Primary biliary cirrhosis: contribution of HLA class II allele DR8

Transcription

1 Quarterly journal oi Medicine, 1993; 86: Primary biliary cirrhosis: contribution of HLA class II allele DR8 W.L. GREGORY 1 ' 2, W. MEHAL 3, A.N. DUNN 4, C. CAVANAGH 4, R. CHAPMAN 3, K.A. FLEMING 3, A.K. DALY 1, J.R. IDLE 1, O.F.W JAMES 2 and M.F. BASSENDINE 2 From the 1 Department of Pharmacological Sciences, University of Newcastle upon Tyne; 2 Department of Medicine, University of Newcastle upon Tyne; 3 Nuffield Department of Pathology and Bacteriology, University of Oxford; 4 Tissue Typing Laboratory, Regional Blood Transfusion Centre, Newcastle upon Tyne Received 7 February 7993; Accepted 22nd March 1993 Summary Primary biliary cirrhosis is a chronic cholestatic disease of unknown aetiology which predominantly affects middle-aged women. It is thought to be autoimmune in nature, but unlike many autoimmune diseases no clear HLA association has been described. Several studies have suggested conflicting associations with HLA class II, although a DR8 association is most frequently described. To test the hypothesis that primary biliary cirrhosis is associated with a certain HLA class II locus we genotyped 0 patients with the disease from the north-east region of England and 363 local healthy controls. HLA-DRB1 and confirmatory DQA and DQB genotypes were determined by Taq\ restriction fragment DNA length polymorphism analysis. In addition, a polymerase chain reaction technique (double ARMS) was used to investigate the DRB3 locus (DR52) in 98 primary biliary cirrhosis patients and 107 local controls. We found an increased frequency of HLA-DR8 (18.5% vs 9.2%, p<0.005, relative risk of 2.0 [ ]) in the primary biliary cirrhosis group. HLA-DR8-positive primary biliary cirrhosis patients had a higher serum bilirubin level (p= 0.03) than DR8-negative patients. There was no difference in the DR52 frequencies and no association with markers of disease severity. These results support earlier serological findings, although the association between primary biliary cirrhosis and DR8 is weaker than previously described. In addition, DR8-positivity may identify a clinical subgroup with a worse prognosis. Introduction The human major histocompatibility complex (MHC) on the short arm of chromosome 6 is highly polymorphic. It comprises three distinct groups of genes, termed human leukocyte antigens (HLA) class I, class II and class III (Figure 1). The class II region, including DR and DQ, encodes a number of transmembrane glycoproteins. These gene products are involved in cell cell interaction with antigen-specific cell surface receptors of T lymphocytes and, therefore, play a pivotal role in initiating the immune cascade in response to foreign antigen presentation. Susceptibility or resistance to a number of diseases thought to be autoimmune in nature, has been found to be associated with specific residues, alleles or haplotypes at class II loci. 1 The mechanism of such associations is not fully understood, but reinforces the proposed immunogenetic basis of these conditions. Address correspondence to Dr M.F. Bassendine, Department oi Medicine, The Medical School, University oi Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, UK Oxford University Press 1993

2 394 W.L. Gregory et al. (i) (ii) (iii) 6q iii i ii tmii C& C4B / \ TNFo TNF 6p subregions and consequently, it is now possible to genotype individuals using molecular biology techniques such as restriction fragment length polymorphism and polymerase chain reaction which give more definitive results. 