Human Immunology 74 (2013) 1134–1140

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Both qualitative and quantitative genetic variation of MHC class IImolecules may influence susceptibility to autoimmune diseases:The case of endemic pemphigus foliaceus

0198-8859/$36.00 - see front matter � 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.humimm.2013.06.008

Abbreviations: CI, confidence interval; CIITA, class II, major histocompatibilitycomplex, transactivator; EPF, endemic pemphigus foliaceus; MHC, major histo-compatibility complex; NE, neutral allele; OR, odds ratio; P, probability; PR,protective allele; SNP, single nucleotide polymorphism; SU, susceptibility allele;Treg, regulatory T cells.⇑ Corresponding author. Address: Departamento de Genética, Universidade

Federal do Paraná, Caixa Postal 19071, 81531-990 Curitiba, Brazil.E-mail address: perler@ufpr.br (M.L. Petzl-Erler).

Bruno Zagonel Piovezan, Maria Luiza Petzl-Erler ⇑Laboratório de Genética Molecular Humana, Departamento de Genética, Universidade Federal do Paraná, Curitiba, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 February 2013Accepted 7 June 2013Available online 15 June 2013

The MHC class II transactivator (CIITA) is a key regulator in expression of the HLA class II genes. It is wellknown that HLA-DRB1 genotypes have a strong influence on the risk of multifactorial autoimmune dis-eases, but the effect of CIITA genotypes remains controversial. We tested in a case–control study whetherCIITA polymorphisms influence the risk of developing endemic pemphigus foliaceus (EPF) and whetherCIITA and HLA-DRB1 interact as regards susceptibility to the disease. The rs4774 SNP is not associatedto EPF, while rs3087456 in the CIITA gene promoter is associated with susceptibility [odds ratio(OR)=2.6, p < 0.001 and OR = 2.0 p = 0.003 for genotypes G/G and G/A, respectively]. We suggest thatthe associations result from the effect of genetically controlled levels of CIITA on expression of the sus-ceptible and protective HLA class II molecules. Remarkably, the interaction between CIITA and HLA-DRB1 genotypes is strong and additive. The OR for individuals having two susceptible HLA-DRB1 allelesis 14.1 in presence of the susceptible CIITA G/G or G/A genotypes and much lower (2.2) in presence ofthe protective CIITA A/A genotype. We conclude that quantitative as well as qualitative variation ofHLA class II molecules have an effect on the risk of an individual developing EPF.� 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights

reserved.

1. Introduction

Pemphigus foliaceus is a bullous autoimmune skin disease char-acterized by antibodies against desmoglein 1, a desmosomal cell-adhesion glycoprotein. Superficial blistering or erosive exfoliationof the skin are characteristic of the disease and result from the lossof adhesion between keratinocytes, a process known as acantholy-sis. Pemphigus foliaceus is rare in most of the world but occurs athigh frequency in some areas of South America. This endemic formof pemphigus foliaceus (EPF), also called Brazilian pemphigus foli-aceus or fogo selvagem (wild fire) is clinically, immunologically andhistologically similar to the sporadic form. The etiology of EPF isnot fully understood, but epidemiologic and genetic data point to-wards a model in which genetically susceptible individuals whenexposed to triggering environmental agent(s) develop pathologic

autoreactivity. The environmental causes remain unidentified,though exposure to bites of hematophagous insects includingsimuliids (black flies), the sand fly Lutzomyia longipalpis and redu-viids (kissing bugs) is suspected. Salivary antigens of these insectsmight trigger the autoimmune disease via molecular mimicry [1].

Susceptibility to EPF is caused by a combination of environmen-tal and genetic factors. The genetic component exerts its effectthrough several genes, each having modest effects. The involve-ment of MHC class II alleles in differential susceptibility to pemphi-gus foliaceus was first recognized in 1989, when associations withHLA-DR and -DQ antigens were described, with DR1–DQ1 andDR4–DQ3 significantly increased in patients and DR7–DQ2 andDR3–DQ2 significantly decreased [2]. The analysis of HLA-DRB1 al-leles and genotypes confirmed previously reported associationswith HLA class II haplotypes and revealed several risk alleles(DRB1⁄01:01, ⁄01:02, ⁄01:03, ⁄04:04, ⁄04:06, ⁄04:10, ⁄14:06 and⁄16:01), as well as alleles that have a significant protective effect(⁄03:01, ⁄07:01, ⁄08:01, ⁄11:01, ⁄11:04 and ⁄14:02) [3]. Additionalsignificant associations were found between EPF and variants ofthe CD40L and IL6 genes [4,5]. Polymorphisms of IL4, CD40, BLYS-(BAFF), PDCD1, CTLA4, CD86, may also contribute to EPF diseasesusceptibility [4–7]. No significant associations were observedwith polymorphisms of the DSG1, TNF, LTA, BAX, TP53, CD28,CD80 and CD19 genes [4,7–10].

