GASTROENTEROLOGY
1992;103:1041-1047
HLA Class II Molecules and Autoimmune Hepatitis Susceptibility in Japanese Patients TAKESHI SEKI, MASAO OTA, SEIICHI FURUTA, HIROHUMI FUKUSHIMA, TOSHIRO KONDO, KUNIHIKO HINO, NOBUHISA MIZUKI, ASAKO ANDO, KIMIYOSHI TSUJI, HIDETOSHI INOKO, and KENDO KIYOSAWA Second Department of Internal Medicine and Department of Legal Medicine, Shinshu University School of Medicine, Matsumoto; Second Department of Internal Medicine, National Defense Medical College, Tokorozawa; and Department of Transplantation Immunology and Department of Molecular Life Science, Tokai IJniversity School of Medicine, Isehara. Japan
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 DRB-associated Dwalleles compared with the DR2-positive controls. No DPBl 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 Bl molecules (Arg on DR2 and His on DR4), contributes to the susceptibility to autoimmune hepatitis among the Japanese. n patients with autoimmune chronic active hepatitis, piecemeal hepatic necrosis is accompanied by hyperglobulinemia and by high serum autoantibody titers.l In the white population, HLA-B8’ and -DR33,4 are significantly associated with autoimmune hepatitis. On the other hand, an increased frequency of DR4 in 17 older white patient? and a secondary association with DR4 in white patients with autoimmune hepatitis6 have been reported. In Japanese, an increased incidence of DR4 and DR2 has been observed among patients with autoimmune hepatitis.7 We previously reported finding a significant association be-
I
tween autoimmune hepatitis and HLA-DR4 in Japanese patients. In that study, most of the patients with autoimmune hepatitis (about 90%) had HLA-DR4.* Two models are used to explain the association between HLA antigens and predisposition to a disease. One is that HLA antigen is only a genetic marker for the non-HLA gene-associated disease susceptibility which is located within the HLA gene region. In this respect, congenital adrenal hyperplasia is a typical disorder caused by a defective mutation in the 2lOH hydroxylase mapped in the class III region of the HLA gene complex.g In white patients with autoimmune hepatitis, a null allele for the complement C4 gene (class III antigen) C4AQ0, which is located between the HLA-B8 (class I antigen) and DR3 (class II antigen) genes on the short arm of sixth human chromosome, is frequently observed, but this extended haplotype does not increase the risk for the disease more than either B8 or DR3.l’ The other model is that specific amino acid sequences of polymorphic HLA antigens have an important role in the immuneresponses to disease onset, especially in autoimmune disorders. Polymorphic HLA-DRBl and/ or DQBl molecules are thought to influence the development of several autoimmune diseases, including rheumatoid arthritis,*‘*” insulin-dependent diabetes mellitus (IDDM),13,14 celiac disease,15 and pemphigus vulgaris.16 Specific amino acid residues in HLA class II antigens have been suggested to contribute directly to the susceptibility and resistance to autoimmune disease.‘2,‘3 We have developed a rapid and efficient method for detective HLA-DQA1,17 -DQB1,18 -DPB1,17 and -DRBl” alleles that involves the digestion of the polymerase chain reaction (PCR)-amplified DNA with allele-specific restriction endonucleases [polymerase chain reaction-restriction fragment length polymor0 1992