18 In addition, many studies of class II association with primary biliary cirrhosis have relied on small sample sizes and inappropriate controls. We have genotyped a large series of primary biliary cirrhosis patients from the north east of England and compared them with a larger series of local unrelated healthy controls using restriction fragment length polymorphism analysis for DRB1 and DQ regions and a polymerase chain reaction technique for the DRB3 region, previously termed DRw52a, b and c phenotypes. B2 «II Al Figure 1. Simplified map of the human major histocompatibility complex (MHC). (i) Location of the MHC within band 6p21.3 of chromosome 6. (ii) Location of HLA class I, II and III gene regions within the MHC: TNF, tumour necrosis factor; "BAT"-"HLA B-associated transcript" genes, 21 OH- 21-hydroxylase, C2, C4 and Bfcomplement genes, (iii) Details of the class II genes. The expressed A genes encode class Ilex (heavy) chains and the expressed B genes encode the highly polymorphic class lip (light) chains. Primary biliary cirrhosis is a chronic cholestatic disorder of unknown aetiology, predominantly affecting middle-aged women. 2 It is thought to be an autoimmune disease because of the presence of circulating autoantibodies, hypergammaglobulinaemia with elevated levels of IgM, altered cellular immune function, and an association with other autoimmune phenomena. Genetic factors are also thought to be important since primary biliary cirrhosis can be familial 3 " 5 and immunological abnormalities may be present in first-degree relatives. 6 ' 7 Unlike many other autoimmune conditions, however, the evidence for an HLA association with primary biliary cirrhosis is conflicting. Most studies to date have used phenotyping methods; two early studies in British patients found no HLA association. 8 ' 9 Several studies have reported an association between primary biliary cirrhosis and HLA-DR8. 10 " However, associations with HLA-DR3, 14 ' 15 HLA-DR4, 16 and HLA-DR2 17 have all been described in various populations. Furthermore, one study found primary biliary cirrhosis patients positive for HLA-DR52 to have a worse prognosis. 10 Until recently, HLA class II typing was exclusively performed using serological and cellular methods. These phenotyping assays are not sensitive enough to resolve the full spectrum of alleles. 18 Molecular cloning has identified the genes within the class II Methods Ten ml of EDTA-whole blood was collected from 363 local healthy volunteers attending the local blood transfusion service. Blood samples were collected from 0 consecutive, unrelated patients with primary biliary cirrhosis. The diagnosis was based on the triad of cholestatic liver blood tests, serum anti-mitochondrial antibody positivity and liver histology diagnostic of, or consistent with, the disease (Table 1). Cenomic DNA from the healthy controls was extracted from whole blood by the salting-out method 19 and for the patient group a 340A nucleic acid extractor (Applied Biosystems, Warrington UK) was used. Restriction fragment length polymorphism genotyping, as described by Bidwell and Bignon 20 was performed by digesting 8.25 u.g DNA with 40 units Taq\ restriction endonuclease, followed by agarose gel electrophoresis and Southern transfer using positively-charged nylon membrane (Hybond-N +, Amersham, UK). The membranes were then hybridized and radiolabelled using the cdna probes (kindly provided by David Wilson, Regional Tissue Typing Laboratory, Glasgow, UK) prtv1 for the DRB1 locus, 20 pll-b-1 for the DQB locus 20 and pdch1 for the DQA locus. 20 Certain Taql DRB1-restriction fragment length polymorphism patterns cannot be separated and therefore, discrimination was based upon known DRB1-DQB- DQA restriction fragment length polymorphism associations due to linkage disequilibrium. 20 In addition, 107 of the healthy controls and 98 of the primary biliary cirrhosis patients were genotyped for the HLA-DRB3 (DR52 serotype) using an amplification refractory mutation system (ARMS) with multiple allele-specific primers as described by Lo et a/. 21 Primers DB3, P-a, P-b, and P-c were ARMS primers designed from published sequences and deliberate mismatches near the 3'-end were

