to be associated with protection from MPA and MPO-AAV in the Japanese population (14).

Although several genes are associated with AAV susceptibility, those associated with relapse have not been well-characterized. In European populations, HLA-DPB1*04:01, the susceptibility allele for GPA, has been shown to be associated with a higher risk of AAV (17) and PR3-AAV relapse (18). However, similar studies have not been conducted in East Asian populations, in which MPA and MPO-AAV account for the majority of AAV cases.

In this study, we focused on MPO-AAV in the Japanese population, and examined whether HLA-DRB1, DQA1, DQB1 and DPB1 alleles are also associated with relapse in MPO-AAV, based on clinical data from Japanese nationwide cohort studies on remission induction therapy in AAV. Data were obtained from “Remission Induction Therapy in Japanese Patients with ANCA-associated Vasculitides” (RemIT-JAV), registered with the University Hospital Medical Information Network Clinical Trials Registry (UMIN000001648) (19) and “Remission Induction Therapy in Japanese Patients with ANCA-associated Vasculitides and Rapidly Progressive Glomerulonephritis” (RemIT-JAV-RPGN) (UMIN000005136) (20) carried out under the initiatives of Japan Research Committee of the Ministry of Health, Labour, and Welfare for Intractable Vasculitis (JPVAS) and Research Committee of Intractable Renal Disease of the Ministry of Health, Labour, and Welfare of Japan.

In this study, we examined whether HLA class II alleles and HLA amino acids were associated with relapse in MPO-AAV, using data from two Japanese nationwide cohort studies on remission induction therapy: RemIT-JAV (19) and RemIT-JAV-RPGN (20). We detected that MPO-ANCA single positive AAV patients carrying HLA-DRB1*09:01, DQA1*03:02 or DQB1*03:03 had nominally higher risk of relapse as compared with those without these alleles, while AAV patients carrying serine residue at position 13 of HLA-DRβ1 had a trend toward lower risk of relapse. By combining these two factors, significant differences were observed in relapse-free survival among the patient subgroups. To the best of our knowledge, this is the first study to demonstrate the association between gene and amino acid variations and AAV relapse in an East Asian population.

We previously identified an association between HLA-DRB1*09:01 and DQB1*03:03 and susceptibility to MPA/MPO-AAV in a Japanese population (12–15). In the present study, we confirmed the association of DQA1*03:02 with susceptibility to MPO-AAV in a Japanese population, which was recently reported in a Chinese population (16). Owing to the tight linkage disequilibrium among these three HLA class II alleles, we could not determine which of the three alleles played a primary causative role.

Currently, the molecular mechanisms underlying the association between HLA class II alleles and MPA/MPO-AAV remain unclear. Among the components of the risk haplotype DRB1*09:01-DQA1*03:02-DQB1*03:03, the polymorphic amino acids encoded by DRB1*09:01 and DQB1*03:03 directly affect antigenic peptide specificity. Because MPO-ANCA has been strongly implicated in MPO-AAV pathogenesis (25), it is possible that specific antigenic peptides derived from human MPO (26) or microbial peptides mimicking human MPO peptides (27) might be preferentially presented by HLA-DRB1*09:01 and/or DQB1*03:03 products, although to our knowledge such peptides have not been identified from the patients for DRB1*09:01 or DQB1*03:03 products, Conversely, the specific amino acid encoded by DQA1*03:02, Asp160, is located in the α2 domain of HLA-DQ molecule, and may be involved in stabilizing HLA class II homodimers, thereby affecting antigenic peptide specificity (16). Thus, it is possible that multiple molecular mechanisms associated with MHC-peptide complex formation and T cell receptor signaling are involved in the genetic association between the risk haplotype and susceptibility to MPO-AAV.

