doi: 10.1111/1346-8138.12321
Journal of Dermatology 2014; 41: 213–220
REVIEW ARTICLE
Genome-wide association studies of atopic dermatitis Mayumi TAMARI, Tomomitsu HIROTA Laboratory for Respiratory and Allergic Diseases, Center for Integrative Medical Sciences, The Institute of Physical and Chemical Research (RIKEN), Kanagawa, Japan
ABSTRACT Atopic dermatitis is a common inflammatory disease caused by a combination of genetic and environmental factors. Genome-wide association study (GWAS) is a comprehensive and unbiased approach to identify the genetic components of human diseases and to discover the cellular pathways underlying them. GWAS and recent immunochip analysis of atopic dermatitis have identified a total of 19 associated loci with a genome-wide level of significance (P < 5 3 10 8). The candidate genes identified by GWAS suggest a role for epidermal barrier functions, innate-adaptive immunity, interleukin-1 family signaling, regulatory T cells, the vitamin D pathway and the nerve growth factor pathway in the pathogenesis of atopic dermatitis. Combinations of these genetic factors may influence a wide range of phenotypes of atopic dermatitis among individuals. Although a more complete collection of associated genes and pathways is needed, genetic components revealed by GWAS provide valuable insights into the pathophysiology of atopic dermatitis.
Key words:
atopic dermatitis, genome-wide association study, immunochip analysis, polymorphisms.
INTRODUCTION Atopic dermatitis is a complex disease caused by a combination of genetic and environmental factors.1,2 Recent advances in genomic medicine have improved our understanding of the human genome’s contribution to health and disease. Genomewide association study (GWAS) is a powerful method for identifying disease susceptibility genes for common human diseases and has begun to reveal underlying cellular pathways and point to new therapeutic approaches.3,4 This review focuses on GWAS and immunochip analysis of atopic dermatitis. Genes located within and around the susceptible loci and their possible roles in pathogenesis of atopic dermatitis are also discussed.
GENOMIC VARIATION AND GWAS Human genetic variants are referred to as either common or rare. Common variants, also called polymorphisms, are defined as variants with a minor allele frequency of at least 1% in the population.5 Single nucleotide polymorphisms (SNP) are the most prevalent class of genetic variations among individuals.5 GWAS comprehensively surveys associations between common SNP in common complex disorders.6 The international HapMap (haplotype map) project (http:// hapmap.ncbi.nlm.nih.gov/) focuses on common SNP with minor allele frequencies of more than 1% and defines these patterns across the entire genome.7–9 The haplotype structure of the human genome implies that a limited set of 1 000 000
SNP can capture approximately 90% of the genetic variation in the population.3 Pairwise linkage disequilibrium (LD) is calculated based on the genotyping data from the international HapMap project, and a set of markers in a haplotype are said to be in LD. Genotyping arrays have also been developed with reference to the LD, and can now assay up to 4.3 million variants. The data from the HapMap project and the development of dense genotyping chips have enabled us to conduct GWAS effectively on a large number of samples. However, if large numbers of SNP are examined simultaneously in a GWAS, the statistical significance threshold must be adjusted to reduce the chance of making a type 1 error (false positive). A genome-wide significance threshold level of 5 9 10 8 is generally used to adjust for 1–2 million independent tests.10 To date, GWAS have identified more than 1000 genetic loci associated with disease susceptibility and other disease-related phenotypes.11 Genotype imputation is a statistical approach deducing the allelic states at polymorphic sites not actually genotyped from the surrounding SNP data, based on LD and likelihood estimates.12 To conduct rigorous statistical and comprehensive GWAS meta-analyses for common disorders, a number of GWAS consortiums have been founded. However, GWAS are often performed using different genotyping platforms. Imputation is useful for merging distinct GWAS and enables powerful combined analyses of GWAS for the discovery of novel associations.12,13 Although recent GWAS have provided valuable findings about genetic factors in common human diseases, they
Correspondence: Mayumi Tamari, M.D., Ph.D., Laboratory for Respiratory and Allergic Diseases, Center for Integrative Medical Sciences, Institute of Physical and Chemical Research (RIKEN), 1-7-22 Suehiro, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. Email: [email protected] Received 19 September 2013; accepted 19 September 2013.
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explain little about heritability and the associated genetic variants have small effect sizes.14,15 Unmapped common and rare variants are considered a source of missing heritability.14,15 Actually, the chips currently used in GWAS contain relatively few rare SNP in the coding and promoter regions. The 1000 Genomes Project aims to find essentially all variants with frequencies of more than 1% across the genome and more than 0.1% in protein-coding regions and expands the catalog of variations for the next generation of GWAS.16 Using data from the 1000 Genomes Project and other available disease-specific re-sequencing data, the immunochip, a custom Illumina Infinium high-density array containing 196 524 variants, has been developed.17,18 The variants on the chip cover intervals with established GWAS.17,18 However, the immunochip does not cover the whole genome and is designed for use in white European populations. Thus, it would seem to be less informative for Asian populations.
GENETIC STUDIES OF ATOPIC DERMATITIS Systemic, well-powered, genome-wide surveys using GWAS and immunochip analysis have explored the relationship between SNP and susceptibility to atopic dermatitis.19–23 It is well established that common loss-of-function variants of filaggrin (FLG) are a major predisposing factor for atopic dermatitis.24,25 Because FLG mutations involved in atopic dermatitis are featured in another review article in this special issue, this review focuses on GWAS and immunochip analysis for atopic dermatitis. To date, a total of 19 genome-wide significant (P < 5 9 10 8) susceptibility loci have been identified through GWAS and immunochip analysis, and Table 1 summarizes the findings.
GWAS for atopic dermatitis The first GWAS of atopic dermatitis was reported in 2009. The GWAS analyzed 939 cases and 975 controls as well as 270 complete nuclear families with two affected siblings.19 A total of 342 303 markers were assessed, and a susceptibility region on chromosome 11q13 located 38 kb downstream of C11orf30 was identified.19 The peak association was observed 68 kb upstream of leucine rich repeat containing 32 (LRRC32). LRRC32 was previously reported to have specific expression in activated human naturally occurring regulatory T cells.26,27 The GWAS also confirmed the important role of FLG mutations in atopic dermatitis. In the study, another association reaching genome-wide significance was observed at rs6661961 in the epidermal differentiation complex (EDC) on chromosome 1q21. Rs6661961 is in high LD with the FLG mutations, and exclusion of FLG mutation carriers abolished the observed association. In 2011, a GWAS reported two new susceptibility loci, 5q22.1 and 20q13.33, for atopic dermatitis in the Chinese Han population.20 The GWAS was conducted using 1012 cases and 1362 controls followed by a replication study in an additional 3624 cases and 12 197 controls. The results were validated using 1806 cases and 3256 controls from Germany. The 5q22.1 region contains transmembrane protein 232 (TMEM232) and solute carrier family 25, member 46 (SLC25A46), and the thymic stromal lymphopoietin (TSLP) gene is located adjacent (~300 kb downstream) to the associated region. TSLP promotes T-helper (Th)2 cell responses associated with immunity to some helminth parasites and the pathogenesis of a number of inflammatory diseases such as atopic dermatitis.28,29 A variety of stimuli, including viral infections, inflammatory cytokines, protease allergens, bacteria and bacterial products, induce TSLP from barrier epithelial cells.28,29 Another study has
Table 1. A total of 19 genome-wide significant susceptibility loci identified by genome-wide association and immunochip studies for atopic dermatitis Population
Analysis
Location
Reported genes
European Chinese
GWAS
11q13.5 1p21.3 5q11.1 20q13.3 11q13 19p13.2 5q31 2q12 3p21.33 3q13.2 6p21.3 7p22 10q21.2 11p15.4 20q13 4q27 11p13 16p13.13 17q21.32
C11ORF30/LRRC32 (GARP) FLG TMEM232/SLC25A46 TNFRSF6B/ZGPAT OVOL1 ACTL9 KIF3A/IL4/IL13 IL1RL1/IL18R1/IL18RAP GLB1 CCDC80 The MHC region CARD11 ZNF365/EGR2 OR10A3/NLRP10 CYP24A1/PFDN4 IL2/IL21 PRR5L CLEC16A/DEXI ZNF652
European Meta
GWAS
Japanese
GWAS
European
Immunochip
Adjacent genes
References 19 20
TSLP 21
22 CCR4
23 TRAF6, RAG1, RAG2 SOCS1 NGFR
GWAS, genome-wide association study.
