Antineutrophil Cytoplasmic Antibody-Associated Vasculitis Update: Genetic Pathogenesis

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is characterized by the inflammation of small and medium vessels and presence of proteinase 3-ANCA or myeloperoxidase-ANCA in the circulation. AAV comprises three clinical subtypes: granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic GPA (EGPA). Although the pathogenesis of AAV is still unclear, genetic and environmental factors and the immune system are thought to be involved. Genetic factors have been confirmed to play an important role in AAV. Genome-wide association studies have identified numerous genetic variants in MHC and non-MHC regions associated with AAV. The strongest evidence of MHC association in AAV is human leukocyte antigen (HLA)-DP. A significant association between AAV and genetic variations in non-MHC regions, such as CTLA-4, FCGR2A, PTPN22, SERPINA1, and TLR9 has also been found. Moreover, different clinical subtypes of AAV have distinct genetic backgrounds. GPA is associated with HLA-DP1, MPA with HLA-DQ, and EGPA with HLA-DRB4. These findings could help elucidate the etiology of AAV and develop new biomarkers for diagnosis and targeted therapy. Herein, we briefly summarize the updates on the genetic pathogenesis and biomarkers of AAV.


INTRODUCTION
Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a complex systemic autoimmune disease presenting with the inflammation of small and medium vessels that results in vascular destruction and tissue necrosis (1,2). AAV is divided into three groups according to clinical features: granulomatosis with polyangiitis (GPA, formerly called Wegener's granulomatosis), microscopic polyangiitis (MPA), and eosinophilic GPA (EGPA, formerly called Churg-Strauss syndrome) (3). The disease is characterized by the presence of proteinase 3 (PR3)-ANCA or myeloperoxidase (MPO)-ANCA in the serum. GPA is predominantly associated with PR3-ANCA, while MPA and EGPA are predominantly associated with MPO-ANCA, but are also occasionally ANCA-negative (4). EGPA is often divided into two subtypes, MPO-ANCA+ EGPA and ANCA-EGPA. Vasculitis can occur in any organ or tissue, commonly affecting the respiratory tract and kidneys, causing life-threatening kidney failure or pulmonary hemorrhage (3).
AAV has an estimated prevalence of 48-184/1,000,000 individuals worldwide, while the incidence and prevalence in Europe are higher than those in other regions (2). The annual incidence in Europe is 4.9-10.6/1,000,000 individuals for GPA, 2.7-11.6/1,000,000 individuals for MPA, and 0.5-3.1/1,000,000 individuals for EGPA. The epidemiological manifestations of AAV differ among geographical regions; GPA and PR3-ANCA AAV are more common in Europeans, while MPA and MPO-ANCA AAV are more common in Asians (5).
Although the pathogenesis of AAV remains elusive, it is believed that both genetic and environmental components are involved. Environmental factors, including silica exposure, bacterial or viral infections, and drugs suggest an association with the occurrence and relapse of AAV (6). Familial studies and genetic association studies have demonstrated that AAV has a background of genetic susceptibility (7)(8)(9). It has been estimated that 20% of AAV risk is due to genetic factors (10).
In this review, we mainly discuss the genetic studies on AAV, focusing on the identified susceptibility genes or loci to enrich our understanding of the disease.

GENETIC APPROACHES FOR AAV
Different genetic approaches have revealed that a large number of genes are related to AAV. First, several candidate gene association studies have been used to identify susceptibility genes associated with AAV. Although these approaches are easy to carry out, they are prone to false-positive results. Human leukocyte antigen (HLA), PTPN22, CTLA-4, IL-10, and TLR9 have been found to be associated with AAV (11,12). The emergence of genome-wide association studies (GWAS) has led to their wide use in exploring the genetic factors involved in various diseases. GWAS is a powerful approach to identify the genetic architecture of complex diseases, and it has made major contributions towards a better understanding of AAV genetics. Four GWAS have been performed in patients with AAV since 2012 (two for MPO and GPA (13,14), one for GPA (15), and one for EGPA (16)). The latest GWAS was on EGPA in 676 cases and 6809 controls with the identification of 11 loci associated with EGPA. The study also revealed that EGPA comprises genetically distinct subgroups, that MPO-ANCA+ subgroup was strongly associated with HLA-DQ and MPO-ANCA-subgroup was associated with non-HLA regions, such as GPA33 and IL5/ IRF1 (16). All these GWAS studies were conducted in populations with European ancestry and have identified more than 20 genes associated with AAV. The findings of GWAS are listed in Tables 1 and 2. In addition, meta-analysis and fine mapping have also been used to identify new gene variations and analyze the effect of the variants (17)(18)(19)(20)(21).