B.Z. Piovezan, M.L. Petzl-Erler / Human Immunology 74 (2013) 1134–1140 1135

The CIITA (major histocompatibility complex class II transacti-vator; C2TA, or MHC2TA) molecule is a transcriptional co-activatorrequired for the expression of all the genes encoding the a and bchains of the MHC class II molecules (HLA-DR, HLA-DP, HLA-DQ,HLA-DM, HLA-DO in humans) as well the gene encoding the Invari-ant chain (Ii or CD74). CIITA does not bind directly to the DNA butrather to transcription factors bound to the promoter region [11]and is the master regulator of both, constitutive and IFNc inducedexpression of these genes in antigen-presenting cells. Mutations inthe CIITA gene are responsible for the bare lymphocyte syndrome, asevere immunodeficiency in which individuals fail to produce MHCclass II molecules [12]. Further, it seems likely that CIITA polymor-phisms are involved in autoimmune disease, although associationsare difficult to replicate for some diseases [13,14].

Because the CIITA molecule is crucial to MHC class II geneexpression and the HLA-DRB1 genotypes play a major role in sus-ceptibility to pemphigus foliaceus, it is possible that polymor-phisms that affect the function and the cellular levels of CIITAhave an impact in susceptibility to the disease. Thereby we wantedto explore whether CIITA polymorphisms influence the risk ofdeveloping EPF. Besides, we tested whether variants of the CIITAgene interact with the HLA-DRB1 genotypes as regards susceptibil-ity to the disease. We tested these hypotheses in a case–controlstudy with patients and controls from the Brazilian population.

2. Methods

2.1. Population samples

In this study, 252 unrelated patients with pemphigus foliaceusand 209 controls without history of the disease were analyzed. Thepatients were diagnosed based on clinical and immunohistochem-ical criteria and were contacted at Hospital Adventista do Pênfigo,in Mato Grosso do Sul state (n = 210); Hospital de DermatologiaSanitária, Paraná state (n = 21), and Hospital das Clínicas da Faculd-ade de Medicina de Ribeirão Preto, São Paulo state (n = 21). Theplaces of origin of patients contacted in the three locations overlapbecause patients travel to hospitals specialized in the treatment ofpemphigus coming from the endemic region. The control subjectswere free of any apparent illness and were contacted mainly at theHospital Adventista do Pênfigo and surroundings (98%) becauseMato Grosso do Sul state is included in the endemic region. Genet-ically unrelated relatives of the patients were preferred. Personaland clinical information were procured through a questionnaireand an interview.

Due to possible differences in allele frequencies among popula-tions, patients and controls were classified according to ancestryand paired in two groups: the Euro-Brazilians (153 patients, 143controls), with predominantly European ancestry; and the Afro-Brazilians (99 patients, 66 controls), with mixed ancestry, predom-inantly African and European. Ancestry was assigned on basis ofinformation obtained in the questionnaire about the geographicorigin of near and distant relatives and the individuals’ phenotype.Written informed consent was obtained from all participants. Thisstudy has received the approval of the Human Research EthicsCommittee of the Federal University of Paraná, in accordance withBrazilian federal laws.

2.2. Genotyping

Genomic DNA was extracted from peripheral blood leukocytesby standard salting out or phenol/chloroform methods.

Two CIITA single nucleotide polymorphisms (SNPs) wereselected: the rs3087456 SNP (�168A>G), located in the promoterregion of the gene and the rs4774 SNP in exon 11 (1632G>C) that

leads to an amino acid change (from glycine to alanine) at position500 of the protein (G500A). Both of them were genotyped in all pa-tients and controls through the SNPlex™ System (Applied Biosys-tems, Foster City, CA, USA). This system consists of theoligonucleotide ligation assay (OLA) combined with a multiplexPCR and capillary electrophoresis to achieve allelic discrimination[15].

