Molecular pathophysiology in Tay–Sachs and
Sandhoff diseases as revealed by gene expression
Rachel Myerowitz1,2, Douglas Lawson1, Hiroki Mizukami2, Yide Mi2, Cynthia J. Tifft2,3and
Richard L. Proia2,*
1Department of Biology, St Mary’s College of Maryland, St Mary’s City, MD 20686, USA,2Genetics of Development
and Disease Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,
Bethesda, MD 20892, USA and3Department of Medical Genetics, Children’s National Medical Center, Washington,
DC 20010, USA
Received February 7, 2002; Revised and Accepted March 19, 2002
Tay–Sachs and Sandhoff diseases are lysosomal storage disorders characterized by the absence of
b-hexosaminidase activity and the accumulation of GM2 ganglioside in neurons. In each disorder, a virtually
identical course of neurodegeneration begins in infancy and leads to demise generally by 4–6 years of age.
Through serial analysis of gene expression (SAGE), we determined gene expression profiles in cerebral
cortex from a Tay–Sachs patient, a Sandhoff disease patient and a pediatric control. Examination of genes
that showed altered expression in both patients revealed molecular details of the pathophysiology of the
disorders relating to neuronal dysfunction and loss. A large fraction of the elevated genes in the patients
could be attributed to activated macrophages/microglia and astrocytes, and included class II histocompat-
ability antigens, the pro-inflammatory cytokine osteopontin, complement components, proteinases and
inhibitors, galectins, osteonectin/SPARC, and prostaglandin D2 synthase. The results are consistent with a
model of neurodegeneration that includes inflammation as a factor leading to the precipitous loss of neurons
in individuals with these disorders.
Lysosomal storage diseases are a group of inherited disorders
that result from defective acid hydrolase function. Tay–Sachs
and Sandhoff diseases are examples of such disorders, and
represent members of a subcategory called the GM2 gang-
liosidoses that are so named because GM2 ganglioside
accumulates in cells owing to its impaired degradation
(reviewed in 1). The absence of b-hexosaminidase A in Tay–
Sachs disease and of b-hexosaminidase A and B in Sandhoff
disease are the primary enzyme deficiencies. Since only
b-hexosaminidase A is able to degrade GM2 ganglioside, the
substrate accumulates similarly in each disease. Affected
individuals with either disease exhibit a virtually identical
clinical course of neurodegeneration leading to death in early
childhood. Apoptosis of neurons is demonstrable in patient
samples and in mouse models (2,3). While it is clear that a
primary insult to neurons is the accumulation of ganglioside
substrates, the exact molecular mechanisms that translate the
primary insultintoneuronal cell deathremaintobedetermined.
Onealluring approachtogaininsightintopathophysiology is
to generate gene expression profiles of the central nervous
system (CNS) in patients affected with these disorders (4).
Suchprofileswouldreveal how geneexpressioninthediseased
state differed from that of the normal. Subsequent scrutiny of
thosegenes exhibiting altered expressioncouldbeawellspring
for hypotheses regarding the pathways leading to the observed
neurodegeneration. Advances, including serial analysis of gene
expression(SAGE) (5) and microarray analysis (6), havemade
the undertaking of such studies feasible and are being used in
the study of neurodegenerativediseases (4). In an earlier study
on a mouse model of Sandhoff disease, we applied microarray
analysis to monitor changes in gene expression. An extensive
upregulation of genes related to an inflammatory process
dominated by activated microglia was found in the disease
model (3). Moreover, activation of microglia was found to
precede massive neuronal death, suggesting that an inflamma-
tory process may participate in neurodegeneration.
Inthepresentstudy, wehaveutilizedtheSAGE methodology
in which 10 base tags obtained from the 30end of each gene
*To whom correspondence should be addressed at: Building 10, Room 9N-314, National Institutes of Health, 10 Center DR MSC 1821, Bethesda,
MD 20892-1821, USA. Tel: þ 1 301 496 4391; Fax: þ 1 301 496 0839; Email: firstname.lastname@example.org
# 2002 Oxford University PressHuman Molecular Genetics, 2002, Vol. 11, No. 11 1343–1350
at University of Portland on May 24, 2011
transcript are concatenated and sequenced to develop gene
expression profiles for the GM2 gangliosidoses. This method,
in principle, can provide both a qualitative and a quantitative
profile of all the genes expressed in the tissues. Genes of
known function, unknown function (expressed sequence tags,
ESTs) and sequences yet to be identified as protein coding
(novel genes) are included in SAGE analysis. The data thus
generated can serve as a reference of the expressed genes in
obtained provide molecular details of the pathophysiology and
demonstrate an intense inflammatory process that may be
directly involved in the neurodegenerative process.
