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First Glimpse of Epigenetic Effects on Pemphigus Foliaceus

2019; Elsevier BV; Volume: 140; Issue: 2 Linguagem: Inglês

10.1016/j.jid.2019.07.691

1523-1747

M Spadoni, Valéria Bumiller‐Bini, Maria Luiza Petzl‐Erler, Danillo G. Augusto, Angelica Beate Winter Boldt,

Feminism, Gender, and Sexuality Studies

Pemphigus foliaceus (PF) is an autoimmune disease, sporadic across the globe, but endemic in Brazil. A significant portion of PF autoantibodies target desmosomal glycoproteins, especially desmoglein-1, which are associated with the occurrence of painful blisters in the granular skin layer (Spindler et al., 2007Spindler V. Drenckhahn D. Zillikens D. Waschke J. Pemphigus IgG causes skin splitting in the presence of both desmoglein 1 and desmoglein 3.Am J Pathol. 2007; 171: 906-916Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Epigenetic mechanisms have been implicated in autoimmune skin diseases. Among them, dysregulated DNA and histone methylation contribute to systemic lupus erythematosus (Hung et al., 2018Hung K.H. Woo Y.H. Lin I.Y. Liu C.H. Wang L.C. Chen H.Y. et al.The KDM4A/KDM4C/NF-κB and WDR5 epigenetic cascade regulates the activation of B cells.Nucleic Acids Res. 2018; 46: 5547-5560Crossref PubMed Scopus (21) Google Scholar), psoriasis (Manczinger and Kemény, 2013Manczinger M. Kemény L. Novel factors in the pathogenesis of psoriasis and potential drug candidates are found with systems biology approach.PLOS ONE. 2013; 8: e80751Crossref PubMed Scopus (27) Google Scholar), pemphigus vulgaris (Zhao et al., 2012aZhao M. Huang W. Zhang Q. Gao F. Wang L. Zhang G. et al.Aberrant epigenetic modifications in peripheral blood mononuclear cells from patients with pemphigus vulgaris.Br J Dermatol. 2012; 167: 523-531Crossref PubMed Scopus (19) Google Scholar), and alopecia areata (Zhao et al., 2012bZhao M. Liang G. Wu X. Wang S. Zhang P. Su Y. et al.Abnormal epigenetic modifications in peripheral blood mononuclear cells from patients with alopecia areata.Br J Dermatol. 2012; 166: 226-273Crossref PubMed Scopus (34) Google Scholar). To investigate the association of genetic polymorphisms in the alteration of these mechanisms in PF, we compared 229 patients to 194 controls (52% women, median age of 43 years old, unrelated, predominantly euro-descendent, and from rural areas), genotyped with microarray hybridization (CoreExome-24, version 1.1, Illumina, San Diego, CA). The Brazilian National Ethics in Research Commission approved the protocol (02727412.4.0000.0096), and all participants gave written informed consent, according to the Declaration of Helsinki. From 1984 to 2015, individuals were recruited in the South and/or Central-Western regions of Brazil, in the Hospital Adventista do Pênfigo (Campo Grande), Lar de Caridade (Uberaba), Hospital de Clínicas (Ribeirão Preto), and Hospital de Clínicas (Curitiba). All patients presented positive Nikolsky signal and sun-exposed lesions (155 localized and 74 generalized); a total of 41 presented active lesions at the moment of blood collection, 87% of the patients were anti-desmoglein-1 positive, and all patients were negative for anti-neutrophil-cytoplasmic, anti-mitochondrial, anti-liver-kidney-microsomal, and anti-gastric-parietal-cells by ELISA (Oliveira et al., 2016Oliveira L.A. Marquart-Filho A. Trevilato G. Timoteo R.P. Mukai M. Roselino A.M. et al.Anti-desmoglein 1 and 3 autoantibody levels in endemic pemphigus foliaceus and pemphigus vulgaris from Brazil.Clin Lab. 2016; 62: 1209-1216Crossref