Autoimmune hepatitis (AIH) is an autoimmune liver disease and cirrhosis is sometimes complicated with AIH at diagnosis, influencing its prognosis. TNFAIP3 gene encodes A20, an inhibitor of nuclear factor-κB pathway, and is a susceptibility gene for autoimmune diseases. We investigated deleterious variants in the coding regions of TNFAIP3 gene of Japanese AIH patients or those with cirrhosis. The deleterious variants in the coding regions of TNFAIP3 gene were analyzed by the cycle sequencing method and the frequencies of deleterious TNFAIP3 alleles of AIH or AIH with cirrhosis were compared with those of Japanese controls. The deleterious alleles in TNFAIP3 were not associated with AIH. A significant association was shown for the deleterious alleles in TNFAIP3 (P = 0.0180, odds ratio (OR) 4.28, 95% confidence interval (CI) 1.53–11.95) with AIH with cirrhosis at presentation. The serum IgM levels in AIH patients with deleterious alleles in TNFAIP3 were tended to be lower than those without (P = 0.0152, Q = 0.1216). The frequency of deleterious alleles in TNFAIP3 was higher in the AIH subset without the DRB1 risk alleles than that with (P = 0.0052, OR 5.10, 95%CI 1.55–16.74). The deleterious alleles in TNFAIP3were associated with AIH with cirrhosis.
Autoimmune hepatitis (AIH) is an autoimmune liver disease with chronic progression1. Liver cirrhosis is sometimes complicated with AIH at diagnosis and influences its prognosis. AIH patients with cirrhosis had a worse survival2. The genetic and environmental factors are involved in the pathogenesis of AIH. A genome-wide association study (GWAS) on European population revealed that human leukocyte antigen (HLA) is the strongest genetic risk factor for AIH3. HLA-DRB1*03:01 and DRB1*04:01 were associated with AIH in European populations4. DRB1*04:01 and DRB1*04:05 were associated with AIH in Japanese populations5,6,7,8. DRB1*08:02 and DRB1*08:03 were also predisposing for the disease, when these alleles were possessed by individuals with DRB1*04:058. The previous GWAS also suggested associations of single nucleotide variants in other genes outside of HLA3. Several candidate-gene approach studies reported weak genetic associations of single nucleotide variants with Japanese AIH in other genes than HLA; STAT49, PTPN2210, ICOS11, TNIP112, and SH2B313.
TNFAIP3 (tumor necrosis factor-α induced protein 3) gene encodes A20, an inhibitor of nuclear factor-κB (NF-κB) activation, and is a susceptibility gene for autoimmune diseases including systemic lupus erythematosus14,15 or rheumatoid arthritis16,17. A20 is a negative regulator of the NLRP3 inflammasome and myeloid cell specific deletion of A20 caused spontaneous arthritis in mice18. Analogically, inflammasome was activated in monocytes of non-transplanted AIH children and liver-transplanted children with de novo autoimmune hepatitis, but increased expression of A20 was observed in monocytes of liver-transplanted children without de novo autoimmune hepatitis19. Thus, A20 plays some important roles against autoimmune diseases. Recent studies reported that loss of function (nonsense or frameshift variants) or deleterious missense variants (variants that changed amino acid residues on positions highly conserved across orthologs) in TNFAIP3 dominantly caused an autoinflammatory disease with Behçet’s disease-like symptoms, the haploinsufficiency of A20 syndrome (HA20)20,21,22. However, the symptoms of HA20 also included those of autoimmune diseases, arthritis, nephritis, vasculitis, or hepatitis23. Thus, we investigated the variants in the coding regions of TNFAIP3 gene of Japanese AIH patients by the cycle sequencing method and tried to compare the frequencies of deleterious TNFAIP3 alleles of AIH or AIH with cirrhosis with those of Japanese controls.