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reported that genetic variants in TSLP are associated with atopic dermatitis, and the association is stronger in patients with atopic dermatitis who have a history of eczema herpeticum.30 The 20q13.33 region contains the tumor necrosis factor receptor superfamily, member 6b (TNFRSF6B) gene that encodes decoy receptor 3 (DcR3). Interestingly, DcR3 binds to LIGHT (TNFSF14), which has been shown to bind a herpesvirus entry mediator (HVEM), and LIGHT is a target for asthmatic airway remodeling.31,32 Herpes simplex viruses (HSV-1 and -2) attach to the cell surface via this herpesvirus entry mediator.31 It has been known that eczema herpeticum, a disseminated viral infection after HSV infection, is a serious complication of atopic dermatitis.33 Although further investigation is required, genetic variants in these loci may influence the immune response to HSV. Large sample sizes are required to ensure sufficient statistical power, and collaborative studies on samples from a large number of cohorts have provided new insights about the genetic bases of common diseases. In 2011, a meta-analysis of GWAS for atopic dermatitis of 5606 cases and 20 565 controls and a validation study in an additional 5419 cases and 19 833 controls were conducted.21 The studies identified a total of three novel risk loci for atopic dermatitis, 11q13.1 (OVOL1), 19p13.2 (ACTL9) and 5q31.1 (KIF3A/IL4/IL13).21 The 11q13.1 locus contains OVO homolog-like 1 (OVOL1), which functions as a c-Myc repressor in superbasal cells and is required for proliferation exit of committed epidermal progenitor cells.34 OVOL1 knockout mice show keratinocyte hyperproliferation, hair shaft abnormalities, kidney cysts and defective spermatogenesis.34,35 The most significant association in the 19p13.2 region was observed between the a disintegrin and metalloproteinase with thrombospondin motifs 10 (ADAMTS10) and actin-like 9 (ACTL9) genes. ADAMTS proteins are secreted zinc-dependent metalloproteinases and play a role in connective tissue remodeling and extracellular matrix turnover.36,37 Actin proteins are important for the maintenance of epithelial morphology and cell migration.38,39 The 5q31.1 region contains a clustered family of Th2 cytokine genes, including IL13 and IL4. The adaptive immune response in atopic dermatitis is associated with increased expression of the Th2 cytokines, interleukin (IL)-4 and IL-13.40 Recent studies have shown a significantly greater number of IL4, IL5 and IL13 mRNA-expressing cells in acute skin lesions and an increased number of Th2 cells expressing IL4 and IL13 mRNA in unaffected skin of atopic dermatitis patients.41,42 This GWAS suggests that atopic dermatitis is caused by both epidermal barrier dysfunctions and immunological features.
GWAS for atopic dermatitis in the Japanese population We conducted a GWAS in the Japanese population with 1472 cases and 7971 controls followed by a replication study in an additional 1856 cases and 7021 controls.22 We assessed a total of 606 164 SNP loci and finally found a total of eight novel susceptibility loci for atopic dermatitis.22 Chromosome 2q12 contains the receptors of the IL-1 family cytokines IL1RL1, IL18R1 and IL18RAP. Many of the IL-1 fam-
© 2014 Japanese Dermatological Association
Chromosome 2q12 Ligands IL33 IL18 IL1F5, ILF6, IL1F8, IL1F9 IL1A, IL1B, IL1RA LD block IL1R2
IL1RL1(ST2)
IL1R1 IL1RL2
IL18R1
IL18RAP LD block
IL-1 receptor cluster ~0.5 Mb
Figure 1. Susceptibility locus on chromosome 2q12 and a cytokine receptor gene cluster. Regional plots of association results in the Japanese population within the 2q12 region.22 Green box shows the susceptibility linkage disequilibrium block (LD) region. The cytokine receptor gene cluster spans approximately 500 kb. Ligands of each receptor are shown in colored circles. ily receptors are clustered in a small region (~0.5 Mb; Fig. 1), and IL1RL1 (also known as ST2) is a component of the IL-33 receptor.43,44 IL-1 family members are abundantly expressed in the skin, and the IL-33/IL-1RL1 axis triggers the release of several pro-inflammatory mediators and induces systemic Th2type inflammation.45,46 IL-33 and its receptors, ST2 and IL1RAcP, are increased in lesional skin of patients with atopic dermatitis, and topical tacrolimus treatment suppresses the increased expression of IL-33 and ST2 caused by irritants, allergens and staphylococcal enterotoxin B.47 Interestingly, expression levels of IL-1a and IL-1b in the stratum corneum are increased in patients with atopic dermatitis with FLG mutations, and these levels are inversely correlated with natural moisturizing factor.48 Associated variants in the loci may influence the pathophysiology of atopic dermatitis through dysfunction of the IL-1, IL-18 and IL-33 signaling pathways. In our study, the most significant association was at rs176095 in the major histocompatibility complex (MHC) class III region, and conditional logistic regression analysis showed two independent association signals in the MHC class I and class III regions. The MHC is associated with a number of autoimmune diseases, and the MHC class I and III regions are the shared GWAS autoimmune loci.49,50 In recent GWAS, the MHC class I region was found to be associated with psoriasis and vitiligo, and the MHC class III region with vitiligo.51–58 Furthermore, immunoglobulin (Ig)E antibodies against keratinocytes and endothelial cells are observed in serum specimens from subjects with severe atopic dermatitis.59 The genetic components in the MHC region may influence the development of autoimmune conditions in chronic inflammation in patients with atopic dermatitis. Chromosomal region 11p15.4 contains nucleotide-binding domain, leucine-rich repeat and pyrin domain containing protein 10 (NLRP10), which belongs to the NALP family but lacks the leucine-rich repeat region.60 A recent study has shown that NALP10 has an essential role to initiate adaptive immunity by dendritic cells. Nlrp10 / mice show a profound defect in
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helper T-cell-driven immune responses to diverse adjuvants and a dendritic cell intrinsic defect in emigration from inflamed tissues.61 This suggests that NLRP10 plays a critical role in diverse types of innate immune stimulation and the immune function of mature dendritic cells.61 The 3p21.33 region is located adjacent to the chemokine (C-C motif) receptor 4 (CCR4) gene, which encodes a Th2associated chemokine receptor for chemokine (C-C motif) ligand 22 (CCL22) and CCL17 (thymus and activation-regulated chemokine [TARC]).62 A keratinocyte-derived TSLP induces dendritic cells to produce TARC, and CCR4 mediates skin-specific recruitment of T cells during inflammation.63 A recent study has shown that the TARC in stratum corneum from patients with atopic dermatitis is correlated with diverse clinical phenotypes: the severity of local skin lesions, the severity scoring of atopic dermatitis (SCORAD) index, serum TARC level, serum IgE level, serum lactate dehydrogenase level, IL-4 producing T-cell ratio and blood eosinophil count.64 Furthermore, CCR4 is a skin-homing receptor of Th22 cells,65 and significant intensification of Th22 and Th2 cytokines is observed in acute and chronic lesions of atopic dermatitis.66 Associated SNP may also play a role in the Th22 immune response in atopic dermatitis. The 3q13.2 locus contains CCDC80, which is involved in induction of C/EBPa and peroxisome proliferator-activated receptor (PPAR).67 C/EBPa and C/EBPb are co-expressed in basal keratinocytes and upregulated while keratinocytes exit the basal layer and undergo terminal differentiation.68 PPARc acts as a negative regulator in immune cells, and a PPARc agonist markedly suppresses both expression of TSLP in the skin and maturation and migration of dendritic cells in a murine model of atopic dermatitis.69 The 7p22 region contains caspase recruitment domain-containing protein 11 (CARD11), which encodes CARD-containing MAGUK protein 1 (CARMA1). CARMA1 plays an essential role in T-cell differentiation and has a critical role in the regulation of JunB and GATA3 transcription factors and subsequent production of Th2 cell-specific cytokines.70 Furthermore, mice homozygous for the Carma-1 mutation show gradual development of atopic dermatitis with hyper-IgE.71 Chromosomal region 10q21.2 contains three genes and, interestingly, contains early growth response protein 2 (EGR2), a key molecule for anergy induction that activates the expression of genes involved in the negative regulation of T-cell proliferation and inflammation.72 A recent study has shown that CD4+CD25–LAG3+ regulatory T cells (Treg) characteristically express Egr-2.73 Interestingly, the presence of environmental microbiota influences the number of CD4+CD25–LAG3+ Treg.73 The 20q13 region includes cytochrome p450, family 24, subfamily A, polypeptide 1 (CYP24A1), which initiates the degradation of 1,5-dihydroxyvitamin D3, the active form of vitamin D3.74 Vitamin D is a modulator of innate and adaptive immune system functions and plays a role in epithelial antimicrobial defense of the skin and intestine.75 An association between vitamin D deficiency and the severity of atopic dermatitis has been reported.76
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Immunochip analysis of atopic dermatitis Rare large-effect mutations have been recognized as causes of many different common medical conditions.77 The immunochip was developed to conduct deep replication of major autoimmune and inflammatory diseases, and fine mapping of established loci identified by GWAS.17,18 A recent immunochip analysis for atopic dermatitis revealed four new susceptibility loci: 4q27 (IL-2/IL21), 11p13 (PRR5L), 16p13.13 (CLEC16A/ DEXI) and 17q21.32 (ZNF652).23 A replication study using a Japanese population confirmed the associations at the 11p13, 16p13.13 and 17q21.32 loci.23 Because the top signal in European populations is not always replicated in Asian populations, further fine-mapping analysis of the locus is necessary in the Japanese population. The 4q27 region contains the IL2 and IL21 genes. IL-2 has pleiotropic immunoregulatory functions and controls the proliferation and survival of regulatory T cells.78 Tacrolimus (FK506) is a well-known medication for atopic dermatitis and blocks IL-2 gene activation in T cells.79 Interestingly, the TNF receptor associated factors (TRAF6) (11p13), recombination-activating gene 1 (RAG1) (11p13), RAG2 (11p13), suppressor of cytokine signaling 1 (SOCS1) (16p13.13) and nerve growth factor receptor (NGFR) (17q21.32) genes are located adjacent to the associated regions.
