Frequency and Nature of Cytokine Gene Polymorphisms in Type 1 Autoimmune Hepatitis

Transcription

1 Frequency and Nature of Cytokine Gene Polymorphisms in Type 1 Autoimmune Hepatitis SHARON COOKSON, 1 PATRIZIA K. CONSTANTINI, 1 MICHAEL CLARE, 1 JAMES A. UNDERHILL, 1 WILL BERNAL, 1 ALBERT J. CZAJA, 2 AND PETER T. DONALDSON 1 Genetic involvement in type 1 autoimmune hepatitis (AIH) is indicated by a marked female preponderance and strong, well-established, human leukocyte antigen (HLA) associations. These associations, however, are not universal and a number of genes outside the major histocompatibility complex may also play a role in susceptibility to type 1 AIH. Prime candidates at present are those polymorphic genes encoding the proinflammatory and immunoregulatory cytokines. The aim of this study was to investigate, for the first time, 2 members of the interleukin-1 (IL-1) family (IL-1B and IL-1RN), 3 polymorphic sites in the interleukin-10 (IL-10) gene promoter (positions 1082, 819, and 592), and 2 polymorphisms in the tumor necrosis factor- (TNF- ) promoter (positions 308 and 238) in type 1 AIH. The study was performed on 2 independently collected DNA banks, each with appropriate controls, and throughout the analysis associations described in the first set were confirmed in the second set. Standard polymerase chain reaction (PCR)-based genotyping techniques were used. Overall there were no significant differences in the distributions of the IL-1B and IL-10 alleles, genotypes, or haplotypes in either study set. In contrast we report a significant association between type 1 AIH and TNF*2 (first set: 34% of controls vs. 49% of patients, Pc.014 and second set: 26% vs. 56%, P.00008). However, TNF*2 is found in strong linkage disequilibrium with the HLA A1-B8-DR3 haplotype and stratification analysis indicates that the association with TNF*2 is interdependent with HLA DRB1*0301. This is an indication that there is more than one susceptibility allele for type 1 AIH on chromosome 6p21.3. (HEPATOLOGY 1999;30: ) Autoimmune hepatitis (AIH) is an unresolving hepatocellular inflammation of unknown cause. The diagnosis requires the presence of interface hepatitis on histological examina- Abbreviations: AIH, autoimmune hepatitis; HLA, human leukocyte antigen; TNF, tumor necrosis factor; PSC, primary sclerosing cholangitis; PBC, primary biliary cirrhosis; IL, interleukin; IL-1RA, interleukin-1 receptor agonist. From the 1 Institute of Liver Studies, King s College Hospital, London, UK; and the 2 Department of Hepatology and Gastroenterology, Mayo Clinic, Rochester, MN. Received April 26, 1999; accepted July 12, P.T.D. s Present address is the Centre for Liver Research, Faculty of Clinical Medical Sciences, The Medical School, University of Newcastle, Newcastle upon Tyne, UK. Address reprint requests to: Peter T. Donaldson, M.D., Centre for Liver Research, Faculty of Clinical Medical Sciences, The Medical School, University of Newcastle, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. P.T.Donaldson@ ncl.ac.uk; fax: (44) Copyright 1999 by the American Association for the Study of Liver Diseases /99/ $3.00/0 tion, hypergammaglobulinemia, marked elevation in serum aminotransferase, and circulating liver-associated autoantibodies. 1 Type 1 (classic) AIH is characterized by the presence of either antinuclear antibodies, smooth muscle antibodies, or both and is the major form of autoimmune hepatitis among white adults. Type 2 AIH is characterized by antibodies to liver-kidney microsome type 1 and it occurs mainly in children. 2,3 Genetic factors are important in the development of most autoimmune diseases including systemic lupus erythematosus, insulin-dependent diabetes mellitus, multiple sclerosis, and rheumatoid arthritis. 4 In type 1 AIH, genetic involvement is indicated by a marked female preponderance and strong, well-established, human leukocyte antigen (HLA) associations. 5,6 Recent studies suggest that approximately 85% of northern European and North American white patients with type 1 AIH are HLA DRB1*0301 and/or DRB1*0401 positive. 