Volume 79 • Number 11

Transcriptomes in Healthy and Diseased

Gingival Tissues
Ryan T. Demmer,* Jan H. Behle,† Dana L. Wolf,† Martin Handfield,‡ Moritz Kebschull,†
Romanita Celenti,† Paul Pavlidis,§ and Panos N. Papapanou†

Background: Clinical and radiographic measures are gold

standards for diagnosing periodontitis but offer little infor-
mation regarding the pathogenesis of the disease. We hy-
pothesized that a comparison of gene expression signatures
between healthy and diseased gingival tissues would provide
novel insights in the pathobiology of periodontitis and would
inform the design of future studies.

A
distinction between states of peri-
Methods: Ninety systemically healthy non-smokers with odontal health and disease is fea-
moderate to advanced periodontitis (63 with chronic peri- sible using a variety of diagnostic
odontitis and 27 with aggressive periodontitis) each contrib- approaches. Among the common clini-
uted at least two diseased interproximal papillae (with cal variables, bleeding on probing (BOP)
bleeding on probing [BOP], probing depth [PD] ‡4 mm, and is considered to best reflect the presence
attachment loss [AL] ‡3 mm) and a healthy papilla, if available of an inflammatory infiltrate adjacent to
(no BOP, PD £4 mm, and AL £2 mm). RNA was extracted, the ulcerated epithelium of the periodon-
amplified, reverse-transcribed, labeled, and hybridized with tal pocket.1,2 Probing depth (PD) ex-
whole genome microarrays. Differential expression was ceeding the typical depth of the healthy
assayed in 247 individual tissue samples (183 from diseased gingival crevice, in the presence of BOP,
sites and 64 from healthy sites) using a standard mixed-effects is also used to signify periodontal pathol-
linear model approach, with patient effects considered ran- ogy, whereas clinical attachment loss
dom with a normal distribution and gingival tissue status con- (AL) describes the cumulative exposure
sidered a two-level fixed effect. Gene ontology analysis to destructive periodontitis.3 Loss of peri-
classified the expression patterns into biologically relevant odontal tissue support can also be as-
categories. sessed radiographically.4
Results: Transcriptome analysis revealed that 12,744 However, clinical and radiographic
probe sets were differentially expressed after adjusting for variables are poor reflections of the un-
multiple comparisons (P <9.15 · 10-7). Of those, 5,295 were derlying pathobiology of the various
upregulated and 7,449 were downregulated in disease forms of periodontitis. The distinct histo-
compared to health. Gene ontology analysis identified 61 dif- logic features of periodontal health and
ferentially expressed groups (adjusted P <0.05), including disease were first documented in a clas-
apoptosis, antimicrobial humoral response, antigen presenta- sic publication by Page and Schroeder.5
tion, regulation of metabolic processes, signal transduction, These investigators described the de-
and angiogenesis. tailed morphologic characteristics of the
Conclusion: Gingival tissue transcriptomes provide a valuable gingival, sulcular, and pocket epithelium;
scientific tool for further hypothesis-driven studies of the pathobi- the underlying connective tissue; and the
ology of periodontitis. J Periodontol 2008;79:2112-2124. types of resident and infiltrating blood
KEY WORDS cells in the initial, early, established, and
advanced periodontal lesion. In parallel,
Gene expression; genomics; infection; microarray;
microbiologic approaches established
periodontitis.
common and distinct constituents of
the periodontal microbiota in health
* Department of Epidemiology, Mailman School of Public Health, Columbia University, New
and disease,6,7 whereas biochemical ap-
York, NY. proaches documented levels of cytokines,
† Division of Periodontics, Section of Oral and Diagnostic Sciences, College of Dental
Medicine, Columbia University.
chemokines, and other inflammatory
‡ Center for Molecular Microbiology and Department of Oral Biology, University of Florida,
Gainesville, FL.
§ Bioinformatics Center, Michael Smith Laboratories, University of British Columbia,
Vancouver, BC. doi: 10.1902/jop.2008.080139

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J Periodontol • November 2008 Demmer, Behle, Wolf, et al.