HLA Region
HLA HLA, located on chromosome 6p21, is the most gene-dense region of the human genome and contains diverse genes involved in key immune responses (22). Molecules encoded by HLA participate in a variety of immune and inflammatory pathways. HLA singlenucleotide polymorphisms (SNPs) are associated with numerous human diseases, especially autoimmune diseases, such as type 1 diabetes, rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE) (23). Since AAV is considered an autoimmune disease, HLA may also be a potential predisposing factor for AAV. Candidate gene studies in HLA regions were performed in different populations, including the Swedish, Germans, and Italians in Europe and Japanese and Chinese in Asia. HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, and HLA-DRB1 were found to be involved in AAV susceptibility.
(1). HLA and AAV Two GWAS were performed to identify the genetic factors associated with AAV. The first GWAS in a European population in 2012 demonstrated that HLA-DP rs3117242 (G) was the strongest signal in the HLA region (P = 1.5×10 −71 , OR = 3.67) (13). Another GWAS in Canadian and American populations conducted in 2017 showed that SNPs rs141530233 and rs1042169 at HLA-DPB1 had the largest effect on the risk of developing AAV (P = 1.13×10 −89 , OR = 2.99; P = 1.12×10 -84 , OR = 2.82, respectively). HLA-DPA1, HLA-DQA1, and HLA-DQB1 are also risk alleles that may cause AAV (14). These studies not only indicated a highly significant association between AAV and HLA regions but also showed genetic distinctions between different clinical phenotypes and ANCA specificity. GPA and PR3-ANCA AAV are associated with HLA-DPB1 and HLA-DPA1, while MPA and MPO-ANCA AAV are associated with HLA-DQB1 and HLA-DQA2. (2). HLA and GPA GWAS for GPA with 492 patients and 1,506 healthy individuals of European descent identified 32 SNPs across the HLA region; among them, HLA-DPB1 rs9277554 and HLA-DPA1 rs9277341 were significantly associated with GPA in the combined cohort (15). In another study with 150 GPA patients and 100 healthy controls conducted in northern Germany, HLA-DPB1*0401 was identified to be associated with GPA (P = 1.51 × 10 -10 , OR = 3.91), and DPB1*0401/ RXRB03 haplotype frequency was significantly increased in patients with GPA (P = 7.13 × 10 -17 , OR = 6.41) (18). The result was replicated in an independent German cohort with 108 patients with GPA (P = 6.4 × 10 -8 ) (20). HLA-DPB1 was also considered a genetic risk factor for GPA in a cohort of 176 Han Chinese patients with AAV (100 with GPA, 76 with MPA) and 485 healthy controls (P = 1.83 × 10 -5 , OR = 2.57).
In this study, the result also showed that the HLA-DPB1 SNP rs3117242 variant T allele was significantly associated with GPA patients (P = 6.24 × 10 -5 , OR = 2.09), but not with MPA patients (24).
. The Italian study also revealed that HLA-DRB1*07 allele frequency was significantly higher in patients with EGPA than in controls (P = 0. 42 × 10 −2 , OR = 2.42) (30). Furthermore, HLA alleles were associated with the severity, prognosis, and relapse of AAV. HLA-DRB1*04:05 was associated with poor renal prognosis; HLA-DRB1*04:02 was associated with high mortality (31); and DPB1*04:01 was significantly associated with an increased risk of relapse (32). AAV is an autoimmune disease. The identification of HLAalleles associated with AAV confirm the centrality of autoreactivity in the development of AAV, help us make a better understanding about its autoimmune pathogenesis, and point to logical therapeutic strategies. The identification of HLAalleles not only can help us to distinguish from cases of GPA, MPA and EGPA and their subtypes, but also to evaluate and predict the severity, prognosis, and relapse of AAV.

