HLA-DRB1 and HLA-DQB1 genes in susceptibility and resistance to cicatricial pemphigoid in French Caucasians

ARTICLE

Cicatricial pemphigoid (CP) is an autoimmune, subepithelial bullous disease, affecting primarily the elderly. CP patients typically present with tense, subepithelial blisters and erosions of mucosae (oral, ocular, and or genital epithelia) with varying degrees of skin involvement and further scar formation [1, 2]. As in other autoimmune skin-blistering diseases, there is in vivo deposition of immunoglobulins (Ig) and complement components at the basement membrane zone (BMZ) of the dermal-epithelial junction. The sera of the majority of patients contain anti-BMZ autoantibodies directed against various antigens mostly located in the anchoring filament region of the dermal-epidermal junction, including BP180 antigen [3-6].

Previous studies have shown a genetic predisposition linked to human leucocyte antigens (HLA): HLA-B12 was first reported to be associated with ocular cicatricial pemphigoid (OCP) [7], but this association has not been subsequently confirmed. More recently OCP was reported to be associated with HLA-DR4/DR5/DQ3 [8] and DQB1*0301 [9]. A DQB1*0301 predisposition appears to be stronger in patients with ocular involvement [6, 10]. The HLA-DQB1*0301 allele, assigned by molecular analysis, is the equivalent of the serologically defined DQ7 (3) antigen. At the 11th HLA International workshop, the HLA-DQB1*0301 genotype frequency was estimated to be 10 to 15% of the European Caucasian population [11]. HLA- DQB1*0301 allele is in linkage disequilibrium with several HLA-DRB1 alleles in Caucasians, as different HLA-DRB1*04 subtypes, HLA-DRB1*11, HLA-DRB1*1303, HLA-DRB1*12. The improvement in HLA genotyping, based on DNA sequences, allows greater precision in assignment as generic and specific HLA-DRB1 and HLA-DQB1 alleles.

Previous studies did not analyze specific DRB1 alleles in CP patients. The aim of our study was to search for an HLA immunogenetic predisposition in CP patients of European Caucasian origin, in comparison with geographically matched, normal individuals, using HLA-DRB1 and HLA-DQB1 DNA typing. Because of the heterogeneous clinical and immunologic characteristics of CP disease, we also searched for an association of different clinical factors with HLA alleles.

Materials and methods

Patients and controls

The twenty-five patients included in this study were unrelated Caucasians from West Central France and were first seen in two centers (n = 14 in Limoges, 11 in Tours) between 1990 and 1996. All patients presented a vesicular and/or bullous eruption that led to erosions of mucosae or the skin. Oral involvement was present in 23/25 cases. Healing and recurrence of the lesions resulted in scarring. Ocular involvement occurred in 9 patients (OCP), including inflammatory conjunctivitis and scarring. Skin lesions were present in 15/25 patients and were primarily restricted to the head, neck, and upper trunk. The diagnosis was confirmed by histologic examination which revealed subepithelial cleavage with moderate to intense inflammatory infiltration in the upper dermis and direct immunofluorescence (IF) analysis which showed linear deposits of Ig and complement at the basement membrane zone. Low titers of circulating IgG anti-BMZ autoantibodies were detected by standard indirect IF in 12/23 cases and were confirmed by indirect IF on salt-split skin, or indirect immunoelectron microscopy in some cases [12]. Autoantibodies against the BP180 and BP230 antigens were tested in sera from18/25 patients, collected at the time of diagnosis, using the immunoblotting technique with protein extracts from normal, heat-separated human epidermis or cultured, normal human keratinocytes [13]. The control group consisted of 106 healthy, unrelated volunteers, of the same geographic origin, who had no history of skin or mucosal blistering disease.

HLA-DRB1 and -DQB1 genotyping

DNA was extracted from whole blood by the standard salting out method [14]. In three cases, because no blood sample could be obtained, DNA was extracted from cryopreserved (at ­ 80° C) skin biopsy specimens: about one milligram of tissue was dissolved overnight, at 37° C, in 1 ml of lysis buffer (NaCl 0.375 M, Na₂ EDTA 0.12 M) with 40 µl SDS 10%, 35 µl of proteinase K 20 mg/ml. After overnight incubation, DNA was extracted by the phenol-chloroform procedure [15].

