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TSLP and IL-7R Variants Are Associated with Persistent Atopic Dermatitis

2020; Elsevier BV; Volume: 141; Issue: 2 Linguagem: Inglês

10.1016/j.jid.2020.05.119

1523-1747

Ronald Berna, Nandita Mitra, Carolyn Lou, Joy Wan, Ole Hoffstad, Bradley Wubbenhorst, Katherine L. Nathanson, David J. Margolis,

Asthma and respiratory diseases

Atopic dermatitis (AD) is a common inflammatory skin disease, which manifests as itchy, red patches appearing early in life (Abramovits, 2005Abramovits W. Atopic dermatitis.J Am Acad Dermatol. 2005; 53: S86-S93Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar; Leung and Bieber, 2003Leung D.Y. Bieber T. Atopic dermatitis.Lancet. 2003; 361: 151-160Abstract Full Text Full Text PDF PubMed Scopus (1145) Google Scholar; Margolis et al., 2014bMargolis J.S. Abuabara K. Bilker W. Hoffstad O. Margolis D.J. Persistence of mild to moderate atopic dermatitis.JAMA Dermatol. 2014; 150: 593-600Crossref PubMed Scopus (167) Google Scholar). Previous studies have associated TSLP, a cytokine that promotes the differentiation of type 2 helper T cells, and specifically the SNP rs1898671 (TSLP:g.110408002C>T) with the prevalence and persistence of AD (Gao et al., 2010Gao P.S. Rafaels N.M. Mu D. Hand T. Murray T. Boguniewicz M. et al.Genetic variants in thymic stromal lymphopoietin are associated with atopic dermatitis and eczema herpeticum.J Allergy Clin Immunol. 2010; 125: 1403-1407.e4Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar; Margolis et al., 2014aMargolis D.J. Kim B. Apter A.J. Gupta J. Hoffstad O. Papadopoulos M. et al.Thymic stromal lymphopoietin variation, filaggrin loss of function, and the persistence of atopic dermatitis.JAMA Dermatol. 2014; 150: 254-259Crossref PubMed Scopus (53) Google Scholar). TSLP exerts its action through a heterodimeric receptor complex composed of IL-7Rα (encoded by the gene IL-7R) and TSLP receptor (encoded by CRLF2) (Fornasa et al., 2015Fornasa G. Tsilingiri K. Caprioli F. Botti F. Mapelli M. Meller S. et al.Dichotomy of short and long thymic stromal lymphopoietin isoforms in inflammatory disorders of the bowel and skin.J Allergy Clin Immunol. 2015; 136: 413-422Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The study of TSLP and its receptor is important for understanding the genetic architecture of AD and identifying potential therapeutic targets (Margolis et al., 2014aMargolis D.J. Kim B. Apter A.J. Gupta J. Hoffstad O. Papadopoulos M. et al.Thymic stromal lymphopoietin variation, filaggrin loss of function, and the persistence of atopic dermatitis.JAMA Dermatol. 2014; 150: 254-259Crossref PubMed Scopus (53) Google Scholar). Previous studies of TSLP and IL-7R have been limited to the examination of tagging SNPs (Gao et al., 2010Gao P.S. Rafaels N.M. Mu D. Hand T. Murray T. Boguniewicz M. et al.Genetic variants in thymic stromal lymphopoietin are associated with atopic dermatitis and eczema herpeticum.J Allergy Clin Immunol. 2010; 125: 1403-1407.e4Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar; Hoffjan et al., 2009Hoffjan S. Beygo J. Akkad D.A. Parwez Q. Petrasch-Parwez E. Epplen J.T. Analysis of variation in the IL7RA and IL2RA genes in atopic dermatitis.J Dermatol Sci. 2009; 55: 138-140Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). In this report, we fully sequenced the TSLP and IL-7R genes in a large longitudinal cohort of white and African American individuals with AD to gain more complete insight into their association with AD persistence. Data were obtained from the Pediatric Eczema Elective Registry, a longitudinal cohort of children with mild-to-moderate AD, for whom disease control was assessed through self-report every 6 months for up to 10 years. The disease was considered persistent if the participant did not report complete disease control over the past 6 months and did not use prescription cream. Multiple measurements of this outcome were recorded over time for each participant. Next-generation sequencing of TSLP and IL-7R was conducted on 739 individuals in Pediatric Eczema Elective Registry. Only the TSLP and IL-7R genes were targeted for sequencing. Association between the outcome measure and individual SNPs was evaluated using generalized estimating equations (Berna et al., 2020Berna R. Mitra N. Hoffstad O. Wan J. Margolis D.J. Identifying phenotypes of atopic dermatitis in a longitudinal United States cohort using unbiased statistical clustering.J Invest Dermatol. 