Gene expression profiling in human age-related nuclear cataract.

Abstract

To identify genes that are differentially expressed in age-related nuclear cataracts compared to transparent human lenses.
Total RNA was extracted from pools of central 5 mm capsulorrhexis epithelial samples microdissected at surgery from eyes with nuclear cataract or from age-matched transparent lenses (post-mortem). mRNA levels in the two samples were compared by hybridization to DNA microarrays (GeneFilter GF211) containing 4,132 known human genes. Only mRNAs consistently modulated over four comparisons were retained for analysis. A subset of the mRNA expression differences thus identified were verified and confirmed by Real-Time RT-PCR. Expressed and modulated genes were categorized according to the Gene Ontology classification.
The data revealed 262 genes that are downregulated and 7 that are upregulated by a factor of 2.5 or more in epithelial samples from cataractous lenses compared with transparent lenses. The highest content of downregulated genes was found in the functional classes "Signal transduction", "Regulation of cell proliferation", and "Protein modification". The "Response to oxidative stress" class was one of the least modulated. Among downregulated genes, we found several mRNAs coding for transcription/translation-related proteins, heat shock proteins 70 and 27, two ubiquitin-conjugating enzymes, two subunits of the cytoskeletal/chaperone protein, tubulin, betaA4-crystallin, and a group of Alzheimer-related proteins, including presenilin 1 and presenilin 2.
An extensive mRNA downregulation accompanies the development of the nuclear type of human age-related cataract. A few genes and classes of genes more prominently displaying this type of response have been identified. Altogether, the data indicate the tendency, in age-related nuclear cataract, towards a shutdown of de novo RNA and protein biosynthesis rather than an upregulation of cell defense components such as chaperones and various kinds of antioxidative or detoxifying proteins.

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Molecular Vision 2003; 9:538-48 <http://www.molvis.org/molvis/v9/a66>
Received 23 June 2003 | Accepted 18 September 2003 | Published 7 October 2003
Age-related cataract still represents the most frequent
worldwide cause of visual impairment and, frequently, of blind-
ness. Even though much is known about the environmental
cues and biochemical changes associated with age-related loss
of lens transparency, the sequence of events ultimately lead-
ing to cataract formation is far from being elucidated. One of
the most popular hypotheses considers oxidative stress as a
possible cause of age-related cataract, and evidence derived
from in vitro analyses as well as from cataractous human eyes
studies have been supportive of this possibility [1-3]. Other
studies, however, came up with inconclusive or even negative
results as to a critical involvement of oxidative stress in
cataractogenesis [4]. The same is largely true for observational
studies aimed at evaluating a possible role of the anti-oxidant
status of human participants in preventing (or at least delay-
ing) the development of age-related lens opacities [5-7].
The identification of genes differentially expressed in age-
related cataract compared with transparent adult human lenses
is likely to provide important insights into the metabolic and
protective processes that characterize the tissue response to
the disease. Although the overall spectrum of gene modula-
tion that characterizes the development of lens opacification
is presently largely unknown, some information has been
gained recently both in animal models of age-onset cataract
and in the human age-related disease. RT-PCR differential dis-
play analysis of lens mRNAs has resulted in the identification
of a limited number of transcripts that appear to be up- or
down-regulated in the lens epithelium of human lenses with
age-related cataract (or corresponding animal disease mod-
els) as compared to epithelial samples from normal transpar-
ent lenses [8-11]. In the Emory mouse, a well-characterized
model of age-dependent cataract, Sheets et al. [8] reported the
downregulation of αA- and βA3/A1-crystallin and the
upregulation of the ARK receptor tyrosine kinase. In human
lens epithelium derived from age-related cataracts, Kantorow
et al. [9-11] observed abnormal changes in the expression lev-
els of a few transcripts, including increased expression of
metallothionein IIA and osteonectin, and decreased expres-
sion of the protein Phosphatase 2A regulatory subunit, as well
as of the mRNAs for ribosomal proteins L21, L15, L13a, and
L7a.