PubMed Scopus (6) Google Scholar) or indirect immunofluorescence (Nisihara et al., 2003Nisihara R.M. de Bem R.S. Hausberger R. Roxo V.S. Pavoni D.P. Petzl-Erler M.L. et al.Prevalence of autoantibodies in patients with endemic pemphigus foliaceus (fogo selvagem).Arch Dermatol Res. 2003; 295: 133-137Crossref PubMed Scopus (18) Google Scholar). A small percentage of patients and controls (0.8–2%) were low-positive and asymptomatic for anti-smooth-muscle, anti-thyroid-microsome, and anti-nuclear-antibodies (Nisihara et al., 2003Nisihara R.M. de Bem R.S. Hausberger R. Roxo V.S. Pavoni D.P. Petzl-Erler M.L. et al.Prevalence of autoantibodies in patients with endemic pemphigus foliaceus (fogo selvagem).Arch Dermatol Res. 2003; 295: 133-137Crossref PubMed Scopus (18) Google Scholar). We selected 1,793 variants in 63 genes that code for lysine methyltransferases (KMT), demethylases (KDM), DNA methyltransferases, and ten-eleven translocation demethylases. We excluded rare variants (minor allele frequencies <0.01), variants that deviated from Hardy-Weinberg equilibrium in controls (P < 0.05), and variants in linkage disequilibrium (r2 ≥ 0.8). A total of 566 polymorphisms remained and were analyzed using logistic regression with correction for two principal components using PLINK, version 1.1.9 (http://zzz.bwh.harvard.edu/plink/). Haplotypes were reconstructed for all 61 single nucleotide polymorphisms, with r2 > 0.8 and evaluated with logistic regression using PLINK, version 1.1.9, and/or STATA, version 9.2. Datasets can be found at http://www.lgmh.ufpr.br/data/Table1.xlsx and http://www.lgmh.ufpr.br/data/Suggestive_results.docx, hosted at the website of the Laboratory of Human Molecular Genetics of the Federal University of Paraná. We found 11 variants in four genes that were associated with PF (P < 0.005). One gene encodes a histone demethylase (KDM4C) and the other three encode histone lysine methytransferases (SETD7/KMT7; MECOM/KMT8E; PRDM16/KMT8F; Table 1).Table 1Gene Polymorphisms of Lysine (De)Methylases Associated with PFGenesSNPMAF%ModelControlsPatientsOR95% CIP-valuesIbControlsPatientsKDM4C9p24.1rs10976056g>A Intron 1216.415.324.0add3/53/13715/79/1331.671.16–2.380.0049rs35826653G>c Exon 118.94.48.8dom16/17840/1872.521.35–4.700.0036rs913588G>a Exon 1540.235.644.3rec22/17252/1742.231.35–4.020.0022SETD74q31.1rs2725790T>c Intron 219.216.725.89add4/56/13213/90/1211.771.24–2.520.0016dom60/132103/1211.841.23–2.760.0029PRDM161p36.32rs1537406C>t Intron 14.29.115.1dom32/16164/1642.051.26–3.320.0036rs731031T>c Intron 123.834.827.5add24/87/8317/89/1180.610.44–0.840.0027MECOM3q26.2rs7652391G>t Intron 216.819.329.5add2/70/12017/97/1081.781.25–2.530.0012rs7625357C>t Intron 134.135.025.1add23/90/8112/91/1260.620.45–0.850.0028rs6797224T>c Intron 217.820.029.5add2/73/11717/100/1101.691.19–2.410.0029rs9852670A>g Intron 27.511.319.3add0/44/1504/80/1441.891.23–2.890.0034rs756644c>A Intron 249.149.244.3rec59/13539/1900.490.31–0.790.0033Logistic associations tests were done with genotype frequencies, frequencies of homozygotes for the minor allele (rec), and summed frequencies of homozygotes and heterozygotes for the minor allele (dom). The minor allele is indicated in lower case (1000 Genomes, http://grch37.ensembl.org/index.html).Abbreviations: add, additive; CI, confidence interval; dom, dominant; Ib, Iberian population; MAF, minor allele frequency; OR, odds ratio; rec, recessive; SNP, single nucleotide polymorphism. Open table in a new tab Logistic associations tests were done with genotype frequencies, frequencies