Abstract
Autoimmune hepatitis (AIH) is an uncommon chronic autoimmune liver disease. Several studies reported the association of polymorphisms between CD28, CTLA4 and ICOS gene cluster in 2q33.2 with autoimmune or inflammatory diseases. The previous genome-wide association study on type 1 AIH in a European population has reported a risk G allele of a single nucleotide polymorphism (SNP), rs4325730, in this region. Here, we conducted an association study of this SNP with type 1 AIH in a Japanese population, as a replication study.An association study of rs4325730 was conducted in 343 Japanese AIH patients and 315 controls.We found that rs4325730 is associated with AIH (P=0.0173, odds ratio (OR) 1.30, 95% confidence interval (CI) 1.05–1.62, under the allele model for G allele, P=0.0070, OR 1.62, 95% CI 1.14–2.31, under the dominant model for G allele). This SNP was strongly associated with definite AIH (P=0.0134, OR 1.36, 95% CI 1.07–1.74; under allele model for G, P=0.0035, OR 1.85, 95% CI 1.22–2.81, under dominant model for G).This is the first replication association study of rs4325730 upstream of ICOS with AIH in the Japanese population and rs4325730G is a risk allele.
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Introduction
Autoimmune hepatitis (AIH) is an uncommon chronic autoimmune liver disease that can cause cirrhosis.1, 2, 3 AIH is characterized by the elevated transaminases and immunoglobulin levels and the presence of auto-antibodies and interface hepatitis. Type 1 AIH is distinguished by the presence of serum anti-nuclear antibodies or anti-smooth muscle antibodies and type 2 AIH by type 1 liver-kidney microsomal antibodies. AIH in Japanese populations is consisted mainly of type 1. The genetic risk factors are thought to be associated with AIH. AIH is associated with human leukocyte antigen (HLA)-DRB1*03:01, and DRB1*04:01 in European populations4 and DRB1*04:05 in Japanese populations.5, 6, 7 Polymorphisms in non-HLA genes also confer the genetic risk of AIH. A recent genome-wide association study of type 1 AIH in a European population has identified genetic risk factors in HLA and non-HLA regions.8 Genetic associations of SNPs in non-HLA genes with AIH in Japanese populations were also investigated; associations were detected in STAT49 and PTPN22,10 but not in CARD10,11 CTLA412 or FCRL3.13
CD28, CTLA4 and ICOS genes are clustered in human chromosome 2q33.2. The products of these genes were CD28-family member molecules and play important roles on the T cell stimulation, transducing co-stimulatory or inhibitory signals. It was predicted that these genes play key roles in autoimmune disease or tumor bearing patients. The previous genome-wide association study on type 1 AIH in a European population has reported a risk G allele of a single nucleotide polymorphism (SNP), rs4325730, in this region.8 This SNP is located upstream of ICOS and is in strong linkage disequilibrium in European with rs4675374 (r2=1)8 that was reported to be associated with celiac disease.14, 15 Here, we conducted an association study of this SNP with type 1 AIH in a Japanese population, as a replication study.
Materials and Methods
Patients and controls
Type 1 AIH patients (n=343; median age (interquartile range), 64 (55–73), 41 male (12.0%)) who satisfied the criteria of International Autoimmune Hepatitis Group (IAIHG score>9)16 without any other types of liver diseases were enrolled from the register of Japanese National Hospital Organization (NHO) Liver Registry. Among 343 AIH patients, 214 were definite AIH patients (IAIHG score>15, 62.4%). The healthy controls (n=315; median age (interquartile range), 36 (31–46), 2 male (0.6%)) were recruited at Sagamihara Hospital or by the Pharma SNP Consortium (Tokyo, Japan).17 All the patients and healthy individuals were native Japanese living in Japan. This study was reviewed and approved by the NHO central Institutional Review Board 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
Genotyping of rs4325730 (G/A) upstream of ICOS was performed using Custom TaqMan SNP Genotyping Assay (Thermo Fisher Scientific Inc., Waltham, MA, USA) on 7500 Fast Real-Time PCR System (Thermo Fisher Scientific Inc.), according to the manufacturer’s instructions. Thermal cycling conditions consisted of initial denaturation at 95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s followed by 60 °C for 30 s. A representative allelic discrimination plot of rs4325730 is shown in Supplementary Figure 1.
