HLA class I and Class II are both associated with the genetic predisposition to primary Sjögren syndrome

HLA Class I and Class II are Both Associated With the Genetic Predisposition to Primary Sjo¨gren Syndrome Pascale Loiseau, Virginia Lepage, Fairouz Djelal, Marc Busson, Ryad Tamouza, Colette Raffoux, Charles Joel Menkes, Olivier Meyer, Dominique Charron, and Danie`le Goldberg ABSTRACT: Primary Sjo¨gren syndrome (pSS) is an autoimmune disease characterized by progressive destruction of the exocrine glands leading to mucosal and conjonctival dryness. It is marked by lymphocytic infiltration of the glands and the accumulation of several types of autoantibodies such as rheumatoid factor (RF), antinuclear, anti-SS-A (anti-Ro) and anti-SS-B (anti-LA) autoantibodies. The susceptibility to pSS and/or the presence of SS-A/SS-B autoantibodies in pSS patients is associated with DRB1*03-DQB1*02 and DRB1*02-DQB1*06 haplotypes, whereas no associations have been described with any HLA class I allele. To define the impact of HLA class I alleles in predisposition to pSS, 46 patients responding to the European criteria and 222 healthy unrelated Caucasians were analyzed for their HLA class I and

INTRODUCTION Primary Sjo¨gren syndrome (pSS) is an autoimmune disease characterized by progressive destruction of the exocrine glands leading to mucosal and conjonctival dryness [1]. pSS generally affects middle-age females, with a prevalence of approximately 1/2500. Patients suffering from pSS present several immune abnormalities including the lymphocytic infiltration of the exocrine glands, predominantly with activated CD4⫹ ␣␤T cells [2– 4] and the oligoclonal B-cell activation From the Laboratoire d’Immunologie et d’Histocompatibilite´ (P.L., V.L., R.T., C.R., D.C.), Hoˆpital Saint-Louis AP-HP, Inserm Unite´ 396 (M.B.), Hoˆpital Saint-Louis Paris France, Service de Rhumatologie (C.J.M.), Hoˆpital Cochin (O.M.), Hoˆpital Bichat (F.D., D.G.), Centre Me´dico-Chirurgical de la Porte de Pantin, Paris, France. Address reprint requests to: Dr. P. Loiseau, Laboratoire d’Immunologie et d’Histocompatibilite´, Hoˆpital Saint-Louis, 1, avenue Claude Vellefaux 75010, Paris, France; Tel: ⫹33 (0) 1-42-49-90-93; Fax: ⫹33 (0)142-49-44-49. Received November 30, 2000; revised February 12, 2001; accepted April 11, 2001. Human Immunology 62, 725–731 (2001) © American Society for Histocompatibility and Immunogenetics, 2001 Published by Elsevier Science Inc.

class II haplotypes. Our results confirm the association of the DRB1*03-DQB1*02 haplotype with SS-A/SS-B autoantibodies positive pSS and demonstrate a significant association of the HLA-A24 with the disease. Moreover, HLA-A24 is more often associated with DRB1*11DQB1*0301 and/or DRB1*0301-DQB1*02 in pSS patients than in the controls. The novel association of HLA class I alleles with susceptibility to pSS provides new insights to the genetic predisposition to this disease and subsequently to its physiopathology. Human Immunology 62, 725–731 (2001). © American Society for Histocompatibility and Immunogenetics, 2001. Published by Elsevier Science Inc. KEYWORDS: HLA; polymorphism; Sjo¨gren syndrome

leading to hypergammaglobulinemia, oligoclonal spikes in protein electrophoresis, and production of autoantibodies. Rheumatoid factors (RF), antinuclear, anti-SS-A (anti-Ro), and anti-SS-B (anti-LA) autoantibodies are among those found in pSS patients. Although detected in 30% to 70% of pSS patients, anti-SS-A antibodies are not disease-specific because they are also detected in systemic lupus erythematosus (SLE) patients. In contrast, the less frequent anti-SS-B antibodies (in 20 to 40% of pSS patients) are more pSS-specific [5]. The etiologic factors that underlay the development of pSS are, so far, unknown, nevertheless a genetic predisposition to the disease was attributed to HLA polymorphism. Two HLA class II haplotypes, the DRB1*03DQB1*02 [6, 7] and DRB1*02-DQB1*06 [8, 9], have been frequently reported as associated with pSS and/or linked to autoantibody responses to SS-A and/or SS-B. Except for one study reporting an increased frequency of the HLA-B8, presumably due to an association with 0198-8859/01/$–see front matter S0198-8859(01)00253-1

