MHC class II sequences of susceptibility and protection in Mexicans with autoimmune hepatitis

Journal of Hepatology 1998; 28: 985–990 Printed in Denmark ¡ All rights reserved Munksgaard ¡ Copenhagen

Copyright C European Association for the Study of the Liver 1998

Journal of Hepatology ISSN 0168-8278

MHC class II sequences of susceptibility and protection in Mexicans with autoimmune hepatitis Miriam Nohemı´ Va´zquez-Garcı´a1,2, Carmen Ala´ez1, Ange´lica Olivo1, He´ctor Debaz1, Elva Pe´rez-Luque1, Ana Burguete1, Silvia Cano1, Gabriela de la Rosa1, Norma Bautista1, Arturo Herna´ndez1, Jose´ Bandera3, Luis Fernando Torres, David Kershenobich3, Fernando Alvarez4 and Clara Gorodezky1 1

Department of Immunogenetics, Instituto Nacional de Diagno´stico y Referencia Epidemiolo´gicos, SSA, 2Biomedicine Molecular Program, Centro de Investigacio´n y Estudios Avanzados, IPN, 3Gastroenterology Service, Instituto Nacional de la Nutricio´n Dr. Salvador Zubira´n, Mexico and 4 Service de Gastroenterologie, Hopital Ste Justine, Montreal, Que´bec, Canada

Background/Aims: Autoimmune hepatitis has a genetic background associated with different HLA DRB1 alleles depending on the ethnic group. The aim of this study was to analyse the immunogenetics of type I autoimmune hepatitis in Mexicans. Methods: Thirty Mexican Mestizo patients and 175 healthy controls were HLA typed as follows: class I antigens were determined by microlymphocytotoxicity and class II typing was done on DNA samples using PCR-SSO and PCR-SSP for DRB1, DQA1 and DQB1 loci. Results: A significant association of autoimmune hepatitis with DRB1*0404 was found, (c2YΩ19.95, pcΩ0.002, RRΩ7.71), suggesting the presence of a susceptibility gene located at the DRB1 locus. Resistance was at least partially due to a DQB1 gene, since a significant decrease in DQB1*0301 was also detected (c2YΩ8.21, pcΩ0.04). Analysis of subgroups

according to age at onset showed an association with DRB1*0404 (c2YΩ4.31, pΩ0.04) in patients with late onset (after 30 years), while DQA1*0501 (c2YΩ5.12, pΩ0.02) was increased in the early onset group. Conclusions: The possible mechanism of HLA association is due to ‘‘shared epitopes’’, since DRB1*0404, and those found in other populations namely, DRB1*0401, *0405 and *0301 share almost the same sequence at position 67–72 (LLEQRR, R or K at 71). Valine-86 is also relevant to the age at onset because DRB1*0404 is increased in the patients with an average age at onset of 32. These findings are relevant in determining which peptides in the liver are targets for T cells.

 with autoimmune hepatitis (AH) have hypergammaglobulinaemia and several circulating autoantibodies. Among the non-organ-specific antibodies are anti-double-stranded DNA (1), anti-nuclear (ANA) and anti-smooth muscle antibodies (SMA) (2). Specific antibodies include anti-liver kidney microsomal type 1 (LKM1) (3,4), anti-asialoglycoprotein receptor (ASGPR) (5,6), anti-liver cytosol antigen type 1

(LC1) (7,8), anti-soluble liver antigen (SLA) (9,10), anti-histones (11) and perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) (12). It is now clear that there are mechanisms which discriminate self from non-self antigens, that maintain the immune system in equilibrium and avoid triggering a pathological autoimmune response against self (13). The Major Histocompatibility Complex (MHC) is a central system in these functions and is therefore also implicated in susceptibility and resistance to many diseases, specially autoimmune diseases, including AH (13). Several associations of HLA antigens with AH have been demonstrated; the first report showed an increase in HLA A1-B8 in Australians (14), but it became clear

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Received 15 December 1997; revised 27 January; accepted 27 January 1998

Correspondence: Clara Gorodezky, Department of Immunogenetics INDRE, SSA, Carpio 470 Col. Santo Toma´s, Mexico D.F. 11340 Mexico. Tel: (525)341-4569. Fax: (525) 538-1884. e-mail: cgorodea/mailer.main.conacyt.mx

Key words: Autoimmune hepatitis; Class II; DNA; HLA association; Immunogenetics; MHC; PCRSSO; Shared epitope; Susceptibility.

