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Genome-wide analysis of copy number variants in alopecia areata in a Central European cohort reveals association with MCHR2

Reference: Fischer Johannes, Degenhardt Franziska, Hofmann Andrea, Blaumeiser Bettina, Nöthen Markus, et al., Redler Silke, Basmanav F. Buket, Heilmann-Heimbach Stefanie, Hanneken Sandra, ....- Genome-wide analysis of copy number variants in alopecia areata in a Central European cohort reveals association with MCHR2 Experimental dermatology - ISSN 0906-6705 - 26:6(2017), p. 536-541 Full text (Publisher's DOI): http://dx.doi.org/doi:10.1111/EXD.13123 To cite this reference: http://hdl.handle.net/10067/1347820151162165141

Institutional repository IRUA Petukhova, Blaumeiser, Giehl, Buket Basmanav, Johannes Fischer, Running Head: a European in cohort Central association reveals with ofcopy analysis nu Genome-wide Article type : Regular Article 08-Jun-2016 : Date Accepted :20-Apr-2016 Date Revised :02-Nov-2015 Date Received This article isprotected Allrights bycopyright. reserved. 10.1111/exd.13123 version andtheVersi this between differences through the copyediting, typesetting, pagination forpublication accepted This articlehasbeen 4 Germany 3 Germany 2 1 Department of Dermatology, University of Düsseldorf, Düsseldorf, D-40225, AcceptedDepartment of Dermatology, University Munich, of Munich, D-80337, Germany Department of Genomics, Life & Brain Center, University Bonn, of Bonn, D-53127, Institute of Genetics, University Bonn, of Bonn, D-53127, Germany Article 4 Hans Wolff, 11 8 Markus Böhm, Markus Angela M. Christiano, Copy number variants in alopecia areata 1 1* Stefanie Heilmann-Heimbach, Franziska Degenhardt, 4 Susanne Moebus, Susanne 9 Natalie Garcia Bartels, Garcia Natalie 11,12 Markus M.Nöthen, and undergone full peer review buthasnotbeen review undergone fullpeer and on of Record. Please cite of Record. Please on 5 mber variants in alopecia areata Roland Kruse, and proofreading process, which may lead to 1,2* Andrea Hofmann, 1,2 Sandra Hanneken, 10 Ulrike Blume-Peytavi, 1,2 6 Gerhard Lutz, Regina C.Betz Regina this article this as doi: 1,2 Silke Redler, MCHR2 3

Kathrin A. A. Kathrin 7 1 Bettina 10 Lynn 1 F. 10 Antwerp, BE-2650, Belgium * York 10032, USA 12 USA 11 and Allergy, Charité-Universitätsmedizin Berlin, Berlin, D-10117, Germany 9 8 7 6 Essen, Duisburg, D-47057, Germany 5 This article is protected by copyright. All rights reserved. copyright. This articleis protectedby E-mail: [email protected] Fax: +49 (0)228 287 51011 Phone: +49 (0)228 287 51023 D-53127 Bonn, Germany Sigmund-Freud-Str. 25 University Bonn of Institute of Human Genetics ReginaProf. C. Betz, MD Correspondence to: contributed equally Department of Dermatology, University Münster, of Münster, D-48149, Germany &Nail, Hair Dermatological Practice, Paderborn, D-33098, Germany Institute of Medical Informatics, Biometry and Epidemiology, University Duisburg- Department of Medical Genetics, University andUniversity Hospital of Antwerp, Department Geneticsof and Development, Columbia University, New York, New ofDermatology Department Skin Hair Science, and Center for Research Clinical Accepted ArticleDepartment of Dermatology, Columbia University, New York, NewYork 10032,

Dermatological Practice, Wesseling, D-50389, Germany

implicated in previous studies of AA pathogenesis. However, previous research has in melanin-concentrating hormone (MCH) signaling controls). The duplications spanned the 342.5 kbassociated region in 6q16.3 (duplications in 4/585 patients; 0/1,340 4q35.2, 6q16.3, 9p23, 16p12.1, and 20p12.1. The most promising finding was a association with AAwas found for CNVs in the following five chromosomal regions: patients and 1,340 controls of Central European origin. A nominally significant and a candidate focused CNV analysis performed in a cohort of 585 AA variants involved in autoimmune other disorders. Here, we report agenome-wide- contribution of copy number variants (CNVs) toAA,a prominent class of genomic common variants atseveral genetic loci. Todate, nostudy hasinvestigated the pathophysiology AAis increasing, of torecent duein part genetic findings implicating often results in pronounced psychological distress. Understanding the of Alopecia areata (AA) is acommon hair loss disorder of autoimmune etiology,which Abstract This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted MCHR2 disorder, alopecia areata, copy number variants, molecular genetics, genetic complex Key words involvement of duplications in MCH signaling. Inconclusion, the present results provide suggestive evidence thefor episode of AA. This might indicate arelationship between AA, pigmentation, and patients frequently shows achange in color when it regrows following anacute melanin aggregation. AApreferentially affects pigmented hairs, and the hair of AA shown that Article