A previous report showed that HLA-DPB1*04:01, the susceptibility allele for GPA, was also associated with an increased risk of AAV relapse in the Dutch and German patients (17). In this study, GPA and PR3-AAV were predominant. In contrast, this association was not observed in a Danish population (28). Although the reason for this discrepancy is unclear, differences in DPB1*04:01 carrier frequency among patients between these studies (77% (17) and 94% (28), respectively) may play a role. A recent study from the United States reported an association between HLA-DPB1*04:01 and relapse in PR3-AAV, but not in MPO-AAV (18).

With respect to MPO-AAV, a small-scale study in a European population did not detect a significant association between relapse and HLA-A, -B, or -DR antigens (29). In our study, HLA-DRB1*09:01, -DQA1*03:02 and -DQB1*03:03, the susceptibility alleles for MPO-AAV in East Asian populations (12–16) showed a trend toward a higher risk of relapse of MPO-AAV in a Japanese population, possibly suggesting that the susceptibility alleles for AAV may also be associated with relapse in each ethnic group.

Unlike a European study (17), this study did not detect an association between DPB1*04:01 and the risk of AAV relapse. This discrepancy likely occurred because this study focused on MPO-ANCA positive AAV, whereas in the previous European study, 51.7% of the subjects were positive for PR3-ANCA (17). In our MPO-AAV data, no significant association was detected between DPB1*04:01 and relapse ( Supplementary Table S4 , https://doi.org/10.6084/m9.figshare.21876159.v3), which is consistent with a report from the United States (18). This difference may not be explained merely by the low carrier frequency of DPB1*04:01 in the Japanese population, because DPB1*04:01 showed a slight tendency toward decrease in patients with relapse (P=0.70, OR: 0.39). In the Japanese population, the protective allele for MPO-AAV susceptibility, DRB1*13:02, is in linkage disequilibrium with DPB1*04:01 (r^{2 =} 0.44), and the allele frequency of DPB1*04:01 was also decreased in MPO-AAV (P=2.1x10^-4, OR: 0.40) due to linkage disequilibrium with DRB1*13:02 (14). Thus, to evaluate the effect of DPB1*04:01 in the Japanese population, PR3-ANCA positive AAV patients with sufficient sample sizes should be investigated in the future.

In contrast to the HLA-DRB1*09:01-DQA1*03:02-DQB1*03:03 haplotype, alleles carrying HLA-DRβ1_13S were found to show a trend toward protection from relapse. HLA-DRβ1_13S is encoded by DR3, DR11, DR13 and DR14. HLA-DRB1 position 13 amino acid is associated with multiple immune system disorders, and was recently shown to be most strongly associated with individual differences in the T cell receptor complementarity determining region 3 (CDR3) repertoire (30). HRs (95% CI) and log-rank P values for relapse-free survival of alleles encoding HLA-DRβ1_13S with minor allele frequency >0.01 in the Japanese population are shown in Supplementary Table S5 . These alleles showed a trend toward protection from relapse, although the differences did not reach statistical significance.

As mentioned above, we previously reported that DRB1*13:02, encoding HLA-DRβ1_13S, exhibited a protective effect against the MPO-AAV development (14). The data on susceptibility to MPO-AAV with HLA-DRβ1_13S, as well as with each allele encoding 13S, are also presented in Supplementary Table S5 . HLA-DRβ1_13S was protective against the development of MPO-AAV (P_uncorr=0.0029, OR 0.71, 95%CI 0.56-.89). Among the HLA-DRβ1_13S encoding alleles, the protective effect of DRB1*13:02 (P_uncorr=6.9x10^-5, OR 0.45, 95%CI 0.30-0.66) appeared to be more striking as compared with other HLA-DRβ1_13S encoding alleles. On the other hand, in the relapse analysis, HR was not much different between DRB1*13:02 and other DRβ1_13S encoding alleles. This could suggest a possibility that the protective effect against relapse may be shared by HLA-DRβ1_13S alleles, while protection from disease occurrence may be mainly ascribed to DRB1*13:02. Further studies with larger sample sizes are needed to validate this hypothesis.