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later that the true association was with class II, DR3 and DR4 alleles (15–20). In fact, A1-B8 is present only because of linkage disequilibrium, while DR3 and DR4 contribute independently to the disease (15–19). The analysis in distinct populations demonstrated different associations. While DR4 was increased in Japanese (18), DR3 (DRB1*0301) was important in Caucasians. However, among the molecular subtypes of DR4, DRB1*0401 variant (20) was the second associated gene in Whites, but DRB1*0405 (19) was found to be increased in Japanese. DR genes are also relevant in the clinical behaviour of AH (21,22). Moreover, DR4 patients respond better to treatment and have a different clinical pattern to those positive for DR3 (17). Among Hispanics, DRB1*0405 and *1301 were increased in Argentinians (22). A report of the same group claimed that class I antigens were also involved, because 88% of the patients with extrahepatic manifestations were DR4-A11, suggesting a synergic effect of the two types of molecules (23). Thus, MHC genes are clearly involved in AH, but very little is known of the HLA pattern of the disease in non-Caucasian groups. Studies of AH have been done in populations of White genetic background, such as Argentinians or Anglo-Saxons, and of Mongoloid origin such as Japanese, but not in Mexicans and other ethnic groups. The genetic composition of Mexican Mestizos is the result of an admixture of genes of Amerindian and Spanish origin with a small gene pool from the Black slaves brought to Mexico by the Spaniards during the 16th Century (24,25). The purpose of this work was to investigate the pattern of MHC genes in Mexicans, since this knowledge is important in understanding the immunogenetic mechanisms underlying the disease, through the localization of the precise class II sequences involved in populations with different ethnicity.

Materials and Methods Patients Thirty unrelated Mexican Mestizo patients were included. They are residents of Mexico City and were born in the highlands of the country. They were classified as AH type 1 according to international criteria (26), with high titres of ANA and or SMA. They were attending the outpatient clinic of the Liver Unit of the National Institute of Nutrition (INN) ‘‘Dr. Salvador Zubira´n’’ and signed an informed consent form. The patients had an average age at onset of x¯Ω32.1 years (12–58 years). All of them had histologically proven active chronic hepatitis. Two patients showed cirrhosis at diagnosis. The minimal duration of symptoms was

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6 months. Patients had 3-fold to 10-fold elevations of serum aminotransferase activity and gammaglobulin levels were twice normal (Table 1). Control group One hundred and seventy-five healthy, non-related adults belonging to the same ethnic group, without autoimmune or chronic disorders, were included for comparison. All were born in and have always lived in Mexico. Immunological tests Autoantibodies were detected with indirect immunofluorescence methods using adequate tissues. The patients were seropositive for ANA, SMA or both (the specificity of these antibodies was against dsDNA and actin), with titers of 1:40 or greater. All patients included in the study were LKM1 seronegative, tested by immunoblot and dot blot using human liver microsome and cytosolic proteins and the P450IID6 peptides representing sequential epitopes (27). This test was done by Dr. Fernando Alvarez at the Service of Gastroenterology of St. Justine Hospital in Montreal, Quebec, Canada. All patients were HBsAg negative (Ausab ELISA kit (hepatitis B surface antigen (human) (subtypes ad and ay); Abbot) and HCV negative (ELISA second-generation test system for the detection of antibody to hepatitis C virus (anti-HCV) in human serum or plasma; Ortho Diagnostics). Serological typing HLA-A, B, C antigens were typed using the standard microcytotoxicity technique (28). Nylon wool was used

TABLE 1 Clinical and immunological features of autoimmune hepatitis in Mexicans Patients (nΩ30) Clinical findings Age (years)a Sex (Female/Male) Extrahepatic manifestations Laboratory findings ASAT (normal, ∞31 U/l)a ALAT (normal, ∞36 U/l)a IgG (normal, ∞3600 U/ml)a Autoantibodies ANAb (x¯Ω1:149) SMAb (x¯Ω1:115) SMA π ANAb LKM1b

32.1∫12.5 27 (90%)/3 (10%) 10 (33%) 292∫511.6 223∫201.3 7470∫206.87 56.7 (17/30) 53.3 (16/30) 33.4 (10/30) 0

ASATΩaspartate aminotransferase; ALATΩalanine aminotransferase; ANAΩanti-nuclear antibodies; SMAΩanti-smooth muscle antibodies; LKMIΩanti-liver kindney microsomal type 1. a Expressed as x¯ bExpressed in %.