GWAS for total IgE levels, allergic sensitization and eosinophil numbers Elevated total IgE levels are considered markers of helminth exposure and allergic inflammation,80,81 and there is a strong genetic contribution to the variability of the total IgE level.82,83 GWAS for total IgE levels have been conducted and identified several susceptibility loci at genome-wide significance levels.84,85 A GWAS of total serum IgE levels found two susceptibility loci, 1q23 (FCER1A) and 5q31 (RAD50) in 1530 individuals from the population-based study following replication studies in four independent population-based study samples (n = 9769).84 The study showed that a functional SNP in the region significantly influenced the cell surface expression of Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide (FCER1A) on basophils and the regulatory mechanism of FCER1A expression via GATA2.84 Another GWAS in the Framingham Heart Study (FHS) reported a total of three loci containing 1q23 (FCER1A), 12q13 (STAT6) and 5q31 (IL13) with genome-wide significance.85 Furthermore, a meta-analysis combining the GWAS of the FHS and replication cohorts identified additional loci, 6p21.3 (HLA-G, HLA-DQA2 and HLA-A) and 1q21 (Duffy blood group, atypical chemokine receptor [DARC]), although the DARC association seems not to be independent of SNP in the nearby FCER1A gene.85 A GWAS of diverse populations including African American and Latino subjects validated findings from previous GWAS (1q23, 5q31, 6p21.3 and 12q13) and found a variant near HLA-DQB1 as a predictor of the total serum IgE levels in multiple race-ethnic groups, but not at the level of genome-wide significance (Pcombined = 2.45 9 10 7).86 Allergen-specific IgE plays a crucial role in the pathogenesis of allergic diseases by binding allergens and initiating immunological responses.87 A meta-analysis of GWAS of allergic
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GWAS of atopic dermatitis
sensitization was conducted for 5789 cases and 10 056 controls, and the top SNP at each of 26 loci was validated using 6114 cases and 9920 controls.87 In the study, the sensitization status was assessed for common food and inhalant allergens evaluated by either the level of allergen-specific IgE in blood or a positive skin prick test. Finally, a total of 10 loci influencing allergic sensitization were identified: 11q13.5 (C11orf30), 12q13.3 (STAT6), 5q22.1 (SLC25A46), 6p21.32 (HLA-DQB1), 2q12.1 (IL1RL1/IL18R1), 4p14 (TLR1/TLR6/TLR10), 3q28 (LPP), 8q24.21 (MYC/PVT1), 4q27 (IL2/ADAD1) and 6p21.33 (HLA-B/ MICA).89 2q12.1, 4q27, 5q22.1, 6q21.3 and 11q13.5 are overlapping susceptibility regions between atopic dermatitis and allergic sensitization (Table 2). Eosinophilic infiltration is a characteristic feature of allergic diseases and parasitic infestation.88 Tissue eosinophilia is a typical characteristic of atopic dermatitis, especially seen in lesions with pronounced epidermal hyperplasia.89 The number of eosinophils in the skin correlates with disease severity, and eosinophils are involved in acute, spongiotic dermatitis.89 A recent GWAS has shown that the 2q12 (IL1RL1), 2q13 (IKZF2), 3q21 (GATA2), 5q31 (IL5) and 12q24 (SH2B3) loci
Table 2. Genetic susceptibility loci overlapping atopic dermatitis and other allergy-related phenotypes identified by GWAS
Chromosome
Locus
2q12
IL1RL1/ IL18R1/ IL18RAP
4q27
ADAD1/IL2/ IL21
5q22
TMEM232/ SLC25A46
5q31.1
RAD50/IL13
6p21.3
The MHC region
11q13.5
C11orf30/ LRRC32 (GARP)
Other allergyrelated phenotypes Atopic dermatitis Asthma Number of eosinophils Allergic sensitization Atopic dermatitis Allergic sensitization Atopic dermatitis Allergic sensitization Atopic dermatitis Asthma Serum total IgE level Atopic dermatitis Asthma Adult asthma Serum total IgE level Allergic sensitization Atopic dermatitis Asthma Allergic rhinitis Allergic sensitization
References 22 25, 92 90 87 23 87 20 87 21 25 84,85 22 25, 93 94 85 87, 95 19 96 95 87
GWAS, genome-wide association study; Ig, immunoglobulin.
© 2014 Japanese Dermatological Association
are associated with the number of blood eosinophils.90 The study reported an association between an SNP at the IL1RL1 locus at 2q12 and asthma with a genome-wide significance level.90
Loci overlapping atopic dermatitis and other allergyrelated phenotypes The allergic march is the natural history of atopic manifestations such as atopic dermatitis, bronchial asthma and allergic rhinitis.91 Recent GWAS have revealed a number of susceptibility loci for bronchial asthma, allergic rhinitis, the number of eosinophils, allergic sensitization and total IgE levels, and there are significant genome-wide susceptibility regions overlapping atopic dermatitis and other allergy-related phenotypes (Table 2).92–96 These genetic components may play a role in the atopic march or atopic phenotypes. The 11q13.5 region, which contains LRRC32 (also called GARP), has been reported to be a susceptibility locus for atopic dermatitis, bronchial asthma, allergic rhinitis, the total serum IgE level and allergic sensitization.19,95,96 Foxp3+ Treg play a crucial role in maintaining tolerance to allergens and inhibiting allergies.97,98 LRRC32 mRNA is highly expressed in activated Foxp3+ regulatory T cells, and LRRC32 is essential for the surface expression of latent transforming growth factor-b on the cells.99 Thus, the findings of GWAS also help to enhance our understanding of the allergic march.
Possible roles of the candidate genes for atopic dermatitis Candidate genes for atopic dermatitis within and around the susceptibility loci suggest roles for epidermal barrier functions, innate and adaptive immunity, IL-1 signaling, the inflammatory response, regulatory T cells, the vitamin D pathway and NGF signaling in the pathogenesis of atopic dermatitis.19–23 Pruritus is a major symptom of atopic dermatitis and the itch–scratch cycle can damage the epidermal keratinocytes.1,40 Recent immunological studies have shown that epithelial-derived cytokines activate innate immune cells such as basophils, mast cells and innate lymphoid cells (ILC).100–103 Interestingly, the candidate genes for atopic dermatitis identified by the GWAS and immunochip are involved with those innate immune cells.19–23 Group 2 ILC (comprising ILC2) contribute to promotion of type 2 responses as potent sources of Th2-associated cytokines.104,105. ILC2-derived cytokines play physiological roles in wound healing and immunity to helminths, and are also involved in allergic diseases.105 Mast cells and basophils, which are also associated with type 2 immune responses, are activated through IL-18R, IL-33R and TSLPR, and these cells produce cytokines such as IL-2, IL-13 and TSLP.106 Various human infections, such as West Nile virus, Japanese encephalitis, Tsutsugamushi disease and malaria parasites, are transmitted via insect bites. Basophils and mast cells release histamine, an important mediator of itching, and itching is the essential hallmark of atopic dermatitis.107 Itching and irritation from skin contact are likely to be a protective response to insect stings. NGF has been suggested to play a role in the pathogenesis of itching in atopic dermatitis.107 Increased
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serum levels of NGF and substance P have been shown to correlate with the severity of atopic dermatitis,108 and increased NGF and its receptors, trkA and NGFR (p75NTR), in atopic dermatitis have been reported in an immunohistochemical study.109 The 17q21.32 region is located adjacent to the NGFR gene. A dysregulated NGF pathway and dysfunctions of mast cells and basophils through the genetic variations may be involved in coordinating the itch–scratch cycle in atopic dermatitis.