MCHR2 affectsthescale color of barfin flounder via fish theinduction of MCHR2 in AA pathogenesis. MCHR2 MCHR2

. These genes have notbeen and MCHR2-AS1 , implicated , implicated warranted. Thedevelopment of novel treatments for AAwill require amore and most are only effective in mild cases, and thus new treatment strategies are in pronounced psychological distress. Currently available therapies are suboptimal, individuals, thestigmatizing potential and unpredictable course of the disorder result characterized by asudden onset and a variable clinical course. In many affected affected site, all hair bearing areas of the body can become involved. AA is often dystrophic hairs (exclamation mark hairs). Although the scalp is thepredominantly substantial increase in the proportion of telogen hairs, and the development of then extend centrifugally andmay coalesce. AA episodes are characterized by botha commence with thedevelopment of non-scarring, isolated hairless patches. These peak incidence in any specific age group (1, 2). Episodes of hair loss typically - 2%(1, 2). Similar rates are observed in both sexes, andno evidence exists for a autoimmune-mediated disorders in . The estimated lifetime risk is around 1.7 Alopecia areata (AA) is acommon hair loss disorder, and oneof the most prevalent Introduction This article is protected by copyright. All rights reserved. copyright. This articleis protectedby development AA.of As is thecase with common variants, associations with rare loci Acceptedcommon variants, rare genetic variants may also play animportant role in the factor (6), andnine other loci of relevance toautoimmunity (6-8). Inaddition to These include loci in theHLA region, with HLA-DR being theprobable key etiological focused candidate gene studies have identifi and (GWAS), studies association genome-wide Recent (3-5). processes biological the development of AA,andthat their identification will elucidate theunderlying Consensus exists among researchers that genetic factors play animportant role in response. However, understanding of this process remains limited. underlying pathomechanism of AA is a T-cell mediated, tissue-specific autoimmune comprehensive understanding pathophysiology. of Research suggests thatthe main Article ed atotal of 12 susceptibility AA. locifor

previously implicated in AA pathogenesis. wide CNV analysis, and second, asubsequent focused analysis of candidate genes European descent. Two separate analyses were performed: first, an initial genome- investigating 585AA patients and 1,340 population based controls of Central The aim of the present studywas toanalyze therelevance of CNVs in AAby performed todate. dermatitis (17). Toour knowledge, nogenome-wide CNV-analysis AAhas of been (10, 11), systemic lupus erythematosus (12, 13), psoriasis (14-16), and atopic number of autoimmune disorders, such as rheumatoid arthritis (9), Crohn's disease genetic variants affecting larger genomic regions, which have been implicated in a characterization ofcopy number variants (CNVs). Theseare a prominent class of genome-wide singlenucleotide polymorphism (SNP) dataallows theefficient array-based of availability studies. The follow-up interms functional of advantages rare variants are typically larger thanfor common variants, theformer may offer may point toimportant stepsin AAdevelopment. Furthermore, since effect sizes for This article is protected by copyright. All rights reserved. copyright. This articleis protectedby help support groups in Germany and the Netherlands. The inclusion criterion was a Acceptedin Belgium andGermany, private dermatology practices in Germany, andAAself- Male and female patients were recruited from the following sources: outpatient clinics Patient cohort inclusion. to prior with theDeclaration of Helsinki. All participants provided written informed consent The study protocol was approved by therespective ethics committees, and complied Sample description Methods and Materials Article