Generic and specific DRB1 and DQB1 alleles were identified either by PCR with reverse-dot hybridisation with Innolipa kits (Innolipa, Murex, Paris, France) or by PCR-SSP (Dynal, Compiègne, France) according to the manufacturer's recommendations. All procedures have been previously reported in detail [16-18]. HLA alleles were assigned according to the international nomenclature [19]. Both laboratories comply with national quality control. HLA haplotypes were deduced from HLA haplotypes validated in the French population [11].

Statistical analysis

The frequencies of independent genotypes, phenotypes or haplotypes were determined by direct counting. Results expressed as genotype frequencies take into account both haplotypes. The P value was defined by the chi-square analysis using a 2 x 2 contingency table or Fisher's exact test if any value was less than five, with the aid of EPIINFO software. For clinical analysis, patients were stratified into two groups according to the presence (+) or absence (­) of autoantibodies (Ab⁺, Ab^­), ocular lesions (Oc⁺, Oc^­), and skin involvement (Sk⁺, Sk^­). HLA allele frequencies were compared for each criterion by a two-sided chi-squared test with a 2 x 2 contingency table between control versus patients, and between patients + versus patients ­. The relative risk (RR) was calculated as recommended by Haldane [20]. The probability obtained (P) was corrected for multiple comparisons according to the number of alleles observed at that locus (Pc). The level of significance was set at 0.05.

Results

The HLA-DQB1*0301 allele confers susceptibility to CP

HLA haplotypes observed in patients and the frequency of generic HLA-DRB1 and specific HLA-DQB1 are summarized in Table I and Table II respectively. HLA-DQB1*0301 genotype frequency was significantly increased in CP patients to 54.0% (27/50) versus 21.7% (46/212) in the control group (Pc = 7 x 10^-5; RR = 4.23). The phenotype frequency of DQB1*0301 was 80% (20/25): 7 patients were homozygous for DQB1*0301, 13 heterozygotes and 5 did not carry DQB1*0301. HLA-DQB1*0301 was associated with an increase of generic HLA-DRB1*11 (16/50 versus 24/212; Pc = 0.008; RR = 3.68) and DRB1*04 (11/50 versus 23/212; Pc not significant). Both alleles are in linkage disequilibrium with HLA-DQB1*0301. Generic HLA-DRB1*11 includes 30 subtypes and HLA-DRB1*04, 24 subtypes, which can be defined by DNA analysis [19]. Only the HLA-DRB1*1101 DQB1*0301 haplotype was significantly increased in CP patients (24.0% versus 6.6%; khi² = 12.3, Pc = 0.002, RR = 4.46). An increase of the DRB1*1104 DQB1*0301 frequency was observed but was not significant when P was corrected (8% vs 0.9%, khi² = 6.2). The frequency of the remaining HLA-DRB1 subtypes in linkage disequilibrium with HLA-DQB1*0301 in French Caucasians (HLA-DRB1*12, DRB*1303, DRB1*04) did not differ from the control population (Table III).

HLA-DQB1*02 is negatively associated with PC

The HLA-DRB1*0701 DQB1*0202 haplotype was absent from the patient population (0% versus 12.7%, khi² = 7.1, Pc = 0.07; RR = 0.06). We observed a non-significant decreased frequency of the DRB1*0301 DQB1*0201 haplotype (4% versus 12.7%). Altogether, the frequency of allele DQB1*02 was significantly decreased (6% versus 25%, khi² = 8.7, Pc < 0.05). DQB1*02/DQB1*0301 or DRB1*11/DRB1*0701 phenotypes frequency did not differ significantly between CP patients and control.

Clinical subtypes

No significant difference in HLA-DQB1*0301 allele frequency was observed whether cutaneous involvement (Sk⁺/Sk^­) or ocular lesions (Oc⁺/Oc^­) were present or not (Table I). The HLA-DQB1*0301 allele was present in 7/8 Ab^­ patients (87.5%) and 7/10 (70%) Ab⁺ (not significant).

Discussion

Our study confirms the genetic predisposition to CP linked to HLA-DQB1*0301 in French Caucasian patients, as previously reported in North American Caucasians [6, 9, 21]. The HLA-DQB1 frequency observed in our French Caucasian population did not differ from that of North American Caucasians reported by these authors. The frequencies of all haplotypes associated with HLA-DQB1*0301 were not equally increased. The association between PC and HLA class II was stronger with the DRB1*1101 DQB1*0301 haplotype. These results suggest that susceptibility to CP might involve HLA-DRB1*1101 alleles as well as the HLA-DQB1*0301 allele. Because we were looking at many phenotypes, we cannot exclude that this might be a chance association even though the Bonifari correction was applied. HLA-DQB1*0301 may interact in synergy with the HLA-DRB1 allele to contribute to the susceptibility to CP as described in juvenile rheumatoid arthritis [22]. HLA genotypes were not associated with clinical subtypes in our study, as has been previously reported [6, 10]; this might be due to the small size of each group and would thus require validation in a larger number of patients.