mediators within the tissues and the gingival crev- Clinical Examination
icular fluid.8-10 All participants underwent a full-mouth examination
Gene-expression profiling, i.e., the systematic cata- of the periodontal tissues at six sites per tooth by a sin-
loging of mRNA sequences in a cell population, organ, gle, calibrated examiner. Variables recorded included
or tissue sample, is a genomic tool that may add to presence/absence of visible dental plaque, presence/
the armamentarium of approaches to study the patho- absence of BOP, PD, and AL. Data were entered chair-
biology of periodontitis. In general, transcriptomes are side into a computer and stored at a central server.
a powerful means of generating comprehen-
sive genome-level data sets on complex dis-
eases and have provided enormous insights, Table 1.
mostly in cancer research,11,12 but also in
conditions such as muscular dystrophy,13 General Characteristics of the Study
Alzheimer’s disease and dementia,14,15 rheu- Participants (N = 90)
matologic disorders,16,17 and asthma.18,19
To our knowledge, a systematic transcrip- Characteristic Mean – SD or % Range
tome-based approach has not been applied
Age (years) 42 – 13 13 to 76
in the study of periodontitis. Our group has
initiated a series of studies to explore whether Female 50
the currently recognized forms of periodonti-
Race
tis are characterized by distinct gene-expres-
20 Black 21
sion profiles in affected gingival tissues. White 37
Our further goal is to explore the feasibility Asian 1
of a novel classification based on similarities Mixed 32
in transcriptional profiles. The aim of this Other 5
first report is to present a comprehensive de- Declined to report 4
scription of the periodontal transcriptome in
Ethnicity
healthy and diseased gingival tissues.
Hispanic 76
Non-Hispanic 23
MATERIALS AND METHODS Declined to report 1
The study was approved by the Columbia
Periodontal diagnosis
University Institutional Review Board.
Chronic periodontitis 70
Subjects Aggressive periodontitis 30
Ninety subjects with moderate to severe peri- Clinical periodontal variables
odontitis (63 with chronic periodontitis and 27 Teeth (n) 28 – 3 22 to 32
with aggressive periodontitis) were recruited Sites with BOP (%) 71 – 0.2 24 to 100
among those referred to the Columbia Uni- PD (mm) 3.9 – 0.7 2.9 to 6.5
versity College of Dental Medicine between Sites/subject with PD ‡5 mm (n) 57 – 25 12 to 156
November 2004 and April 2007. Eligible pa- AL (mm) 4.1 – 0.9 2.7 to 6.5
tients were >13 years old, had ‡24 teeth, had Sites/subject with AL ‡5 mm (n) 54 – 30 10 to 150
no history of systematic periodon-
tal therapy other than occasional
prophylaxis, had received no sys- Table 2.
temic antibiotics or anti-inflam-
matory drugs for ‡6 months, had Distribution of Tissue Samples According to PD
at least four teeth with radio- and Clinical AL
graphic bone loss, did not have
diabetes or any systemic condi- Tissue Samples in Specified PD Range (%) Tissue Samples in Specified AL Range (%)
tion that entailed a diagnosis of
1 to 2 mm 19 1 to 2 mm 18
‘‘periodontitis as a manifestation
of systemic diseases,’’21 were not 3 to 4 mm 14 3 to 4 mm 18
pregnant, and were not current
5 mm 31 5 mm 21
users of tobacco products or
nicotine-replacement medication. ‡6 mm 36 ‡6 mm 41
Signed informed consent was ob-
Non-readable 2
tained prior to enrollment.

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Figure 1.
Visualization of the top 50 probe sets with increased expression in diseased tissue relative to healthy gingival tissue (A) and of the top 50 probe sets
with decreased expression in diseased tissue relative to healthy gingival tissue (B). Gingival tissue samples are grouped according to clinical periodontal
status with diseased tissues on the left (red horizontal bar) and healthy tissues on the right (green bar). The color of each pixel represents the gene
expression level, with darker colors indicating lower relative expression values. Columns correspond to individual tissue samples, and rows correspond to
probe sets. Fold change (FC) describes the ratio of mean expression in diseased tissue over the mean expression in healthy tissue. Note that multiple
probe sets map to a single gene. Because of space limitations, only one gene symbol and gene name per probe set are identified. A complete list of
gene symbols and names per probe set is provided in the online supplementary Table A.

