U.S. patent application number 13/226640 was filed with the patent office on 2012-03-15 for use of serum amyloid a gene in diagnosis and treatment of glaucoma and identification of anti-glaucoma agents.
This patent application is currently assigned to NOVARTIS AG. Invention is credited to Abbot F. CLARK, Loretta Graves McNATT, Wan-Heng WANG.
Application Number | 20120064532 13/226640 |
Document ID | / |
Family ID | 34710163 |
Filed Date | 2012-03-15 |
United States Patent
Application |
20120064532 |
Kind Code |
A1 |
CLARK; Abbot F. ; et
al. |
March 15, 2012 |
Use of Serum Amyloid A Gene in Diagnosis and Treatment of Glaucoma
and Identification of Anti-Glaucoma Agents
Abstract
The present invention provides compositions and methods for
treating glaucoma, methods for diagnosing glaucoma, and methods for
identifying agents which may be useful in the treatment of
glaucoma. More specifically, the present invention describes the
use of agents that modulate the expression of serum amyloid A.
Inventors: |
CLARK; Abbot F.; (Arlington,
TX) ; WANG; Wan-Heng; (Grapevine, TX) ;
McNATT; Loretta Graves; (Hurst, TX) |
Assignee: |
NOVARTIS AG
Basel
CH
|
Family ID: |
34710163 |
Appl. No.: |
13/226640 |
Filed: |
September 7, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12038673 |
Feb 27, 2008 |
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13226640 |
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11000757 |
Dec 1, 2004 |
7357931 |
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12038673 |
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60530430 |
Dec 17, 2003 |
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Current U.S.
Class: |
435/6.12 ;
435/7.92 |
Current CPC
Class: |
A61K 31/385 20130101;
A61K 38/046 20130101; A61P 27/06 20180101; A61P 27/02 20180101;
G01N 2500/02 20130101; A61P 27/00 20180101; G01N 2800/168 20130101;
G01N 33/5023 20130101; G01N 2333/4709 20130101 |
Class at
Publication: |
435/6.12 ;
435/7.92 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/566 20060101 G01N033/566 |
Claims
1. A method for diagnosing glaucoma, said method comprising: c)
obtaining a biological sample from a patient; and d) analyzing said
sample for the aberrant level, bioactivity or mutations of the gene
encoding serum amyloid A protein (SAA), its promoter region, or
gene products, wherein said gene encoding SAA comprises the
sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, said promoter
region comprises the sequence set forth in SEQ ID NO:12 or SEQ ID
NO:13, and wherein SAA comprises the sequence set forth in SEQ ID
NO:2 or SEQ ID NO:4; wherein the aberrantly high level or
bioactivity or mutations of the SAA genes or the gene products
indicates a diagnosis of glaucoma.
2. The method of claim 1, wherein the biological sample is ocular
tissue, tears, aqueous humor, cerebrospinal fluid, nasal or cheek
swab or serum.
3. The method of claim 2, wherein the biological sample comprises
trabecular meshwork cells.
4. A method for diagnosing glaucoma in a patient, said method
comprising: e) collecting cells from a patient; f) isolating
nucleic acid from the cells; g) contacting the sample with one or
more primers which specifically hybridize 5' and 3' to at least one
allele of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:12, or SEQ ID NO:13,
under conditions such that hybridization and amplification of the
allele occurs; and h) detecting the amplification product; wherein
aberrant level or mutations of SEQ ID NO:1 or SEQ ID NO:3 in the
sample indicates a diagnosis of glaucoma.
Description
[0001] The present invention is a divisional of U.S. application
Ser. No. 12/038,673 filed Feb. 27, 2008; which is a divisional of
11/000,757 filed Dec. 1, 2004 (now U.S. Pat. No. 7,357,931), which
claims priority to U.S. provisional application No. 60/530,430,
filed Dec. 17, 2003.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the field of diagnosis and
treatment of glaucoma. More specifically, the invention provides
methods and compositions for diagnosing and treating glaucoma and
for identifying agents potentially useful for the treatment of
glaucoma.
[0004] 2. Description of the Related Art
[0005] There are a number of ocular conditions that are caused by,
or aggravated by, damage to the optic nerve head, degeneration of
ocular tissues, and/or elevated intraocular pressure. For example,
"glaucomas" are a group of debilitating eye diseases that are a
leading cause of irreversible blindness in the United States and
other developed nations. Primary Open Angle Glaucoma ("POAG") is
the most common form of glaucoma. The disease is characterized by
the degeneration of the trabecular meshwork, leading to obstruction
of the normal ability of aqueous humor to leave the eye without
closure of the space (e.g., the "angle") between the iris and
cornea (Vaughan, D. et al., (1992)). A characteristic of such
obstruction in this disease is an increased intraocular pressure
("IOP"), resulting in progressive visual loss and blindness if not
treated appropriately and in a timely fashion. The disease is
estimated to affect between 0.4% and 3.3% of all adults over 40
years old (Leske, M. C. et al. (1986); Bengtsson, B. (1989);
Strong, N. P. (1992)). Moreover, the prevalence of the disease
rises with age to over 6% of those 75 years or older (Strong, N.
P., (1992)).
[0006] Glaucoma affects three separate tissues in the eye. The
elevated IOP associated with POAG is due to morphological and
biochemical changes in the trabecular meshwork (TM), a tissue
located at the angle between the cornea and iris. Most of the
nutritive aqueous humor exits the anterior segment of the eye
through the TM. The progressive loss of TM cells and the build-up
of extracellular debris in the TM of glaucomatous eyes leads to
increased resistance to aqueous outflow, thereby raising IOP.
Elevated IOP, as well as other factors such as ischemia, cause
degenerative changes in the optic nerve head (ONH) leading to
progressive "cupping" of the ONH and loss of retinal ganglion cells
and axons. The detailed molecular mechanisms responsible for
glaucomatous damage to the TM, ONH, and the retinal ganglion cells
are unknown.
[0007] Twenty years ago, the interplay of ocular hypertension,
ischemia and mechanical distortion of the optic nerve head were
heavily debated as the major factors causing progression of visual
field loss in glaucoma. Since then, other factors including
excitotoxicity, nitric oxide, absence of vital neurotrophic
factors, abnormal glial/neuronal interplay and genetics have been
implicated in the degenerative disease process. The consideration
of molecular genetics deserves some discussion insofar as it may
ultimately define the mechanism of cell death, and provide for
discrimination of the various forms of glaucoma. Within the past 10
years, over 15 different glaucoma genes have been mapped and 7
glaucoma genes identified. This includes six mapped genes
(GLC1A-GLC1F) and two identified genes (MYOC and OPTN) for primary
open angle glaucoma, two mapped genes (GLC3A-GLC3B) and one
identified gene for congenital glaucoma (CYP1B1), two mapped genes
for pigmentary dispersion/pigmentary glaucoma, and a number of
genes for developmental or syndromic forms of glaucoma (FOXC1,
PITX2, LMX1B, PAX6).