2020; 140: 477-479Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). Full methods are described in Supplementary Materials and Methods. Written informed consent was obtained from the parents or guardians of all enrolled children. This study was approved by the Institutional Review Board of the University of Pennsylvania (Philadelphia, PA). A total of 703 individuals described themselves as either white (n = 379) or African American (n = 324). Participant demographics are presented in Table 1. For the TSLP gene, 161 distinct SNPs were identified, 30 with minor allele frequency (MAF) of ≥1%. Within the IL-7R gene, 428 distinct SNPs were identified, 144 with MAF of ≥1%. Figure 1 presents the associations of TSLP and IL-7R SNPs with the persistence of AD over time in the full cohort and separately by race. The rs10073816 SNP (TSLP:g.110413489G>A) retained statistical significance after multiple testing corrections in the full cohort (P = 0.001). rs61423440 (TSLP:g.110408482G>C) and rs60340825 (TSLP:g.110408483T>A) also retained significance after correction in the full cohort (P = 0.001 and P = 0.001, respectively). Within IL-7R, the rs11567725 SNP (IL7R:g.35865962A>G) was associated with increased AD persistence in the full cohort after correction (P = 0.0003).Table 1Participant DemographicsDemographicsFull CohortAfrican AmericanWhiteP-value (African American Vs White)Number703324379—Age at AD onset, y, mean (SD)1.98 (2.74)2.14 (2.85)1.84 (2.65)0.162Sex, male, n (%)332 (47.23)136 (41.98)196 (51.72)0.012Asthma, n (%)382 (54.34)182 (56.17)200 (52.77)0.408Seasonal allergies, n (%)494 (70.27)228 (70.37)266 (70.18)∼1Observation time in mo, mean (95% CI)117.3 (115.8–118.8)116.9 (115.2–118.8)117.7 (115.8–119.4)0.549Abbreviations: AD, atopic dermatitis; CI, confidence interval. Open table in a new tab Figure 1Evaluation of TSLP and IL-7R SNPs. (a) Association of TSLP SNPs with outcome, ordered by the number of allele occurrences. Only variants with raw P-values in the pooled analysis ≤0.05 and MAF ≥1% are reported. The presented P-values are all raw. Bolded SNPs have P-values, which satisfy Bonferroni correction in the pooled analysis (adjusted P = 0.00167). Genomic placements are from sequence GRCh37.p13 chr 5. (b) Schematic representation of human TSLP based on Fornasa et al., 2015Fornasa G. Tsilingiri K. Caprioli F. Botti F. Mapelli M. Meller S. et al.Dichotomy of short and long thymic stromal lymphopoietin isoforms in inflammatory disorders of the bowel and skin.J Allergy Clin Immunol. 2015; 136: 413-422Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar (hg 19), with locations of SNPs from the table above. Red regions are coding. (c) Association of IL-7R SNPs with AD persistence. Only variants with raw P-values in the pooled analysis ≤0.05 and MAF ≥1% are reported. Presented P-values are all raw. Bolded SNPs have P-values, which satisfy Bonferroni correction in the pooled analysis (adjusted P = 0.00035). Genomic placements are from sequence GRCh37.p13 chr 5. (d) Schematic representation of human IL-7R based on the NCBI gene database (hg 19), with locations of SNPs from the table above. Red regions are coding. AD, atopic dermatitis; CI, confidence interval; lfTSLP, long isoform of TSLP; MAF, minor allele frequency; NCBI, National Center for Biotechnology Information; hg, human genome; sfTSLP, short isoform of TSLP.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Abbreviations: AD, atopic dermatitis; CI, confidence interval. rs10073816 was associated with more persistent AD (R2 > 0.6) and is not in strong linkage disequilibrium with other TSLP SNPs with MAF of ≥ 1%, with the exception of rs2289277 (TSLP:g.110409067C>G) (R2 = 0.61), an SNP not associated with AD persistence (Supplementary Figure S1). Expression quantitative trait locus analysis provided in genotype-tissue expression demonstrates that rs10073816 is negatively associated with TSLP mRNA expression in sun-exposed skin, non–sun-exposed skin, and cultured fibroblasts (P = 8.6e-39, 1.1e-35, 5.3e-18, respectively). TSLP variant rs10073816 was more strongly associated with AD persistence when controlling for IL-7R variant rs11567725 in both the full cohort (OR = 0.646, 95% confidence interval = 0.525–0.796; P = 3.961e-5) and African American population (OR = 0.609, 95% confidence interval = 0.409–0.908; P = 0.0148). Our study demonstrates considerable variation in