Genetics of Microscopic Polyangiitis in the Japanese Population

Naoyuki Tsuchiya, MD, PhD

The epidemiology of ANCA-associated vasculitis is substantially different between Caucasians and Japanese, which may be related to differences in genetic backgrounds. In this review, I discussed our findings on the genetics of microscopic polyangiitis (MPA) in Japanese. Analysis of HLA genes revealed a significant increase in the HLA-DRB1*09:01-DQB1*03:03 haplotype MPA. This is one of the most frequent haplotypes in Japanese, but is nearly absent in Caucasians, and has been shown to be associated with multiple autoimmune diseases. Analysis of KIR genes revealed significant decreases in the carrier frequency of an activating receptor KIR2DS3 in MPA. When KIRs were analyzed in combination with HLA ligands, the proportion of individuals carrying KIR3DL1 and HLA-Bw4 but not KIR3DS1, the most inhibitory of all KIR3DS1/3DL1/ HLA-B combinations, was significantly increased in MPA. These results suggested that decreased activation of NK and/or T cells may cause a predisposition to MPA. LILRA2 is an activating receptor involved in granulocyte and macrophage activation. LILRA2 SNP rs2241524 G >A, which disrupts the intron 6 splice acceptor site, was significantly associated with MPA. The risk allele produces an LILRA2 isoform lacking three amino acids in the linker region. These findings, when confirmed by larger-scale studies, will shed light on the molecular mechanisms of MPA. (*English Translation of J Jpn Coll Angiol 2009; 49: 31-37.)

Keywords: microscopic polyangiitis, genetics, HLA, KIR, LILRA2

Introduction populations are associated with certain genetic and environmental factors. A possible environmental factor is mong anti-neutrophil cytoplasmic antibody microbial infection. Studies on human ANCA-associated A(ANCA)-associated vasculitides, granulomatosis vasculitis have suggested the involvement of Staphylo- with polyangiitis (GPA) is frequently observed in West- coccus aureus1) or cytomegalovirus (CMV) infection.2) ern countries, particularly in northern Europe, whereas Another article of this special feature introduces studies microscopic polyangiitis (MPA) is frequently observed suggesting a microbial contribution in model animals in Japan. It is speculated that such differences among with vasculitis. At present, it may be appropriate to consider that individual differences in responses to en- Molecular and Genetics Epidemiology Labratory, Faculty of vironmental factors such as microorganisms are involved Mecicine, University of Tsukuba, Tsukuba, Ibaraki, Japan in the development of ANCA-associated vasculitis. Disorders such as ANCA-associated vasculitis have Received: July 23, 2012; Accepted: July 30, 2012 Corresponding author: Naoyuki Tsuchiya, MD, PhD. Molecular and few clues to their pathogenesis, as such, the identification Genetic Epidemiology Laboratory, Faculty of Medicine, University of of disease susceptibility genes is expected to provide clues Tsukuba, 1-1-1, Tennodai, Tsukuba, Ibaraki 305-8575, Japan to clarify pathogenesis and essential pathology and de- Tel: +81-29-853-3071, Fax: +81-29-853-3071 velop treatment methods. However, since their incidences E-mail: [email protected] *This article is English Translation of J Jpn Coll Angiol 2009; 49: are low, it has been difficult to collect disorder-prone 31-37. families necessary for genome-wide linkage analysis