3 Alkaline phosphatase* (IU/I) 392 ( ) ' biliary cirr 'losis and HLA-DR8 Table 1 Clinical details of patients with PBC Age* (years) Sex (female/ male) Histological stage Prothrombin* time (sees) Albumin* (g/d Bilirubin* (Hmol/I) Alanine transaminase* (IU/I) PBC (n=0) 59 (35-82) 122/8 I = 35 ll/lll = 59 IV = 36 (11-17) 40 (28-45) 12 (3-421) 62 (8-258) 'Median value (range)

4 396 W.L. Gregory et al. Results As can be seen in Figure 3, DR8 typing with this method is very accurate. The resultant single AAT (360) b (188) c (128) a (95) Figure 2. HLA-DRB3 typing by double ARMS. Lane 1, polymerase chain reaction product from an individual with DRw52a; Lane 2, no DRB3 alleles (null); Lane 3, DRw52c; Lane 4, DRw52b; Lane 5, DRw52a; Lane 6, DNA molecular weight marker VIII (Boehringer Mannheim, Lewes, East Sussex, UK). AAT, a, b and c indicate positions of a^antitrypsin positive control, polymerase chain reaction products from the DRB3*0101 (DRw52a), DRB3*O2O1/2 (DRw52b) and DRB3'0301 (DRw52c) respectively. Numbers in parentheses indicate sizes of product in base pairs. LANE SIZE (kb) 14.8 v#r ^ 7.8-" m mm <m DR8 m 0 5A Figure 3. HLA-DRB1 typing by Taq I restriction length polymorphism. Each lane contains a DNA fragment pattern which corresponds to particular HLA DRB1 genotype(s). Fragment sizes are taken from Bidwell and Bignon.20 In lane 3 the single 9.6 kb fragment (8.66 kb in the Tenth Histocompatibility Workshop) is specific to DR8 and the remaining four fragments correspond to DR4. The individual is thus heterozygous for DR4/8. Similarly, lane 1 = heterozygous DR7 /14b, lane 2 = DRK'BrW1 and lane 4 = DRK'BrO/ kb fragment associated with the DR8 genotype is unique and easily recognized. This fragment corresponds to the 8.66 kb band defined by the Tenth International Histocompatibility Workshop. Table 2 shows the frequencies of HLA-DRB1 alleles in both the control and patient groups. There was an increased frequency of DR8 in the primary biliary cirrhosis group (18.5%) compared with the control group (9.1%), giving a relative risk of 2.0 ( ). Furthermore, DR8-positive primary biliary cirrhosis patients had a significantly higher (p = 0.03) serum bilirubin level [median 18 mmol/l, (range 5-125)] compared with DR8-negative patients [median 11 mmol/l, (range 3-421)]. No differences were detected between other markers of disease severity: histological stage, serum liver function tests (alkaline phosphatase, transaminases and albumin) and prothrombin time. DRB1-restriction fragment length polymorphism fragments 72/9 and a1 were increased in the control group (Table 2). The Taql restriction frag- introduced in P-a, P-b and P-c to increase specificity. Primer DB3 is DRB3-specific and primers P-a, P-b and P-c distinguish between the alleles DRB3*0101 (DRw52a), DRB3*0201 and DRB3*0202 (DRw52b) and DRB3*0301 (DRw52c) respectively. AAT-1 and AAT-2 are internal positive controls and produced a 360-base pair polymerase chain reaction product from exon III of the o^-antitrypsin gene.21 Polymerase chain reaction was done in 100 \i\ reaction volume with five units Taq polymerase. To allow multiplex allele-specific polymerase chain reaction, all six primers were present in the single reaction vessel. Thirty cycles of polymerase chain reaction (denaturation, 94 C 1 min; annealing, 54 C 1 min; extension, 72 C 1 min) were done on 1 ug genomic DNA (Figure 2). Statistical evaluation was performed using %2 analysis with Yates's correction. As one aim of the study was to confirm or refute a disease association with HLA-DR8, as described previously,10" the probability values given are not corrected for the number of antigens tested with regard to this allele. Differences in prognostic indicators for the various HLA genotypes in the primary biliary cirrhosis patient group were compared using the Mann-Whitney U test.

