Transcriptional Profiling in Alopecia Areata Defines Immune and Cell

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Genomics 96 (2010) 146–153

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Genomics

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Transcriptional profiling in alopecia areata defines immune and cell cycle control related genes within disease-specific signatures

Raghunandan Dudda Subramanya, Alvin B. Coda, Animesh A. Sinha ⁎

Center for Investigative Dermatology, Division of Dermatology and Cutaneous Sciences, College of Human Medicine, East Lansing, MI, USA

article info abstract

Article history: Alopecia areata (AA) is a non-scarring inflammatory hair loss disease with a complex autoimmune Received 18 January 2010 etiopathogenesis that is poorly understood. In order to investigate the pathogenesis of AA at the molecular Accepted 8 May 2010 level, we examined the gene expression profiles in skin samples from lesional (n=10) and non-lesional sites Available online 28 May 2010 (n=10) of AA patients using Affymetrix Hu95A-v2 arrays. 363 genes were found to be differentially expressed in AA skin compared to non-lesional skin; 97 were up-regulated and 266 were down-regulated. Keywords: Functional classification of the differentially expressed genes (DEGs) provides evidence for T-cell mediated Alopecia Microarray immune response (CCL5, CXCL10, CD27, ICAM2, ICAM3, IL7R, and CX3CL1), and a possible humoral Pathogenesis mechanism (IGHG3, IGHM, and CXCR5) in AA. We also find modulation in gene expression favoring cellular proliferation arrest at various levels (FGF5, FGF18, EREG, and FOXC2) with apoptotic dysregulation (LCK, TNF, TRAF2, and SFN) and decreased expression of hair follicle structural proteins. Further analysis of patients with AAT (b1 year duration, n=4) and AAP (N1 year duration, n=6) of disease revealed 262 DEGs distinctly separating the 2 groups, indicating the existence of gene profiles unique to the initial and later stages of disease. © 2010 Elsevier Inc. All rights reserved.

Introduction (DR11), and DQ3 in AA and HLA-DR5 (DR11) and DQ7 in long- standing AT/AU [3]. Within the context of genetic predisposition, the Alopecia areata (AA), a non-scarring inflammatory hair loss onset of AA may be triggered by several possible mechanisms disease, is a complex disease determined by multiple genetic loci. It including environmental factors [including a viral etiology] [5,6] and involves immune dysregulation leading to hair loss with an estimated psychological stress [7,8]. Suggestion for autoimmune disease lifetime incidence rate of 1.7% in the general population [1].AA mechanisms stems from a peribulbar and intrafollicular mononuclear presents most frequently as patchy hair loss on the scalp (and cell infiltrate (consisting of primarily CD4+ and CD8+ cells as well as sometimes other hair-bearing areas) that is either transient (AAT) or macrophages and Langerhans cells) which is closely associated with persistent (AAP), but may progress into total scalp hair loss, alopecia dystrophic anagen stage hair follicles [9,10], and the increased totalis (AT), or even complete body hair loss, alopecia universalis incidence of other autoimmune diseases (including thyroid disorders, (AU). pernicious anemia and vitiligo) observed in probands as well as While the exact etiopathogenesis of AA remains unclear, there is unaffected relatives [4,11]. considerable evidence suggesting autoimmune [2], genetic [3], and Despite strong evidence supporting a genetic basis for AA, environmental factors in disease pathology. Various signaling mole- including a recent genomewide scan linkage analysis [12], there are cules mediate the transition through phases of the hair cycle involving no confirmed genetic susceptibility genes or markers of disease anagen (growth), catagen (regression), telogen (rest) and exogen induction and progression. The extent and mechanisms of genetic (shedding). The complex interplay between these factors may also contribution to the etiopathogenesis of AA remain elusive [3].We account for the extensive clinical heterogeneity seen in the disorder. have examined gene expression directly in the skin tissues of patients Evidence for a genetic basis in the pathogenesis of AA is derived from with AA to search for molecular links within the cascade of genetic the increased frequency of family history (reported by 20 to 42 % of elements and immunologic interactions that coordinate the initiation patients) [4], a 55% concordance rate among monozygotic twins, and and progression of the disease process. We report patterns of gene increased associations of specific HLA alleles, including HLA-DR4, DR5 expression in 10 alopecia patients using oligonucleotide microarrays that distinguish AA affected vs. unaffected skin and identify distinct signatures of gene expression that further differentiate early vs. later stages of disease. Examination of specific sequences within these ⁎ Corresponding author. 4179 Biomedical and Physical Sciences Building, East Lansing, MI 48824-1320, USA. Fax: +1 517 353 8957. signatures provides evidence for immune activation, cell cycle E-mail address: [email protected] (A.A. Sinha). disruption, and apoptosis-related mechanisms of disease.