When the combined effect of DQA1*03:02 and DRβ1_13S was examined, time to relapse was significantly shorter in the highest risk combination (DQA1*03:02 positive and DRβ1_13S negative) when compared with the lowest risk combination (DQA1*03:02 negative and DRβ1_13S positive), even after correction for multiple testing (Q=0.033). The HR was 4.02, which was higher than that for HLA-DPB1*04:01 in Dutch and American studies (17, 18).

In view of our results, it could be hypothesized that susceptibility alleles other than HLA may also be associated with the risk of MPO-AAV relapse. To date, no gene other than HLA has been established as a susceptibility gene for MPO-AAV in Asian populations. In a European GWAS, PTPN22 rs2476601 was reported to be associated with GPA/PR3-AAV and MPA/MPO-AAV (10); however, the risk allele of PTPN22 is almost absent in Asian populations. Recently, the association of a BACH2 variant was identified using target sequencing (31). According to the Genome Aggregation Database (gnomAD) (https://gnomad.broadinstitute.org/) (32), the risk allele in the BACH2 gene is not detected in East Asian populations. In a European population, it was reported that -463 A allele (rs2333227) in the MPO gene, which is associated with lower expression of MPO, was also associated with the risk of relapse in patients with MPO-AAV (33), which needs to be replicated in independent studies.

This study had several limitations. AAV is a rare disease, with an annual incidence of 22.6/million people in Japan (2). Because of the limited sample size and lack of independent replication cohort, it should be emphasized that this study is of an exploratory nature. For example, the attenuation of a trend toward relapse in DRB1*08:02 after conditioning ( Table 5 ) could possibly be caused by insufficient detection power (0.589) due to the lower carrier frequencies of DRB1*08:02 compared to other alleles ( Table 4 ). In fact, the HR of DRB1*08:02 remained >2.0 even after conditioning ( Table 5 ). Further studies are required to draw a definitive conclusions.

Additionally, because this study was based on observational cohort studies conducted between 2009 and 2016 (19, 20), the potential effects of currently available treatments such as rituximab and avacopan could not be addressed. In this study, relapse was defined as the recurrence or new onset of clinical signs and symptoms attributable to active vasculitis, similar to previous studies on Japanese patients (6, 23). Although some previous studies employed strict definition, including a requirement for treatment escalation (17), we did not use this definition, because we thought that whether treatment is reinforced or not partly depends on each physician’s decision, and might possibly cause a bias. Finally, the classification of AAV is based on the EMA algorithm (1), and the compatibility of our findings with the 2022 American College of Rheumatology/European Alliance of Associations for Rheumatology Classification Criteria (34–36) needs to be validated. These limitations should be addressed in future studies.

In the present study, we demonstrated that DRB1*09:01, DQA1*03:02 and DQB1*03:03, susceptibility alleles for MPO-AAV, are also nominally associated with the risk of relapse in MPO-AAV in the Japanese population. In addition, we found that the carriers of HLA-DRβ1 allele with serine residue at position 13 are nominally associated with lower risk of relapse. By combining these two factors, a statistically significant difference in the time to relapse was observed. These findings may be relevant in detecting individuals at high risk for AAV relapse of after remission, and adjusting treatment.

The datasets presented in this study can be found in Figshare: https://doi.org/10.6084/m9.figshare.21876159.v1.

The studies involving human participants were reviewed and approved by The Faculty of Medicine ethics committee, University of Tsukuba, as well as by all the institutes involved in the recruitment of subjects, as listed in the Materials and Methods. The patients/participants provided their written informed consent to participate in this study.

AK and NTs designed the study, interpreted the data and wrote the manuscript. K-ES, KY, HM, YA and MH coordinated the cohorts. AK, PAK performed genotyping and statistical analysis. K-ES, FH. SK, KN, TS, NO, TF, MK, NTa, YK, KI and TS recruited the participants and collected clinical data. All authors contributed to the article and approved the submitted version.