Full text of DOI:10.1038/sj.tpj.6500067

The Pharmacogenomics Journal (2001) 1, 272–287
 2001 Nature Publishing Group All rights reserved 1470-269X/01 $15.00
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ORIGINAL ARTICLE
Molecular classification of psoriasis disease-
associated genes through pharmacogenomic
expression profiling
JL Oestreicher1
ABSTRACT
Psoriasis is recognized as the most common T cell-mediated inflammatory
IB Walters3
disease in humans. Genetic linkage to as many as six distinct disease loci has
T Kikuchi3
been established but the molecular etiology and genetics remain unknown.
P Gilleaudeau3
To begin to identify psoriasis disease-related genes and construct in vivo path-
J Surette2
ways of the inflammatory process, a genome-wide expression screen of mul-
U Schwertschlag2
tiple psoriasis patients was undertaken. A comprehensive list of 159 genes
that define psoriasis in molecular terms was generated; numerous genes in
AJ Dorner1
this set mapped to six different disease-associated loci. To further interpret
the functional role of this gene set in the disease process, a longitudinal phar-
macogenomic study was initiated to understand how expression levels of
these transcripts are altered following patient treatment with therapeutic
agents that antagonize calcineurin or NF-␬B pathways. Transcript levels for
a subset of these 159 genes changed significantly in those patients who
responded to therapy and many of the changes preceded clinical improve-
ment. The disease-related gene map provides new insights into the patho-
genesis of psoriasis, wound healing and cellular-immune reactions occurring
in human skin as well as other T cell-mediated autoimmune diseases. In
addition, it provides a set of candidate genes that may serve as novel thera-
peutic intervention points as well as surrogate and predictive markers of treat-
ment outcome. The Pharmacogenomics Journal (2001) 1, 272–287.
JG Krueger3
WL Trepicchio1
1Division of Molecular Medicine, Genetics
Institute/Wyeth-Ayerst Research, Cambridge,
MA, USA; 2Department of Experimental
Medicine, Genetics Institute/Wyeth-Ayerst
Research, Cambridge, MA, USA; 3Department
of Investigative Dermatology, Rockefeller
University, New York, NY, USA
Correspondence:
WL Trepicchio, Genetics Institute/Wyeth-
Ayerst Research, 1 Burtt Road, Andover,
MA 01810, USA
Tel: +1 978 247 1332
Keywords: psoriasis; inflammation; gene expression profiling; microarrays; recom-
binant human Interleukin 11 (rhIL-11); Cyclosporin A
Fax: +1 978 247 1333
E-mail: wtrepicchioȰgenetics.com
JG Krueger, Department of Investigative
Dermatology, The Rockefeller University,
1230 York Avenue, New York, New York
10021, USA
INTRODUCTION
Psoriasis is a chronic cutaneous inflammatory disease characterized by hyperplas-
tic regenerative epidermal growth, and infiltration of immune cells, particularly
T cells into the dermis and epidermis resulting in the release of proinflammatory
molecules.1 The therapeutic benefit of immunosuppressive drugs, eg, Cyclosporin
A, FK506, anti-CD4 antibody and IL-2 diphtheria toxin conjugate supports the
hypothesis that activated immune cells, particularly T cells are pathogenic effec-
tors of psoriasis.2–4 More recently, treatment of psoriasis patients with the immu-
nomodulatory drug, recombinant human interleukin-11, resulted in a downreg-
ulation of type 1 cytokines suggesting a role for Th1-specific cells in disease
progression.5 As a result of these studies, there is broad interest among researchers
in using psoriasis as a ‘model’ inflammatory disease mediated by Type 1 T-cells
and many new anti-inflammatory or immune-modulating drugs are now tested
for the first time in humans in this disease.
Tel: +1 212 327 7730
Fax: +1 212 327 8232
E-mail: kruegej@mail.rockefeller.edu
The precise etiology of psoriasis remains elusive, although genetic and cellular
factors are being elucidated.6 In addition to environmental factors, there is a
genetic component to the disease. About one-third of patients report a family
member with the disease and family studies estimate the risk to first-degree rela-
tives at between 8–23%. There is a 65–70% concordance in monozygotic twins,
Received 6 July 2001
Revised 28 September 2001
Accepted 17 October 2001

Pharmacogenomic expression profiling of psoriasis
JL Oestreicher et al
273
compared to 15–20% in dizygotic twins (reviewed in Bethesda, MD, USA). In all instances throughout these stud-
Bhalerao and Bowcock7). Several psoriasis susceptibility loci ies, individual patient samples were hybridized to arrays. A
have been mapped: PSORS1 on 6p21.3, PSORS2 on 17q, global comparison of gene expression identified 1295–1858
PSORS3 on 4q, PSORS4 on 1cen-q21, PSORS5 on 3q21 and genes (19–26% of sampled genome) expressed in unin-
PSORS6 on 19p13.7
volved skin and 1352–2587 genes (19–38% of sampled
Despite the identification of disease-associated loci, spe- genome) expressed in lesional skin (Table 1). The number
cific genes that play a causative role in disease progression of genes expressed in normal skin varied from 1383–2375
have yet to be identified. Mutations within coding regions (20–22%) indicating no overt differences in expression pro-
affecting protein function or levels have traditionally been files between normal and disease tissue at this global level.
examined for disease-related genes. However, mutations
affecting transcriptional regulation or mRNA stability can were between lesional and uninvolved skin types and across
also play a role in disease initiation or progression. To begin unrelated individuals, unsupervised cluster analysis
To determine how similar or dissimilar expression profiles
a
to identify such genes and/or pathways affected at the approach was taken.8 This method, which clusters samples
mRNA transcript level, a genome-wide scan of uninvolved based on a hierarchical correlation coefficient, grouped
and lesional psoriatic skin as well as inflamed skin from expression patterns of normal and uninvolved skin together
non-psoriasis patients was performed using oligonucleotide while expression patterns of lesional skin formed a separate
arrays containing over 7000 human genes. A large number cluster (Figure 1a). A dendrogram of these relationships
of differentially regulated genes were identified that dis- illustrates greater inter-patient similarities in expression pro-
tinctly classified psoriasis lesions from other uninvolved files for like skin types than intra-patient similarities
skin types. The functional significance of these expression between uninvolved and lesional skin types (Figure 1a). An
patterns was determined by experimentally manipulating illustrative cluster image analysis identified a group of genes
human skin in vivo by inducing a cellular immune response whose expression levels were elevated in lesional skin com-
or a wound healing response, as well as through therapeutic pared to normal or uninvolved skin (Figure 1a). This result
treatment of patients with immunosuppressant or immuno- suggested that significant differences in expression profiles
modulatory drugs that impact the calcineurin or NF-␬B between uninvolved and lesional skin could be observed in
pathways. These studies indicate that complex genomic a differential comparison of gene expression frequency
expression patterns in inflammatory disease can be resolved levels.
into immune-associated vs reactive cellular components by
functional manipulation coupled with genome-wide assess- Identification of a Psoriasis Disease Gene Classification
ments. Furthermore, differentially regulated gene sets and Set
pathways identified by these studies may play a causative A differential mRNA expression profile of uninvolved to
role in disease progression or may be early markers of clini- lesional skin was performed. Comparisons of gene fre-
cal efficacy.