Results ated by the DAVID database and PubMed literature searches (Fig. 1). 62 differentially modulated genes were found to be involved in immune Differential gene expression in AA lesional skin and inflammatory response. Among them we found evidence for active T-cell immunity at the site of pathology. For instance, genes CD27, A total of 20 human skin biopsies, lesional (n=10) and non- ICAM2 (CD102), and ICAM3 (CD50) are reported to be essential for lesional (n=10) skin from patchy alopecia areata patients, were used dendritic cell–T-cell adherence and T-cell activation [14].Theup- for gene expression analysis (Table 1). After normalization per gene regulation of LCK and fractalkine (CX3CL1) suggest an IFN-γ-mediated per chip (see Materials and methods), statistical comparison of Th1 type of response, while the up-regulation of IRF7 and ISG15 suggest lesional alopecia skin and non-lesional control skin returned 363 a role for IFN-alpha in lesional AA skin tissue. ISG15 is one of the earliest sequences differentially expressed between the 2 groups at Pb0.05. and most predominant proteins to be induced in mammals following We include all DEGs with a statistically significant differential fold IFN-alpha/beta stimulation [15], while IRF7 is essential for the change in our analysis since even minimal differences in gene transcriptional activation of IFN-alpha genes [16]. Up-regulation of expression may be biologically significant [13]. 97 DEGs were up- CD4 antigen (an accessory protein for MHC class-II antigen/T-cell regulated in the lesional samples (fold changes ranging from +1.1 to receptor interaction) [17] and down-regulation of MRC2 (a mannose +3.0) and 266 genes were down-regulated (fold changes ranging receptor on macrophages involved in endocytic protein clearance) [18] from −1.1 to −6.7) (Supplemental Table 1). Gene KRT81 (keratin suggest an enhanced antigen presentation activity in the lesional AA. 81), located on the chromosome 12, showed the greatest fold change Along with these indications of an enhanced T-cell response, we with a down-regulation of 6.7-fold in AA skin. Several other acidic and also observe an up-regulation of CXCR5 (CD185), a cytokine receptor basic hair keratins were also found to be significantly down-regulated that is essential in B-cell migration and localization. IGHG3 (immu- (KRT14, KRT31, KRT32, KRT33A, KRT33B, KRT34, KRT35, KRT4, KRT83, noglobulin heavy constant-γ-3/G3m marker; +2.7 fold) and IGHM KRT85, KRT86, and KRTAP26-1). The DEGs with the greatest up- (immunoglobulin heavy locus; +1.7 fold) were also up-regulated in regulated fold change in AA skin were ADAM3A (Adam metallopepti- AA skin compared to normal controls, suggesting active immuno- dase domain 3a, +3.0 fold) and KLRD1 (Killer cell lectin-like receptor globulin production in lesional AA skin. subfamily D member 1, +2.8 fold). Another 27 differentially regulated genes participate in cell cycle To confirm the direction of the change in gene expression observed processes, including 7 genes involved in mitosis at various points and by the microarray analysis, the fold changes for 4 genes – KRT81, KRT86, 6 involved in DNA repair and replication. Among the genes involved in HSPA2 and DTD (SLC26A2) – between paired lesional and non-lesional mitotic processes are FGF5, FGF18, cyclin M2, CDC27, SPC25, EREG, samples determined by microarray data and real-time RT-PCR were and PPP2R1B. Positive signals for cell division like PPP2R1B, CDC27, compared (Supplemental Fig. 1). We included both up- and down- FGF5, FGF18, and cyclin M2 are down-regulated in AA skin. Six DEGs regulated, as well as minimally and highly differentially expressed participating in DNA repair and replicative processes were down- sequences (as determined by microarray) for analysis (KRT81: −11 regulated, including RAD51L3, RPA3, SFN (stratifin), DNA2, and