Cytotoxic T-lymphocyte antigen 4 gene +49A/G polymorphism significantly associated with susceptibility to primary biliary cirrhosis: A meta-analysis

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1 Journal of Digestive Diseases 2011; 12; doi: /j x Meta analysis Cytotoxic T-lymphocyte antigen 4 gene +49A/G polymorphism significantly associated with susceptibility to primary biliary cirrhosis: A meta-analysis Rui Rui CHEN, Zhe Yi HAN, Jin Ge LI, Yong Quan SHI, Xin Min ZHOU, Jing Bo WANG, Xi Qiang CAI, Xue Chang WANG, Ying HAN & Dai Ming FAN Institute of Digestive Diseases, Xijing Hospital, State Key Laboratory of Cancer Biology, Fourth Military Medical University, Xi an, Shaanxi Province, China OBJECTIVE: To evaluate comprehensively the association of cytotoxic T-lymphocyte antigen 4 (CTLA-4) +49A/G polymorphism with susceptibility to primary biliary cirrhosis (PBC). METHODS: PubMed was used to search for the relevant published articles. The risk of PBC association with the CTLA-4 +49A/G polymorphism was estimated for each study in a random-effects model. Odds ratio (OR) and 95% confidence interval (CI) were estimated for each study. Risks to PBC were estimated by stratified analysis in patients with different ethnicity and antimitochondrial antibody (AMA) status, as well as histological stages. RESULTS: A total of 12 articles were included in the study. An association between PBC and CTLA-4 G allele was found, overall OR = 1.20, 95% CI (P = 0.02). However, stratification by ethnicity indicated a significant association between the G allele and PBC in Asians (OR = 1.36, 95% CI , P = 0.002), but not in Caucasians (OR = 1.15, 95% CI , P = 0.15). Moreover, AMA positive patients carrying G allele were more susceptible to PBC compared with AMA negative patients (OR = 1.23, 95% CI , P = 0.007; OR = 0.98, 95% CI , P = 0.88, respectively). CONCLUSIONS: Polymorphism in exon 1 of CTLA-4 gene at position 49 may act as a candidate of susceptibility locus to PBC. However, larger studies with participants of varying ethnicity and stratified by clinical and laboratory characteristics are needed to validate our findings. KEY WORDS: meta-analysis. cytotoxic T-lymphocyte antigen 4, single nucleotide polymorphism, primary biliary cirrhosis, Correspondence to: Ying HAN, Institute of Digestive Diseases, Xijing Hospital, State Key Laboratory of Cancer Biology, Fourth Military Medical University, 127 West Changle Rd, Xi an, Shaanxi Province , China. hanying1@fmmu.edu.cn Journal of Digestive Diseases 2011 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Blackwell Publishing Asia Pty Ltd. INTRODUCTION Primary biliary cirrhosis (PBC) is an autoimmune disease characterized by female preponderance and destruction of intrahepatic bile ducts that often results in cirrhosis and hepatic failure. 1 4 Twin and family aggregated data suggest that there is a significant genetic predisposition for PBC, and the complex features in etiology are thought to be resulted from the interaction between multiple genetic and environmental factors. 2,5 428

2 Journal of Digestive Diseases 2011; 12; CTLA-4 gene and PBC disease 429 Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a negative regulator of T-cell immune response and is expressed predominantly in activated regulatory T-cells. 6 8 The absence of gene in knockout or CTLA- 4-deficient mice leads to massive lymphoproliferative disorders, fatal multi-organ destruction and early death. 2,3 The apparent association between pathological T-cell response and the development of PBC 9,10 suggests that CTLA-4 might be an attractive and candidate facilitator of the PBC susceptibility gene. Variants in the gene encoding immunoregulatory CTLA-4 were examined for the candidate susceptibility loci for PBC. CTLA-4 gene is located on chromosome 2q33; the adjacent gene encoding another two immunoregulatory proteins: inducible co-stimulator and its stimulatory counterpart (CD28). 11 Significant amino-acid replacement resulted from single nucleotide polymorphisms (SNPs) has been described. For example, the T and C alleles of CTLA-4 318C/T and CTLA-4 CT60 have been widely screened for autoimmune disease association. 