A more comprehensive picture of the mRNA expression
profile of a tissue and of the changes in expression levels that
can take place during disease development (or maintenance,
as in late onset chronic diseases such as age-related cataract)
may be obtained with the use of DNA microarrays, a tech-
© 2003 Molecular Vision
Gene expression profiling in human age-related nuclear cataract
Roberta Ruotolo,1 Francesca Grassi,2 Riccardo Percudani,1 Claudio Rivetti,1 Davide Martorana,3 Giovanni
Maraini,2 Simone Ottonello1
1Dipartimento di Biochimica e Biologia Molecolare, University of Parma, Parma, Italy; 2Dipartimento di Scienze Otorino Odonto
Oftalmologiche e Cervico Facciali, University of Parma, Parma, Italy; 3Dipartimento di Clinica Medica, Nefrologia e Scienze della
Prevenzione, University of Parma, Parma, Italy
Purpose: To identify genes that are differentially expressed in age-related nuclear cataracts compared to transparent
human lenses.
Methods: Total RNA was extracted from pools of central 5 mm capsulorrhexis epithelial samples microdissected at
surgery from eyes with nuclear cataract or from age-matched transparent lenses (post-mortem). mRNA levels in the two
samples were compared by hybridization to DNA microarrays (GeneFilter GF211) containing 4,132 known human genes.
Only mRNAs consistently modulated over four comparisons were retained for analysis. A subset of the mRNA expression
differences thus identified were verified and confirmed by Real-Time RT-PCR. Expressed and modulated genes were
categorized according to the Gene Ontology classification.
Results: The data revealed 262 genes that are downregulated and 7 that are upregulated by a factor of 2.5 or more in
epithelial samples from cataractous lenses compared with transparent lenses. The highest content of downregulated genes
was found in the functional classes “Signal transduction”, “Regulation of cell proliferation”, and “Protein modification”.
The “Response to oxidative stress” class was one of the least modulated. Among downregulated genes, we found several
mRNAs coding for transcription/translation-related proteins, heat shock proteins 70 and 27, two ubiquitin-conjugating
enzymes, two subunits of the cytoskeletal/chaperone protein, tubulin, βA4-crystallin, and a group of Alzheimer-related
proteins, including presenilin 1 and presenilin 2.
Conclusions: An extensive mRNA downregulation accompanies the development of the nuclear type of human age-
related cataract. A few genes and classes of genes more prominently displaying this type of response have been identified.
Altogether, the data indicate the tendency, in age-related nuclear cataract, towards a shutdown of de novo RNA and
protein biosynthesis rather than an upregulation of cell defense components such as chaperones and various kinds of
antioxidative or detoxifying proteins.
Correspondence to: Giovanni Maraini, Ophthalmology, University
of Parma, Via Gramsci 14, 43100 Parma, Italy; Phone: +39 0521
703098; FAX: +39 0521 994820; email: maraini@unipr.it
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nique that allows the simultaneous analysis of thousands of
genes within a single test [12]. To our knowledge, no
microarray analysis of human cataract has been reported so
far. We have utilized microarrays containing 4,132 human
cDNAs of known function to compare the mRNA expression
profiles of human lens central epithelium samples obtained at
surgery from pure nuclear age-related cataracts with those of
central lens epithelial samples surgically microdissected from
adult transparent human lenses. The main outcome of this study
is that a fairly extensive downregulation (affecting more than
40% of the 612 genes in our array that turned out to be ex-
pressed in the lens) is the main gene expression change asso-
ciated with the development of age-related cataract in man. A
few functional classes of genes more prominently display this
type of response. A general resemblance between cataract and
other age-related diseases (most notably Alzheimer) has been
identified.