Associations of deleterious alleles in TNFAIP3 with AIH with cirrhosis
The exons and its boundaries of TNFAIP3 gene were amplified by 9 primer sets to detect variants by the direct sequencing method. Seven single nucleotide variants were found in the coding regions of TNFAIP in 360 AIH patients. No variant was detected in the splice sites of TNFAIP3 gene and no insertion or deletion was found. Two (rs200595071 and rs769014911) of these variants were synonymous and the other five were non-synonymous. Of these five non-synonymous variants, three variants [c.116A > G (p.H39R, chr6:137871343 in GRCh38), c.305A > G (rs146534657, p.N102S, chr6:137874854), and c.1897G > C (p.E633Q, chr6:137879342)] were predicted to be deleterious (probably damaging, affect protein function, disease causing, or deleterious) by all five protein prediction algorithms, and two [c.380T > G (rs2230926, p.F127C, chr6:137874929) and c.2140C > T (rs369155845, p.P714S, chr6:137881086)] were predicted to be neutral (benign, tolerated, polymorphism, or neutral) by all. Thirteen alleles of these three deleterious variants (one c.116A > G, eleven c.305A > G, and one c.1897G > C, Fig. S1) were detected in twelve AIH patients and one was estimated to be a compound heterozygote for c.116A > G and c.305A > G.
Deleterious allele frequencies in the AIH patients and the Japanese controls are shown in Table 1. No significant association with AIH was detected for the deleterious alleles in TNFAIP3. When AIH patients with cirrhosis at presentation were compared with the Japanese controls, a significant association was shown for the deleterious alleles in TNFAIP3 (P = 0.0180, odds ratio (OR) 4.28, 95% confidence interval (CI) 1.53–11.95, Table 1).
Clinical features of the AIH patients with or without deleterious alleles in TNFAIP3
The demographic features of AIH patients with or without deleterious alleles in TNFAIP3 were analyzed (Table 2). The serum levels of IgM in AIH patients with deleterious alleles in TNFAIP3 were tended to be lower than those without (P = 0.0152, Q = 0.1216).
Associations of deleterious alleles in TNFAIP3 with AIH subsets with or without the DRB1 risk alleles
The gene-gene interaction between TNFAIP3 and DRB1 was also investigated (Table 3). The frequency of deleterious alleles in TNFAIP3 was higher in the AIH subset without the DRB1 risk alleles than that with (P = 0.0052, OR 5.10, 95%CI 1.55–16.74).
The present study revealed that deleterious variants in TNFAIP3 were predisposing for AIH with cirrhosis in a Japanese population. TNFAIP3 encodes A20, an inhibitor of the NF-κB signaling pathway, and is a susceptibility gene for autoimmune diseases and HA2014,15,16,17,20,21,22,23. A20 is a negative regulator of the NLRP3 inflammasome and plays some important roles against autoimmune diseases18,19. TNIP1 is a predisposing gene in AIH12, and encodes an adaptor protein binding to A20. These data suggested the common signaling pathways in the pathogenesis of AIH, other autoimmune diseases, and HA20.
The progression pattern of AIH patients with cirrhosis at presentation would be smoldering and latent and the prognosis of AIH with cirrhosis was worse2. Cirrhosis at presentation was also a risk factor of development of hepatocellular carcinoma24. However, an age at onset was older in AIH patients with cirrhosis at presentation2, but that of AIH patients with deleterious variants in TNFAIP3 was not older (Table 2), suggesting the existence of differential subsets of AIH. Deleterious variants in TNFAIP3 were revealed to be risk factors for AIH with cirrhosis (Table 1). Thus, deleterious variants in TNFAIP3 would modulate the progression pattern of AIH, contribute to configure the subset of AIH, and influence the prognosis of AIH. Further observation studies were needed to clarify the prognosis and the progression pattern of AIH patients with deleterious variants in TNFAIP3. In the present study, three deleterious variants in TNFAIP3 (c.116A > G, c.305A > G, and c.1897G > C) were detected in Japanese AIH patients, but none of them were reported to cause HA20. One of the deleterious variants in TNFAIP3 (c.305A > G) was appeared to be related to the poor clinical outcome of rheumatoid arthritis16, suggesting that deleterious variants in TNFAIP3 are modulating symptoms of autoimmune diseases.