studies: KORA (Cooperative Health Research in theRegion of Augsburg, Germany) Population-based controls were drawn from the following three epidemiological Control cohort (7.5%); AT AU (9.6%); or AT/AU(14.4%). patientsof (68.5%) had patchy AA. The remaining patients were classified as having: universalis (AU)was defined as 100% loss of both scalp and body hair. The majority was defined as 100% loss of scalp hair with variable loss of body hair. Alopecia totalis (AT) was defined as100% loss scalp of hair without loss of body hair. AT/AU Patchy AAwas defined as one or more circumscribed patches of hair loss. Alopecia participants, the mean age of onset was 29.1 years (standard deviation (SD) = 17.2). After quality control (QC) 585patients remained in the dataset. For these syndrome were excluded. descent according toself-reported ancestry. Patients with Down syndrome or Turner dermatologist-assigned diagnosis AA.All of participants were of Central European This article is protected by copyright. All rights reserved. copyright. This articleis protectedby described elsewhere (21): SNPs with acall rateof < 97% andindividuals with aDNA Acceptedto avoid technical artifacts, the following stringent QC criteria were applied, as both array types were analyzed (n = 546,356 SNPs). To ensure high data quality and CA,US Diego, Inc.,San (Illumina microarray San Diego, CA, USA). Controls weregenotyped onIllumina’s HumanHap550v3 Patients were genotyped onIllumina’s Human660W-Quad microarray (Illumina Inc., Genotyping andquality contro (20). After QC, 1,340 controls remained in the study. (HeinzNixdorf RECALL [Risk Evaluation factors, of coronary calcium and lifestyle]) (18), PopGen (Populat Article ion-Based Ge Recruitment for l priortoCNVdetection A). Only markers that were common to

netics Research) (19),andHNR analysis. both QS and PC were included in the genome-wide- andthe candidate gene exceeded 0.30, ascalculated over all SNPs. Only thoseCNVs that were detectedby MHC region was analyzed. Individuals were excluded if their SD from the log R ratio http://www.openbioinformatics.org/penncnv/) (23). Neither the X-chromosome nor the http://www.well.ox.ac.uk/QuantiSNP) (22), and PennCNV (PC, 2011June16 version; Putative CNVs were identified using QuantiSNP (QS, version 2.1, CNV detectionandquality control defined according to multi-dimensional scalingwith HapMap phase 2 data. removed as well ascryptic related individuals (IBS sample duplicates identified by identity bystate (IBS) estimates (IBS = 2) were between X-chromosomally inferred and phenotypic sexwere excluded and DNA call rate of < 97%were removed from the analysis, individuals with adifference This article is protected by copyright. All rights reserved. copyright. This articleis protectedby AcceptedSNPs > 10). criteriawere relaxed (30> lBF/confidence value > 10;30> number consecutive of To allow the inclusion smallerof aberrations in the associated CNV regions, the filter regions were required toshare at least three consecutive SNPs in all CNV carriers. rearrangements in CNV regions mayvary between individuals, theassociated bothCNV callsfrom calling programs. Sincetheposition andextent genomic of associated CNV regions were required toshow nominal significance according tothe QS) or aconfidence value (PC) of All CNVs were required to span at least 30 SNPs, and have a log Bayes Factor (lBF; Genome-wide CNV analysis Article ≥ 30. To be included in further analyses, the

≥ 1.6) andpopulationoutliers, as presently applied microarray types, all Genome Viewer. Inaprevious CNV analysis byour group, which involved the candidate genes were visually inspected All CNVs in theassociated regions (genome-wide CNV analysis) andin the Experimental verificat used. chromosomal regions/candidate gene regions, Fisher’s exact test (two-sided) was (PLINK version 1.07) (25). To test associationfor between AA and CNVs in specific The datasets generated by QSand PC were analyzed separately using PLINK Statistical analysis of CNVs coverage with > 10SNPs in the genome-wide microarrays. the study, the candidate gene region (RefSeq gene boundary + 20kb) required nearest upstream and downstream gene for each intergenic SNP. To be included in list comprised all genes with anintragenic genome-wide significant SNP, and the selected, and a list of candidate genes was generated (supplementary table 1). This Genome-wide significant SNPs (p < 5x 10 Selection ofca QS) or aconfidence value (PC) of assigned RefSeq gene, span at least 10 SNPs, and have a log Bayes Factor (lBF; within 20 lie to required were CNVs NCBI build 36 and the UCSCGenome Browser (http://genome.ucsc.edu/) (24).All The transcription start and end position of each RefSeq gene was determined using Candidate gene This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Article ndidate genes CNV analysis ion ofpredictedCNVs ≥ 10.