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HLA class II HLA-DRB1*03 [10], no HLA class I associations with pSS has been reported. To further define the impact of HLA class I and II alleles in the predisposition to pSS, 46 patients and 222 healthy unrelated Caucasians were analyzed. MATERIALS AND METHODS HLA Typing HLA-A and -B typing were performed using a standard microlymphocytotoxicity method. Allosera that detected 17 HLA-A and 28 HLA-B specificities were obtained from the French national histocompatibility reference laboratory. All the HLA class I defined as HLA-A24 by serology, were confirmed by DNA typing using a PCRSSP kit (Dynal, Oslo, Norway). For HLA class II typing, HLA-DRB1 and HLA-DQB1 medium resolution genotyping was performed using polymerase chain reaction– single-strand oligonucleotide (PCR-SSO) reverse dot blot kits (InnoLipa DRB key and InnoLipa DQB kits, respectively; Innogenetics, Zwijndrecht, Belgium). Patients Forty-six French patients fulfilling the following criteria were included in the study: (a) their pSS diagnosis are based on the European criteria [11]; (b) do not have secondary ethiologies; (c) signed an agreement for both biomedical and ethical disclosure. The mean age of participating patients at the onset of the disease was 57 years old (range 34 –76 years old) with male/female sex ratio of 0.07 (3 males and 43 females). The sicca syndrome was identified in all patients by an ocular and buccal dryness and by a positive Schirmer test (three patients did not undergo the test). A salivary scintigraphia, performed in 41 patients, demonstrated a glandular attack in 15 patients (36%). The immunologic abnormalities observed in the patients were: (a) a lymphocytic infiltration at the histology of the accessory salivary glands (grade 肁 3) in 75% of the patients (33/44); (b) anti-SS-A and/or anti-SS-B autoantibodies in 33% of the patients (15/46); (c) rheumatoid factors and/or antinuclear antibodies in 80% of the patients (33/41).

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(pc) multiplying by the number of tested alleles at each considered locus. Odds ratios were also calculated for each allele. In order to detect the strongest association when alleles from different loci were found significantly associated with the disease, we used the methodology proposed by Svejgaard et al. [12]. RESULTS HLA Class I Antigens Patients and healthy volunteers were analyzed for their HLA-A and HLA-B antigens and the allelic and phenotypic frequencies of these specificies are shown in Tables 1 and 2. Statistical analysis showed that the HLA-A24 antigen was the only HLA class I antigen that showed increased frequency with a phenotypic frequency of 33% versus 12% in controls (pc ⫽ 0.009) (Tables 1 and 2). Odds ratios calculations indicated that the risk of developing pSS is 3.5 times more frequent in HLA-A24positive individuals as compared with HLA-A24-negative patients. In contrast, we did not observe any association between HLA-A24 and the secretion of any of the autoantibodies reported as frequent among pSS patients.

Controls A total of 222 healthy unrelated Caucasians from Paris, France, were analyzed as the control population.

Class II Alleles Although the association of the HLA class II haplotypes (HLA-DRB1*03-HLA-DQB1*02 and HLA-DRB1*15HLA-DQB1*06) and the HLA-DRB1*03/HLADRB1*15 heterozygote genotype have been previously associated with susceptibility to pSS or with anti-SS-A/ SS-B autoantibodies production [6 –9, 13, 14], our results did not confirm these associations (Tables 3 and 4). In our patient’s panel, except for the association of HLADRB1*03 HLA-DQB1*02 haplotype with anti-SS-A/ SS-B autoantibodies production (47% versus 19%, p ⫽ 0.05; Table 4), no other association was defined. Moreover, the HLA class I HLA-A24 was more frequently associated with HLA-DRB1*03 or HLADRB1*11 in pSS patients than in controls (Svejgaard’s test pc ⫽ 0.0013 and 0.0015, respectively). Tables 5 and 6 illustrate that the frequency of HLA-A24-DQB1*03 or HLA-A24-DRB1*11 individuals was higher in patients than in controls (11% versus 1.3%, pc ⫽ 0.02, OR ⫽ 8.9, confidence interval [CI] 95% ⫽ 1.64 –58.8; 13% versus 1.8% pc ⫽ 0.008, OR ⫽ 8.18 CI 95% ⫽ 1.82– 40.72, respectively).

