Paulo Lisboa Bittencourt

Simplified criteria for the diagnosis of autoimmune hepatitis

Diagnosis of autoimmune hepatitis (AIH) may be challenging. However, early diagnosis is important because immunosuppression is life‐saving. Diagnostic criteria of the International Autoimmune Hepatitis Group (IAIHG) were complex and purely meant for scientific purposes. This study of the IAIHG aims to define simplified diagnostic criteria for routine clinical practice. Candidate criteria included sex, age, autoantibodies, immunoglobulins, absence of viral hepatitis, and histology. The training set included 250 AIH patients and 193 controls from 11 centers worldwide. Scores were built from variables showing predictive ability in univariate analysis. Diagnostic value of each score was assessed by the area under the receiver operating characteristic (ROC) curve. The best score was validated using data of an additional 109 AIH patients and 284 controls. This score included autoantibodies, immunoglobulin G, histology, and exclusion of viral hepatitis. The area under the curve for prediction of AIH was 0.946 in the training set and 0.91 in the validation set. Based on the ROC curves, two cutoff points were chosen. The score was found to have 88% sensitivity and 97% specificity (cutoff ≥6) and 81% sensitivity and 99% specificity (cutoff ≥7) in the validation set. Conclusion: A reliable diagnosis of AIH can be made using a very simple diagnostic score. We propose the diagnosis of probable AIH at a cutoff point greater than 6 points and definite AIH 7 points or higher. (HEPATOLOGY 2008.)

Genetic heterogeneity in susceptibility to autoimmune hepatitis types 1 and 2

OBJECTIVES:Susceptibility to autoimmune hepatitis (AIH) type 1 has been associated with DRB1*03, DRB1*04, and DRB3 alleles in European and North-American whites, with DRB1*04 in Japan, and with DRB1*04 and DRB1*13 in Latin America. Very few studies have been performed on AIH type 2. The aim of the present study was to evaluate the association of AIH types 1 and 2 with HLA-DR and DQ loci.METHODS:We performed HLA-DRB and -DQB1 typing by polymerase chain reaction amplification with sequence-specific primers (PCR-SSP) in 139 AIH patients. Most had AIH type 1 associated with circulating anti-smooth muscle antibody with F-actin specificity or antinuclear antibody. Twenty-eight patients presented AIH type 2 with anti-liver/kidney microsome type 1 or anti-liver cytosol type 1 antibodies.RESULTS:We observed a significant increase of DRB1*13 (70%vs 26% of controls, p < 0.00001) and DRB3 (93%vs 69% of controls, p < 0.00001) in AIH type 1 patients. Analysis of patients without DRB1*13 disclosed a secondary association with DRB1*03 (70%vs 30% of controls, p= 0.0001) and either the DRB1*13 or the DRB1*03 alleles were present in the majority of these patients (91%vs 48% of controls, p= 0.001). Comparison of DRB1*13- and DRB1*03-positive subjects revealed that the former alleles conferred susceptibility to younger patients with AIH type 1. DQB1 typing showed a significant increase in DQB1*06 (68%vs 41% of controls, p= 0.00007) in strong linkage disequilibrium with DRB1*13, and a decrease in DQB1*0301 (8%vs 47% of controls, pc= 0.0003). On the other hand, HLA typing of patients with AIH type 2 disclosed a significant increase in the DRB1*07 (68%vs 20% of controls, pc < 0.00014), DRB4 (79%vs 43% of controls, pc= 0.004), and DQB1*02 (86%vs 42%, p= 0.00002) alleles. After exclusion of DRB1*07, a secondary association with HLA-DRB1*03 was further observed in these patients (78%vs 30%, p= 0.007) and most of them had either DRB1*07 or DRB1*03 (93%vs 44% of controls, pc < 0.0001).CONCLUSIONS:Our data indicate that predisposition to AIH types 1 and 2 is associated, respectively, with the DRB1*13 or DRB1*03 and DRB1*07 or DRB1*03 alleles, and suggest that protection against type 1 disease may be conferred by DQB1*0301. In addition, the cluster of DRB1*13 in children with AIH type 1 also supports the concept that different HLA alleles might influence the onset of the disease.