by the American Gastroenterological 0016-5085/92/$3.00
Association
1042 SEKI ET AL.
phisms (PCR-RFLP)]. This method eliminates the need for radioisotopes and sequence-specific oligonucleotide probes. In a previous study,” we applied this new method only to DR4-genotyping in patients with autoimmune hepatitis and found no significant difference in the Dw frequencies between DR4-positive patients vs. DR4-positive controls. In the present study, we performed HLA genotyping with respect to DRBl, DQAl, DQBl, and DPBl in Japanese patients with autoimmune hepatitis using the PCR-RFLP method to investigate the relationship between the distribution of the HLA allele and the susceptibility to autoimmune hepatitis. Materials and Methods Patients A total of 53 patients, 6 men and 47 women, who satisfied the diagnostic criteria for autoimmune hepatitis, were enrolled in this study. They were followed up at Shinshu University Hospital and the National Defense Medical College Hospital from January 1978 to October 1990. None had a history of blood transfusion before the onset of hepatitis. Age at disease onset ranged from 32 to 72 years (median, 50.1 years). All patients were Japanese who were born in Japan, were heterosexual, had not lived abroad, had no previous contact with hepatitis patients, had no exposure to parenteral blood products, and denied any abuse of drug or alcohol. All were negative for hepatitis B surface antigen (HBsAg) and for antibody to hepatitis B core antigen (anti-HBc). All were also negative for the antibody to hepatitis C antigen (RIBA test) and had negative results in the test using the PCR technique for identifing hepatitis C virus (HCV) genomic material in sera. All patients showed increased serum levels of aspartate aminotransferase (AST) (>200 IU/L),immunoglobulin G (IgG) (>2600 mg/dL), and of nonorganic-specific autoantibodies. In all cases, liver biopsy specimens showed severe piecemeal necrosis and the presence of inflammatory mononuclear cell infiltration in the portal tract. Healthy Japanese who were unrelated to the patients and who had normal serum levels of AST (~40 IU/L) served as controls. Methods HLA serological typing. HLA antigens were serologically typed using antibodies from One Lambda Inc. (Los Angeles, CA). HLA genotyping. Genotyping for HLA class II alleles was performed in 49 of 53 patients whose genomic DNAs could be obtained. DNA samples. Genomic DNAs from patients with autoimmune hepatitis and healthy indiviudals were isolated by phenolic extraction of sodium dodecyl sulfate-lysed and proteinase K-treated cells, as described previously.21 PCR amplification. Genomic DNA was amplified by the PCR procedure with 2.5U of Taq DNA polymerase (Perkin
Elmer Cetus Inc., Kyoto, Japan).” The reaction mixture was subjected to 30 cycles consisting of 1 minute of dena-
GASTROENTEROLOGYVol.103,No.3
turing, 1 minute of annelaing, and 2 minutes of extension by an automated PCR thermal sequencer (Iwaki Glass Inc., Tokyo, Japan). Temperature conditions and specific primers for DRBl, DQAl, DQBl, and DPBl were those previously described.‘7-‘g The first domains of the DRBl, DQAl, DQBl, and DPBl genes were selectively amplified by generic- and/or group-specific primers.‘7-‘Q Digestion with restriction endonucleases. After amplification by PCR, 5yL aliquots of the reaction mixture were digested with allele-specific restriction endonucleases”-I9 at optimal temperature for 3 hours after the addition of an appropriate incubation buffer. Acrylamide gel electrophoresis. Samples of the restriction enzyme-cleaved, amplified DNAs were subjected to electrophoresis in 12% polyacrylamide gel in an electrophoretic apparatus (Mupid, Cosmo Bio Co. Ltd., Tokyo, Japan). The DNA genotype was defined on the basis of the RFLP patterns obtained by digested fragments according to standard patterns.‘7-‘g Serological tests for antibodies. HBsAg and anti-HBc were measured by enzyme immunoassay using the Enzygnost kit (Behringwerke AG, Marburg, Germany). Serum autoantibodies, including antinuclear antibody (ANA), antismooth-muscle antibody (ASMA), and antimitochondrial antibody (AMA), were analyzed by an indirect fluorescent antibody technique using Hep 2 cells for ANA and cryostat sections of rat kidney and stomach as a substrate for ASMA and AMA. Titers of 21:80were considered positive for ANA, ASMA, and AMA. Anti-HCV levels were measured by an enzyme immunoassay kit (Abbott Laboratories, North Chicago, IL). Tests for anti-HCV were performed on pretreatment serum samples that had been stored at -20°C. The cutoff value was set at an optical density derived from the mean of the negative controls (NCx) and the mean of the positive controls (PCx) (cutoff value = NCx + 0.25X PCx). The PCR technique was to identify HCV genomic material in the pretreatment sera of all patients. Statistical analysis. Data were analyzed by the standard statistical procedure of x2 contingency table analysis with Yates’ correction. Corrected P values were calculated using the Bonferroni inequality method. Relative risk was obtained by the cross-product ratio of the entries in the 2 X 2 table. A level of P < 0.05was accepted as statistically significant.
Results Serologically
Defined HLA Antigens
Autoimmune
Hepatitis
in
The frequency of HLA class I and II antigens in patients with autoimmune hepatitis compared with that in healthy individuals is shown in Table 1. Among the class I antigens, HLA-Bw 54 was significantly increased in patients with autoimmune hepatitis vs. the healthy individuals. Among the class II antigens, HLA-DR4, -DR53, and
-DQ4 were significantly associated with autoimmune hepatitis. DR4 was the marker most frequently associated
with
autoimmune
hepatitis
among
the
September
HLA
1992
Table 1.