HLA class II DNA in autoimmune hepatitis TABLE 2 HLA DRB1 alleles in Mexicans with autoimmune hepatitis Locus DRB1

*0101 *0102 *03011 *03012 *0403 *0404 *0407 *0701 *0802 *1301

Controls nΩ175 PF

Patients nΩ30 PF

n% 9 (5.14) 18 (10.29) 10 (5.71) 0 (0.00) 6 (3.43) 13 (7.43) 52 (29.71) 24 (13.71) 44 (25.14) 9 (5.14)

n% 5 (16.66) 3 (10.00) 5 (16.66) 2 (6.66) 2 (6.66) 11 (36.66) 7 (23.33) 4 (13.33) 7 (23.33) 4 (13.33)

c2Y

p

pc

RR

EF

19.95

0.00008

0.002

7.71

0.32

PFΩpopulation frequency; pΩprobability; pcΩcorrected probability; RRΩrelative risk; EFΩetiologic fraction. *0402, *0405, *0801, *0803, *0901, *1101, *1102, *1304, *1305, *1312, *1401, 1402 and *1502 are absent in the patients. *0401, *0411, *1001, *1104, *1201, *1302, *1303, *1406, *1501, *1503, *1601 and *1602 are present in the patients at frequency of 1.7%.

for T cell isolation from ACD blood samples. HLA typing was done with 218 reagents from the local Bank of the Department of Immunogenetics, which includes sera and monoclonal antibodies obtained locally and by international exchange. DNA typing Genomic DNA was obtained from peripheral blood leukocytes using proteinase K, phenol-chloroform extraction and isopropanol precipitation according to the protocols of the Twelfth International Histocompatibility Workshop (12W) (29). DNA was PCR amplified for the polymorphic second exon of DRB1, DQA1 and DQB1 genes using the primers recommended by the 12W. Generic as well as group-specific amplifications were done for DRB1-1, DRB1-2, DRB1-4 and DR52 associated alleles. A final volume of 100 ml was used containing 1 mg of DNA, dNTPs 200 mM, MgCl2 1.5 mM, 2U of Taq-polymerase (PROMEGA) and different primer concentrations depending on each region. The reaction was done for 35 cycles in a MJR Thermocycler denaturing for 60øø at 95æC, annealing for 60øø (the temperature varied according to the region) and an extension of 60øø at 72æC was done. A final extension was performed for 10 min at 72æC. PCR products were denatured, dot-blotted onto nylon membranes and immobilised using UV irradiation. The membranes were hybridised with the set of sequence-specific digoxigenin-labelled oligonucleotide probes recommended by the 12W (150 probes were used for the three loci). The membranes were washed with SSPE (2¿ and 5¿) at the Tm of each probe, they were incubated with phosphatase anti-DIG antibody (Boheringer-Mannheim No. 1363905) for 60 min. After removing the excess, the hybridisation patterns were visualised with

chemiluniscence detection on x-ray films using CSPD as a substrate (Boehringer-Mannheim No. 1655884). PCR amplification with sequence-specific primers (PCR-SSP) was done to confirm certain DRB1 (DRB1*1001, *0701, *0901, *0403 and *0404), DQA1 and DQB1 alleles (30). A final volume of 25 ml contain-

TABLE 3 Distribution of DQB1 in Mexicans with autoimmune hepatitis Allele

*0201 *0301 *0302 *0303 *0402 *0501 *0602 *0603 *0604

Controls nΩ175 PF

Patients nΩ30 PF

n% 33 (18.85) 85 (48.57) 88 (50.28) 3 (1.71) 50 (28.57) 29 (16.57) 26 (14.86) 9 (5.14) 6 (3.43)

n% 11 (36.66) 5 (16.66) 20 (66.66) 1 (3.33) 7 (23.33) 10 (33.33) 2 (6.66) 3 (10.00) 1 (3.33)

c2Y

p

8.21

0.004 0.04

pc

PrF

0.29

PFΩpopulation frequency; pΩprobability; pcΩcorrected probability; PrFΩprotective fraction. DQB1 *0502, *0503, *0504 *0601 are absent in the patients.

