Takeshi Seki

HLA class II molecules and autoimmune hepatitis susceptibility in Japanese patients

To investigate the association between autoimmune hepatitis and HLA alleles in Japanese patients, serological typing and class II genotyping were performed using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method. Serological typing showed that HLA-B54, -DR4, -DR53, and -DQ4 were significantly more frequent in patients with autoimmune hepatitis than in controls. HLA-DR4 was most frequently associated with autoimmune hepatitis (88.7%). In PCR-RFLP typing, the frequency of DRB1*0405 was significantly higher in autoimmune hepatitis than in controls. However, there was no significant difference in the frequency of Dw between the patients and the controls who were DR4-positive. The significant increase observed in DQA1*0301 and DQB1*0401 was explained by a linkage disequilibrium with DR4. Six DR4-negative patients had DR2, but there was no significant difference in the frequency of the DR2-associated Dw-alleles compared with the DR2-positive controls. No DPB1 allele was significantly associated with autoimmune hepatitis. These findings suggest that the basic amino acid at position 13, which is present only on the DR2 and DR4 B1 molecules (Arg on DR2 and His on DR4), contributes to the susceptibility to autoimmune hepatitis among the Japanese.

Demographics of anti-asialoglycoprotein receptor autoantibodies in autoimmune hepatitis

BACKGROUND/AIMS The asialoglycoprotein receptor (ASGPR) is an established, liver-specific autoantigen. This multicenter study investigated the specificity of anti-ASGPR autoantibodies for autoimmune hepatitis (AIH) in different ethnic groups. METHODS Nine hundred fourteen sera from European, Japanese, and North American (U.S.) patients with chronic inflammatory liver disorders were tested. An enzyme-immunoassay using human ASGPR and a radioimmunoassay against rabbit ASGPR, performed independently on coded sera, were compared. RESULTS The highest frequency (76%) of anti-human ASGPR was found in AIH patients (11/24 U.S.; 21/25 European; 28/30 Japanese), particularly in those with active disease before treatment (53/62, 85%), and decreased in titer with response to immunosuppressive therapy. These antibodies were found at low titers in 43 (11%) of 385 patients with viral hepatitis and in 25 (7.6%) of 328 patients with other chronic inflammatory liver disorders (P < 0.0005 compared with all AIH patients). Twenty of 37 sera tested by enzyme-immunoassay and radioimmunoassay were positive, and nine were negative for anti-ASGPR by both assays (78% concordance); six sera were exclusively positive on human substrate. CONCLUSIONS Circulating anti-ASGPR autoantibodies are closely associated with autoimmune hepatitis independent of geographic or ethnic criteria. Two anti-ASGPR assays currently in use show high reliability.

A possible association between basic amino acids of position 13 of DRB1 chains and autoimmune hepatitis

Fifty-one patients with autoimmune hepatitis have been studied for HLA association by conventional serology and also by modified polymerase chain reaction-restriction fragment lenght polymorphism (PCR-RFLP) genotyping.HLA-DR4 was significantly associated with autoimmmune hepatitis (46 of 51 patients, 90.2%). DNA typing of the DRB1 gene for 43 DR4-positive patients by using the PCR-RFLP technique revealed that of 43 patients, 33 had DRB1*0405 (Dw15), five had DRB1*0406 (DwKT2), four had DRB1*0403 (Dw13a), two had DRB1*0401 (Dw4), two of 43 had DRB1*0407 (Dw13b) and one had DRB1*0408 (Dw14b). Thus, there was no significant difference in Dw frequencies between DR4-positive patients and DR4-positive healthy subjects. These findings suggest that the DR4-specific sequence (Val 11 and His 13 at amino acid positions 11 and 13, respectively), but not particular Dw-associated DR4 sequence, in the first domain of the DRB1 chain contributes to susceptibility to autoimmune hepatitis among Japanese. Interestingly, all five of the DR4-negative patients had the DR2 specificity (DRB11502 or 1601). Taken together, these results imply that the basic amino acids at position 13, which is present only on the DR2 and DR4 B1 molecules (Arg on DR2 and His on DR4), are most important for determining the predisposition to autoimmune hepatitis.