7,8 Thus 55% of northern European and 51% of North American patients have DRB1*0301 compared with 24% and 19% of controls respectively and, 54% and 55% of DRB1*0301 negative patients from each population, respectively, have DRB1*0401 compared with 23% and 17% of DRB1*0301 negative controls. 7,8 The genetic bases for susceptibility to type 1 AIH are not fully understood, but the HLA alleles DRB1*0301 and DRB1*0401 share a common 6 amino acid motif, LLEQKR, at positions of the DR polypeptide of the expressed DR molecule. 7,8 Within this motif the critical amino acid appears to be a lysine residue at position 71. This residue, which is on the lip of the antigen-binding groove, may influence both the steric configuration of the antigen-presenting complex and its subsequent recognition by immunocytes. 8 One explanation for the association with this motif is that it may preferentially bind and present an (as yet unidentified) autoantigenic peptide. Not all patients with type 1 AIH share these same risk factors and type 1 AIH in patients from Argentina, 9 Mexico, 10 and Japan 11 is associated with different HLA alleles. A number of genes outside the major histocompatibility complex may also play a role in susceptibility to type 1 AIH, including the T cell receptor constant region genes, 12 the T cell receptor variable region genes, 13 and those of the immunoglobulin heavy chain constant region. 14 None of these, however, account for all instances of disease and other genetic and/or environmental factors must be involved. Prime candidates include those polymorphic genes encoding the proinflammatory and immunoregulatory cytokines. Recently polymorphisms in the tumor necrosis factor (TNF- ) promoter gene sequence at position 308 have been associ- 851

2 852 COOKSON ET AL. HEPATOLOGY October 1999 ated with primary sclerosing cholangitis (PSC) 15 and primary biliary cirrhosis (PBC). 16 However, neither PSC nor PBC appear to be associated with the 3 known polymorphisms of the interleukin-10 (IL-10) promoter (Donaldson PT, unpublished data), 17 and although polymorphisms of the IL-1 gene family have been extensively studied in inflammatory bowel disease, they have not been assessed in AIH, PSC, or PBC. IL-1 and TNF- are the key cytokines in the inflammatory response. The IL-1 gene family on chromosome 2q13 20 encodes 3 proteins, including IL-1, IL-1, and the IL-1 receptor antagonist (IL-1RA). IL-1RA competes with IL-1 (and IL-1 ) for binding to the IL-1 receptors and is a potent inhibitor of IL-1 activity. 21 Four nucleotide substitutions of the IL-1 gene (IL-1B) have been described, but only one, which forms a Taq1 restriction fragment length polymorphism at position 3953 in exon 5, has been widely investigated for its effect on protein production. The presence of the less common allele 2 (IL-1B*2) at this site has been associated with increased IL-1 secretion in vitro. 22 Studies of the gene encoding IL-1RA (which is referred to as IL-1RN), the second intron of which contains a variable number of tandem repeat sequences, have also indicated that genetic polymorphism may influence biological activity. 23 Five alleles have been described, corresponding to 2 to 6 copies of the sequence, but only 2 are commonly found, the 4-repeat (IL-1RN*1) and the 2-repeat (IL-1RN*2) variants. In vitro studies have shown that the IL-1RN*2 allele is associated with high IL-1RA production in healthy volunteers. 24 Linkage has also been recognized between IL-1RN*2 and IL-1B*1, which is associated with high IL-1 production. 25,26 The gene encoding TNF- lies in the HLA class III region within the chromosome 6p21.3 band. 27 Seven polymorphisms of this gene have been described, including 2 in the promoter sequence at positions 308 and ,29 The rare 308A allele (TNF*2) may be associated with increased transcriptional activity compared with 308G (TNF*1),30 but the effect of TNF*2 on TNF- production in vitro is unclear. Some studies have found no difference in the level of TNF- secreted between TNF*1 and TNF*2 carriers, 31,32 whereas others have reported increased TNF- production in TNF*2 carriers. 