We observed a significantly decreased frequency of HLA-DRB1*0701. In Caucasians, HLA-DRB1*0701 is associated with HLA-DQB1*0202, and rarely with DQB1*0303. Both haplotypes were absent from our patient population. HLA-DQB1*0202 is only found in linkage desequilibrium with HLA-DRB1*0701, either allele (or both) could be involved in a negative association. A non-significant decrease of the HLA-DRB1*0301-DQB1*0201 haplotype occurred. The sequencing data of the entire coding region of DQB1 alleles showed that DQB1*0201 differs from DQB1*0202 only at codon 135, GAC and GGC respectively [23] and cannot be distinguished by serological typing (HLA DQ2). Therefore we suggest that DQ2 molecules might be responsible for a negative association with CP.

Polymorphic residues in the MHC class II peptides binding groove have been shown to be linked to susceptibility to many human diseases [24]. Two polymorphic position in HLA class II ß chain are critical for selective binding protein: position 71 of DRß and position 57 of DQß chain [25]. Pemphigus vulgaris, an autoimmune blistering disease whose target is desmoglein 3, is associated with HLA-DRB1*0402. Analysis of the charge residue of the DRB1*0402 cleft has been used to localize candidate T cell epitopes of desmoglobein 3: only seven peptides from this 999 amino acid protein matched the motif able to bind the DRB1*0402 pocket [26].

The confirmation of positive and negative HLA class II associations in cicatricial pemphigoid should be useful to identify residues of BMZ autoantigens capable of selective binding to class II molecules. DQB1*0301 differs from others DQ molecule by the presence of a negatively charge aspartic acid (Asp) at DQß57, which replaces the uncharged valine or alanine. Because DQß57 is a critical position in selective peptide binding, we can speculate that amino acid 57 of the DQß chain may have a potential role in the selection of BMZ peptides in CP and that a peptide with a positive charge at the matching position would be expected to bind to the CP-associated DQB1*0301 molecule.

REFERENCES

1. Mutasim DF, Pelc NJ, Anhalt GJ. Cicatricial pemphigoid. Dermatol Clin 1993; 11: 499-510.

2. Caux FA, Giudice GJ, Diaz LA, Fairley JA. Cicatricial pemphigoid. J Geriat Dermatol 1996; 4: 42-6.

3. Bernard P, Prost C, Durepaire N, Basset-Seguin N, Didierjean L, Saurat JH. The major cicatricial pemphigoid antigen is a 180-kD protein that shows immunologic cross-reactivities with the bullous pemphigoid antigen. J Invest Dermatol 1992; 99: 174-9.

4. Kirtschig G, Marinkovich P, Burgeson R, Yancey K. Anti-basement membrane autoantibodies in patients with anti-epiligrin cicatricial pemphigoid bind the subunit of laminin 5. J Invest Dermatol 1995; 105: 543-8.

5. Balding S, Prost C, Diaz A, Bernard P, Bedane C, Aberdam D, Giudice G. Cicatricial pemphigoid autoantibodies react with multiple sites on the BP180 extracellular domain. J Investig Dermatol 1996; 106: 141-6.

6. Chan LS, Hammerberg C, Cooper KD. Significantly increased occurrence of HLA-DQB1*0301 allele in patients with ocular cicatricial pemphigoid. J Investig Dermatol 1997; 108: 129-32.

7. Mondino BJ, Brown SI, Bighan AW. HLA antigens in ocular cicatricial pemphigoid. Arch Ophthalmol 1979; 97: 479.

8. Zaltas MM, Ahmed R, Foster CS. Association of HLA-DR4 with ocular cicatricial pemphigoid. Current Eye Research 1989; 8: 189-91.

9. Ahmed AR, Foster S, Zaltas M, Notani G, Awdeh Z, Alper CA, Yunis EJ. Association of DQw7 (DQB1*0301) with ocular cicatricial pemphigus. Proc Natl Acad Sci USA 1991; 88: 11579-82.