METHODS
Isolation of epithelium-capsule samples from human lenses:
A few days before surgery, cataractous lenses were classified
for type and severity of the opacity at the slit-lamp by an ex-
perienced observer according to LOCS II [13]. Only lenses
with a nuclear opalescence grade greater than or equal to 2
and a cortical and/or posterior sub-capsular opacity grade less
than or equal to 1 were selected for the present study. Written
informed consent was obtained and the principles of the Dec-
laration of Helsinki were observed during this study. Epithe-
lium-capsule samples (approximately 5 mm in diameter) ob-
tained immediately after capsulorrhexis in the operating room
were carefully washed to eliminate possible contamination by
blood and fiber cells, and immediately stored at -80 °C. Two
pools of cataractous epithelial tags (64% females) were thus
set up (pool 1: n=14, mean age 75 ±7.5 years; pool 2: n=14,
mean age 75.3 ±6.7 years) and utilized for the preparation of
two distinct cDNA probes that were employed for two inde-
pendent microarray hybridization experiments (see below). A
single pool of normal lens epithelia (n=14, 52% females) was
set up from adult donors (mean age 62.9 ±8.9 years) with the
same surgical technique under an operating microscope dur-
ing multi-organ explant procedures. Epithelial lens tags were
obtained immediately after corneal removal, which was usu-
ally the last of the multi-organ explants to be performed. The
time elapsed between the interruption of mechanical ventila-
tion and ocular explant was thus always less than one h, ex-
cept for one case in which the interval was 6 h. Only epithelia
from transparent lenses having no sign of opacity were col-
lected.
RNA extraction and probe preparation: Total RNA was
prepared from epithelial tag pools using the RNAzol B ex-
traction method (Biotech Italia, Roma, Italy) according to the
manufacturer’s recommendations, except for a final 4 M LiCl
precipitation that was followed by an ethanol wash of the pel-
let and by an additional sodium acetate/ethanol precipitation.
The amount of RNA in each sample was quantified by both
spectrophotometric analysis and gel-electrophoresis, followed
by analysis of ethidium bromide-stained gels with the Quan-
tity One software (BioRad, Hercules, CA). Radioactively la-
belled, single-stranded cDNA probes were produced by re-
verse-transcription (90 min at 37 °C) of total RNA (2 µg) in
reaction mixtures containing 2.0 µg of oligo-dT16-18, 6 µl of
5X first-strand buffer, 1.5 µl of reverse transcriptase (Super-
script II, Invitrogen, Paisley, UK), 1.5 µl of a dNTP mixture
containing dATP, dGTP, dTTP (each at a 20 mM concentra-
tion), and 250 µCi of [33P]-dCTP (2,500 Ci/mmol; Amersham
Biosciences Corp, Piscataway, NJ). The resulting cDNA probes
were purified on Bio-Spin 6 columns (BioRad) and quanti-
fied using a scintillation counter.
Microarray hybridization: Human “Named Genes”
GeneFilters Microarrays (GF211, Release 1), consisting of
4,132 cDNAs (IMAGE consortium collection, greater than or
equal to 1 kbp in length) coding for proteins of known func-
tion, spotted onto nylon membranes (Invitrogen, Carlsbad,
CA), were used for hybridization analysis according to the
manufacturer’s instructions (Research Genetics Invitrogen
Corporation, Gene Filters® Mammalian Microarrays Techni-
cal Handbook). Filters were prehybridized with 5.0 µg of de-
natured Cot-1 DNA, 5.0 µg of poly-dA and 4 ml of MicroHyb
hybridization solution (Invitrogen) for 2 h at 42 °C, followed
by the addition of radiolabeled probes and hybridization for
18 h at 42 °C. After a double-wash at 50 °C with 2X SSC, 1%
sodium dodecyl sulfate (SDS, 20 min each), plus a 15 min
wash at 55 °C (0.5X SSC, 1% SDS), membranes were aligned
on a phosphorimaging screen (Super Resolution SR,
PerkinElmer, Branchburg, NJ), wrapped with a lead envelope,
and exposed for various lengths of time at room temperature.
The screens were scanned with a Cyclone phosphorimager
(PerkinElmer) at a resolution of 42 µm and saved as digital
images using the OptiQuant software. Two GF211 filters from
different batches (hereby designated F1 and F2) were sepa-
rately hybridized with radiolabeled cDNA probes containing
the same amount of incorporated [33P]-dCMP radioactivity.