HLA-DRB1 genotypes were known for 206 patients and 203 con-trols. 95 patients and 157 controls were typed in a previous study[3]. The others (111 patients, 46 controls) were typed using med-ium-resolution Reverse SSO (LABType, One Lambda, Canoga Park,CA, USA). For these subjects, allele-level genotypes were inferredusing the medium-resolution SSO results and information aboutthe frequencies of HLA alleles and haplotypes worldwide. When-ever it wasn’t possible to assign with confidence a specific allele,only the allelic lineage (group) was used.

2.3. Statistical analysis

Allelic and genotypic frequencies and the frequencies of indi-viduals having an allele were obtained by direct counting. Haplo-type frequencies for CIITA SNPs were estimated after inference ofthe gametic phase using the ELB algorithm implemented in theARLEQUIN v3.11 software package [16]. The same software wasused to test for the Hardy–Weinberg equilibrium by Guo andThompson’s method [17].

For the association analyses between the disease and the poly-morphisms, odds ratios (OR) and the 95% confidence intervals (CI)were calculated by the Mantel–Haenszel’s method [18] and exact Pvalues via the metropolis algorithm [19]. For CIITA, associationanalyses were done for the Afro- and the Euro-Brazilian samplesseparately and for the total population sample.

To check if the allelic frequencies of the Afro- and Euro-Brazil-ian population strata differed, population differentiation tests wereperformed using 2 � 2 contingency tables and estimating exact Pvalues via the metropolis algorithm [19]. When a significant differ-ence between the allele frequencies of Afro- and Euro-Brazilianswas detected, then the proportions of Afro- and Euro-Braziliansin the total population sample were adjusted, in order to attainsimilar ancestry for both the patient and the control groups. Asso-ciation analysis for HLA-DRB1 and the stratified analysis to test forinteractions between the HLA-DRB1 and CIITA genotypes were per-formed considering only the adjusted total sample.

3. Results

The SNP rs3087456 (�168A>G) located at the CIITA promoter re-gion was associated with pemphigus foliaceus (Table 1 and Fig. 1).The G allele frequency was significantly increased in patients. Bothhomozygosity and heterozygosity for the G allele, as assessed bythe odds ratio (OR), result in increased disease risk, in comparisonto the A/A genotype. Because the OR of the G/A genotype is inter-mediate between the OR of both homozygotes, the most likely alle-lic interaction is incomplete dominance, with the G/G genotypehaving the highest risk. The significant association with SNPrs3087456 was observed in the adjusted total sample and in theEuro-Brazilian population stratum. For the Afro-Brazilians a similarpattern was observed, however, statistical significance was notreached. No significant difference between patients and controlswas observed when comparing allelic and genotypic frequenciesof SNP rs4774 (1632G>C), a missense substitution resulting inreplacement of a glycine for an alanine (G500A) (Table 1).

Because the combined effect of different polymorphisms in thecis configuration may not be seen when analyzing each polymor-phism individually, association analyses were performed also for

Table 1Frequencies of the CIITA rs3087456 and rs4774 SNPs alleles and genotypes in patients and controls and results of the association analysis.

Total Afro-Brazilian Euro-Brazilian

Patients % Controls % ORa 95 CIb pc Patient % Controls % ORa 95 CIb pc Patients % Controls % ORa 95 CIb pc

rs3087456 n = 226 n = 206 n = 97 n = 66 n = 153 n = 140Allele A 47.1 59.7 0.60 0.46–0.79 p < 10�3 40.2 47.8 0.74 0.47–1.15 0.201 51.3 65.4 0.56 0.40–0.78 p < 10�6

Allele G 52.9 40.3 1.66 1.27–2.18 59.8 52.3 1.36 0.87–2.12 48.7 34.6 1.79 1.28–2.50A+ 72.1 81.6 0.59 0.37–0.92 0.014 63.9 71.2 0.72 0.36–1.41 0.391 76.5 86.4 0.51 0.28–0.94 0.033G+ 77.9 62.1 2.15 1.41–3.27 p < 10�3 83.5 75.8 1.62 0.74–3.53 0.247 73.9 55.7 2.25 1.37–3.67 0.002A/A 22.1 37.9 1.00 16.5 24.2 1.00 26.1 44.3 1.00G/A 50.0 43.7 1.96 1.25–3.07 0.003 47.4 47.0 1.86 0.74–4.68 0.392 50.3 42.1 2.02 1.20–3.41 0.010G/G 27.9 18.4 2.59 1.51–4.42 p < 10�3 36.1 28.9 2.27 0.85–6.08 0.252 23.5 13.6 2.94 1.48–5.82 0.002HWd 1.00 0.192 1.00 0.629 1.00 0.45