5 Primary biliary cirrhosis and HLA-DR8 397 Table 2 Frequencies of HLA-DRB1 alleles in healthy controls and PBC patients using TaqI restriction fragment length polymorphism HLA-DR serological specificity DRB1 RFLP Controls (%) (n = 363) PBC (%) (n = 0) P value (uncorrected) 1 & 'BR' & ( not significant) 1/Br / a r a 3 a 4 b 14a 14b (26.4) 4(36.9) 61 (16.8) 59 (16.3) 33 (9.1) 5 (1.4) 31 (8.5) (3.6) 20 (5.5) 24 (6.6) 14 (3.9) 9 (2.5) 12 (3.3) 7(1.9) 95 (26.2) 8 (2.2) 45 (12.4) 12 (3.3) 1 (0.3) ment length polymorphism method cannot differentiate between fragments 7 2 and However, the statistical significance for both is low and as such associations have not been described before, their probabilities require correction for multiple testing. This renders them insignificant. There was no difference between the frequencies of the class II DRB3 alleles (HLA-DR52) in both groups (Table 3). Furthermore, there were no differences in the markers of disease severity (histological stage, serum liver function tests and prothrombin time) between DR52-positive and DR52-negative patients. 39 (30.0) 52 (40.0) 23 (17.7) 11 (8.5) 24 (18.5) 2(1.5) 12 (9.2) 2 (1.5) 1 (0.8) 9 (6.9) 5 (3.8) 0(0) 8 (6.2) 3 (2.3) 31 (23.8) 0(0) 16 (12.4) 5 (3.8) 1 (0.8) Discussion <0.05 < <0.05 Our data confirm an association between primary biliary cirrhosis and HLA-DR8 in a homogeneous British Caucasian population. The strength of the association is comparable to that recently described in a similar study using genotyping methods on an equally large British series of patients. 22 However, the association is weaker than that described using less definitive phenotypic methods. 10 " Therefore, unlike many other autoimmune conditions, such as chronic active hepatitis 23 and insulin-dependent diabetes mellitus, 24 Table 3 Frequencies of HLA-DRB3 alleles in healthy controls and PBC patients using PCR HLA-DR serological specificity DRB3* Allele Controls (%) (n = 107) PBC (%) (n = 98) P value (uncorrected) Null Total DR52 positive - 51 (47.6) 58 (54.2) 59 (60.2) 40 (40.8) DRw52a DRw52b DRw52c & (27.1) 18 (16.8) 11 (10.3) 16 (16.3) 20 (20.5) 4 (4.1) ( not significant)

6 398 W.L. Gregory et al. there appears to be a relatively small contribution from the class II region of the major hostocompatibility complex to susceptibility to primary biliary cirrhosis. As recombination between DR/DQ and DP occurs with a frequency of less than 3% 25 one can predict that an association of similar magnitude with DP also exists. It is interesting to speculate whether the higher frequency of DR8-positivity in both our patient and control groups (19% vs. 9%) compared to a similar study 22 of patients largely from southern England (11% vs. 4%) may explain the apparent increased incidence of primary biliary cirrhosis in the north east of England. 26 Our investigations have shown that primary biliary cirrhosis patients positive for DR8 have a significantly higher serum bilirubin level than DR8- negative individuals. Serum bilirubin levels are considered to be one of the main prognostic indicators in primary biliary cirrhosis. 27 " 29 One can speculate that DR8-positivity may identify a clinical subgroup of patients within a heterogeneous disease, who have a worse prognosis. This may prove to be useful clinically and further prospective studies are required to substantiate this. Our data support the hypothesis that, at least in a subgroup of primary biliary cirrhosis patients, there is an immunogenetic predisposition to either developing the disease and/or having a worse prognosis. Within this subgroup either DR8 could be in linkage disequilibrium with the true disease susceptibility gene(s) on chromosome 6 or this allele itself could be responsible for an altered immune response to exogenous or endogenous antigen. Sequence analysis of the DR8 allele to date has identified seven separate allelic variants showing microheterogeneity within this locus 30 and these are amenable to polymerase chain reaction typing techniques. It is now becoming apparent that single base changes and therefore, single amino acid residue changes in certain class II gene products are important in the development of other autoimmune diseases such as chronic active hepatitis and insulin-dependent diabetes mellitus. 