Annals of Vascular Diseases Vol.5, No.3 (2012) 289 Tsuchiya N. and samples from a thousand or more patients necessary [OR], 2.44).8) In addition, HLA-DQB1, DPB1, B, and C for reliable genome wide association studies. At pres- were evaluated and a significant association between ent, candidate gene approaches based on the results of MPA and DQB1*03:03 (OR, 2.35) was observed. Due to pathological analysis in humans and results from animal the presence of marked linkage disequilibrium between models have been mainly performed. HLA-DRB1*09:01 and DQB1*03:03, it was impossible Immune system genes are major factors for biological to determine which of them is the primary susceptibility responses to environments and may be promising can- gene. On the other hand, MPA was not clearly associated didate genes since marked functional diversity has been with HLA-B, C, or DPB1. Thus, in the Japanese popula- acquired due to microorganisms as selection pressure tion, the HLA-DRB1*09:01-DQB1*03:03 haplotype was in human populations. Concerning the genetic back- found to be a significant genetic factor for MPA.9) ground of ANCA-associated vasculitis in Japanese, we The HLA-DRB1*09:01-DQB1*03:03 haplotype has two as members of “The Research Committee of Intractable unique characteristics. One is that this haplotype is fre- Vasculitis Syndrome” of the Ministry of Health, Labor, quently observed in Asian populations such as Japanese and Welfare of Japan evaluated immune system genes but is rarely present in European or African populations. as candidate genes. Although this research is a multi- The other is the association between this haplotype and institutional joint study, due to the low incidences of various autoimmune diseases such as type I diabetes disorders, the number of samples that could be analyzed mellitus, juvenile myasthenia gravis, anti-phospholipid was only 50 for MPA, 8 for GPA, and 8 for eosinophilic antibody production, and anti-CCP antibody-negative granulomatosis with polyangiitis (EGPA). Therefore, rheumatoid arthritis in Japanese.10–13) This is like a mir- statistical analysis was mainly focused on MPA. Thus far, ror image of the HLA-DRB1*03:01 haplotype, which is relatively small number of studies have been published on frequently observed in populations in northern Europe, genetic predispositions for ANCA-associated vasculitis, but is absent in Japanese and is associated with various most of which came from Western countries.. In addition, autoimmune diseases. although ANCA-associated vasculitis is mostly observed The function of HLA-DR and DQ is antigen presenta- in Japan and Western countries, its epidemiology consid- tion to T cells. Therefore, one possible molecular mecha- erably differs between the two regions. Therefore, in this nism that can explain the association between the HLA- paper, we do not provide general remarks about ANCA- DRB1*09:01-DQB1*03:03 haplotype and MPA is specific associated vasculitis, but introduce the results from our presentation of a certain pathogenic antigen peptide by research committee. one of the following molecules: HLA-DR9 molecule, which is a heterodimer of the HLA-DRB1*09:01 allele Human Leukocyte Antigen (HLA) Genes product and HLA-DRA1 allele product, the HLA-DQ9 molecule, which is a heterodimer of HLA -DQB1*03:03 HLA molecules present antigens to T cells and show and the DQA1*03 allele products encoded by this haplo- extensive polymorphism directly affecting immune type, and the HLA-DR53 molecule, which is a heterodi- responses. Since HLA genes are the most established mer of HLA-DRB4*01 and DRA1 products (Fig. 1). disease susceptibility genes in many immunological However, it may be difficult to explain the association disorders and infections, we initially analyzed them as between HLA genes and diverse disorders based on bind- candidate genes. ing of a specific antigen peptide.HLA genes are encoded Studies on the association between HLA and ANCA- on human 6p21.3. On this region, genes other than HLA associated vasculitis have shown associations between genes (TNFα, C4, and TAP genes) are also encoded and GPA and HLA-DR4 as well as DR1 in Europe.3–5) In linkage with HLA is observed. Considering that this hap- Japan, before our study, studies in a few cases have shown lotype is associated with various autoimmune diseases, an increase in HLA-DR9 in GPA and MPO-ANCA- it is more likely that polymorphism of immune system associated glomerulonephritis.6,7) genes (other than HLA genes), which are encoded on this We performed association studies between ANCA- haplotype, rather than presentation of a specific antigen associated vasculitis and the HLA-DRB1 genotype and peptide is causally associated with MPA (Fig. 1). The observed HLA-DRB1*09:01 in 29.1% of the healthy two possibilities should be taken into consideration for control group but 50% of the MPA group, showing a clarification of the molecular mechanism. significant increase in the latter P( = 0.037; odds ratio In addition, as described above, the prevalence of GPA