rs4774 n = 246 n = 200 n = 96 n = 62 n = 150 n = 138Allele G 67.0 71.0 0.81 0.60–1.07 0.147 66.1 71.8 0.77 0.47–1.26 0.333 67.3 71.4 0.83 0.58–1.18 0.337Allele C 33.0 29.0 1.24 0.93–1.66 33.9 28.2 1.30 0.80–2.13 32.7 28.6 1.21 0.85–1.73G+ 90.0 93.0 0.67 0.34–1.32 0.326 91.7 91.9 0.97 0.30–3.10 1.000 88.7 93.5 0.55 0.24–1.27 0.216C+ 56.0 50.0 1.28 0.88–1.86 0.216 59.4 48.4 1.56 0.82–2.97 0.200 54.0 50.7 1.14 0.72–1.81 0.629G/G 44.0 50.0 1.00 40.6 51.6 1.00 46.0 49.3 1.00G/C 46.0 43.0 1.22 0.82–1.80 0.357 51.0 40.3 1.61 0.82–3.15 0.192 42.7 44.2 1.03 0.64–1.68 0.905C/C 10.0 7.0 1.65 0.81–3.36 0.217 8.3 8.1 1.31 0.39–4.41 0.765 11.3 6.5 1.86 0.78–4.46 0.195HWd 0.452 0.488 0.255 1.00 0.71 0.41

n: number of individuals. The OR of the reference genotypes is 1, by definition. A+, C+ and G+: individuals which are either homozygous or heterozygous for the A, C and Gallele. respectively.

a Odds ratio.b Confidence interval.c Probability.d Hardy–Weinberg equilibrium probability values.

0.10 1.00 10.00

G/A

G/G

G/A

G/G

G/A

G/G

0.10 1.00 10.00

A carriers

G carriers

A carriers

G carriers

A carriers

G carriers

A B

Fig. 1. Association analyses for the CIITA rs3087456 SNP. Dots represent the odds ratio (OR); horizontal lines the 95% confidence intervals of the OR. The ordinate axisindicates OR = 1. Triangles correspond to total population sample, squares to the Afro-Brazilian population sample and circles to the Euro-Brazilian population sample. (A)Shows the results for the G/G and G/A genotypes. (B) Shows the results for the presence of each allele either in homozygosity or heterozygosity (A carriers: genotypes A/A plusG/A; G carriers: genotypes G/G plus G/A).

1136 B.Z. Piovezan, M.L. Petzl-Erler / Human Immunology 74 (2013) 1134–1140

the haplotypes formed by the two SNPs of the CIITA gene. The hap-lotype frequencies differed among patients and controls (data notshown). Linkage disequilibrium was low (D0 = 0.2727, r2 = 0.0366,p < 0.01) and the SNP rs3087456 alone could account for the ob-served associations.

To reevaluate the previously described associations betweenEPF susceptibility and HLA-DRB1 genotypes and alleles, we com-pared the frequencies of the specific alleles, allelic lineages(groups) and genotypes (after grouping of susceptible, protectiveand neutral alleles – see below) between the patient and controlsamples (Tables 2 and 3). We used a sample that partially overlapswith that of Pavoni et al. [3]. (please refer to Section 2.2). In thepresent study, new patients and controls were included andhealthy control individuals not living in the endemic regions wereexcluded from the analysis. The findings of Pavoni et al. [3] wereconfirmed, apart from minor differences accounted for by thelow frequency of some alleles and the ensuing proportionally

larger sampling error. Alleles DRB1⁄01:01, ⁄01:02, ⁄04:04 and⁄04:06 were significantly increased among patients and allelesDRB1⁄03:01, ⁄07:01, ⁄11:01, ⁄11:04, ⁄13:01, ⁄13:03 were significantlydecreased. Trends of increase could be noticed for allelesDRB1⁄01:03, ⁄04:02 and 14:06 as well as a trend of decrease forDRB1⁄15:02. When grouping alleles pertaining to the same lineage,DRB1⁄01, DRB1⁄04 and DRB1⁄16 were significantly more frequentand DRB1⁄07, DRB1⁄08, DRB1⁄11 and DRB1⁄13 were significantlyless frequent in the patients sample, apart from a non-significantdecrease of the lineage DRB1⁄15. All observed alleles of the lineagesDRB1⁄01, DRB1⁄08 and DRB1⁄11 seem to contribute to differencesbetween patients and controls. Conversely, the differences ob-served for the other allelic lineages resulted from associations withparticular alleles. These results confirm the associations reportedin a previous study of the same population [3].