fold, KRT86: −4.9 fold, HSPA2: −1.6 fold and DTD: +2.0 fold). The TERF2. TERF2 is known to protect against end-to-end fusion of relative fold changes for these 4 genes obtained by real-time PCR for the chromosomes and plays a role in successful progression through the skin samples analyzed corresponded well with fold changes determined cell division cycle. Overall, these observations suggest inhibition of by microarray analysis. Correlation coefficient (r2) observed between cellular proliferation in AA affected skin. the 2 methods was 0.9455 for gene KRT81, 0.9899 for gene KRT86, Genes encoding heat shock and related proteins (HSPA2 and 0.7681 for gene HSPA2 and 0.6471 for DTD (SLC26A2), thus validating HSPA5) were found to be down-regulated in AA, as well as the stress- our gene chip data. related gene MT4 (metallothionein 4), which may regulate zinc metabolism during differentiation of stratified epithelia [19]. Genes participating as signals in hair follicle morphogenesis and cycling, AA lesional skin signature reveals differential expression of genes FGF5, FZD1, and BMP2, were down-regulated in AA lesional skin involved in immunity, cell cycle, apoptosis, and structural hair follicle [20,21]. The reduced expression of these genes suggests arrested genes follicular development and cycles. Seventeen down-regulated DEGs code for structural and cytoskeletal proteins, including several acidic To evaluate the alterations in gene expression that differentiate and basic hair keratins and tubulins. We additionally report 24 DEGs lesional vs. non-lesional AA skin, we annotated the 363 DEGs into as miscellaneous since the exact functions of these genes are not known functional categories and performed pathway analyses gener- identified yet. Finally, it should be noted that the functional annotations reported overlap for several genes. For instance, the gene EREG (epiregulin) is involved in immune system mechanisms, Table 1 although the primary functions include stimulating localized cell Subjects participating in this study. Median age=38 years; median duration of b N proliferation and/or angiogenesis. disease=3 years; early stage ( 1 year duration) =4 subjects, late stage ( 1 year fi duration) =7 subjects; Caucasians=6 subjects, African Americans=1 subject, His- As discussed above, a previous report identi ed several mouse and panic=4 subjects. All patients were off therapy for more than 4 weeks and none had human genes through microarray which may be relevant to disease documented therapy at the site of biopsy. Patient AA3006, however, was noted to have pathogenesis [22]. In comparing our list of DEGs with the previous completed a course of intralesional steroid at the site of biopsy 4 weeks prior to skin study, we found 11 overlapping DEGs with corresponding dysregula- sampling. tion in diseased skin compared to normal skin. These genes include six Patient Age (yr) Sex Ethnicity Type down-regulated hair keratin genes and up-regulation of immune AA1008 38 M Caucasian AAT system-related genes (LCK and ISG15). AA3001 37 F Hispanic AAT Overall, functional classification DEGs suggest active immune AA3007 39 F Hispanic AAT mechanisms of disease; there is evidence for active antigen AA3008 38 F Hispanic AAT presentation, T-cell (Th1) mediated immunity, and humoral activity. AA1005 35 F African-American AAP AA1006 42 F Caucasian AAP Further, we report disruption of cell cycle pathways within lesional AA1007 38 F Caucasian AAP skin, with inhibition of cellular proliferation and up-regulation of AA3006 39 F Hispanic AAP apoptosis. Finally, there is evidence for an arrest of hair follicle cycling AA1009 56 M Caucasian AAP and regeneration with the resultant down-regulation of hair-related AA1010 28 F Caucasian AAP keratins. Fig. 2 provides a visual representation of the sequence of 148 R.D. Subramanya et al. / Genomics 96 (2010) 146–153