Statistical Analysis Analysis was done by HLASTAT software. Gene frequencies were estimated by maximum likelihood. Comparisons for phenotype frequencies were made between groups, allele by allele, and each p value was corrected

DISCUSSION To evaluate the implication of HLA class I and class II alleles in the genetic predisposition to pSS, 46 patients and 222 controls were typed for HLA class I (HLA-A and

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TABLE 1 Frequencies of HLA-A specificities in pSS patients and controls Patients (n ⫽ 46)

Controls (n ⫽ 222)

Specificity HLA-A

Number of cases

Phenotypic frequency (%)

Allelic frequency (%)

Number of cases

Phenotypic frequency (%)

Allelic frequency (%)

p c Value

01 02 03 11 23 24 25 26 28 29 30 31 32 33 66 34 36

9 16 9 7 2 15 0 4 4 6 3 3 1 2 0 0 0

19.6 34.8 19.6 15.2 4.3 32.6 0 8.7 8.7 13.0 6.5 6.5 2.2 4.3 0 0 0

10.3 21.7 11.9 7.6 2.2 17.4 0 5.4 5.4 7.6 3.3 3.3 1.1 2.2 0 0 0

49 108 53 26 18 27 6 17 17 22 25 11 21 6 4 0 0

22.1 48.6 23.9 11.7 8.1 12.2 2.7 7.7 7.7 9.9 11.3 4.9 9.5 2.7 1.8 0 0

12.2 29.5 12.2 6.1 4.0 6.1.1 1.3 4.0 3.8 5.2 5.8 2.5 4.7 1.3 0.9 0 0

NS NS NS NS NS 0.009 NS NS NS NS NS NS NS NS NS NS NS

OR

3.5

p c ⫽ p value corrected; OR ⫽ odds ratio; pSS ⫽ primary Sjo¨gren syndrome; HLA-A ⫽ human leukocyte antigen A; NS ⫽ not significant.

HLA-B) by serology and for HLA class II (HLA-DR, HLA-DQ) and HLA class I HLA-A24 by molecular typing. Our analysis of HLA class I and class II alleles predisposition to primary SS confirmed the previously increased frequency of DRB1*03-DQB1*02 in patients producing anti-SS-A/SS-B autoantibodies (p ⫽ 0.05) [6 –9, 13, 14] and demonstrated, for the first time, an increased frequency of HLA-A24 in pSS patients. Until now, only one study has reported an increased frequency of HLA-B8 presumably due to an association with DRB1*03. Two explanations could be given to explain the absence, so far, of HLA class I associations with the disease. First, the SS disease is heterogeneous and the criteria for pSS diagnosis were not well defined until recently [11], leading to nonhomogenous group of patients. Second, because most of the autoimmune diseases are associated with HLA class II alleles, only one study was done for HLA class I typing of primary SS patients [10]. HLA-A24 molecules are different from the other HLA-A molecules since they possess, in a quite specific manner, a serine (S), an asparagine (N), an isoleucine (I), an alanine (A), and a phenylalanine (F) at positions 9, 77, 80, 81, and 99, respectively, and in a specific manner, a glutamate (E) and a glycine (G) at positions 62 and 65 of the ␣ chain [15]. These amino acids (AA; except AA at positions 62 and 65) are involved in the structure of the pockets of the peptide binding groove (especially pockets B and F) of the HLA class I molecules in which are

loaded the AA side chains of the bound peptide. The glutamate and the glycine at positions 62 and 65 of the HLA-A24 molecules are located in the vicinity of AA 63 and 66, involved in the composition of the pockets A and B, and thereby could influence the characteristics of the pockets. The specifications of these AA underlay the binding constraints of the peptide loaded in the groove of HLA-A24 molecules (tyrosine and isoleucine, leucine or phenylalanine for the second and nineth AA of the peptide, respectively) already described by Rammensee et al [16]. Therefore, the association of HLA-A24 with the pSS disease might provide some insights to the physiopathology of this disease. It has been recently reported that in the lacrymalglands of pSS patients the aquaporin 5 protein (AQP 5), a water specific membrane channel protein, accumulates in the cytoplasm instead of reaching the apical membrane of the acinar cells [17]. The cytoplasmic accumulation of AQP5 protein could increase the presentation of AQP5 derived peptides by HLA class I molecules. Among them, one perfectly fulfills the constraints of binding to HLA-A24 (tyrosine in position 2 and leucine in position 9). The increased frequency of HLA-A24 has been also reported for two other diseases: the scleroderma [18] and the chronic toxic oil syndrome (TOS) [19]. The latter consists of a syndrome with systemic involvement, including a sclerodermalike neuromuscular disease, sicca syndrome, Raynaud’s phenomenon, and pulmonary hypertension. The etiology of the pSS, scleroderma, and