In order to compare genotype frequencies of patients and con-trols and to access the interaction between HLA-DRB1 genotypes

Table 2HLA-DRB1 allele frequencies in patients and controls and results of the association analysis.

HLA-DRB1 alelle/group Patients (N = 206) Controls (N = 203) ORa 95 CIb pc

n % n %

⁄01 125 30.3 37 9.1 4.34 2.92–6.47 <10�6

⁄01:01 33 8.0 19 4.7 1.77 0.99–3.17 0.046⁄01:02 57 13.8 8 2.0 7.99 3.76–16.97 <10�6

⁄01:03 7 1.7 1 0.2 7.00 0.86–57.16 0.070⁄03 25 6.1 37 9.1 0.64 0.38–1.09 0.109⁄03:01 10 2.4 24 5.9 0.40 0.19–0.84 0.015⁄03:02 13 3.2 6 1.5 2.17 0.82–5.77 0.168⁄04 102 24.8 51 12.6 2.29 1.58–3.31 <10–3

⁄04:01 4 1.0 5 1.2 0.79 0.21–2.95 0.748⁄04:02 9 2.2 2 0.5 4.51 0.97–21.01 0.065⁄04:03 3 0.7 2 0.5 1.48 0.25–8.91 1.000⁄04:04 28 6.8 14 3.4 2.04 1.06–3.94 0.039⁄04:05 6 1.5 4 1.0 1.49 0.42–5.30 0.752⁄04:06 6 1.5 0 0.0 13.00 0.73–231.53 0.032⁄04:07 0 0.0 4 1.0 0.11 0.01–2.02 0.123⁄04:08 0 0.0 1 0.2 ND ND ND⁄04:10 2 0.5 1 0.2 ND ND ND⁄04:11 13 3.2 10 2.5 1.29 0.56–2.98 0.672⁄07 9 2.2 51 12.6 0.16 0.08–0.32 <10�6

⁄07:01 7 1.7 45 11.1 0.14 0.06–0.31 <10�6

⁄08 11 2.7 27 6.7 0.39 0.19–0.79 0.006⁄08:01 1 0.2 4 1.0 0.25 0.03–2.20 0.213⁄08:02 5 1.2 10 2.5 0.49 0.17–1.44 0.200⁄08:03 0 0.0 3 0.7 0.14 0.01–2.71 0.125⁄08:04 2 0.5 3 0.7 0.66 0.11–3.94 0.684⁄08:07 0 0.0 1 0.2 ND ND ND⁄09 3 0.7 8 2.0 0.36 0.10–1.39 0.143⁄09:01 2 0.5 1 0.2 ND ND ND⁄10 4 1.0 8 2.0 0.49 0.15–1.63 0.267⁄10:01 4 1.0 8 2.0 0.49 0.15–1.63 0.261⁄11 5 1.2 41 10.1 0.11 0.04–0.28 <10�6

⁄11:01 1 0.2 19 4.7 0.05 0.01–0.37 <10�6

⁄11:02 2 0.5 3 0.7 0.66 0.11–3.94 0.681⁄11:03 0 0.0 3 0.7 0.14 0.01–2.71 0.246⁄11:04 1 0.2 11 2.7 0.09 0.01–0.68 0.006⁄12 9 2.2 5 1.2 1.79 0.60–5.39 0.416⁄12:01 5 1.2 4 1.0 1.24 0.33–4.63 1.000⁄12:02 2 0.5 1 0.2 ND ND ND⁄12:03 1 0.2 0 0.0 ND ND ND⁄13 33 8.0 63 15.5 0.47 0.30–0.74 0.001⁄13:01 5 1.2 21 5.2 0.23 0.08–0.60 0.002⁄13:02 21 5.1 22 5.4 0.94 0.51–1.73 0.880⁄13:03 0 0.0 6 1.5 0.07 0.00–1.33 0.014⁄14 22 5.3 15 3.7 1.47 0.75–2.88 0.318⁄14:01 9 2.2 9 2.2 0.99 0.39–2.51 1.000⁄14:02 1 0.2 4 1.0 0.25 0.03–2.20 0.214⁄14:04 2 0.5 0 0.0 ND ND ND⁄14:06 5 1.2 0 0.0 10.97 0.61–199.1 0.064⁄15 30 7.3 45 11.1 0.63 0.39–1.02 0.066⁄15:01 16 3.9 18 4.4 0.87 0.44–1.73 0.724⁄15:02 0 0.0 4 1.0 0.11 0.01–2.02 0.059⁄15:03 10 2.4 11 2.7 0.89 0.38–2.13 0.824⁄16 34 8.3 18 4.4 1.94 1.08–3.49 0.032⁄16:01 11 2.7 8 2.0 1.37 0.54–3.43 0.644⁄16:02 11 2.7 10 2.5 1.09 0.46–2.59 0.646