Table 1 Summary of gene expression data
RESULTS
Patient Demographics and Global Expression Analysis
of Uninvolved, Psoriatic and Normal Skin
Patient
No.
Skin type
Gene chip data
To identify disease-specific genes that were differentially
regulated in psoriatic lesions, 6-mm full thickness punch
biopsies were obtained from uninvolved and lesional skin
of 24 patients (13 females and 11 males) with moderate to
severe chronic plaque psoriasis. For comparison, biopsies
from normal skin of non-psoriatic patients were also
obtained. Biopsies were bisected with one half going
towards histological analysis and one half flash frozen and
used for RNA expression analysis. All patients enrolled in the
study had chronic psoriasis vulgaris affecting at least 10% of
their body surface area as measured by a combination of
clinical and histological parameters.5 On average patients
were affected with psoriasis for 24 years (range 4–44 years)
while only one patient had disease for less than 10 years.
Patients were primarily of Caucasian descent and had a
mean age of 41 years (range of 23–63 years).
No. present
nonlesion
No. present
lesion
Differential
expression
201
202
203
205
212
222
301
304
401
402
403
501
503
504
psoriasis
1295
1428
1671
1653
1776
1743
1858
1680
1383
1534
1502
2187
2375
1612
1352
2059
1847
2587
1868
1812
2027
1773
1619
1683
1724
2336
2514
2331
519
878
416
1321
543
340
454
349
309
62
psoriasis
psoriasis
psoriasis
psoriasis
psoriasis
psoriasis
psoriasis
tape strip
tape strip
tape strip
DTH
283
492
925
782
DTH
DTH
RNA was prepared from lesional and uninvolved skin
from an initial group of eight psoriasis patients and com-
pared to normal skin. Samples were analyzed on oligonucle-
otide arrays containing approximately 7000 human genes
RNA was prepared from one-half of a 6-mm punch biopsy of uninvolved skin and
lesional skin obtained from patients prior to treatment with rhIL-11 or Cyclosporin
A or following a DTH or tape-stripping reaction. RNA was hybridized to oligonu-
cleotide arrays containing over 7000 human genes. The number of genes called
‘present’ on each array is shown. The number of differentially regulated genes
(Unigem collection; National Biotechnology Information, between uninvolved and lesional skin is presented under differential expression.
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quencies were made between uninvolved and lesional skin tially expressed transcripts between uninvolved and lesional
from the same individual in a pairwise fashion in order to skin was observed across patients (Table 1). This finding was
reduce the possibility of confounding demographic para- somewhat unexpected especially since the disease severity
meters. A large number of differentially regulated transcripts of the affected tissue across patients was relatively homo-
were identified. Expression of 340–1321 genes was found to geneous. A likely explanation for these findings is that glo-
vary by an average factor of two-fold or greater between bal analysis of gene expression using relatively small sample
lesional and uninvolved skin within individual patients sizes has the potential to identify differentially regulated
(Table 1). To give an overall picture of gene expression genes that not only correspond to the disease state but also
changes in a variety of skin types, off center diagonal plots correspond to demographic differences (age, sex or ethnic
of the gene expression measurements from each sample origin) as well as environmental exposure differences (diet,
were plotted against all other samples and correlation coef- antigen exposure, temporal gene expression patterns)
ficients were calculated (Figure 1b). Comparison of lesional within the starting patient populations. To minimize the
to uninvolved skin types from the same individuals indi- inclusion of differentially regulated genes unrelated to the
cated a higher degree of unrelatedness (r2 = 0.87) than com- disease-state, a statistical approach to the data analysis was
parisons of normal to normal skin (r2 = 0.96), uninvolved to taken. Average frequency gene expression values between
uninvolved skin (r2 = 0.94) and lesional to lesional skin types uninvolved and lesional skin were calculated and a paired
(r2 = 0.98) between unrelated individuals (Figure 1b). Com- t-test was performed. Four hundred and seventy-six genes
parison of normal skin to uninvolved psoriasis skin also with transcripts levels statistically different between these
indicated a high degree of similarity (correlation coefficient two defined groups with a confidence level of 95% or greater
0.95) with only 34 genes differing by two-fold or greater were identified. This list was further refined to 159 genes by
(Figure 1b). These results validate the consistency of the pro- selecting only those genes whose transcript levels differed
filing method and indicate that uninvolved and normal skin on average by two-fold or greater between uninvolved and
from different individuals is more similar to each other than lesional skin. To further confirm the significance of these
to lesional tissue.
findings, we performed 100 random permutations of the 16
A large degree of heterogeneity in the number of differen- samples into two groups. This resulted in only 28–102 genes
that were statistically significant between two random
groups of eight arrays and only 6–15 genes differed by as
Figure 1 Global expression analysis. Total RNA obtained from
much as two-fold or greater between the two groups (Figure
lesional and uninvolved skin biopsies from eight psoriasis patients
1c). Class prediction analysis using supervised classification
and normal skin from three normal patients were analyzed on oli-
metrics indicated that this 159 gene set can be used to pre-
gonucleotide arrays containing 7000 full length human genes. In
total, 19 samples were analyzed on individual arrays. In no case
were RNAs pooled. (a) Unsupervised hierarchical cluster analysis of
dict with 100% accuracy expression patterns unique to
normal/uninvolved skin vs lesional skin (Figure 1d). These
uninvolved and involved psoriasis skin lesions and normal skin. 159 genes therefore comprise a disease classification set for
Cluster analysis was performed using the methods of Eisen et al.8
A normal skin sample was used as a common baseline. A dendro-
chronic plaque psoriasis.
gram of sample relatedness is shown. Patient or volunteer identifi-
cation numbers followed by the designated skin type are shown
The 159 differentially regulated genes were categorized into
below the dendrogram. Red colors indicate genes that are elevated
relative to the baseline and green colors are genes that are
decreased relative to the baseline. (b) Representative of diagonal
Characterization of Psoriasis Classification Genes
functional groups. The expression levels of these genes are
presented as the fold-change of the frequency levels of
plots and correlation coefficients (r2) of frequency values of unin-
volved to lesional psoriatic skin from a single patient, normal to
normal skin from unrelated donors, uninvolved to uninvolved skin
from unrelated patients, lesional to lesional skin from unrelated
patients and normal to uninvolved skin from unrelated patients.