kb up- or downstream of the boundaries of the in silico in using Illumina's Genotyping Module 1.9.4 CNVs the fulfilling above mentioned -8 ) from previous AAstudies were

nominally significant association with the following five chromosomal regions was included in the downstream analyses. For both the QS and the PC dataset, a The samples of atotal of 585 patients and 1,340 controls passed QC and were Genome-wide analysis Results no experimental verification of this CNV was performed. identified in thepresent candidate gene CNV analysis spanned more than 30 SNPs, in comparison tothree housekeeping genes ( their sequences are obtainable upon request. Relative copy numbers were measured SYBR®Green (Life Tec duplications were verified experimentally using real-time quantitative PCR and Power of duplications in 6q16.3, which constituted the most promising finding. These a proof of principle for thepresent study, we selectivelyverified all four occurrences filter criteria thegenome-widefor CNV analysiswereverified experimentally (21). As This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Acceptedare obtainable upon request. Forwhose CNVs putative breakpoints differed CNV detected in the present patient cohort. Individual genotype andintensity data Supplementary table 2shows thechromosomal position and sizeof each associated 0.041). None these of associations withstood correction for multiple testing. 16p12.1 (p = 0.028), and deletions in a 25.8 kb region onchromosome 20p12.1 (p = deletions); deletions and one duplication in a474.6 kb region onchromosome only) onchromosome 9p23 (p = 0.005 for duplications and deletions; p= 0.01 for one duplication in a 99.7 kb (duplications and deletions) or 84.8 kb region (deletions and deletions (p=0.008), 6q16.3 on region kb ina342.5 duplications found (Tab. 1): deletions in a 130.2 kb region onchromosome 4q35.2 (p = 0.028), Article hnologies, Carlsbad,CA).Five BNC1

, CFTR primer pairs primer , RPP38 ). Since theCNV were us ed, and Candidate gene the associatedregion. duplication affected neither (67.0 kb) was detected within the associated region in one control subject. This AS1; 2( factor hormone concentrating region spans 342.5 kb (100.40 - 100.75 Mb), andincludes the genes melanin- breakpoints, and their sizes varied between 341.7 kb and 361.1 kb. The associated no CNV was detected in the control cohort. The four CNVs had differing putative In 6q16.3, four patients carried duplications (0.68%). Using our primary filter criteria, CNVs in6q16.3 reported. according to PC and QS, predicted CNV sizes common to both programs are This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted SNPs (111.11 - 111.57 Mb). No CNV was detected in the control cohort. serine/threonine kinase ( the genes acyl-CoA oxidase-like ( This duplication in 2q13 was found in one patient (0.17 %, P> 0.05), and spanned Within the eleven remaining candidate gene regions, only oneCNV was detected. excluded from the downstream analysis (supplementary table 3). lacked sufficient coverage onthemicroarrays (< 10SNPs), andwere therefore A total of 20 genes were selected for the candidate gene analysis. Of these, nine Article figure 1 ). After application of the relaxed filter criteria, asmaller duplication

based analysis BUB1 MCHR2 ). Theduplication spans 462.3 kb, and includes 91 MCHR2 nor ACOXL MCHR2-AS1 ) andMCHR2antisense RNA 1( )

and BUB1 mitotic checkpoint

, and did not reduce thesize of MCHR2- is similar tothatdetected in our controls (0%), andtherefore supports thepresent In total, 12duplications were observed in 57,328individuals (0.02%). This frequency least oneduplication affecting Genomic Variants (DGV; http://dgv.tcag.ca/dg duplications spanned the genes 6q16.3. Here, duplications were detected 20p12.1. Themost promising association was observed for a342.5kbregion in Nominal significance was found for CNVs in4q35.2, 6q16.3, 9p23, 16p12.1, and increase. CNVs. This strategy reduces type I errors, whereas type II errors are expected to po have a CNVs,which oflarger selection application of stringent filter criteria in thegenome-wide CNV analysis ensured the analysis in a cohort of 585 AA patients and 1,340 population-based controls. The The present study involved agenome-wide- andacandidate gene focused CNV Discussion This article is protected by copyright. All rights reserved. copyright. This articleis protectedby dosage and might thus enhance, or otherwise interfere with, the function of this gene Acceptedspeculatethat AA. We frequently changes tograywhite or when it regrows followingacute episode an of Interestingly, AApreferentially affectspigmented hairs, and the hair ofAA patients lighter scale color in barfin flounder viafish the induction of melanin aggregation (32). feeding and energy homeostasis (e.g.(31)), ligand MCH, themelanin-concentrating hormone. Besides its important roles in characterized in some detail. Although the precise function of patient cohort). finding of an association between duplications in 6q16.3 and AA (0.68% in our Article MCHR2 MCHR2 MCHR2 MCHR2 affecting duplications lead to an increase in gene MCHR2-AS MCHR2 is aG- coupled receptor for its primary (27-30)http://www.1000genomes.org/home). and in four patients butno controls. These 1 remains unclear, tentially stronger impact than smaller than impact stronger tentially v/app/home) (26) five studiesreport at MCHR2 MCHR2-AS1