23 ' 24 It has been suggested that such minor substitutions cause conformational changes in these glycoproteins which are sufficient to alter peptide and/or T cell receptor interactions, thereby altering the immune response. 24 This may apply to the DR8-positive primary biliary cirrhosis subgroup and further sequence analysis of this region may be fruitful. In the case of insulin-dependent diabetes, the strongest association appears to be with the polymorphic residues on the DQ3.2 P chain, 24 and this is now thought to be one of the susceptibility candidate genes in the development of autoimmunity. Our restriction fragment length polymorphism analysis of DQA and DQB alleles was performed to confirm the DRB1-restriction fragment length polymorphism typing by linkage disequilibrium 20 and therefore, too limited to be of use with regard to disease association. However, Underhill et al., 22 using polymerase chain reaction and sequence specific oligonucleotide analysis of DQB genotypes in primary biliary cirrhosis patients found an increased frequency (11% vs. 3%) in the DQB1*0402 allele which is itself linked to DR8, suggesting that the DQ locus may be as important as DR8 but not more so. Primary sclerosing cholangitis is a disease of the liver with many similarities to primary biliary cirrhosis. 2 The two diseases have clinical and pathological features in common and an immunological cause has been postulated for primary scerosing cholangitis. One study recently found a very strong association between primary sclerosing cholangitis and the HLA-DRw52a phenotype (DRB3*0101 allele) 11 and although not as strong, this association was confirmed using gene amplification and sequence-specific oligonucleotide probing. 31 In addition, using phenotyping techniques the Mayo Clinic group found DR52-positive patients with primary biliary cirrhosis have a worse prognosis. 10 Using a polymerase chain reaction method we have found no association between alleles within the DRB3 locus and primary biliary cirrhosis. Furthermore, there was no correlation between markers of disease severity and the presence of DR52 antigens. The conflicting evidence previously published with regard to primary biliary cirrhosis and HLA class II alleles presumably reflects small patient sample size, or conversely, large control population size in some studies, together with possible racial variation in the patient and control populations and the inherent limitations of previous phenotyping methods. In summary, our study has shown an association between primary biliary cirrhosis and the HLA class II allele DR8; although only one in five subjects within our patient population carry this allele, these patients may have a worse prognosis. However, primary biliary cirrhosis is clinically a heterogeneous disease and therefore, almost certainly of multifactorial aetiology. A number of mechanisms operating differently in different patient subgroups may be important. The genetic contribution itself may be polygenic and is more likely to exert an influence rather than be the sole determining factor. The short arm of chromosome 6 is now known to contain numerous gene regions many of which have unknown or poorly understood function. 32 Clues to the elucidation of the pathogenesis of primary biliary cirrhosis may lie

7 Primary biliary cirrhosis and HLA-DR8 399 there, or perhaps the search for candidate genes conferring disease susceptibility should be widened and take into account the female preponderance of this enigmatic disease. Acknowledgements WLG is in receipt of an MRC Training Fellowship and WM is a Wellcome Medical Graduate Fellow. References 1. Todd JA, Acha-Orbea H, Bel! Jl, et a/. A molecular basis for MHC class II associated autoimmunity. Science 1988; 240: Sherlock S, Dooley J. Diseases ol the Liver and Biliary System, 9th Edn. Oxford: Blackwell Scientific Publications, Walker JC, Bates D, Doniach D, Ball PAJ, Sherlock S. Chronic liver disease and