Green triangles represent genes whose transcript levels do not
change in a two-way comparison between the samples. Blue tri-
angles represent genes whose transcript levels increase in a com-
parison of one sample vs another. Red triangles represent genes
whose transcript levels decrease in comparison of one sample rela-
tive to another. (c) Supervised cluster analysis. The correlation
measurements of the 1000 most statistically significant genes of
defined classes (uninvolved to lesion) compared to randomly per-
muted class distinctions are plotted. The top 1%, 5% and median
distance measurements of 100 randomly permuted classes com-
pared to the observed distance measurements for uninvolved and
lesional classes are plotted. (d) Histogram of class prediction
strengths of normal, uninvolved and psoriasis lesions. Scores range
from +1, corresponding to a perfect prediction score of normal or
uninvolved skin to −1, corresponding to a perfect prediction score
of lesional skin.
lesional over uninvolved skin (Figure 2). The heterogeneous
nature of the cells within the skin biopsies can result in a
dilutional effect of messages from rare cell types. Our experi-
ence has indicated that small fold-changes in low frequency
genes can actually be quite large changes in rare populations
of cells such as lymphocytes. Alternatively, they can rep-
resent small changes in high frequency cells such as kera-
tinocytes. Therefore, to facilitate a clearer biological under-
standing of the gene expression changes in the context of
these different cell populations, genes are further color
coded according to their frequency levels (Figure 2).
Genes involved in functions as diverse as transcriptional
regulation, metabolic control, protein processing, intracellu-
lar signaling, cell cycle control, lymphocyte regulation and
extracellular matrix destruction were identified (Figure 2).
Many of these genes were previously reported to be differen-
tially regulated in psoriasis lesional skin such as psoriasin
(S100A7), fatty acid binding protein (FABP5), elafin (SKALP,
PI3), retinoic acid binding protein (CRAB2), squamous cell
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carcinoma antigen (SCCA), beta defensin-2 (DEFB2), keratin Comparative Expression Profiles with Other Cutaneous
17 (KRT17) and keratin 16 (KRT16).5,9,10,11 This finding vali- Inflammatory Skin Conditions
dates our microarray comparison approach (Figure 2).
Psoriasis can be viewed as both a focus of T cell-mediated
Many genes not previously known as being differentially inflammation and as a local wound-healing reaction. To
regulated in psoriatic lesions were also identified. For begin to characterize the role of the differentially regulated
example, overexpression of S100A12 (calgizarin C, psoriasis gene set in the pathophysiology of psoriasis,
ENRAGE), matrix metalloproteinases (MMP-12) and heparin expression profiles were generated for other cutaneous
binding protein 17 (HBP-17) were observed (Figure 2). A inflammatory/hyperproliferative conditions whose mechan-
number of genes such as keratin 2, Apolipoprotein E (APOE), istic components comprised a subset of the psoriasis pheno-
GATA3, RB1, calponin 1 (CNN1), Cystatin 6 (CST6), TIMP- type. These conditions included antigen-induced inflam-
3 and TNXA were downregulated in the psoriatic lesions vs mation as occurs in the delayed type hypersensitivity
uninvolved skin as indicated by their negative fold-change reaction (DTH) and regenerative epidermal hyperplasia
values (Figure 2). Other genes involved in inflammation and induced by tape stripping. The DTH reaction was intended
immune regulation such as the IL-4R, CD2, CD24, CD47, as a surrogate of a T cell-driven cellular immune response
STAT-1, IFI27, IFI56, MX1, MnSOD, and SCYA1 (MCP1) in skin while tape stripping was intended to activate regen-
were elevated in lesional vs uninvolved tissue (Figure 2).
erative epidermal hyperplasia. From past work it is known
The oligonucleotide array findings for a number of differ- that tape-stripping induces K16 expression, keratinocyte
entially regulated transcripts were extended to a larger pso- hyperplasia and up-regulation of some inflammatory cyto-
riasis patient population using a high throughput quantitat- kines but inflammation as described here is ‘non-specific’ in
ive RT-PCR approach. Analysis of 16 additional psoriasis the sense that it is not antigen-driven nor T cell-depen-
patients confirmed elevation of S100A12, K16, HBP17, IL- dent.11 To induce a DTH reaction, three volunteers were sen-
4R, CCNF, LAD1, MAPKK3, MMP-12 and DSG3 mRNA levels sitized with DNCB and 72 h later challenged with a second
in lesional skin (data not shown). Lower levels of CST6, dose of DNCB. A comparison of a biopsy obtained at the site
TNXA, ID4, TIMP-3, GATA3, and ApoE in lesional skin vs of the DTH reaction to an uninvolved region identified a
uninvolved skin were also confirmed (data not shown). large number of genes that were differentially regulated
Comparable fold changes were observed between the RT- between normal and inflamed skin (Table 1). On average,
PCR approach and the oligo arrays for most genes. Better 259 genes differed by two-fold or greater in two out of three
correlation was observed for genes expressed at high levels volunteers (Figure 3a). Likewise biopsies were obtained from
in both lesional and uninvolved samples. Somewhat normal skin and tape stripped skin from three different sub-
reduced fold changes were observed for genes expressed at jects. Significant differences in gene expression levels were
lower levels. This was due to the greater sensitivity of the observed between normal vs irritated skin in the three vol-
RT-PCR approach that resulted in a more accurate quantit- unteers (Table 1). One hundred and sixty-one genes were
identified that were differentially regulated on average two-
fold or greater in two out of three tape-stripped samples
(Figure 3a).
ation of gene expression levels in the uninvolved samples.
Since transcript levels are not always correlative with pro-
tein levels, immunohistochemical analysis of a number of
differentially regulated genes was conducted. Consistent
with the elevated mRNA levels, protein levels of KRT16,
S100A12 and MMP-12 were elevated in lesional vs unin-
volved skin of these patients (data not shown).
A comparison of the gene sets obtained from the psoriasis
patients, DTH reaction and tape stripping showed some
overlap of the mRNA expression profiles (Figure 3a). Thir-
teen transcripts including KRT16, S100A2, S100A7, S100A9,
PI3 and DEFB2 were found to be differentially regulated in
all three inflammatory/hyperproliferative conditions (Figure
3b). Expression of 23 transcripts including STAT-1, IL-4R
and SCYA2 were differentially regulated in psoriasis and fol-
lowing a DTH reaction (Figure 3c). mRNA transcript levels
of 30 genes including KRT17, KRT2A and HBP17 were differ-
entially regulated in psoriasis and following tape stripping
(Figure 3d). Subtraction of these gene sets from the psoriasis
gene set identified 94 transcripts including S100A12
(ENRAGE), RAGE and GATA3 that were differentially regu-
lated only in psoriasis. Finally, 11 differentially expressed
mRNA transcripts were in common between DTH and tape
stripped samples but did not change between lesional and
uninvolved psoriasis samples (data not shown).
Figure 2 Histograms of the 159 statistically significant differen-
tially expressed genes between uninvolved and involved psoriasis
skin. Average frequency values and standard deviations from eight
paired uninvolved and lesional samples were calculated. A paired
students t-test was performed to identify statistically significant dif-
ferences between these samples and P values are presented. P
values less than 0.05 were considered statistically significant. Fold
changes in gene expression were calculated as the ratio of the aver-
age frequency of lesional skin compared to uninvolved skin change
and this value is found at the end of the histograms. Increases in
transcript levels in lesions relative to uninvolved skin are presented
as positive numbers and decreases in transcript levels are presented
as negative values. Histograms are color coded according to the
average frequency value. Genes are grouped according to func-
tional significance. Gene names and accession numbers are pro-
vided.