Clinical distinctions and pathogenic implications of type 1 autoimmune hepatitis in Brazil and the United States

BACKGROUND/AIMS Type 1 autoimmune hepatitis has a strong genetic predisposition that varies among different ethnic groups. Our aims were to determine if the clinical manifestations differed between patients with type 1 autoimmune hepatitis from Brazil and the United States and if classical disease could be associated with region-specific susceptibility markers. METHODS The clinical manifestations and genetic risk factors of 161 patients from the United States were compared to those of 115 patients from Brazil. RESULTS The patients from Brazil had earlier disease onset, lower frequency of concurrent immune diseases, higher serum levels of aspartate aminotransferase and gamma-globulin, greater occurrence of smooth muscle antibodies, and lower frequency of antinuclear antibodies than the patients from the United States. Human leukocyte antigen (HLA) DR13 and DRB1*1301 occurred more commonly in the Brazilian patients and HLA DR4 less often. Normal subjects from each country had similar frequencies of HLA DR13 and DR3. CONCLUSIONS Type 1 autoimmune hepatitis in Brazil has different features at presentation than the disease in Caucasoid patients from the United States, and it is associated with HLA DR13. Background populations in each country have similar frequencies of HLA DR13 and DR3, and region-specific etiologic factors may determine the HLA association.

Analysis of HLA haplotypes in autoimmune hepatitis type 1: identifying the major susceptibility locus

Susceptibility to autoimmune hepatitis type I (AIH-1) has been associated with HLA-DR3, DR52, and DR4 antigens in Caucasian and Oriental patients. However, in Brazil, disease susceptibility is primarily linked to DR13 and DR52. In this highly admixed population, we find different DR13-associated haplotypes, presenting a unique opportunity to discriminate relevant genes within a tightly linked genomic region. To identify the primary susceptibility locus, we sequenced DR13 alleles of 39 patients with AIH-1 and 22 controls. Patients were almost exclusively DRB1*1301, but half of controls typed DRB1*1302. HLA-DQ haplotypes were varied. Oligotyping of DRB3 locus of all patients and also within the HLA-DR13 positive group showed an allele distribution comparable to controls, confirming that the stronger association lies in the DRB1 locus. On the other hand, if DRB1*1301 is the major susceptibility factor in our sample, the only amino acid different from DRB1*1302 in position 86, corresponding to pocket 1 in the peptide-presenting groove, may be important. We propose that peptide presentation leading to pathogenesis of AIH-1 may be quite stringent, but will also be affected by other strong genetic or environmental susceptibility factors, which would explain the various HLA molecules associated to the disease in the different populations.

Autoimmune hepatitis, HLA and extended haplotypes.

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease. Characteristic liver-infiltrating immune cells in portal and periportal areas, hypergammaglobulinemia and typical autoantibodies indicate an ongoing autoimmune reaction against liver self antigens, which lead to irreversible cellular damage and ultimately to severe hepatic failure. A significant part of adult, but not pediatric AIH patients, exhibit concurrent autoimmune diseases, further strengthening the immunological etiology of the disease. Genetic susceptibility to autoimmune hepatitis is strongly associated with HLA-DRB1 alleles. In Caucasian European and North American patients, AIH-1 is associated with the presence of DRB1*0301, DRB3*0101 and DRB1*0401 alleles, while AIH-2 is associated with DRB1*0301 or DRB1*07. In Brazil, the primary susceptibility allele for AIH-1 is DRB1*1301, but a secondary association with DRB1*0301 has also been identified. We looked for additional susceptibility factors in the extended MHC region. We genotyped 107 AIH-1 children and up to 326 healthy subjects for TNFA G-308A, TNFA G-238A, LTA A+252G, LTA A+80C, NFKBIL1 T-63A, BAT1 C-348T, BAT1 G-22C, MICA, and HLA-B polymorphisms. The TNFA-308 A allele was significantly increased in AIH-1 when compared with healthy controls, confirming data from other studies. Linkage disequilibrium analysis was carried out. The ancestral haplotype comprising TNFA-308A, TNFA-238G, LTA+252G, LTA+80C, NFKBIL1-63A, BAT1-348C, BAT1-22C, HLA-B*08, MICA*08 was more common in DRB1*03 positive patients than in controls (40% vs. 14%), showing a seven-fold increased risk for the disease [OR=7.8 (95%CI 2.04-29.9.2, p=0.0021). In contrast, the remaining patients carrying DRB1*03 exhibited varied haplotypes. Finally, a variety of class III haplotypes was also present in HLA-DRB1*13 patients, without a predominant pattern. The most common of the 98 haplotypes present in patients were completely absent in controls. The extended haplotype analysis in this sample of AIH-1 patients highlights not only the genetic diversity present in the Brazilian population, but is also in accordance with the previously documented microdiversity within the MHC region. The present knowledge of AIH suggests that the same or a very similar disease can be induced by yet unknown, but different, triggers followed by presentation on different HLA-DR molecules of the epitopes derived from the corresponding autoantigens, characterizing a much more complex disease than previously thought.