of homozygotes for the minor allele (rec), and summed frequencies of homozygotes and heterozygotes for the minor allele (dom). The minor allele is indicated in lower case (1000 Genomes, http://grch37.ensembl.org/index.html). Abbreviations: add, additive; CI, confidence interval; dom, dominant; Ib, Iberian population; MAF, minor allele frequency; OR, odds ratio; rec, recessive; SNP, single nucleotide polymorphism. The KDM4C non-synonymous variants rs35826653*C (p.Ser179Thr) and rs913588*A (p.Val602Ile) are associated with higher KDM4C expression levels in thyroid, testis, and musculoskeletal tissues (P < 10–6; https://gtexportal.org/home/snp/rs35826653 and https://gtexportal.org/home/snp/rs913588). These variants presented dominant (odds ratio [OR] = 2.52, P = 0.0036) and additive (OR = 2.23, P = 0.0022) susceptibility effects to PF, respectively, in addition to the intronic rs10976056*A variant (OR = 1.67, 95% confidence interval [CI] = 1.16–2.38]). Additive effects were observed for variant combinations as follows: rs913588*A + rs35826653*C (OR = 7.04, 95% CI = 1.55–65.45, P = 0.0033); rs913588*A + rs10976056*A (OR = 4.90, 95% CI = 2.02–13.71, P = 0.0001); and rs10976056*A + rs35826653*C (OR = 3.35, 95% CI = 1.46–8.43, P = 0.0024). KDM4C demethylates lysine 9 on histone 3 (trimethylated to dimethylated H3K9) in the nucleosomes of genes whose products increase cell proliferation (Gregory and Cheung, 2014Gregory B.L. Cheung V.G. Natural variation in the histone demethylase, KDM4C, influences expression levels of specific genes including those that affect cell growth.Genome Res. 2014; 24: 52-63Crossref PubMed Scopus (14) Google Scholar; Figure 1a). The recognition of follicular T helper cell-derived CD40L by the CD40 receptor on B cells pregulates KDM4C expression. KDM4C binds to NF-κB, activating WD40-repeat protein to trimethylate H3K4, increasing cyclin-dependent kinase gene expression needed for proper B-cell activation (Hung et al., 2018Hung K.H. Woo Y.H. Lin I.Y. Liu C.H. Wang L.C. Chen H.Y. et al.The KDM4A/KDM4C/NF-κB and WDR5 epigenetic cascade regulates the activation of B cells.Nucleic Acids Res. 2018; 46: 5547-5560Crossref PubMed Scopus (21) Google Scholar; Figure 1b). Interestingly, CD40_rs3092945*T and CD40L_rs1883832*T (associated with increased CD40L expression in sun-exposed skin; https://gtexportal.org/home/snp/rs1883832) were associated with protection against PF (Malheiros and Petzl-Erler, 2009Malheiros D. Petzl-Erler M.L. Individual and epistatic effects of genetic polymorphisms of B-cell co-stimulatory molecules on susceptibility to pemphigus foliaceus.Genes Immun. 2009; 10: 547-558Crossref PubMed Scopus (26) Google Scholar). In contrast, KDM4C was downregulated in systemic lupus erythematosus (Hung et al., 2018Hung K.H. Woo Y.H. Lin I.Y. Liu C.H. Wang L.C. Chen H.Y. et al.The KDM4A/KDM4C/NF-κB and WDR5 epigenetic cascade regulates the activation of B cells.Nucleic Acids Res. 2018; 46: 5547-5560Crossref PubMed Scopus (21) Google Scholar). This agrees with the opposite effects observed in systemic lupus erythematosus for PF-associated variants within genes of the complement system (Bumiller-Bini et al., 2018Bumiller-Bini V. Cipolla G.A. de Almeida R.C. Petzl-Erler M.L. Augusto D.G. Boldt A.B.W. Sparking fire under the skin? Answers from the association of complement genes with pemphigus foliaceus.Front Immunol. 