The association of deleterious variants in TNFAIP3 was also analyzed between patients with or without the DRB1 risk alleles. The frequency of deleterious variants in TNFAIP3 was higher in the AIH patients without the DRB1 risk alleles than those with. These data suggested that deleterious variants might not predispose AIH in individuals with the strongest genetic risk factor, the DRB1 risk alleles. It was also possible that deleterious variants would be readily found in the individuals without the DRB1 risk alleles. Serum IgM levels in AIH with deleterious variants in TNFAIP3 were tended to be lower than those without (Table 2). Since AIH patients with higher serum IgM levels were suspected to be overlapped with primary biliary cholangitis, the AIH patients with lower serum IgM levels would be patients with lower probability of the overlap. It was reported that IgM levels of AIH patients with DRB1*04:05, one of the DRB1 risk alleles, were higher than those without6,8, explaining the decreased IgM levels in AIH patients with deleterious variants. Since DRB1 and TNFAIP3 were distantly located on the different arms of chromosome 6 (DRB1: 6p21.32, 32578769–32589848 in GRCh38, TNFAIP3: 6q23.3, 137867188–137883312), linkage disequilibrium was not strong between these two loci (r2 = 0.003, between the DRB1 risk alleles and deleterious variants in TNFAIP3 in AIH patients), suggesting that deleterious variants in TNFAIP3 and the DRB1 risk alleles would be independently associated with AIH. However, the independence could not be confirmed by logistic regression analysis, because the genotypes of the Japanese controls were not available.
To the best of our knowledge, this is the first report of the association of deleterious variants in TNFAIP3 with Japanese AIH with cirrhosis. Since the sample size of this study was limited and the frequencies of deleterious variants were low, the association was modest. The associations should be confirmed in future large scale studies.
Materials and Methods
Patients and controls
A total of 360 AIH patients were recruited from National Hospital Organization (NHO) hospitals. The clinical information of the enrolled patients was obtained and 38 had cirrhosis at presentation2,24. All the AIH patients were native Japanese living in Japan and satisfied the criteria of International Autoimmune Hepatitis Group for diagnosis of AIH25. The allele frequencies of single nucleotide variants in TNFAIP3 gene in Japanese population were referred to 3.5KJPN panel from the genome cohort study of Tohoku Medical Megabank Organization (https://ijgvd.megabank.tohoku.ac.jp/)26,27,28. The distribution pattern of the age and gender of the controls was described elsewhere (https://ijgvd.megabank.tohoku.ac.jp/statistics/statistics-3-5kjpn-all/). This study was reviewed and approved by NHO Central IRB and University of Tsukuba Research Ethics Committee. Written informed consent was obtained from each individual. This study was conducted in accordance with the principles expressed in the Declaration of Helsinki.
Genotyping of TNFAIP3 gene was performed by the direct sequencing method of PCR products amplified with previously reported primers16 for exon 2 to exon 9 and BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc., Waltham, MA) using Applied Biosystems 3130xl Genetic Analyzer (Thermo Fisher Scientific Inc.). Deleterious alleles were defined by missense variants annotated as deleterious by all five protein prediction algorithms of PolyPhen-2 HumDiv (http://genetics.bwh.harvard.edu/pph2/index.shtml, probably damaging), PolyPhen-2 HumVar (http://genetics.bwh.harvard.edu/pph2/index.shtml, probably damaging), SIFT (http://sift.bii.a-star.edu.sg/, affect protein function), Mutation Taster (http://www.mutationtaster.org/, disease causing), and Likelihood Ratio Test Predictions (http://www.genetics.wustl.edu/jflab/lrt_query.html, deleterious)29,30,31,32,33,34. DRB1 genotyping results of the AIH patients were reported previously7,8. DRB1*04:01, *04:05, *08:02, or *08:03 were designated as the DRB1 risk alleles for AIH8.
The distribution of deleterious allele frequencies in AIH patients or AIH patients with cirrhosis was compared with those in Japanese controls by Fisher’s exact test using 2 × 2 contingency tables under the allele model35. The clinical phenotypes of AIH patients with deleterious alleles were compared with those without by Fisher’s exact test using 2 × 2 contingency tables or Mann-Whitney’s U Test. The distribution of deleterious allele frequencies was compared between AIH patients with or without the DRB1 risk alleles by Fisher’s exact test using 2 × 2 contingency tables under the allele model. Correction for multiple testing was performed by calculating false discovery rate Q-value36.
All relevant data are within the manuscript and its Supplementary Information files.
Wang, Q. et al. The clinical phenotypes of autoimmune hepatitis: A comprehensive review. J Autoimmun 66, 98–107 (2016).
Migita, K. et al. Evaluation of risk factors for the development of cirrhosis in autoimmune hepatitis: Japanese NHO-AIH prospective study. J Gastroenterol 46(Suppl 1), 56–62 (2011).
de Boer, Y. S. et al. Genome-wide association study identifies variants associated with autoimmune hepatitis type 1. Gastroenterology 147, 443–452 e445 (2014).