Cytotoxic T lymphocyte antigen–4 gene polymorphisms do not confer susceptibility to autoimmune hepatitis types 1 and 2 in Brazil

OBJECTIVES:Susceptibility to autoimmune hepatitis (AIH) has been linked to different HLA-DR antigens in distinct populations. Recently, an A–G polymorphism in exon 1 of the cytotoxic T lymphocyte antigen-4 (CTLA-4) gene was associated with predisposition to AIH type 1 (AIH-1) in white individuals in North America. This polymorphism has been associated with several other autoimmune diseases, presumably because of its effect in the expression of CTLA-4, an adhesion molecule that downregulates peripheral T cell responses. The aims of this study were to assess the frequency of CTLA-4 genotypes in Brazilian patients with AIH-1 and AIH type 2 (AIH-1), as well as to investigate the influence of these genotypes in disease expression.METHODS:Determination of CTLA-4 genotypes was carried out in 106 patients with AIH-1, 26 subjects with AIH-2, and 67 healthy control subjects by polymerase chain reaction (PCR)–based techniques.RESULTS:No difference in the distribution of CTLA-4 genotypes was observed in subjects with AIH-1 and AIH-2 as compared to healthy controls. Patients with AIH-1 and AIH-2 with the GG genotype exhibited lower γ-globulin and ALT levels, respectively.CONCLUSIONS:Susceptibility to AIH-1 and AIH-2 in Brazilian patients is not influenced by exon 1 CTLA-4 gene polymorphisms at position 49.

FREQUENCY OF CONCURRENT AUTOIMMUNE DISORDERS IN PATIENTS WITH AUTOIMMUNE HEPATITIS: EFFECT OF AGE, GENDER, AND GENETIC BACKGROUND

Background Concurrent autoimmune disorders (CAIDs) have been shown to occur in 22% to 34% of the patients with autoimmune hepatitis (AIH). Their presence has been linked to female gender, older age, and to certain HLA antigens, namely HLA-A11, DRB1*04, and DRB4*01. Aims To assess the frequency and nature of CAID in Brazilian patients with AIH types 1 (AIH-1) and 2 (AIH-2) and to investigate the influence of age, gender, and genetic background in their occurrence. Patients and Methods The presence and nature of CAID was studied in 143 patients [117 females, median age 11 (1.3 to 69)] with AIH-1 (n=125) and AIH-2 (n=28). HLA typing and tumor necrosis factor α gene promoter and exon 1 cytotoxic T lymphocyte associated antigen 4 (CTLA-4) gene polymorphisms were determined by polymerase chain reaction-based techniques. Results The frequency of CAID was similar in patients with AIH-1 (14%) and AIH-2 (18%), but their nature was shown to vary. Arthritis was seen in half of the patients (n=8) with CAID and AIH-1 and in none of those with AIH-2. Subjects with AIH-1 and CAID were shown to be older [24 (1.3 to 61) vs. 11 (1.3 to 69) y, P=0.02] and to have more often circulating antinuclear antibody (76% vs. 40%, P=0.008) and less frequently antiactin antibodies (33% vs. 75%, P=0.008) when compared with their counterparts without CAID. No particular HLA-DR and DQ alleles, as well as tumor necrosis factor α and CTLA-4 genotypes, were associated with CAID. Conclusions The nature, but not the frequency, of CAID was shown to vary in AIH-1 and AIH-2. In subjects with AIH-1, CAID was linked to older subjects and to the presence of antinuclear antibody. No predisposition to CAID was associated to HLA-DRB1*04 or DDB4*01 alleles. The observed lower frequency of CAID could be attributed to the lower age of disease onset in Brazilians and to differences in HLA-encoded susceptibility to AIH-1 observed in South America.