Multiple clinical phenotypes of atopic dermatitis It is known that there are multiple clinical phenotypes of atopic dermatitis.40 There are great differences in disease severity between individuals, and mild atopic dermatitis can be outgrown in later childhood. Furthermore, some patients have the atopic march, and some patients are prone to skin infections such as Staphylococcus aureus, eczema herpeticum and Malassezia.40 Approximately 80% of patients with atopic dermatitis have elevated serum IgE and/or immediate skin test reactivity to allergens.40 To date, a total of 19 susceptibility loci have been identified at a genome-wide level of significance (Table 1). Combinations of these genetic factors may influence diverse phenotypes of atopic dermatitis among individuals.
CONCLUSION Recent GWAS that comprehensively assess genes related to multifactorial diseases in a non-biased manner across the whole genome have improved our understanding of human atopic dermatitis. The susceptibility loci contain a number of genes encoding markers of immune cells and inflammation that are suppressed by topical corticosteroids and calcineurin inhibitors. There may be a novel therapeutic target gene within or around the associated regions. Further studies to elucidate the functional links between associated variants and phenotypic traits are important to improve our understanding of atopic dermatitis. The findings of GWAS are also helpful to highlight the genes and establish an experimental animal model that mimics human atopic dermatitis. Cross-disciplinary studies combining genetics, immunology, bioinformatics, epidemiology and clinical allergology are necessary for translation of the research into clinical practice.
CONFLICT OF INTEREST:
There is no conflict of interest
in this study.
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31 Montgomery RI, Warner MS, Lum BJ, Spear PG. Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/ NGF receptor family. Cell 1996; 87: 427–436. 32 Doherty TA, Soroosh P, Khorram N et al. The tumor necrosis factor family member LIGHT is a target for asthmatic airway remodeling. Nat Med 2011; 17: 596–603. 33 Beck LA, Boguniewicz M, Hata T et al. Phenotype of atopic dermatitis subjects with a history of eczema herpeticum. J Allergy Clin Immunol 2009; 124: 260–269. 34 Nair M, Teng A, Bilanchone V, Agrawal A, Li B, Dai X. Ovol1 regulates the growth arrest of embryonic epidermal progenitor cells and represses c-myc transcription. J Cell Biol 2006; 173: 253– 264. 35 Dai X, Schonbaum C, Degenstein L, Bai W, Mahowald A, Fuchs E. The ovo gene required for cuticle formation and oogenesis in flies is involved in hair formation and spermatogenesis in mice. Genes Dev 1998; 12: 3452–3463. 36 Apte SS. A disintegrin-like and metalloprotease (reprolysin-type) with thrombospondin type 1 motif (ADAMTS) superfamily: functions and mechanisms. J Biol Chem 2009; 284: 31493–31497. 37 Porter S, Clark IM, Kevorkian L, Edwards DR. The ADAMTS metalloproteinases. Biochem J 2005; 386: 15–27. 38 Pollard TD. The cytoskeleton, cellular motility and the reductionist agenda. Nature 2003; 422: 741–745. 39 Pollard TD, Borisy GG. Cellular motility driven by assembly and disassembly of actin filaments. Cell 2003; 112: 453–465. 40 Leung DY. New insights into atopic dermatitis: role of skin barrier and immune dysregulation. Allergol Int 2013; 62: 151–161. 41 Hamid Q, Boguniewicz M, Leung DY. Differential in situ cytokine gene expression in acute versus chronic atopic dermatitis. J Clin Invest 1994; 94: 870–876. 42 Hamid Q, Naseer T, Minshall EM, Song YL, Boguniewicz M, Leung DY. In vivo expression of IL-12 and IL-13 in atopic dermatitis. J Allergy Clin Immunol 1996; 98: 225–231. 43 Sims JE, Smith DE. The IL-1 family: regulators of immunity. Nat Rev Immunol 2010; 10: 89–102. 44 Liew FY, Pitman NI, McInnes IB. Disease-associated functions of IL-33: the new kid in the IL-1 family. Nat Rev Immunol 2010; 10: 103–110. 45 Johnston A, Xing X, Guzman AM et al. IL-1F5, -F6, -F8, and -F9: a novel IL-1 family signaling system that is active in psoriasis and promotes keratinocyte antimicrobial peptide expression. J Immunol 2011; 186: 2613–2622. 46 Schmitz J, Owyang A, Oldham E et al. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity 2005; 23: 479–490. 47 Savinko T, Matikainen S, Saarialho-Kere U et al. IL-33 and ST2 in atopic dermatitis: expression profiles and modulation by triggering factors. J Invest Dermatol 2012; 132: 1392–1400. 48 Kezic S, O’Regan GM, Lutter R et al. Filaggrin loss-of-function mutations are associated with enhanced expression of IL-1 cytokines in the stratum corneum of patients with atopic dermatitis and in a murine model of filaggrin deficiency. J Allergy Clin Immunol 2012; 129: 1031–1039. 49 Horton R, Wilming L, Rand V et al. Gene map of the extended human MHC. Nat Rev Genet 2004; 5: 889–899. 50 Zhernakova A, van Diemen CC, Wijmenga C. Detecting shared pathogenesis from the shared genetics of immune-related diseases. Nat Rev Genet 2009; 10: 43–55. 51 Capon F, Bijlmakers MJ, Wolf N et al. Identification of ZNF313/ RNF114 as a novel psoriasis susceptibility gene. Hum Mol Genet 2008; 17: 1938–1945. 52 Liu Y, Helms C, Liao W, Zaba LC et al. A genome-wide association study of psoriasis and psoriatic arthritis identifies new disease loci. PLoS Genet 2008; 4: e1000041. 53 Nair RP, Duffin KC, Helms C et al. Genome-wide scan reveals association of psoriasis with IL-23 and NF-kappaB pathways. Nat Genet 2009; 41: 199–204.
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54 Genetic Analysis of Psoriasis Consortium & the Wellcome Trust Case Control Consortium 2, Strange A, Capon F et al. A genomewide association study identifies new psoriasis susceptibility loci and an interaction between HLA-C and ERAP1. Nat Genet 2010; 42: 985–990. 55 Ellinghaus E, Ellinghaus D, Stuart PE et al. Genome-wide association study identifies a psoriasis susceptibility locus at TRAF3IP2. Nat Genet 2010; 42: 991–995. 56 Jin Y, Birlea SA, Fain PR et al. Variant of TYR and autoimmunity susceptibility loci in generalized vitiligo. N Engl J Med 2010; 362: 1686–1697. 57 Quan C, Ren YQ, Xiang LH et al. Genome-wide association study for vitiligo identifies susceptibility loci at 6q27 and the MHC. Nat Genet 2010; 42: 614–618. 58 Jin Y, Birlea SA, Fain PR et al. Genome-wide analysis identifies a quantitative trait locus in the MHC class II region associated with generalized vitiligo age of onset. J Invest Dermatol 2011; 131: 1308–1312. 59 Altrichter S, Kriehuber E, Moser J et al. Serum IgE autoantibodies target keratinocytes in patients with atopic dermatitis. J Invest Dermatol 2008; 128: 2232–2239. 60 Wang Y, Hasegawa M, Imamura R et al. PYNOD, a novel Apaf-1/ CED4-like protein is an inhibitor of ASC and caspase-1. Int Immunol 2004; 16: 777–786. 61 Eisenbarth SC, Williams A, Colegio OR et al. NLRP10 is a NOD-like receptor essential to initiate adaptive immunity by dendritic cells. Nature 2012; 484: 510–513. 62 Horikawa T, Nakayama T, Hikita I et al. IFN-gamma-inducible expression of thymus and activation-regulated chemokine/CCL17 and macrophage-derived chemokine/CCL22 in epidermal keratinocytes and their roles in atopic dermatitis. Int Immunol 2002; 14: 767–773. 63 Soumelis V, Reche PA, Kanzler H et al. Human epithelial cells trigger dendritic cell-mediated allergic inflammation by producing TSLP. Nat Immunol 2002; 3: 673–680. 64 Morita E, Takahashi H, Niihara H et al. Stratum corneum TARC level is a new indicator of lesional skin inflammation in atopic dermatitis. Allergy 2010; 65: 1166–1172. 65 Fujita H. The role of IL-22 and Th22 cells in human skin diseases. J Dermatol Sci 2013; 72: 3–8. rez-Farin ~as M et al. Progressive activa66 Gittler JK, Shemer A, Sua tion of T(H)2/T(H)22 cytokines and selective epidermal proteins characterizes acute and chronic atopic dermatitis. J Allergy Clin Immunol 2012; 130: 1344–1354. 67 Tremblay F, Revett T, Huard C et al. Bidirectional modulation of adipogenesis by the secreted protein Ccdc80/DRO1/URB. J Biol Chem 2009; 284: 8136–8147. 68 Lopez RG, Garcia-Silva S, Moore SJ et al. C/EBPalpha and beta couple interfollicular keratinocyte proliferation arrest to commitment and terminal differentiation. Nat Cell Biol 2009; 11: 1181– 1190. 69 Jung K, Tanaka A, Fujita H et al. Peroxisome proliferator-activated receptor c-mediated suppression of dendritic cell function prevents the onset of atopic dermatitis in NC/Tnd mice. J Allergy Clin Immunol 2011; 127: 420–429. 70 Blonska M, Joo D, Zweidler-McKay PA, Zhao Q, Lin X. CARMA1 controls Th2 cell-specific cytokine expression through regulating JunB and GATA3 transcription factors. J Immunol 2012; 188: 3160–3168. 71 Jun JE, Wilson LE, Vinuesa CG et al. Identifying the MAGUK protein Carma-1 as a central regulator of humoral immune responses and atopy by genome-wide mouse mutagenesis. Immunity 2003; 18: 751–762. 72 Safford M, Collins S, Lutz MA et al. Egr-2 and Egr-3 are negative regulators of T cell activation. Nat Immunol 2005; 6: 472– 480. 73 Okamura T, Fujio K, Shibuya M et al. CD4 + CD25-LAG3+ regulatory T cells controlled by the transcription factor Egr-2. Proc Natl Acad Sci USA 2009; 106: 13974–13979.
