Mucous membrane pemphigoid (MMP) is an autoimmune mucosal scarring disease having severe ocular morbidity [1]. Disease susceptibility is associated with increased frequencies of human leukocyte antigen (HLA)-DQB1*03:01, HLA-DRB1*11, and HLA-DRB1*04 and decreased frequencies of DQB1*02 [2]. To explore correlations between clinical involvement and HLA-class-II alleles, we prospectively phenotyped a cohort of 55 British MMP patients, and 41 age/sex-matched controls (ethics approval reference 09/H0721/54).
Inclusion criteria have been described [3] and were pre-agreed clinical criteria with or without a positive direct immunofluorescence (DIF) study. HLA-typing used allele-specific sequencing protocols (Protrans S3 HLA-DRB1* and HLA-DQB1* Cyclerstrips, Protrans, Hockenheim, Germany). Statistical analysis used Fisher’s exact test (p), with Benjamini–Hochberg correction (pc) defined as significant when pc < 0.05.
The study dataset, with clinical and laboratory results, is provided in Table and Legend S1. Fifteen patients, 14 of them with ocular involvement, were DIF-negative of whom seven had antibodies to basement membrane zone epitopes. MMP-affected sites varied: eight were ocular only, ten oral only, 15 oral and ocular only and 22 multisite. Exons 2 and 3 in HLA-DQB1 were fully analysed in 54/55 patients and 39/41 controls (Table 1a). The frequency of HLA-DQB1*03:01 was increased (pc = < 0.01) in 36/54 (67%) of MMP patients (13 homozygous and 23 heterozygous) compared to 13/39 (33%) controls (two homozygous and 11 heterozygous, Table 1a). Exon 2 of HLA-DRB1 was also fully analysed in 54/55 patients and 40/41 controls; HLA-DRB1*03:01 was decreased (p = < 0.01, pc = 0.045, Table 1b). Additionally, we compared the frequency of HLA-DQB1*0301 with controls for different sites of involvement showing that HLA-DQB1*0301 was increased in all subgroups except for ocular only MMP. Compared to controls, DIF-positive MMP had significantly increased HLA-DQB1*0301 (p = 0.00009) but this difference was not significant for either DIF-negative MMP (p = 0.113) or for DIF-positive ocular only MMP.
Table 1a.
Distribution of HLA-DQB1 among patients and controls
| Patients | Controls | p-value | p c | |||
|---|---|---|---|---|---|---|
| (n = 54) | in % | (n = 39) | in % | |||
| HLA-DQB1 | ||||||
| HLA-DQB1*02 | 9 | 16.6 | 18 | 46.2 | <0.01 | <0.01 |
| HLA-DQB1*03 | 48 | 88.9 | 20 | 51.3 | <0.01 | <0.01 |
| HLA-DQB1*04 | 4 | 7.4 | 6 | 15.4 | 0.31 | 0.39 |
| HLA-DQB1*05 | 13 | 24 | 11 | 28.2 | 0.81 | 0.81 |
| HLA-DQB1*06 | 13 | 24 | 18 | 46.2 | 0.04 | 0.07 |
| HLA-DQB1*02 | ||||||
| HLA-DQB1*02:01 | 6 | 11.1 | 9 | 23.1 | 0.15 | ND |
| HLA-DQB1*02:02 | 3 | 5.5 | 7 | 17.9 | 0.09 | ND |
| HLA-DQB1*03 | ||||||
| HLA-DQB1*03:01 | 36 | 66.6 | 13 | 32.1 | <0.01 | 0.01 |
| HLA-DQB1*03:02 | 7 | 13 | 2 | 5.1 | 0.29 | 0.63 |
| HLA-DQB1*03:03 | 4 | 7.4 | 2 | 5.1 | 1 | 1 |
| HLA-DQB1*03:05 | 0 | 0 | 1 | 2.6 | 0.42 | 0.63 |
| HLA-DQB1*03:19 | 1 | 1.8 | 0 | 0 | 1 | 1 |
| HLA-DQB1*03:22 | 0 | 0 | 1 | 1.3 | 0.42 | 0.63 |
Table 1b.