[0008] Thus, each form of glaucoma may have a unique pathology and
accordingly a different therapeutic approach to the management of
the disease may be required. For example, a drug that effects the
expression of enzymes that degrade the extracellular matrix of the
optic nerve head would not likely prevent RGC death caused by
excitotoxicity. In glaucoma, RGC death occurs by a process called
apoptosis (programmed cell death). It has been speculated that
different types of insults that can cause death may do so by
converging on a few common pathways. Targeting downstream at a
common pathway is a strategy that may broaden the utility of a drug
and increase the probability that it may have utility in the
management of different forms of the disease. However, drugs that
effect multiple metabolic pathways are more likely to produce
undesirable side-effects. With the advent of gene-based diagnostic
kits to identify specific forms of glaucoma, selective
neuroprotective agents can be tested with the aim of reducing the
degree of variation about the measured response.
[0009] Glaucoma is currently diagnosed based on specific signs of
the disease (characteristic optic nerve head changes and visual
field loss). However, over half of the population with glaucoma are
unaware they have this blinding disease and by the time they are
diagnosed, they already have irreversibly lost approximately 30-50%
of their retinal ganglion cells. Thus, improved methods for early
diagnosis of glaucoma are needed.
[0010] Current glaucoma therapy is directed to lowering IOP, a
major risk factor for the development and progression of glaucoma.
However, none of the current IOP lowering therapies actually
intervenes in the glaucomatous disease process responsible for
elevated IOP and progressive damage to the anterior segment
continues. This is one possible reason why most patients become
"resistant" to conventional glaucoma therapies. Thus, what is
needed is a therapeutic method for altering (by inhibiting or even
reversing) the disease process.
SUMMARY OF THE INVENTION
[0011] The present invention overcomes these and other drawbacks of
the prior art by providing methods to diagnose and compositions to
treat glaucoma. In one aspect, the present invention provides a
method for treating glaucoma by administering to a patient in need
thereof a therapeutically effective amount of a composition
comprising an agent that interacts with a gene encoding serum
amyloid A protein (SAA), or with the gene's promoter sequence. The
interaction between the agent and the gene encoding SAA, or with
its promoter sequence, modulates the expression of SAA, such that
the patient's glaucomatous condition is treated. In preferred
embodiments, the agent will be a protein, peptide, peptidomimetic,
small molecule or nucleic acid.
[0012] In another aspect, the present invention provides a method
for treating glaucoma by administering to a patient in need thereof
a therapeutically effective amount of a composition comprising an
agent that inhibits interaction of the serum amyloid A protein
(SAA) with its receptor. Preferably, the agent will be a peroxisome
proliferator-activated receptor .alpha. (PPAR.alpha.) agonists,
tachykinin peptides and their non-peptide analogs or .alpha.-lipoic
acid. Most preferably, the agent will be fenofibrate, Wy-14643,
(4-chloro-6-(2,3-xylidino)-2-pryrimidinylthiol)-acetic acid),
ciprofibrate, 2-bromohexadecanoic acid, bezafibrate and
ciglitizone, bafilomycin, concanamycin or pseudolaric acid B.
[0013] The present invention further provides a pharmaceutical
composition for treating glaucoma comprising a therapeutically
effective amount of a serum amyloid A protein (SAA) antagonist and
a pharmaceutical carrier. The antagonist contained in the
composition may be any of the compounds identified above.
[0014] In yet another embodiment, the present invention provides a
method for diagnosing glaucoma, by the following steps: [0015] a)
obtaining a biological sample from a patient; and [0016] b)
analyzing said sample for an aberrant level, aberrant bioactivity
or mutations of the gene encoding serum amyloid A protein (SAA) or
its promoter region or its gene products, wherein said gene
encoding SAA comprises the sequence set forth in SEQ ID NO:1 or SEQ
ID NO:3, wherein its promoter region comprises the sequence set
forth in SEQ ID NO:12 or SEQ ID NO:13, and wherein SAA comprises
the sequence set forth in SEQ ID NO:2 or SEQ ID NO:4; [0017]
wherein the aberrantly high level, aberrantly high bioactivity or
mutations of the SAA genes or the gene products indicates a
diagnosis of glaucoma.
[0018] In preferred aspects, the biological sample is ocular
tissue, tears, aqueous humor, cerebrospinal fluid, nasal or cheek
swab or serum. Most preferably, the biological sample comprises
trabecular meshwork cells.
[0019] Alternatively, the present invention provides a method for
diagnosing glaucoma in a patient, by the steps: [0020] a)
collecting cells from a patient; [0021] b) isolating nucleic acid
from the cells; [0022] c) contacting the sample with one or more
primers which specifically hybridize 5' and 3' to at least one
allele of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:12, or SEQ ID NO:13
under conditions such that hybridization and amplification of the
allele occurs; and [0023] d) detecting the amplification product;
[0024] wherein aberrant level or mutations of SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:12, or SEQ ID NO:13, in the sample indicates a
diagnosis of glaucoma.
[0025] The present invention also provides a method for identifying
agents potentially useful for treating glaucoma, by the steps:
[0026] a) obtaining cells expressing SAA (SEQ ID NO:1 or SEQ ID
NO:2) or cells containing SAA promoter/reporter gene such that the
reporter gene is expressed; [0027] b) admixing a candidate
substance with the cells; and [0028] c) determining the level of
SAA protein (SEQ ID NO:2 or SEQ ID NO:4) or the level of gene
expression in the cells; [0029] wherein an increase or decrease of
the production of SAA protein or gene expression in the presence of
said candidate substance indicates an agent potentially useful for
the treatment of glaucoma.
[0030] In another aspect, the present invention provides a method
for identifying an agent potentially useful for treating glaucoma,
by the steps: [0031] a) forming a reaction mixture comprising:
[0032] (i) an SAA protein or a cell expressing SAA or a reporter
gene driven by an SAA promoter; [0033] (ii) an SAA protein binding
partner; and [0034] (iii) a test compound; and [0035] b) detecting
interaction of the SAA protein and binding partner or level of
reporter gene products in the presence of the test compound and in
the absence of the test compound;
[0036] wherein a decrease or increase in the interaction of the SAA
protein with its binding partner in the presence of the test
compound relative to the interaction in the absence of the test
compound indicates a potentially useful agent for treating
glaucoma.
