Genes and Immunity (2010) 11, 531–541 & 2010 Macmillan Publishers Limited All rights reserved 1466-4879/10 www.nature.com/gene ORIGINAL ARTICLE Peripheral blood gene expression in alopecia areata reveals molecular pathways distinguishing heritability, disease and severity AB Coda, V Qafalijaj Hysa, K Seiffert-Sinha and AA Sinha Center for Investigative Dermatology, Division of Dermatology and Cutaneous Sciences, College of Human Medicine, East Lansing, MI, USA Alopecia areata (AA) is an autoimmune hair loss disorder in which systemic disturbances have been described, but are poorly understood. To evaluate disease mechanisms, we examined gene expression in the blood of defined clinical subgroups (patchy AA persistent type, AAP, n ¼ 5; alopecia universalis, AU, n ¼ 4) and healthy controls (unaffected relatives, UaR, n ¼ 5; unaffected non-relatives, UaNR, n ¼ 4) using microarrays. Unsupervised hierarchical clustering separates all four patient and control groups, producing three distinct expression patterns reflective of ‘inheritance’, ‘disease’ and ‘severity’ signatures. Functional classification of differentially expressed genes (DEGs) comparing disease (AAP, AU) vs normal (UaR) groups reveals upregulation in immune response, cytokine signaling, signal transduction, cell cycle, proteolysis and cell adhesion- related genes. Pathway analysis further reveals the activation of several genes related to natural killer-cell cytotoxicity, apoptosis, mitogen activated protein kinase, Wnt signaling and B- and T-cell receptor signaling in AA patients. Finally, 35 genes differentially expressed in AA blood overlap with DEGs previously identified in AA skin lesions. Our results implicate innate and adaptive immune processes while also revealing novel pathways, such as Wnt signaling and apoptosis, relevant to AA pathogenesis. Our data suggest that peripheral blood expression profiles of AA patients likely carry new biomarkers associated with disease susceptibility and expression. Genes and Immunity (2010) 11, 531–541; doi:10.1038/gene.2010.32; published online 10 June 2010 Keywords: alopecia areata; alopecia; microarray; pathogenesis; genetics Introduction highlighting the role of immune dysfunction as a crucial feature in AA pathogenesis. Alopecia areata (AA) is an autoimmune skin disorder The etiopathogenesis of AA is not completely under- typically characterized by the spontaneous onset of stood. The most widely accepted hypothesis describes patchy hair loss of the scalp. Although 80% of patients AA as a T-cell mediated autoimmune response targeting present with a minimal number of patches (AA; patchy an unknown antigen in anagen-stage hair follicles.8,9 persistent, AAP or transient, AAT), 7% of cases progress It has also been postulated that compromise of immune in clinical severity to total scalp-hair loss (alopecia totalis; privilege in normal hair follicles, mediated in part by AT) or total body hair loss (alopecia universalis, AU). interferon-gamma (INF-g),10 has a role in the suscep- The prevalence of AA in the United States is reported to tibility and pathogenesis of AA.11,12 Considerable evi- be 0.1–0.2% in the general population, with a lifetime risk dence indicates that genetic elements have a significant of 1.7%.1,2 The histopathological hallmark of AA is the role in the etiology of AA, as evidenced by the recent presence of a perifollicular and intrafollicular lympho- identification of a number of suggested susceptibility cytic infiltrate. T lymphocytes (predominantly CD4 þ loci,13 the high incidence of positive family history in cells), as well as Langerhans cells, macrophages and affected individuals14,15 and high twin concordance natural killer (NK) cells are found in the dermal papilla rates.16–20 Many epidemiologic studies have shown and matrix of the involved hair follicles.3,4 Lymphocytic a higher prevalence of autoimmune diseases, such as infiltration of the anagen hair bulb and dermal papilla is vitiligo, thyroid disease and collagen vascular disease, in accompanied by aberrant expression of human leukocyte AA patients and unaffected relatives (UaR)21,22 suggest- antigen class I and class II antigens and intercellular ing a genetic predisposition to autoimmunity associated adhesion molecule-1 on the follicular epithelium,5–7 with familial background of AA.23 These findings imply that systemic disturbances contribute to this T-cell Correspondence: Dr AA Sinha, Division of Dermatology and dependent, organ-specific disease. Overall, AA is a Cutaneous Sciences, Michigan State University, 4179 Biomedical complex, polygenic disease likely initiated by the and Physical Sciences Building, East Lansing, MI 48824-1320, USA. E-mail: [email protected] interactions between genetic and environmental factors Received 1 