Generation of SAGE data
SAGE libraries were constructed from mRNAs isolated from
cerebral cortex tissue samples derived from a normal child, a
child with Tay–Sachs disease and a child with Sandhoff
disease. A total of 107976 tags were generated; 34137 from
normal, 38940 from Tay–Sachs and 34899 from Sandhoff
tissues (Table1). Sequence analysis showed that between 39%
and 47% of the tags in each of the libraries were unique and
corresponded to known genes, ESTs or potentially novel
transcripts. Disregard for sequence tags that matched to more
than onegene in each SAGE library allowed us to discern that
at most 9231 genes were represented in the pediatric control,
11679 intheTay–Sachs and11111 intheSandhoff. Whilethe
majority of these corresponded to a sequence in GenBank,
approximately 30% in either the Tay–Sachs or Sandhoff
libraries did not match any GenBank entry and constitute a
cadre of potentially novel transcripts awaiting characterization.
Comparison of geneexpression profilesin normal and GM2
gangliosidosis cerebral cortex tissues
Results summarized in Table 2 show that of the 11679 genes
and novel tags expressed in the Tay–Sachs SAGE library, 631
(5.4%) showed differential expression at a statistically
significant level (P<0.05). About an equivalent number were
elevated in expression as were depressed, and 10% of these
showed a 10-fold or greater differential expression when
comparedwiththepediatric normal. Of the16transcriptsinthe
Tay–Sachs library displaying the highest fold elevation of
expression (between 30 and 100), 9 belong to the novel tag
category. Similar results were obtained with the Sandhoff
library: 4.9% of the expressed genes and novel tags showed
significant differential expression, withanapproximately equal
number elevated as depressed. To increase the probability of
focusing on genes related to the pathophysiology of the GM2
gangliosidosesratherthanindividual variation, weconfinedour
scrutiny to those genes whose expression was altered in both
the Tay–Sachs and Sandhoff disease patients relative to the
normal individual. Of the gene tags displaying differential
both patients. This represents more than 50% of genes
displaying differential expression in each of the disease
libraries. These gene expression profiles can be found in the
Supplementary Material. Thosegeneswithelevatedexpression
in the patients that could be functionally classified are listed
in Table 3. Assignation of genes to a particular functional
category is indicated by its color code. A category containing
the largest fraction of the overexpressed genes included those
associated with inflammation and injury responses. Many of
these genes were associated with activated macrophages/
microglia and astrocytes, the cell types that mediate inflamma-
tion and injury responses in the CNS. Genes that have been
reported to be expressed by activated macrophages include
cartilage gp-39 (7), galectin 3 (8), glycoprotein nmb (9,10),
HLA-DR (7) and the complement component 1q b chain
(11,12) (Table3). A tag corresponding to CD68 (13), a classic
macrophagemarker, was also elevated in both diseaselibraries
(Tay–Sachs, 13; Sandhoff, 4; normal, 0). Theexpressionof two
genes characteristic of activated astrocytes – glial fibrillary
acidic protein (GFAP) and vimentin (14) – were both very
highly elevated. Other genes elevated in both disease libraries
andgenerally associated withastrocytes includedaB-crystallin
(15), apoliproteinE (15,16), calcyclin(17) andthegapjunction
protein, connexin 43 (14). Elevated genes related to inflam-
matory responsesandreportedtobeexpressedby activatedglia
were prostoglandin D2synthase (18), clusterin (12), cathepsin
B (7) and the proinflammatory cytokine, osteopontin (19).
Osteonectin (SPARC), a gene found to be upregulated in
astrocytes after neural damage was also elevated in both
Inthecategory related to stress/apoptosis, several heat-shock
proteins and the ubiquitin-conjugating enzyme E2H were
elevated. Also of potential interest was the elevation of death-
associated protein 6 (DAXX), encoding a protein that
associates with the Fas receptor to mediate activation of Jun
N-terminal kinase (JNK) and programmed cell death induced
by Fas (22).