Frequencies of HLA-Bw54, -DR, -DRw, and -DQw Antigens in Patients With Autoimmune Hepatitis and Healthy Controls Autoimmune hepatitis
Local controls
National controls
(%) (n = 53)
(%) (n = 100)
(%) (n = 472)
x2
B54
39.6
11
14.0
17.2
1992;103:1041-1047
HLA Class II Molecules and Autoimmune Hepatitis Susceptibility in Japanese Patients TAKESHI SEKI, MASAO OTA, SEIICHI FURUTA, HIROHUMI FUKUSHIMA, TOSHIRO KONDO, KUNIHIKO HINO, NOBUHISA MIZUKI, ASAKO ANDO, KIMIYOSHI TSUJI, HIDETOSHI INOKO, and KENDO KIYOSAWA Second Department of Internal Medicine and Department of Legal Medicine, Shinshu University School of Medicine, Matsumoto; Second Department of Internal Medicine, National Defense Medical College, Tokorozawa; and Department of Transplantation Immunology and Department of Molecular Life Science, Tokai IJniversity School of Medicine, Isehara. Japan
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 DRB-associated Dwalleles compared with the DR2-positive controls. No DPBl 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 Bl molecules (Arg on DR2 and His on DR4), contributes to the susceptibility to autoimmune hepatitis among the Japanese. n patients with autoimmune chronic active hepatitis, piecemeal hepatic necrosis is accompanied by hyperglobulinemia and by high serum autoantibody titers.l In the white population, HLA-B8’ and -DR33,4 are significantly associated with autoimmune hepatitis. On the other hand, an increased frequency of DR4 in 17 older white patient? and a secondary association with DR4 in white patients with autoimmune hepatitis6 have been reported. In Japanese, an increased incidence of DR4 and DR2 has been observed among patients with autoimmune hepatitis.7 We previously reported finding a significant association be-
I
tween autoimmune hepatitis and HLA-DR4 in Japanese patients. In that study, most of the patients with autoimmune hepatitis (about 90%) had HLA-DR4.* Two models are used to explain the association between HLA antigens and predisposition to a disease. One is that HLA antigen is only a genetic marker for the non-HLA gene-associated disease susceptibility which is located within the HLA gene region. In this respect, congenital adrenal hyperplasia is a typical disorder caused by a defective mutation in the 2lOH hydroxylase mapped in the class III region of the HLA gene complex.g In white patients with autoimmune hepatitis, a null allele for the complement C4 gene (class III antigen) C4AQ0, which is located between the HLA-B8 (class I antigen) and DR3 (class II antigen) genes on the short arm of sixth human chromosome, is frequently observed, but this extended haplotype does not increase the risk for the disease more than either B8 or DR3.l’ The other model is that specific amino acid sequences of polymorphic HLA antigens have an important role in the immuneresponses to disease onset, especially in autoimmune disorders. Polymorphic HLA-DRBl and/ or DQBl molecules are thought to influence the development of several autoimmune diseases, including rheumatoid arthritis,*‘*” insulin-dependent diabetes mellitus (IDDM),13,14 celiac disease,15 and pemphigus vulgaris.16 Specific amino acid residues in HLA class II antigens have been suggested to contribute directly to the susceptibility and resistance to autoimmune disease.‘2,‘3 We have developed a rapid and efficient method for detective HLA-DQA1,17 -DQB1,18 -DPB1,17 and -DRBl” alleles that involves the digestion of the polymerase chain reaction (PCR)-amplified DNA with allele-specific restriction endonucleases [polymerase chain reaction-restriction fragment length polymor0 1992