December 2009; revised and accepted 20 April 2010; triggering the immune dysfunction that results in published online 10 June 2010 autoimmune hair loss. Peripheral blood gene expression in alopecia areata AB Coda et al 532 Global gene-expression profiling by DNA microarray Results has become a valuable tool for the study of autoimmune and/or inflammatory diseases.24 Microarray studies on Unsupervised hierarchical clustering separates sample groups organ-specific autoimmune diseases have advanced our by familial inheritance, absence or presence of disease and understanding of autoimmune disease pathogenesis, and clinical subtype revealed targets for clinical biomarkers, prognostic We used Affymetrix U133 Plus 2.0 microarray chips factors and subtype distinction for individualized ther- (Affymetrix, Santa Clara, CA, USA) to generate gene apy.25–27 Our recent microarray analysis in AA patient expression profiles from buffy coat samples obtained skin samples,28 along with a previous microarray study in 5 alopecia areata, patchy persistent (AAP) patients, in murine and human AA skin,29 help to substantiate 4 alopecia universalis (AU) patients, 5 unaffected relatives AA as a Th1-cell-mediated autoimmune disorder with (UaR) and 4 unaffected non-relatives (UaNR) (Table 1). To a late secondary humoral response. No study, however, explore the similarities and differences in global gene has previously used gene microarray technology as a expression patterns among the samples without apriori large-scale screening tool to investigate gene regulatory knowledge of sample identity, we used an unsupervised alterations in the peripheral blood of AA patients. As hierarchical clustering method using those 551 probe sets with many organ-specific diseases, the systemic envi- with the highest variation across samples. This unbiased ronment in AA patients is likely to be critical in the approach was used to determine the extent to which global generation and regulation of the immune response gene expression analysis can be used to distinguish clinically and inflammatory cascade that is ultimately directed to defined subgroups (class prediction), as well as identify novel hair follicles. or unexpected gene expression patterns (class discovery). The aims of the present study were to (1) establish We report that unsupervised hierarchical clustering peripheral gene expression profiles associated with AA reveals distinct separation of AA patients from controls and AA subgroups and (2) examine AA-associated (Figure 1). Upon further evaluation, three unique transcriptional signatures to identify functional path- patterns emerge. First, our data reveal that AA patients ways and genes relevant to AA. On the basis of the and first-degree relatives of AA patients (AAP, AU and global microarray analysis in AA patients and healthy UaR) cluster distinctly from the healthy controls with no control blood, we report three distinct transcriptional familial connection to AA (UaNR), conceivably produ- signatures relevant to (i) the genetic predisposition to cing an ‘inheritance’ signature. Second, within this disease, (ii) the clinical expression of disease and (iii) the ‘inheritance’ cluster, AAP and AU samples further degree of phenotypic severity. Evaluation of each of these separate from UaR controls, with the exception of a signatures implicates several immune-related and single AU (‘AU 4’) sample, to produce a ‘disease’ signaling pathways in AA pathogenesis. signature. Third, within the ‘disease’ cluster, AAP Table 1 Demographic data for study subjects Sample code Race Age (years) Gender Disease type Length of disease (years) Unaffected non-relatives (UaNR) UaNR 1 Caucasian 40 F NA NA UaNR 2 Caucasian 32 F NA NA UaNR 3 Hispanic 33 F NA NA UaNR 4 Asian 37 M NA NA Unaffected relatives (UaR) UaR 1 Caucasian 27 F UaR NA UaR 2 Caucasian 57 M UaR NA UaR 3 Caucasian 22 M UaR NA UaR 4 African–American 42 F UaR NA UaR 5 Caucasian 48 F UaR NA Alopecia areata, patchy persistent (AAP) AAP 1 Caucasian 34 F AAP 9 AAP 2 Caucasian 25 F AAP 21 AAP 3 Hispanic 37 F AAP 13 AAP 4 African–American 42 F AAP 1 AAP 5 Caucasian 54 M AAP 29 Alopecia universalis (AU) AU 1 Caucasian 66 F AU 47 AU 2 Caucasian 62 M AU 47 AU 3 Hispanic 31 F AU 26 AU 4 Caucasian 23 M AU 11 Abbreviations: AAP, alopecia areata, patchy persistent; AU, alopecia universalis; F, female; M, male; UaNR, unaffected non-relatives; UaR, unaffected relatives. Genes and Immunity Peripheral blood gene expression in alopecia areata AB Coda et al 533 samples split from the AU samples to define a ‘severity’ signature. It should be noted, however, that one AAP patient (AAP 5) did share gene expression characteristics with control samples. It is noteworthy that this patient was the only one in this study who was undergoing