[0037] In another aspect, the present invention provides a method
for identifying an agent potentially useful for treating glaucoma,
by the steps: [0038] a) forming a reaction mixture comprising:
[0039] (i) cells comprising SAA recombinant protein (SEQ ID NO:2 or
SEQ ID NO:4) or cells comprising expression vectors comprising SEQ
ID NO:1 or SEQ ID NO:3; and [0040] (ii) a test compound; and [0041]
b) detecting the effect on downstream signalling (IL-8) in the
presence of the test compound and in the absence of the test
compound; wherein a decrease or increase in the downstream
signalling in the presence of the test compound relative to the
interaction in the absense of the test compound indicates a
potentially useful agent for treating glaucoma.
[0042] In preferred aspects, the cells containing the SAA protein
or expression vectors will be HL-60 cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to these drawings in combination with the
detailed description of specific embodiments presented herein.
[0044] FIG. 1. QPCR analysis of SAA expression in 12 glaucoma vs.
11 normal TM tissues. NTM and GTM represent average expression
level of the gene in normal and glaucoma groups, respectively.
[0045] FIG. 2A. QPCR analysis of SAA expression in TM cell lines.
NTM and GTM represent average expression level of the gene in
normal and glaucoma groups, respectively.
[0046] FIG. 2B. QPCR analysis of SAA expression in optic nerve head
tissues. NTM and GTM represent average expression level of the gene
in normal and glaucoma groups, respectively.
[0047] FIG. 3. SAA protein in TM tissues from normal and glaucoma
donors (n=6). A significant increase (3-fold) in SAA was observed
in glaucoma TM tissues compared to normal tissue (p=0.031). The
bars show mean+/-s.e.m.
[0048] FIG. 4. SAA protein determined by ELISA in human aqueous
humor from normal and glaucomatous individuals. The values are
expressed as the average SAA in ng/ml of aqueous humor, +/-s.e.m.
(p=0.005).
[0049] FIG. 5. IL-8 secretion by HL-60 cells in response to
increasing concentrations of rhSAA.
DETAILED DESCRIPTION PREFERRED EMBODIMENTS
[0050] Glaucoma is a heterogeneous group of optic neuropathies that
share certain clinical features. The loss of vision in glaucoma is
due to the selective death of retinal ganglion cells in the neural
retina that is clinically diagnosed by characteristic changes in
the visual field, nerve fiber layer defects, and a progressive
cupping of the ONH. One of the main risk factors for the
development of glaucoma is the presence of ocular hypertension
(elevated intraocular pressure, IOP). IOP also appears to be
involved in the pathogenesis of normal tension glaucoma where
patients have what is often considered to be normal IOP. The
elevated IOP associated with glaucoma is due to elevated aqueous
humor outflow resistance in the trabecular meshwork (TM), a small
specialized tissue located in the iris-corneal angle of the ocular
anterior chamber. Glaucomatous changes to the TM include a loss in
TM cells and the deposition and accumulation of extracellular
debris including proteinaceous plaque-like material. In addition,
there are also changes that occur in the glaucomatous optic nerve
head (ONH). In glaucomatous eyes, there are morphological and
mobility changes in ONH glial cells. In response to elevated IOP
and/or transient ischemic insults, there is a change in the
composition of the ONH extracellular matrix and alterations in the
glial cell and retinal ganglion cell axon morphologies.
[0051] The present inventors have discovered that the expression of
Serum Amyloid A (SAA) mRNA and protein are significantly
upregulated in glaucomatous TM tissues and cells. The inventors
have verified the differential mRNA expression seen using
Affymetrix gene chips by real time quantitative polymerase chain
reaction (QPCR) and increased SAA protein levels by SAA ELISA. This
is the first time SAA has been shown to be expressed in the TM.
[0052] Human SAA comprises a number of small, differentially
expressed apolipoproteins encoded by genes localized on the short
arm of chromosome 11. There are four isoforms of SAAs. SAM (SEQ ID
NO:2), encoded by SEQ ID NO:1, and SAA2 (SEQ ID NO:4), encoded by
SEQ ID NO:3, are known as acute phase reactants, like C-reactive
protein, that is, they are dramatically upregulated by
proinflammatory cytokines The 5'UTR promoter regions of SAM and
SAA2 genes are also provided (SEQ ID NO:12 and SEQ ID NO:13,
respectively). SAA3 (SEQ ID NO:5) is a pseudogene and SAA4 (SEQ ID
NO:6) is a low level constitutively expressed gene encoding
constitutive SAA4 (SEQ ID NO:7). SAA2 has two isoforms, SAA2a (SEQ
ID NO:9), encoded by SEQ ID NO:8, and SAA213 (SEQ ID NO:11),
encoded by SEQ ID NO:10, which differ by only one amino acid. SAM
and SAA2 proteins are 93.5% identical at the amino acid level (SEQ
ID NO:2 and SEQ ID NO:4, respectively) and these genes are 96.7%
identical at the nucleotide level (SEQ ID NO:1 and SEQ ID NO:3,
respectively).
[0053] SAA is an acute-phase reactant whose level in the blood is
elevated approximately 1000-fold as part of the body's responses to
various injuries, including trauma, infection, inflammation, and
neoplasia. As an acute-phase reactant, the liver has been
considered to be the primary site of expression. However,
extrahepatic SAA expression was described initially in mouse
tissues, and later in cells of human atherosclerotic lesions
(O'Hara et al. 2000). Subsequently, SAA mRNA was found widely
expressed in many histologically normal human tissues. Localized
expression was noted in a variety of tissues, including breast,
stomach, small and large intestine, prostate, lung, pancreas,
kidney, tonsil, thyroid, pituitary, placenta, skin epidermis, and
brain neurons. Expression was also observed in lymphocytes, plasma
cells, and endothelial cells. SAA protein expression co-localized
with SAA mRNA expression has also been reported in histologically
normal human extrahepatic tissues. (Liang et al. 1997;
Urieli-Shoval et al. 1998).
[0054] SAA isoforms are apolipoproteins that become a major
component of high-density lipoprotein (HDL) in the blood plasma of
mammals and displaces A-I (ApoA-I) and phospholipid from the HDL
particles (Miida et al. 1999). SAA binds cholesterol and may serve
as a transient cholesterol-binding protein. In addition,
over-expression of SAA1 or SAA2 leads to the formation of linear
fibrils in amyloid deposits, which can lead to pathogenesis (Uhlar
and Whitehead 1999; Liang et al. 1997). SAA plays an important role
in infections, inflammation, and in the stimulation of tissue
repair. SAA concentration may increase up to 1000-fold following
inflammation, infection, necrosis, and decline rapidly following
recovery. Thus, serum SAA concentration is considered to be a
useful marker with which to monitor inflammatory disease activity.