Gingival Tissue Donor Areas and Tissue tation. After local anesthesia, submarginal incisions
Sample Collection were performed, mucoperiosteal flaps were reflected,
A specially developed software identified periodon- and the portion of each interproximal gingival papilla
tally diseased and healthy tooth sites based on the that adhered to the root surface was carefully dis-
clinical data. Diseased sites showed BOP and had in- sected. This section comprised the ulcerated epithe-
terproximal PD >4 mm and concomitant AL ‡3 mm. lial lining of the interproximal periodontal pockets
Healthy sites showed no BOP and had PD £4 mm and the underlying connective tissue. After dissec-
and AL £2 mm. Next, the software identified maxillary tion, the gingival tissue specimens were thoroughly
diseased and healthy interdental papillae, based on rinsed with sterile normal saline solution and trans-
the above criteria, and pairs of diseased interdental ferred into Eppendorf tubes containing a liquid RNA
papillae with similar clinical presentation (PD and stabilization reagent.i A minimum of two diseased
AL within 2 mm of each other). A posterior maxillary papillae were harvested from each sextant and, when-
sextant encompassing a pair of qualifying diseased ever available, a healthy tissue specimen was obtained
interdental papillae was identified. from an adjacent site. After collection of the specimens,
Periodontal surgery was performed at the identified
sextant with no prior supra- or subgingival instrumen- i RNAlater, Ambion, Austin, TX.

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Figure 1.
Continued

pocket elimination/reduction periodontal surgery was reagents for in vitro transcription.‡‡ The cRNA yield
completed according to standard procedures. All pa- was determined spectrophotometrically at 260 nm.
tients received additional periodontal therapy accord- The cRNA was fragmented by incubation in fragmen-
ing to their individual needs. tation buffer at 94C for 35 minutes and stored at
-80C until hybridizations.
RNA Extraction, Reverse Transcription, and
In Vitro cRNA Synthesis Gene Chip Hybridizations
The tissue specimens were stored in a liquid RNA Human genome arrays§§ were used, including 54,675
stabilization reagent¶ overnight at 4C, snap-frozen, probe sets to analyze >47,000 transcripts, including
and stored in liquid nitrogen. All further processing 38,500 well-characterized human genes. Hybridiza-
occurred simultaneously for gingival biopsies origi- tions, probe array scanning, and gene-expression
nating from the same donor. Specimens were homog- analysis were performed at the Gene Chip Core Facil-
enized in a liquid buffer.# After incubation with ity, Columbia University Genome Center. Each sam-
chloroform and centrifugation at 12,000 · g, RNA col- ple was hybridized once, and each subject contributed
lected in the upper aqueous phase was precipitated two to four (median, three) samples.
by mixing with isopropyl alcohol and additional cen-
trifugation, and it was washed in 75% ethanol. The ex- ¶ RNAlater, Ambion.
tracted RNA was purified using a total RNA isolation # TRIzol, Invitrogen Life Technologies, Carlsbad, CA.
** RNeasy, Qiagen, Valencia, CA.
kit** and quantitated spectrophotometrically; 7.5 mg †† GeneChip Expression 39 amplification one-cycle cDNA synthesis kit,
total RNA was reverse-transcribed using a one-cycle Affymetrix, Santa Clara, CA.
‡‡ GeneChip Expression 39 amplification reagents for IVT labeling kit,
cDNA synthesis kit.†† Synthesis of biotin-labeled Affymetrix.
cRNA was performed using appropriate amplification §§ Human Genome U133 Plus 2.0 array, Affymetrix.