Fig. 1. Differential expression of genes reveals ontologies and pathways active in lesional skin. 363 genes were found to be differentially expressed in AA lesional skin as compared to non-lesional skin with a P-valueb0.05, followed by functional annotation by DAVID. Bars indicate fold change levels along the x-axis. pathogenetic events and correlative gene expression changes that areata, patchy transient (b1 year; AAT) (n=4) and alopecia areata, may contribute to hair loss. patchy persistent (N1 year; AAP) (n=6). To explore the similarities and differences in global gene expression patterns among the samples Comparison of transient vs. persistent AA lesional skin reveals without a priori knowledge of sample identity, we employed an differential expression of epidermal differentiation, cell proliferation, unsupervised hierarchical clustering method using the 98 probe sets and apoptosis with the highest variation across samples (Fig. 3). This unbiased approach was able to differentiate transient (AAT) vs. persistent To better understand mechanisms related to disease progression, (AAP) lesional skin, indicating that gene expression analysis may be we next analyzed gene expression differences between alopecia able to classify samples by disease evolution. R.D. Subramanya et al. / Genomics 96 (2010) 146–153 149

Fig. 2. Possible pathogenetic events supported by microarray data. Immune dysregulation underlying arrested follicular development, ultimately leading to loss of hair keratins and clinical hair loss. Genes up-regulated in AA are shown in red and those down-regulated are shown in blue.

Furthermore, AA lesional samples showed a differential expression disease [26]. Our microarray data support the concept of immune of 262 genes at Pb0.05 depending on duration of disease; 129 dysregulation in AA. Nearly 22% of the DEGs encode immune sequences were up-regulated in AAT and 133 were up-regulated in regulators and pro-inflammatory proteins, providing evidence for AAP samples (Supplemental Table 2). GJB3 (connexin 31) had the both cellular and humoral immunity involvement in AA. highest fold change in AAT (+11.8); it is expressed in the stratum Perivascular, peribulbar, and intrabulbar inflammatory infiltrates granulosum of the epidermis with a suggested role in late keratino- in areas of hair loss are comprised of T cells (CD4NCD8), macrophages, cyte differentiation [23]. On the other hand, CD72 antigen (Lyb-2) had B cells, and NK cells, with increased infiltration in the early, the highest fold change in AAP (+8.3); it plays a key role in B-cell progressive phase of disease as compared to the later, stabilized proliferation and differentiation by associating with CD5 [24]. LOR phase. Thus, it is likely that infiltrating lymphocytes contribute to the (loricrin), FLG (filaggrin), KLK7 (kallikrein 7), and KLK13 (kallikrein differential gene expression differences found between lesional and 13) are all down-regulated in AAP, perhaps representing markers of non-lesional skin. Accordingly, we do find up-regulation of genes such disease progression from early to later stages. Finally, several as LCK, a lymphocyte-specific tyrosine kinase, and CD4 surface antigen fibroblast growth factors (FGF6, FGF9, FGF13 and FGF18) were up- in lesional that demonstrate active T-cell specific gene expression. regulated in AAP. While the role of FGFs on hair follicles are poorly Cytokines (CX3CL1, CXCL1, CCL5, and CXCL10) involved in cellular understood, studies have shown a suppression in follicle initiation and immunity were overexpressed in AA skin. CX3CL1 is not only induced development causing delayed hair cycling, as well as a decreased by IFN-γ, but also acts as an amplification circuit of polarized Th1 anagen:telogen ratio [25]. responses [27], indicating a Th1 type of response in AA skin. Increased expression of CCL5 (RANTES), an IL-1 beta and TNF-alpha inducible Discussion gene, also suggests Th1 pathway activity [28]. Up-regulation of ICAM2 and ICAM3 in AA skin supports a mechanism allowing enhanced In this study we examined gene expression profiles in alopecia antigen-specific immune responses through increased dendritic cell areata to define disease-specific signatures that may advance our (DC) migration and DC–T-cell interaction [29] in lesional skin. Our understanding of the pathogenesis and progression of disease. This observation in human AA skin tissue is consistent with the study provides a foundation for unraveling the, so far, poorly defined observation of a Th1 response in the C3H/HeJ murine AA model [22]. pathogenetic mechanisms operative in AA. Due to the absence of MHC class I (MHC I) expression in the distal A previous study [22] reported gene expression patterns in a total hair follicle epithelium, it has been suggested that the human anagen of 4 patients [2 AT, an AU, and an androgenetic alopecia patient], along hair bulb is a site of immune privilege [30]. Our data reveals decreased with an analysis of induced alopecia areata in the C3H/HeJ murine expression of TGFB1 (transforming growth factor, beta 1), a potent model using Affymetrix mouse 11K and HuFL gene chips (6800 suppressor of MHC I expression, in lesional skin of AA patients. Our genes). Our investigation using Affymetrix Hu95A chip (12,627 data also support a possible role for IFN-α in AA, evidenced by the up- genes) focused on patients with confirmed AA and was performed regulation IFN-α related genes (ISG15 and IRF7) in AA lesional tissue on a larger sample size (n=20). In comparing gene lists, only 11 DEGs [31]. Furthermore, lesional AA skin showed increased expression of were found to be common between the studies. These 11 overlaps TLR3, which, in conjunction with IFN-α, triggers MHC I up-regulation reveal an analogous up-regulation of immune system-related genes and subsequent T-cell autoreactivity [32]. IFN-α inducible pathways and down-regulation of hair keratin genes in both datasets that have been recently documented in lupus erythematosus and have supports an active immune process and loss of structural proteins been postulated to play a role in breaching immune tolerance that essential to hair follicles in AA. The overall paucity of matching genes may be related to the regulation of dendritic cell maturation [33]. between the studies may be a function of the smaller sample size and Thus, impaired immunosuppression and enhanced expression of gene chip in the previous study, and/or patient heterogeneity. MHCI in lesional skin likely contributes to the collapse of immune The suggestion that AA is an autoimmune disease is derived from privilege at the hair follicle, and thus, recognition of self-antigens several sources of evidence [2], including the intrafollicular infiltra- leading to overt autoimmune disease. tion of anagen follicles by activated T cells, macrophages, and Although our data clearly support a cellular immune response, Langerhans cells, as well as the efficacy of immunomodulators on there has also been data to indicate that humoral mechanisms may 150 R.D. Subramanya et al. / Genomics 96 (2010) 146–153