TABLE 4 Relationship between HLA class II genes and extrahepatic manifestations Allele

Patients with allele

Patients with allele with associated autoimmune disorders

DRB1 *0301

6

2 (33%)

DRB1 *0404 DQB1 *0301

11 5

5 (45%) 1 (10%)

1 Thyroiditis/1 Fisher Evans 4 SLE/1 Thyroiditis SLE

SLEΩsystemic lupus erythematosus.

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M. N. Va´zquez-Garcı´a et al. TABLE 5 Sequences involved in susceptibility to autoimmune hepatitis Population

Allele

Caucasian (a) Caucasian (a) Japanese (b) Argentinian (c)

DRB1 DRB1 DRB1 DRB1 DRB1 DRB1

Mexican

*0301 *0401 *0405 *0405 *1301 *0404

Age at onset

67

68

69

70

71

72

86

∞30 47 51 42 34 32

L L L L I L

L L L L L L

E E E E E E

Q Q Q Q D Q

K K R R E R

R R R R R R

V G G G V V

(a) Doherty et al. 1994 (20); (b) Seki et al. 1992 (19); (c) Fainboim et al. 1997 (22).

ing 1 mg of DNA, dNTPs 200 mM, MgCl2 1.5 mM, 0.5U of Taq-polymerase (PROMEGA) and different primer concentrations depending on each region was used for amplification. The reaction was done for 35 cycles in a MJR Thermocycler under the following conditions: denaturalization 60øø at 95æC; annealing for 60øø, (the temperature varied according to the region); and extension for 60øø at 72æC. A final extension was done at 72æC for 10 min. With these tests, 155 alleles were identified. Statistical methods Statistical analysis was performed according to the following methods (31): allele frequencies were calculated using Haldane’s method. The frequencies between patients and controls were compared using the c2 test with Yates correction. When an allele was found to be significantly increased in the patients, the relative risk (RR) and etiologic fraction (EF) were calculated, to assess the strength of the association. The preventive fraction (PF) was also calculated when an allele was found to be significantly decreased in the patients, in order to assess how much of the protection was due to the marker.

Results AH was more prevalent in women (90%). Aminotransferases and immunoglobulins were significantly increased; all patients had ANA and/or SMA but not LKM1 antibodies (Table 1). Thirty-three percent (10/ 30) had other autoimmune disorders: systemic lupus erythematosus (SLE) (5), thyroiditis (3), Sjo¨gren syndrome (1) and Fisher-Evans (1). DRB1*0404 was found to be significantly associated (18.3% in AH vs. 3.8% in controls; c2YΩ19.95 with a p-value of 0.00008 that remained significant after correction, multiplying the p-value by 22 alleles (pcΩ 0.002). The RR was 7.71 and the EFΩ0.32 (Table 2). A significant decrease in DQB1*0301 was also detected (8.3% in patients vs 28.3% in controls; c2YΩ8.21), that also remained significant after correction multiplying

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by the 9 DQB1 alleles (pΩ0.004; pcΩ0.04) with a PFΩ 0.29 (Table 3), suggesting that the susceptibility is located to the DRB1 locus, and resistance is linked to the DQB1 region. DRB1*0404 was present in 45% of the patients with other autoimmune diseases, namely SLE (clinical and immunological international criteria for diagnosis were followed (32)) and thyroiditis, but DQB1*0301 was present only in one of eight patients with other autoimmune disorders (Table 4). The power of the association was 84%, indicating that the size sample is sufficient and the association is real. No association was found with any class I antigen. An analysis subdividing the patients by age at onset was also done, separating patients before and after 30 years of age at onset. A significant increase in DRB1*0404 (c2YΩ4.31; pΩ0.04) was found in those patients with an onset of the disease after age 30, while in those with early onset, an increase of DQA1*0501 was shown (c2YΩ5.12; pΩ0.02). Both associations lost their significance upon correction. However, these results are important, because, DQA1*0501 is in linkage disequilibrium with the DRB1*0301 haplotype (DRB1*0301–DQA1*0501–DQB1*0201), which is involved in susceptibility to AH in Caucasians with an early onset (16,17).

