Susceptibility to primary biliary cirrhosis is associated with human leukocyte antigen DRB1*0803 in Japanese patients

Internutinul

Hepatologv ELSEVIER

International Hepatology Communications 2 (1994) 263-270

communi~tions

Susceptibility to primary biliary cirrhosis is associated with human leukocyte antigen DRB1*0803 in Japanese patients Hikaru

Oguri*, Sakae Oba, Hider0 Ogino, Yutaka Inagaki, Masashi Unoura, Kenichi Kobayashi

Shuichi Kaneko,

First Department of Internal Medicine, Kanazawa University School of Medicine, 13-l. Takaramachi, Kanazawa. 920, Japan

(Received 3 December 1993; accepted 7 January 1994)

Abstract An association of primary biliary cirrhosis (PBC) with human leukocyte antigen (HLA) class II has been reported in previous studies based on the results of serological HLA typing. To evaluate the association between PBC and HLA class II more precisely, we performed HLADRBl and DPBl genotyping in 53 Japanese patients with PBC using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. In DRBl genotyping, the frequency of the *0803 allele was significantly higher in patients with PBC than that in control subjects. Twenty out of 53 patients were *0803 positive (37.7%), whereas only five out of 60 controls (8.3%) had the allele (relative risk = 6.67; P c 0.001; corrected P -z 0.05). In DPBl genotyping, there was no significant difference in the frequency of the DPBl alleles between patients with PBC and controls. Amino acid analysis of the DR@ chain revealed that the frequency of leucine at position 74 was significantly higher in patients with PBC than that in controls. These results suggest that HLA-DRB1*0803 allele and a subsequent amino acid substitution encoded by the polymorphic regions of the allele may play an important role in the pathogenesis of PBC. Key words: Primary biliary cirrhosis; Human leukocyte antigen; HLA genotyping; PCR-RFLP method

1. Introduction Primary biliary cirrhosis (PBC) is a chronic inflammatory disease that is characterized by the destruction of small intrahepatic bile ducts. Although etiology of PBC remains unknown, autoimmune pathogenesis may be suspected because of the pres*Corresponding author. 0928-4346/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0928-4346(94)00009-T

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ence of circulating autoantibodies, increased serum IgM level, alternations of T-cell function and association of other autoimmune disorders such as rheumatoid arthritis, Sjiigren’s syndrome and Hashimoto’s thyroiditis. On the other hand, the occasional familial occurrence and marked female preponderance indicate that some genetic factors may be also important in the pathogenesis of PBC. Furthermore, the fact of increased expression of human leukocyte antigen (HLA) class II in the bile duct epithelium suggests an important role of HLA class II in the progression of the disease. HLA class II, which acts as receptors for antigen presentation in immunocompetent cells, has been demonstrated to be related to some autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Several investigators have also paid attention to the association of PBC with HLA class II. In the previous studies, an association of HLA-DR8 has, been reported in the United States [l], Germany [2] and Japan [3]. On the other hand, two reports of small series from Spain [4] and Japan [5] have suggested an association of DR3 and DR2, respectively, and other studies failed to find any association with HLA-DR antigens [6,7]. These variations among each study may be due to the problems in HLA serotyping. All of these studies determined HLA types by a serological method. In the serological typing, some antigens are difficult to be defined because of cross-reactivity, limitations in the availability of monospecific antisera and injury of surface antigens by the various medications. Recently, HLA genotyping has been developed on the basis of difference in DNA sequences. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method can detect directly the difference in DNA sequences by digestion of amplified DNA fragments with allele-specific restriction endonucleases. This new technique used for HLA genotyping is more accurate and allows to detect a lot of alleles that can not be detected by standard serological methods. To evaluate the relationship.between the HLA class II and PBC more precisely, we performed HLA-DRBl and DPBl genotyping using the PCR-RFLP method in 53 Japanese patients with PBC.

2. Materials and methods Patients and controls Fifty-three Japanese patients with PBC living in Ishikawa and Toyama prefectures were studied. They were 40 women and 13 men whose ages ranged from 36 to 75 years (mean of 57.4 years). They were diagnosed as PBC on the basis of standard clinical, histological and immunological criteria. Sixty healthy Japanese people whose sex, age and origin were matched with PBC group were subjected to the study as controls. Methods DRBl genotyping was performed using the PCR-RFLP method (Sumitest HLADR genotyping kit; Sumitomo Metal Inc., Tokyo, Japan). The method for DRBl typing by amplification of the DRBl gene and digestion with allele-specific restriction endonucleases has been described in detail elsewhere [8,9]. Briefly, this method con-