contribute to AA. We found that CD27 is up-regulated in lesional skin suggesting active T-cell mediated B-cell activation and immunoglobulin synthesis. The participation of humoral mechanisms is further supported by the increased expression of IGHG3 (immunoglobulin heavy constant-γ-3) and IGHM (immunoglobulin heavy constant mu) in lesional AA skin compared to normal skin. Taken together, our data supports the notion that humoral mechanisms may be at play alongside cellular immunity. Nevertheless, whether antibody response in AA is relevant in disease induction and/or progression or simply represents an immunological epiphenomenon is at this point unclear. Despite the clear importance of lymphocyte activation in AA, the identification of autoantigen triggers of disease and/or targets of autoimmune attack has remained elusive. Our data do, however, provide direct evidence that an active antigen processing and presentation is operational in AA lesional skin. MRC2 (also called ENDO180), a marker of the mannose-receptor family shown to facilitate phagocytosis, is suggested to play a role in tethering particles and engaging a classical phagocytic receptor to drive phagocytic machinery [34]. The down-regulation of MRC2 seen in AA perhaps suggests decreased antigen clearance from the matrix of the skin, which may promote autoimmune recognition and subsequent activation. Alternatively, the overexpression of MRC2 in non-lesional sites may imply a protective mechanism through enhanced antigen clearance. While immune attack is clearly evident, the final effector mechanisms that result in target tissue damage in AA are not known. Our study provides evidence for a global inhibition of cellular proliferation in skin. There is down-regulation of genes involved in mitosis, including EREG (epiregulin), FGF18 and PPP2R1B. Epiregulin stimulates keratinocyte proliferation [35], while FGF18 and PPP2R1B (via down- regulation of MAPK kinase pathway) are known to relay signals for cellular proliferation [36]. Down-regulation of CDC27 suggests a block in cell cycle progression through disruptions in mitotic spindle formation and anaphase induction. We also found a potential impairment of DNA repair based on down-regulation of TERF2 (telomeric repeat binding factor 2) and SFN (stratifin). SFN acts at the G2 check point, preventing mitotic death with its inactivation influencing telomere behavior and ionizing radiation-induced chro- mosomal instability [37]. Decreased expression of SFN implicates increased DNA errors in daughter cells which can lead to “mitotic- catastrophe” and apoptosis [38]. The PAK2 gene (p21 protein (Cdc42/ Rac)-activated kinase 2) was also down-regulated in lesional AA samples. PAK2, expressed in response to stress stimulants, stimulates cell survival by inhibiting phosphorylation and inhibition of pro- apoptotic genes [39]. To add to this, the down-regulation of heat shock proteins (HSPA2 and HSPA5) also suggests reduced inhibition of apoptosis [40]. These findings parallel the systemic up-regulation of apoptosis-related genes in AA patient blood [51]. Thus, we show evidence for an overall inhibition of cellular proliferation and execution of apoptosis in AA skin. Whether apoptosis in AA is occurring in infiltrating immune cells or hair follicular cells is unclear. The specific localization of apoptotic events and their role in disease induction and/or target damage remains to be determined. We also find evidence for disruption of the follicular developmental cycle and follicular morphogenesis in AA skin. Actions of canonical WNT (Wingless-type MMTV) proteins in the skin are essential for generation of the first dermal message in hair follicle formation, even in postnatal life [41,42]. FZD1 (frizzled homolog 1), up-regulated in AA lesional skin, is a receptor for both canonical and non-canonical WNT signaling