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Table 3.
Gene Ontology Groups Differentially Expressed in Diseased and Healthy Gingival
Tissues at P <0.05

Group Name ID P Value Probes (n) Genes (n)

Induction of apoptosis GO:0006917 1.26E-09 360 152

Negative regulation of cell proliferation GO:0008285 1.76E-05 373 170
Protein metabolic process GO:0019538 5.19E-05 441 180

Negative regulation of apoptosis GO:0043066 1.00E-04 368 169

Regulation of cellular process GO:0050794 1.36E-04 360 163
Antimicrobial humoral response (sensu Vertebrata) GO:0019735 2.50E-04 145 87
Antimicrobial humoral response GO:0019730 2.53E-04 142 85

Regulation of Ras protein signal transduction GO:0046578 3.10E-04 275 99

Cell motility GO:0006928 3.57E-04 382 180
Antigen processing and presentation of peptide antigen GO:0002474 4.04E-04 163 52
via MHC class I
Taxis GO:0042330 5.74E-04 189 110

Lipid biosynthetic process GO:0008610 6.29E-04 211 98

Positive regulation of apoptosis GO:0043065 9.02E-04 321 138
Chemotaxis GO:0006935 9.72E-04 194 114
Rho protein signal transduction GO:0007266 9.92E-04 260 96

Protein kinase cascade GO:0007243 1.11E-03 305 117

Enzyme linked receptor protein signaling pathway GO:0007167 1.16E-03 250 90
Protein complex assembly GO:0006461 1.18E-03 372 146

Regulation of growth GO:0040008 1.53E-03 217 98

rRNA metabolic process GO:0016072 2.16E-03 98 53
Induction of programmed cell death GO:0012502 2.21E-03 263 114

Lymphocyte activation GO:0046649 2.41E-03 103 52

Regulation of apoptosis GO:0042981 2.63E-03 342 143
Anti-apoptosis GO:0006916 2.75E-03 326 145

Localization of cell GO:0051674 3.28E-03 267 130

MAPKKK cascade GO:0000165 4.21E-03 170 72
Transmembrane receptor protein tyrosine kinase signaling GO:0007169 4.37E-03 338 127
pathway
Blood vessel morphogenesis GO:0048514 4.57E-03 147 59
Cellular defense response GO:0006968 4.70E-03 184 95

Angiogenesis GO:0001525 5.68E-03 147 62

Antigen processing and presentation of peptide antigen GO:0048002 5.72E-03 153 49
Tissue development GO:0009888 5.74E-03 214 121

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Table 3. (continued)
Gene Ontology Groups Differentially Expressed in Diseased and Healthy Gingival
Tissues at P <0.05

Group Name ID P Value Probes (n) Genes (n)

Dephosphorylation GO:0016311 5.79E-03 377 164

Endocytosis GO:0006897 6.32E-03 339 144
Regulation of signal transduction GO:0009966 6.32E-03 298 129

Regulation of Rho protein signal transduction GO:0035023 7.89E-03 221 80

Response to DNA damage stimulus GO:0006974 7.99E-03 388 182
Anatomical structure formation GO:0048646 8.97E-03 139 56
Macromolecule complex assembly GO:0065003 9.55E-03 333 127

Regulation of programmed cell death GO:0043067 0.014246 239 99

Actin filament-based process GO:0030029 0.014402 272 99
Cell growth GO:0016049 0.014682 281 124

Actin cytoskeleton organization and biogenesis GO:0030036 0.01542 345 125

Negative regulation of signal transduction GO:0009968 0.0155 168 65
Ectoderm development GO:0007398 0.017542 137 78

RNA metabolic process GO:0016070 0.017803 215 93

Ribosome biogenesis and assembly GO:0042254 0.021545 110 59
Positive regulation of transcription from RNA polymerase II GO:0045944 0.021767 139 45
promoter
Phospholipid metabolic process GO:0006644 0.02178 122 64
Positive regulation of transcription, DNA dependent GO:0045893 0.021971 309 113

rRNA processing GO:0006364 0.021976 106 57

Cytoskeleton organization and biogenesis GO:0007010 0.028137 258 96
Epidermis development GO:0008544 0.030145 119 71