Retinoid X Receptor Beta
RXRB, located on 6p21.32, encodes a member of the retinoid X receptor family of nuclear receptors. Strong associations of RXRB with GPA were revealed through the markers located in the HLA region and RXRB loci. Because RXRB located in the HLA region near HLA-DPB1, HLA-DPB1*0401/RXRB03 haplotype was found to be strongly associated with GPA (P = 7.13 × 10 -17 , OR = 6.41) (18). RXRB SNP rs6531 (P = 5.20 × 10 -5 , OR = 1.88) was significantly increased among patients with GPA compared to controls (19). The result was confirmed in a later meta-analysis, which included 140 genetic variants associated with AAV (17).

Ring Finger Protein 1
RING is also located in the HLA region, near HLA-DPB1. RING is a member of the polycomb repressive complex 1 that mediates histone H2A polyubiquitination and monoubiquitination, regulating its gene expression. Three SNPs in the region near RING1 (rs213210, rs213209, and rs213208) were found to be associated with GPA in German patients (20). The HLA-DPB1/ RING1 haplotype is strongly associated with GPA in ANCApositive subjects.

Non-HLA Region
Genes Encoding ANCA Associated Proteins Serpin family A Member 1 SERPINA1, located on 14q32.13, encodes a1-antitrypsin, which is the major inhibitor of PR3 and is thought to limit the damage to local tissues (33). In AAV, the genetic variants of SERPINA1 may lead to the decreased function of a1-antitrypsin, potentially resulting in ANCA generation as PR3 accumulates in tissues, causing the inflammation of blood vessels. The first AAV GWAS reported that the SERPINA1 Z allele along with the rs7151526 risk allele were significantly associated with AAV and were the most prominent non-HLA regions associated with both PR3-ANCA and GPA subgroups (13). Another GWAS confirmed the result with the identification of SERPINA1 rs28929474, which was associated with AAV (P = 3.09 × 10 −12 , OR = 2.18), especially associated with GPA (P = 3.53 × 10 −13 , OR = 2.35) (14). Several studies have investigated the role of the Z allele in AAV and showed that heterozygous patients for the Z variant of SERPINA1 have an increased risk of developing GPA than the general population (34,35).

Proteinase 3 (PRTN3)
PRTN3, located on 19p13.3, encodes PR3. PR3 is a neutrophil intracellular protease that is the main antigen of ANCA autoantibodies. PR3 is located on the plasma membrane of a subset of neutrophils and stored in the neutrophil azurophilic granules. The membrane-bound form interacts directly with ANCA (33), decreasing neutrophil activation and endothelial adhesion.
In a Swedish cohort of 79 patients with GPA and 129 controls, the coding and promoter sequences of PRTN3 were investigated. An association with GPA was demonstrated by PRTN3 A564G variation in the promoter region affecting a transcription factorbinding site (P < 0.00001, OR = 4.2) (36). The association of PRTN3 has been reported in two GWAS of AAV. Subgroup analysis revealed that PRTN3 SNP rs62132295 and rs62132293 were significantly associated with AAV, especially PR3-AAV or GPA (P = 2.6 × 10 −5 , OR = 0.78; P = 8.60 × 10 −11 , OR = 1.29) (13,14). As observed for the above-mentioned HLA association, the strength of the PRTN3 SNP signal increased in the PR3-ANCApositive subgroup, independent of the clinical diagnosis (P = 2.6 × 10 −7 , OR = 0.73). No association between PRTN3 and patients with MPA or MPO-AAV was found, which indicates that PRTN3 only plays a crucial role in the pathogenesis of anti-PR3positive AAV.