33 The secretion of TNF- and IL-1 is regulated by IL-10, 34 which has been mapped to chromosome Recent studies have identified 3 single base pair exchange polymorphisms at positions 1082, 819, and 592 in the IL-10 promoter. One of these, the substitution adenosine (A) for cytosine (C) at position 1082, correlates with low IL-10 production in vitro. 36 The aim of this study was to investigate the relationship between these candidate genes and susceptibility to type 1 AIH. We chose to study 2 members of the IL-1 gene family: IL-1B and IL-1RN, 3 polymorphic sites in the IL-10 gene promoter ( 1082, 819, 592), and 2 polymorphisms in the TNF- promoter ( 308, 238). The study was performed on two independently collected DNA banks with associations described in the first set being confirmed in the second set. PATIENTS AND METHODS Study Population. Two independently collected series were studied, the first from King s College Hospital and the second from the Mayo Clinic. Each series included only patients who fulfilled the diagnostic criteria recommended by the International Autoimmune Hepatitis Group. 37 All patients were classified as having type 1 AIH based on antibody profiles and were of northern European white background. 3 The first study population (King s College Hospital) consisted of 84 adult patients. The median age of presentation was 45 years (range, 16 to 68 years) and 58 (69%) were female. Hepatitis B surface antigen and antibodies to hepatitis C virus were undetectable by second generation enzyme-linked immunosorbent assays. Control DNA samples were drawn from a bank of 134 geographically and racially matched health care workers, 51 of whom were women (38%) and none of whom had a history of liver disease or autoimmune disease. The second study population consisted of 86 adult patients seen by 1 investigator (A.J.C.) at the Mayo Clinic, Rochester, MN. The mean age at presentation was 45 years (range, 13 to 73 years) and 67 were women (78%). Each lacked serological evidence of infection with hepatitis B and C viruses by second generation enzyme-linked immunosorbent assays. Control DNA was drawn from a bank of 102 healthy white adults in the Mayo Clinic volunteer blood donor program. The mean age of the control group was 40 years (range, 26 to 68) and 59 were women (58%). Each had indicated the absence of major illness on the standard blood donation questionnaire. All were residents of Olmstead County, MN, and their racial backgrounds were consistent with those of the patient group. Determination of Gene Polymorphisms. One hundred nanograms of genomic DNA that had been extracted from whole blood was amplified in reaction mixtures containing 200 µmol/l each of datp, deoxycytidine triphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate (Amersham Pharmacia-Biotech, St. Albans, UK), 1.5 mmol/l MgCl 2, 10 mmol/l Tris-HCl ph8.3, 50 mmol/l KCl, 0.01% gelatin, 0.5 µmol/l of each primer (Table 1) and 2 to 2.5 U Taq Polymerase (Perkin Elmer, Norwalk, CT) on a Perkin-Elmer GeneAmp 9600 according to the following protocols. Polymorphisms of IL-1B. A 249-bp fragment of the fifth exon encoding IL-1B (positions 3816 to 4066) was amplified in a 25-µL reaction mixture. The conditions for amplification were as follows: 96 C for 5 minutes; 3 cycles of 96 C for 90 seconds, 53 C for 90 seconds, 72 C for 90 seconds; 35 cycles of 96 C for 60 seconds, 53 C for 60 seconds, 72 C for 60 seconds, and a single final extension at 72 C for 10 minutes. After amplification, 15 µl of the amplicon were digested with 6-8 units of Taq 1 restriction endonuclease TABLE 1. The Sequences of the Primers and Probes Used in the Analysis of the IL-1B, IL-1RA, IL-10, and TNFA Genes IL-1B Primers IL-1B primer 1 5 -gttgtcatcagactttgacc-3 IL-1B primer 2 5 -TTCAgTTCATATggACCAgA-3 IL-1RA Primers IL-1RN primer 1 5 -CTCAgCAACACTCCTAT-3 IL-1RN primer 2 5 -TCCTggTCTgCAggTAA-3 IL-10 Primers IL-10 primer 1 5 -ATCCAAgACAACACTACTAA-3 IL-10 primer 2 5 -TAAATATCCTCAAAgTTCC-3 Probes 1082 G 5 -TTCTTTgggAgggggAAg A 5 -ACTTCCCCTTCCCAAAGAA C 5 -CAggTgATgTAACATCTCTgTgC T 5 -gcacagagatattacatcacctgt C 5 -CCgCCTgTCCTgTAggAA A 5 -TTCCTACAgTACAggCggg-3 TNFA Primers TNFA primer 1 5 -CAAACACAggCCTCAggACTC-3 TNFA primer 2 5 -AgggAgCgTCTgCTggCTg-3 Probes 308G(TNFA*1) 5 -AggggCATggggACggg-3 308A(TNFA*2) 5 -AggggCATgAggACggg G 5 -CCCTgCTCCgATTCCgAG A 5 -CCTCggAATCAgAgCAggg-3