Strettell, M. D. et al. Allelic basis for HLA-encoded susceptibility to type 1 autoimmune hepatitis. Gastroenterology 112, 2028–2035 (1997).
Umemura, T. et al. Human leukocyte antigen class II haplotypes affect clinical characteristics and progression of type 1 autoimmune hepatitis in Japan. PLoS One 9, e100565 (2014).
Furumoto, Y. et al. Evaluation of the role of HLA-DR antigens in Japanese type 1 autoimmune hepatitis. BMC Gastroenterol 15, 144 (2015).
Maeda, Y. et al. Association between Anti-Ganglionic Nicotinic Acetylcholine Receptor (gAChR) Antibodies and HLA-DRB1 Alleles in the Japanese Population. PLoS One 11, e0146048 (2016).
Oka, S. et al. HLA-DRB1 and DQB1 alleles in Japanese type 1 autoimmune hepatitis: The predisposing role of the DR4/DR8 heterozygous genotype. PLoS One 12, e0187325 (2017).
Migita, K. et al. Association of STAT4 polymorphisms with susceptibility to type-1 autoimmune hepatitis in the Japanese population. PLoS One 8, e71382 (2013).
Umemura, T. et al. Genetic Association of PTPN22 Polymorphisms with Autoimmune Hepatitis and Primary Biliary Cholangitis in Japan. Sci Rep 6, 29770 (2016).
Higuchi, T. et al. Association of a single nucleotide polymorphism upstream of ICOS with Japanese autoimmune hepatitis type 1. J Hum Genet 62, 481–484 (2017).
Oka, S. et al. Association of a single nucleotide polymorphism in TNIP1 with type-1 autoimmune hepatitis in the Japanese population. J Hum Genet 63, 739–744 (2018).
Umemura, T. et al. Association of autoimmune hepatitis with Src homology 2 adaptor protein 3 gene polymorphisms in Japanese patients. J Hum Genet (2017).
Nair, R. P. et al. Genome-wide scan reveals association of psoriasis with IL-23 and NF-kappaB pathways. Nat Genet 41, 199–204 (2009).
Kawasaki, A. et al. Association of TNFAIP3 polymorphism with susceptibility to systemic lupus erythematosus in a Japanese population. J Biomed Biotechnol 2010, 207578 (2010).
Zhu, L. et al. Characteristics of A20 gene polymorphisms and clinical significance in patients with rheumatoid arthritis. J Transl Med 13, 215 (2015).
Zhang, L. et al. Associations Between TNFAIP3 Gene Polymorphisms and Rheumatoid Arthritis Risk: A Meta-analysis. Arch Med Res 48, 386–392 (2017).
Vande Walle, L. et al. Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis. Nature 512, 69–73 (2014).
Arterbery, A. S. et al. Inflammasome Priming Mediated via Toll-Like Receptors 2 and 4, Induces Th1-Like Regulatory T Cells in De Novo Autoimmune Hepatitis. Front Immunol 9, 1612 (2018).
Zhou, Q. et al. Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease. Nat Genet 48, 67–73 (2016).
Shigemura, T. et al. Novel heterozygous C243Y A20/TNFAIP3 gene mutation is responsible for chronic inflammation in autosomal-dominant Behcet’s disease. RMD Open 2, e000223 (2016).
Aksentijevich, I. & Zhou, Q. NF-kappaB Pathway in Autoinflammatory Diseases: Dysregulation of Protein Modifications by Ubiquitin Defines a New Category of Autoinflammatory Diseases. Front Immunol 8, 399 (2017).
Kadowaki, T. et al. Haploinsufficiency of A20 causes autoinflammatory and autoimmune disorders. J Allergy Clin Immunol 141, 1485–1488 e1411 (2018).
Migita, K. et al. Hepatocellular carcinoma and survival in patients with autoimmune hepatitis (Japanese National Hospital Organization-autoimmune hepatitis prospective study). Liver Int 32, 837–844 (2012).
Alvarez, F. et al. International Autoimmune Hepatitis Group Report: review of criteria for diagnosis of autoimmune hepatitis. J Hepatol 31, 929–938 (1999).
Kuriyama, S. et al. The Tohoku Medical Megabank Project: Design and Mission. J Epidemiol 26, 493–511 (2016).
Yamaguchi-Kabata, Y. et al. iJGVD: an integrative Japanese genome variation database based on whole-genome sequencing. Hum Genome Var 2, 15050 (2015).