290 Annals of Vascular Diseases Vol.5, No.3 (2012) Genetics of MPA in the Japanese Population

Fig. 1 Possible mechanisms of association between HLA-DRB1*09:01- DQB1*03:03 haplotype with MPA. HLA-DRB1*09:01-DQB1*03:03 is one of the haplotypes that be- long to the DR53 group haplotype, which carries the HLA-DRB4 locus. Three HLA-class II molecules, HLA-DR9, DQ9, and DR53, are produced from this haplotype. It is possible that an antigenic peptide specifically presented by any of these molecules is causally involved in diseases. Another possibility is that an as of yet unidentified functional polymorphism specifically encoded on this haplotype is causally associated with diseases, and HLA represents a proxy of such causal alleles. MPA: microscopic polyangiitis

or MPA markedly differs among populations. Further domains and a long intracellular domain, and KIR2DS1 studies in various populations are necessary to determine has two extracellular Ig domains and a short intracellular whether these differences are caused by differences in domain. In general, the long domain (DL) group includes HLA among populations. inhibitory receptors with immunoreceptor tyrosine-based inhibitory motifs (ITIM) in the intracellular domain while Killer Cell Immunoglobulin-Like Receptor the short domain (DS) group has a positively charged (KIR) Genes amino acid (Lys) in the membrane spanning domain and is coupled to immunoreceptor tyrosine-based activation Human NK cells express many activating and inhibi- motif (ITAM)-containing DAP12 as an adaptor molecule, tory receptors. Inhibitory receptors recognize self HLA- transmitting activating signals. However, only KIR2DL4 class I molecules expressed in most cells and transmit in the DL group has positively-charged Arg in the mem- inhibitory signals, preventing the self-cytotoxicity of NK brane spanning domain and is coupled to FcRγ, inducing cells. In situations such as the presence of tumor cells and IFNγ production.15) virus-infected cells in which self HLA-class I expression KIR, unlike T cell receptors, recognizes subgroups is inhibited, inhibitory signals decrease, resulting in of HLA-class I, classified according to the amino acid cytotoxicity against such cells (missing self-theory).14) sequence motif (Table 1). KIR, belonging to the immunoglobulin superfamily, is KIR genes show not only base sequence polymorphism expressed on NK cells and some subpopulations of T but also extensive polymorphism due to the presence or cells and there are both activating and inhibitory types. absence of loci themselves. As a result, the number of KIR is encoded on 14 genetic loci adjacent to leukocyte KIR genes in the genome markedly varies (7–14) among Ig-like receptor (LILR) genes in the leukocyte receptor individuals. For details of this variation, refer to other complex (LRC) located on human chromosome 19q13.4. reviews.16–18) KIR molecules are termed according to the number of the KIR and HLA are encoded on difference chromosomes extracellular Ig domain and the length of the intracellular and, therefore, are inherited independently. Combina- domain. For example, KIR3DL1 has three extracellular Ig tions of KIR and HLA as a ligand, both of which are