Other functional categories included genes involved in
protein synthesis, cytoskeleton, signaling/gene expression and
metabolism/housekeeping. A neuron-specific genesignificantly
elevated in both patients was growth-associated protein 43
Table 1. Summary of SAGE analysis
Tissue sampleTotal tags Distinct tags Genesa
aAs defined by matches to GenBank entries.
Table2. Summary of differentially expressedgenesinTay–SachsandSandhoff
diseases compared with pediatric normala
Tissue sampleKnown genesESTs Novel tagsElevatedb
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(GAP43), a phosphoprotein expressed in the elongating
terminals of neurites (23). However, GAP43 appeared to be
unique as a gene with specific neuronal expression that was
elevated in the patients’ libraries. A number of tags with
specific neuron expression appeared to be significantly
depressed in the patients’ gene profiles (Table 2 in Supple-
mentary Material). These included neuronal-specific enolase
(NSE, enolase 2) (24), b-synuclein (25), neuronal pentraxin
receptor (26), metabotropic glutamate receptor 3 (27), neuro-
granin(28), complexin1(29) andhippocalcin(30). Themyelin
Table 3. Classified genes elevated in both Tay–Sachs and Sandhoff diseases
Human Molecular Genetics, 2002, Vol. 11, No. 111345
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Figure1. Histologic analysis of neurons, microgliaandastrocytes. Sections of cerebral cortex fromcontrol (A, D, G, J ), theTay–Sachs patient (B, E, H, K) and
theSandhoff patient(C, F, I, L) wereimmunostainedwithantibody toneuronal-specific enolase(NSE) todetect neurons, glial fibrillary acidic protein(GFAP) to
detectastrocytesandCD68todetectmicroglia/macrophages. Staining withantibody tophosphotyrosinewasusedtodetectactivatedmicroglia/macrophages. Note
the low density of neurons and the enlarged astrocytes and microglia/macrophages in the patients’ sections. Bar¼50mm.
Figure2. Confirmationof SAGE by immunostaining of cerebral cortex sections. Sectionsof cerebral cortex fromcontrol (A, D, G, J, M), theTay–Sachspatient(B,
E, H, K, N) and the Sandhoff patient (C, F, I, L, O) were immunostained with antibody to cathepsin B, aB-crystallin, ferritin, osteopontin and GAP43.
1346Human Molecular Genetics, 2002, Vol. 11, No. 11
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basic protein gene, expressed by oligodendrocytes, was also
significantly depressed in the patients’ SAGE libraries.
Confirmation of SAGE data
To confirm the results of our SAGE library data, we checked
the expression of several genes through immunohistochemical
staining of paraffin-embedded sections of cortex (Fig. 1).
Antibody toNSE, agenefoundtohavedecreasedexpressionin
the Tay–Sachs and Sandhoff SAGE libraries, demonstrated
less intense staining in the patients’ sections compared with
the control (Fig. 1A–C). The patients’ sections appeared to
contain fewer neurons, and those that were present had an
altered, swollen morphology. Immunostaining for glial fibril-
lary acidic protein (GFAP) showed astrocytes that were clearly
enlarged in the patients’ sections, characteristic of an activated
state (31) (Fig. 1D–F). CD68 immunostaining indicated a
significantly elevated number of microglia in the patients’
sections (P<0.001) (Fig. 1G–I). The patients’ microglia
were amoeboid with minimal processes, a morphology
associated with their activation (32). Their activation was
confirmedby intensestaining withantibody tophosphotyrosine
(Fig. 1J–L) (3).
The protein products of several other genes with altered
expression were assessed by immunostaining. Cathepsin B
expression was elevated in the patients’ sections, and appeared
to be expressed by the amoeboid microglia (Fig. 2A–C).
Staining with aB-crystallin was more intense in the patients’
sections than in the control, consistent with the SAGE results,
and appeared to be localized to the enlarged astrocytes and
neurons (Fig. 2D–F). Ferritin immunostaining was also
enhanced in patients’ samples, as predicted from the elevated
expression of ferritin heavy- and light-chain genes (Table 3),
and appeared to be localized to amoeboid microglia as well as
other cell types (Fig. 2G–I). Osteopontin was found to be
highly overexpressed in the patients’ sections (Fig. 2J–L).