Low prevalence of hepatitis C virus infection in patients with auto-immune hepatitis type 1.

Hepatitis C virus (HCV) antibodies were measured in 28 patients with auto‐immune hepatitis type 1 using six different assay kits, three for C100–3 antibody and three for second generation HCV antibody, and two confirmatory tests to determine the prevalence of HCV infection in auto‐immune hepatitis. These patients were confirmed to have human leucocyte antigen DR 4 or 2 which is susceptible to auto‐immune hepatitis in Japanese. Of the 28 patients, four (14.3%) were positive for HCV antibody in all assays and reacted positively in at least one of the two confirmatory tests, indicating a true positive finding. Eight were positive for HCV antibody only by the Ortho ELISA kit and were negative in both confirmatory tests. The cut‐off level for these results was low and became negative soon after the patients received corticosteroid treatment. Thus, these eight patients are presumed to be false‐positive reactors. Hepatitis C virus RNA was detected in the serum of two of the four patients with HCV antibody and in none of 24 patients without HCV antibody. No significant difference was observed between the patients with and without HCV antibody in terms of clinical background, liver function tests and auto‐antibodies. Our results showed that the prevalence of a past or present HCV infection in patients with auto‐immune hepatitis in Japan is low; thus, auto‐immune hepatitis is thought to be distinct from hepatitis type C. However, it is also suggested that HCV infection can potentially trigger auto‐immune hepatitis.

Comparison of the clinical and immunogenetic features between patients with autoimmune hepatitis and patients with type C chronic active hepatitis

SummaryWe clarified the clinical and immunogenetical differences between patients with autoimmune hepatitis (AI-CAH), and patients with type C chronic active hepatitis (C-CAH) and type B chronic active hepatitis (B-CAH) who were positive for autoantibodies and hyperglobulinemia. While histories of blood transfusion, intravenous drug abuse and tattoo were seen frequently in patients with type C-CAH, they were rare in patients with AI-CAH. The severe subjective symptoms including anorexia, lethargy, icterus, high fever and extrahepatic manifestations, and severe abnormality of biochemical data were seen in AI-CAH predominantly. Ongoing or past infection of HCV was seen in only 14% of patients with AI-CAH. HLA-DR4 was the most frequently associated with AI-CAH (89%) and 6 DR4-negative patients were positive for DR2. HLA-DNA typing showed that there was no significant difference in the frequency of DR4-associated Dwalleles between the patients and controls who were positive for DR4. These findings suggest that the basic amino acid at position 13, which is present only on the DR2 and DR4 B1 molecules (Arg on DR2 and His on DR4), may contribute to the susceptibility to autoimmune hepatitis of Japanese. Thus, we conclude that AI-CAH is a genetically restricted, disease, and different from CCAH which is a viral infectious disease.

Application of HLA-class II Genotyping by the Modified PCR-RFLP Method to the Forensic Science

The HLA class II antigens(HLA-DR, -DQ, -DP), located in the short arm of chromosome 6, show a great deal of polymorphisms. These antigens have been usually defined by serological procedures (for DR and DQ) using alloantisera or monoclonal antibody and cellular assay procedure (for Dw and DP). Definition of HLA class II antigens became possible at the DNA level using Southern blot hybridization technique with class II antigen cDNA probes. More recently, detection of nucleotide sequence polymorphisms in the HLA class II region has become feasible with the advent of polymerase chain reaction (PCR) technique. The PCR method permits precise and direct analysis of allelic variations with as little as 1 ng of genomic DNA. We earlier reported the modified PCR-RFLP methods (1–3), which were legible and easy to use in the accurate definition of HLA class II (DQA1, DQB1, DRB1 and DPB1) alleles. This method allows discrimination of 36, 91, 946 and 190 combinations, including homozygotes and heterozygotes of the DQA1 (8 alleles), DQB1 (13 alleles), DRB1 (43 alleles) and DPB1 (18 alleles) respectively. In this study, we examined the possibility of HLA-class II genotyping by the modified PCR-RFLP method for DNA samples extracted from hairs, a small volume of whole blood, fresh or old dental pulp tissues.