has been reported to cause . In theDatabase of MCHR2 MCHR2 has been region of association. To our knowledge, none of these genes has been implicated associated region. None of the deletions or duplications in the DGV span the entire CDR2 contains the genes CNVs in a475kbregion 16p12.1 of were also associated with AA.This region gene in the pathogenesis of AA. results provide suggestive evidence for the involvement of asecond pigmentation STX17, AA of GWAS (8), asignificant associationwas with found common variants in constitutive risk toattractautoreactive CD8+ Tcells (34-37). Moreover, in thefirst autoantigens generated during active hair shaftpigmentation demonstrate a discussed aswell asexperimentally shown, that melanogenesis-associated MCH signaling, andAA. Already in previous AAstudies, it was theoretically Together, these observations might indicate a relationship between pigmentation, in combination with the additional, synergistic effects of environmental hazards (33). This article is protected by copyright. All rights reserved. copyright. This articleis protectedby MACROD2 Accepteddeletions are part of the allelic spectrum of this gene. conclude We thatdeletions in lists numerous deletions affecting false positive associations arising as a result the of selected filter criteria. The DGV detected among the patients. This demonstrates the need to be aware ofpotentially CNVs were smaller several as criteria, filter relaxed the of application following cohort using our primary filter criteria. Notably, the association signal was lost deletions were detected in our control cohort, no deletion was detected in the patient In 20p12.1, deletions located within disorder. However, their involvement in the pathogenesis of AAcannot be excluded. previously in thepathogenesis of either AAor any other common autoimmune Article . The DGV lists several inversions, duplications, and deletions within the a gene associated with the gray hair phenotype in horses (38). The present are unlikely tobeof relevance to the AA phenotype. UQCRC2 , PDZD9 MACROD2 MACROD2 , C16orf52 , thusdemonstrating thatcommon were associated with AA. 11While ,

VWA3A , EEF2K , POLR3E , and warranted to elucidate the relevance of CNVs in these AA candidate genes. cohorts, and in cohorts genotyped on more densely covered SNP arrays, are genes lacked sufficient coverage onthe microarrays. Therefore, studies in larger analysis was restricted to11 out of 20 AA candidate genes, since the remaining nine overlooked due to alack statistical of power. Itis of note that our focused CNV duplications contributing tothe allelic spectrum of these candidate genes were wecandidate genes.However, cannotexcl No association was detected between AA and CNVs in any of the investigated essential in order to evaluate their relevance to the pathogenesis of AA. analyses ofall identified associated CNV regions in independent cohorts are were unable to compare our results with those of previous reports. Replication Since the present study represents the first genome-wide CNV analysis ofAA, we excluded. be AAcannot to relevance biological CNVsare of these possibility that the However, The CNVs detected in 4q35.2 and9p23 had nodirect effect on any RefSeq gene. This article is protected by copyright. All rights reserved. copyright. This articleis protectedby findings. Fourth, we excluded the MHC region from our CNV analysis, asit is prone Acceptedour stringent filter criteria led tothe exclusion of smaller, and potentially genuine, < 20%, theimpact of unscreened versus screened control status is low (39). Third, mathematical calculations have shown that may have led to false-negative findings genuinelyfor associated regions. However, for AAis 1- 2%,some the of control CNV carriers may have been affected, and this none of the controls were screened for AA. Given that the approximate lifetime risk replication is necessary toprove or disprove theobserved associations. Second, none of the association findings survived correction for multiple testing. Independent variants with high levels significance of was limited. It is therefore unsurprising that The present study had several limitations. First, the power of our cohort to detect rare Article ude the possibility that for diseases with alifetime prevalence of