Double immunostaining revealed that the majority of osteo-
pontin expression was confined to microglia (not shown).
GAP43, a neuron-specific gene, was more intensely expressed
in patients’ sections and appeared in a punctuate pattern
Both Tay–Sachs and Sandhoff diseases are characterized by a
deficiency of b-hexosaminidases that results in the accumula-
tion of GM2 ganglioside in lysosomes. As a consequence of
a similar biochemical defect, the two diseases are virtually
indistinguishable in their neurodegenerative courses.
Although much is known about the enzymology and
molecular genetics of these diseases (1), there is considerably
less information regarding how the primary cellular insult –
lipid storage – leads to massive neuronal cell death and what
cellular and molecular mechanisms participate in this process.
We hypothesized that gene expression profiles of cerebral
tissue from patients with the GM2 gangliosidoses could
provide clues that would prove valuable in elucidating the
pathological mechanisms operating in these diseases. Toward
that goal, we employed the SAGE methodology to obtain
global gene expression profiles for cerebral cortex tissue from
a Tay–Sachs and a Sandhoff disease patient. We confined our
attention to those genes with significantly elevated or
decreased expression in both the Tay–Sachs and Sandhoff
disease patients to focus our attention on genes mostly likely
to be relevant to the neurodegenerative process rather than
Decreased expression of neuron-specific genes was char-
acteristic of bothpatientlibraries. Immunostaining withNSE, a
genespecifically depressed inthepatient libraries, confirmed a
decreased density of neurons in the disease samples. These
findings underscore the neuronal loss that occurs during the
disease process. Apoptosis of neurons has been demonstrated
inboth Tay–Sachs and Sandhoff diseasepatients and inmouse
models, and is thought to be the cause of the precipitous
neuronal loss (2,3). Of potential relevance to the neuronal cell
deathinthedisorders istheelevatedexpressionof cathepsinB,
a lysosomal protease that has been shown to be released by
activated microglia and to directly cause neuronal apoptosis
(33). In Alzheimer’s disease, high levels of cathepsin B are in
senileplaques(34). ProstoglandinD2synthase, elevatedinboth
patients’ SAGE libraries, has also been demonstrated to
directly induce apoptosis in neuronal cells (35).
The elevation of GAP43 expression in the patients may be
indicative of dysfunction or damage to neurons during the
pathogenesis of disorder. GAP43 is a phosphoprotein that
promotes neurite formation and is expressed during axonal
growthandregeneration(23,36). A characteristic featureinthe
GM2 gangliosidoses is the presence of ‘meganeurites’ – areas
of swollen neuronal processes with abnormally high densities
of neurites (37). An elevation of GAP43 expression provides
a molecular explanation for this enhanced neuritogenesis, a
process that has been suggested to cause neuronal dysfunction
in the GM2 gangliosidoses (37). Abnormal neurite sprouting
attributed to GAP43 expression has also been found in
Alzheimer disease neurons (38). In addition, increased
expression of GAP43 has been observed in neurons in
amyotropic lateral sclerosis patients (39).
Inflammation is believed to be a central factor causing
neuronal cell deathinanumberof neurodegenerativedisorders,
including Alzheimer disease, Parkinson disease, amyotropic
lateral sclerosis and HIV dementia (40,41). We previously
provided evidence that inflammation contributes to the
neurodegeneration in the GM2 gangliosidoses using a mouse
model of Sandhoff disease (3). Through cDNA microarray
analysis, wefound that a large fraction of overexpressed genes
in the CNS from the disease-model mice were related to an
inflammatory process. Our results with SAGE show a
remarkably similar profile of increased expression of inflam-
matory genes. Elevated genes in common between the mouse
and human gene profiles included class II histocompatability
antigens, the pro-inflammatory cytokine osteopontin, comple-
ment components, proteinases and inhibitors, galectin-3, and
prostaglandin D2 synthase. Both activated macrophages/
microglia and activated astrocytes are known mediate inflam-
matory responses in the CNS (40,42). In the SAGE study, the
In addition to mediating inflammation, astrocytes along with
microglia respond to neuronal damage by aprocess of reactive
gliosis where the cells proliferate and migrate to sites of
Human Molecular Genetics, 2002, Vol. 11, No. 111347
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damage to preserve tissue integrity (14,32). The elevated
expression of genes concerned with protein synthesis and the
and migratory activity of the glia. The heightened expression
gene encoding osteonectin (SPARC), an extracellular matrix
protein involved in tissue remodeling, is likely related to an
injury response in the patients (20,43).