Discussion Autoimmune hepatitis in Mexicans occurs as a classical type I autoimmune hepatitis with female prevalence, presence of ANA and or SMA and LKM1 negative. Although clinically similar to other populations, the differences found in the genetic profile demonstrate the importance of analysing ethnic groups other than Caucasians. While in Japanese the susceptibility allele was DRB1*0405 (19), in Argentinians DRB1*0405 and DRB1*1301 (22) were present and in Mexicans the association was with DRB1*0404. These data are consistent with the fact that in the Mexican population mainly Spanish and Indian genes are present. Moreover, DRB1*0301, has a very low frequency in Mexico

HLA class II DNA in autoimmune hepatitis

(24,25), and DRB1*0404 in Mexicans is associated with other autoimmune diseases such as rheumatoid arthritis (33). Concerning severity, an increase of autoimmune disorders, mainly thyroiditis has been reported in Caucasians (62%) (34), but in Mexicans, SLE was the primary associated disease (33%), (Table 4). The increase in DRB1*0404 in patients with secondary diseases suggests that this gene may be involved in the expression of associated autoimmune disorders, but other factors are involved, since not all DRB1*0404 patients showed secondary diseases. The analysis regarding late vs early onset was carried out because age may be relevant to severity, with tragic consequences early in life. The late average age at onset in Japanese and Caucasians was associated with DRB1*0405 and DRB1*0401, respectively (19,20). DRB1*0301 Caucasian carriers express the disease at a younger age (16,17). Our results suggest that DRB1*0404 is associated with a late onset and DQA1*0501 with an early onset (∞30 years), although more patients are needed to confirm these associations because the p-value became non-significant after correction. DQA1*0501 is present in DR3, 11, 12, 13 and 14 haplotypes that are DR52 associated (DRB3-locus). In fact, it has been previously shown that the DRB3 locus may be relevant to AH in young Caucasians (35). The involvement of the DQB1 locus in protection was shown here, suggesting that a specific aminoacid sequence in the DQB1*0301 allele may play an important role in protection. Data from Argentinians, Brazilians and Mexicans are in agreement (22). The aminoacid sequence of the third hypervariable region of DRB1 gene of DRB1*0301, *0401, *0404 and *0405 (Table 5) shares the same ‘‘motif ’’ from 67– 72 except for the exchange of K to R at 71 in DRB1*0404 and *0405, but both are basic residues; thus, this difference may not be functionally important. However, it has recently been claimed that K at position 71 may be the major risk factor in Caucasians (21). Moreover, position 86 shows a relevant difference: DRB1*0401 and *0405 have a glycine (G) (polar aminoacid) and DRB1*0301 and *0404 have a substitution for valine (V), a non-polar residue. The patients with DRB1*0401 (Caucasians) and *0405 (Japanese) have a late onset and the patients with DRB1*0301 (Caucasians) and *0404 (Mexicans) have an early onset. This substitution could be central to the recognition of the ‘‘hepatogenic peptide’’ and consequently the onset of disease may be driven by the associated autoantigens, since the residues of the DRB1 molecule play a critical role in antigen presentation to the TCR and to the induction of the immune response. Interest-

ingly, DRB1*1301 carried by Argentinian AH patients has two important substitutions at 70 (Q/D) and 71 (R/E) which may bind different liver peptides. Thus, the aminoacids at position 67–72 may be critical for selecting the specificity of the TCR repertoire, leading to the autoimmune response. Some substitutions like those discussed here, are implicated in susceptibility to different diseases such as insulin-dependent diabetes mellitus (36). In addition, in a recent report the importance of pocket 4 in viral peptide recognition was also emphasised (37). We propose the ‘‘shared epitope’’ hypothesis as the responsible mechanism of susceptibility of AH since the LLEQRR ‘‘motif ’’ at position 67–72 is identical in DRB1*0405 (susceptibility allele in Japanese) and DRB1*0404 (susceptibility allele in Mexicans); in Caucasians the associated DRB1*0401 and DRB1*0301 have only the difference already mentioned at position 71. Thus, the importance resides in having the same ‘‘motif ’’, even if the HLA molecules are different. Finally, position 86 influences peptide binding and may determine an early or a late onset because, as shown here, V is present in both alleles involved in an early average age at onset of Caucasians and Mexicans, while G is present at a later onset in the Caucasian DRB1*0401 and the Japanese DRB1*0405 (Table 5). These results should stimulate analysis of patients from different ethnic groups and open up a complete area of research into the hierarchy of class II-peptide affinities, enabling determination of the exact sequences of resistance and susceptibility, and focusing on prediction and prevention of AH.

Acknowledgements We are grateful to Gloria Garcı´a for typing the manuscript and to Eduardo Infante for help with computer analysis. This work was partly supported by CONACyT, grant no. 115763.

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