3 HEPATOLOGY Vol. 30, No. 4, 1999 COOKSON ET AL. 853 (Amersham Pharmacia-Biotech) at 65 C. Digested restriction fragments were visualized on a 3% (wt/vol) agarose gel with appropriate commercially available size markers (Amersham Pharmacia- Biotech). The presence of a base-exchange substitution at position 3953 creates the Taq1 restriction site in allele 1 but not allele 2; therefore, Taq 1 digestion of the 249-bp IL-1B amplicon resulted in fragments of 114 and 135 bp (allele 1) and/or intact amplicon (allele 2). Polymorphisms of IL-1RN. A penta-allelic polymorphic site containing variable numbers of an 86-bp tandem repeat sequence in intron 2 of the IL-1 receptor antagonist gene (IL-1RN) was amplified in a 25-µL reaction mixture. The conditions for amplification were as follows: 96 C for 2 minutes, 30 cycles of 96 C for 60 seconds, 58 C for 60 seconds, 72 C for 60 seconds, and a final extension at 72 C for 10 minutes. After amplification the amplicon was visualized on a 2% (wt/vol) agarose gel with appropriate commercially available size markers (Amersham Pharmacia-Biotech). Five alleles were assigned based on amplicon size: allele 1 (4 repeats) 410 bp, allele 2 (2 repeats) 240 bp, allele 3 (3 repeats) 325 bp, allele 4 (5 repeats) 500 bp, and allele 5 (6 repeats) 595 bp. Polymorphisms of IL-10. Polymorphisms at 3 positions of the IL-10 promoter gene sequence ( 1082, 819, and 592) were determined by a modification of the methods of Turner et al. 36 A 587-bp fragment of the IL-10 promoter ( 1115 to 528) was amplified in a single 30-mL reaction, using the following conditions: 96 C for 10 minutes, 35 cycles of 96 C for 30 seconds, 54 C for 60 seconds, 72 C for 60 seconds, and a single final extension at 72 C for 10 minutes. After amplification, 3 µl of the IL-10 amplicon were blotted onto 6 positively charged nylon membranes (Boehringer Mannheim, Lewes, UK) and fixed by exposure to 0.25 J/cm 2 ultraviolet light. Each of the membranes were probed with 1 of 6 digoxygenin labeled oligonucleotide probes (Table 1). Stringency washes were performed in 3 mol/l tetra methyl ammonium chloride at 55 C for the 1082 and 592 probes and 65 C for the 819 probes. Bound probe was detected using a commercial chemiluminescent detection system (Boehringer Mannheim) and exposure to X-ray film. The resulting photographs were interpreted independently by 2 observers. Polymorphisms of TNF-. Polymorphisms at position 308 and 238 of the TNF- promoter sequence (TNFA) were determined by a modification of the methods of McGuire et al. 38 and D Alfonso et al. 39 A 519-bp fragment of the TNFA promoter was amplified using the following conditions: 96 C for 2 minutes, 10 cycles of 96 C for 60 seconds, 65 C for 60 seconds, 72 C for 60 seconds, and 20 cycles of 96 C for 10 seconds, 65 C for 50 seconds, 72 C for 30 seconds. After amplification, 3 µl of the TNFA amplicon were blotted onto 4 positively charged nylon membranes (Boehringer Mannheim) and fixed by exposure to 0.25 J/cm 2 ultraviolet light. Each of the 4 membranes were probed with 1 of 4 digoxygenin labeled oligonucleotide probes (Table 1). Stringency washes were performed in 3 mol/l tetra methyl ammonium chloride at 55 C. Bound probe was detected using a commercial chemiluminescent detection system (Boehringer Mannheim) and exposure to X-ray film. The resulting photographs were interpreted independently by 2 observers. Statistical Analyses. Allele and genotype frequencies were compared using 2 and Fisher s exact probability tests, as appropriate. All analyses were performed using the Epistat statistical software (CDC, Atlanta, GA) and comparisons of allele frequencies were made at the phenotypic level (i.e., counting individuals rather than chromosomes) following the recommendations of Svejgaard and Ryder. 