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Unsuspected Associations of Variants within the Genes NOTCH4 and STEAP2-AS1 Uncovered by a GWAS in Endemic Pemphigus Foliaceus

2021; Elsevier BV; Volume: 141; Issue: 11 Linguagem: Inglês

10.1016/j.jid.2021.04.017

1523-1747

Danillo G. Augusto, Rodrigo Coutinho de Almeida, Ticiana D.J. Farias, Wagner C. S. Magalhães, Danielle Malheiros, Maria Fernanda Lima‐Costa, Maurício L. Barreto, Bernardo Lessa Horta, Vinod Kumar, Michael Wittig, André Franke, Hauke Busch, Enno Schmidt, Ana Maria Ferreira Roselino, Eduardo Tarazona‐Santos, Angelica Beate Winter Boldt, Maria Luiza Petzl‐Erler,

Urticaria and Related Conditions

Pemphigus foliaceus is a blistering autoimmune disease of the skin representing a public health issue in Brazil, where it is endemic and neglected. Sporadic cases are reported across the globe. Nevertheless, an astonishing prevalence of more than 3% was reported for endemic pemphigus foliaceus (EPF) in some Brazilian regions (Schmidt et al., 2019Schmidt E. Kasperkiewicz M. Joly P. Pemphigus.Lancet. 2019; 394: 882-894Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), the highest ever reported for an autoimmune disease worldwide. Although the reasons for its endemicity are not clear, it has been suggested that environmental factors, such as agricultural activities, insect bites, and others, may trigger the disease in genetically susceptible individuals (Aoki et al., 2015Aoki V. Rivitti E.A. Diaz L.A. Cooperative Group on Fogo Selvagem Research. Update on fogo selvagem, an endemic form of pemphigus foliaceus.J Dermatol. 2015; 42: 18-26Crossref PubMed Scopus (51) Google Scholar; Qian et al., 2016Qian Y. Culton D.A. Jeong J.S. Trupiano N. Valenzuela J.G. Diaz L.A. Non-infectious environmental antigens as a trigger for the initiation of an autoimmune skin disease.Autoimmun Rev. 2016; 15: 923-930Crossref PubMed Scopus (22) Google Scholar). We previously described strong associations with alleles of HLA class II genes (Pavoni et al., 2003Pavoni D.P. Roxo V.M.M.S. Marquart Filho A. Petzl-Erler M.L. Dissecting the associations of endemic pemphigus foliaceus (fogo selvagem) with HLA-DRB1 alleles and genotypes.Genes Immun. 2003; 4: 110-116Crossref PubMed Scopus (52) Google Scholar; Petzl-Erler and Santamaria, 1989Petzl-Erler M.L. Santamaria J. Are HLA class II genes controlling susceptibility and resistance to Brazilian pemphigus foliaceus (fogo selvagem)?.Tissue Antigens. 1989; 33: 408-414Crossref PubMed Scopus (59) Google Scholar). Furthermore, through candidate gene association studies, we revealed polymorphisms of immune-related genes altering susceptibility to EPF (Petzl-Erler, 2020Petzl-Erler M.L. Beyond the HLA polymorphism: a complex pattern of genetic susceptibility to pemphigus.Genet Mol Biol. 2020; 43e20190369Crossref PubMed Scopus (10) Google Scholar). Although the results based on candidate gene association studies established a strong role of genetic factors in EPF pathogenesis, this approach cannot reveal unsuspected susceptibility loci. Here, we present a GWAS in EPF. This study was approved by the Human Research Ethics Committee of the Federal University of Paraná and the Brazilian National Human Research Ethics Committee (CONEP), protocol number CAAE 02727412.4.0000.0096, under the Brazilian Federal laws. All individuals gave written informed consent following the Declaration of Helsinki. The discovery cohort was composed of 234 patients approached in the endemic areas of Mato Grosso do Sul State, in a reference hospital specialized in EPF (Pemphigus Adventist Hospital, Campo Grande), and 5,658 controls that are part of the EPIGEN-Brasil initiative, which is based on three well-defined population-based cohorts from Brazilian regions (Kehdy et al., 2015Kehdy F.S.G. Gouveia M.H. Machado M. Magalhães W.C.S. Horimoto A.R. Horta B.L. et al.Origin and dynamics of admixture in Brazilians and its effect on the pattern of deleterious mutations.Proc Natl Acad Sci USA. 2015; 112: 8696-8701Crossref PubMed Scopus (160) Google Scholar; Magalhães et al., 2018Magalhães W.C.S. Araujo N.M. Leal T.P. Araujo G.S. Viriato P.J.S. Kehdy F.S. et al.EPIGEN-Brazil initiative resources: a Latin American imputation panel and the scientific workflow.Genome Res. 2018; 28: 1090-1095Crossref PubMed Scopus (14) Google Scholar). All patients enrolled in this study were diagnosed by experienced dermatologists on the basis of clinical features, histopathological features, immunofluorescence, and evaluation of antidesmoglein autoantibodies. Individuals were genotyped for SNPs with the Illumina platform (Illumina, San Diego, CA) using microarray chips CoreExome-24 v1.1 for patients and HumanOmni2.5 for controls. Quality control was performed as described previously (Anderson et al., 2010Anderson C.A. Pettersson F.H. Clarke G.M. Cardon L.R. Morris A.P. Zondervan K.T. Data quality control in genetic case-control association studies.Nat Protoc. 2010; 5: 1564-1573Crossref PubMed Scopus (746) Google Scholar; Kehdy et al., 2015Kehdy F.S.G. Gouveia M.H. Machado M. Magalhães W.C.S. Horimoto A.R. Horta B.L. et al.Origin and dynamics of admixture in Brazilians and its effect on the pattern of deleterious mutations.Proc Natl Acad Sci USA. 2015; 112: 8696-8701Crossref PubMed Scopus (160) Google Scholar). In summary, we eliminated related individuals and those with large-scale differences in ancestry (Supplementary Figure S1). Further, we excluded markers whose genotypes deviated from Hardy-Weinberg equilibrium (P < 0.001) and those with strong linkage disequilibrium (r2 > 0.8). We also excluded markers with minor allele frequency <0.10 and call rate <96%. Principal component analysis was used to control for outliers and to merge datasets further. After quality control, 204,967 markers remained for the logistic regression analysis, assuming an additive model using four principal components as covariates to correct for possible population stratification. We observed minimal overall inflation of the genome-wide statistical results (λGC = 1.05; Supplementary Figure S1). The strongest association signals were within the major histocompatibility complex, specifically within intergenic regions in the HLA class II region (Figure 1). In addition, we found a suggestive association (P < 5 × 10–5) with the intronic rs2854050 in NOTCH4, a non-HLA gene within the major histocompatibility complex (Table 1). According to the Genotype Tissue Expression portal (https://gtexportal.org), this SNP has an expression quantitative trait loci effect on 13 different genes in 15 tissues (including skin and whole blood). More specifically, it is associated with lower HLA-C gene expression in sun-exposed skin and higher HLA-DQA2 expression in whole blood (P < 10–6). Variation in NOTCH4 has been strongly associated with alopecia, also an autoimmune skin disease (Petukhova et al., 2010Petukhova L. Duvic M. Hordinsky M. Norris D. Price V. Shimomura Y. et al.Genome-wide association study in alopecia areata implicates both innate and adaptive immunity.Nature. 2010; 466: 113-117Crossref PubMed Scopus (537) Google Scholar). Another non-HLA suggestive association was with rs6968049, located at the long noncoding RNA gene STEAP2-AS1 on chromosome 7. Detailed annotation of all variants with the most significant P-values is given in Supplementary Tables S1 and S2. Results for all 204,967 variants are in Supplementary Table S3.Table 1Associations of Genetic Variants with Three Independent Pemphigus CohortsChrAnnotationSNPAlleleDiscovery - EPF234 Patients and 5,658 ControlsReplication - EPF95 Patients and 1,000 ControlsValidation – PV153 Patients and 1,000 ControlsORL95U95P-ValueORL95U95P-ValueORL95U95P-Value6HLA class II1Intergenic between HLA-DQB1 and HLA-DQA2.rs9275523A5.263.637.621.82E−186.914.7810.001.37E−264.203.285.391.14E−296HLA class II2Intergenic between HLA-DRB1 and HLA-DQA1.rs9271170T3.222.294.521.42E−116HLA class II1Intergenic between HLA-DQB1 and HLA-DQA2.rs10947332A4.062.686.163.74E−113.352.394.693.27E−110.360.220.612.03E−056HLA class II2Intergenic between HLA-DRB1 and HLA-DQA1.rs17533090T3.462.345.125.58E−102.421.693.464.32E−061.621.222.160.0026HLA class II3Intergenic between HLA-DRA and HLA-DRB5.rs1964995G2.721.973.759.18E−106NOTCH44Intronic variant.rs2854050T4.212.417.354.08E−072.021.163.510.01830.920.541.570.8945Intergenic5Between the genes ATP6AP1L and LOC105379050.rs1032757T0.310.190.525.83E−067STEAP2-AS16Long