Cytokine and chemokine mediated signaling pathway GO:0019221 0.034265 45 25

Regulation of cell growth GO:0001558 0.03479 282 130
Cell migration GO:0016477 0.036679 227 92

Carboxylic acid transport GO:0046942 0.036845 76 37

Protein amino acid dephosphorylation GO:0006470 0.037447 343 146
DNA replication GO:0006260 0.037785 257 125

Cellular lipid metabolic process GO:0044255 0.038116 313 144

Membrane invagination GO:0010324 0.048082 206 85
MHC = major histocompatibility complex; rRNA = ribosomal RNA; MAPKKK = mitogen activated protein kinase kinase kinase.
Number of probe sets and number of genes refer to the number of probe sets and genes represented in each ontology group. Analysis was carried out using a
24
gene set enrichment software (ermineJ, University of British Columbia).

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Figure 2.
Ontology analysis of selected pathways. A) Mitogen-activated protein kinase (MAPK) signaling pathway. B) Cytokine–cytokine receptor interaction.
C) Cell-adhesion molecules. D) Apoptosis. Genes shown in red are overexpressed and genes shown in blue are underexpressed in diseased gingival
tissues compared to healthy tissues. Genes in green are unchanged at the P <0.05 significance level. O = other molecule, mostly chemical compound;
+p = phosphorylation event; -p = dephosphorylation event; ? = receptors that are yet to be identified. A full description of the abbreviations of genes
listed in Figure 2 is provided in the online Journal of Periodontology as a supplementary file (Table B).

Data Analysis Resampling method. P values were used as input to

Two statistical analysis packagesii¶¶ were used identify biologically relevant groups of genes showing
throughout. Expression data were normalized and differential expression in health and disease. Gene
summarized using the log-scale robust multiarray symbols and descriptions were derived from a data-
analysis22 with default settings. Differential expres- base and software system for the meta-analysis of
sion was assayed using a standard mixed-effects lin- gene expression data.***
ear model approach, with patient effects considered Additional ontology analysis of all genes with a
random with a normal distribution and gingival tissue q-value <0.05 was carried out using a second gene
status considered a two-level fixed effect (healthy ver- set enrichment software,25††† in which the differen-
sus diseased). Statistical significance for each probe set tially expressed genes were mapped to the Kyoto En-
was determined using the Bonferroni criterion and cyclopedia of Genes and Genomes pathways.
q-value.23 For each probe set, a fold-change was com- Experimental details and results following the Min-
puted by dividing the raw expression values among dis- imum Information About a Microarray Experiment
eased tissue samples by the raw expression values
ii Reversion 2.3.1 for Linux OS, Free Software Foundation, Boston, MA.
among healthy samples. Therefore, fold-change values ¶¶ SAS for PC version 9.1, SAS Institute, Cary, NC.
represent relative RNA levels in disease versus health. ## ermineJ, University of British Columbia.
*** GEMMA, University of British Columbia.
Gene ontology analysis was performed using a ††† Pathway Express, Intelligent Systems and Bioinformatics Laboratory,
gene set enrichment software24## with the Gene Score Wayne State University, Detroit, MI.