40 Throughout the analysis the King s series was evaluated as a first set and the Mayo Clinic series as a second set. The probability values obtained were corrected for multiple testing (Bonferroni s correction) as follows: for the first series (King s College), a correction factor of 7 was used, representing the total number of biallelic polymorphisms tested, and for the second series (Mayo Clinic), only those alleles not significant in the analysis of the first series were corrected. Because TNF*2 is in linkage with the HLA A1-B8- DRB1*0301 haplotype, the method of Svejgaard and Ryder 40 was used to determine the relative strength of the association with TNFA-308 compared with both HLA B8 and DRB1*0301. This study design was chosen because previous studies of HLA alleles in these same patients and control groups suggested that, although both sets of patients and controls were drawn from the same racial group, this is a heterogeneous grouping, and there were some marked differences in gene distribution between the 2 sets. This study design has 2 other advantages: we can be more confident that associations described do not simply reflect the founder population (founder effect) and by analyzing the data as we have done we can avoid falsely rejecting weak associations because these may be confirmed in the second set without the need for Bonferroni s correction. RESULTS There were no significant differences in the distributions of the IL-1B and IL-10 alleles, genotypes, or haplotypes in either study population compared with controls (Tables 2 and 3). Overall, there was a weak significant difference in the IL-1RN genotype distribution comparing King s AIH patients with controls (P.042). This difference, however, was not significant after correction for multiple testing, and it was not found in the Mayo Clinic series. In contrast, both TNFA-238 and TNFA-308 were both associated with an increased susceptibility to type 1 AIH in the King s series (Table 4). This was because of an increased frequency of the TNF-308 allele TNFA*2 allele in patients compared with controls (49% vs. 34%, P.0066) and a lower frequency of the rare A nucleotide at TNFA-238 (4.5% vs. 13.9%, P.048). After correction for multiple testing only one of these, the association with TNFA*2, remained significant (34% vs. 49%, Pc.046) and this association was highly significant in the Mayo Clinic series (26% vs. 56%, P.00006). Stratification analysis (Table 5) in the second data set for TNFA*2 versus HLA B8 and TNFA*2 versus HLA DRB1*0301, the 2 most closely linked HLA genes, showed that the TNFA*2 association was dependent on both B8 and DRB1*0301. In addition neither association was independent of the other. This reflects the high degree of linkage disequilibrium between the TNFA locus and both HLA B8 and HLA DRB1*0301 within the extended haplotype B8-TNFA*2- DRB1*0301. DISCUSSION The present study reports 3 original observations. First, we have identified a highly significant association between type 1 TABLE 2. IL-1B and IL-1RN Genotypes in AIH n 96 Genotype Frequency Number (%) n 84 n 102 n 86 IL-1B genotype 1,1 53 (55) 44 (52) 60 (60) 47 (57) 1,2 36 (38) 35 (42) 37 (37) 31 (37) 2,2 7 (7) 5 (6) 4 (4) 5 (6) failed IL-1RN genotype 1,1 63 (66) 39 (48) 51 (50) 46 (53) 1,2 22 (22) 32 (40) 40 (39) 28 (33) 2,2 6 (6) 6 (7) 7 (7) 8 (9) other 5 (5) 4 (5) 4 (4) 4 (5) failed

4 854 COOKSON ET AL. HEPATOLOGY October 1999 Bases at Position TABLE 3. IL-10 Genotypes and Haplotypes in AIH n 78 Genotype Frequency Number (%) n 84 n 102 n AA 21 (28) 26 (31) 29 (29) 23 (27) AG 35 (45) 42 (50) 54 (54) 41 (48) GG 22 (27) 16 (19) 18 (18) 21 (25) 819 CC 42 (54) 53 (63) 59 (59) 49 (58) CT 31 (40) 28 (33) 38 (38) 31 (36) TT 5 (6) 3 (4) 4 (4) 5 (6) 592 AA 5 (6) 3 (4) 4 (4) 5 (6) AC 31 (40) 28 (33) 38 (38) 31 (36) CC 42 (54) 53 (63) 59 (59) 49 (58) failed Haplotype n 156 Haplotype Frequency Number (%) n 168 n 202 n 170 G--C--C 77 (49) 94 (56) 91 (45) 83 (48) A--C--C 38 (24) 40 (24) 65 (32) 46 (27) A--T--A 41 (26) 34 (20) 46 (23) 41 (24) failed AIH and the TNFA-308 allele 2 (TNFA*2). The second and third observations were both negative, thus none of the IL-1 (IL-1B or IL-1RN) or IL-10 alleles that were studied were associated with susceptibility to or resistance from type 1 AIH. The association between type 1 AIH and TNFA*2 has not been reported elsewhere. The TNFA*2 allele has been shown to be in strong linkage disequilibrium with the autoimmune HLA haplotype A1-B8-DR3. 