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Table 1 KIRs and their ligands cells was reported.27) This finding supports the above KIR ligand results that resistance to virus infection decreases in KIR2DL1 HLA-Cgroup2 individuals assumed to have intense inhibitory signals KIR2DL2/3 HLA-Cgroup1 mediated by KIR. KIR2DL4 HLA-G In view of this background, we analyzed the presence KIR2DL5 or absence of KIR genes and KIR/HLA ligand combina- 28) KIR3DL1 HLA-Bw4 tions in Japanese subjects with MPA. Initially, the presence or absence of 14 KIR loci was KIR3DL2 HLA-A3, A11 determined in 443 Japanese patients with MPA and 239 KIR3DL3 healthy controls. An activating receptor KIR2DS3 was KIR2DS1 HLA-Cgroup2 positive in 16.7% of healthy controls but 4.7% of patients KIR2DS2 (HLA-Cgroup1) with MPA, showing a significant decrease in the latter KIR2DS3 (P = 0.038). KIR2DS4 Subsequently, KIR/HLA ligand combinations were KIR2DS5 evaluated. No significant differences were observed KIR3DS1 (HLA-Bw4) in the frequency of HLA-B or HLA-C alone between HLA-Cgroup2 consists of HLA-Cw alleles possessing Asn77 MPA and control groups. However, the frequency of the and Lys80, such as Cw2, 4, 5, and 6. HLA-Cgroup1 includes combination of HLA-Bw4+, KI3DL1+, and KIR3DS1-, HLA-Cw1, 3, 7, and 8, which contain Ser77 and Asn80. HLA- which is considered to be the most inhibitory among the Bw4 is defined by position 77-83 amino acids and includes combinations between KIR3DL1/3DS1 and HLA-Bw4, HLA-B13, B27, B44, B51, B52, and A24. A weak interaction was significantly higher P( = 0.014) in the MPA group has been postulated between KIR2DS2 and HLA-Cgroup1 (46.5%) than that in the control group (27.0%) with an and between KIR3DS1 and HLA-Bw4, which has not been conclusively established. odds ratio of 2.35 (Fig. 2). Furthermore, HLA-C was also incorporated as a ligand into this analysis . Combinations that are considered to extensively polymorphic, cause genetic individual dif- be more inhibitory showed a more marked increase in ferences in signal transmission mediated by HLA-KIR. the risk of developing MPA. These results suggest an With this background, an association between KIR/HLA association between inhibition of NK cells and T cells combinations and virus infection particularly that due to mediated by KIR and the risk of developing MPA. HIV,19) HCV, 20) and human papillomavirus,21) or autoim- These results together with the above previous reports mune diseases such as rheumatoid artiritis,22) psoriasis concerning autoimmune diseases and virus infection vulgaris,23) psoriatic arthritis,24) type I diabetes melli- suggest that combinations associated with decreased tus,25) and scleroderma26) has been reported. The results resistance to virus rather than autoimmune diseases of previous studies can be summarized as follows. The are susceptible to MPA. As described at the beginning, risk of developing autoimmune diseases tends to increase some studies have suggested the causal involvement of when activating signals assumed by HLA-KIR combina- Staphylococcus aureus or CMV in ANCA-associated tions are intense while the risk of virus infection tends to vasculitides.1,2) In addition, MPA develops at advanced increase when inhibitory signals are intense. However, age and is often complicated by severe intractable infec- in uterine cervix cancer associated with virus infection, tion such as CMV pneumonia during its course. These inhibitory combinations decrease the risk of developing findings may allow the proposal of the following hypoth- this disease, which is considered to be because persistent esis: Combination of an age-related decrease in resistance inflammatory reactions in the host to virus infection are and a genetic decrease in resistance to infection due to associated with carcinogenesis. the KIR genotype causes persistence and aggravation of Each NK cell in individuals expresses their KIR fam- virus infection, and this condition, together with mul- ily gene clusters in a random fashion to some extent. tiple genetic predispositions and environmental factors, Therefore, in the same individuals, there are NK cells eventually leads to ANCA production and results in the expressing and those not expressing a certain type of development of vasculitis. KIR. In a child with repeated CMV infection, expression of an inhibitory receptor KIR2DL1 as in all NK

292 Annals of Vascular Diseases Vol.5, No.3 (2012) Genetics of MPA in the Japanese Population

Fig. 2 HLA-Bw4, KIR3DL1, and KIR3DS1 combinations in Japanese MPA and healthy controls. Each individual can be grouped into 6 groups according to the presence/ absence of KIR3DL1, KIR3DS1, and HLA-Bw4, the ligand of KIR3DL1 (and possibly of KIR3DS1). KIR3DL1 contains ITIM and transmits inhibitory signals, while KIR3DS1 associates with DAP12, which contains ITAM and transmits activation signals. The frequencies of each group in Japanese patients with MPA and controls are shown on the right. The most inhibitory combination, HLA-Bw4+, KIR3DL1+, KIR3DS1- (top), was significantly increased in MPA.28) Because the ligand of KIR3DS1 has not been identified, here we assumed an unknown ligand expressed in all individuals. It should be noted that there is a possibility that KIR3DS1 weakly interacts with HLA-Bw4, as described in Table 1. However, this does not significantly affect the above interpretation. OR: odds ratio; CI: confidence interval