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sists of two steps. The first step is to determine the thirteen DR groups, and the second step is to determine the DRBl alleles in detail. In the first step, genomic DNA was prepared from 10 ml of peripheral blood sample using phenol-chloroform extraction and ethanol precipitation. Two hundred nanograms of genomic DNA was amplified by PCR using DR group-specific primers. Amplified products were subjected to electrophoresis on a 10% polyacrylamide gel, and were stained with ethidium bromide. Thirteen DR groups were able to be identified according to the patterns of the amplified DNA band. In the second step, closer examination of DRBl alleles was performed. Genomic DNA was amplified by PCR using each DR group-specific primers. Amplified products were digested with allele-specific restriction endonucleases. Samples of the digested DNA were subjected to electrophoresis on a 10% polyacrylamide gel. Forty-seven DRBl alleles were able to be identified on the basis of RFLP patterns. DPBl genotyping was performed in the same way according to DRBl genotyping method as previously reported [lo]. Statistical analysis Allele frequencies in patients and controls were compared using? analysis with Yates’ correction. P values were calculated byX* values, and corrected P values were calculated by multiplying the P values by the number of alleles tested. Relative risk was calculated according to the crossed products ratio method. A corrected P value of less than 0.05 was considered as statistically significant. The clinical variables in groups of patients were compared using 2 analysis and Student’s t test, and the frequencies of amino acid substitutions in patients and controls were compared using 2 analysis with Yates’ correction.

3. Results Genotyping of the HLA-DRBl in patients with PBC The results of HLA genotyping using the PCR-RFLP method for the DRBl gene are shown in Fig. 1. The DRBl*O803 allele was detected most frequently among the patients with PBC. The frequency of the *0803 allele was significantly higher in patients with PBC than that in controls. Twenty out of 53 patients were *0803 positive (37.7%), whereas only five out of 60 controls (8.3%) had the allele (relative risk = 6.67; 2 = 12.47; P < 0.001; corrected P C 0.05). Frequencies of other DRBl alleles in the patients were not significantly different from those in controls. Genotyping of the HLA-DPBl in patients with PBC The results of HLA genotyping using the PCR-RFLP method for the DPBl gene are shown in Fig. 2. There was no significant difference in the frequencies of the HLA-DPBl alleles between patients with PBC and controls. Relationship between clinicalfeatures and the presence of DRBI *0803 allele inpatients with PBC We examined the relationship between DRB1*0803 allele and the following clinical

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allele 0101 1501 1502 1602 0302 0401 0403 0405 0406 0410 1101 1201 1202 1302 1304 1401 1403 1405 1406 1407 0701 0802

m

PBC (nd53) Controls (n=60)

0803

Fig. 1. Genotyping of the HLA-DRBl in patients with PBC and controls.

variables: sex, age, symptoms, histological stage, titer of anti-mitochondrial antibody, serum levels of alkaline phosphatase, y-glutamyl transpeptidase and IgM. We found no statistical difference in these variables between the patients with and without DRBl*O803 allele (Table 1). Substitution of amino acid residues of the DR/lchain A sequence analysis of DRBI DNA revealed that fifteen or more amino acid residues of DR/I chain encoded by DRBl*O803 were different from those encoded by the greater part of the other alleles. We investigated the frequencies of these unique amino acid substitutions of DR/? chain in patients with PBC and controls. Of these amino acid residues, the frequency of leucine at position 74 was significantly higher in patients with PBC than that in controls. DRj3 chains containing leucine at position 74 were found in 43.3% of the patients and 21.4% of the controls (relative risk = 2.77, ,$ = 5.16; P < 0.05) (Table 2).

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allele 0201 0202 0301 0401 0402 0501 0601 0901 1301 B PBC (n=53) EZE'JControls(n=60)

1401 1501 1701 0

10

20

30

40

50

60

70 %

Fig. 2. Genotyping of the HLA-DPBl in patients with PBC and controls.

4. Discussion

We have investigated the relationship between HLA class II and PBC. We found a significant association of PBC with DRB1*0803 allele. These data not only confirmed the results of previous studies showing DR8 associations using serological method

Table 1 Relationship between clinical features and the presence of DRB1*0803 allele in patients with PBC Clinical features

Male (%) Mean age (yr) Symptomatic (%) Histological stage I-II (%) III-IV (%) AMA (< 20) (%) (20-80) (%) (2 160) (%) ALP (IV/l) J-GTP (IU/l) IgM (mg/dl) AMA, anti-mitochondrial

PBC (n = 53)

25 51.3 17 81 19 26 21 53 442 202 530

*0803 positive (n =20) 30 59.1 25 15 25 30 25 45 384 149 441

*0803 negative (n = 33) 21 55.9 12 85 15 24 18 58 458 232 573

antibody; ALP, alkaline phosphatase; y-GTP, y-glutamyl transpeptidase.