Hepatic biosynthesis of SAA is up-regulated by pro-inflammatory
cytokines, leading to an acute phase response. Chronically elevated
SAA concentrations are a prerequisite for the pathogenesis of
secondary amyloidosis, a progressive and sometimes fatal disease
characterized by the deposition in major organs of insoluble
plaques composed principally of proteolytically cleaved SAA. This
same process also may lead to atherosclerosis. There is a
requirement for both positive and negative SAA control mechanisms
to maintain homeostasis. These mechanisms permit the rapid
induction of SAA expression to fulfill host-protective functions,
but they also must ensure that SAA expression is rapidly returned
to baseline levels to prevent amyloidosis. These mechanisms include
modulation of promoter activity involving, for example, the inducer
nuclear factor kB (NF-kB) and its inhibitor IkB, up-regulation of
transcription factors of the nuclear factor for interleukin-6
(NF-IL6) family, and transcriptional repressors such as yin and
yang 1 (YY1). Post-transcriptional modulation involving changes in
mRNA stability and translation efficiency permit further up- and
down-regulatory control of SAA protein synthesis to be achieved. In
the later stages of the AP response, SAA expression is effectively
down-regulated via the increased production of cytokine antagonists
such as the interleukin-1 receptor antagonist (IL-1Ra) and of
soluble cytokine receptors, resulting in less signal transduction
driven by pro-inflammatory cytokines (Jensen and Whitehead
1998).
[0055] There are several reports suggesting that primary
amyloidosis may be associated with glaucoma. For example, it was
found that amyloid was deposited in various ocular tissues
including the vitreous, retina, choroid, iris, lens, and TM in
primary systemic amyloidosis patients (Schwartz et al. 1982).
Ermilov et al. (1993) reported that in 478 eyes of 313 patients,
aged 25 years to 90 years, with cataracts, glaucoma, and/or
diabetes mellitus, 66 (14%) of the eyes contained
amyloid-pseudoexfoliative amyloid (PEA). Krasnov et al. (1996)
reported that 44.4% of 115 patients with open-angle glaucoma
revealed extracellular depositions of amyloid. Amyloidosis was
revealed in the sclera in 82% of the cases and in the iris in 70%
of the cases. A number of clinical conditions, including
Alzheimer's disease, exhibit aberrant amyloid tissue deposits
associated with disease. However, amyloids are molecularly
heterogeneous and encoded by different amyloid genes. The previous
reports are unclear regarding which amyloid(s) might be associated
with glaucoma. The present inventors have shown, for the first
time, that SAA gene expression is elevated significantly in
glaucomatous TM tissues. Increased SAA may be involved in the
generation of elevated IOP and damage to the optic nerve leading to
vision loss in glaucoma patients. The present invention provides
methods of using a finding of increased SAA expression to diagnose
glaucoma. The present invention further provides methods for
screening for agents that alter SAA expression or function in order
to identify potentially anti-glaucomatous agents. In another
aspect, the present invention provides methods and compositions of
using agents that antagonize SAA actions and/or interactions with
other proteins for the treatment of glaucoma.
Diagnosing Glaucoma
[0056] Based on the inventors' finding that certain subjects with
glaucoma have increased levels of SAA expression, the present
invention provides a variety of methods for diagnosing glaucoma.
Certain methods of the invention can detect mutations in nucleic
acid sequences that result in inappropriately high levels of SAA
protein. These diagnostics can be developed based on the known
nucleic acid sequence of human SAA, or the encoded amino acid
sequence (see Miller 2001). Other methods can be developed based on
the genomic sequence of human SAA or of the sequence of genes that
regulate expression of SAA. Still other methods can be developed
based upon a change in the level of SAA gene expression at the mRNA
level.
[0057] In alternative embodiments, the methods of the invention can
detect the activity or level of SAA signaling proteins or genes
encoding SAA signaling proteins. For example, methods can be
developed that detect inappropriately low SAA signaling activity,
including for example, mutations that result in inappropriate
functioning of SAA signaling components, including SAA induction of
IL-8. In addition, non-nucleic acid based techniques may be used to
detect alteration in the amount or specific activity of any of
these SAA signaling proteins.
[0058] A variety of means are currently available to the skilled
artisan for detecting aberrant levels or activities of genes and
gene products. These methods are well known by and have become
routine for the skilled artisan. For example, many methods are
available for detecting specific alleles at human polymorphic loci.
The preferred method for detecting a specific polymorphic allele
will depend, in part, upon the molecular nature of the
polymorphism. The various allelic forms of the polymorphic locus
may differ by a single base-pair of the DNA. Such single nucleotide
polymorphisms (or SNPs) are major contributors to genetic
variation, comprising some 80% of all known polymorphisms, and
their density in the human genome is estimated to be on average 1
per 1,000 base pairs. A variety of methods are available for
detecting the presence of a particular single nucleotide
polymorphic allele in an individual. Advancements in the field have
provided accurate, easy, and inexpensive large-scale SNP
genotyping. For example, see U.S. Pat. No. 4,656,127; French Patent
2,650,840; PCT App. No. WO91/02087; PCT App. No. WO92/15712; Komher
et al. 1989; Sokolov 1990; Syvanen et al. 1990; Kuppuswamy et al.
1991; Prezant et al. 1992; Ugozzoli et al. 1992; Nyren et al. 1993;
Roest et al. 1993; and van der Luijt et al. 1994).
[0059] Any cell type or tissue may be utilized to obtain nucleic
acid samples for use in the diagnostics described herein. In a
preferred embodiment, the DNA sample is obtained from a bodily
fluid, e.g., blood, obtained by known techniques (e.g.
venipuncture), or buccal cells. Most preferably, the samples for
use in the methods of the present invention will be obtained from
blood or buccal cells. Alternately, nucleic acid tests can be
performed on dry samples (e.g. hair or skin).
[0060] Diagnostic procedures may also be performed in situ directly
upon tissue sections (fixed and/or frozen) of patient tissue
obtained from biopsies or resections, such that no nucleic acid
purification is necessary. Nucleic acid reagents may be used as
probes and/or primers for such in situ procedures (see, for
example, Nuovo 1992).
[0061] In addition to methods which focus primarily on the
detection of one nucleic acid sequence, profiles may also be
assessed in such detection schemes. Fingerprint profiles may be
generated, for example, by utilizing a differential display
procedure, Northern analysis and/or RT-PCR.
[0062] A preferred detection method is allele specific
hybridization using probes overlapping a region of at least one
allele of an SAA signaling component that is indicative of glaucoma
and having about 5, 10, 20, 25 or 30 contiguous nucleotides around
the mutation or polymorphic region. In a preferred embodiment of
the invention, several probes capable of hybridizing specifically
to other allelic variants involved in glaucoma are attached to a
solid phase support, e.g., a "chip" (which can hold up to about
250,000 oligonucleotides). Oligonucleotides can be bound to a solid
support by a variety of processes, including lithography. Mutation
detection analysis using these chips comprising oligonucleotides,
also termed "DNA probe arrays" is described e.g., in Cronin et al.