Fig. 3. Unsupervised hierarchical clustering differentiates early vs. late AA skin. Unsupervised hierarchical clustering of the 98 genes of highest variance across samples from 10 AA lesional and non-lesional skin samples. Each row signifies one of the 98 genes, while the columns represent samples in processed by a Euclidean distance algorithm in dChip. Expression value intensities are illustrated by the color scale with a range of −3 to 3 on a log2 scale. R.D. Subramanya et al. / Genomics 96 (2010) 146–153 151 pathways that is maintained at the bulbous peg stage in early anagen, suggest that the self-directed immune response in AA leads to a block with highest expression in cells of the developing outer root sheath [21]. in the progression of hair follicular cycle, perhaps related to increased While the exact role is uncertain, this expression may reflect poorly apoptosis in the follicular unit with a cessation of morphogenesis such coordinated hair follicle cycling, and thus, follicular arrest. FGF18, highly that the formation of new hair follicles is inhibited. expressed in hair follicles and capable of inducing anagen from telogen stage hair follicles [43], is a direct target and known marker of the Wnt Materials and methods signaling pathway that is down-regulated in AA lesional skin. Recent work from our group in AA patient blood also showed down-regulation Tissue procurement and handling of the Wnt/β-catenin signaling pathway through up-regulated Wnt suppressors [51], suggesting that a systemic overexpression of Wnt/β- Subjects were identified to have AA based on established clinical catenin signaling inhibitors may predispose an individual to developing and histological criteria [50] at the Dermatology outpatient clinic of AA. Finally, FGF5, important for determining the timing of active hair New York Presbyterian Hospital – Cornell University. Informed growth (anagen) and transition into follicular regression (catagen) [44], consent was obtained in each case (IRB# 0998-398) and two punch is down-regulated in AA lesional skin. Taken together, our data provide biopsies of 6 mm each were performed on each of 10 patients under solid support for dysfunctional coordination of follicular cycling and local anesthesia (1% lidocaine with 1/100,000 epinephrine). The skin development. tissues so procured corresponded to lesional alopecic skin to Genes related to cellular structure and encoding cytoskeleton represent the diseased sample and paired site-matched non-lesional components form a substantial group of the DEGs observed in AA. skin as a normal control sample from the same individual. Specimens Decreased expression of hair structure-related genes is likely to were snap-frozen in liquid nitrogen immediately after biopsy. Ten represent the final marker of effective hair loss. Decreased expression patients had alopecia areata (AA) and one had alopecia universalis of KRT81 (basic hair keratin 1) and KRT86 (basic hair keratin 6) are (AU) (Table 1). Thus, a total of 20 biopsies (10 lesional and 10 non- also seen in Monilethrix [45], where patients present with alopecia and lesional) were used for gene expression analysis (Table 1). We further perifollicular hyperkeratosis (OMIM IDs: 602153 and 601928 respec- classified the 10 AA patients to be AAT (n=4) (within 1 year of the tively). Among the other structural/cytoskeleton related sequences onset of first patch), and AAP (n=6) (greater than 1 year after disease that are down-regulated are several acidic and basic hair keratins and onset) based on published investigational assessment guidelines [50]. alpha- and beta-tubulins. The CRIP2 gene (previously reported to be All patients were off therapy for at least 4 weeks and none had one of the keratin associated proteins possibly involved in the human documented therapy at the site of biopsy (subject AA3006 completed trichothiodystrophy) [46] and another skin structure-related gene a course of intralesional steroid therapy at the