raredel etions and Acknowledgments Acknowledgments investigations. both confirmation in large, independent cohorts, and functional follow-up involved in pigmentation might be of relevance toAA. Thepresent findings require contains AA, andgenerated s In summary, thepresent study represents the first genome-wide CNV investigation of investigate therelevance CNVs of in the HLA region to AA. arthritis, and type 1 diabetes (11). Therefore, further studies are warranted to several CNVs in the HLA region were associated with Crohn’s disease, rheumatoid date, which did notinclude AA, usedapurpose-designed array andfound that to false positive findings. Thelargest CNV study ondiverse autoimmune disorders to This article is protected by copyright. All rights reserved. copyright. This articleis protectedby AcceptedThe author contributions are as follows: funding (BONFOR) to JF, FD, and RCB. Research Foundation (DFG, BE2346/5-1). This work was supported in part by local Halbach-Stiftung. RCB was arecipient a of Heisenberg Professorship of the German ImmunoSensation. MMN received support from the Alfried Krupp von Bohlen und cohort. RCB and MMN are members of theDFG-funded Excellence Cluster Prof. S.Schreiber for providing accesstotheSNP-chip data from the PopGen control H.-E. Wichmann for supplying theSNP-chip data from the KORA control cohort, and to The authors thank allpatients for their participation in thestudy. are gratefulWe toProf. Article MCHR2,

might beimplicated. This supports the hypothesis that genes uggestiv e evidence that duplications

in the6p16re gion, which References References The authors have no conflicts of interest to declare. Conflicts ofinterest disclosure or tools, Petukhova, Giehl, F. BuketBasmanav, . Petukhova L, Christiano A M. The genetic archit 3. 134: 1141-1142. SA,Schrum risk A incidence of RR. alopecia G, MD, Torgerson Lifetime Mirzoyev Davis areata estimated at 2.1% by Rochester Epidemiolo 2. Safavi K H, Muller 628-633. 70: 1995: Proc Clin Mayo S 1989. through A,1975 Minnesota, County, Suman Olmsted in V J, Moshell A N, Melton L J, 3rd.1. Incidence of alopecia areata 1 Blaumeiser, Johannes Fischer, This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Craddockcases of eight common diseases and 3,000 shared controls. Nature 2010: 464: 713-720. N, Hurles11. M E, Cardin N, etHumGenet 2006:439-448. 79: al. Genome-wideStange E,SchaeffelerE, et K, FellermannD J Am colon. the of disease Crohn to predisposes number copy gene 2 beta-defensin human low with association study10. is NCF1 gene the of variation number al. Copy et AK, A,Lindqvist of Nerstedt M, L Olsson CNVs in 16,000associated with rheumatoid arthritis. 9. Petukhova L, Duvic M, Hordinsky M,et al. Ge implicates both innate and adaptive immunity. Nature 2010: 466: 113-117. 8. wide significance. J Invest Dermatol 2012: 132: 2192-2197. first of the study genome-wide al.Follow-up FF,et S, Brockschmidt D, Redler Jagielska andKIAA0 IL13 scaninalopecia areata: association 7. Betz RC,Petukhova L,Ripke S, etal.Genome-wide meta-analysis in alopecia areata resolves HLA associations andreveals two new susceptibility loci.NatCommun 6: 2015: 5966. 6. B, Blaumeiser van der GootI,Fimmers R, et al.Familial aggregation alopeciaof areata. J Am AcadDermatol 2006: 54: 627-632. 5. 425. Jackow infectioncytomegalovirus in twins: genes versus 4. C, PufferSymp Proc 2013: 16:S16-22. N, Hordinsky M, Nelson J, Tarrand J, Duvic M. Alopecia areata and Accepted Articleperformed the research, 3 Hans Wolff, 4 analysed the data, 3 3 Angela M. Christiano, Markus Markus Böhm, 1,2,4,5 3 4 Susanne Moebus, Stefanie Heilmann-Heimbach, Stefanie Franziska Degenhardt, 2 designed theresearch study, 5 wrote the manuscript 3 Natalie Bartels, Natalie Garcia Antioxid Redox Signal 2012: 16: 71-78. 3 Markus M.Nöthen, Markus environment? J Am Acad Dermatol 1998: 38: 418- gy Project, 1990-2009. J Invest Dermatol 2014: 2014: Dermatol JInvest 1990-2009. gy Project, 3 350 as susceptibility loci supported assusceptibility loci with350 genome- al. A chromosome 8 gene-cluster polymorphism al.A 8gene-cluster chromosome Roland Kruse, nome-wide study association inalopeciaareata ecture of alopecia areata. J Investig Dermatol 1,2,4,5 Andrea Hofmann,

4 2, 5 2, Sandra Hanneken, 3 3 Ulrike Blume-Peytavi, Regina C. Regina Betz contributed essential reagents 3 Gerhard Lutz, 2,3,4 2, 4, 5 2, Silke Redler, 3 Kathrin A. Kathrin