Probes designated C1 and C2 were prepared from RNA
samples extracted from two different cataractous epithelial tag
pools (a biological replicate carried out with pools 1 and 2,
see above). Two cDNA probes, independently prepared from
RNA derived from a single pool of transparent epithelial tags
(designated N1 and N2, a technical replicate), were utilized
for “normal lens” hybridizations. N1 and C1, and N2 and C2
© 2003 Molecular Vision
Molecular Vision 2003; 9:538-48 <http://www.molvis.org/molvis/v9/a66>
TABLE 1. CONCORDANT SPOTS FROM TWO INDEPENDENT SETS OF
PAIRWISE HYBRIDIZATIONS
Above background Above background Background
regular spots irregular spots level spots
(flag 0) (flag 3) (flag 2)
---------------- ---------------- -----------
N-probe
hybridization 348 264 2,988
C-probe
hybridization 92 91 3,224
Data refer to the total number of concordantly flagged spots obtained
from independent N1 versus N2 and C1 versus C2 hybridizations
carried out on filter 1 (N1 and C1) or 2 (N2 and C2). The total num-
ber of non-concordant spots was 532 and 725, for N-probe and C-
probe hybridizations, respectively. Spots tagged as flag 3 in at least
one of the two hybridizations (N1 versus N2, or C1 versus C2) are
included in this flag pair class.
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were hybridized with filters 1 and 2, respectively. Filters were
utilized no more than three-times. Prior to re-utilization they
were stripped in hot 0.5% SDS for 1 h.
Data analysis: Digitized images of comparable overall
signal intensity as measured with the program Quantity One
(BioRad) were used for hybridization analysis. Individual
images, as well as various pairwise combinations (N versus
N, C versus C, and N versus C, n=6), were initially analyzed
with the GF211-dedicated software Pathways 4 (Invitrogen).
A more detailed, and quantitatively rigorous analysis was then
performed with the program ImaGene 5.0 (Biodiscovery,
Marina Del Rey, CA). For each spot, the program calculates
the mean signal and the mean local background intensity. Spots
with a signal intensity (Is) exceeding local background inten-
sity (Ib) by at least one standard deviation of the mean local
background measured across the entire array are tagged by
the program as flag 0. For each of such spots, the hybridiza-
tion intensity (Is-Ib) was calculated and normalized with re-
spect to the mean of all the hybridization intensities of flag 0
spots. Spots with a signal intensity differing from the local
background by less than one standard deviation of the back-
ground measured across the entire array are tagged by the pro-
gram as flag 2. These spots were by far the most abundant and
were regarded as background level. When required (eg, for
the determination of an arbitrary expression ratio in an N ver-
sus C comparison) they were assigned an intensity value cor-
responding to the minimum normalized intensity in that par-
ticular hybridization. Spots with a local background differing
from the background distribution measured across the entire
image and/or with an irregular shape are tagged as flag 3. These
included some of the most strongly hybridizing cDNAs in the
entire array (see Results). Therefore, cDNAs corresponding
to flag 3 spots were assigned an average local background.
Normalization was performed by considering the mean inten-
sity of all above-background spots. Spots flagged as 0/0, 0/3,
3/0, or 3/3 in two independent N versus N (or C versus C)
hybridizations were deemed as “concordant” and representa-
tive of expressed genes. By the same criterion, concordant
flag 2 spots were considered as representative of non-expressed
genes. The total number of concordant spots obtained from
four independent hybridizations carried out with the N and
the C probes is reported in Table 1. Concordance between
hybridizations performed on filter 1 and filter 2 was estimated,
in pairwise comparisons, from the percentage of concordant
spots over the total number of cDNAs. It was 87% and 83%
for N1 versus N2 and C1 versus C2, respectively, and a lower
overall hybridization intensity was consistently observed with
filter 2. For N versus C comparisons (N=4), data analysis was
conducted on hybridizations performed with the same filter
(N1 versus C1 and N2 versus C2) as well as across filters (N1
versus C2 and N2 versus C1). Spots labeled as flag 0, 3, or 2
in N and 0 or 3 in C, with a normalized signal intensity ratio
(IC/IN) greater than or equal to 1.5 were judged as upregulated
in C. Spots labeled as flag 0 or 3 in N and 0, 3, or 2 in C, with
an (IN/IC) greater than or equal to 1.5 were judged as