12 Several other SNP in promoter region have also been described, such as SNP at +49A/G in exon 1 of the gene. 4,9,12 18 In recent years, intensive work has been performed to elucidate such genetic predisposition to autoimmune diseases However, different studies had different conclusions about the association between the +49A/G polymorphism and susceptibility to PBC. Some studies reported that patients carrying the CTLA-4 G allele were more susceptible to the development of PBC compared with controls, 9,12 18,22 while some other studies did not support this result. 4,18 Hence, a comprehensive retrieval of the relevant literature was performed to evaluate whether the genetic polymorphisms are associated with PBC susceptibility. The aim of the study was to assess the association of CTLA-4 +49A/G polymorphism with PBC via a meta-analysis, as well as to quantify the inter-study heterogeneity. MATERIALS AND METHODS Literature search A computer-assisted search using PubMed databases was conducted to identify potentially relevant published articles in English (last update literature searched being March, 2010) using combinations of key words and search terms autoimmune disease, PBC, primary biliary cirrhosis, SNPs, Single nucleotide polymorphisms, CTLA-4, cytotoxic T- lymphocyte antigen 4, polymorphism, and variant. Meanwhile, reference lists of relevant publications were searched manually for additional articles. Study inclusion criteria and data extraction This meta-analysis included randomized controlled trials (RCTs) and cohort studies. In all cases, diagnosis of PBC was based on the internationally accepted criteria: (i) positive serum antimitochondrial antibody (AMA) ( 1:40); (ii) biochemical features of cholestasis, based mainly on alkaline phosphatase elevation for more than 6 months; 23 and (iii) histological changes consistent with PBC, which consist of nonsuppurative destructive cholangitis and destruction of interlobular bile ducts. Diagnosis of PBC could be established when two of these three criteria were fulfilled. Histological lesions were divided into four stages: stages I II were classified as early PBC and stages III IV as advanced PBC. 24 The latter were also defined by a history of major complications related to portal hypertension. 25 All eligible studies needed to provide descriptions on patient selection, allocation, study design and to be human studies. Articles without data of allele or genotyping frequency or without healthy controls were excluded. The data extracted were compared and any disagreement between the two authors (Rui Rui CHEN and Zhe Yi HAN) was resolved by consensus. Meta-analysis of relative risk Analysis was conducted with Review Manager (The Cochrane Collaboration, Oxford, UK). The results were obtained using SAS 13.0 (SAS Institute Inc., Cary, NC, USA). All P values were two-sided. The risk (odds ratio, OR) of PBC association with the CTLA-4 +49A/G polymorphism was estimated for each study in a random-effects model. OR and 95% confidence interval (CI) were estimated for each study. Furthermore, risks to PBC were estimated by stratified analysis in patients with different ethnicity and AMA status, as well as histological stages. Heterogeneity among studies was examined using the c 2 -based Q and I 2 statistics. A P value < 0.05 was considered statistically significant for the c 2 -based Q, and the I 2 statistic was interpreted as the proportion of total variation contributed by inter-study variation. If there was a significant heterogeneity (P < 0.05), then the random-effects model was used to pool the data. To make certain the summary OR, cumulative meta-analysis was performed as more data was accumulated. 26,27

3 430 RR Chen et al. Journal of Digestive Diseases 2011; 12; RESULTS Literature search A total of 12 appropriate articles were included for this meta-analysis. 4,9,12 18,25,28,29 We established a database in accordance with the information extracted from these articles. One article was excluded because it overlapped other two selected articles with larger sample size. 22 Articles did not include allele or genotyping frequency (n = 1) 11 for OR calculation were excluded as well. Unfortunately, a large-scale genetic study reported by Hirschfield et al. 30 as a supplement in Nature Genetics 2010 reporting findings on various gene associations, including CTLA-4, could not be included because of lacking of data we needed. Table 1 showed the characteristics of these articles, all of which were published in English. Nine articles were studies on Caucasians, 4,9,15 18,25,28,29 and three were on Asians, including two on Japanese 12,14 and one on Chinese. 