Immunoregulatory Genes for AAV Protein Tyrosine Phosphatase, Non-Receptor Type 22
PTPN22, located on 1p13.2, encodes lymphoid tyrosine phosphatase (Lyp), a member of the non-receptor class 4 subfamily of the protein tyrosine phosphatase family. PTPN22 620W variation causes the substitution of arginine with tryptophan at amino acid residue 620, disturbing the function of PTPN22, which is involved in B-cell receptor and T cell receptor signaling, causing autoimmune diseases. It is associated with type 1 diabetes, RA, SLE, and other autoimmune disorders (37,38).
The 620W allele conferred susceptibility to AAV in a German cohort (P = 0.002, OR = 1.75), and the allele frequency was significantly increased in ANCA-positive GPA (P < 0.0002) (39). This result has been confirmed in a British cohort (P = 1.4 × 10 -4 , OR = 1.40) (40) and an Italian cohort (P = 0.005, OR = 1. 91) (41). A meta-analysis of four studies in 1,399 patients of European descent found the association between the PTPN22 620W allele and the occurrence and development of AAV (42).

Cytotoxic T Lymphocyte-Associated Antigen-4
CTLA4, located on 2q33.2, encodes an inhibitory glycoprotein expressed on activated T cells, which transmits an inhibitory signal to T cells. CTLA4 competes with CD28 to bind to CD80 and CD86, which function antagonistically. CTLA4 is a negative regulator of T cell activation and inhibits the immune response, while CD28 transmits a stimulatory signal. The expression of CTLA4 on CD4 T cells is increased in GPA (43). Mutations on this gene have been associated with Graves' disease, celiac disease, SLE, and other autoimmune diseases.

Toll-Like Receptors
TLRs, located on chromosome 3p21.2, encode a family of innate receptors whose specificities are predetermined in the germline. TLRs can recognize microbiological structures and activate immune responses that participate in the development of autoimmune diseases (46). Infection, particularly Staphylococcus aureus infection, is considered a potential trigger of AAV, and TLR9 signaling may be involved in disease pathology, favoring models of infectious agents triggering AAV development (47). SNPs in TLR9 were genotyped in a German cohort comprising 863 patients with AAV and 1,344 healthy controls to investigate the contribution of genetic polymorphisms of TLR9 to the susceptibility and clinical manifestation. Strong association of TLR9 genotypes and haplotypes with the subgroups of GPA and MPA was observed, but no association was observed with EGPA. In a cohort of 426 Dutch and British AAV cases, the findings were not replicated (21). TLR9 signaling may be involved in disease pathology, favoring models of infectious agents triggering AAV development.

Fc Gamma Receptors
FCGRs are a group of proteins expressed on the surface of different cell types with different affinities for the Fc portions of different IgG subclasses. The key role of Fc receptors is the regulation of inflammatory and immune responses. FCGR3B encodes FcgRIIIB, which is expressed by neutrophils and eosinophils and is important in the tethering of neutrophils to immune complexes and the clearance of immune complexes (48). The FCGR3B copy number may play a role in the development of SLE or other autoimmune diseases (49).
In a study consisted with two independent cohorts of individuals with GPA from Britain and France, strong association between FCGR3B copy numbers and risk of GPA was shown. In British patients, low FCGR3B copy number was found to be associated with GPA (P = 0.015, OR = 2.46), but in French GPA patients, the association with high FCGR3B copy number was identified (P = 0.002, OR = 0.28) (50). The study also explored the relationship between FCGR3B and MPA in a separate British cohort and observed a significant association between low FCGR3B copy number and MPA(P = 0.013, OR = 2.56). Later, the results were replicated in a large Chinese cohort with 139 AAV patients and 564 controls (P = 0.040, OR = 1.72) with the result that low FCGR3A CNVs were significantly associated with AAV susceptibility (P = 0.042, OR = 2.64) (51).
Possible associations of SNPs of FCGR2A, FCGR2B, FCGR3A and the FCGR3B haplotype NA1/NA2 and EGPA have been discussed in a recent study, which included 130 EGPA patients and 181 controls. FCGR3B haplotype NA1/ NA2 was found to be associated with relapse-free survival of EGPA that relapse-free survival was significantly lower in carriers of FCGR3B haplotype NA1-NA1 than the others (P = 0.029). MPO-ANCA+ EGPA subgroup was also associated with relapse(P = 0.032) while no association was observed in MPO-ANCA-EGPA subgroup(P = 0.68) (52).