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Figure 2.
Continued

standards26 are available at the Gene Expression ferentially regulated (P <9.15 · 10-7; 5,295 were upre-
Omnibus (accession number GSE 10334). gulated and 7,449 were downregulated in disease
compared to health). The complete list of differentially
RESULTS regulated probe sets can be viewed in the online Journal
The mean age of the patients was 42 years (range, 13 of Periodontology (supplementary Table A).
to 76 years; Table 1). Based on self-reported race/ Fold-changes in expression ranged between 5.73
ethnicity, 37% of the patients were white, 21% were and 3.89 (all P values <1.1 · 10-16) for the top 50
black, 32% were of mixed race, and 76% were His- probe sets, with increased expression in diseased tis-
panic. According to the 1999 International Workshop sue samples relative to healthy tissue samples (Fig.
for a Classification of Periodontal Diseases and Con- 1A), and between 4.35 and 2.13 (all P values <1.1 ·
ditions criteria,27 70% of the patients had chronic peri- 10-16) for the top 50 probe sets, with decreased ex-
odontitis and 30% had aggressive periodontitis. On pression in diseased samples (inverse values of the
average, study participants had 28 teeth present, 57 strongest [0.23] and weakest [0.47] fold-change
sites with PD ‡5 mm, 54 sites with AL ‡5 mm, and values quoted; Fig. 1B).
71% of sites with BOP. Among the 247 harvested gin- Gene ontology analysis identified 61 differentially
gival tissue samples (183 from diseased sites and 64 expressed groups at P <0.05, including apoptosis, an-
from healthy sites), 67% had PD ‡5 mm, and 62% had timicrobial humoral response, antigen presentation,
AL ‡5 mm (Table 2). No healthy gingival tissues sam- regulation of metabolic processes, signal transduc-
ples were available from 26 subjects. tion, and angiogenesis (Table 3). Four selected differ-
Transcriptome analysis revealed that 32,598 probes entially regulated pathways identified by a second
sets were differentially expressed between healthy and gene set enrichment software‡‡‡ are illustrated in Fig-
diseased tissue samples at q<0.05. Of those, 51% were ure 2. The top 50 pathways identified by this analysis
upregulated and 49% were downregulated in disease are listed in Table 4.
compared to health. Applying the Bonferroni correction
for 54,675 comparisons, 12,744 probe sets were dif- ‡‡‡ Pathway Express, Intelligent Systems and Bioinformatics Laboratory.

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Figure 2.
Continued

DISCUSSION First, we studied a relatively large sample of well-

To the best of our knowledge, this was the first study to characterized, patients with periodontitis who were
systematically describe the transcriptomes of healthy free of confounding exposures, such as systemic dis-
and diseased gingival tissues in patients with destruc- ease, medications, and smoking. Second, our gene
tive periodontal diseases. The primary aim of this expression data were generated by a large number
report was to provide a comprehensive description of arrays representing strictly defined clinical con-
that will serve as an information resource for investi- ditions and multiple sites per subject. Third, our
gators interested in the pathobiology of periodontitis. gingival tissue samples were obtained prior to any
Clearly, the presented gene expression data need to therapeutic manipulation of the gingival tissues.
be subjected to additional verification steps before Lastly, by allowing direct access to our raw data,
their exact biologic significance is fully appreciated. we enable independent investigators to conduct fo-
These may include confirmation by independent tech- cused analyses targeting individual genes and path-
niques on the mRNA level, such as real time reverse ways of particular interest to them.
transcription-polymerase chain reaction, and by pro- We would like to draw attention to a number of
teomic analyses. Therefore, at this point, the pre- points that will facilitate a correct interpretation of
sented data are not meant to provide unequivocal our findings: it must be realized that the reported tran-
evidence for the involvement of any particular gene scriptomes represent the composite gene expression
in the disease process, but rather to identify broad of a variety of cells that constitute and populate
consortia of genes and pathways that are likely dif- the healthy and diseased gingival tissues, including
ferentially expressed in states of gingival health and epithelial cells, connective tissue fibroblasts, and in-
disease. filtrating cells. Although the assayed tissue samples
Our study has several strengths relevant to its am- were deemed to be diseased or healthy based on
bition to serve as a high-quality research resource. accepted clinical signs of gingival inflammation, the