13 The number of patients and controls ranged from 45 to 506 and 73 to 1248, respectively. In total, 2137 cases were histologically confirmed while 1111 were not. The controls were mainly of a healthy group. Juran et al. published three articles 15,18,22 on PBC and CTLA-4 gene polymorphisms, however, the result of +49A/G alleles and genotypes in one article 18 was overlapped with the result of other two articles 15,22 with larger size of participants. Hence, we used the most recent article. The distribution of genotypes in the controls of most articles was in agreement with the Hardy Weinberg equilibrium (goodness of fit, c 2 test, degree of freedom [d.f.] =1). Quantitative synthesis The overall OR and 95% CI showed that patients carrying the CTLA-4 G allele had a slightly increased risk of developing PBC (OR = 1.20, 95% CI , P = 0.02) (Fig. 1), especially when stratified by ethnicity. It was shown that the G allele was significantly associated with PBC in Asians (OR = 1.36, 95% CI , P = 0.002). However, no association was found in Caucasians (OR = 1.15, 95% CI , P = 0.15). Table 2 lists the associations between G allele and AMA status or histological stages. First, we stratified the analysis according to whether the PBC patients were AMA positive. In patients with G allele, those who were AMA positive had a slightly increased risk of PBC (OR = 1.23, 95% CI , P = 0.007), while those who were AMA negative did not (OR = 0.98, 95% CI , P = 0.88) (Fig. 2). No association between CTLA-4 +49A/G polymorphism and PBC was observed when stratified by histological stages (stages I II, OR = 1.22, 95% CI , P = 0.07; stages III IV, OR = 1.18, 95% CI , P = 0.36) (Fig. 3). Test of heterogeneity and publication bias Each time one of the studies involved in the metaanalysis was deleted to examine the influence of the individual data set on pooled OR, and the corresponding pooled OR was not materially altered. For the CTLA-4 +49A/G allele and homozygote comparisons, heterogeneity was observed (c 2 = 48.72, d.f. = 12, P < ). There was also heterogeneity among studies of Caucasian participants when evaluated by ethnicity (c 2 = 42.84, d.f. = 8, P < ) but not among studies of Asian participants (c 2 = 3.09, d.f. = 3, P = 0.38). There was no heterogeneity with respect to histological stage (c 2 = 5.53, d.f. = 7, P = 0.60) and AMA status (c 2 = 5.92, d.f. = 7, P = 0.55). DISCUSSION Allele variation at position 49 of CTLA-4 exon 1 was significantly associated with PBC. Polymorphisms in CTLA-4 gene have been reported to be associated with numerous autoimmune diseases. 15,17 19 There are a series of studies on CTLA-4 +49A/G polymorphism in Graves disease. Kouki et al. and Maurer et al. found a correlation of +49A/G genotype with reduced inhibitory function of CTLA-4, and suggested that this particular polymorphism was actually of a disease-associated allele. 31,32 In this metaanalysis, we found a strong association between allele variation at position 49 of CTLA-4 exon 1 and PBC in Asian population. The frequency of G allele increased in patients with PBC compared with that in controls, and the G allele conferred susceptibility to PBC in Asians. These findings are consistent with the role of CTLA-4 gene variants in predisposing to PBC and with the notion that CTLA-4 region is a shared susceptibility locus for autoimmunity and therefore is a potential candidate of therapeutic target. In stratification by ethnicity, we found a significant association between CTLA-4 +49A/G allele with

4 Journal of Digestive Diseases 2011; 12; CTLA-4 gene and PBC disease 431 Table 1. Allele and genotype frequencies of patients and controls of the studies included in the meta-analysis First author Year Country/ethnicity Genotype GF (%) GAF (%) HWE (P value) PBC Control PBC Control PBC Control Kanno Japan/Asian A/A 5 (11) 14 (19) A/G 20 (44) 33 (45) G/G 20 (44) 26 (36) Agarwal UK/Caucasian A/A 57 (28.5) 99 (49.5) G/A 106 (53) 80 (40) G/G 37 (18.5) 21 (10.5) Juran USA/Caucasian A/A 131 (37) 79 (39) A/G 161 (46) 99 (48) G/G 59 (17) 27 (13) Fan China/Asian A/A 6 (7.8) 23 (14.4) A/G 34 (44.2) 93 (58.1) G/G 37 (48) 44 (27.5) Walker USA/Caucasian A/A 162 (33.6) 493 (39.5) A/G 223 (46.4) 577 (46.2) G/G 96 (20) 178 (14.3) Bittencourt Brazilian/Caucasian A/A 23 (46) 29 (43) A/G 23 (46) 30 (45) G/G 4 (8) 8 (12) Juran USA/Caucasian A/A 58 (16.1) 43 (10.6) (Mayo) A/G 