Interleukin 2 Receptor Subunit Alpha
IL2RA encodes the high-affinity IL-2 receptor, which is represented not only on the surface of T cells, but also on activated B cells, NK cells, and monocytes (57). The IL2-IL2RA pathway is critical for the physiological function of the immune system; survival, proliferation, and activation of T cells and development of a normal Treg repertoire (58). An association has been reported in a UK cohort of 670 patients with AAV for the IL2RA SNP rs41295061 (P = 1.22× 10 −2 ) (59), but no correlation between IL12RA and AAV was found during the replication stage of the first GWAS validation.

Other Susceptible Genes Defensin Beta 4A
DEFB4 is an antibiotic peptide. The copy number variation of DEFB4 reportedly causes susceptibility to inflammatory disorders (60). Higher DEFB4 copy number was demonstrated to be significantly higher in a small cohort of 112 Chinese patients with AAV than in controls, which supported the role of the defensin system in autoimmunity (61).

Thymic Stromal Lymphopoietin
TSLP is located on 5q22.1. Its product, TSLP, is released by stromal and epithelial cells in response to inflammatory stimuli, which drives eosinophilia and enhances TH2 responses that are associated with immunity in various inflammatory diseases, including asthma, allergic inflammation, and chronic obstructive pulmonary disease (65). TSLP SNP rs1837253 which lies immediately upstream of TSLP was observed to be EGPA susceptibility variant (P = 5.2 × 10 -11 , OR = 1.42) (16).
ETS Proto-Oncogene 1 ETS1, located on chromosome 11q24.3, encodes a member of the ETS family of transcription factors. ETS1 inhibits the differentiation of Th17 cells and induces the development of Tregs. Genetic variation may cause an increase in Th17 in patients with GPA, causing inflammation of small vessels. A GWAS in a Chinese population reported that ETS1 SNP rs1128334 was significantly associated with SLE (66). In order to observe whether this gene was associated with other autoimmune diseases, a Japanese study which contained 466 patients with AAV and 1099 healthy controls was carried out. ETS1 SNP rs1128334 was genotyped and found to be associated with GPA (P = 0.030, OR = 1.54) and PR3-ANCA AAV (P = 0.021, OR = 1.72) (67).

Telomerase Reverse Transcriptase and Desmoplakin
TERT is the catalytic subunit of telomerase and contributes to the maintenance of telomere length (68). TERT rs2736100A is associated with shorter leukocyte telomere length (69). TERT also has an anti-apoptotic effect, and a decreased expression of TERT associated with the risk allele may result in the enhancement of apoptosis (70). With the evidence that a proportion of T cells show short telomeres in patients with GPA and an enhancement in neutrophil apoptosis in patients with AAV, TERT has been proposed as a susceptibility gene for AAV (71). In a Japanese cohort comprising 544 patients with AAV and 5558 controls, the frequency of TERT rs2736100A alleles was significantly increased in MPA (P = 2.3 × 10 −2 , OR= 1.38) and MPO-AAV (P = 0.15× 10 −2 , OR = 1.33) (72). DSP rs2076295G showed similar results to TERT rs2736100A (P = 0.69× 10 -2 , OR = 1.32) in MPO-AAV (P = 0.011, OR = 1.26) (72). DSP reportedly modulates Wnt/b-catenin signaling, which is involved in cell proliferation, differentiation, immune response, and carcinogenesis. Wnt signaling plays a role in autoimmune diseases and may also be associated with the onset of AAV.
All the genes associated with AAV were summarized in Table 3.

CONCLUSION
In the past 20 years, genetic studies, including GWAS and candidate gene studies, have identified numerous key loci that are associated with the risk of AAV. These genes have not only provided novel etiological clues to deepen our understanding of AAV but also novel therapeutic and prophylactic biomarkers for new approaches to treat and prevent AVV. Biomarker-targeting therapies and preventing recurrence in the future may be more effective. With the increasing number of GWAS being conducted, it is desirable to combine these findings and improve the statistical power. To date, GWAS for AAV have been performed in the European population; in the future, more GWAS with large samples in different populations and different subgroups of AAV are required to reveal the pathogenesis of the disease and identify reliable biomarkers for precision medicine.
contributed to the collection of data, and reporting of the work described. All authors contributed to the article and approved the submitted version.