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Figure 2.
Continued

extent of the inflammatory infiltrate, the degree of health and disease, may reflect varying states of dis-
vascularization, and the epithelial/connective tissue ease activity among clinically homogeneous sites.
content of each gingival tissue sample were unknown There are additional potential explanations for this
and likely variable. In future studies, the use of cell- heterogeneity, such as differential bacterial coloniza-
capture techniques may facilitate the study of homo- tion patterns across diseased sites. Future analyses
geneous cell subpopulations and may generate data from our group will incorporate data on bacterial
that can be directly comparable to those stemming colonization patterns and will be informative in this
from well-defined in vitro systems, such as the re- regard. Lastly, although a potential effect of infiltra-
cently reported transcriptional profiles of cultured oral tion anesthesia on gene expression is conceivable,
epithelial cells challenged by specific periodontal there is little reason to expect differential anesthe-
pathogens and commensals,28-30 or the in vivo regu- sia-mediated effects in diseased versus healthy sam-
lation of specific proteins in rodent junctional and ples and, thus, a systematic bias in the reported
pocket epithelia.31 With respect to the clinical status comparisons.
of the obtained gingival tissue samples, the transcrip- This report does not include an in-depth discussion
tomes of healthy and intact gingival tissues of indi- of specific differentially regulated pathways in health
viduals with periodontitis may not necessarily be and disease but rather provides examples that under-
identical to those of healthy sites in subjects who score the usefulness of the expression data. At first
have not experienced destructive periodontitis. Con- glance, one can view the transcriptome findings as
sequently, because our data are based exclusively on largely confirmatory of anticipated differences based
a cohort of patients with periodontitis, our findings on earlier histologic or biochemical analyses. For ex-
cannot identify susceptibility genes. Furthermore, ample, the vast majority of the top genes with in-
the observed heterogeneity in expression among dis- creased expression in disease compared to health
eased tissue samples, even for genes that were, on av- are immunoglobulin-related genes. However, genes
erage, undisputedly differentially regulated between far less readily associated with periodontitis were also

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Table 4.
Ontology Analysis of the Top 50 Differentially Expressed Pathways in Diseased and
Healthy Gingival Tissues

Input Genes/Pathway
Impacted Pathway* Impact Factor† Genes (%)‡ P Value
Antigen processing and presentation 42.2 39.0 0.102874871
Cell adhesion molecules 40.5 53.0 6.38E-05

B-cell receptor signaling pathway 16.1 68.3 8.03E-07

Adherens junction 14.0 63.6 3.30E-06
Leukocyte transendothelial migration 13.1 56.0 2.02E-05
Natural killer cell–mediated cytotoxicity 11.9 51.9 5.12E-05

Circadian rhythm 11.8 41.7 0.402801166

Focal adhesion 10.8 50.8 9.47E-05
Renal cell carcinoma 10.6 60.9 7.58E-05

Regulation of actin cytoskeleton 10.4 49.0 1.61E-04

Phosphatidylinositol signaling system 10.0 35.1 0.689660919
Colorectal cancer 8.8 55.3 4.88E-04

T-cell receptor signaling pathway 8.3 52.7 0.001630673

MAPK signaling pathway 8.1 47.3 9.90E-04
Tight junction 6.9 49.6 0.004079315

VEGF signaling pathway 6.5 52.9 0.004965142

GnRH signaling pathway 6.3 50.5 0.006525075
Fc epsilon RI signaling pathway 6.3 52.0 0.00785814

Cytokine–cytokine receptor interaction 6.2 43.4 0.008217367

Small cell lung cancer 6.1 51.2 0.007222158
Wnt signaling pathway 5.7 46.3 0.010884856
Chronic myeloid leukemia 5.7 51.3 0.010359917

Notch signaling pathway 5.7 53.2 0.010958518

Glioma 5.7 50.0 0.029273605
Alzheimer’s disease 5.6 63.6 0.011980061

Epithelial cell signaling in Helicobacter pylori infection 5.5 50.7 0.019515233

Insulin signaling pathway 5.4 47.4 0.012739089
Jak-STAT signaling pathway 5.3 45.1 0.021944981

Pancreatic cancer 5.1 49.3 0.027534648

Prostate cancer 4.8 48.8 0.022130731
ErbB signaling pathway 4.8 48.3 0.027500726

Toll-like receptor signaling pathway 4.7 46.7 0.041239323

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Table 4. (continued)
Ontology Analysis of the Top 50 Differentially Expressed Pathways in Diseased and
Healthy Gingival Tissues

Input Genes/Pathway
Impacted Pathway* Impact Factor† Genes (%)‡ P Value
Long-term depression 4.7 47.4 0.05266026
ECM-receptor interaction 4.2 46.0 0.057512646