142 (39.4) 179 (44.3) G/G 160 (44.4) 182 (45) Juran America/Caucasian A/A 83 (16.4) 36 (10.1) (Toronto) A/G 215 (42.5) 153 (42.9) G/G 208 (41.1) 168 (47.1) Donaldson Italy and UK/Caucasian A/A 115 (36.4) 160 (41.0) A/G 159 (50.3) 177 (45.4) G/G 42 (13.3) 53 (13.6) Oertelt Italy/Caucasian A/A 27 (17.5) 45 (26.7) A/G 87 (56.5) 72 (44.3) G/G 40 (26) 49 (28.9) Schott NA/Caucasian A/A 58 (32.2) 78 (38.6) A/G 90 (50.0) 87 (43.1) G/G 32 (17.8) 37 (18.3) Poupon French/Caucasian A/A A/G G/G Joshita Japan/Asian A/A (Shinshu) A/G G/G Joshita Japan/Asian A/A (Nagasaki) A/G G/G GAF, G allele frequency; GF, genotype frequency; HWE, Hardy Weinberg equilibrium; NA, not available; PBC, primary biliary cirrhosis. PBC in Asians, but not in Caucasians. The data suggested that certain effects of the genetic polymorphism appear to be population-specific. Different linkage disequilibrium (LD) patterns might contribute to the discrepancy. This polymorphism might be in LD with a nearby causal variant seen in one ethnic group but not in another, 26 which could probably explain why CTLA-4 +49A/G polymorphisms contribute to PBC risk mainly in Asian populations. Interestingly, the analysis revealed a slightly increased risk of AMA positive PBC in patients with G allele. This

5 432 RR Chen et al. Journal of Digestive Diseases 2011; 12; Figure 1. Odds ratio (OR, log scale) of primary biliary cirrhosis (PBC) associated with cytotoxic T-lymphocyte antigen 4 (CTLA-4) gene +49A/G allele in different ethnicities. The estimate of OR and its 95% confidence interval (CI) is plotted with a box and a horizontal line; ( ) pooled OR and its 95% CI. Figure 2. Odds ratio (OR, log scale) of primary biliary cirrhosis (PBC) of antimitochondrial antibody (AMA) positive and AMA negative patients associated with cytotoxic T-lymphocyte antigen 4 (CTLA-4) gene +49A/G alleles in the studies. The estimate of OR and its 95% confidence interval (CI) is plotted with a box and a horizontal line; ( ) pooled OR and its 95% CI.

6 Journal of Digestive Diseases 2011; 12; CTLA-4 gene and PBC disease 433 Figure 3. Odds ratio (OR, log scale) of primary biliary cirrhosis (PBC) of early stage and late stage associated with cytotoxic T-lymphocyte antigen 4 (CTLA-4) gene +49A/G alleles in the studies. The estimate of OR and its 95% confidence interval (CI) is plotted with a box and a horizontal line; ( ) pooled OR and its 95% CI. finding is in agreement with the Caucasian study by Juran et al. 18 and thus constitutes a promising susceptibility gene candidate. It was reported that 49 minor allele frequencies reduced in AMA positive PBC patients compared to those in AMA negative patients in three studies. 12,14,15 Additionally, CTLA-4 had a noteworthy impact on risk of PBC and might play a role in mediating AMA development according to a haplotype analysis. 22 So we considered seriously the disease sub-phenotypes like AMA status in the analysis. It should be very careful when the predominant autoreactive antibody is detected in serum of an individual carrying CTLA-4 +49G allele, considering it has a high sensitivity and specificity to PBC. However, the number of patients showing AMA negativity was too small to evaluate. We had to use as much data as we could, for example, we used the work of Juran et al. 18 to increase the number of allele and genotype in our analysis, and the work of Juran et al. 15 to increase the number of AMA subgroups of patients with PBC. In this respect we need more studies to gain greater insight into the genetic basis of this perplexing disease. No statistical significance was found with regard to disease progression and this was incompatible with previous studies. 12 More evidence for this was derived from an analysis of patients who had received orthotropic liver transplantation (OLT) probably due to advanced disease stage. Notably, a significant increase in autoimmune-risk G allele and G/G genotype of +49A/G was observed in patients who had received OLT than in those who had not. 15 Considering that +49A/G might affect cell surface expression of CTLA-4, it is reasonable to assume that the G allele could have an impact on T-cell function as well as disease progression. The risk with CTLA-4 +49G allele for PBC patients in both early and late stage had increased, although it did not reach statistical significance. This was possibly due to the small number of study samples. A recent