(1996). In one embodiment, a chip comprises all the allelic
variants of at least one polymorphic region of a gene. The solid
phase support is then contacted with a test nucleic acid and
hybridisation to the specific probes is detected. Accordingly, the
identity of numerous allelic variants of one or more genes can be
identified in a simple hybridization experiment.
[0063] These techniques may further include the step of amplifying
the nucleic acid before analysis. Amplification techniques are
known to those of skill in the art and include, but are not limited
to, cloning, polymerase chain reaction (PCR), polymerase chain
reaction of specific alleles (ASA), ligase chain reaction (LCR),
nested polymerase chain reaction, self sustained sequence
replication (Guatelli et al. 1990), transcriptional amplification
system (Kwoh et al. 1989), and Q-Beta Replicase (Lizardi, et al.
1988).
[0064] Amplification products may be assayed in a variety of ways,
including size analysis, restriction digestion followed by size
analysis, detecting specific tagged oligonucleotide primers in the
reaction products, allele-specific oligonucleotide (ASO)
hybridization, allele specific 5' exonuclease detection,
sequencing, hybridization, SSCP, and the like.
[0065] PCR based detection means can include multiplex
amplification of a plurality of markers simultaneously. For
example, it is well known in the art to select PCR primers to
generate PCR products that do not overlap in size and can be
analyzed simultaneously. Alternatively, it is possible to amplify
different markers with primers that are differentially labeled and
thus can each be differentially detected. Of course, hybridization
based detection means allow the differential detection of multiple
PCR products in a sample. Other techniques are known in the art to
allow multiplex analyses of a plurality of markers.
[0066] In a merely illustrative embodiment, the method includes the
steps of (i) collecting a sample of cells from a patient, (ii)
isolating nucleic acid (e.g., genomic, mRNA or both) from the cells
of the sample, (iii) contacting the nucleic acid sample with one or
more primers which specifically hybridize 5' and 3' to at least one
allele of SAA that is indicative of glaucoma under conditions such
that hybridization and amplification of the allele occurs, and (iv)
detecting the amplification product. These detection schemes are
especially useful for the detection of nucleic acid molecules if
such molecules are present in very low numbers.
[0067] In a preferred embodiment of the subject assay, aberrant
levels or activities of SAA that are indicative of glaucoma are
identified by alterations in restriction enzyme cleavage patterns.
For example, sample and control DNA is isolated, amplified
(optionally), digested with one or more restriction endonucleases,
and fragment length sizes are determined by gel
electrophoresis.
[0068] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
allele. Exemplary sequencing reactions include those based on
techniques developed my Maxim and Gilbert (1977) or Sanger (1977).
It is also contemplated that any of a variety of automated
sequencing procedures may be utilized when performing the subject
assays, including sequencing by mass spectrometry (see, for example
WO94/16101; Cohen et al. 1996; Griffin et al. 1993). It will be
evident to one of skill in the art that, for certain embodiments,
the occurrence of only one, two or three of the nucleic acid bases
need be determined in the sequencing reaction. For instance,
A-track or the like, e.g., where only one nucleic acid is detected,
can be carried out.
[0069] In a further embodiment, protection from cleavage agents
(such as a nuclease, hydroxylamin or osmium tetraoxide and with
piperidine) can be used to detect mismatched bases in RNA/RNA or
RNA/DNA or DNA/DNA heteroduplexes (Myers et al. 1985b; Cotton et
al. 1988; Saleeba et al. 1992). In a preferred embodiment, the
control DNA or RNA can be labeled for detection.
[0070] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes).
For example, the mutY enzyme of E. coli cleaves A at G/A mismatches
and the thymidine DNA glycosylase from HeLa cells cleaves T and G/T
mismatches (Hsu et al. 1994; U.S. Pat. No. 5,459,039).
[0071] In other embodiments, alterations in electrophoretic
mobility will be used to identify aberrant levels or activities of
SAA that are indicative of glaucoma. For example, single strand
conformation polymorphism (SSCP) may be used to detect differences
in electrophoretic mobility between mutant and wild type nucleic
acids (Orita et al. 1989; Cotton 1993; Hayashi 1992; Keen et al.
1991).
[0072] In yet another embodiment, the movement of alleles in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (DGGE) (Myers et al.
1985a). In a further embodiment, a temperature gradient is used in
place of a denaturing agent gradient to identify differences in the
mobility of control and sample DNA (Rosenbaum and Reissner
1987).
[0073] Examples of other techniques for detecting alleles include,
but are not limited to, selective oligonucleotide hybridization,
selective amplification, or selective primer extension. For
example, oligonucleotide primers may be prepared in which the known
mutation or nucleotide difference (e.g., in allelic variants) is
placed centrally and then hybridized to target DNA under conditions
which permit hybridization only if a perfect match is found (Saiki
et al. 1986; Saiki et al. 1989). Such allele specific
oligonucleotide hybridization techniques may be used to test one
mutation or polymorphic region per reaction when oligonucleotides
are hybridized to PCR amplified target DNA or a number of different
mutations or polymorphic regions when the oligonucleotides are
attached to the hybridizing membrane and hybridized with labeled
target DNA.
[0074] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation or
polymorphic region of interest in the center of the molecule (so
that amplification depends on differential hybridization) (Gibbs et
al. 1989) or at the extreme 3' end of one primer where, under
appropriate conditions, mismatch can prevent, or reduce polymerase
extension (Prossner 1993). In addition it may be desirable to
introduce a novel restriction site in the region of the mutation to
create cleavage-based detection (Gasparini et al. 1992). It is
anticipated that in certain embodiments amplification may also be
performed using Taq ligase for amplification (Barany 1991). In such
cases, ligation will occur only if there is a perfect match at the
3' end of the 5' sequence making it possible to detect the presence
of a known mutation at a specific site by looking for the presence
or absence of amplification.
[0075] In another embodiment, identification of an allelic variant
is carried out using an oligonucleotide ligation assay (OLA), as
described, E.g., in U.S. Pat. No. 4,998,617 and in Landegren et al.
1988). Nickerson et al. have described a nucleic acid detection
assay that combines attributes of PCR and OLA (Nickerson et al.
1990). In this method, PCR is used to achieve the exponential
amplification of target DNA, which is then detected using OLA.
[0076] Several techniques based on this OLA method have been
developed and can be used to detect aberrant levels or activities
of SAA that are indicative of glaucoma. For example, U.S. Pat. No.
5,593,826 and To be et al. (1996), describe such techniques that
are frequently used.
[0077] In one embodiment, fenofibrate, a peroxisome
proliferator-activated receptor .alpha. (PPAR.alpha.) agonist, may
be formulated in a pharmaceutically acceptable composition and used
to treat glaucoma by modulating SAA expression. Studies have shown
that fenofibrate and WY 14643 treatment reduces plasma SAA
concentration (Yamazaki et al. 2002). It is believed that other
PPAR.alpha. agonists, such as ciprofibrate, 2-bromohexadecanoic
acid, bezafibrate, ciprofibrate and ciglitizone may also be useful
for the treatment of glaucoma.