Pharmacological Treatment of Patients Identifies
Pharmacogenomic Classes of Responding Genes
There is a tacit assumption that changes in gene expression
are causally related to disease states. However, changes in
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mRNA expression levels may also be a consequence of the were treated with Cyclosporin A for 8 weeks. At the end of
disease process. We hypothesized that changes in gene this treatment period, patients were monitored for an
expression that precede clinical improvement may play a additional 4-week follow-up period. Response rates of these
more causal role in disease progression compared with those patients were typical of those previously reported.2,5 Sixty
genes whose expression changes mirror clinical improve- per cent of rhIL-11-treated and 90% of Cyclosporin A-
ment or do not change despite clinical improvement. To treated patients were considered to have responded to ther-
better understand possible functional relationships between apy as defined by changes in several clinical and histopatho-
differentially regulated genes and disease, lesion biopsy logical criteria.5
samples were obtained from psoriasis patients before, dur-
ing, and after pharmacological treatment with therapies pre- the course of drug treatment, comparisons in expression
viously shown to be effective. profiles of lesional skin were made between responding and
To identify genes whose expression patterns change over
Patients were treated with the experimental immunomod- non-responding patients prior to drug treatment and at
ulatory cytokine, rhIL-11, or with the immunosuppressant weeks 1, 4, 8 and 12 following initiation of therapy. Gene
drug, Cyclosporin A. Interleukin-11 has shown activity in chip analysis was initially performed on six rhIL-11-treated
psoriasis patients to improve clinical and histopathological (five responding and one non-responding) and three Cyclo-
scores and these changes correlate with a reduction over sporin-treated (two responding and one nonresponding)
time in the expression levels in the skin of type I cytokines patients. The expression levels of the 159 genes that were
such as IFN-␥ and IL-12p40.5 In addition, stable and/or over- significantly different between lesional and uninvolved skin
expression of type II cytokines such as IL-4 and IL-5 were were monitored over the course of drug treatment in these
observed in the same lesion biopsies.5 rhIL-11 has been nine patients.
shown to reduce proinflammatory cytokine production in
Self-organizing maps (SOMs) were employed to aid in the
part through the inhibition of NF-␬B nuclear translocation identification of pharmacogenomic expression patterns.15
in activated macrophages but does not appear to regulate For this analysis, the level of expression of the lesional skin
the p38/JNK pathway.12 Cyclosporin A is an immunosup- prior to therapy was normalized to a value of one and the
pressant drug active in psoriasis that also inhibits proin- change in expression of the 159 gene set over the course of
flammatory cytokine mRNA production in psoriatic treatment was calculated relative to this baseline level. Four
lesions.2,5 Mechanistically Cyclosporin A is distinct from patterns of gene expression changes were identified in
rhIL-11 with inhibitory effects on T cell activation parti- responding patients over the course of drug treatment
cularly through blockade of the calcineurin/NFAT and (Figure 4). In contrast, no significant change in expression
p38/JNK signaling pathways.13,14 Comparisons of gene patterns for this gene set was observed over the course of
expression levels between uninvolved and lesional skin fol- drug treatment in nonresponding patients. Clusters I, II, and
lowing treatment with rhIL-11 or Cyclosporin A cover a III correspond to genes whose levels of expression were elev-
diverse spectrum of pathways of therapeutic intervention ated in lesional compared to uninvolved skin. Cluster IV
for inflammatory and autoimmune diseases.
corresponds to genes whose expression levels were lower in
rhIL-11 was administered subcutaneously to patients at lesional compared to uninvolved skin. Of particular note, 27
either doses of 2.5 ␮g kg−1 day−1 and 5 ␮g kg−1 day−1 or a differentially expressed genes mapped to the six identified
single 1 mg or 2 mg dose administered once weekly. Cyclo- psoriatic susceptibility loci (Figure 4). The transcript levels
sporin A was administered at a dose of 5 ␮g kg−1 day−1.2 Fif- of the 41 genes in cluster I were found to change on average
teen patients were treated with rhIL-11 and nine patients from 2–8 fold in the rhIL-11- and/or Cyclosporin A-treated
responding patients as early as one week following the
initiation of drug therapy (Figure 4, cluster I). Interestingly,
Figure 3 Comparison of differentially expressed genes in DTH,
tape strip, and psoriasis samples. Total RNA was prepared from
affected and unaffected skin of three patients following tapestrip-
ping and three patients following induction of a DTH reaction and
analyzed on oligonucleotide arrays containing 7000 full length
human genes. Genes differentially regulated by two-fold or greater
between uninvolved and lesional psoriasis skin, normal and DTH
skin and normal and tape-stripped skin are presented. (a) Venn dia-
gram comparing the overlap in differential gene expression
between the various sample types. (b) Histograms of 13 genes dif-
ferentially regulated following a DTH reaction, tape stripping (TS)
and in psoriasis lesions. (c) Histograms of 23 genes differentially
regulated following a DTH reaction and in psoriasis lesions. (d) His-
tograms of 30 genes differentially regulated following tape strip-
ping and in psoriasis lesions. Data are graphed as fold change and
are color coded according to the average frequency value. Average
fold change values are depicted at the end of the histograms. Histo-
grams are color coded according to the average frequency value.
Gene names and accession numbers are provided.
these changes in expression levels preceded significant clini-
cal improvement as measured by changes in the average PSI
or PASI scores. The average PSI score declined only modestly
from 9.0 to 8.7 after 1 week of therapy (Figure 4). These
changes were not observed in the non-responding patients
examined. Twelve genes in this cluster including S100A12,
ID4, MTX and HBP17 mapped to five of the six known pso-
riasis-susceptibility loci with the exception of the PSOR5 loci
(Figure 4).
Cluster II corresponded to 91 genes whose transcript levels
returned to those found in uninvolved skin but only at the
end of drug therapy and not prior to clinical improvement
(Figure 4, cluster II). Included in this cluster were 11 genes
that mapped to all six psoriatic disease loci. Cluster III
included 17 genes whose expression levels did not change
over the course of therapy despite clinical improvement in
the patients skin lesions (Figure 4, cluster III). Only one of
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these genes, KPNB1, mapped to a known psoriasis locus observed in either rhIL-11- or Cyclosporin A-treated nonre-
sponding patients (data not shown).
(PSORS2). Finally, cluster IV contained 10 genes whose tran-
script levels were lower in lesional compared to uninvolved
skin. The transcript levels of these genes increased over the
course of drug treatment and approached the levels found
in uninvolved skin. Changes in the expression levels of
some of these genes such as TNXA, RAGE, ID4 and GATA3
were elevated as early as 1 week following the start of ther-
apy and these changes also preceded clinical improvement
(Figure 4, cluster IV). Both RAGE and TNXA map to the
PSORS1 locus and CNN1 maps to the PSORS6 locus. Interest-
ingly, a ligand for the RAGE receptor, S100A12, mapped to
the PSORS4 locus at 1q21.