meta-analysis by Miyake et al. 33 suggested that the CTLA-4 +49A/G polymorphism may be associated with susceptibility to PBC, which is consistent with our results. However, there are some differences between the two meta-analysis. First, Miyake et al. 33 tested the association of genotype of CTLA-4 +49A/G polymorphisms with PBC, but we did not. Second, risks to PBC were estimated by a stratified analysis in patients with different ethnicity, AMA status as well as histological stages in our metaanalysis, however, Miyake et al. studied subgroups by different ethnicity only. 33 In conclusion, we found that there was a modest but significant association between the CTLA-4 +49A/G polymorphism and susceptibility to PBC in this metaanalysis. However, data with respect to CTLA-4 polymorphisms and PBC are limited and were generally

7 434 RR Chen et al. Journal of Digestive Diseases 2011; 12; Table 2. Clinico-laboratory findings in primary biliary cirrhosis (PBC) patients according to the genotype variations at exon 1 position 49 on CTLA-4 gene AMA+ PBC AMA- PBC PBC stage I/II PBC stage III/IV Controls GF (%) GAF (%) GF (%) MAF (%) GF (%) MAF (%) GF (%) MAF (%) GF (%) MAF (%) Country/ ethnicity Genotype First author Year Kanno Japan/Asian A/A 4 (80) (20) (80) (20) (19) 58.2 A/G 14 (72) 6 (28) 17 (85) 3 (15) 33 (45) G/G 14 (70) 6 (30) 11 (55) 9 (45) 26 (36) Juran America/ A/A 112 (37) (43) (39) 37.3 Caucasian A/G 134 (44) 24 (55) 99 (48) G/G 57 (19) 1 (2) 27 (13) Joshita Japan/Asian A/A A/G G/G Oertelt Italy A/A 25 (18.9) (9.1) (15.8) (19.2) (26.7) 51.2 A/G 71 (53.8) 16 (72.7) 43 (56.6) 44 (56.4) 72 (44.3) G/G 36 (27.3) 4 (18.2) 21 (27.6) 19 (24.4) 49 (28.9) Bittencourt Brazilian/ A/A 10 (53) (57) (43) 34.3 Caucasian A/G 6 (32) 6 (29) 30 (45) G/G 3 (16) 3 (14) 8 (12) AMA, anti-mitochondrial antibody; GAF, G allele frequency; GF, genotype frequency; HWE, Hardy Weinberg equilibrium; MAF, minor allele frequency. obtained from relatively small sample size studies. Only 12 studies were included in this study. This paucity of data may cause genetic heterogeneity. Meanwhile, the precise function of SNP remains undefined. All data mentioned above should be interpreted very carefully. Sequencing the entire gene and assessing the functional role of SNP will be required. The association between genetic variants and function of CTLA-4 gene remains to be elucidated in future investigations. ACKNOWLEDGEMENT This study was approved in part by the Chinese National Nature Science Foundation (No , , , ), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, China (No. HG3505), and the Scientific Technology Innovation Foundation from Shaanxi Province, China (No. 2009ZDKG-71). REFERENCES 1 Selmi C, Zuin M, Biondi ML et al. Genetic variants of endothelial nitric oxide synthase in patients with primary biliary cirrhosis: association with disease severity. J Gastroenterol Hepatol 2003; 18: Lee JC, Bridger S, McGregor C, Macpherson AJ, Jones JE. Why children with inflammatory bowel disease are diagnosed at a younger age than their affected parent. Gut 1999; 44: Donaldson P, Agarwal K, Craggs A, Craig W, James O, Jones D. HLA and interleukin 1 gene polymorphisms in primary biliary cirrhosis: associations with disease progression and disease susceptibility. Gut 2001; 48: Bittencourt PL, Palacios SA, Farias AQ et al. Analysis of major histocompatibility complex and CTLA-4 alleles in Brazilian patients with primary biliary cirrhosis. J Gastroenterol Hepatol 2003; 18: Jones DE, Watt FE, Metcalf JV, Bassendine MF, James OF. Familial primary biliary cirrhosis reassessed: a geographically-based population study. J Hepatol 1999; 30: Ueda H, Howson JM, Esposito L et al. Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 2003; 423: Thompson CB, Allison JP. The emerging role of CTLA-4 as an immune attenuator. Immunity 1997; 7: Kristiansen OP, Larsen ZM, Pociot F. CTLA-4 in autoimmune diseases a general susceptibility gene to autoimmunity? Genes Immun 2000; 1: Agarwal K, Jones DE, Daly AK et al. CTLA-4 gene polymorphism confers susceptibility to primary biliary cirrhosis. J Hepatol 2000; 32: Krawitt EL. Can you recognize autoimmune hepatitis? Postgrad Med 1998; 104: Ramirez-Soriano A, Lao O, Soldevila M, Calafell F, Bertranpetit J, Comas D. Haplotype tagging efficiency in worldwide populations in CTLA4 gene. Genes Immun 2005; 6:

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