Statistical analysis
The distribution of allele and genotype frequencies was compared between AIH patients and healthy controls by chi-square analysis using 2 × 2 contingency tables. Overall AIH or definite AIH subset were compared with healthy control. Association of clinical features of the AIH patients with or without G allele of rs4325730 was tested by chi-square analysis using 2 × 2 contingency tables or Mann–Whitney's U Test. This study had 80% statistical power to detect associations when the genotype relative risk was higher than 1.36.18
Results
Association analysis of rs4325730 upstream of ICOS with AIH
The genotyping of rs4325730 was performed in Japanese AIH patients and healthy controls. No deviation from Hardy-Weinberg equilibrium was detected in the controls (P=0.8790) or overall AIH patients (P=0.0573). The number of heterozygout AIH patients tended to be higher than expected. A significant association was found for rs4325730 (P=0.0173, odds ratio (OR) 1.30, 95% confidence interval (CI) 1.05–1.62, Table 1) under the allele model for G allele. This SNP was also significantly associated with the overall AIH (P=0.0070, OR 1.62, 95% CI 1.14–2.31) under the dominant model for G allele. We further explored associations between rs4325730 and definite AIH and rs4325730 was more significantly associated with definite AIH (P=0.0134, OR 1.36, 95% CI 1.07–1.74; under allele model for G, P=0.0035, OR 1.85, 95% CI 1.22–2.81, under dominant model for G). Thus, the association of rs4325730 with AIH was detected in the Japanese population and more important roles of the SNP in definite AIH were suggested.
Finally, we analyzed the clinical phenotypes of AIH patients with or without G allele of rs4325730. No significant difference of demographic features of the AIH patients with or without G allele of rs4325730 was detected (Table 2).
Discussion
The previous genome-wide association study reported an association of SNPs in non-HLA regions, rs3184504 in SH2B3 and rs6000782 in CARD10, with type I AIH.8 Polymorphism of rs3184504 was not detected in Japanese and rs6000782 was not associated with AIH in Japanese.11 The present study showed that rs4325730G is a risk allele for type I AIH in the Japanese population. To the best of our knowledge, we were the first to replicate the association of SNPs in non-HLA region reported in the genome-wide association study with Japanese AIH.
Several studies reported the association of polymorphisms between CD28, CTLA4 and ICOS gene cluster in 2q33.2 with autoimmune or inflammatory diseases. However, most of the disease-associated variants reported were located between CD28 and CTLA4.19, 20, 21, 22 A few studies identified disease-associated SNPs in ICOS gene.14, 15 These studies reported a celiac disease associated SNP, rs4675374, located in the first intron of ICOS. The location of rs4325730 is 3 kbps upstream of ICOS gene. Two SNPs, rs4675374 and rs4325730 are in strong linkage disequilibrium in European (r2=1)8 and Japanese (r2=0.981, http://www.ensembl.org/), indicating the shared susceptible SNPs between celiac disease and type I AIH. These data suggested the common signaling pathways in the pathogenesis of these diseases. Future fine mapping studies of this region should be performed.
AIH shares some clinical features with systemic lupus erythematosus (SLE)23, 24 and also shares some susceptible genes, HLA4, 5, 6, 7, 25 and STAT4.9, 26 In the present study, rs4325730 upstream of ICOS was associated with AIH, but the association of SNPs in CTLA4 was not detected in Japanese AIH.12 On the other hand, it was reported that SNPs upstream of ICOS was not associated with SLE in Japanese.27 An association was reported between SLE and SNPs located in CTLA4.28 These results suggested that SLE and type I AIH do not share susceptible SNPs in this region.