downregulated in C. Only spots with an average signal inten-
sity ratio greater than or equal to 2.5 were considered as rep-
resentative of modulated mRNAs. Genes were categorized
according to GeneOntology [14]. Functional classification of
genes was carried out with the DAVID program [15]. Statisti-
cal analysis of gene distribution in the different functional
© 2003 Molecular Vision
Molecular Vision 2003; 9:538-48 <http://www.molvis.org/molvis/v9/a66>
TABLE 2. PRIMER SEQUENCES, SIZES AND MELTING TEMPERATURES FOR
REAL-TIME PCR AMPLICONS
Amplicon Tm
Target gene Primer sequence length (bp) (°C)
------------------------- --------------------------------- ----------- ----
Crystallin βA4 For.: 5'-GAAGTGCTGAGTGGAGCGTG-3' 51 80
Rev.: 5'-CCTTGGAAGCCAGCATGCT-3'
Glucosidase 1 For.: 5'-CCTCACGTCTACTTCGGCATGA-3' 63 82
Rev.: 5'-CCACATCAGTCCGGTGAGGA-3'
Gelsolin For.: 5'-GAAGACCTGGCAACGGATGA-3' 77 81
Rev.: 5'-TGAGAATCCTTTCCAACCCAGA-3'
Presenilin 1 For.: 5'-GCTCAGGAGAGAAATGAAACGC-3' 80 76
Rev.: 5'-CCTTCTGCCATATTCACCAACC-3'
Polymerase (RNA) II For.: 5'-GTCCCTGAGCACGTCGTCAT-3' 80 83
polypeptide E Rev.: 5'-GGCAGCTGGTTCTCTCGGA-3'
Dhydropyrimidinase-like 2 For.: 5'-GGACCCCGACAGCGTTAAA-3' 83 80
Rev.: 5'-GGCACTCCATGCCTTCAAAG-3'
β-2-microglobulin For.: 5'-GTATGCCTGCCGTGTGAACC-3' 86 80
Rev.: 5'-CCCTCCATGATGCTGCTTACAT-3'
The Tm of each amplicon was determined by melting curve analysis.
Figure 1. Typical Real-time PCR data. The amplification curves for
presenilin 1, obtained with three 10-fold serial dilutions of a catarac-
tous lens-derived cDNA (A), and the corresponding melting curves
(B) are shown. An 80 bp-long amplicon was produced (see Table 2
and Material and Methods for details on Real-Time PCR analysis).
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classes was carried out with the EASE program [16]. Lens
and human fetal eye EST collections were retrieved from the
NEIBank web site.
Real-Time PCR: Real-Time PCR was conducted on reversed-
transcribed first strand cDNA derived from the same (N and
C1) total RNA samples utilized for microarray hybridization.
An independent sample of transparent lens RNA (kindly pro-
vided by Marc Kantorow, Department of Biology, University
of West Virginia) was utilized as a test RNA to set up Real-
Time RT-PCR analysis. Total RNA (1 µg in a final reaction
mixture volume of 20 µl) was used for cDNA synthesis, which
was carried out at 42 °C for 90 min, followed by thermal inac-
tivation of reverse transcriptase (70 °C for 10 min). All other
reaction conditions were the same as those described previ-
ously for probe preparation. Real-Time-PCR was performed
with a TaqMan ABI PRISM 7700 Sequence Detector System
(PE Applied Biosystems, Foster City, CA) according to the
manufacturer’s instructions. Reaction mixtures (25 µl final
volume) were assembled with the following components: 2.5
µl of 10-fold serial dilutions of the two cDNAs, individually
optimized amounts of each primer set (300 nM for GCS1 and
POLR2E, 50 nM for all the other amplifications), 2X SYBR
Green PCR Master Mix (PE Applied Biosystems), containing
AmpliTaq GOLD® DNA Polymerase, reaction buffer, dNTPs,
Passive Reference and SYBR Green I. The housekeeping
βactin [17] and β-2-microglobulin [18] genes served as inde-
pendent internal standards. To avoid interference by contami-
nating genomic DNA, all primers (reported in Table 2) were
designed to cross exon-intron boundaries using the Primer
Express software (PE Applied Biosystems). β-Actin primers
were from PE Appled Biosystems (βactin Control reagents,
part number 401846). All primer pairs produced only one
amplification band (ranging in size from 50 to 90 bp) when
tested in conventional RT-PCR. The specificity of individual
Real-Time PCR products was assessed by melting curve analy-
sis [19], carried out immediately after PCR completion fol-
lowing the manufacturer’s instructions. Melting curves for
individual PCR products displayed a single peak. Melting tem-
peratures (Tm) were determined with the Dissociation Curve
software (PE Applied Biosystems). All sets of reactions were
conducted in triplicate, and each included a non-template con-
trol (NTC). A representative amplification plot and the corre-
sponding melting curve are shown in Figure 1. The threshold
cycle (Ct) was used to calculate relative gene expression lev-
els using the “Comparative Ct method” (PE Applied
Biosystems, User Bulletin number 2), which is based on the
assumption that the efficiency of amplification of target and
reference cDNAs are approximately the same. The validity of
the above assumption was verified for each primer set using
serial cDNA dilutions.