3 Bettina Bettina 3 Lynn 3

31. Chee J, Chee M Pissios P, Prasad D, E. Maratos-Flier Expression of melanin-concentrating hormone 81-88. 155: 2014: in Endocrinology mice. male againstobesity diet-induced protects 2 receptor 31. Cooper G M, CoeB P, Girirajan S,et al.A copy number variation morbidity map of developmentaldelay. Nat Genet 2011: 43: 838-846. 30. B, S, Walker Thiruvahindrapuram UddinM, Genet 2014. Med genetics. for clinicalresource andpopulation 29. number copy variation JA,et Bal. Refining K, of Rosenfeld analyses P, Witherspoon Coe identifies specific genes associated with developmental Nat delay. Genet 2014: 46: 1063-1071. 28. incontrol population Copy-number SW. variation Scherer C,Feuk L, D, Marshall Pinto cohorts. Hum Mol Genet 2007: 16 Spec No. 2: R168-173. 27. 992. a Variants: Genomic of Database The W. S Scherer L, Feuk RK, R,Yuen JR,Ziman MacDonald ofstructural collection Nucleic Acids humangenome. variationcurated inthe Res 2014: 42: D986- 26. al.PLIN et K, B,Todd-Brown S,Neale Purcell 81: 559-575. Am JHum Genet 2007: analyses. linkage population-based 25. Res at UCSC.Genome browser al.The S,et T Furey CW, J,Sugnet W Kent 2002: 12: 996-1006. 24. Res 2007: 17: 1665-1674. for designed model Markov hidden anintegrated al.PennCNV: et D, Hadley M, Li K, Wang Genome SNP detection genotyping data. number copy in variation whole-genome high-resolution 23. 2007: 35: 2013-2025. Colella S,Yau C, Taylor J M, et al. QuantiSNP: an Objective Bayes Hidden-Markov Model to AcidsRes Nucleic data. genotyping number variation using SNP and accurately detect map copy 22. Genet 2012: 159B:263-273. in16p11.2 variants number copy Herms between al.Association F, L, S,et Priebe Degenhardt BNeuropsychiatr Genet JMed case-control Am sample. inaGerman disorder depressive and major 21. Factors, Evaluation of Coronary Calcium and Lifestyle. Am Heart J 2002: 144: 212-218. Risk Study. RECALL Nixdorf Heinz the of and design rationale subjects: middle-aged in healthy al. A,et S,Stang A,Mohlenkamp Schmermund risk for factors pr andnovel andestablished disease 20. 55-61. Genet 9: 2006: Community relationships. phenotype Krawczak M, Nikolaus S, von Eberstein H,Crou genotype- of analysis for complex andcontrols the patients recruitment of population-based 19. HE, Wichmann Gieger C,Illig T.KORA-gen--r S26-30. Suppl 1: 67 Gesundheitswesen disease of phenotypes. spectrum broad 18. 132: 98-104. number within filaggrinvariation copy al.Intragenic K,SandilandsA,et SJ, Kroboth Brown 2012: Dermatol JInvest effect. adose-dependent with dermatitis risk the of to atopic contributes 17. 984. Huffmeier U,Bergboer J G,Becker T,etal.Replication ofLCE3C-LCE3B CNV asariskfactorfor and 979- 130: genetic factors.psoriasis analysis of risk interaction2010: withJInvest Dermatol other 16. E,Carretero-Iglesia Pedrosa L,BoadaA,etal.CCL4L andCCL4/CCL4Lpolymorphisms serum levels are associated withpsoriasis severity 15. LCE3B envelope late cornified the of al. E, P L, et Deletion CidR,Riveira-Munoz de Zeeuwen and LCE3C genes asa susceptibility factor psoriasis.for NatGenet 2009: 211-215.41: 14. 1037-1054. 80: 2007: AmJHum Genet Americans. risk factor fornumber andhighcopy isa protec polymorphisms andassociated variation copy-number al.Gene et L, Y Wu ChungEK, Y, Yang is number (SLE):copy low a systemic lupuserythematosus inhuman component C4 complement of 13. McKinney C,Merriman T confirms for R.Meta-analysis deletion arole in inFCGR3B 2370-2376. 21: 2012: Genet Mol Hum phenotypes. autoimmune 12. This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Article . J Invest Dermatol 2011: 131: 1830-1837. 1830-1837. 131: 2011: Dermatol Invest J . tive factor againstSLE susceptibility in European edicting myocardial infarction andcardiacdeath myocardial edicting K: a tool K: atool whole-genomeset for association and esource genetics, population for controls and a et al. A high-resolution copy-number variation al.A et high-resolution cher P J, El Mokhtari N E, Schreiber S. PopGen: Assessment of clinically silent atherosclerotic silent clinically of Assessment