Quantitative RT-PCR was used to analyze gene expression
changes for the early responding genes in clusters I and IV
in a larger patient population. Expression levels of genes for
S100A12, KRT16, SCYA2, PRKMK3, CST6, TNXA, CCNF and
GATA3 were analyzed in 15 patients treated with rhIL-11
and nine patients treated with Cyclosporin A (Figure 5a–b
and data not shown). Consistent with gene chip analysis in
the smaller patient population, similar changes in gene
expression in response to Cyclosporin A or rhIL-11 were
observed as early as 1 week following drug therapy in
responding patients but not in nonresponding patients.
Changes in gene expression continued to decline over the
course of drug treatment. This change in gene expression
preceded clinical improvement in these patients. Following
discontinuation of drug treatment at week 8, mRNA
expression levels for some of these genes such as KRT16,
S100A12 and SCYA2 began to rebound at week 12 towards
those found in untreated lesions (Figure 5a).
rhIL-11 and Cyclosporin A treatment had differential
effects on the transcript levels of 11 genes. Interestingly,
many of these differences were observed in genes in cluster
IV. For example, only rhIL-11-treatment resulted in signifi-
cant elevation of GATA3, CRIP1 and TNXA. Cyclosporin A
treatment did not significantly modulate the transcript lev-
els of these genes. Alternatively, Cyclosporin A significantly
reduced MMP-12, CCNF and HBP17 levels following treat-
ment while rhIL-11 had no significant effect on these tran-
script levels. These differential effects of rhIL-11 and Cyclo-
sporin A were examined in a larger treatment group using
quantitative RT-PCR (Figure 5b). Nine rhIL-11-treated
patients who responded to therapy were compared to eight
Cyclosporin A-treated patients who responded to therapy.
Elevation of GATA3 and TNXA were observed as early as 1
week following rhIL-11 treatment but these changes were
not observed in the Cyclosporin A treated patients (Figure
5b). Following termination of rhIL-11 administration these
transcripts returned to levels observed prior to treatment
and these changes in gene expression preceded a clinical
worsening of disease. Inhibition of HBP17 and CCNF (cyclin
F) was observed in Cyclosporin A-treated responding
patients but was not observed in rhIL-11-treated responders
(Figure 5b). Again, upon termination of Cyclosporin A
DISCUSSION
The pathophysiology of psoriasis is complex involving mul-
tiple cell types and is characterized by epidermal hyperplasia
with differentiation of keratinocytes along a regenerative
pathway.1 The underlying disease mechanism involves an
immune response with epidermal and dermal infiltration of
activated CD4 and CD8 T cells.16 We have used a pharmaco-
genomic mRNA expression analysis approach to identify
genes linked to these pathogenic processes and cell types in
order to understand better the molecular basis of the disease.
A comparison of gene expression profiles from psoriatic
lesions and uninvolved skin from multiple patients ident-
ified over 159 differentially regulated genes that comprise a
predictor set of the disease-state as determined by nearest
neighbor analysis. As validation of our experimental
approach, most genes previously identified by orthogonal
methods as being differentially regulated in psoriasis lesions,
such as S100A7 (psoriasin), PI3 (elafin), FABP5, DEFB2 (beta
defensin-2), KRT16 and SCCA1, were also identified in the
current studies.5,9,10,11
In addition to these known differentially expressed genes,
a large number of genes were identified with no previous
link to psoriasis and/or inflammatory conditions. Many of
these genes fell into clusters that define a number of clinical
and histological components of psoriasis such as hyperkera-
tosis, metabolic dysfunction and immune deregulaton. For
example, altered expression of a number of genes involved
in protein synthesis, degradation and metabolism were
observed. Members of this functional cluster include a group
of genes involved in lipid and sterol metabolism (FABP5,
fatty acid binding protein 5; APOE, apolipoprotein E; SQLE,
squalene epoxidase; ALDH10, fatty aldehyde dehydrogen-
ase; LDLR, low density lipoprotein receptor). Some of these
metabolic enzymes are associated with skin diseases. For
example, deficiency in fatty aldehyde dehydrogenase is asso-
ciated with Sjogren–Larsson syndrome that is characterized
by ichthyosis and hyperkeratosis.17 In addition, the LDLR
gene maps to the PSORS5 locus supporting a potential link
in deregulated lipid metabolism with disease development.
In support of this, engulfment of excess lipid molecules by
macrophages can result in the subsequent activation of this
cell type resulting in the release of proinflammatory cyto-
kines.18
Another functional cluster of genes can be associated with
the regenerative phenotype and hyperkeratosis observed in
psoriatic plaques. Genes in this cluster include KLK7
(stratum
(squamous cell carcinoma antigen-2), SPRR2A and SPRR1B
(small proline-rich proteins), TGM1 (keratinocyte
corneum
chymotryptic
enzyme),
SCCA2
transglutaminase), TGM3 (transglutaminase E3) and mul-
tiple keratins (Keratin 16, 17, 2 and 6E), PI3 (elafin) and
S100A7 (psoriasin). Many of these genes are components of
the stratified squamous epithelium, which are cross-linked
administration these transcripts returned to levels observed by transglutaminases or involved in associated proteolytic
in the lesions prior to treatment. These changes were not processes.19
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Figure 5 Quantitative RT-PCR analysis of treatment-responsive genes in a larger psoriasis patient population. Skin biopsies were obtained
from 24 patients treated with rhIL-11 (n = 15) or Cyclosporin A (n = 9) at baseline, week 1, week 4, week 8 of treatment and 4 weeks
following the end of treatment (week 12). In addition, a single biopsy of uninvolved skin was obtained at baseline. (a) Patients were
divided into treatment responders (R; n = 17) and nonresponders (NR; n = 7) based on a combination of clinical and histopathological
criteria.5 Quantitative RT-PCR was performed on the selected genes. Average fold change and standard deviations were calculated from
baseline (lesion) for each group at the indicated time points and are presented as line graphs. Statistically significant differences (P Ͻ
0.05) are represented by *. (b) Patients were divided into rhIL-11 responding (n = 9) and Cyclosporin A responding (n = 8) groups.
Quantitative RT-PCR was performed on the indicated genes. Average fold change and standard deviations from baseline (lesion) for each
group at the indicated time points were calculated and are presented as line graphs.
Finally, gene sets have also been identified related to an ogy of psoriasis and inflammation in general, the differen-
activated immune response and inflammation. Genes in this tial gene expression profiles allow for the formation of new
set include RAGE and the S100 family of RAGE ligands hypotheses to further explain the underlying pathology of
which activate and are activated in part by NF-␬B.20 Several this multi-genic disease. For example, parallels can be drawn
interferon inducible genes such as IFI56 and IFI27SEP also between genes that are differentially expressed in tumor
fall into this category and are consistent with our previous cells as well as in psoriasis lesions such as CTSB (cathepsin
findings that IFN-␥ and IL-12 are elevated in psoriatic B), CST6 (cystatin 6), and HBP17 (heparin binding protein
lesions.5 In addition, reduced levels of the TH2-specific tran- 17). CTSB is a lysosomal cysteine protease that plays a role
scription factor, GATA3, and elevated levels of IL-4R and the in antigen processing. In the neoplastic state, CTSB is over-
T cell surface molecule, CD2, as well as molecules involved expressed in multiple tumor types and its localization corre-
in leukocyte trafficking such as CD24 and CD47 were lates with areas of angiogenesis, inflammation and
observed in this study. GATA3 is a transcription factor, necrosis.23 It is thought to play a role in the loss of contact
abundantly found in T lymphocytes, that positively regu- inhibition of tumor cells. Expression of cystatin 6, an anti-
lates CD4 cells to a type-2 phenotype.21 We have previously protease inhibitor of cathepsin B, is downregulated in tumor
identified a relative deficiency of type-2 T cells in psoriatic samples and this downregulation has been correlated with
patients.22
tumor progression.24 Overexpression of cathepsin B and
In addition to supporting and enhancing the known etiol- downregulation of cystatin 6 in the epidermis of psoriasis
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patients may contribute to deregulated keratinocyte growth reduction in apoptosis.32 Reduction of ID4 expression in the
and differentiation. Likewise, HBP17, a heparin-binding pro- epidermis, through either inherited mutations or following
tein for basic fibroblast growth factor (bFGF), is over- insult, may lead to deregulation of a number of downstream
expressed in squamous cell carcinoma.25 Ribozyme-specific bHLH transcription factors that control keratinocyte
targeting of HBP17 resulted in the decreased growth and growth. Another gene, S100A12 (calgranulin C) located on
angiogenesis of xenograft tumors in mice. Interestingly, 1q21, is a soluble ligand for the RAGE receptor which has
HBP17 is upregulated by p38 MAP kinase and inhibition of been implicated in activation of the proinflammatory NF-
p38 results in a loss of HBP17 production, further demon- ␬B pathway.20 The RAGE gene maps to the PSORS1 locus on
strating a role for the p38 pathway in the progression of 6p21.3. Finally, PRKMK3 is mitogen-activated protein kinase
disease.26 A deregulated p38 MAP kinase pathway in pso- kinase 3 (MKK3), an upstream regulator of p38 MAPK.33 p38
riasis in addition to effects on the inflammatory process MAPK is activated by proinflammatory molecules such as
would result in upregulation of HBP17 in the epidermis, TNF-␣ and plays a role in the inflammatory process. Block-
resulting in increased angiogenesis and aberrant kera- ing p38 activity results in a reduction in the inflammatory
tinocyte growth. Interestingly, despite these similarities, state.34
conversion of a psoriatic plaque to squamous cell carcinoma
is rare, suggesting the presence of additional tumor sup-
pressor pathways that are not deregulated in the lesions.27 Comparison of the Psoriasis Gene Set to Other
This hypothesis can be tested with further functional Cutaneous Inflammatory Conditions
analysis.
A comparison of the 159 gene set to expression profiles from
inflamed skin following a DTH response or tape stripping
has allowed us to construct genomic maps that resolve pso-
riasis into immune-associated vs reactive cellular compo-
Association of Differentially Regulated Genes to
Psoriasis-susceptibility Loci
Understanding the multifactorial contribution of an nents. Some of the genes that comprise these maps are pso-
unknown number of genes interacting with each other and riasis-specific while others are shared in common with
with the environment has complicated the identification of general skin inflammatory conditions. Furthermore, some of
individual risk-associated genes in multi-genic diseases in these genes are only found differentially regulated in either
general and in psoriasis in particular. Interestingly, 27 genes DTH or tape-stripped samples but not both. Functional
in the psoriasis gene set identified in the current studies map characterization of the genes shared and/or specific to these
to one of the six known psoriasis-susceptibility loci. We and experimental conditions was consistent with the known
others have hypothesized that deregulated genes that reside biology of the systems. For example, many of the differen-
in chromosomal disease loci may play a fundamental role tially regulated transcripts shared between psoriasis and
in the etiology of the disease. Autosomal or somatic DTH samples are immune-specific genes expressed in vari-
mutations in regions of these 27 genes that affect transcrip- ous cell types of the immune system such as monocytes, B
tional regulation or message stability may contribute to cells and T cells or are induced by proinflammatory immun-
increased risk of developing psoriasis. In addition, some of omodulatory cytokines such as IL-12 or IFN-␥. Genes shared
these genes code for transcription factors whose aberrant between psoriasis and tape stripped samples are expressed
expression may serve to deregulate other downstream path- predominantly in cells of epithelial origin, such as kera-
ways that further contribute to development of the disease. tinocytes, consistent with the shared wound healing compo-
This may contribute to observed epistatic interactions nent of both tissue types. Psoriasis-specific genes crossed the
between different psoriasis disease loci.28 In addition, spectrum of cell types such as neutrophils, leukocytes and
environmental factors that impact regulation of these tran- keratinocytes. These genes also crossed the spectrum of
scription factors may also contribute to disease development cellular functions from roles in metabolism (ATP1AL1,
and progression. These findings could explain the difficulty HAL), transcriptional regulation (ID1, ID4), immune modu-
in identifying disease alleles in the coding region of candi- lation and inflammation (S100A12, IFI56) and cornified
date genes within psoriasis disease loci.29 Furthermore, a envelope development and keratinocyte growth regulation
nonrandom cluster of autoimmune susceptibility loci (TGM1, GJB2, SPRR2A).
resides on 6p21 and suggests that the genes identified here
Genes activated in an antigen-specific manner during a
may play a role in other autoimmune diseases as well.30 DTH response may play a role in a variety of inflammatory
Expression analysis of other complex inflammatory/ conditions such as rheumatoid arthritis and Crohn’s disease
autoimmune diseases will validate this approach.
while genes activated during a tape-strip reaction may play
These 27 genes have a variety of cellular functions that a more fundamental role in generalized wounding of the
support a role for the protein products of these transcripts skin. In support of this, a differentially regulated gene ident-
in the etiology of the disease. For example, ID4, located in ified in the current studies as overexpressed during the DTH
the HLA locus on 6p21.3, is a dominant/negative regulator response, MMP-12, has previously been shown through
of the basic helix-loop-helix (bHLH) family of transcription microarray analysis to be deregulated in Crohn’s and RA
factors and serves as a general antagonist of cellular differen- tissue.35 Other genes overexpressed in tape-stripped samples,
tiation and proliferation in a variety of cell lineages.31 Cellu- such as HBP17, have been shown to play a role in angiogen-
lar injury results in a downregulation of ID4 leading to a esis and wound healing.25
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Pharmacogenomic Profiling Identifies Disease Gene
Candidates and Novel Pathways for Pharmacological
Intervention
cular fragility and poor wound healing.38 Cyclosporin A
does not impact the expression of these transcript levels;
rather it specifically upregulates transcript levels for several
Changes in gene expression can be causally related to the genes such as CCNF and HBP17. Induction of HBP17 is regu-
disease state or may be a consequence of the disease process. lated by the p38/JNK pathway that is blocked by Cyclospo-
A general problem with genome-wide differential expression rin A.39 Understanding the impact of different therapeutic
analysis is the difficulty in differentiating between these two treatments on these pathways may help differentiate
possibilities. For example, the gene DEFB2, a defensin, is an between desirable (specific and efficacious) and undesirable
antimicrobial protein induced during inflammation whose (toxic) drug activities.