Dorso-ventral axis formation 4.1 53.6 0.044417147

Melanogenesis 4.1 45.1 0.053503025
Endometrial cancer 4.1 50.0 0.046475053
TGF-beta signaling pathway 3.8 44.0 0.085106723

Axon guidance 3.7 43.8 0.09158817

Ubiquitin-mediated proteolysis 3.6 48.9 0.081701274
Type I diabetes mellitus 3.4 43.2 0.131289041

Adipocytokine signaling pathway 3.3 44.4 0.121801571

Apoptosis 3.3 42.9 0.190763082
Huntington’s disease 3.2 50.0 0.114527344

Neurodegenerative disorders 3.1 50.0 0.114527344

Melanoma 2.9 43.7 0.177477033
Calcium signaling pathway 2.9 40.6 0.198877948

Gap junction 2.9 42.4 0.178991356

Thyroid cancer 2.9 41.9 0.374566966
Long-term potentiation 2.8 43.5 0.190074619
MAPK = mitogen-activated protein kinase; VEGF = vascular endothelial growth factor; GnRH = gonadotropin-releasing hormone; RI = receptor I; Wnt =
wingless-type; Jak = Janus kinase; STAT = signal transducer and activator of transcription; ErbB = V-erb-b erythroblastic leukemia viral oncogene; ECM =
extracellular matrix; TGF = transforming growth factor.
25
* Analysis carried out by means of a gene set enrichment software (Pathway Express, Intelligent Systems and Bioinformatics Laboratory) using all genes
with false discovery rate <0.05, mapped to Kyoto Encyclopedia of Genes and Genomes pathways and ranked according to impact factor.
† The impact factor identifies the relatively most affected pathways by considering and integrating the proportion of differentially regulated genes, the
perturbation factors of all pathway genes, and the consistency of the propagation of these perturbations throughout the pathway.
‡ Ratio of the number of regulated genes in the pathway/total number of genes currently mapped to the pathway.

observed to be least expressed in disease or, alterna- ing future focused studies of the pathobiology of peri-
tively, most expressed in health (e.g., desmocollin 1, odontitis.
arylacetamide deacetylase-like 2, and guanylate cy-
clase C). Likewise, the most expressed chemokine in ACKNOWLEDGMENTS
disease (by 3.85-fold, P <10-18) was CXCL6 (granu- This study was supported by grant DE015649 from the
locyte chemoattractant protein 2), a molecule known National Institutes of Health (NIH), Bethesda, Mary-
to be involved in inflammatory bowel diseases32 but land, to Dr. Papapanou. Dr. Pavlidis was supported
not earlier associated with gingival inflammation. by NIH grant 20R48583 and a Michael Smith Founda-
Lastly, confirming and extending recent preliminary tion for Health Career Investigator Award, Vancouver,
findings,33 our data showed that matrix metallopro- BC. Dr. Handfield was supported by NIH grant
teinases-1, -2, -3, -7, -9, -13, -14, and -28, and tissue DE16715. Dr. Kebschull was partly supported by
inhibitor of MMP (TIMP)-2 and -3, are significantly up- a stipend from Neue Gruppe Wissenschaftsstiftung,
regulated in diseased tissues. The above examples Wangen/Allgäu, Germany. We are indebted to Ms.
illustrate the usefulness of transcriptional data in guid- Elena Schwartz, RDH, Division of Periodontics,

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Columbia University College of Dental Medicine, for 18. Burke W. Genomics as a probe for disease biology. N
examining the patients. Technical assistance was Engl J Med 2003;349:969-974.
provided by Mr. Jun Yang, Division of Periodontics, 19. Izuhara K, Saito H. Microarray-based identification
of novel biomarkers in asthma. Allergol Int 2006;55:
Columbia University College of Dental Medicine. The 361-367.
authors report no conflicts of interest related to this 20. Papapanou PN, Abron A, Verbitsky M, et al. Gene ex-
study. pression signatures in chronic and aggressive periodon-
titis: A pilot study. Eur J Oral Sci 2004;112:216-223.
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