10. Yunis JJ, Mobini N, Yunis EJ, Alper CA, Deulofeut R, Rodriguez A, Foster CS, Marcus-Bagley D, Good RA, Ahmed AR. Common major histocompatibility complex class II markers in clinical variants of cicatricial pemphigoid. Proc Natl Acad Sci USA 1994; 91: 7747-51.

11. Tsuji K, Aizawa M, Sasazuki T. HLA 1991. Proceedings of the Eleventh International Histocompatibility Workshop and Conference. Oxford Science Publications, 1992.

12. Bedane C, McMillan JR, Balding SD, Bernard P, Prost C, Bonnetblanc JM, Diaz LA, Eady RAJ, Giudice GJ. Bullous pemphigoid and cicatricial pemphigoid autoantibodies react with ultrastructurally separable epitopes on the BP180 ectodomain: evidence that BP180 spans the lamina lucida. J Invest Dermatol 1997; 108: 901-7.

13. Bernard P, Prost C, Lecerf V, Intrator L, Combemale P, Bedane C, Roujeau JC, Revuz J, Bonnetblanc JM, Dubertret L. Studies of cicatricial pemphigoid autoantibodies using direct immunoelectron microscopy and immunoblot analysis. J Invest Dermatol 1990; 94: 630-5.

14. Miller S, Dykes D, Polesky H. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acid Res 1988; 16: 1215-8.

15. Maniatis T, Fritsch EF, Sambrocok J. Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Laboratory, Cold Spring Harbor Laboratory, 1982.

16. Buyse L, Decorte R, Baens M, Cuppens H, Semana G, Emonds MP, Maryenen P, Cassiman JJ. Rapid DNA typing of class II HLA antigens using the polymerase chain reaction and reverse dot blot hybridisation. Tissue Antigen 1993; 41: 1-14.

17. Olerup O, Zetterqist H. HLA-DR typing by PCR amplification with sequence-specific primer (PCR-SSP) in 2 hrs: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation. Tissue Antigens 1992; 39: 225-35.

18. Olerup O, Aldener A, Fogdell A. HLA-DQB1 and DQA1 typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hrs. Tissue Antigens 1993; 41: 119-34.

19. Bodmer J, Marsh S, Albert E, Bodmer W, Bontrop R, Chrron D, Dupont B, Erlich H, Fauchet R, Mach B, Mayr W, Parham P, Sasazuki T, Schreuder G, Strominger J, Svelgaard A, Terasaki P. Nomenclature for factors of the HLA system, 1996. Tissue Antigens 1997; 49: 297-321.

20. Haldane JBS. The estimation and significance of the logarithm of a ratio frequencies. Am Hum Genet 1956; 20: 309-11.

21. Delgado JC, Turbay D, Yunis EJ, Yunis JJ, Morton ED, Bhol K, Norman R, Alper CA, Good RA, Ahmed R. A common major histocompatibility complex class II allele HLA-DQB1*0301 is present in clinical variants of pemphigoid. Proc Natl Acad Sci 1996; 93: 8569-71.

22. Ploski R, McDowell T, Symons J, Flatø B, Gordon Wd, Thorsby E, Øystein Førre. Interaction between HLA and interleukin-1alpha in juvenile rheumatoid arthritis indicates heterogeneity of pathologic mechanisms of the disease. Hum Immunol 1995; 42: 343-7.

23. Hall MA, Lanchbury JS, Lee JS, Welsh KI, Ciclittiria P. HLA-DQ2 second-domain polymorphisms may explain increased trans-associated risk in coeliac disease and dermatitis herpetiformis. Hum Immunol 1993; 38: 284-92.

24. Todd JA, Acha-Orbea H, Bell JI, Chao N, Froneck Z, Jacob CO, McDermott M, Sinha AA, Timmerman L, Steinman L. A molecular basis for MHC class II-associated autoimmunity. Science 1988; 240: 1003-9.

25. Wucherpfenning KW, Strominger JL. Selective binding of self peptides to disease-associated major histocompatibility complex (MHC) molecules: a mechanism for MHC-linked susceptibility to human autoimmunity diseases. J Exp Med 1995; 181: 1597-8.

26. Wucherpfenning KW, Yu B, Bhol K, Monos DS, Argyris E, Karr RW, Ahmed AR, Strominger JL. Structural basis for major histocompatibility complex (MHC)-linked susceptibility to autoimmunity: charged residues of a single MHC binding pocket confer selective presentation of self-peptides in pemphigus vulgaris. Proc Natl Acad Sci USA 1995; 92: 11935-9.