LILRA2 ducted an association study on autoimmune rheumatic diseases, and during this process, found an association For LILR (also called ILT or CD85) genes adjacent to between SNP rs2241524 replacing the splice accep- KIR genes in LRC, there are 13 genetic loci including tor site at the intron 6-exon 7 junction of LILRA2 and those for 2 pseudogenes. LILR molecules are classified systemic lupus erythematosus (SLE) as well as MPA.29) into activating (LILRA1, -A2, -A4, -A5, and –A6), in- The A/A genotype was observed in 7.0% of the healthy hibitory (LILRB1, -B2, -B3, -B4, and –B5), and soluble control group but 12.1% of the SLE group (P = 0.041; (LILRA3, -A5s) types. Although the cell type distribution OR compared with G/G genotype, 1.82) and 16.0% in the differs among LILR types, these receptors are generally MPA group (P = 0.049; OR, 2.52), showing significant expressed in the leukocyte line, and some of them use increases. HLA-class I or UL18 of CMV as a ligand. Since this SNP was expected to change the splicing LILRA2 is involved in the activation of eosinophils, site, the cDNA sequence was evaluated for each geno- basophils, and macrophages. This type has also been type. When an A allele is present, the cryptic splice site reported to exert an inhibitory action on immune re- 9 nucleotides downstream of the original splice acceptor sponses by shifting IL-12 production to IL-10 production site is used, and, therefore, translation to protein with in monocytes and inhibiting TLR signals. deletion of 3 amino acids corresponding to positions Regarding LILR genes as candidate genes, we con- 419–421 in the amino acid sequence was considered

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Fig. 3 LILRA2 rs2241524 associated with MPA and SLE activates a cryptic splice acceptor site and causes an inframe deletion of three amino acids in the linker region. SNP rs2241524 G >A (arrow) disrupts the consensus splice acceptor motif and activates the cryptic splice site 9 nucleotides downstream of the original splice acceptor site.29) This leads to an inframe deletion of 3 amino acids (419–422) in the linker region of LILRA2. The splice sites employed in each allele are underlined and exons are shaded. to occur (Fig. 3). In individuals with the A/A genotype, the clarification of pathogenesis and drug discovery. If 100% of mRNAs showed this Δ419–421 isoform. Posi- hundreds of samples can be collected from Japanese with tions 419–421 correspond to the linker region that is ANCA-associated vasculitis, a large-scale susceptibility present between the extracellular Ig-like domain and gene analysis becomes possible by genome wide associa- membrane spanning domain. Studies using monoclo- tion studies. Joint research systems at the national level nal antibodies have confirmed that this isoform is also and in Asian populations are necessary in the future. expressed on the monocyte surface. Further studies are necessary on changes in LILRA2 signals due to this Acknowledgments deletion of 3 amino acids. I conducted these studies as a member of “The Re- Conclusions search Committee of Intractable Vasculitis Syndrome,” with a support by a Grant-in-Aid, Scientific Research from the Ministry of Health, Labor, and Welfare of Ja- Concerning the genetic predispositions for MPA, we pan. I express deep gratitude to Dr. Hiroshi Hashimoto have mainly evaluated multigene families showing ex- (Juntendo University), who was the Chairman of this Re- tensive functional polymorphisms (HLA, KIR, and LILR) search Committee and provided me with the opportunity among gene clusters in the immune system. MPA is a to perform this study, Dr. Shoichi Ozaki (St. Marianna rare disease and only an extremely few cases have been University School of Medicine), and many doctors for evaluated in our studies as genetic studies. Therefore, their instructions and cooperation in this study. further studies are necessary on the reproducibility of results. However, our and other previous studies have References shown an association between each of these genes and 1) Kallenberg CG, Rarok A, Stegeman CA, et al. New other autoimmune diseases or infections, which supports insights into the pathogenesis of antineutrophil cyto- the possibility that these polymorphisms have functional plasmic autoantibody-associated vasculitis. Autoim- significance.. mun Rev 2002; 1: 61-6. [Medline] [CrossRef] In the future, the associations found in this study and 2) Meyer MF, Hellmich B, Kotterba S, et al. Cytomega- lovirus infection in systemic necrotizing vasculitis: molecular mechanisms should be further evaluated for

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