The results obtained by SAGE allow new detail to be added
to our previous understanding of the pathophysiology of the
GM2 gangliosidoses. A proposed model is shown in Fig. 3.
Neuronal storage of ganglioside, due to the b-hexosaminidase
deficiency, is the primary insult in these diseases, and leads to
neuronal dysfunction and damage. The molecular events
involved in these early processes are still poorly understood.
The overexpression of GAP43 and the formation of ectopic
dendrites may beoneof theseevents (37), but has not yetbeen
determined experimentally. Both microglia and astrocytes
become activated by sensing neuronal damage (14,32) or
through their own accumulation of glycolipid, which may
activate cells directly (44). The activated microglia and
astrocytes express inflammatory proteins, including cytokines,
proteases and complement proteins, and monocytes are
recruited from the blood (45). The inflammatory milieu in
the CNS causes an additional insult to the already compro-
mised neurons, leading to a phase of rapid neuronal apoptosis.
Demyelination, prominentinthedisorders, occursasaresultof
the loss of axons (46). The severe neuronal cell death also
triggersreactivegliosisandmay inducetheexpressionof genes
related to tissue remodeling such as osteonectin/SPARC.
A number of genes elevated in GM2 gangliosidosis patients
neurodegenerativedisorders (19,47–51). Themolecular simila-
rities between the disorders of diverse etiology suggest
common mechanisms for neurodegeneration, regardless of
the nature of the primary insult. In many of these disorders,
inflammation has been implicated as a central mechanism
leading to neurodegeneration.
In addition to establishing molecular details of the patho-
physiology of neurodegeneration in the GM2 gangliosidoses,
the gene profiles can provide potential markers for monitoring
the clinical progression of the disorders. The ability to
assess the neurodegenerative course would be critical for the
development of therapies for these disorders.
MATERIALS AND METHODS
Samples of cerebral cortex from a 2-year-old male suffering
from Sandhoff disease (#2081) (frozen <24 hours post-
mortem) and from a normal 9-month-old male who died in
an accident (#2844) (frozen 20 hours postmortem) were
provided by the University of Miami Brain and Tissue Bank
for Developmental Disorders. This represents a joint effort of
the University of Miami and the University of Maryland
Brain and Tissue Banks through NICHD Contract N01-HD-
8-3284. The Tay–Sachs cerebral cortex sample was frozen
1.5 hours following the death of a 32-month-old male and
was obtained from the Children’s National Medical Center,
Washington, DC. A recent study has indicated that there was
little degradation in human brain RNA up to 96 hours
Total cellularRNA from5g of eachcerebral cortex samplewas
isolated using Trizol reagent (Gibco BRL). Poly(A) RNA was
thenpurifiedfromtotal RNA using theOligotex (Quiagen) spin
Construction and sequencing of SAGE libraries
The SAGE method was performed essentially as previously
described (5) and is accessible on the SAGE Home Page
Figure 3. A proposed model for the pathophysiology of the GM2 gangliosidoses.
1348 Human Molecular Genetics, 2002, Vol. 11, No. 11
at University of Portland on May 24, 2011
(www.sagenet.org/). Briefly, 5mm of poly(A) RNA isolated
from the Tay–Sachs and Sandhoff cerebral cortex samples
was converted to double-stranded DNA using a cDNA
synthesis kit (Gibco BRL) according to the protocol described
by the manufacturer, except that a poly(A) biotinylated
primer, 50-T18 biotin, was used in the synthesis. The cDNA
was cleaved with NlaIII (anchoring enzyme) and the resulting
30-terminal cDNA fragments were bound to streptavidin-
coated beads (Dynal). These bound fragments were divided
into two pools. Each was ligated to one of two types of
linkers that contained a BsmFI (New England Biolabs)
restriction site. The ligations were followed by digestion with
BsmF1 (tagging enzyme) to release 14bp sequence tags from
the streptavidin-bead-bound cDNAs. Following fill-in of the
SAGE tag overhangs with Klenow, the two pools of tags were
ligated to each other to form ditags still bound to the linkers.