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Table 2 Amino acid substitutions of the DRB chain Position

13 31 51 70 74

Amino acid

Gly Tyr Ser Asp Leu

Incidence PBC (%)

controls (%)

41.2 60.4 52.8 58.5 43.4

28.3 45.0 33.3 51.7 21.7

Relative risk

2

P

2.26 1.86 2.24 1.32 2.71

3.51 2.09 3.62 0.29 5.16

NS NS NS NS < 0.05

“Gly, glycine; Tyr, tyrosine; Ser, serine; Asp, aspartic acid; Leu, leucine. NS, not statistically significant (P 2 0.05).

[l-3] but also revealed in more detail the importance of DRB 1*0803 allele among DR8 subtypes by means of HLA genotyping. There have been three reports concerning the association of PBC with certain HLA genotypes [l l-l 31. First, an association of DRS-DQB1*0402 haplotype was noted in United Kingdom [l 11.They showed a significantly high frequency of DR8 in patients with PBC using Taql RFLP analysis. However, they defined DR types indirectly based on the RFLP patterns of DRB gene linked to a polymorphic region, and failed to detect DR alleles in detail. The second report from Denmark, however, found no relationship between HLA class II genes including DRBl*O8 and PBC using RFLP analysis [12]. The possible reason for no association may be that their patients with PBC consisted of a small population, and they also defined DR types indirectly according to the RFLP patterns. The third report using PCR-RFLP analysis was done in Japan, which indicated an association of PBC with DPBl*O501 allele [13]. However, we failed to find a significant association of PBC with DPB1*0501 allele in this study. DPB1*0501 is the most common allele in Japan; more than half of Japanese (55.3% of controls in their study and 63.3% of our controls) have DPB1*0501 allele. Since PBC is a relatively rare disease, it is unlikely that more than half of Japanese have the gene which plays an important role in the pathogenesis of such a rare disease. We have shown an association between PBC and DRB1*0803 allele using a detail analysis of HLA genotyping. An association of DRB1*0803 with PBC using HLA genotyping has never been reported in the previous studies. In our study, we used 60 local controls whose sex, age and origin were matched with PBC group. The allelic frequencies of our local controls were not statistically different from those of 493 Japanese controls which were reported in the 1lth International Histocompatibility Workshop and Conference in 199 1. Even when using these reported national controls instead of our own, it dose not alter the results; the frequency of DRBl*O803 allele in patients with PBC is significantly higher than that in the national controls. Three hypotheses can be drawn to explain for the high incidence of DRB1*0803 allele in PBC patients. First, the structure of *0803 molecule may be similar to that of

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some pathogenic agents. It has been reported that HLA-B27, which is well known for its association with ankylosing spondylitis, has similar amino acid sequence to Klebsiella [14], and certain DQB molecule which is associated with insulin dependent diabetes mellitus has similar structure to Ebstein-Bar viral protein [15]. When the patient is infected with a trigger foreign antigen which has similar structure to his own HLA, the immune reaction to the foreign antigen may lead to an autoimmune response. However, no pathogenic agent has been shown to trigger the onset of PBC. The second hypothesis is that DRB1*0803 allele is a genetic marker tightly linked to susceptibility gene which is located near the DRBl gene. DRBl gene locus is located on the short arm of human chromosome 6. Around the DRBl locus, there are a lot of non-HLA genes which are related to the immune system such as C4, Bf, C2, heat shock protein, tumor necrosis factor (TNF), large multifunctional protease (LMP), transporter of antigen peptides (TAP). Among these genes C4B and TNF-CY genes have been reported to be associated with PBC [16,17]. These genes as well as DRBl may be linked to an unknown gene which is responsible for susceptibility to PBC. Finally, specific amino acid residues encoded by the polymorphic regions of DRBl*O803 may play a direct role in the immune response at the onset of PBC. Some specific amino acid residues in the Dw chain have been suggested to influence the development of several autoimmune diseases. For example, basic amino acid residues at positions 70 and 71 of the Dv chain were reported to contribute to rheumatoid arthritis susceptibility in white and Japanese patients [18,19]. On the other hand, leucine at position 38 of the DR/J chain was reported to contribute to the susceptibility to primary sclerosing cholangitis [20]. In our study, leucine at position 74 of the Dw chain was found more frequently among patients with PBC than the control subjects. These data suggest that the amino acid residue at this position may play an important role in the development of PBC by changing either the affinity of peptide binding or the recognition by the T-cell receptor. However, since the relative risk of this amino acid substitution was lower than that of DRBl*O803 allele, other factors including the difference in three dimensional structure of DR/? chain should be also considered. Together with the fact that DR8 association with PBC has been reported in various races and countries [ l-3,1 11,the presence of DRBl*O803 allele may be a common risk factor for the development of PBC. Further studies using national and international populations are needed to elucidate the pathogenic role of the DRB1*0803 allele in PBC.