Antismooth Muscle and Antiactin Antibodies Are Indirect Markers of Histological and Biochemical Activity of Autoimmune Hepatitis

Reactivity and titers of autoantibodies vary during the course of autoimmune hepatitis (AIH), and some autoantibodies have been associated with disease activity and adverse outcomes after treatment. The aim of this study was to assess the autoantibody behavior in AIH and its significance as predictors of biochemical and histological remission. A total of 117 patients with AIH (mean age 18.6 [4‐69] years) were evaluated and tested for autoantibodies at disease onset and successively (mean 3.2 [2‐6] times) after a mean follow‐up evaluation of 70 [20‐185] months. Antismooth muscle (ASMA), antiliver kidney microsome type 1 (anti‐LKM1), antiliver cytosol type 1 (anti‐LC1), antimitochondrial, antinuclear (ANA), and antiactin antibodies (AAA) were determined at disease onset and 379 other times during the follow‐up evaluation through indirect immunofluorescence in rodent tissues, HEp‐2 cells, and human fibroblasts. Anti‐SLA/LP were assessed 45 times in the follow‐up evaluation of 19 patients using enzyme‐linked immunosorbent assay (ELISA). Upon admission, AIH types 1 and 2 were observed in 95 and 17 patients, respectively. Five subjects had AIH with anti‐SLA/LP as the sole markers. Patients initially negative for AAA did not develop these antibodies thereafter. ANA were detected de novo in six and three subjects with AIH types 1 and 2, respectively. After treatment, only ASMA (>1:80) and AAA (>1:40) were significantly associated with biochemical (76.9% and 79.8%) and histological features (100% and 100%) of disease activity (P < 0.001). Conclusion: With the exception of ANA, the autoantibody profile does not markedly vary in the course of AIH. The persistence of high titers of ASMA and/or AAA in patients with AIH is associated with disease activity. (Hepatology 2014;59:592–600)

Analysis of major histocompatibility complex and CTLA-4 alleles in Brazilian patients with primary biliary cirrhosis

Background and Aims:  Predisposition to primary biliary cirrhosis (PBC) has been classically linked to HLA‐DRB1 locus. However, the presence of the HLA‐DRB1*08 antigen has been reported in less than one‐third of PBC patients from Northern Europe and Japan. Recently, polymorphisms in the tumor necrosis factor alpha (TNFA) gene promoter at position −308 and in exon 1 of the cytotoxic T lymphocyte antigen‐4 (CTLA‐4) gene at position 49 have been associated with susceptibility to PBC in Caucasians. In addition, the presence of HLA‐DRB1*08 and the TNFA*1 allele was also linked to progression to end‐stage liver disease. The aims of the present study were to investigate the frequencies of HLA‐DR and DQ antigens and TNFA and CTLA‐4 alleles in PBC patients from a different genetic background, as well as to assess the role of TNFA alleles and HLA‐DR antigens in disease progression.

Autoimmune hepatitis in Brazilian patients is not linked to tumor necrosis factor α polymorphisms at position −308

BACKGROUND/AIMS Susceptibility to autoimmune hepatitis (AIH) has been linked to different HLA-DR antigens. Recently, AIH type 1 was associated with polymorphisms in the tumor necrosis factor alpha gene promoter (TNFA) at position -308. In this respect, the frequency of the TNFA*2 allele, in linkage disequilibrium with HLA-DRB1*0301, was shown to be significantly increased in whites with AIH type 1. The aim of this study was to assess the role of TNFA alleles in conferring susceptibility to AIH, studying a population where the disease is not primarily associated with HLA-DRB1*03. METHODS The determination of HLA-DRB1 and TNFA alleles was performed in 92 patients with AIH type 1, 29 subjects with AIH type 2 and 83 healthy controls by polymerase chain reaction-based techniques. RESULTS The distribution of TNFA alleles was similar in patients with AIH types 1 and 2, when compared with controls. In addition, the TNFA*2 allele was identified in patients carrying HLA-DR antigens other than HLA-DRB1*03. Interestingly, higher gammaglobulin levels were observed in TNFA*2 positive patients. CONCLUSIONS Our data indicate that susceptibility to AIH remains primarily linked to the HLA-DRB1 locus, and suggest that the association of AIH with TNFA*2 previously observed in whites might be secondary to a linkage disequilibrium with HLA-DRB1*0301.