site of biopsy 4 weeks KRT14 (whose mutation was noted in epidermolysis bullosa) were prior to skin sampling). also down-regulated in AA skin [47]. In addition to defining a disease-associated signature, we Target preparation compared the AA affected samples that were obtained from subjects within 1 year of AA onset (AAT) and those from more than 1 year Total RNA was prepared from each sample using TRIzol reagent as onset (AAP) in order to understand the kinetics of molecular events recommended by the manufacturer (GIBCO, Carlsbad, CA) and that occur in the different stages of AA. Unsupervised hierarchical subsequently purified using the RNeasy Mini kit (Qiagen, Chatsworth, clustering analysis of expression profiles between AAT and AAP CA). Quantity and quality of total RNA were assessed by spectropho- patients clearly distinguishes the samples for duration (AAT vs. AAP), tometry. 16 µg total RNA per sample was used to synthesize cDNA but also separates samples based on ethnicity. The effect of patient serving as a template for generation of biotinylated cRNA. Fragmented variables, such as ethnicity, on gene expression in human skin is cRNA was hybridized to Human Genome U95A arrays (Affymetrix, poorly understood and likely to be subtle [48,49], thus not obscuring Santa Clara, CA) for 16 h at 45 °C. Chips were then washed, stained the AA disease process from valid analysis. Ultimately, future studies and scanned as per GeneChip Expression Analysis Manual protocol with larger cohorts will be required to validate the presence of distinct (Affymetrix). gene expression profiles based on disease duration. Evaluation of genes that are preferentially expressed in the initial Analysis stages of the disease focuses examination on events related to disease induction and early pathogenesis. Of specific interest are: FGFs 6, 9, Affymetrix Human Genome U95A microarrays were used for 13, and 18, leukocyte membrane antigen (CD300A), and IFN-alpha 21, hybridization and screening of 12,627 genes. Data obtained (by which are up-regulated in AAP, and structure-related genes FLG, scanning) were first analyzed using Affymetrix's MicroArray Suite v5 CDSN, and LOR (encoding the structural proteins filaggrin, corneo- (MAS5). Metrics files were then loaded into dChip for further analysis. desmosin, and loricrin, respectively), and KLK7 (kallikrein7) and KLK13 (kallikrein13), which are down-regulated in AAP. However, Quality control and per chip-per gene normalization our current data is a cross-sectional representation of early vs. late Before data normalization, consistency in quality of all 20 (10 stage AA skin samples. Studies at sequential points in time, with skin lesional and 10 non-lesional) was checked using scatter plots, samples from the same subjects collected prospectively will be correlation tests, and sample trees in order to eliminate any outlier needed to fully understand how the gene expression profile changes sample. There was a strong correlation in global gene expression with clinical deterioration or improvement of AA. among all subjects analyzed (r2 N0.79). Median polishing method was In summary, our study of gene expression in AA was designed to applied to normalize raw gene expression values. First, all of the gain a better understanding of the molecular pathways involved in the measurements on each chip were divided by the 50th percentile pathogenesis of AA through a non-reductionist approach. Our data value. The bottom 10th percentile was used as a test for correct support the hypothesis that disease is initiated and perpetuated by background subtraction. Second, each gene was normalized to its unknown autoantigens, perhaps localized within hair follicles, median. Further, consistency in the quality of samples was analyzed presented to T cells via skin resident antigen presenting cells i.e. the using scatter plots and correlation tests. disease is antigen driven locally. 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