RESULTS & DISCUSSION
Overall representation of lens epithelium-expressed genes
in the microarray: The GF211 microarray utilized in the
present study contains cDNAs from various human cDNA li-
braries, none of which was of lens origin. Therefore, we ini-
tially looked for some basic information regarding the pro-
portion of lens-expressed genes that were represented in the
GF211 array. To this end, we determined the number of spot-
ted cDNAs with a unigene code (a total of 3,983 entries) that
match sequences previously isolated as lens ESTs (NEIBank
Human Lens cDNA library [20], 2,275 distinct unigene codes;
dbEST Human Lens cDNA library, 2,319 distinct unigene
codes). We found that 531 (23.3%) and 555 (23.9%) lens ESTs
from such databases match cDNA sequences that are present
in the GF211 array. To compare expression levels in transpar-
ent human lenses deduced from either EST sampling or cDNA
hybridization, we determined the number of sequence matches
found in each of our three flag classes. As shown in Table 3,
using aggregate concordant data from two independent “nor-
mal lens” hybridizations, we found that the percentage of
© 2003 Molecular Vision
Molecular Vision 2003; 9:538-48 <http://www.molvis.org/molvis/v9/a66>
Figure 2. Representative scatterplot of the signal intensities derived
from an N versus C comparison. The data shown are from hybrid-
izations conducted with filter 2. Normalized signal intensity calcula-
tions and graph construction were performed with the Pathways 4
program. Dots lying above and below the blue dashed line (IC/IN=1)
correspond to normalized signal intensities that are, respectively,
higher and lower in cataractous than in transparent lens.
TABLE 3. COMPARISON BETWEEN MICROARRAY HYBRIDIZATION AND
LENS EST DATA
EST Library
-----------------------------------------
NEIBank dbEST
human lens human lens dbEST human
cDNA library cDNA library fetal eye
------------ ------------ -----------
Flag 0 23% 22% 12%
Flag 2 15% 15% 11%
Flag 3 22% 20% 11%
Unigene sequences in the GF211 filter, concordantly flagged in two
independent normal lens epithelim (N-probe) hybridizations (see
Materials and Methods, and Table 1 for details), were compared with
unigene lens ESTs from the NEIBank (2,275 entries) and from the
dbEST (2,329) Humal Lens cDNA libraries. A human fetal eye EST
database (11,029 unigene sequences) was used as reference for this
comparison. Since different numbers of ESTs are available in the
different databases, the number of matches obtained from individual
pairwise comparisons was normalized with respect to the lowest num-
ber of ESTs (2,275), as found in the NEIBank cDNA library.