N: number of individuals; n: number of alleles; ND: not done. The frequencies of the allele lineages correspond to the sum of the frequencies the specific (four digit) allelesplus the frequency of alleles pertaining to the same group, which could not be identified.

a Odds ratio.b Confidence interval.c Probability.

B.Z. Piovezan, M.L. Petzl-Erler / Human Immunology 74 (2013) 1134–1140 1137

and CIITA alleles, HLA-DRB1 alleles were grouped in three catego-ries: susceptibility (SU), neutral (NE) and protective (PR). Theywere grouped following the criterion 2 of Pavoni et al. [3], whichstates that any given allele that is significantly increased in pa-tients or that belongs to and allelic linage significantly increasedin patients is classified as a SU allele. Likewise, any allele that is sig-nificantly decreased in patients or that belongs to and allelic linagesignificantly decreased in patients is classified as a PR allele. Theremaining alleles are classified as NE. The highest predisposing ef-fect was observed for the SU/SU genotype (OR = 8.1, p < 10�6). The

OR values gradually decreases through SU/NE, NE/NE SU/PR, andPR/NE and finally PR/PR, which is the most protective genotype(OR = 0.15) (Table 3). This result is compatible with a dose-depen-dent effect of the SU and the PR alleles. Interestingly, the SU/PRgenotype results in a neutral phenotype in what refers to diseasesusceptibility, as evidenced by the OR that is similar to that ofthe NE/NE genotype and does not differ significantly from the va-lue 1. This corroborates the results of Pavoni et al. [3].

In search of interactions between CIITA and HLA-DRB1, the CIITAgenotypes were analyzed in each of the strata defined by the

Table 3Association analysis after grouping HLA-DRB1 alleles into susceptible, protective and neutral.

HLA-DRB1 genotype Patients (n = 204) Controls (n = 197)

n % n % ORa 95 CIb pc

SU/SU 57 27.9 9 4.6 8.06 2.80–23.2 <10�6

SU/NE 74 36.3 22 11.2 4.28 1.70–10.76 <10–6SU/PR 38 18.6 47 23.9 1.03 0.42–2.53 0.243NE/NE 11 5.4 14 7.1 1.00 0.535NE/PR 18 8.8 55 27.9 0.42 0.16–1.08 <10�6

PR/PR 6 2.9 50 25.4 0.15 0.05–0.49 <10�6

n: number of individuals. For reference genotypes, the OR corresponds to 1 by definition. SU: susceptible alleles; PR: protective alleles; NE: neutral alleles.a Odds ratio.b Confidence interval.c Probability.

Table 4HLA-DRB1 and CIITA genotype frequencies in patients and controls and results of the association analysis, showing the additive effect of the two genes on disease susceptibility.

HLA-DRB1 genotype CIITA genotype Patients (n = 204) Controls (n = 197)

n % n % ORa 95% CIb pc

SU/SU A/A 11 5.4 5 2.5 2.19 0.75–6.42 0.211G+ 46 23.0 4 2.0 14.05 4.95–39.87 <10–6

SU/NE A/A 19 9.3 6 3.0 3.27 1.28–8.37 0.014G+ 55 27.0 16 8.0 4.18 2.30–7.59 <10�6

SU/PR A/A 10 4.9 18 9.1 0.51 0.23–1.14 0.118G+ 28 14.0 29 15.0 0.92 0.53–1.61 0.779

NE/NE A/A 2 1.0 7 3.6 0.27 0.06–1.31 0.101G+ 9 4.0 7 4.0 1.25 0.46–3.43 0.795

NE/PR A/A 3 1.5 20 10.2 0.13 0.04–0.45 <10�3

G+ 15 7.0 35 18.0 0.37 0.19–0.70 0.002PR/PR A/A 2 1.0 17 8.6 0.11 0.02–0.46 <10�4

G+ 4 2.0 33 17.0 0.10 0.03–0.29 <10�6

n: number of individuals. SU: susceptible alleles; PR: protective alleles; NE: neutral alleles. G+: G/G and A/G genotypes.a Odds ratio.b Confidence interval.c Probability.