The ditags were amplified via PCR, and the products were
isolated from a 12% polyacrylamide gel and treated with
NlaIII to produce 24bp ditags freed of linkers. Following
isolation via a 12% acrylamide gel, the 24bp ditags were
concatenated using T4 ligase (Gibco BRL) and the con-
catenates were purified on an 8% acrylamide gel. Concate-
nates migrating on the gel in the 400–800bp range were
isolated and ligated into the SphI site of pZero vector
(Invitrogen). Transfection of one-sixth of this ligation mixture
into ElectroMAXDH10B (Gibco BRL) via electroporation
yielded about 500 colonies. The colonies were screened for
inserts via PCR utilizing M13F and M13R as primers. Several
hundred colonies displaying inserts greater than 400 bases in
size were selected for sequencing via the ABI Prism Big Dye
Terminator Cycle Sequencing Ready Reaction Kit (PE
Biosystems), with 721M13 serving as the primer, and were
analyzed on an ABI Prism 377 automated sequencer. This
analysis was done to assess the fitness of the libraries. High-
throughput sequencing was subsequently performed on
minipreps of the pZero plasmids containing ditag concate-
nated inserts by the NIH Intramural Sequencing Center
(NISC). The pediatric normal SAGE library was constructed
using the I-SAGE Kit (Invitrogen).
Data analysis and comparison of SAGE libraries
Tag sequences and their count in the libraries were organized
and tallied via eSAGE software developed by Margulies and
Innis (52). In order to eliminate the potential PCR bias in
amplification of ditags during the SAGE protocol, we counted
any ditag that was found more than one time in any given
library only once for that library. Library comparisons were
performed using eSAGE 2000 software. This software
comparedtherelativeabundanceof eachtag betweenadisease
and the control library and produced a P-value for each
comparison. Only tags that had P<0.05 were considered
statistically significant. Tags that matched to more than one
gene were considered ambiguous and excluded from the
analysis. Matching tags to specific genes in GenBank was
achieved by comparing tags for each library with a SAGE tag
to a Unigene map that had been downloaded from the NCBI
SAGE website. The classification of function of individual
genes was based in part on information obtained from the
OMIM (www.ncbi.nlm.nih.gov/OMIM/) and LocusLink
Antibodies were from the following sources: ferritin, NSE,
CD68 and GFAP from DAKO (Carpinteria, CA), phosphotyr-
osine from Santa Cruz Biotechnology Inc. (Santa Cruz, CA),
cathepsin B from Oncogene Research Products (Cambridge,
MA), GAP43 from Sigma (St Louis, MO), Osteopontin from
IBL (Fujioka-shi, Gunnma, Japan) and aB-crystallin from
Stressgen (Victoria, British Columbia). Antibodies to CD68
andGFAP wereconjugatedtoperoxidase. Forimmunostaining,
paraffin sections were deparaffinized and rehydrated; in some
cases, antigen retrieval was accomplished by trypsin and
microwave treatment. Sections were incubated with primary
antibodies overnight at 4?C. When appropriate, the washed
sections were incubated with labeled polymer peroxidase-
conjugated mouse and rabbit secondary antibodies (DAKO
Corp., Carpenteria, CA). The peroxidase reaction was
visualized by diaminobenzidine and hydrogen peroxide.
For Supplementary Material, please refer to HMG Online.
Wethank JenniferRhodeforassistancewithstatistical analysis,
preliminary sequencing. We are grateful to Jeff Touchman and
GerardBouffardat at NISC for thehigh-throughput sequencing
of the SAGE libraries. This research was funded in part by a
grant to R.M. from the Mathew Forbes Romer Foundation, an
Affiliate of National Tay–Sachs and Allied Diseases Associa-
tion, and the Evan Lee Ungerleider Foundation, National
Tay–Sachs and Allied Diseases Association NY Area.
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at University of Portland on May 24, 2011