41 This haplotype is not only associated with AIH, 5 but also with several other autoimmune diseases including celiac disease, Graves disease, myasthenia gravis, primary Sjogren s syndrome, systemic lupus erythematosus, and insulin-dependent diabetes mellitus. 4,42 Because TNFA*2 is part of an extended haplotype it is difficult to determine the relative contribution of this allele to disease susceptibility. In the present study stratification analysis has only partly answered this question, thus although the risk of disease is more strongly associated with HLA DRB1*0301 (odds ratio 4.7 vs. OR 3.6 for the second set) it is not independent of TNFA*2. In PSC the TNFA*2 association is much stronger than that for HLA DR3 Base and (genotype) Position TABLE 4. TNF 308 and 238 Genotypes in AIH n 93 Genotype Frequency Number (%) n 67 n 102 n AA (A2, A2) 1 (1) 8 (12) 2 (2) 10 (44) AG (A1, A2) 31 (33) 24 (37) 25 (25) 38 (45) GG (A1, A1) 61 (66) 33 (51) 75 (74) 37 (12) failed AA 2 (2) AG 11 (12) 3 (4) 7 (7) 5 (6) GG 80 (86) 64 (96) 94 (93) 80 (94) failed TABLE 5. Stratification Analysis of TNFA*2 in Comparison With HLA-A1, in HLA-B8, and HLA-DRB1*0301 in AIH Patients and Controls Phenotype Patients Controls Comparison OR P TNFA*2 HLA-A1 (n 83) (n 98) vs vs. NS 4 9 vs. NS TNFA*2 HLA-B8 (n 83) (n 98) vs vs. NS 0 3 vs. NS TNFA*2 HLA-DRB1*0301 (n 85) (n 102) vs vs. NS 4 3 vs. NS 71 4 Abbreviations: OR, odds ratio; NS, not significant. (DRB1*0301) and is independent of DRB1*0301 suggesting that the TNFA locus may be the primary susceptibility locus in PSC. 15 In contrast the present data may indicate a more complex role for this extended haplotype in type 1 AIH, such that both TNFA and DRB1 may contribute to disease susceptibility. One way to prove a relationship between TNFA and type 1 AIH would be to show that the TNFA gene polymorphism influences TNF- production. It has been shown that TNFA*2 is associated with increased transcription of TNF-, but whether or not protein levels are increased is currently controversial. Multiple studies have suggested that individuals can be classed as high, intermediate, or low TNF- responders when their cells are stimulated in vitro. 32,43 Low responders, who also have lower levels of TNF- messenger RNA, are at an increased risk of fatal outcome after meningococcal disease. 32 The TNFA*2 allele has also been associated with an increased risk for fatal cerebral malaria, presumably because of consequences of increased TNF- transcription and a more severe inflammatory response. 38 In addition, HLA DR3 and DR4 positive individuals have been shown to exhibit higher levels of TNF- production than those who are HLA DR2 positive. 44 This is important in type 1 AIH because both DR3 and DR4 are known susceptibility alleles for type 1 AIH while the DR2 allele (DRB1*1501) is associated with a reduced risk of disease. 5-8 The controversy surrounding the relationship between TNFA polymorphism and TNF- production results from differences in the methodologies used to establish this link. For example, there are differences in the type of cells stimulated (monocytes, lymphocytes, or whole blood) and the stimulant used (varying concentrations of lipopolysaccharide, phorbol myristate acetate or anti-cd3). In addition it is unlikely that cells from normal healthy individuals will respond in the same way as those isolated from patients with active or treated disease. This raises the more fundamental question over the validity of many of these studies, and for these reasons we have not assessed protein production as part of the present study. The 2 other major findings of this study were both negative. Neither the IL-1B, IL-1RN, nor the IL-10 alleles studied were associated with susceptibility to or resistance