mitochondnal antibodies. Br Med 11972; i: Fagan EA, Williams R, Cox S Primary biliary cirrhosis in mother and daughter Br Med 11977; ii: Kato Y, Suzuki K, Kumagai M, et a/. Familial primary biliary cirrhosis. Am I Castroenterol 1981; 75: Tsuji H, Mwai K, Akagi K, Fujishima M. Familial primary biliary cirrhosis associated with impaired Concanavalin A-induced lymphocyte transformation in relatives. Two family studies. Dig Dis So 1992; 37: Calbraith RM, Smith M, Mackenzie RM, Tee DE, Doniach D, Williams R. High prevalence of seroimmunologic abnormalities in relatives of patients with active chronic hepatitis or primary biliary cirrhosis. N Engl I Med 1974; 290: Hamlyn AN, Adams D, Sherlock S. Primary or secondary sicca complex? Investigation in primary biliary cirrhosis by histocompatibility testing. BM11980; 281: Bassendine MF, Dewar PJ, James OFW. HLA-DR antigens in primary biliary cirrhosis: lack of association. Cut 1985, 26: Cores CJ, Moore SB, Fisher LD, Powell FC, Dickson ER. Primary biliary cirrhosis: associations with class II major histocompatibility complex antigens. Hepatology 1987; 7: Prochazka EJ, Terasaki PI, Park MD, Goldstein LI, Busuttil RW. Association of primary sclerosing cholangitis with HLA-DRw52a. N Engl I Med 1990; 322: Manns MP, Bremm A, Schneider PM, et al. HLA DRw8 and complement C4 deficiency as risk factors in primary biliary cirrhosis. Castroenterology 1991; 101: Maeda T, Onishi S, Saibara T, Iwasaki S, Yamamoto Y. HLA DRw8 and primary biliary cirrhosis. Castroenterology 1992; 103: Ercilla C, Pares A, Arriaga F, et al. Primary biliary cirrhosis associated with HLA-DRw3. Tissue Antigens 1979; 14: Morling N, Dalhoff K, Fugger L, et al. DNA polymorphisms of HLA class II genes in primary biliary cirrhosis. Immunogenetics 1992; 35: Johnston DE, Kaplan MM, Miller KB, Connors CM, Milford EL. Histocompatibility antigens in primary biliary cirrhosis. Am I Castroenterol 1987; 82: Miyamori H, Kato Y, Kobayashi K, Hattori N. HLA antigens in Japanese patients with primary biliary cirrhosis and autoimmune hepatitis. Digestion 1983; 26: Mytilineos J, Scherer S, Opelz C. Comparison of RFLP-DR beta and serological HLA-DR typing in 1500 individuals. Transplantation 1990; 50: Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Adds Res 1988; 16: Bidwell JL, Bignon JD. DNA-RFLP methods and interpretation scheme for HLA-DR and DQ typing. Eur I Immunogenet 1991; 18: Lo Y-MD, Mehal WZ, Wordsworth BP, et al. HLA typing by double ARMS. Lancet 1991; 338: Underhill J, Donaldson P, Bray C, Doherty D, Portmann B, Williams R. Susceptibility to primary biliary cirrhosis is associated with HLA-DR8-DQBT0402 haplotype. Hepatology 1992; 16: Donaldson PT, Doherty DC, Hayllar KM, McFarlane IC, Johnson PJ, Williams R. Susceptibility to autoimmune chronic active hepatitis: human leukocyte antigens DR4 and A1-B8-DR3 are independent risk factors. Hepatology 1990; : Todd JA. Genetic control of autoimmunity in type 1 diabetes. Immunol Today 1990; 11: Ronningen KS, Spurkland A, Markussen G, Iwe T, Vardtal F, Thorsby E Distribution of HLA class II alleles among Norwegian Caucasians. Hum Immunol 1990; 29: Myszor M, James OFW The epidemiology of primary biliary cirrhosis in North-east England: an increasingly common disease? Q / Med 1990; 276: Shapiro JM, Smith H, Schnaffner F. Serum bihrubin. a prognostic indicator in primary biliary cirrhosis. Gut 1979; 20: Dickson ER, Crambsch PM, Fleming TR, Fisher LD, Langworthy A. Prognosis in primary biliary cirrhosis: model for decision making. Hepatology 1989; 10: Rydning A, Schrumdf E, Abdelnoor M, Elgjo K, Jenssen E. Factors of prognostic importance in primary biliary cirrhosis. Scand I Castroenterol 1990; 25: Bodmer JC, Marsh SGE, Albert ED et al. Nomenclature for factors of the HLA system, Vox Sang 1992; 63: Farrant JM, Doherty DC, Donaldson PT, et al. Amino acid substitutions at position 38 of the DRp 1 polypeptide confer susceptibility to and protection from primary sclerosing cholangitis. Hepatology 1992; 16: Ziegler A, Field LL, Sakaguchi AY. Report of the committee on the genetic constitution of chromosome 6. Cytogenet Cell Cenet 1991; 58: