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

Open ArchivePublished:May 12, 2021DOI:https://doi.org/10.1016/j.jid.2021.04.017

      Abbreviations:

      EPF (endemic pemphigus foliaceus), PV (pemphigus vulgaris)
      To the Editor
      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 E.
      • Kasperkiewicz M.
      • Joly P.
      Pemphigus.
      ), 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 V.
      • Rivitti E.A.
      • Diaz L.A.
      Cooperative Group on Fogo Selvagem Research. Update on fogo selvagem, an endemic form of pemphigus foliaceus.
      ;
      • Qian 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.
      ).
      We previously described strong associations with alleles of HLA class II genes (
      • Pavoni 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.
      ;
      • Petzl-Erler M.L.
      • Santamaria J.
      Are HLA class II genes controlling susceptibility and resistance to Brazilian pemphigus foliaceus (fogo selvagem)?.
      ). Furthermore, through candidate gene association studies, we revealed polymorphisms of immune-related genes altering susceptibility to EPF (
      • Petzl-Erler M.L.
      Beyond the HLA polymorphism: a complex pattern of genetic susceptibility to pemphigus.
      ). 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 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.
      ;
      • Magalhães 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.
      ). 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 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.
      ;
      • Kehdy 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.
      ). 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 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.
      ). 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.
      Figure thumbnail gr1
      Figure 1GWAS identifies variants associated with endemic pemphigus foliaceus. (a) Manhattan plot of the GWAS on endemic pemphigus foliaceus. The genome-wide significance level is set at 5 × 10–8, and a threshold of 5 × 10–5 was set to indicate variants suggestively associated. Each dot represents an SNP. Y-axis shows the negative logarithm of the association P-value, and numbers in the X-axis indicate chromosomes. (b) Detailed visualization of variants within the HLA region. Each dot represents an SNP, color-coded according to linkage disequilibrium patterns with other variants. Y-axis shows the negative logarithm of the association P-value for the genes that are shown in the X-axis. Adapted from LocusZoom online tool (http://http://locuszoom.org/). chr, chromosome.
      Table 1Associations of Genetic Variants with Three Independent Pemphigus Cohorts


      Chr
      AnnotationSNPAlleleDiscovery - EPF

      234 Patients and 5,658 Controls
      Replication - EPF

      95 Patients and 1,000 Controls
      Validation – PV

      153 Patients and 1,000 Controls
      ORL95U95P-ValueORL95U95P-ValueORL95U95P-Value
      6HLA class II
      Intergenic between HLA-DQB1 and HLA-DQA2.
      rs9275523A5.263.637.621.82E−186.914.7810.001.37E−264.203.285.391.14E−29
      6HLA class II
      Intergenic between HLA-DRB1 and HLA-DQA1.
      rs9271170T3.222.294.521.42E−11
      6HLA class II
      Intergenic between HLA-DQB1 and HLA-DQA2.
      rs10947332A4.062.686.163.74E−113.352.394.693.27E−110.360.220.612.03E−05
      6HLA class II
      Intergenic between HLA-DRB1 and HLA-DQA1.
      rs17533090T3.462.345.125.58E−102.421.693.464.32E−061.621.222.160.002
      6HLA class II
      Intergenic between HLA-DRA and HLA-DRB5.
      rs1964995G2.721.973.759.18E−10
      6NOTCH4
      Intronic variant.
      rs2854050T4.212.417.354.08E−072.021.163.510.01830.920.541.570.894
      5Intergenic
      Between the genes ATP6AP1L and LOC105379050.
      rs1032757T0.310.190.525.83E−06
      7STEAP2-AS1
      Long non-coding RNA gene.
      rs6968049A2.021.482.771.13E−051.651.232.230.00100.980.771.260.899
      21DSCAM
      Intronic variant.
      rs2837819G0.400.270.601.19E−050.660.421.030.0720.700.501.000.052
      2Intergenic
      Between the genes TMEFF2 and PCGEM1.
      rs2357149T2.091.492.932.07E−051.471.032.100.0411.020.771.360.886
      2Intergenic
      Between the genes TMEFF2 and PCGEM1.
      rs1601324G1.981.442.732.57E−05
      6IL20RA
      Intronic variant.
      rs1744061T0.470.330.673.28E−05
      11SERGEF
      Intronic variant.
      rs1548528A1.811.362.415.24E−051.180.851.640.3160.820.641.040.110
      18Intergenic
      Between the genes DCC and MBD2.
      rs12962837A2.791.694.616.05E−051.080.701.660.7360.980.711.350.935
      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.
      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.
      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 C.M.
      • Stanley J.R.
      Recent advances in understanding pemphigus and bullous pemphigoid.
      ;
      • Vodo D.
      • Sarig O.
      • Sprecher E.
      The genetics of pemphigus vulgaris.
      ), 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 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.
      ). 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 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.
      ;
      • Gil 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.
      ;
      • Petzl-Erler M.L.
      Beyond the HLA polymorphism: a complex pattern of genetic susceptibility to pemphigus.
      ), 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.

      Data availability statement

      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.

      ORCIDs

      Wagner C. S. Magalhães: http://orcid.org/0000-0003-3575-8068
      Maria Fernanda Lima-Costa: http://orcid.org/0000-0002-3474-2980
      Eduardo Tarazona-Santos: http://orcid.org/0000-0003-3508-3160
      Maria Luiza Petzl-Erler: http://orcid.org/0000-0002-0345-5276

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      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.

      Author Contributions

      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

      Supplementary Material

      References

        • Abi-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; 13e0206512
        • Anderson 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: 1564-1573
        • Aoki 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: 18-26
        • Brochado 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: 19-24
        • Gil 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: 557-562
        • Hammers C.M.
        • Stanley J.R.
        Recent advances in understanding pemphigus and bullous pemphigoid.
        J Invest Dermatol. 2020; 140: 733-741
        • Kehdy 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: 8696-8701
        • Magalhães 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: 1090-1095
        • Pavoni 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: 110-116
        • Petukhova 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: 113-117
        • Petzl-Erler M.L.
        Beyond the HLA polymorphism: a complex pattern of genetic susceptibility to pemphigus.
        Genet Mol Biol. 2020; 43e20190369
        • Petzl-Erler M.L.
        • Santamaria J.
        Are HLA class II genes controlling susceptibility and resistance to Brazilian pemphigus foliaceus (fogo selvagem)?.
        Tissue Antigens. 1989; 33: 408-414
        • Qian 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: 923-930
        • Schmidt E.
        • Kasperkiewicz M.
        • Joly P.
        Pemphigus.
        Lancet. 2019; 394: 882-894
        • Vodo D.
        • Sarig O.
        • Sprecher E.
        The genetics of pemphigus vulgaris.
        Front Med (Lausanne). 2018; 5: 226