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TABLE 2 Frequencies of HLA-B specificities in pSS patients and controls Patients (n ⫽ 46)

Controls (n ⫽ 222)

Specificity HLA-B

Number of cases

Phenotypic frequency (%)

Allelic frequency (%)

Number of cases

Phenotypic frequency (%)

Allelic frequency (%)

pc Value

07 08 13 14 17 18 21 27 35 37 38 39 41 42 44 45 47 51 52 53 55 56 60 61 62 63 67 70

9 10 3 6 4 7 3 3 6 2 0 1 1 0 8 2 1 11 0 0 2 0 1 0 5 0 0 0

20.0 22.2 6.7 13.3 8.9 15.6 6.7 6.7 13.3 4.4 0 2.2 2.2 0 17.8 4.4 2.2 24.4 0 0 4.4 0 2.2 0 11.1 0 0 0

10.0 12.2 3.3 6.7 5.6 7.8 3.3 3.3 6.7 2.2 0 1.1 1.1 0 11.5 3.3 1.1 12.2 0 0 2.2 0 1.1 0 5.6 0 0 0

46 35 13 14 16 18 14 19 36 12 12 8 5 1 63 1 3 28 4 10 12 2 19 11 21 3 1 2

20.7 15.8 5.8 6.3 7.2 8.1 6.3 8.6 16.2 5.4 5.4 3.6 2.2 0.4 28.4 0.4 1.3 12.6 1.7 4.5 5.4 0.9 8.6 4.9 9.5 1.3 0.4 0.9

10.8 8.1 2.9 3.4 3.6 4.0 3.4 4.3 9.0 2.7 2.7 1.8 1.1 0.2 15.1 0.2 0.7 6.8 0.9 2.2 2.7 0.4 4.3 2.5 4.7 0.7 0.2 0.4

NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS

p c ⫽ p value corrected; HLA-B ⫽ human leukocyte antigen B; pSS ⫽ primary Sjo¨gren syndrome; NS ⫽ not significant.

chronic TOS diseases is unknown even if in the latter a causative agent (xenobiotic) is involved. It was suggested that scleroderma may be a final pathologic and symptomatic stage of different processes triggered by different chemicals (xenobiotics) [19] and/or metabolic or hormonal factors in susceptible people. The question is to know if pSS, scleroderma, and TOS with increased HLAA24 allele frequency and common genetic background

are the final step of different processes triggered by at least one of the previously cited factors. Although little is known about the physiopathology of pSS, it is an autoimmune disease characterized by lymphocytic infiltration into the lacrimal and salivary glands. Immunohistochemical studies of the lacrimal and salivary glands revealed that most of the infiltrating lymphocytes are CD4⫹ ␣␤ T cells. These infiltrating T

TABLE 3 HLA class II associations previously reported to be associated with pSS and their frequencies in patient and control populations

DRB1*03 ⫺ DQB1*02 DRB1*11 ⫺ DQB1*0301 DRB1*15 ⫺ DQB1*06 DRB1*03 ⫺ DRB1*15 heterozygote

Patients (n ⫽ 46)

Controls (n ⫽ 222)

p c Value

13 (28%) 9 (19%) 13 (28%) 4 (9%)

44 (20%) 48 (22%) 44 (20%) 8 (4%)

NS NS NS NS

p c ⫽ p value corrected; HLA ⫽ human leukocyte antigen; pSS ⫽ primary Sjo¨gren syndrome; NS ⫽ not significant.

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TABLE 4 HLA class II associations previously reported to be associated with SS-A/SS-B autoantibody and their frequencies in patient and control populations SS-A and/or SS-B positive patients (n ⫽ 15)

SS-A and/or SS-B negative patients (n ⫽ 31)

DRB1*03 ⫺ DQB1*02

7 (47%)

6 (19%)

DRB1*15 ⫺ DQB1*06 DRB1*03 ⫺ DRB1*15 heterozygote

5 (33%) 2 (13%)

8 (26%) 2 (6.4%)

p Value 0.05 (p c ⫽ NS) NS NS

NS ⫽ not significant; HLA ⫽ human leukocyte antigen; SS-A ⫽ anti-Ro autoantibody; SS-B ⫽ anti-LA autoantibody.