TSLP and IL-7R; however, little is associated with AD persistence. We identify that variants, to our knowledge, previously unassociated with AD, including rs61423440 and rs60340825 (MAF < 5%), associated with a 4-fold increase in AD persistence, and rs10073816 (MAF = 50.1%), associated with a 1.4-fold increase in AD persistence. The rs10073816 association was only observed in the full cohort, likely because this population is larger than the African American or white populations alone. Our rs10073816, rs61423440, and rs60340825 associations have not, to our knowledge, been observed in other studies, even though a number of analyses of TSLP have been conducted (Gao et al., 2010Gao P.S. Rafaels N.M. Mu D. Hand T. Murray T. Boguniewicz M. et al.Genetic variants in thymic stromal lymphopoietin are associated with atopic dermatitis and eczema herpeticum.J Allergy Clin Immunol. 2010; 125: 1403-1407.e4Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar; Lou et al., 2019Lou C. Mitra N. Wubbenhorst B. D'Andrea K. Hoffstad O. Kim B.S. et al.Association between fine mapping thymic stromal lymphopoietin and atopic dermatitis onset and persistence.Ann Allergy Asthma Immunol. 2019; 123: 595-601.e1Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar; Margolis et al., 2014aMargolis D.J. Kim B. Apter A.J. Gupta J. Hoffstad O. Papadopoulos M. et al.Thymic stromal lymphopoietin variation, filaggrin loss of function, and the persistence of atopic dermatitis.JAMA Dermatol. 2014; 150: 254-259Crossref PubMed Scopus (53) Google Scholar; Ziegler, 2012Ziegler S.F. Thymic stromal lymphopoietin and allergic disease.J Allergy Clin Immunol. 2012; 130: 845-852Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar). The primary difference between our study and previous reports is that we used next-generation sequencing to identify variants. The identified TSLP variants are located in noncoding regions, suggesting that TSLP variation in AD is linked to altered protein quantity, not structure. The TSLP gene is transcribed into two proteins: a long isoform, which is proinflammatory, and a short isoform, which is anti-inflammatory (Fornasa et al., 2015Fornasa G. Tsilingiri K. Caprioli F. Botti F. Mapelli M. Meller S. et al.Dichotomy of short and long thymic stromal lymphopoietin isoforms in inflammatory disorders of the bowel and skin.J Allergy Clin Immunol. 2015; 136: 413-422Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The fact that rs10073816 is associated with decreased TSLP mRNA production and not in linkage disequilibrium with any variants equally associated with AD persistence suggests that its effect on AD persistence may be causal. Because AD is thought to be an inflammatory disorder and rs10073816 is associated with decreased production of TSLP, it is likely that rs10073816 is associated with decreased production of TSLP's short isoform. It is reasonable to think that IL-7R, a component of the TSLP receptor, could be associated with AD persistence. One SNP, rs11567725 (MAF of ∼1%), was associated with increased AD persistence. Our data imply that further study of IL-7R may not uncover the variants, at a clinically important frequency, associated with AD. The fact that variants in IL-7R do modulate AD persistence suggests that this receptor protein may still be a valuable therapeutic target in AD. This report's primary limitation is that SNPs in the TSLP and IL-7R genes with MAF of 700 individuals), these rare variants were difficult to analyze owing to limited statistical power. However, our primary interest was the identification of clinically actionable mutations; rare variants are less useful in clinical practice. We therefore a priori only evaluated common SNPs. In summary, we have conducted next-generation sequencing of the TSLP and IL-7R genes and identified that TSLP variants rs10073816, rs61423440, and rs60340825 are associated with more persistent AD. The rs10073816 variant is very common in the population associated with decreased TSLP mRNA production and not in strong linkage disequilibrium with any variants equally likely to affect AD persistence. IL-7R had only one mutation, rs11567725, associated with AD persistence. IL7R variants may modulate the effect of TSLP variants because the association of rs10073816 with AD persistence is strengthened when controlling for rs11567725. This study strongly supports additional investigation of the TSLP pathway in AD, encourages