expression levels are increased as much as 200 fold in psori-
These studies present for the first time a genome-wide
atic lesions.36 DEFB2 may be upregulated as an effect of the expression response to pharmacological intervention in a
wound healing process. However, recent studies indicate longitudinal study and indicate that this method has utility
that defensins are also chemoattractive for lymphocytes and in prioritizing disease-associated genes for further functional
may contribute to adaptive immunity by mobilizing T cells genomic analysis. Pharmacogenomic gene expression analy-
and dendritic cells.37 Thus, descriptions of the biological sis of a complex multi-genic disease such as psoriasis has the
role of these differentially expressed genes are not sufficient potential to identify novel regulators of the disease-state.
to determine candidates for further functional analysis. This One hundred and fifty-nine genes were identified that are
is particularly the case when the differentially expressed differentially expressed in the disease state. Of these, 41
gene is relatively uncharacterized. To address this issue in transcripts are modulated rapidly by inhibitors of the cal-
an in vivo human setting, we analyzed psoriasis biopsy tissue cineurin and NF-␬B pathways and 12 of these genes map to
from patients following pharmacological intervention.
known psoriasis susceptibility loci. Further analysis of these
Drugs such as rhIL-11 and Cyclosporin A are known to genes will aid in elucidating the etiology of psoriasis and
block the NF-␬B and calcineurin/p38 pathways, respectively. perhaps other inflammatory diseases such as rheumatoid
We have hypothesized that changes in gene expression that arthritis, multiple sclerosis and Crohn’s disease where NF-
precede clinical improvement may play a more causal role ␬B and Type I cytokines also play a predominant role. They
in disease progression as opposed to genes whose expression may also serve to identify novel therapeutic intervention
changes mirror clinical improvement or do not change points or clinical markers of drug efficacy.
despite clinical improvement. This approach identified a
subset of 41 differentially regulated genes that returned to METHODS
normal or uninvolved levels following therapeutic inter- Study Design and Psoriasis Patient Entry Criteria
vention with rhIL-11 or Cyclosporin A at time points that These studies were approved by The Rockefeller University
preceded clinical improvement. Members of this group Hospital Institutional Review Board, New York, NY. Thirty
included 12 genes such as ID4, HBP-17, KRT16, S100A2, adult patients (16 female, 14 male) with chronic psoriasis
S100A9, S100A12, GNA15, MTX, PRKMK3, and SCYA2 that vulgaris affecting at least 10% of their body surface area, as
all localized to psoriasis susceptibility loci. Sequence analysis measured by a combination of clinical and histological para-
of these genes in families with a history of psoriasis may meters as previously described, signed informed consent
help to identify specific affected alleles at susceptibility loci. forms and were enrolled in this study.5 Patients had stable
Pharmacogenomic analysis also can serve to identify path- chronic disease as evidenced by a median duration of disease
ways suitable for pharmacological intervention. Of parti- of 24 years (range 4–44 years). Patients were primarily of
cular interest is the finding that 11 IFN-␥-regulated genes are Caucasian descent with a mean age of 41 years (range of 23–
aberrantly expressed in psoriasis lesions. Thus, agents that 63 years).
target the IFN-␥ or Type I cytokine pathway may have future
Global clinical assessment of each patient before, during
therapeutic potential. For example, neutralization of the and after therapy was based on the Psoriasis Area and Sever-
IFN-␥ inducer, IL-12, or inhibition of IL-12 production from ity Index (PASI) as described previously.2 The grading system
mature Langerhans cells may provide effective treatment. In used to measure local clinical disease activity was the Pso-
addition, blockade of Type I cytokine production from acti- riasis Severity Index (PSI). The PSI index grades individual
vated CD8+ Tc1 cells or CD4+ Th1 cells may also be effective. psoriasis lesions for scale, erythema, and induration on a 0–
This approach has also allowed us to begin to identify in 6 point scale for each parameter (0, absent; 1, trace; 2, mild;
vivo pathways that are modified in a drug-specific manner. 3, mild to moderate; 4, moderate; 5, moderate to severe; and
For example, 11 genes were differentially regulated between 6, severe). The final score sums the individual parameters
rhIL-11- and Cyclosporin A-treated patients. Blockade of the for a range of 0–18.40
NF-␬B pathway by rhIL-11 resulted in specific upregulation
Patients were treated on an outpatient basis with rhIL-11
of a number of genes including the type II helper T cell tran- (n = 21) or Cyclosporin A (n = 9) for 8 weeks. The dose and
scriptional regulator GATA3 and the lymphocyte differen- schedule selection of rhIL-11 (2.5 ␮g kg−1 day−1, 5 ␮g kg−1
tiation and maturation molecule CRIP1. Also, rhIL-11 upreg- day−1, and 1 mg once weekly and 2 mg once weekly) and
ulates the extracellular matrix protein, TNXA. TNXA Cyclosporin A (5 ␮g kg−1 day−1) was as previously described.5
deficiency has been associated with Ehlers–Danlos syn- At the start of enrollment, a psoriatic plaque was chosen for
drome that is characterized by skin hyperextensibility, vas- weekly assessment of lesion severity scores by the study
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coordinator. A 6-mm punch biopsy was taken of this included in vitro synthesized transcripts of 11 bacterial genes
lesional skin prior to rhIL-11 or Cyclosporin A treatment in each hybridization reaction as described by Hill et al.42
and at weeks 1, 4, 8 and 12 during rhIL-11 or Cyclosporin The abundance of these transcripts ranged from 1:300000
A treatment. In addition, prior to initiation of treatment, a (3 ppm) to 1:1000 (1000 ppm) stated in terms of the number
6 mm punch biopsy was taken from uninvolved skin at a of control transcripts per total transcripts. As determined by
location of the patient’s choosing. Biopsies were equally div- the signal response from these control transcripts, the sensi-
ided for immunohistochemical analysis and for RNA prep- tivity of detection of the arrays ranged between ෂ1:300000
aration. Normal skin was obtained from healthy volunteers. and 1:100000 copies per million. Labeled probes were
Six-micrometer cryostat sections were made from frozen denatured at 99°C for 5 min and then 45°C for 5 min and
biopsies, were reacted with antibodies to CD3, CD8, keratin hybridized to oligonucleotide arrays comprised of over 7000
16, Ki67, ICAM-1, or HLA-DR and processed for immunohis- human genes (HuGeneFL, Affymetrix, Santa Clara, CA,
tochemistry as previously described.5 Computer-assisted USA). Arrays were hybridized for 16 h at 45°C with rotation
image analysis was used to quantify epidermal thickness and at 60 rpm. After hybridization, probes were removed and the