[0078] The present inventors further postulate that agents that
prevent amyloid-induced cell death may be useful for protecting TM
and other ocular cells in the anterior uvea and at the back of the
eye, especially the retina and optic nerve head.
[0079] The Compounds of this invention, can be incorporated into
various types of ophthalmic formulations for delivery to the eye
(e.g., topically, intracamerally, or via an implant). The Compounds
are preferably incorporated into topical ophthalmic formulations
for delivery to the eye. The Compounds may be combined with
ophthalmologically acceptable preservatives, surfactants, viscosity
enhancers, penetration enhancers, buffers, sodium chloride, and
water to form an aqueous, sterile ophthalmic suspension or
solution. Ophthalmic solution formulations may be prepared by
dissolving a Compound in a physiologically acceptable isotonic
aqueous buffer. Further, the ophthalmic solution may include an
ophthalmologically acceptable surfactant to assist in dissolving
the Compound. Furthermore, the ophthalmic solution may contain an
agent to increase viscosity, such as, hydroxymethylcellulose,
hydroxyethylcellulose, hydroxypropylmethylcellulose,
methylcellulose, polyvinylpyrrolidone, or the like, to improve the
retention of the formulation in the conjunctival sac. Gelling
agents can also be used, including, but not limited to, gellan and
xanthan gum. In order to prepare sterile ophthalmic ointment
formulations, the active ingredient is combined with a preservative
in an appropriate vehicle, such as, mineral oil, liquid lanolin, or
white petrolatum. Sterile ophthalmic gel formulations may be
prepared by suspending the Compound in a hydrophilic base prepared
from the combination of, for example, carbopol-974, or the like,
according to the published formulations for analogous ophthalmic
preparations; preservatives and tonicity agents can be
incorporated.
[0080] The Compounds are preferably formulated as topical
ophthalmic suspensions or solutions, with a pH of about 4 to 8. The
establishment of a specific dosage regimen for each individual is
left to the discretion of the clinicians. The Compounds will
normally be contained in these formulations in an amount 0.01% to
5% by weight, but preferably in an amount of 0.05% to 2% and most
preferably in an amount 0.1 to 1.0% by weight. The dosage form may
be a solution, suspension microemulsion. Thus, for topical
presentation 1 to 2 drops of these formulations would be delivered
to the surface of the eye 1 to 4 times per day according to the
discretion of a skilled clinician.
[0081] The Compounds can also be used in combination with other
agents for treating glaucoma, such as, but not limited to,
13-blockers, prostaglandins, carbonic anhydrase inhibitors, a.sub.2
agonists, miotics, and neuroprotectants.
[0082] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Increased Expression of SAA1 and SAA2 in Glaucomatous TM Cells and
Tissues
[0083] RNA pools of TM tissues from 13 normal donors vs. 9 glaucoma
donors was used to determine gene expression using the Affymetric
GeneChips set (HG-U133). Amyloid A2 expression was identified to
increase 4 fold in glaucoma comparing to that in normal TM tissues.
To confirm this result, QPCR was conducted using individual RNA
from 12 glaucoma and 11 normal TM tissues. Five from 12 glaucoma TM
tissues (42%) showed significant increase in SAA1/2 expression.
Average of SAA expression in the 12 glaucoma TM was 5.4 fold to
that in the 11 normal TM (FIG. 1). In addition, a similar trend of
SAA differential expression was observed in glaucoma TM cells or
glaucoma optic nerve head tissues. There was an average increase of
5.4-fold in glaucoma TM cells (14 glaucoma vs. 11 normal TM cell
lines, FIG. 2A) and 118-fold in glaucoma optic nerve head tissues
(14 glaucoma vs. 12 normal, FIG. 2B) compared to normals,
respectively. ELISA of SAA in TM tissues from 6 normal and 6
glaucoma donors showed that SAA protein was also significantly
increased in glaucoma TM tissues compared to normals. There was a
3-fold difference in SAA concentration in glaucomatous tissue
compared to normal tissue (11.3 and 3.8 .mu.g/mg protein
respectively). These data are shown in FIG. 3.
[0084] An association of increased expression of SAA with glaucoma
was further demonstrated in human aqueous humor. SAA protein was
measured by ELISA in aqueous humor from 16 normal and 20
glaucomatous individuals. SAA was found to be almost 3 times higher
in glaucomatous aqueous humor than in normal samples (10.0 ng/ml
vs. 3.7 ng/ml respectively). The results are shown in FIG. 4.
Example 2
Formulation of Fenofibrate for Topical Application
[0085] 1% Fenofibrate suspension for topical application to
decrease SAA and lower IOP in the eye.
TABLE-US-00001 Description Conc. Units Purpose Fenofibrate
(AL18543), 1% W/V % active NOC ingredient hydroxypropyl 0.5% W/V %
viscosity methylcellulose modifier (2910) (E4M), USP dibasic sodium
phosphate 0.2% W/V % buffering agent (anhydrous), usp sodium
chloride, usp 0.75% W/V % tonicity agent disodium edta 0.01% W/V %
chelating agent (edetate disodium), usp polysorbate 80, nf 0.05%
W/V % wetting agent benzalkonium chloride, nf 0.01% W/V %
preservative sodium hydroxide, nf q.s. pH W/V % pH adjust
hydrochloric acid, nf q.s. pH W/V % pH adjust purified water, usp
q.s. 100% W/V % vehicle
Example 3
Procedure for Screening and Identifying Compounds that Alter the
Expression of SAA mRNA or SAA Proteins
[0086] One method that can be used for screening for agents that
alter SAA expression and function is to determine changes in SAA
protein levels. Kits for in vitro assay for quantitative
determination of Serum Amyloid A (SAA) in animal or human sera,
plasma, buffered solutions, cell culture media, and tissue or cell
extracts are commercially available. The assay is a solid phase
sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA). A monoclonal
antibody specific for SAA has been coated onto the wells of a
microtiter plate. Samples, including standards of known SAA
content, or unknowns, are added to these wells along with a
secondary antibody conjugated to alkaline phosphatase or
peroxidase. The antibodies are constructed such that neither one
interferes with the binding epitope of the other. The SAA is both
captured on the plate by the immobilized antibody and labeled with
the conjugated second antibody in a one step procedure. After an
incubation period, the plate is washed to remove all unbound
material and a substrate (PNPP or peroxide) is added. The intensity
of the colored product is proportional to the concentration of SAA
present in the unknown sample.