2018; 9: 695Crossref PubMed Scopus (13) Google Scholar). Thus, higher levels of KDM4C may foster PF autoantibody production by increasing B-cell activation. This is further supported by the association with the SETD7 (KMT7) rs2725790*C variant (OR = 1.77, P = 0.0016). SETD7 monomethylates H3K4 and K37 of NF-κB-light-chain-enhancer p65, promoting the activation of pro-inflammatory genes (Batista and Helguero, 2018Batista I.A.A. Helguero L.A. Biological processes and signal transduction pathways regulated by the protein methyltransferase SETD7 and their significance in cancer.Signal Transduct Target Ther. 2018; 3: 19Crossref PubMed Scopus (55) Google Scholar). Interestingly, skin exposure to ultraviolet radiation, a known PF risk factor, increases reactive oxygen species production and enhances p65 monomethylation (Ea and Baltimore, 2009Ea C.K. Baltimore D. Regulation of NF-kappaB activity through lysine monomethylation of p65.Proc Natl Acad Sci U S A. 2009; 106: 18972-18977Crossref PubMed Scopus (178) Google Scholar). The co-occurrence of SETD7_ rs2725790*C and any KDM4C variant was associated with increased PF susceptibility (rs35826653*C: OR = 5.66, 95% CI = 1.80–23.63, P = 0.0008; rs913588*A: OR = 4.80, 95% CI = 1.81–14.96, P = 0.0004; and rs10976056*A: OR = 3.20, 95% CI = 1.65–6.39, P = 0.0002). PF etiology has been related to phagocytosis and presentation of an environmental antigen (e.g., a saliva protein of hematophagous insects) to T helper cells, stimulating B-cell autoantibody production (Spindler et al., 2007Spindler V. Drenckhahn D. Zillikens D. Waschke J. Pemphigus IgG causes skin splitting in the presence of both desmoglein 1 and desmoglein 3.Am J Pathol. 2007; 171: 906-916Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Target recognition by autoantibodies is associated with keratinocytes’ death, exposing skin self-antigens that will feedback the autoimmune response. In this context, we found the PRMD16 variant rs1537406*T, which is associated with a dominant effect increasing PF susceptibility (OR = 2.05, P = 0.0036). This allele is predicted to reduce binding affinity of several microRNAs (hsa-miR-2964a-5p, hsa-miR-3150b-3p, and hsa-miR-4689), which would increase mRNA levels and the abundance of this H3K9 methyltransferase (Liu et al., 2012Liu C. Zhang F. Li T. Lu M. Wang L. Yue W. et al.MirSNP, a database of polymorphisms altering miRNA target sites, identifies miRNA-related SNPs in GWAS SNPs and eQTLs.BMC Genomics. 2012; 13: 661Crossref PubMed Scopus (218) Google Scholar). This variant is also associated with increased ARHGEF16 expression in sun-exposed skin (https://gtexportal.org/home/snp/rs1537406). ARHGEF16 promotes phagocytic engulfment of apoptotic cells and may remove acantholytic keratinocyte debris (Lee et al., 2014Lee J. Park B. Kim G. Kim K. Pak J. Kim K. et al.Arhgef16, a novel Elmo1 binding partner, promotes clearance of apoptotic cells via RhoG-dependent Rac1 activation.Biochim Biophys Acta. 2014; 1843: 2438-2447Crossref PubMed Scopus (22) Google Scholar). Furthermore, carriers of the PRDM16 allele rs731031*C exhibit greater resistance to PF (OR = 0.61, P = 0.0027; Figure 1c). Acantholysis caused by PF demands skin regeneration. MECOM (KMT8E) is a PRMD16 paralog, encoding a H3K9 monomethyltransferase deemed responsible for increasing psoriatic keratinocyte proliferation (Manczinger and Kemény, 2013Manczinger M. Kemény L. Novel factors in the pathogenesis of psoriasis and potential drug candidates are found with systems biology approach.PLOS ONE. 2013; 8: e80751Crossref PubMed Scopus (27) Google Scholar). The following MECOM variants were associated with an additive effect for both PF susceptibility and