5 HEPATOLOGY Vol. 30, No. 4, 1999 COOKSON ET AL. 855 from type 1 AIH. Despite weak associations with IL-1RN*1 and also with TNF-238 in the King s series, neither of these associations was confirmed in the Mayo Clinic series. Further analysis did, however, highlight an interesting anomaly regarding IL-1RN in the Mayo Clinic series, whereby there was a significant imbalance in the proportion of IL-1RN*2 positive patients carrying the IL-1B*2 allele. This is contrary to the expected pattern of linkage for the IL-1RN IL-1B haplotype but at present the significance of this observation, which was not shown in the King s series, is unclear. Population differences between the 2 series and the testing of a large number of different alleles at several gene loci may have influenced this finding. Such minor variations further emphasize the importance of using 2 separate cohorts of patients to confirm candidate gene associations and further validate our study design. The IL-1B*1 and IL-1RN*2 alleles have been linked with susceptibility to inflammatory bowel disease, 20,45 systemic lupus erythematosus, 46 and severe forms of Sjogren s syndrome, 47 whereas polymorphisms in the IL-10 promoter have been linked with susceptibility to rheumatoid arthritis 36 and asthma. 48 Studies so far have failed to link the IL-1 gene family and IL-10 promoter polymorphisms with susceptibility to other liver diseases including the autoimmune disorders PSC (Donaldson PT, unpublished data) and PBC, 17 advanced alcohol-related liver disease, 49 or chronic hepatitis C virus infection (Donaldson PT, unpublished data). The link between candidate genes and disease may be either functional or secondary to linkage disequilibrium. Studies evaluating in vitro cytokine production by liver infiltrating T cells from patients with and without type 1 AIH suggest that the cytokine responses vary and are disease specific Lymphocytes from patients with type 1 AIH have a predominantly Th2 cytokine response (producing more IL-4 and/or IL-10 than interferon- and/or IL-2), whereas T cells from patients with chronic viral hepatitis have mainly a Th1 cytokine response. 52 A predominance of Th2 cytokine producing T cells in type 1 AIH may lead to an imbalance in cytokine immunoregulation, which may predispose individuals towards more severe disease and/or other autoimmune reactions. 53 Considering the degree and range of genetic variability now characterized within the cytokine genes, it is not surprising that a genetic explanation for these imbalances is being sought. However, because cytokines are pleitropic molecules with complex regulatory networks, 54 the identification of a single candidate gene among the many polymorphic cytokine genes may not fully explain either disease susceptibility or variation in disease severity. Consequently in the present study we have chosen to investigate a number of different but inter-related cytokine genes in a single study rather than to evaluate each one separately. In future studies the role of these candidate genes in susceptibility to variant forms of AIH must also be considered. Although there is controversy within the international autoimmune hepatitis study group regarding subgroups of AIH, at least 2 other less common variant forms of AIH are thought to exist. Type 2 AIH characterized by liver-kidney microsomal antibodies type 1 and type 3 characterized by antibodies to cytosolic antigens (soluble liver antigen antibody and/or liver pancreas antibody). 2,3 Immunogenetic data on type 3 AIH is not available, but type 2 AIH has been associated with HLA DR3 and DQ2, as well as the null C4A allele C4A*Q0. 55 In summary, we have addedtnfa*2 to the list of candidate susceptibility genes in type 1 AIH and removed other polymorphisms associated with the IL-1B, IL-1RN, and IL-10 genes. It is unclear at present whether this association is caused by linkage or is functional. There are many more potentially influential and polymorphic immunoregulatory genes still to be investigated in type 1 AIH and further studies are warranted. REFERENCES 1. Krawitt EL. Autoimmune hepatitis. N Engl J Med 1996;334: Homberg JC, Abauf N, Bernard O, Islam S, Alvarez F, Khalil SH, Poupon R, et al. 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