Example 4
Induction of SAA in Cultured Cell Lines for Screening Compounds
that Alter the Expression of SAA mRNA or Protein
[0087] The human hepatoma cell line, HepG2, is widely used for
studies on SAA induction by cytokines, for transfection with
plasmids, and reporter assays. SAA mRNA and protein synthesis can
be induced by various cytokines in several human hepatoma cell
lines including PCL/PRF/5, HepB and HepG2 (Uhlar and Whitehead
1999). SAA synthesis by human aortic smooth muscle cells (HASMC) is
induced by glucocorticoid hormones and not by the proinflammatory
cytokines, IL-1, IL-6, and TNF-.alpha., which stimulate the
production of SAA by hepatocytes (Kumon et al. 2002b; Kumon et al.
2001; Thorn and Whitehead 2002). SAA stimulated the chemotactic
migration of HASMC in a dose dependent manner when assayed using a
Chemotaxicell culture chamber (Kumon et al. 2002a). SAA mRNA
expression and protein production was demonstrated in primary
cultures of rheumatoid arthritis synoviocytes (O'Hara et al.
2000).
Example 5
Functional Analysis of SAA in Cultured Cells
[0088] Cytokine-like properties of SAA include induction of IL-8
secretion by neutrophils. (Furlaneto and Campa, 2002; He et al.
2003). HL-60 cells, a promyelocytic cell line, was identified that
responds to SAA with increased IL-8 secretion, and can be used for
in vitro assays of SAA function. HL-60 cells were treated for four
hours with increasing concentrations of recombinant human SAA, and
IL-8 was measured in the media by ELISA. IL-8 secretion increased
in a dose dependent manner (FIG. 5). HL-60 cells can be used as a
surrogate cell line for functional assays to identify agents that
alter SAA function and expression levels.
[0089] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and structurally related may be
substituted for the agents described herein to achieve similar
results. All such substitutions and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
REFERENCES
[0090] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by
reference.
United States Patents
Books
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Sequence CWU 1
1
131369DNAhomo sapiens 1atgaagcttc tcacgggcct ggttttctgc tccttggtcc
tgggtgtcag cagccgaagc 60ttcttttcgt tccttggcga ggcttttgat ggggctcggg
acatgtggag agcctactct 120gacatgagag aagccaatta catcggctca
gacaaatact tccatgctcg ggggaactat 180gatgctgcca aaaggggacc
tgggggtgtc tgggctgcag aagcgatcag cgatgccaga 240gagaatatcc
agagattctt tggccatggt gcggaggact cgctggctga tcaggctgcc
300aatgaatggg gcaggagtgg caaagacccc aatcacttcc gacctgctgg
cctgcctgag 360aaatactga 3692122PRThomo sapiens 2Met Lys Leu Leu Thr
Gly Leu Val Phe Cys Ser Leu Val Leu Gly Val1 5 10 15Ser Ser Arg Ser
Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala 20 25 30Arg Asp Met
Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile 35 40 45Gly Ser
Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Lys 50 55 60Arg
Gly Pro Gly Gly Val Trp Ala Ala Glu Ala Ile Ser Asp Ala Arg65 70 75
80Glu Asn Ile Gln Arg Phe Phe Gly His Gly Ala Glu Asp Ser Leu Ala
85 90 95Asp Gln Ala Ala Asn Glu Trp Gly Arg Ser Gly Lys Asp Pro Asn
His 100 105 110Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr 115
1203570DNAhomo sapiens 3agggacccgc agctcagcta cagcacagat cagcaccatg
aagcttctca cgggcctggt 60tttctgctcc ttggtcctga gtgtcagcag ccgaagcttc
ttttcgttcc ttggcgaggc 120ttttgatggg gctcgggaca tgtggagagc
ctactctgac atgagagaag ccaattacat 180cggctcagac aaatacttcc
atgctcgggg gaactatgat gctgccaaaa ggggacctgg 240gggtgcctgg
gccgcagaag tgatcagcaa tgccagagag aatatccaga gactcacagg
300ccatggtgcg gaggactcgc tggccgatca ggctgccaat aaatggggca
ggagtggcag 360agaccccaat cacttccgac ctgctggcct gcctgagaaa
tactgagctt cctcttcact 420ctgctctcag gagacctggc tatgaggccc
tcggggcagg gatacaaagt tagtgaggtc 480tatgtccaga gaagctgaga
tatggcatat aataggcatc taataaatgc ttaagaggtc 540aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 5704122PRThomo sapiens 4Met Lys Leu Leu Thr
Gly Leu Val Phe Cys Ser Leu Val Leu Ser Val1 5 10 15Ser Ser Arg Ser
Phe Phe Ser Phe Leu Gly Glu Ala Phe Asp Gly Ala 20 25 30Arg Asp Met
Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile 35 40 45Gly Ser
Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Lys 50 55 60Arg
Gly Pro Gly Gly Ala Trp Ala Ala Glu Val Ile Ser Asn Ala Arg65 70 75
80Glu Asn Ile Gln Arg Leu Thr Gly His Gly Ala Glu Asp Ser Leu Ala
85 90 95Asp Gln Ala Ala Asn Lys Trp Gly Arg Ser Gly Arg Asp Pro Asn
His 100 105 110Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr 115
12054286DNAhomo sapiens 5gatggttgac aactcccctc ctcttccccc
tcttctactg tctactcctg ggaccaagtg 60agccacgcca gctcagatac tacactgacc
acagggaatc ccaccttttc caaggaatgg 120aagttgtgta gggaatattc
aaatgttgct tagcattgcc ttagataaga accaaaggga 180cagggaaatc
ctctgacagc tatctgcctt ataactttca ttttactgtg cctaaaatat
240gctcagaacc cagaaagagg cataattcct aattttggca ggctctaatc
taaaataatg 300attctcaaac atggtgtgac ttttgtctat ttgctttatc
ctgggtcact gctcctcttc 360tgtcagatac tgggattcca atgagacaaa