further study of therapeutics targeting TSLP and IL-7R, and promotes the case for including TSLP in genetic risk models for AD persistence. Datasets related to this article can be found within the online data repository hosted by Mendeley at doi:10.17632/sgnkksb36x.1 under the title "Datasets for 'Thymic Stromal Lymphopoietin and IL7R Variants are Associated with Persistent Atopic Dermatitis.'" Ronald Berna: http://orcid.org/0000-0003-0520-1218 Nandita Mitra: http://orcid.org/0000-0002-7714-3910 Carolyn Lou: http://orcid.org/0000-0002-7604-833X Joy Wan: http://orcid.org/0000-0002-0189-0442 Ole Hoffstad: http://orcid.org/0000-0002-0261-903X Bradley Wubbenhorst: http://orcid.org/0000-0001-8489-3659 Katherine L. Nathanson: http://orcid.org/0000-0002-6740-0901 David J. Margolis: http://orcid.org/0000-0002-0506-8085 DJM is a consultant for Pfizer, Leo, and Sanofi with respect to studies of atopic dermatitis and serves on an advisory board for the National Eczema Association. JW receives research funding from Pfizer for work unrelated to this study. The remaining authors state no conflicts of interest. This project has been funded in whole or in part by R01-AR060962 and R01-AR070873 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (Principal Investigator DJM). The Pediatric Eczema Elective Registry study is funded by Valeant Pharmaceuticals (Principal Investigator DJM). The sponsors did not have a role in the design of the study; the collection, analysis, or interpretation of the data; the preparation of this report; or the decision to submit this report for publication. Conceptualization: RB, DJM; Data Curation: RB, OH, BW; Formal Analysis: RB, DJM; Funding Acquisition: DJM; Investigation: RB, DJM; Methodology: RB, DJM, NM, CL, KLN; Project Administration: RB, DJM; Resources: DJM, KLN; Software: RB, OH, DJM, BW; Supervision: DJM, NM; Validation: RB; Visualization: RB, DJM; Writing - Original Draft Preparation: RB, DJM; Writing - Review and Editing: RB, DJM, NM, JW, KLN The Pediatric Eczema Elective Registry study has been previously described (Chang et al., 2017Chang J. Mitra N. Hoffstad O. Margolis D.J. Association of filaggrin loss of function and thymic stromal lymphopoietin variation with treatment use in pediatric atopic dermatitis.JAMA Dermatol. 2017; 153: 275-281Crossref PubMed Scopus (20) Google Scholar; Kapoor et al., 2008Kapoor R. Menon C. Hoffstad O. Bilker W. Leclerc P. Margolis D.J. The prevalence of atopic triad in children with physician-confirmed atopic dermatitis.J Am Acad Dermatol. 2008; 58: 68-73Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar; Margolis et al., 2014Margolis D.J. Kim B. Apter A.J. Gupta J. Hoffstad O. Papadopoulos M. et al.Thymic stromal lymphopoietin variation, filaggrin loss of function, and the persistence of atopic dermatitis.JAMA Dermatol. 2014; 150: 254-259Crossref PubMed Scopus (60) Google Scholar, Margolis et al., 2012Margolis D.J. Apter A.J. Gupta J. Hoffstad O. Papadopoulos M. Campbell L.E. et al.The persistence of atopic dermatitis and filaggrin (FLG) mutations in a US longitudinal cohort.J Allergy Clin Immunol. 2012; 130: 912-917Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). To be eligible for the Pediatric Eczema Elective Registry study, a child must have been diagnosed with atopic dermatitis (AD), be between 2 years and 17 years of age at enrollment, and be exposed to pimecrolimus cream 1% for at least 6 weeks of the 6 months before enrollment (Chang et al., 2017Chang J. Mitra N. Hoffstad O. Margolis D.J. Association of filaggrin loss of function and thymic stromal lymphopoietin variation with treatment use in pediatric atopic dermatitis.JAMA Dermatol. 2017; 153: 275-281Crossref PubMed Scopus (20) Google Scholar; Kapoor et al., 2008Kapoor R. Menon C. Hoffstad O. Bilker W. Leclerc P. Margolis D.J. The prevalence of atopic triad in children with physician-confirmed atopic dermatitis.J Am Acad Dermatol. 2008; 58: 68-73Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar; Margolis et al., 2014Margolis D.J. Kim B. Apter A.J. Gupta J. Hoffstad O. Papadopoulos M. et al.Thymic stromal lymphopoietin variation, filaggrin loss of function, and the persistence of atopic dermatitis.JAMA Dermatol. 2014; 150: 254-259Crossref PubMed Scopus (60) Google Scholar, Margolis et al., 2012Margolis D.J. Apter A.J. Gupta J. Hoffstad O. Papadopoulos M. Campbell L.E. et al.The persistence of atopic dermatitis and filaggrin (FLG) mutations in a US longitudinal cohort.J Allergy Clin Immunol. 