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© 2003 Molecular Vision
Molecular Vision 2003; 9:538-48 <http://www.molvis.org/molvis/v9/a66>
TABLE 4. GENES DIFFERENTIALLY EXPRESSED IN CATARACTOUS LENS EPITHELIA COMPARED TO TRANSPARENT EPITHELIA
Downregulated genes
Accession Flag Flag
number Title Gene Chromosome N1/N2 IN C1/C2 IC IN/IC
--------- ------------------------ --------- ---------- ----- ---- ----- ---- -----
AA291490 glucosidase I GCS1 2 0/0 3.29 2/2 0.20 16.29
H53703 growth factor GRB7 17 0/0 2.43 2/2 0.20 12.06
receptor-bound protein 7
AA701860 follistatin FST 5 0/0 2.07 2/2 0.20 10.24
AA027042 Pol (RNA) II (DNA POLR2E 19 0/0 2.06 2/2 0.20 10.20
directed) polypeptide E
AA454218 TBP associated TAF1C 16 0/0 1.92 2/2 0.20 9.51
factor.RNA polI.C
AA443547 v-rel avian RELA 11 0/0 1.85 2/2 0.20 9.16
reticuloendotheliosis
viral oncogene
AA147640 phosphorylase, glycogen PYGL 14 0/0 1.72 2/2 0.20 8.54
T72202 signal transducer and STAT6 12 0/0 1.57 2/2 0.20 7.78
activator of
transcription 6
N64051 Werner syndrome WRN 8 0/0 1.53 2/2 0.20 7.59
AA164440 pericentriolar material1 PCM1 8 0/0 1.51 2/2 0.20 7.50
N81029 collagen, type XVIII, COL18A1 21 0/0 1.49 2/2 0.20 7.39
alpha 1
AA430512 serine (or cysteine) SERPINB9 6 0/0 1.49 2/2 0.20 7.37
proteinase inhibitor
AA463924 coagulation factor F8A X 0/0 2.33 0/2 0.32 7.33
VIII-associated
AA884403 cardiotrophin 1 CTF1 16 0/0 1.44 2/2 0.20 7.15
AA099169 phosphofructokinase PFKM 12 0/0 1.42 2/2 0.20 7.06
AA443039 heat shock 70 kD HSPA1B 6 0/0 1.42 2/2 0.20 7.02
protein 1B
AA454854 amylase, alpha 2A AMY2A 1 0/0 1.38 2/2 0.20 6.85
H79353 Fc fragment of IgE FCER1G 1 0/0 1.35 2/2 0.20 6.66
AA644128 nuclear autoantigenic NASP 1 0/0 1.31 2/2 0.20 6.51
protein
(histone-binding)
AA134814 TRAF family TANK 2 0/0 1.29 2/2 0.20 6.41
member-associated NFKB
activator
T55558 colony stimulating CSF1 1 0/0 1.28 2/2 0.20 6.34
factor 1
AA488622 signal transducing STAM 10 0/0 1.28 2/2 0.20 6.32
adaptor moleculeSH3
domain
R44005 glutamate decarboxylase2 GAD2 10 0/0 1.26 2/2 0.20 6.26
AA877347 putative b.b- B-DIOX-II 11 0/0 1.25 2/2 0.20 6.18
carotene-9',
10'-dioxygenase
AA844124 Homo sapiens, clone 14 0/0 1.24 2/2 0.20 6.13
IMAGE:4070070
AA496792 ESTs 9 0/0 1.22 2/2 0.20 6.03
AA127100 ribophorin I RPN1 3 0/0 1.21 2/2 0.20 6.00
AA431795 hypothetical protein EDAG-1 9 0/0 1.21 2/2 0.20 6.00
EDAG-1
AA449753 capping protein (actin CAPZA1 1 0/0 1.18 2/2 0.20 5.83
filament) muscle Z-line,
alpha 1
AA401972 RAB2, member RAS RAB2L 6 0/0 1.71 0/2 0.31 5.53
oncogene family-like
AA427906 beclin 1 BECN1 17 0/0 1.11 2/2 0.20 5.49
AA421783 zinc finger protein 263 ZNF263 16 0/0 1.11 2/2 0.20 5.49
AA478724 insulin-like growth IGFBP6 12 0/0 1.11 2/2 0.20 5.48
factor binding protein 6
AA458630 renin REN 1 0/0 1.10 2/2 0.20 5.44
AA458982 sodium channel, SCNN1A 12 0/0 1.09 2/2 0.20 5.39
nonvoltage-gated 1 alpha
N51278 chemokine (C-X3-C) CX3CR1 3 0/0 1.09 2/2 0.20 5.38
receptor 1
542