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74 Hart PH, Gorman S, Finlay-Jones JJ. Modulation of the immune system by UV radiation: more than just the effects of vitamin D? Nat Rev Immunol 2011; 11: 584–596. 75 Gallo RL, Hooper LV. Epithelial antimicrobial defence of the skin and intestine. Nat Rev Immunol 2012; 12: 503–516. 76 Peroni DG, Piacentini GL, Cametti E, Chinellato I, Boner AL. Correlation between serum 25-hydroxyvitamin D levels and severity of atopic dermatitis in children. Br J Dermatol 2011; 164: 1078–1082. 77 McClellan J, King MC. Genetic heterogeneity in human disease. Cell 2010; 141: 210–217. 78 Boyman O, Sprent J. The role of interleukin-2 during homeostasis and activation of the immune system. Nat Rev Immunol 2012; 12: 180–190. 79 Schreiber SL, Crabtree GR. The mechanism of action of cyclosporin A and FK506. Immunol Today 1992; 13: 136–142. 80 Cooper PJ, Ayre G, Martin C, Rizzo JA, Ponte EV, Cruz AA. Geohelminth infections: a review of the role of IgE and assessment of potential risks of anti-IgE treatment. Allergy 2008; 63: 409–417. 81 Gould HJ, Sutton BJ. IgE in allergy and asthma today. Nat Rev Immunol 2008; 8: 205–217. 82 Jacobsen HP, Herskind AM, Nielsen BW, Husby S. IgE in unselected like-sexed monozygotic and dizygotic twins at birth and at 6 to 9 years of age: high but dissimilar genetic influence on IgE levels. J Allergy Clin Immunol 2001; 107: 659–663. 83 Strachan DP, Wong HJ, Spector TD. Concordance and interrelationship of atopic diseases and markers of allergic sensitization among adult female twins. J Allergy Clin Immunol 2001; 108: 901– 907. 84 Weidinger S, Gieger C, Rodriguez E et al. Genome-wide scan on total serum IgE levels identifies FCER1A as novel susceptibility locus. PLoS Genet 2008; 4: e1000166. 85 Granada M, Wilk JB, Tuzova M et al. A genome-wide association study of plasma total IgE concentrations in the Framingham Heart Study. J Allergy Clin Immunol 2012; 129: 840–845. 86 Levin AM, Mathias RA, Huang L et al. A meta-analysis of genomewide association studies for serum total IgE in diverse study populations. J Allergy Clin Immunol 2013; 131: 1176–1184. 87 Bønnelykke K, Matheson MC, Pers TH et al. Meta-analysis of genome-wide association studies identifies ten loci influencing allergic sensitization. Nat Genet 2013; 45: 902–906. 88 Simon D, Wardlaw A, Rothenberg ME. Organ-specific eosinophilic disorders of the skin, lung, and gastrointestinal tract. J Allergy Clin Immunol 2010; 126: 3–13. 89 Kiehl P, Falkenberg K, Vogelbruch M, Kapp A. Tissue eosinophilia in acute and chronic atopic dermatitis: a morphometric approach using quantitative image analysis of immunostaining. Br J Dermatol 2001; 145: 720–729. 90 Gudbjartsson DF, Bjornsdottir US, Halapi E et al. Sequence variants affecting eosinophil numbers associate with asthma and myocardial infarction. Nat Genet 2009; 41: 342–347. 91 Spergel JM, Paller AS. Atopic dermatitis and the atopic march. J Allergy Clin Immunol 2003; 112: S118–S127.
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92 Torgerson DG, Ampleford EJ, Chiu GY et al. Meta-analysis of genome-wide association studies of asthma in ethnically diverse North American populations. Nat Genet 2011; 43: 887–892. 93 Noguchi E, Sakamoto H, Hirota T et al. Genome-wide association study identifies HLA-DP as a susceptibility gene for pediatric asthma in Asian populations. PLoS Genet 2011; 7: e1002170. 94 Hirota T, Takahashi A, Kubo M et al. Genome-wide association study identifies three new susceptibility loci for adult asthma in the Japanese population. Nat Genet 2011; 43: 893–896. 95 Ramasamy A, Curjuric I, Coin LJ et al. A genome-wide meta-analysis of genetic variants associated with allergic rhinitis and grass sensitization and their interaction with birth order. J Allergy Clin Immunol 2011; 128: 996–1005. 96 Ferreira MA, Matheson MC, Duffy DL et al. Identification of IL6R and chromosome 11q13.5 as risk loci for asthma. Lancet 2011; 378: 1006–1014. 97 Allan SE, Broady R, Gregori S et al. CD4 + T-regulatory cells: toward therapy for human diseases. Immunol Rev 2008; 223: 391– 421. 98 Strickland DH, Holt PG. T regulatory cells in childhood asthma. Trends Immunol 2011; 32: 420–427. 99 Tran DQ, Andersson J, Wang R, Ramsey H, Unutmaz D, Shevach EM. GARP (LRRC32) is essential for the surface expression of latent TGF-beta on platelets and activated FOXP3+ regulatory T cells. Proc Natl Acad Sci USA 2009; 106: 13445–13450. 100 Lambrecht BN, Hammad H. The airway epithelium in asthma. Nat Med 2012; 18: 684–692. 101 Fort MM, Cheung J, Yen D et al. IL-25 induces IL-4, IL-5, and IL13 and Th2-associated pathologies in vivo. Immunity 2001; 15: 985–995. 102 Schneider E, Petit-Bertron AF, Bricard R et al. IL-33 activates unprimed murine basophils directly in vitro and induces their in vivo expansion indirectly by promoting hematopoietic growth factor production. J Immunol 2009; 183: 3591–3597. 103 Neill DR, Wong SH, Bellosi A et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 2010; 464: 1367–1370. 104 Scanlon ST, McKenzie AN. Type 2 innate lymphoid cells: new players in asthma and allergy. Curr Opin Immunol 2012; 24: 707–712. 105 Walker JA, Barlow JL, McKenzie AN. Innate lymphoid cells—how did we miss them? Nat Rev Immunol 2013; 13: 75–87. 106 Voehringer D. Protective and pathological roles of mast cells and basophils. Nat Rev Immunol 2013; 13: 362–375. 107 Yosipovitch G, Papoiu AD. What causes itch in atopic dermatitis? Curr Allergy Asthma Rep 2008; 8: 306–311. 108 Toyoda M, Nakamura M, Makino T, Hino T, Kagoura M, Morohashi M. Nerve growth factor and substance P are useful plasma markers of disease activity in atopic dermatitis. Br J Dermatol 2002; 147: 71–79. € mer L, Emtestam L, Johansson O. Increased 109 Dou YC, Hagstro nerve growth factor and its receptors in atopic dermatitis: an immunohistochemical study. Arch Dermatol Res 2006; 298: 31–37.
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REVIEW ARTICLE
Genome-wide association studies of atopic dermatitis Mayumi TAMARI, Tomomitsu HIROTA Laboratory for Respiratory and Allergic Diseases, Center for Integrative Medical Sciences, The Institute of Physical and Chemical Research (RIKEN), Kanagawa, Japan
ABSTRACT Atopic dermatitis is a common inflammatory disease caused by a combination of genetic and environmental factors. Genome-wide association study (GWAS) is a comprehensive and unbiased approach to identify the genetic components of human diseases and to discover the cellular pathways underlying them. GWAS and recent immunochip analysis of atopic dermatitis have identified a total of 19 associated loci with a genome-wide level of significance (P < 5 3 10 8). The candidate genes identified by GWAS suggest a role for epidermal barrier functions, innate-adaptive immunity, interleukin-1 family signaling, regulatory T cells, the vitamin D pathway and the nerve growth factor pathway in the pathogenesis of atopic dermatitis. Combinations of these genetic factors may influence a wide range of phenotypes of atopic dermatitis among individuals. Although a more complete collection of associated genes and pathways is needed, genetic components revealed by GWAS provide valuable insights into the pathophysiology of atopic dermatitis.
