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Programa de Pós-Graduação em Genética, Universidade Federal do Paraná, Curitiba, BrazilDepartment of Biomedical Data Sciences, Section Molecular Epidemiology, Leiden University Medical Center, Leiden, The Netherlands
Núcleo de Ensino e Pesquisa, Instituto Mário Penna, Belo Horizonte, BrazilDepartamento de Genética, Ecologia e Evolução, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The NetherlandsDepartment of Internal Medicine, Radboud University Medical Center, Nijmegen, The NetherlandsRadboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, The NetherlandsNitte (Deemed to be University), Division of Infectious Diseases, Nitte University Centre for Science Education and Research (NUCSER), Paneer Campus, Deralakatte, Mangaluru, India
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 (
), 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 (
). 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 (
). 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 (
). 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 (
). 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 Cohorts
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 (
), 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 (
). 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 (
), 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.
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.