Distribution of HLA-DRB1 among patients and controls
| Patients | Controls | p-value | p c | |||
|---|---|---|---|---|---|---|
| (n = 54) | in % | (n = 39) | in % | |||
| HLA-DRB1 | ||||||
| HLA-DRB1*01 | 10 | 18.5 | 12 | 30 | 0.23 | 0.48 |
| HLA-DRB1*0301a | 7 | 13 | 15 | 37.5 | <0.01 | 0.046 |
| HLA-DRB1*04 | 20 | 37 | 7 | 17.5 | 0.04 | 0.15 |
| HLA-DRB1*05 | 0 | 0 | 1 | 2.5 | 0.43 | 0.59 |
| HLA-DRB1*07 | 5 | 9.3 | 10 | 25 | 0.05 | 0.14 |
| HLA-DRB1*08 | 3 | 5.5 | 4 | 10 | 0.45 | 0.59 |
| HLA-DRB1*09 | 3 | 5.5 | 0 | 0 | 0.27 | 0.48 |
| HLA-DRB1*11 | 25 | 40.7 | 7 | 17.5 | <0.01 | 0.046 |
| HLA-DRB1*12 | 3 | 5.5 | 1 | 2.5 | 0.63 | 0.75 |
| HLA-DRB1*13 | 13 | 24 | 6 | 17.5 | 0.31 | 0.5 |
| HLA-DRB1*14 | 1 | 1.8 | 0 | 0 | 1 | 1 |
| HLA-DRB1*15 | 9 | 16.6 | 14 | 35 | 0.05 | 0.14 |
| HLA-DRB1*16 | 1 | 1.8 | 0 | 0 | 1 | 1 |
| HLA-DRB1*11 | ||||||
| HLA- DRB1*11:01/11:97 | 12 | 22.2 | 5 | 12.5 | 0.29 | ND |
| HLA-DRB1* 11:01/11:97 or 11:04 |
2 | 3.7 | 0 | 0 | 0.5 | ND |
| HLA-DRB1*11:02 or 11:36 or 11:48 | 1 | 1.85 | 0 | 0 | 1 | ND |
| HLA-DRB1*11:03 | 2 | 3.7 | 0 | 0 | 0.5 | ND |
| HLA-DRB1*11:03 or 11:11 | 2 | 3.7 | 0 | 0 | 0.5 | ND |
| HLA-DRB1*11:04 | 6 | 11.1 | 2 | 5 | 0.46 | ND |
In the first step of the analysis the gene frequencies for the allel groups of HLA-DQB1 (2–6) and HLA-DRB1 (1, 3–5, 7–9, and 11–16) were compared and in the second step, only significant alleles were further analysed regarding specific HLA-protein (e.g., HLA-DRB1*11) and compared. The p-value is given for comparisons of HLA gene frequencies, between cases and controls, using Fisher's exact test for each allele. The pc value is the probability value after using the Benjamini–Hochberg correction for multiple testing.
ND not done
a All patients with HLA-DRB1*03 expressed the specific protein HLA-DRB1*0301.
Some studies have described a correlation of HLA-DQB1*03:01 with ocular and oral MMP or in ocular only MMP, whereas others have shown HLA-DQB1*03:01 to be associated with multisite MMP [2]. In this study, the association of HLA-DQB1*0301 with MMP was lower than in the largest reported study [2] (pc < 0.00000028), possibly due to our inclusion of eight patients without detectable tissue-bound or serum antibodies, who were excluded from the latter study [2]. In ocular only MMP 50% are DIF-negative but have a phenotype that both progresses and responds to therapy in the same way as DIF-positive cases [3, 4]. DIF-negative ocular MMP may result from inadequate test sensitivity, because of the small volumes of tissue involved, to dominance of an autoreactive T-cell mediated over a autoantibody-mediated disease [4] or because this is a different disease subset. We included our DIF-negative cases because to leave these out of this analysis disregards a group of cases which do not fit criteria for any other disease.
In our prospectively characterized cohort the association with HLA-DQB1*0301, HLA-DQB1*02, and HLA-DRB1*11 was corroborated, whereas HLA-DRB1*0301 was identified as a potentially protective allele, which requires confirmation in a larger study.
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Funding
From anonymous donors to John Dart’s undesignated research funds, through a Grant from the Moorfields Eye Hospital Special Trustees MEH Special Trustees Reference ST 11 11 B and from the NIHR Biomedical Research Centre for Ophthalmology, Major Award to support translational research initiatives (Ref.: BMRC 045). Part of J.D. salary is funded by the National Institute for Health Research (NIHR) Biomedical Research Centre at the Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology.
MMP Study Group 2009
Moorfields Eye Hospital, London: Debbie Booth, Elaina Reid, Nicole Carnt, Stefano Gugliemetti, Vijay Shanmuganathan, Martin Watson, Valerie Saw, Mark Wilkins, Vicky McCudden, Saj Ahmad,Catey Bunce. The UCL Institute of Ophthalmology, London: Virginia Calder, Waseem Naushin, Beverly Scott.Guys and Thomas’s Hospital, London: Stephen Challacombe, Mike Gleeson, Sarah Ali, William Gage. Epivision (Ophthalmic Epidemiology Consultants), Penn, UK: Darwin Minassian. The Royal National ENT Hospital, London: Valerie Lund, Guri Sandhu.
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Conflict of interest
The authors declare that they have no conflict of interest.
Electronic supplementary material
The online version of this article (10.1038/s41433-018-0092-5) contains supplementary material, which is available to authorized users.
References
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