Key words:
atopic dermatitis, genome-wide association study, immunochip analysis, polymorphisms.
INTRODUCTION Atopic dermatitis is a complex disease caused by a combination of genetic and environmental factors.1,2 Recent advances in genomic medicine have improved our understanding of the human genome’s contribution to health and disease. Genomewide association study (GWAS) is a powerful method for identifying disease susceptibility genes for common human diseases and has begun to reveal underlying cellular pathways and point to new therapeutic approaches.3,4 This review focuses on GWAS and immunochip analysis of atopic dermatitis. Genes located within and around the susceptible loci and their possible roles in pathogenesis of atopic dermatitis are also discussed.
GENOMIC VARIATION AND GWAS Human genetic variants are referred to as either common or rare. Common variants, also called polymorphisms, are defined as variants with a minor allele frequency of at least 1% in the population.5 Single nucleotide polymorphisms (SNP) are the most prevalent class of genetic variations among individuals.5 GWAS comprehensively surveys associations between common SNP in common complex disorders.6 The international HapMap (haplotype map) project (http:// hapmap.ncbi.nlm.nih.gov/) focuses on common SNP with minor allele frequencies of more than 1% and defines these patterns across the entire genome.7–9 The haplotype structure of the human genome implies that a limited set of 1 000 000
SNP can capture approximately 90% of the genetic variation in the population.3 Pairwise linkage disequilibrium (LD) is calculated based on the genotyping data from the international HapMap project, and a set of markers in a haplotype are said to be in LD. Genotyping arrays have also been developed with reference to the LD, and can now assay up to 4.3 million variants. The data from the HapMap project and the development of dense genotyping chips have enabled us to conduct GWAS effectively on a large number of samples. However, if large numbers of SNP are examined simultaneously in a GWAS, the statistical significance threshold must be adjusted to reduce the chance of making a type 1 error (false positive). A genome-wide significance threshold level of 5 9 10 8 is generally used to adjust for 1–2 million independent tests.10 To date, GWAS have identified more than 1000 genetic loci associated with disease susceptibility and other disease-related phenotypes.11 Genotype imputation is a statistical approach deducing the allelic states at polymorphic sites not actually genotyped from the surrounding SNP data, based on LD and likelihood estimates.12 To conduct rigorous statistical and comprehensive GWAS meta-analyses for common disorders, a number of GWAS consortiums have been founded. However, GWAS are often performed using different genotyping platforms. Imputation is useful for merging distinct GWAS and enables powerful combined analyses of GWAS for the discovery of novel associations.12,13 Although recent GWAS have provided valuable findings about genetic factors in common human diseases, they
Correspondence: Mayumi Tamari, M.D., Ph.D., Laboratory for Respiratory and Allergic Diseases, Center for Integrative Medical Sciences, Institute of Physical and Chemical Research (RIKEN), 1-7-22 Suehiro, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. Email: [email protected] Received 19 September 2013; accepted 19 September 2013.
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explain little about heritability and the associated genetic variants have small effect sizes.14,15 Unmapped common and rare variants are considered a source of missing heritability.14,15 Actually, the chips currently used in GWAS contain relatively few rare SNP in the coding and promoter regions. The 1000 Genomes Project aims to find essentially all variants with frequencies of more than 1% across the genome and more than 0.1% in protein-coding regions and expands the catalog of variations for the next generation of GWAS.16 Using data from the 1000 Genomes Project and other available disease-specific re-sequencing data, the immunochip, a custom Illumina Infinium high-density array containing 196 524 variants, has been developed.17,18 The variants on the chip cover intervals with established GWAS.17,18 However, the immunochip does not cover the whole genome and is designed for use in white European populations. Thus, it would seem to be less informative for Asian populations.
GENETIC STUDIES OF ATOPIC DERMATITIS Systemic, well-powered, genome-wide surveys using GWAS and immunochip analysis have explored the relationship between SNP and susceptibility to atopic dermatitis.19–23 It is well established that common loss-of-function variants of filaggrin (FLG) are a major predisposing factor for atopic dermatitis.24,25 Because FLG mutations involved in atopic dermatitis are featured in another review article in this special issue, this review focuses on GWAS and immunochip analysis for atopic dermatitis. To date, a total of 19 genome-wide significant (P < 5 9 10 8) susceptibility loci have been identified through GWAS and immunochip analysis, and Table 1 summarizes the findings.
GWAS for atopic dermatitis The first GWAS of atopic dermatitis was reported in 2009. The GWAS analyzed 939 cases and 975 controls as well as 270 complete nuclear families with two affected siblings.19 A total of 342 303 markers were assessed, and a susceptibility region on chromosome 11q13 located 38 kb downstream of C11orf30 was identified.19 The peak association was observed 68 kb upstream of leucine rich repeat containing 32 (LRRC32). LRRC32 was previously reported to have specific expression in activated human naturally occurring regulatory T cells.26,27 The GWAS also confirmed the important role of FLG mutations in atopic dermatitis. In the study, another association reaching genome-wide significance was observed at rs6661961 in the epidermal differentiation complex (EDC) on chromosome 1q21. Rs6661961 is in high LD with the FLG mutations, and exclusion of FLG mutation carriers abolished the observed association. In 2011, a GWAS reported two new susceptibility loci, 5q22.1 and 20q13.33, for atopic dermatitis in the Chinese Han population.20 The GWAS was conducted using 1012 cases and 1362 controls followed by a replication study in an additional 3624 cases and 12 197 controls. The results were validated using 1806 cases and 3256 controls from Germany. The 5q22.1 region contains transmembrane protein 232 (TMEM232) and solute carrier family 25, member 46 (SLC25A46), and the thymic stromal lymphopoietin (TSLP) gene is located adjacent (~300 kb downstream) to the associated region. TSLP promotes T-helper (Th)2 cell responses associated with immunity to some helminth parasites and the pathogenesis of a number of inflammatory diseases such as atopic dermatitis.28,29 A variety of stimuli, including viral infections, inflammatory cytokines, protease allergens, bacteria and bacterial products, induce TSLP from barrier epithelial cells.28,29 Another study has
Table 1. A total of 19 genome-wide significant susceptibility loci identified by genome-wide association and immunochip studies for atopic dermatitis Population
Analysis
Location
Reported genes
European Chinese
GWAS
11q13.5 1p21.3 5q11.1 20q13.3 11q13 19p13.2 5q31 2q12 3p21.33 3q13.2 6p21.3 7p22 10q21.2 11p15.4 20q13 4q27 11p13 16p13.13 17q21.32
C11ORF30/LRRC32 (GARP) FLG TMEM232/SLC25A46 TNFRSF6B/ZGPAT OVOL1 ACTL9 KIF3A/IL4/IL13 IL1RL1/IL18R1/IL18RAP GLB1 CCDC80 The MHC region CARD11 ZNF365/EGR2 OR10A3/NLRP10 CYP24A1/PFDN4 IL2/IL21 PRR5L CLEC16A/DEXI ZNF652
European Meta
GWAS
Japanese
GWAS
European
Immunochip
Adjacent genes
References 19 20
TSLP 21
22 CCR4
23 TRAF6, RAG1, RAG2 SOCS1 NGFR
GWAS, genome-wide association study.
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reported that genetic variants in TSLP are associated with atopic dermatitis, and the association is stronger in patients with atopic dermatitis who have a history of eczema herpeticum.30 The 20q13.33 region contains the tumor necrosis factor receptor superfamily, member 6b (TNFRSF6B) gene that encodes decoy receptor 3 (DcR3). Interestingly, DcR3 binds to LIGHT (TNFSF14), which has been shown to bind a herpesvirus entry mediator (HVEM), and LIGHT is a target for asthmatic airway remodeling.31,32 Herpes simplex viruses (HSV-1 and -2) attach to the cell surface via this herpesvirus entry mediator.31 It has been known that eczema herpeticum, a disseminated viral infection after HSV infection, is a serious complication of atopic dermatitis.33 Although further investigation is required, genetic variants in these loci may influence the immune response to HSV. Large sample sizes are required to ensure sufficient statistical power, and collaborative studies on samples from a large number of cohorts have provided new insights about the genetic bases of common diseases. In 2011, a meta-analysis of GWAS for atopic dermatitis of 5606 cases and 20 565 controls and a validation study in an additional 5419 cases and 19 833 controls were conducted.21 The studies identified a total of three novel risk loci for atopic dermatitis, 11q13.1 (OVOL1), 19p13.2 (ACTL9) and 5q31.1 (KIF3A/IL4/IL13).21 The 11q13.1 locus contains OVO homolog-like 1 (OVOL1), which functions as a c-Myc repressor in superbasal cells and is required for proliferation exit of committed epidermal progenitor cells.34 OVOL1 knockout mice show keratinocyte hyperproliferation, hair shaft abnormalities, kidney cysts and defective spermatogenesis.34,35 The most significant association in the 19p13.2 region was observed between the a disintegrin and metalloproteinase with thrombospondin motifs 10 (ADAMTS10) and actin-like 9 (ACTL9) genes. ADAMTS proteins are secreted zinc-dependent metalloproteinases and play a role in connective tissue remodeling and extracellular matrix turnover.36,37 Actin proteins are important for the maintenance of epithelial morphology and cell migration.38,39 The 5q31.1 region contains a clustered family of Th2 cytokine genes, including IL13 and IL4. The adaptive immune response in atopic dermatitis is associated with increased expression of the Th2 cytokines, interleukin (IL)-4 and IL-13.40 Recent studies have shown a significantly greater number of IL4, IL5 and IL13 mRNA-expressing cells in acute skin lesions and an increased number of Th2 cells expressing IL4 and IL13 mRNA in unaffected skin of atopic dermatitis patients.41,42 This GWAS suggests that atopic dermatitis is caused by both epidermal barrier dysfunctions and immunological features.