2012; 130: 912-917Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Children were recruited nationwide through physicians' offices, which made the diagnosis of AD (Kapoor et al., 2008Kapoor R. Menon C. Hoffstad O. Bilker W. Leclerc P. Margolis D.J. The prevalence of atopic triad in children with physician-confirmed atopic dermatitis.J Am Acad Dermatol. 2008; 58: 68-73Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Demographic and disease-specific information was received from the child's physician and caregiver at enrollment. At 6-month intervals, information about the severity and medication use was collected through a survey from the caregiver or child when age appropriate. A subgroup of children provided a sample of DNA. Previous studies have shown that no clinically significant differences existed between children who provided DNA samples and those who did not (Margolis et al., 2011Margolis D.J. Papadopoulos M. Apter A.J. McLean W.H. Mitra N. Rebbeck T.R. Obtaining DNA in the mail from a national sample of children with a chronic non-fatal illness.J Invest Dermatol. 2011; 138: 1765-1767Abstract Full Text Full Text PDF Scopus (9) Google Scholar). This study was approved by the Institutional Review Board of the University of Pennsylvania, and informed consent was obtained from the parents or guardians of all enrolled children. DNA was collected with Oragene DNA collection kits (DNA Genotek, Ottawa, Canada). Massively parallel sequencing of the TSLP and IL-7R genes were conducted on the 739 Pediatric Eczema Elective Registry individuals with sufficient DNA. Raw sequencing data were aligned and mapped to the reference genome GRCh37 using the Burrows-Wheeler Aligner (http://bio-bwa.sourceforge.net/; Li and Durbin, 2010Li H. Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler Transform.Bioinformatics. 2010; 26: 589-595Crossref PubMed Scopus (5951) Google Scholar). Single nucleotide variant and indel calling were accomplished using the Genome Analysis Toolkit HaplotypeCaller (Broad Institute, https://gatk.broadinstitute.org/hc/en-us), after following Genome Analysis Toolkit Best Practices realignment and recalibration (DePristo et al., 2011DePristo M.A. Banks E. Poplin R. Garimella K.V. Maguire J.R. Hartl C. et al.A framework for variation discovery and genotyping using next-generation DNA sequencing data.Nat Genet. 2011; 43: 491-498Crossref PubMed Scopus (6319) Google Scholar; Poplin et al., unpublished data, 2018; Van der Auwera et al., 2013Van der Auwera G.A. Carneiro M.O. Hartl C. Poplin R. Del Angel G. Levy-Moonshine A. et al.From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline.Curr Protoc Bioinformatics. 2013; 43: 11.10.1-11.10.3Google Scholar). We a priori decided to examine only variants with >1% minor allele frequency in the genotyped population. Determination of race was based on self-report. Within this cohort, self-reported race and genetically determined race (using ancestry informative markers) were previously determined to be highly correlated (Lou et al., 2019Lou C. Mitra N. Wubbenhorst B. D'Andrea K. Hoffstad O. Kim B.S. et al.Association between fine mapping thymic stromal lymphopoietin and atopic dermatitis onset and persistence.Ann Allergy Asthma Immunol. 2019; 123: 595-601.e1Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Disease persistence was evaluated on the basis of the self-reported outcome of whether or not a child's skin was AD symptom free without the use of topical medication (e.g., corticosteroids or calcineurin inhibitors) during the previous 6 months (Lou et al., 2019Lou C. Mitra N. Wubbenhorst B. D'Andrea K. Hoffstad O. Kim B.S. et al.Association between fine mapping thymic stromal lymphopoietin and atopic dermatitis onset and persistence.Ann Allergy Asthma Immunol. 2019; 123: 595-601.e1Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar; Margolis et al., 2014Margolis D.J. Kim B. Apter A.J. Gupta J. Hoffstad O. Papadopoulos M. et al.Thymic stromal lymphopoietin variation, filaggrin loss of function, and the persistence of atopic dermatitis.JAMA Dermatol. 2014; 150: 254-259Crossref PubMed Scopus (60) Google Scholar). Symptom free was defined as reporting complete disease control over the past 6 months, and medication free was defined as reporting no need for a prescription cream or ointment to treat this child's itchy rash and/or eczema during the last six months (Margolis et al., 2012Margolis D.J. Apter A.J. Gupta J. Hoffstad O. Papadopoulos M. Campbell L.E. et al.The persistence of atopic dermatitis and filaggrin (FLG) mutations in a US longitudinal cohort.J Allergy Clin Immunol. 