protection: rs7652391*T (OR = 1.78, P = 0.0012), rs6797224*C (OR = 1.69, P = 0.0029), rs9852670*G (OR = 1.89, P = 0.0034), rs7625357*T (OR = 0.62, P = 0.0028), and rs756644*C, with a recessive effect (OR = 0.49, P = 0.0033; Figure 1d). There was also a MECOM haplotype containing rs9852670*G and rs6797224*C, which was associated with PF susceptibility (OR = 1.93, 95% CI = 1.23–3.05, P = 0.004; Supplementary Figures S1 and S2). Thus, as judged by the associated genetic polymorphisms, dysregulated histone (de)methylation seems to play a major role in PF. This overcomes the expected effects of dysregulated DNA (de)methylation, which has been historically privileged in epigenetic disease-association studies. We predict a beneficial effect from the therapeutic use of specific KDM and/or KTM inhibitors, as suggested by others (Thaler and Mercurio, 2016Thaler F. Mercurio C. Compounds and methods for inhibiting histone demethylases: a patent evaluation of US20160102096A1.Expert Opin Ther Pat. 2016; 26: 1367-1370Crossref PubMed Scopus (2) Google Scholar, Westaway et al., 2016Westaway S.M. Preston A.G.S. Barker M.D. Brown F. Brown J.A. Campbell M. et al.Cell penetrant inhibitors of the KDM4 and KDM5 families of histone lysine demethylases. 1. 3-Amino-4-pyridine carboxylate derivatives.J Med Chem. 2016; 59: 1357-1369Crossref PubMed Scopus (42) Google Scholar), in order to reduce tissue damage and B-cell activation, and increase skin renewal. Mariana Basso Spadoni: https://orcid.org/0000-0002-6496-7048 Valéria Bumiller-Bini: https://orcid.org/0000-0003-4000-2417 Maria-Luiza Petzl-Erler: https://orcid.org/0000-0002-0345-5276 Danillo Gardenal Augusto: https://orcid.org/0000-0001-5665-8547 Angelica Beate Winter Boldt: https://orcid.org/0000-0002-0902-9622 The authors state no conflict of interest. We are deeply grateful to the patients who participated in this study and to the staff of the Laboratory of Human Molecular Genetics of the Federal University of Paraná for their assistance with DNA extraction. This work was supported by grants from the following funding agencies: Fundação Araucária (protocol number 39894.413.43926.1904/2013), Conselho Nacional de Desenvolvimento Científico e Tecnológico (protocol number 470483/2014-8), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (a post-doc scholarship with bench rate for DGA, protocol number 400648/2014-8). The sponsors had no role in the study design, collection, analysis and interpretation of data, and in the writing of the letter and the decision to submit it for publication. The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The data used for the analyses described in this manuscript were obtained from: https://gtexportal.org/home/snp/rs35826653, https://gtexportal.org/home/snp/rs913588, https://gtexportal.org/home/snp/rs1883832, https://gtexportal.org/home/snp/rs1537406 of the GTEx Portal on 02/01/19. Supplementary Figure S2Phylogenetic haplotype tree of the MECOM gene. Phylogenetic tree of MECOM haplotypes formed by block 1 of the LD plot (Supplementary Figure S1), inferred using the Maximum Parsimony method. Highlighted, the haplotype associated with PF (rs9852670*G, rs16853318*C, rs13085351*G, rs6797224*C, rs1352340*T; with OR = 1.93, 95% CI = 1.23–3.05, P = 0.004). The phylogenetic tree was rooted on the haplotype of PAN. Evolutionary analyses were conducted in MEGA7. CI, confidence interval; LD, linkage disequilibrium; OR, odds ratio; PAN, Pan troglodytes; PF, pemphigus foliaceus.View Large Image Figure ViewerDownload Hi-res image Download (PPT)