1138 B.Z. Piovezan, M.L. Petzl-Erler / Human Immunology 74 (2013) 1134–1140

HLA-DRB1 genotypes (Table 4). We observed a major effect of theHLA-DRB1 genotypes and an additive effect of the two genes. Thehighest OR values were seen for the HLA-DRB1 SU/SU plus CIITAG/G and G/A genotypes (OR = 14.0). The same pattern was observedwhen the sample was stratified according to the CIITA genotypes(results not shown).

4. Discussion

MHC class II molecules are essential for T cell- and antibody-mediated immune responses through presentation of antigenicpeptides to CD4+ T cells. In pemphigus, the activation of desmog-lein-specific B cells and secretion of autoantibodies depends onthe interaction between the T cell receptor and classical MHC classII molecules [20]. In EPF, variation of the MHC class II gene HLA-DRB1 has a strong effect on differential susceptibility [3]. Herewe examined genotypes of CIITA, whose product is a regulatoryprotein essential for transcriptional regulation of HLA-DRB1 andother MHC class II genes, for association with susceptibility tothe disease. We also wanted to explore whether the CIITA andHLA-DRB1 genes interact regarding the risk of developing EPF. Dif-ferently from other transcription factors, which have hundreds ifnot thousands of target genes, a high degree of specificity was ob-served for CIITA. It is uniquely dedicated for MHC class II genes anda handful of other genes also implicated in antigen presentation[11].

Our result show that the SNP rs3087456 located in the CIITApromoter region is associated to EPF susceptibility. Relative to

the A/A genotype, the A/G and G/G genotypes result in a twofoldand threefold increase of susceptibility, respectively. The associa-tion was detected in Euro-Brazilians and in the total populationsample. We also observed increased frequency of allelers3087456 G in both genotypes, G/G and G/A, in Afro-Brazilian pa-tients when compared to their respective controls, but that differ-ence was not significant. The lack of significance may be due to lowstatistical power, because this population sample was small andalso because the allele and genotype frequencies differed less be-tween the Afro-Brazilian patients and controls than between theEuro-Brazilian patients and controls.

The other polymorphism analyzed in CIITA, the rs4774 SNP, isnot associated with pemphigus foliaceus susceptibility. This SNPpredicts an amino acid change (from glycine to alanine, G500A)in the nucleotide binding domain of the protein. Glycine is thesmallest amino acid. For the R group, there is a hydrogen (H). Ala-nine is the next in size, with a methyl group (CH3) as the R group.Because of the physicochemical properties of the two amino acids,the replacement of a glycine for an alanine is not considered radi-cal [21] and may not have a major effect on the structure and func-tion of the CIITA molecule. Furthermore, the lack of a statisticallysignificant association between the disease and the rs4774 SNP isexplained by the low linkage disequilibrium with the disease-asso-ciated rs3087456 SNP.

As observed here for pemphigus foliaceus, the CIITA rs3087456allele G was associated to increased susceptibility to other autoim-mune diseases, including Addison’s disease [22], rheumatoidarthritis [13] and Löfgren’s syndrome [23]. Different from EPF,rs4774 allele C has been associated to increased susceptibility to