the number of CD3+, CD8+, or Ki67+ cells in tissue sections cartridges washed extensively with 6 × SSPET and stained
as described.5
with phycoerythrin coupled to streptavidin as described.41
DTH and Tapestrip Induction
Gene Expression Data Reduction
Patients were sensitized with 2 mg of dinitrochlorobenzene Data analysis was performed on raw fluorescent intensity
(DNCB, 0.2 ml of 1% solution) to an area of normal skin. values using GENECHIP 3.2 software (Affymetrix). Gene-
The DNCB was placed into a 2-cm hole in a Teflon template Chip 3.2 software uses an algorithm to calculate the likeli-
and slowly evaporated under a steady stream of nitrogen hood as to whether a gene is ‘absent’ or ‘present’ as well as
gas. The area was covered with a sterile bandage. Patients a specific hybridization intensity value or ‘average differ-
returned in 2 weeks for challenge with 50 ␮g DNCB (0.1 ml ence’ for each transcript represented on the array. The algor-
of 0.05% solution) in the same fashion as described above. ithms used in these calculations are described in the Affyme-
If no reaction occurred within 96 h, a second challenge was trix GeneChip Analysis Suite User Guide (Affymetrix). The
placed with 100 ␮g (0.2 ml of 0.05% solution). A 6-mm full ‘average difference’ for each transcript was normalized to
thickness punch biopsy was taken from the middle of the ‘frequency’ values according to the procedures of Hill et al.42
erythematous plaque (DTH-reaction) for histological and This was accomplished by referring the average difference
RNA preparation 72 h following challenge. For tape strip- values on each chip to a calibration curve constructed from
ping, a 1 × 3 inch area of normal skin was repetitively the average difference values for the 11 control transcripts
stripped with adhesive tape as previously described.11 When with known abundance that were spiked into each hybridiz-
a smooth glistening erythematous epidermis was reached, ation solution. This process also served to normalize
the stripping was stopped; the wound was covered with a between arrays.42
sterile bandage. After 24 h, a full thickness 6 mm punch
biopsy was taken for histological and RNA preparation.
Specific transcripts were evaluated further if they met the
following criteria. First, genes that were designated ‘absent’
by the GENECHIP 3.2 software in all samples were excluded
from the analysis; 3320 (49%) of the transcripts met this
criteria. Second, in comparisons of transcript levels between
Isolation of RNA and Preparation of Labeled Microarray
Targets
Total RNA was isolated from one-half of a 6-mm full thick- arrays, a gene was required to be present in at least 50% of
ness skin biopsy using the RNeasy mini kit (Qiagen, Valen- the arrays comprising one or more groups. Third, for com-
cia, CA, USA). Labeled target for oligonucleotide arrays was parisons of transcript levels between groups, a t-test was
prepared using a modification of the procedure described by applied to identify a subset of transcripts that had a signifi-
Lockhart et al.41 Two micrograms total RNA were converted cant (P Ͻ 0.05) difference in frequency values. Fourth, aver-
to cDNA by priming with an oligo-dT primer containing a age fold changes in frequency values across this statistically
T7 DNA polymerase promoter at the 5Ј end. The cDNA was significant subset of genes was required to be 2-fold or
used as the template for in vitro transcription using a T7 greater. These criteria were established based on numerous
DNA polymerase kit (Ambion, Woodlands, TX, USA) and replicate experiments performed in our lab that estimated
biotinylated CTP and UTP (Enzo, Farmingdale, NY, USA). the intra-assay reproducibility (data not shown). Quantitat-
Labeled cRNA was fragmented in 40 mM Tris-acetate pH 8.0, ive RT-PCR was also performed on a select subset of genes
100 mM KOAc, 30 mM MgOAc for 35 min at 94°C in a final to further validate these criteria (data not shown).
volume of 40 ␮l.
Quantitative Reverse Transcriptase-polymerase Chain
Hybridization to Affymetrix Microarrays and Detection
of Fluorescence
Reaction (RT-PCR)
Quantitative RT-PCR was performed as previously
Ten micrograms of labeled target were diluted in 1 × MES described.5 RNA samples from patient biopsies were treated
buffer with 100 ␮g ml−1 herring sperm DNA and 50 ␮g ml−1 with 10 U of RQ1 DNase I (Promega, Madison, WI, USA) for
acetylated BSA. To normalize arrays to each other and to 30 min at 37°C. rTth DNA polymerase was used to reverse
estimate the sensitivity of the oligonucleotide arrays, we transcribe and amplify 10 ng of total RNA in a single tube
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ance in data analysis and experimental design. This work was supported
in part by grant GM-42461, from the National Institutes of Health. This
work has previously been presented in abstract form at the 61st Annual
Meeting of the Society for Investigative Dermatology, Chicago, IL, May
10–14, 2000.
assay using the TaqMan EZ RT-PCR kit (ABI, Foster City, CA,
USA) with gene specific sense and anti-sense primers and
a probe fluorescently labeled at the 5Ј end with 6-carboxy-
fluorescein (6-FAM). Amplification was performed using the
ABI Prism 7700 sequence detection system as described by
the manufacturer (ABI). Primers and fluorescently labeled
probes were generated using Primer Express software (ABI).
Sequence-specific amplification was detected as an increased
fluorescent signal of 6-FAM during the amplification cycle.
Quantitation of gene-specific message levels was based on a
comparison of the fluorescent intensity in the unknown
mRNA sample to the fluorescent intensity from a standard
curve of known mRNA levels. Amplification of the gene for
human acidic ribosomal protein (HARP) was performed on
all samples tested to control for variations in RNA
amounts.43 All genes were subsequently normalized to HARP
mRNA levels. Levels of gene-specific messages were graphed
as normalized message units as determined from the stan-
dard curve. A no template control was included in each
amplification reaction to control for contaminating tem-
plates.
DUALITY OF INTEREST
None declared.
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Statistical Analysis
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Note Added in Proof
Since submission of this manuscript for publication, Bow-
cock et al have also described differential expression profile
analysis of psoriasis lesions (Bowcock AM, Shannon W, Du
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ACKNOWLEDGEMENTS
The authors would like to thank the many patients and volunteers who
donated biopsy samples for these studies. Expert technical assistance
was provided by Mary Bozza, Yared Tekabe, John P Kim and Lori Casci-
otti. We would also like to thank Maryann Whitley, William Mounts,
John Ryan and Gene Brown for many thoughtful discussions and assist-
The Pharmacogenomics Journal

Pharmacogenomic expression profiling of psoriasis
JL Oestreicher et al
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