References [l] Grobes GJ, Moore SB, Fisher LD, Powell FC. Dickson ER. Primary biliary cirrhosis: association with class II major histocompatibility antigens. Hepatology 1987;7:889-892. [2] Manns MP, Bremm A, Schneider PM, et al. HLA DRw8 and complement C4 deficiency as risk factors in primary biliary cirrhosis. Gastroenterology 1991 ;lOl : 1367-1373. [3] Maeda T, Onishi S, Saibara T, Iwasaki S, Yamamoto Y. HLA DRw8 and primary biliary cirrhosis. Gastroenterology 1992;103:1118.

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[4] Ercilla G, Pares A, Arriaga F, et al. Primary biliary cirrhosis associated with HLA-DRw3.

Tissue Antigens 1979;14;449-452. [S] Miyamori H, Kato Y, Kobayashi K, Hattori, N. HLA antigens in Japanese patients with primary biliary cirrhosis and autoimmune hepatitis. Digestion 1983;26:213-2 17. [6] Bassendine MF, Dewar PJ, James OFW. HLA DR antigens in primary biliary cirrhosis: lack of association. Gut 1985;26:625628. [A Johnston DE, Kaplan MM, Miller KB, Conners CM, Milford EL. Histocompatibility antigens in primary biliary cirrhosis. Am J Gastroenterol 1987;82:1127-1129. [8] Uryu N, Maeda M, Ota K, Tsuji K, Inoko, H. A simple and rapid method for HLA-DRB and DQB typing by digestion of PCR-amplified DNA with allele specific restriction endonucleases. Tissue Antigens 1990;35:20-31. [9] Ota M, Seki T, Fukushima N, Tsuji K, Inoko H. Modified PCR-RFLP method for HLA-DRBl genotyping. Tissue Antigens 1992;39:187-202. [IO] Ota M, Seki T, Nomura N, et al. Modified PCR-RFLP method for HLA-DPBl and -DQAl genotyping. Tissue Antigens 1991;38:60-71. [l I] Underhill J, Donaldson P, Bray G, Doherty D, Portmann B, Williams R. Susceptibility to primary biliary cirrhosis is associated with the HLA-DRS-DQB1*0402 haplotype. Hepatology 1992;16:1404 1408. [12] Morling N, Dalhoff K, Fugger L, et al. DNA polymorphism of HLA class II genes in primary biliary cirrhosis. Immunogenetics 1992;35:112-116. [13] Seki T, Kiyosawa K, Ota M, et al. Association of primary biliary cirrhosis with human leukocyte antigen DPB1*0501 in Japanese patients. Hepatology 1993;18:73-78. [14] Schwimmbeck PL, Yu DTY, Oldstone MBA. Autoantibodies to HLA-B27 in the sera of HLA-B27 patients with ankylosing spondylitis and Relter’s syndrome. Molecular mimicry with Klebsiella pneumoniae as potential mechanism of autoimmune disease. J Exp Med 1987;166:173-18 1. [15] Horn G, Bugawan T, Long C, Erlich H. Allelic sequence variation of the HLA-DQ loci: Relationship to serology and to insulin-dependent diabetes susceptibility. Proc Nat1 Acad Sci USA 1988;85:60126016. [16] Briggs DC, Donaldson PT, Hayes P, Welsh KI, Williams R, Neuberger JM. A major histocompatibility complex class III allotype (C4B2) associated with primary biliary cirrhosis (PBC). Tissue Antigens 1987;29:141-145. [17] Fugger L, Morling N, Ryder LP, et al. NcoI RFLP of tumor necrosis factor alpha region in primary biliary cirrhosis in healthy Danes. Stand J Immunol 1989;30:185-189. [18] Todd JA, Acha-Orbea H, Bell JI, et al. A molecular basis for MHC class II-associated autoimmunity. Science 1988;240:1003-1009. [19] Watanabe Y, Tokunaga K, Matsuki K, et al. Putative amino acid sequence of HLA-DRB chain contributing to rheumatoid arthritis susceptibility. J Exp Med 1989;169:2263-2268. [20] Farrant JM, Doherty DG, Donaldson PT, et al. Amino acid substitutions at position 38 of the DRB polypeptide confer susceptibility to and protection from primary sclerosing cholangitis. Hepatology 1992:16:390-395.