by the American Gastroenterological 0016-5085/92/$3.00
Association
1042 SEKI ET AL.
phisms (PCR-RFLP)]. This method eliminates the need for radioisotopes and sequence-specific oligonucleotide probes. In a previous study,” we applied this new method only to DR4-genotyping in patients with autoimmune hepatitis and found no significant difference in the Dw frequencies between DR4-positive patients vs. DR4-positive controls. In the present study, we performed HLA genotyping with respect to DRBl, DQAl, DQBl, and DPBl in Japanese patients with autoimmune hepatitis using the PCR-RFLP method to investigate the relationship between the distribution of the HLA allele and the susceptibility to autoimmune hepatitis. Materials and Methods Patients A total of 53 patients, 6 men and 47 women, who satisfied the diagnostic criteria for autoimmune hepatitis, were enrolled in this study. They were followed up at Shinshu University Hospital and the National Defense Medical College Hospital from January 1978 to October 1990. None had a history of blood transfusion before the onset of hepatitis. Age at disease onset ranged from 32 to 72 years (median, 50.1 years). All patients were Japanese who were born in Japan, were heterosexual, had not lived abroad, had no previous contact with hepatitis patients, had no exposure to parenteral blood products, and denied any abuse of drug or alcohol. All were negative for hepatitis B surface antigen (HBsAg) and for antibody to hepatitis B core antigen (anti-HBc). All were also negative for the antibody to hepatitis C antigen (RIBA test) and had negative results in the test using the PCR technique for identifing hepatitis C virus (HCV) genomic material in sera. All patients showed increased serum levels of aspartate aminotransferase (AST) (>200 IU/L),immunoglobulin G (IgG) (>2600 mg/dL), and of nonorganic-specific autoantibodies. In all cases, liver biopsy specimens showed severe piecemeal necrosis and the presence of inflammatory mononuclear cell infiltration in the portal tract. Healthy Japanese who were unrelated to the patients and who had normal serum levels of AST (~40 IU/L) served as controls. Methods HLA serological typing. HLA antigens were serologically typed using antibodies from One Lambda Inc. (Los Angeles, CA). HLA genotyping. Genotyping for HLA class II alleles was performed in 49 of 53 patients whose genomic DNAs could be obtained. DNA samples. Genomic DNAs from patients with autoimmune hepatitis and healthy indiviudals were isolated by phenolic extraction of sodium dodecyl sulfate-lysed and proteinase K-treated cells, as described previously.21 PCR amplification. Genomic DNA was amplified by the PCR procedure with 2.5U of Taq DNA polymerase (Perkin
Elmer Cetus Inc., Kyoto, Japan).” The reaction mixture was subjected to 30 cycles consisting of 1 minute of dena-
GASTROENTEROLOGYVol.103,No.3
turing, 1 minute of annelaing, and 2 minutes of extension by an automated PCR thermal sequencer (Iwaki Glass Inc., Tokyo, Japan). Temperature conditions and specific primers for DRBl, DQAl, DQBl, and DPBl were those previously described.‘7-‘g The first domains of the DRBl, DQAl, DQBl, and DPBl genes were selectively amplified by generic- and/or group-specific primers.‘7-‘Q Digestion with restriction endonucleases. After amplification by PCR, 5yL aliquots of the reaction mixture were digested with allele-specific restriction endonucleases”-I9 at optimal temperature for 3 hours after the addition of an appropriate incubation buffer. Acrylamide gel electrophoresis. Samples of the restriction enzyme-cleaved, amplified DNAs were subjected to electrophoresis in 12% polyacrylamide gel in an electrophoretic apparatus (Mupid, Cosmo Bio Co. Ltd., Tokyo, Japan). The DNA genotype was defined on the basis of the RFLP patterns obtained by digested fragments according to standard patterns.‘7-‘g Serological tests for antibodies. HBsAg and anti-HBc were measured by enzyme immunoassay using the Enzygnost kit (Behringwerke AG, Marburg, Germany). Serum autoantibodies, including antinuclear antibody (ANA), antismooth-muscle antibody (ASMA), and antimitochondrial antibody (AMA), were analyzed by an indirect fluorescent antibody technique using Hep 2 cells for ANA and cryostat sections of rat kidney and stomach as a substrate for ASMA and AMA. Titers of 21:80were considered positive for ANA, ASMA, and AMA. Anti-HCV levels were measured by an enzyme immunoassay kit (Abbott Laboratories, North Chicago, IL). Tests for anti-HCV were performed on pretreatment serum samples that had been stored at -20°C. The cutoff value was set at an optical density derived from the mean of the negative controls (NCx) and the mean of the positive controls (PCx) (cutoff value = NCx + 0.25X PCx). The PCR technique was to identify HCV genomic material in the pretreatment sera of all patients. Statistical analysis. Data were analyzed by the standard statistical procedure of x2 contingency table analysis with Yates’ correction. Corrected P values were calculated using the Bonferroni inequality method. Relative risk was obtained by the cross-product ratio of the entries in the 2 X 2 table. A level of P < 0.05was accepted as statistically significant.
Results Serologically
Defined HLA Antigens
Autoimmune
Hepatitis
in
The frequency of HLA class I and II antigens in patients with autoimmune hepatitis compared with that in healthy individuals is shown in Table 1. Among the class I antigens, HLA-Bw 54 was significantly increased in patients with autoimmune hepatitis vs. the healthy individuals. Among the class II antigens, HLA-DR4, -DR53, and
-DQ4 were significantly associated with autoimmune hepatitis. DR4 was the marker most frequently associated
with
autoimmune
hepatitis
among
the
September
HLA
1992
Table 1.
Frequencies of HLA-Bw54, -DR, -DRw, and -DQw Antigens in Patients With Autoimmune Hepatitis and Healthy Controls Autoimmune hepatitis
Local controls
National controls
(%) (n = 53)
(%) (n = 100)
(%) (n = 472)
x2
B54
39.6
11
14.0
17.2