cells exhibit a limited clonality with a restricted junctional usage in the TCR CDR3 region. This supports the notion that infiltrating T cells expand by antigen-driven stimulation and recognize limited epitopes on autoantigens in the context of HLA [20]. These CD4⫹ ␣␤ T cells could give help to autoantibody production by B cells or to cytotoxic activity by T cells. It has also been reported that epithelial cells from salivary glands of SS patients exhibited an activated status with expression of B7 molecules, indicating a capacity for presenting antigens to T cells [21]. The demonstration of an association between HLA-A24 and pSS could suggest that a cytotoxic response restricted by HLA-A24 could be a process of glandular destruction. The autoantigen recognized by the auto-reactive T cells remains unknown. The RO/SS-A protein, a ribonucleoprotein particle distributed mainly in the cytoplasm of all cells, but also expressed on the cell surface, could be a good candidate. Indeed, Namekawa et al. [22, 23] reported a high frequency of RO/SS-A 52 kD reactive T cells in labial salivary glands from SS patients. On the other hand, autoantibodies anti-RO/SS-A and anti-La/ SS-B (which exists as part of the ribonucleoproteine RO/SS-A) are detected in 30% to 70% of primary SS patients. The humoral response does not seem to be directly implicated in the pathogenesis of the primary SS

or at least, it is not the unique pathologic process of this polymorphic syndrome. Nevertheless, some arguments exist in favor of the implication of these autoantibodies in the pathogenesis of the disease: (a) the presence of anti-SS-A/SS-B antibodies seems to be associated with more severe glandular and extraglandular manifestations [24 –26]; and (b) these antibodies are probably implicated in the pathogenesis of congenital heart block [27]. Our results demonstrated that HLA-A24 (11 of 15) is associated with HLA-DRB1*11 or HLA-DRB1*0301 in pSS patients (Svejgaard’s test pc ⫽ 0.0015 and 0.0013, respectively). Such associations were not observed in our control population (Tables 5 and 6). Indeed, the DRB1*11 frequency alone was not increased in the patients, suggesting that this HLA class II allele exerts only a synergistic activity with the HLA-A24. However, an increased frequency of HLA-DRB1*11 and HLADQB1*0301 were already described in Israeli Jewish and Greek patients suffering from pSS [28, 29], although these patients were not typed for HLA class I. In conclusion, our results demonstrated that the frequency of HLA class I HLA-A24 increases among pSS patients and that the HLA-A24 is associated with DRB1*03 or DRB1*11 in these patients. The associa-

TABLE 5 Frequency and OR for the risk of individuals positive for both HLA-A24 and DRB1*03 HLA alleles A24

DRB1*03

Patientsa (n ⫽ 46)

⫺ ⫹ ⫺ ⫹

⫺ ⫺ ⫹ ⫹

23 (50%) 10 (22%) 8 (17%) 5 (11%)

a

Controlsa (n ⫽ 222) 154 (69.3%) 24 (11%) 41 (18.4%) 3 (1.3%)

ORb

8.90 (CI 95%: 1.64–58.8)

Individuals who have (⫹) or do not have (⫺) the HLA alleles. OR was calculated using the group of individuals who were negative for both A24 and DRB1*03. p c ⫽ p value corrected; HLA ⫽ human leukocyte antigen; OR ⫽ odds ratio; CI 95% ⫽ confidence interval 95%; NS ⫽ not significant. b

p c Value NS NS NS 0.02

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TABLE 6 Frequency and OR for the risk of individuals positive for both HLA-A24 and DRB1*11 HLA Alleles A24

DRB1*11

⫺ ⫹ ⫺ ⫹

⫺ ⫺ ⫹ ⫹

% Patientsa (n ⫽ 46) 28 (61%) 9 (19.5%) 3 (6.5%) 6 (13%)

% Controlsa (n ⫽ 222) 167 (75.2%) 23 (10.4%) 28 (12.6%) 4 (1.8%)

ORb

8.18 (CI 95%: 1.82–40.72)

p c Value NS NS NS 0.008

a

Individuals who have (⫹) or do not have (⫺) the HLA alleles. OR was calculated using the group of individuals who were negative for A24 and DRB1*11. p c ⫽ p value corrected; HLA ⫽ human leukocyte antigen; OR ⫽ odds ratio; CI 95%: ⫽ confidence interval 95%; NS ⫽ not significant. b

tion with HLA-A24 suggests new insights to the physiopathology of pSS. ACKNOWLEDGMENTS

We thank N. David, N. Delva, M. Dourche, M. Duchange, F. Fecamp, G. Henry, and V. Se´ite´ for technical work and S. Laurent for secretarial assistance. This work was supported by the “Association de Recherche sur la Polyarthrite.”

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