non-coding RNA gene.rs6968049A2.021.482.771.13E−051.651.232.230.00100.980.771.260.89921DSCAM 4Intronic variant.rs2837819G0.400.270.601.19E−050.660.421.030.0720.700.501.000.0522Intergenic7Between the genes TMEFF2 and PCGEM1.rs2357149T2.091.492.932.07E−051.471.032.100.0411.020.771.360.8862Intergenic7Between the genes TMEFF2 and PCGEM1.rs1601324G1.981.442.732.57E−056IL20RA4Intronic variant.rs1744061T0.470.330.673.28E−0511SERGEF4Intronic variant.rs1548528A1.811.362.415.24E−051.180.851.640.3160.820.641.040.11018Intergenic8Between the genes DCC and MBD2.rs12962837A2.791.694.616.05E−051.080.701.660.7360.980.711.350.935Abbreviations: Chr, chromosome, EPF, endemic pemphigus foliaceus; L95, lower endpoint of the 95% confidence interval; PV, pemphigus vulgaris; U95, upper endpoint of the 95% confidence interval.1 Intergenic between HLA-DQB1 and HLA-DQA2.2 Intergenic between HLA-DRB1 and HLA-DQA1.3 Intergenic between HLA-DRA and HLA-DRB5.4 Intronic variant.5 Between the genes ATP6AP1L and LOC105379050.6 Long non-coding RNA gene.7 Between the genes TMEFF2 and PCGEM1.8 Between the genes DCC and MBD2. Open table in a new tab Abbreviations: Chr, chromosome, EPF, endemic pemphigus foliaceus; L95, lower endpoint of the 95% confidence interval; PV, pemphigus vulgaris; U95, upper endpoint of the 95% confidence interval. For replication and validation, we analyzed an independent cohort of 95 EPF and 153 pemphigus vulgaris (PV) patients, recruited at the University Hospital of the Ribeirão Preto Medical School of the University of São Paulo, Brazil. These cohorts were genotyped with the iPLEX MassARRAY System (Agena Bioscience, Inc., San Diego, CA) and compared with an independent subset of 1,000 controls from EPIGEN-Brasil. We replicated six of nine associations in the independent EPF cohort (Table 1 and Supplementary Figure S2). These results point to the importance of closely looking at suggestive associations because we replicated the associations of NOTCH4 and STEAP2-AS1 variants in the independent cohort. Considering the similarities of pemphigus foliaceus and PV regarding their pathogenesis (Hammers and Stanley, 2020Hammers C.M. Stanley J.R. Recent advances in understanding pemphigus and bullous pemphigoid.J Invest Dermatol. 2020; 140: 733-741Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar; Vodo et al., 2018Vodo D. Sarig O. Sprecher E. The genetics of pemphigus vulgaris.Front Med (Lausanne). 2018; 5: 226Crossref PubMed Scopus (39) Google Scholar), we checked if variants associated with EPF were also associated with PV (Table 1). We found three variants associated with EPF and PV. The variant rs10947332∗A was associated with an increased risk of EPF and decreased risk of PV. We provide further insights into this result by analyzing all publicly available HLA and SNP genotyping data of 2,214 individuals of the 1000 Genomes Project populations (Abi-Rached et al., 2018Abi-Rached L. Gouret P. Yeh J.H. Di Cristofaro J. Pontarotti P. Picard C. et al.Immune diversity sheds light on missing variation in worldwide genetic diversity panels.PLoS One. 2018; 13e0206512Crossref PubMed Scopus (31) Google Scholar). We found that DRB1∗01-DQB1∗05:01 is the only HLA haplotype in strong linkage disequilibrium with rs10947332∗A (D' = 0.96), whereas rs10947332∗G occurs in all other DRB1-DQB1 haplotypes. Haplotype DRB1∗01-DQB1∗05:01 is strongly associated with increased risk of EPF and decreased risk of PV (Brochado et al., 2016Brochado M.J. Nascimento D.F. Campos W. Deghaide N.H. Donadi E.A. Roselino A.M. Differential HLA class I and class II associations in pemphigus foliaceus and pemphigus vulgaris patients from a prevalent Southeastern Brazilian region.J Autoimmun. 2016; 72: 19-24Crossref PubMed Scopus (40) Google Scholar; Gil et al., 2017Gil J.M. Weber R. Rosales C.B. Rodrigues H. Sennes L.U. Kalil J. et al.Study of the association between human leukocyte antigens (HLA) and pemphigus vulgaris in Brazilian patients.Int J Dermatol. 