Nagasaki, M. et al. Rare variant discovery by deep whole-genome sequencing of 1,070 Japanese individuals. Nat Commun 6, 8018 (2015).
Adzhubei, I. A. et al. A method and server for predicting damaging missense mutations. Nat Methods 7, 248–249 (2010).
Vaser, R., Adusumalli, S., Leng, S. N., Sikic, M. & Ng, P. C. SIFT missense predictions for genomes. Nat Protoc 11, 1–9 (2016).
Schwarz, J. M., Cooper, D. N., Schuelke, M. & Seelow, D. MutationTaster2: mutation prediction for the deep-sequencing age. Nat Methods 11, 361–362 (2014).
Chun, S. & Fay, J. C. Identification of deleterious mutations within three human genomes. Genome Res 19, 1553–1561 (2009).
Li, Q. et al. Gene-specific function prediction for non-synonymous mutations in monogenic diabetes genes. PLoS One 9, e104452 (2014).
Do, R. et al. Exome sequencing identifies rare LDLR and APOA5 alleles conferring risk for myocardial infarction. Nature 518, 102–106 (2015).
Stuart, B. D. et al. Exome sequencing links mutations in PARN and RTEL1 with familial pulmonary fibrosis and telomere shortening. Nat Genet 47, 512–517 (2015).
Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Statist Soc B 57, 289–300 (1995).
The members of the NHO-AIH study group are: Kiyoshi Migita (Fukushima Medical University School of Medicine), Hideo Nishimura (NHO Asahikawa Medical Center), Hironori Sakai (NHO Beppu Medical Center), Toshihiko Kaneyoshi (NHO Fukuyama Medical Center), Eiichi Takezaki (NHO Higashi Hiroshima Medical Center), Noboru Hirashima, Hironao Takahashi (NHO Higashinagoya National Hospital), Noriaki Naeshiro (NHO Higashihiroshima Medical Center), Yukio Oohara (NHO Hokkaido Medical Center), Hajime Ohta (NHO Kanazawa Medical Center), Takeaki Sato (NHO Kokura Medical Center), Kazuhiro Sugi (NHO Kumamoto Medical Center), Hiroshi Kohno (NHO Kure Medical Center and Chugoku Cancer Center), Motoyuki Kohjima, Makoto Nakamuta (NHO Kyushu Medical Center), Michio Kato, Iwao Yabuuchi (NHO Minami Wakayama Medical Center), Minoru Nakamura, Seigo Abiru, Sung Kwan Bae, Shigemune Bekki, Satoru Hashimoto, Hiromi Ishibashi, Yuka Jiuchi, Atsumasa Komori, Shinya Nagaoka, Masashi Ohtani, Katsumi Yamasaki, Hiroshi Yatsuhashi (NHO Nagasaki Medical Center), Masaaki Shimada (NHO Nagoya Medical Center), Fujio Makita (NHO Nishigunma National Hospital), Toyokichi Muro (NHO Oita Medical Center), Haruhiro Yamashita (NHO Okayama Medical Center), Eiji Mita (NHO Osaka Medical Center), Taizo Hijioka (NHO Osaka Minami Medical Center), Eiji Mita (NHO Osaka National Hospital), Yoko Nakamura, Yukio Watanabe (NHO Sagamihara National Hospital), Minoru Tomizawa (NHO Shimoshizu National Hospital), Kaname Yoshizawa (NHO Shinshu Ueda Medical Center), Atsushi Naganuma (NHO Takasaki General Medical Center), Masahiro Kikuchi (NHO Tokyo Medical Center), Hiroshi Kamitsukasa, Michiyasu Yagura (NHO Tokyo National Hospital), Keisuke Ario (NHO Ureshino Medical Center), Tatsuji Komatsu (NHO Yokohama Medical Center), Michio Yasunami (Saga-ken Medical Centre Koseikan, Life Science Institute), and Hiroshi Furukawa (University of Tsukuba, Faculty of Medicine). The work was supported by Grants-in-Aid for Clinical Research from National Hospital Organization.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Higuchi, T., Oka, S., Furukawa, H. et al. Role of deleterious single nucleotide variants in the coding regions of TNFAIP3 for Japanese autoimmune hepatitis with cirrhosis. Sci Rep 9, 7925 (2019). https://doi.org/10.1038/s41598-019-44524-5