B.Z. Piovezan, M.L. Petzl-Erler / Human Immunology 74 (2013) 1134–1140 1139

multiple sclerosis [24] and systemic lupus erythematosus [25]. Inany case the effect of the CIITA risk genotype was low (OR = 1.1–1.7)and difficult to replicate [24,26]. The discrepancies betweenassociations with distinct diseases might result from the complexregulation of CIITA gene expression in connection with differentmechanisms operating in the pathogenesis of these diseases. Thegene has four promoters, which control its expression in differentcell types [27]. The SNP rs3087456 is located at promoter III that isresponsible for constitutive expression of CIITA in B cells that playan important role in pemphigus. It has been shown that this poly-morphism can affect promoter III functionality. When peripheralblood cells from different individuals were stimulated ex vivo withIFNc, the ones with genotype G/G had lower levels of CIITA andMHC class II mRNA than genotypes A/A and A/G [13]. This observa-tion leads us to suggest that lower expression of the MHC class IIalleles is associated to higher susceptibility to the disease. Thereaf-ter, at least two not mutually exclusive mechanisms could beresponsible for the association: (i) higher levels of MHC class IImolecules protect against the disease, or (ii) lower levels of MHCclass II molecules promote the disease. In fact, several HLA-DRB1alleles are associated with decreased risk of pemphigus foliaceus,the most prominent being DRB1⁄07:01 (OR = 0.14, p < 10�6) andDRB1⁄03:01, (OR = 0.4 p = 0.01). It is reasonable to think that higherlevels of these protective HLA-DR molecules or of other protectiveMHC class II molecules associated to them (e.g., HLA-DQ2, whosebeta chain is encoded by DQB1⁄02 alleles in strong linkage disequi-librium with both the DRB1⁄07:01 and DRB1⁄03:01 alleles) help toprevent the disease. On the other hand, lower levels of MHC classII molecules may result in less efficient antigen presentation to reg-ulatory T cells (Treg), a specialized population of T cells that cansuppress the activation of the antigen-specific immune responses,helping to maintain homeostasis and inducing tolerance to self-antigens. Actually, mRNA of the Treg markers FOXP3, CTLA-4 andGITR, are up-regulated in lesional skin compared to uninvolvedskin of pemphigus foliaceus patients and it has been proposed thatthese cells are insufficient and/or inefficient to control the autoim-mune response in the susceptible individuals, resulting in activa-tion and expansion of pathogenic T cells (Malheiros et al.,personal communication). Moreover, the proportion of these cellsamong peripheral blood mononuclear cells of pemphigus vulgarispatients is severely reduced [28]. Moreover impairment in Tregfunction due to lower expression of class II MHC molecules result-ing in less efficient antigen presentation to Treg cells can also ex-plain the rs3087456 association.

Seeking for interactions between CIITA and HLA-DRB1 in ende-mic pemphigus foliaceus susceptibility, the CIITA genotypes wereanalyzed in each of the strata defined by the HLA-DRB1 genotypes.Because high resolution HLA-DRB1 genotypes are individually toorare for statistical analysis, we grouped the HLA-DRB1 alleles intosusceptible (SU), protective (PR) and neutral (NE) according toPavoni et al. [3]. The effect of the susceptible CIITA G/G and G/Agenotypes was not significant in the presence of protective andneutral HLA-DRB1 genotypes, in accordance with the major effectof HLA-DRB1 on the risk of developing EPF. Yet, the lowest ORvalues were observed for the strata having protective genotypesof both the HLA and the CIITA genes. Most impressive, thepopulation strata with both the HLA and the CIITA susceptibilitygenotypes presented significantly higher OR values than thoseobserved for each gene individually. So, the odds ratio for theHLA-DRB1 SU/SU is of 14.05 in the sample with the susceptibleCIITA G+ genotypes and much lower (2.19) in presence of theprotective CIITA A/A genotype. This reveals an additive effect ofthe HLA and CIITA genotypes on disease susceptibility. Interactionbetween HLA-DRB1 and CIITA alleles or genotypes has beenreported also for multiple sclerosis [24] but not for rheumatoidarthritis [29].

In conclusion, we show here that the SNP rs3087456 located inthe CIITA gene promoter is associated with susceptibility to pem-phigus foliaceus and suggest that the association results from theeffect of genetically controlled levels of CIITA on the expressionof the susceptible and protective HLA class II molecules. On basisof these results we conclude that quantitative as well as qualitativevariation of HLA class II molecules have an effect on disease risk.Further functional studies are needed to investigate how geneticvariation of CIITA may affect the expression of MHC class II genes.

Acknowledgments

We thank the staff of the Laboratory of Human MolecularGenetics at the Department of Genetics of the Federal Universityof Paraná for assistance and support. This project received financialsupport from the Conselho Nacional de Desenvolvimento Científicoe Tecnológico (CNPq), PRONEX (Programa de Apoio a Núcleos deExcelência – Fundação Araucária and CNPq) and Coordenação deAperfeiçoamento de Pessoal de Nível Superior (CAPES).

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