Moskvina V, Holmans P, KSchmidt M, Craddock N. Design case-controlsof studies with unscreened controls. Ann Hum Genet 2005: 69: 566-576. 39. causes al.mutation regulatory A,Sundstrom E, et Acis-acting G, Golovko Pielberg Rosengren premature hairgraying andsusceptibility to melano 38. bulb? Yale J Biol Med 1993: 66: 541-554. abnormal by class exposed MHC hair anagen Iexpression , inthe melanogenesis-related Paus R,37. Slominski McElwee K J,609-626. Gilhar A, Tobin D J, et A, al. Paus R, Nickoloff What BJ, Ito T. A 'hairy' privilege. Trends Immunol causes 2005: 26: Czarnetzki 32-40. alopecia36. areata? Exp Dermatol Gilhar A, Etzioni A, Paus R. Alopecia areata. N Engl 2013: J Med 2012: 366: 1515-1525. 22: 35. B M. Is34. al protein duplication: dosage after I evolution gene expression B.Complexity Rogozin of 516508. 2014: Int 2014: Res Genet rebalancing. 33. 234. 229- 181: 2013: Endocrinol Comp Gen Verasper moseri. barfinflounder from emerging roles change: between A.Interrelation Y, Saito Takahashi T, Yamanome K, Kobayashi Y, Mizusawa color body inphysiological hormone andmelanin-concentrating hormone melanocyte-stimulating 32. association after applying our relaxed filter criteria. using Fisher'sexact test (two-sided). “-“ interval; dup, duplication; del, deletion. All P values and odds ratios were calculated according to NCBI built 36/hg18. p, P value; OR, odds ratio; CI, 95% confidence Only atleast nominally significant Pvalues are displayed. All positions given Sixassociated regions identified in genome-wide CNV analysis Table 1 This article is protected by copyright. All rights reserved. copyright. This articleis protectedby relaxed filter criteria (11,820,879 – 11,909,732); associated region for dup+ del, associated region is reduced after applying our h soitdrgo ains(=8)cnrl n130 p- controls (n=1,340) patients(n=585) associated region chr 62,5,2-231191dp0dp008- (0.9- 0.028 0dup 1dup 20 21,856,623-22,331,199 16 9,8,8-9,1,6 u u .2 - (0.9- 0.028 Accepted - (1.5- 0.008 0dup 2dup 4dup 9 0dup 100,402,745-100,745,258 6 190,385,789-190,515,967 4 Article 14,729,684-14,755,487 11,820,879-11,905,692 11,810,083-11,909,732 3 2 1 u u .4 - (0-0.9) 0.041 intergenic between 5.4 (1.2-32.4) 0.011 6.2 (1.5-36.2) 0dup 0.005 0 dup 0dup 1 dup 2 del 0 del 0 del 2 7 del 5 del 5 del 7 0 del 11 del 11 0 del 0 del 0 del 0 del 0 del 0 del 3 dup+del OR (CI)-dup+del p-dup OR (CI)-dup p-del OR (CI)-del affected gene affected (CI)-del OR p-del (CI)-dup OR p-dup (CI)-dup+del OR dup+del refers to null observation in one group. ma inthehorse. Nat Genet 2008: 40: 1004-1009. opecia areata anautoimmune-response against ∞ ) 2

associated region del; for ∞ ) ∞ intergenic between ) EEF2K, POLR3E, CDR2 UQCRC2, PDZD9, C16orf52, VWA3A, MACROD2 MCHR2, MCHR2-AS1 MCHR2, and LINC01262 3 no TYRP1 TYRP1 LINC01060 1 and PTPRD

was generated using UCSCgenome browse SYBR Green verification were designed (black vertical lines). This schematic drawing associated region (black bar) spanned approximately 342.5kb. Five primer pairs for did control inthe duplication The bar). blue Duplications were detected in four patients (dark blue bars) andin onecontrol (light 6q16.3 in Duplications 1Figure legends Figure This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Article r (http://genome.ucsc.edu/); NCBI36). not fulfill the primary filter criteria. The criteria. The primary filter the fulfill not