GWAS for atopic dermatitis in the Japanese population We conducted a GWAS in the Japanese population with 1472 cases and 7971 controls followed by a replication study in an additional 1856 cases and 7021 controls.22 We assessed a total of 606 164 SNP loci and finally found a total of eight novel susceptibility loci for atopic dermatitis.22 Chromosome 2q12 contains the receptors of the IL-1 family cytokines IL1RL1, IL18R1 and IL18RAP. Many of the IL-1 fam-
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Chromosome 2q12 Ligands IL33 IL18 IL1F5, ILF6, IL1F8, IL1F9 IL1A, IL1B, IL1RA LD block IL1R2
IL1RL1(ST2)
IL1R1 IL1RL2
IL18R1
IL18RAP LD block
IL-1 receptor cluster ~0.5 Mb
Figure 1. Susceptibility locus on chromosome 2q12 and a cytokine receptor gene cluster. Regional plots of association results in the Japanese population within the 2q12 region.22 Green box shows the susceptibility linkage disequilibrium block (LD) region. The cytokine receptor gene cluster spans approximately 500 kb. Ligands of each receptor are shown in colored circles. ily receptors are clustered in a small region (~0.5 Mb; Fig. 1), and IL1RL1 (also known as ST2) is a component of the IL-33 receptor.43,44 IL-1 family members are abundantly expressed in the skin, and the IL-33/IL-1RL1 axis triggers the release of several pro-inflammatory mediators and induces systemic Th2type inflammation.45,46 IL-33 and its receptors, ST2 and IL1RAcP, are increased in lesional skin of patients with atopic dermatitis, and topical tacrolimus treatment suppresses the increased expression of IL-33 and ST2 caused by irritants, allergens and staphylococcal enterotoxin B.47 Interestingly, expression levels of IL-1a and IL-1b in the stratum corneum are increased in patients with atopic dermatitis with FLG mutations, and these levels are inversely correlated with natural moisturizing factor.48 Associated variants in the loci may influence the pathophysiology of atopic dermatitis through dysfunction of the IL-1, IL-18 and IL-33 signaling pathways. In our study, the most significant association was at rs176095 in the major histocompatibility complex (MHC) class III region, and conditional logistic regression analysis showed two independent association signals in the MHC class I and class III regions. The MHC is associated with a number of autoimmune diseases, and the MHC class I and III regions are the shared GWAS autoimmune loci.49,50 In recent GWAS, the MHC class I region was found to be associated with psoriasis and vitiligo, and the MHC class III region with vitiligo.51–58 Furthermore, immunoglobulin (Ig)E antibodies against keratinocytes and endothelial cells are observed in serum specimens from subjects with severe atopic dermatitis.59 The genetic components in the MHC region may influence the development of autoimmune conditions in chronic inflammation in patients with atopic dermatitis. Chromosomal region 11p15.4 contains nucleotide-binding domain, leucine-rich repeat and pyrin domain containing protein 10 (NLRP10), which belongs to the NALP family but lacks the leucine-rich repeat region.60 A recent study has shown that NALP10 has an essential role to initiate adaptive immunity by dendritic cells. Nlrp10 / mice show a profound defect in
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helper T-cell-driven immune responses to diverse adjuvants and a dendritic cell intrinsic defect in emigration from inflamed tissues.61 This suggests that NLRP10 plays a critical role in diverse types of innate immune stimulation and the immune function of mature dendritic cells.61 The 3p21.33 region is located adjacent to the chemokine (C-C motif) receptor 4 (CCR4) gene, which encodes a Th2associated chemokine receptor for chemokine (C-C motif) ligand 22 (CCL22) and CCL17 (thymus and activation-regulated chemokine [TARC]).62 A keratinocyte-derived TSLP induces dendritic cells to produce TARC, and CCR4 mediates skin-specific recruitment of T cells during inflammation.63 A recent study has shown that the TARC in stratum corneum from patients with atopic dermatitis is correlated with diverse clinical phenotypes: the severity of local skin lesions, the severity scoring of atopic dermatitis (SCORAD) index, serum TARC level, serum IgE level, serum lactate dehydrogenase level, IL-4 producing T-cell ratio and blood eosinophil count.64 Furthermore, CCR4 is a skin-homing receptor of Th22 cells,65 and significant intensification of Th22 and Th2 cytokines is observed in acute and chronic lesions of atopic dermatitis.66 Associated SNP may also play a role in the Th22 immune response in atopic dermatitis. The 3q13.2 locus contains CCDC80, which is involved in induction of C/EBPa and peroxisome proliferator-activated receptor (PPAR).67 C/EBPa and C/EBPb are co-expressed in basal keratinocytes and upregulated while keratinocytes exit the basal layer and undergo terminal differentiation.68 PPARc acts as a negative regulator in immune cells, and a PPARc agonist markedly suppresses both expression of TSLP in the skin and maturation and migration of dendritic cells in a murine model of atopic dermatitis.69 The 7p22 region contains caspase recruitment domain-containing protein 11 (CARD11), which encodes CARD-containing MAGUK protein 1 (CARMA1). CARMA1 plays an essential role in T-cell differentiation and has a critical role in the regulation of JunB and GATA3 transcription factors and subsequent production of Th2 cell-specific cytokines.70 Furthermore, mice homozygous for the Carma-1 mutation show gradual development of atopic dermatitis with hyper-IgE.71 Chromosomal region 10q21.2 contains three genes and, interestingly, contains early growth response protein 2 (EGR2), a key molecule for anergy induction that activates the expression of genes involved in the negative regulation of T-cell proliferation and inflammation.72 A recent study has shown that CD4+CD25–LAG3+ regulatory T cells (Treg) characteristically express Egr-2.73 Interestingly, the presence of environmental microbiota influences the number of CD4+CD25–LAG3+ Treg.73 The 20q13 region includes cytochrome p450, family 24, subfamily A, polypeptide 1 (CYP24A1), which initiates the degradation of 1,5-dihydroxyvitamin D3, the active form of vitamin D3.74 Vitamin D is a modulator of innate and adaptive immune system functions and plays a role in epithelial antimicrobial defense of the skin and intestine.75 An association between vitamin D deficiency and the severity of atopic dermatitis has been reported.76
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Immunochip analysis of atopic dermatitis Rare large-effect mutations have been recognized as causes of many different common medical conditions.77 The immunochip was developed to conduct deep replication of major autoimmune and inflammatory diseases, and fine mapping of established loci identified by GWAS.17,18 A recent immunochip analysis for atopic dermatitis revealed four new susceptibility loci: 4q27 (IL-2/IL21), 11p13 (PRR5L), 16p13.13 (CLEC16A/ DEXI) and 17q21.32 (ZNF652).23 A replication study using a Japanese population confirmed the associations at the 11p13, 16p13.13 and 17q21.32 loci.23 Because the top signal in European populations is not always replicated in Asian populations, further fine-mapping analysis of the locus is necessary in the Japanese population. The 4q27 region contains the IL2 and IL21 genes. IL-2 has pleiotropic immunoregulatory functions and controls the proliferation and survival of regulatory T cells.78 Tacrolimus (FK506) is a well-known medication for atopic dermatitis and blocks IL-2 gene activation in T cells.79 Interestingly, the TNF receptor associated factors (TRAF6) (11p13), recombination-activating gene 1 (RAG1) (11p13), RAG2 (11p13), suppressor of cytokine signaling 1 (SOCS1) (16p13.13) and nerve growth factor receptor (NGFR) (17q21.32) genes are located adjacent to the associated regions.