2017; 56: 557-562Crossref PubMed Scopus (15) Google Scholar; Petzl-Erler, 2020Petzl-Erler M.L. Beyond the HLA polymorphism: a complex pattern of genetic susceptibility to pemphigus.Genet Mol Biol. 2020; 43e20190369Crossref PubMed Scopus (10) Google Scholar), and the linkage disequilibrium pattern explains the differential association of rs10947332 with these two diseases. None of the non-HLA variants associated with EPF were associated with PV, suggesting that their effect is restricted to EPF. In summary, our results confirmed that HLA class II variants are the strongest genetic factors involved in EPF etiology. We also showed that EPF and PV share some intergenic susceptibility variants in the HLA class II region. The associations with intergenic variants may contribute to identifying causal variants; understanding the mechanism underpinning the associations between HLA class II genotypes and EPF; and exploring the differences and similarities of EPF, sporadic pemphigus foliaceus, and PV in the future. In addition, we identified at least two associations of EPF with possibly regulatory variants in non-HLA genes. Hopefully, this study will add a step ahead in comprehending this complex and unique disease that affects thousands of individuals worldwide. All data are available either in Supplementary Material or on request. Data from EPIGEN-Brasil are at https://www.ebi.ac.uk/ega/datasets/EGAD00010000787. Danillo G. Augusto: http://orcid.org/0000-0001-5665-8547 Rodrigo C. de Almeida: http://orcid.org/0000-0001-7966-056X Ticiana D. J. Farias: http://orcid.org/0000-0002-3606-2428 Wagner C. S. Magalhães: http://orcid.org/0000-0003-3575-8068 Danielle Malheiros: http://orcid.org/0000-0002-2528-9707 Maria Fernanda Lima-Costa: http://orcid.org/0000-0002-3474-2980 Maurício L. Barreto: http://orcid.org/0000-0002-0215-4930 Bernardo L. Horta: http://orcid.org/0000-0001-9843-412X Vinod Kumar: http://orcid.org/0000-0002-2775-6049 Michael Wittig: http://orcid.org/0000-0003-4240-4356 Andre Franke: http://orcid.org/0000-0003-1530-5811 Hauke Busch: http://orcid.org/0000-0003-4763-4521 Enno Schmidt: http://orcid.org/0000-0002-1206-8913 Ana Maria Roselino: http://orcid.org/0000-0002-2709-1825 Eduardo Tarazona-Santos: http://orcid.org/0000-0003-3508-3160 Angelica B. W. Boldt: http://orcid.org/0000-0002-0902-9622 Maria Luiza Petzl-Erler: http://orcid.org/0000-0002-0345-5276 The authors state no conflict of interest. We warmly thank all the individuals who voluntarily enrolled in this study. Special thanks to Hospital Adventista do Pênfigo for kindly opening their doors for our lab members and for treating patients with pemphigus with so much care and respect. We thank the staff of the Laboratório de Genética Molecular Humana, Universidade Federal do Paraná for their support. We also thank the statistician Chao Zhao for revision and advice with metanalysis. This work was supported by grants from the following funding agencies: Fundação Araucária (PRONEX FA/CNPq protocolo 50530 convenio 116/2018 and 9894.413.43926.1904/2013), Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (470483/2014-8), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES (400648/2014-8 and finance Code 001). DGA acknowledges funding from Young Talent Attraction, Science without Borders Program (CAPES 88881.067970/2014-01). ABWB acknowledges the Conselho Nacional de Desenvolvimento Científico e Tecnológico fellowship (314288/2018-0). TDJF received a scholarship under the International Sandwich Doctorate Program (Capes - PDSE 88881.132221/2016-01) and housing assistance from the German Academic Exchange Service, Deutscher Akademischer Austauschdienst - DAAD. The EPIGEN-Brasil project was funded by Departamento de Ciência, Tecnologia e Inovacão of the Brazilian Ministry of Health. HB, AF, and ES acknowledge funding by the Deutsche Forschungsgemeinschaft, German Research Foundation under Germany's Excellence Strategy – EXC 22167-390884018. Conceptualization: DGA, RCDA, DM, ABWB, MLPE; Data Curation: RCDA, WCSM; Formal Analysis: RCDA, TDJF, WCSM, DGA; Investigation: DGA; Methodology: VK; Resources: DGA, MFLC, MLB, BLH, MW, AF, HB, ES, AMR, ETS, ABWB, MLPE; Supervision: MLPE; Writing - Original Draft Preparation: DGA, MLPE, TDJF; Writing - Review and Editing: DGA, RCDA, TDJF, WCSM, DM, MFLC, MLB, BLH, VK, MW, AF, HB, ES, AMR, ETS, ABWB, MLPE Download .pdf (.76 MB) Help with pdf files Supplementary Figures Download .xlsx (12.88 MB) Help with xlsx files Supplementary Material