We first reported the association of polymorphisms upstream of ICOS with Japanese AIH. However, functional influences and pathological roles of the SNP in AIH are still unknown. The SNP, rs4325730, might be in histone marks sites (http://www.broadinstitute.org/mammals/haploreg/haploreg.php)29 and influence the expression pattern of CD28 and ICOS genes (http://www.genenetwork.nl/bloodeqtlbrowser/).30 ICOS molecules are expressed on activated T cells and play important roles in the maintenance of the T cell activation. Since ICOS-deficient mice have reduced germinal center formation,31 elevated expression levels of ICOS molecules may change the development of T follicular helper cells and the formation of germinal centers, leading to the altered development of autoreactive B cells.
We also detected a stronger association of rs4325730 with definite AIH patients than overall AIH. It was suggested that the predisposing effects of rs4325730 was enhanced in AIH patients diagnosed more strictly, though other each clinical manifestation of AIH is not correlated with the presence of rs4325730 risk allele (Table 2). Since rs4325730G allele frequencies in other ethnic populations are different, the association of the SNP with AIH in other populations should be replicated. Although it is difficult to increase the sample size, the associations of rs4325730 should be confirmed in future large scale studies.
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Acknowledgements
The members of the NHO-AIH study group are: KM (Department of Rheumatology, School of Medicine, Fukushima Medical University), Masashi Ohtani, SA, Yuka Jiuchi, SN, Sung Kwan Bae, AK, Masashi Ohtani, SH, Shigemune Bekki, Katsumi Yamasaki, HY, Hiromi Ishibashi (NHO Nagasaki Medical Center), Minoru Nakamura (Department of Hepatology, Nagasaki University Graduate School of Biomedical Sciences), Michio Yasunami (Life Science Institute, Saga-ken Medical Centre Koseikan), Yukio Watanabe, Yoko Nakamura (NHO Sagamihara National Hospital), Michiyasu Yagura (NHO Tokyo National Hospital), Tatsuji Komatsu (NHO Yokohama Medical Center), MS (NHO Nagoya Medical Center), Hiroshi Kouno (NHO Kure Medical Center), Taizo Hijioka (NHO Osaka Minami Medical Center), Motoyuki Kohjima (NHO Kyushu Medical Center), Michio Kato (NHO Minami Wakayama Medical Center), KY (NHO Shinshu Ueda Medical Center), Hajime Ohta (NHO Kanazawa Medical Center), Eiichi Takezaki (NHO Higashi Hiroshima Medical Center), HN (NHO Asahikawa Medical Center), Takeaki Sato (NHO Kokura Medical Center), KA (NHO Ureshino Medical Center), Noboru Hirashima (NHO Higashi Nagoya National Hospital), Yukio Oohara (NHO Hokkaido Medical Center), HY (NHO Okayama Medical Center), AN (NHO Takasaki General Medical Center), Toyokichi Muro (NHO Oita Medical Center), Hironori Sakai (NHO Beppu Medical Center), Eiji Mita (NHO Osaka Medical Center), Kazuhiro Sugi (NHO Kumamoto Medical Center), FM (NHO Nishigunma National Hospital). The work was supported by Grants-in-Aid for Clinical Research from National Hospital Organization. The funders had no role in study design, data collection and analysis, decision to publish, or preparing the manuscript.
Author contributions
Conceived and designed the experiments: HF, AK, NT and KM, Performed the experiments: TH, SO and HF, Analyzed the data: TH and HF, Contributed reagents/materials/analysis tools: HF, MN, AK, SA, SN, SH, AN, NN, KY, MS, HN, MT, MK, FM, HY, KA, HY, ST and KM. Wrote the manuscript: TH, HF, AK, NT and KM.
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Higuchi, T., Oka, S., Furukawa, H. et al. Association of a single nucleotide polymorphism upstream of ICOS with Japanese autoimmune hepatitis type 1. J Hum Genet 62, 481–484 (2017). https://doi.org/10.1038/jhg.2016.155
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DOI: https://doi.org/10.1038/jhg.2016.155
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