GWAS for total IgE levels, allergic sensitization and eosinophil numbers Elevated total IgE levels are considered markers of helminth exposure and allergic inflammation,80,81 and there is a strong genetic contribution to the variability of the total IgE level.82,83 GWAS for total IgE levels have been conducted and identified several susceptibility loci at genome-wide significance levels.84,85 A GWAS of total serum IgE levels found two susceptibility loci, 1q23 (FCER1A) and 5q31 (RAD50) in 1530 individuals from the population-based study following replication studies in four independent population-based study samples (n = 9769).84 The study showed that a functional SNP in the region significantly influenced the cell surface expression of Fc fragment of IgE, high affinity I, receptor for; alpha polypeptide (FCER1A) on basophils and the regulatory mechanism of FCER1A expression via GATA2.84 Another GWAS in the Framingham Heart Study (FHS) reported a total of three loci containing 1q23 (FCER1A), 12q13 (STAT6) and 5q31 (IL13) with genome-wide significance.85 Furthermore, a meta-analysis combining the GWAS of the FHS and replication cohorts identified additional loci, 6p21.3 (HLA-G, HLA-DQA2 and HLA-A) and 1q21 (Duffy blood group, atypical chemokine receptor [DARC]), although the DARC association seems not to be independent of SNP in the nearby FCER1A gene.85 A GWAS of diverse populations including African American and Latino subjects validated findings from previous GWAS (1q23, 5q31, 6p21.3 and 12q13) and found a variant near HLA-DQB1 as a predictor of the total serum IgE levels in multiple race-ethnic groups, but not at the level of genome-wide significance (Pcombined = 2.45 9 10 7).86 Allergen-specific IgE plays a crucial role in the pathogenesis of allergic diseases by binding allergens and initiating immunological responses.87 A meta-analysis of GWAS of allergic
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GWAS of atopic dermatitis
sensitization was conducted for 5789 cases and 10 056 controls, and the top SNP at each of 26 loci was validated using 6114 cases and 9920 controls.87 In the study, the sensitization status was assessed for common food and inhalant allergens evaluated by either the level of allergen-specific IgE in blood or a positive skin prick test. Finally, a total of 10 loci influencing allergic sensitization were identified: 11q13.5 (C11orf30), 12q13.3 (STAT6), 5q22.1 (SLC25A46), 6p21.32 (HLA-DQB1), 2q12.1 (IL1RL1/IL18R1), 4p14 (TLR1/TLR6/TLR10), 3q28 (LPP), 8q24.21 (MYC/PVT1), 4q27 (IL2/ADAD1) and 6p21.33 (HLA-B/ MICA).89 2q12.1, 4q27, 5q22.1, 6q21.3 and 11q13.5 are overlapping susceptibility regions between atopic dermatitis and allergic sensitization (Table 2). Eosinophilic infiltration is a characteristic feature of allergic diseases and parasitic infestation.88 Tissue eosinophilia is a typical characteristic of atopic dermatitis, especially seen in lesions with pronounced epidermal hyperplasia.89 The number of eosinophils in the skin correlates with disease severity, and eosinophils are involved in acute, spongiotic dermatitis.89 A recent GWAS has shown that the 2q12 (IL1RL1), 2q13 (IKZF2), 3q21 (GATA2), 5q31 (IL5) and 12q24 (SH2B3) loci
Table 2. Genetic susceptibility loci overlapping atopic dermatitis and other allergy-related phenotypes identified by GWAS
Chromosome
Locus
2q12
IL1RL1/ IL18R1/ IL18RAP
4q27
ADAD1/IL2/ IL21
5q22
TMEM232/ SLC25A46
5q31.1
RAD50/IL13
6p21.3
The MHC region
11q13.5
C11orf30/ LRRC32 (GARP)
Other allergyrelated phenotypes Atopic dermatitis Asthma Number of eosinophils Allergic sensitization Atopic dermatitis Allergic sensitization Atopic dermatitis Allergic sensitization Atopic dermatitis Asthma Serum total IgE level Atopic dermatitis Asthma Adult asthma Serum total IgE level Allergic sensitization Atopic dermatitis Asthma Allergic rhinitis Allergic sensitization
References 22 25, 92 90 87 23 87 20 87 21 25 84,85 22 25, 93 94 85 87, 95 19 96 95 87
GWAS, genome-wide association study; Ig, immunoglobulin.
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are associated with the number of blood eosinophils.90 The study reported an association between an SNP at the IL1RL1 locus at 2q12 and asthma with a genome-wide significance level.90
Loci overlapping atopic dermatitis and other allergyrelated phenotypes The allergic march is the natural history of atopic manifestations such as atopic dermatitis, bronchial asthma and allergic rhinitis.91 Recent GWAS have revealed a number of susceptibility loci for bronchial asthma, allergic rhinitis, the number of eosinophils, allergic sensitization and total IgE levels, and there are significant genome-wide susceptibility regions overlapping atopic dermatitis and other allergy-related phenotypes (Table 2).92–96 These genetic components may play a role in the atopic march or atopic phenotypes. The 11q13.5 region, which contains LRRC32 (also called GARP), has been reported to be a susceptibility locus for atopic dermatitis, bronchial asthma, allergic rhinitis, the total serum IgE level and allergic sensitization.19,95,96 Foxp3+ Treg play a crucial role in maintaining tolerance to allergens and inhibiting allergies.97,98 LRRC32 mRNA is highly expressed in activated Foxp3+ regulatory T cells, and LRRC32 is essential for the surface expression of latent transforming growth factor-b on the cells.99 Thus, the findings of GWAS also help to enhance our understanding of the allergic march.
Possible roles of the candidate genes for atopic dermatitis Candidate genes for atopic dermatitis within and around the susceptibility loci suggest roles for epidermal barrier functions, innate and adaptive immunity, IL-1 signaling, the inflammatory response, regulatory T cells, the vitamin D pathway and NGF signaling in the pathogenesis of atopic dermatitis.19–23 Pruritus is a major symptom of atopic dermatitis and the itch–scratch cycle can damage the epidermal keratinocytes.1,40 Recent immunological studies have shown that epithelial-derived cytokines activate innate immune cells such as basophils, mast cells and innate lymphoid cells (ILC).100–103 Interestingly, the candidate genes for atopic dermatitis identified by the GWAS and immunochip are involved with those innate immune cells.19–23 Group 2 ILC (comprising ILC2) contribute to promotion of type 2 responses as potent sources of Th2-associated cytokines.104,105. ILC2-derived cytokines play physiological roles in wound healing and immunity to helminths, and are also involved in allergic diseases.105 Mast cells and basophils, which are also associated with type 2 immune responses, are activated through IL-18R, IL-33R and TSLPR, and these cells produce cytokines such as IL-2, IL-13 and TSLP.106 Various human infections, such as West Nile virus, Japanese encephalitis, Tsutsugamushi disease and malaria parasites, are transmitted via insect bites. Basophils and mast cells release histamine, an important mediator of itching, and itching is the essential hallmark of atopic dermatitis.107 Itching and irritation from skin contact are likely to be a protective response to insect stings. NGF has been suggested to play a role in the pathogenesis of itching in atopic dermatitis.107 Increased
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serum levels of NGF and substance P have been shown to correlate with the severity of atopic dermatitis,108 and increased NGF and its receptors, trkA and NGFR (p75NTR), in atopic dermatitis have been reported in an immunohistochemical study.109 The 17q21.32 region is located adjacent to the NGFR gene. A dysregulated NGF pathway and dysfunctions of mast cells and basophils through the genetic variations may be involved in coordinating the itch–scratch cycle in atopic dermatitis.
Multiple clinical phenotypes of atopic dermatitis It is known that there are multiple clinical phenotypes of atopic dermatitis.40 There are great differences in disease severity between individuals, and mild atopic dermatitis can be outgrown in later childhood. Furthermore, some patients have the atopic march, and some patients are prone to skin infections such as Staphylococcus aureus, eczema herpeticum and Malassezia.40 Approximately 80% of patients with atopic dermatitis have elevated serum IgE and/or immediate skin test reactivity to allergens.40 To date, a total of 19 susceptibility loci have been identified at a genome-wide level of significance (Table 1). Combinations of these genetic factors may influence diverse phenotypes of atopic dermatitis among individuals.
CONCLUSION Recent GWAS that comprehensively assess genes related to multifactorial diseases in a non-biased manner across the whole genome have improved our understanding of human atopic dermatitis. The susceptibility loci contain a number of genes encoding markers of immune cells and inflammation that are suppressed by topical corticosteroids and calcineurin inhibitors. There may be a novel therapeutic target gene within or around the associated regions. Further studies to elucidate the functional links between associated variants and phenotypic traits are important to improve our understanding of atopic dermatitis. The findings of GWAS are also helpful to highlight the genes and establish an experimental animal model that mimics human atopic dermatitis. Cross-disciplinary studies combining genetics, immunology, bioinformatics, epidemiology and clinical allergology are necessary for translation of the research into clinical practice.
CONFLICT OF INTEREST:
There is no conflict of interest
in this study.
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