2012; 130: 912-917Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). This outcome has been shown to be a reasonable marker of long-term disease severity (Chang et al., 2017Chang J. Mitra N. Hoffstad O. Margolis D.J. Association of filaggrin loss of function and thymic stromal lymphopoietin variation with treatment use in pediatric atopic dermatitis.JAMA Dermatol. 2017; 153: 275-281Crossref PubMed Scopus (20) Google Scholar). Multiple measurements of the persistence outcome were reported over time for each participant. The association between these outcomes and individual SNPs was evaluated using generalized estimating equations for binary outcomes, assuming an exchangeable working correlation structure with empirical standard errors (Berna et al., 2020Berna R. Mitra N. Hoffstad O. Wan J. Margolis D.J. Identifying phenotypes of atopic dermatitis in a longitudinal United States cohort using unbiased statistical clustering.J Invest Dermatol. 2020; 140: 477-479Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar; Lou et al., 2019Lou C. Mitra N. Wubbenhorst B. D'Andrea K. Hoffstad O. Kim B.S. et al.Association between fine mapping thymic stromal lymphopoietin and atopic dermatitis onset and persistence.Ann Allergy Asthma Immunol. 2019; 123: 595-601.e1Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar; Margolis et al., 2018Margolis D.J. Mitra N. Gochnauer H. Wubbenhorst B. D'Andrea K. Kraya A. et al.Uncommon filaggrin variants are associated with persistent atopic dermatitis in African Americans.J Invest Dermatol. 2018; 138: 1501-1506Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). This model evaluates the odds of healing over time, which is equivalent to a measure of disease persistence. Higher ORs indicate less persistent disease. The association of individual TSLP and IL-7R variants with AD was assessed with separate generalized estimating equation models, adjusted for age and sex. Reported P-values are reported as uncorrected for multiple testing, but we mention which associations meet statistical significance after Bonferroni correction. Our interest is on the effect of genetic variation on the longitudinal activity of AD in the general population. As a result, an a priori decision was made to formally analyze only common variants (i.e., minor allele frequency of >1%). All SNP association tests were conducted on the full cohort and then separately within the white and African American subgroups. The decision for how to correct for multiple testing is a complex statistical and philosophical issue. Because TSLP and IL-7R were independently previously associated with AD persistence, their SNPs were treated separately for correction purposes. Furthermore, because we only examined SNPs with a minor allele frequency of >1%, only these SNPs were used to compute the Bonferroni correction factor. A total of 30 tests were corrected for with TSLP and 144 with IL-7R. GnomAD (Broad Institute, https://gnomad.broadinstitute.org/), a database aggregating genome sequencing data from numerous large sequencing projects, was used to annotate the variants identified in TSLP and IL-7R. Interaction effects were investigated between TSLP SNPs and IL-7R SNPs by controlling for rs11567725 within the generalized estimating equation models associating TSLP SNP rs10073816 with AD persistence. Expression quantitative trait locus analyses associate variants with mRNA expression (GTEx Consortium, 2013GTEx ConsortiumThe genotype-tissue expression (GTEx) project.Nat Genet. 2013; 45: 580-585Crossref PubMed Scopus (3473) Google Scholar). We obtained expression quantitative trait loci from genotype-tissue expression (https://gtexportal.org/) for rs10073816. The genotype-tissue expression portal provides both a P-value for association and a normalized effect size. The normalized effect size has no biological interpretation; it merely informs the directionality of the association (i.e., whether the variant is associated with increased or decreased mRNA production). With this in mind, we present the P-values but not the normalized effect size for the associations. All analyses were implemented in Stata, version 15.1 (StataCorp, https://www.stata.com/) and R, version 3.6.1.