tggaaatgga gacgtagacc ctctgacctt 420ctatctttta tctatacaca
tacacctgtg tgtgtgtgtg tgtgtgtgtg tgtgcgtgtg 480taaaaccgag
tgggtttttt tcttggaatg aaagaatgga ctaacattac aaaaaataaa
540aacttgaaac agaatgtgta ttatccttgg ttgtgtttcc ttggccctgc
agcaggatga 600agctctccac tggcatcatt ttctgctccc tggtcctggg
tgtcagcagc caaggatggt 660taacattcct caaggcagct ggccaaggtg
aggtccacag gatagggggc aggaggctgc 720ttctggctgc ccccaggatg
cagctgagca gaggccacat ccccactggg caaaggtgct 780agtgatgcca
cagatggata gagaaggggc atggtttttc ataagcgtgg ttcctcatgc
840ttttctggac agctttgaca ctcttctatg aggatcctcc agccgaggtc
gcataaggtg 900tgagctgcct cttttcagca ggaccatgag agagatgtgg
agttgagggg tgcatgttcc 960cataataccg gtggggctct actgccccct
agtgggaaat ctgggacagt tcatgtctat 1020gtctcctggg aagccaggaa
gcaggtggat caaaagtgtg aggcgagtcc atggggaagc 1080tgaacggagc
caaccgtccc cataaaaaca accaagctta gctgagattt taatacgtac
1140taggcactgt ttaaatgtac taatgaattg gtttccatca tttagtccta
tgatgcaagc 1200agcattatcc cttaacagag aagctaacac acacacacac
acacacacac taacacacac 1260acacacacac acacacacac aaaccccaag
atacgtaaag aagttccaaa gcagagcagg 1320attaacccag gcagtcttgc
tctgcagaac ttgctcttaa tcaaggtact ctgctgcttt 1380caaaacaaga
gtttcggatt tgtgaacaca tagctcatcc tttatctaag aaatggcaaa
1440taggatgtgg tgcctttgga aggtaagtct agctccactt atcccagtaa
aacctacagt 1500gaattacctt gatggtggtt ctactggggc ttatatatgg
ccaggaaact gctagcaaga 1560gaaatatacc ccgagggctg ggcacagtgg
ctcacacctg taatcccagc actttgggag 1620gctgaggtgg gcagatcacc
tgaggtcaag agttcgagac cagcctggcc aacatggcga 1680aatcctgtct
ctactaaaaa tacagaaatt agccgggtgt ggtggcatgc gcctataatc
1740ccagcctctc gggaggctga gggagaagaa ttgcttgaac tcaggaggca
gaggttgcag 1800tgagctgtga tcacaccact gcactccagc ctaggagaca
gagcaagact ccatctagag 1860agacagagag agagagagag ggagaaatat
accccactag ccataataaa gtggcaaaat 1920tttgttttca gaatgcagta
ttttaaattt caggtattat tatttttctg agtctctgaa 1980aaatggtttt
aaggatttgc ttttaatcct atttacatgt tcacacactc aactacaaat
2040atctttcatt ccttaggtta atatttttca aagggttgtt ctgggaccac
ttgcgtgaga 2100atcacctgga ttctgggatg ctttgtgaaa tgaaatgaag
attcccgggt ccatacccta 2160ccccctgccc ccaacagcca cagtctcttg
ggacagagcc tagaaatctt gcctttgcta 2220agcacctcgg tagattttta
tgcacagcaa aggttgagaa ccactacctc ttgttttgct 2280gctgaaagtg
ataaaatgtg ccaggaattt tggaagtact tattaagcca atctgaacat
2340caaggagcca tttaagtcag taactcagag gaataagtag agtaaaaatg
tcataaactc 2400tcaataaaag caatcaattt aacaccagga gtaataaatg
cataaaatga agatgagtta 2460tctaatagag aaattatata aaccatgatt
ataactctat atttgagttc ccccttttcc 2520gtaatcagtt aattttctaa
aaaatcttcg tcacttaatt ctagcttgat cagatccctt 2580cagtccgtaa
ctccctgctc ctcatcttag tttagccctt cttttttctt atgccacctt
2640tcctaaggac cagagaagtg aaatgataat atattggcca cctacaatgt
tctagacatc 2700atacatgtat tttctctgct cttctgcata atcactgtga
ggcaggcaat actcctccat 2760ttcattgggg aggacattga ggttctgaac
tagtgggtca gttgtccttt ttctgaattt 2820gattacccag tagtataaag
ctttcttagg taactcacct ttatcacttg ctgactgaat 2880tctgacagat
gtcagtttct aattatagcc tggacattca gatgtattca ggaccaagtt
2940gtcctcactc tacctacagg catgaatttc tctcattgac taggttagga
gcgccatatg 3000tctgcagcct ccctcagaat cccctgtgtt ctcacaccag
ggaactgagg gttccctggg 3060tccttccagg tagaagttca ttgtacaatg
aaacatccct taaggaccat ttcatctctt 3120ctttaggtgc atcacacatg
gttaaaacaa agtaataaca gaacttagaa tggaatcaaa 3180cagaatgaaa
cttacaccaa gtacaattct cattacatta acccagagaa gtgaaaagta
3240gaagaatatt tatttcaagc caatataatt tccaagggct ttgttgaagg
ctgaaatctt 3300cgggaggaaa gtagtgagaa gaaaactgtt cattcctcta
ttttcccagt atataattgt 3360tttgatcatt ttctttcctt tccagggact
aaagacatgt ggaaagccta ctctgacatg 3420aaagaagcca attacaaaaa
attcagacaa atacttccat gcttggggga actatgatgc 3480tgtacaaagg
gggcttgggg ctgtctgggc tacagaagtg atcaggtaat gcacattcct
3540gatgttgcca ggaatgagtg agcagagctt gactgccttg gacagtcagg
agagaggtaa 3600gctccttgca gagaagttag aggctgcagc ccctcctcct
cttgccctct ctctgcctgt 3660gtgcttagtg cgagggtctg agtggatggt
agaagtgagt gattcctcac cctccctctc 3720tgggtgctgt tcatccagcc
taggggtgcc cagcctggct gagtggggca gtgcccaggc 3780agggtcattg
ttttcacccc tccttccttg gccttcctgg gcttctccca gagtcctccc
3840ttggaaagca gagaatggga aggtgggctg ttgctcactg gcctggtgat
taatctcctt 3900gcttgcctgg actacagcga tgccagagag aacgtccaga
gactcacagg agaccatgca 3960gaggattcgc tggctggcca ggctaccaac
aaatggggcc agagtggcaa agaccccaat 4020cacttccgac ctgctggcct
gccagagaaa tactgagctt ccttttcaat ctgctctcag 4080gagacctggc
tgtgagcccc tgagggcagg gacatttgtt gacctacagt tactgaattc
4140tatatcccta gtacttgata tagaacacat aaaaatgctt aataaatgct
tgtgaaatcc 4200agtttgttat tggaatctgg aagcagaata tgacagtctt
cctgggatca tgggcctgtt 4260tagtaccata gggatgacca ataaac
42866193DNAhomo sapiens 6gttttctgct ccttggtcct gggtgtcagc
agccgaagct tcttttcgtt ccttggcgag 60gcttttgatg gggctcggga catgtggaga
gcctactctg acatgagaga agccaattac 120atcggctcag acaaatactt
ccatgctcgg gggaactatg atgctgccaa aaggggacct 180gggggtctgg gct
193764PRThomo sapiens 7Val Phe Cys Ser Leu Val Leu Gly Val Ser Ser
Arg Ser Phe Phe Ser1 5 10 15Phe Leu Gly Glu Ala Phe Asp Gly Ala Arg
Asp Met Trp Arg Ala Tyr 20 25 30Ser Asp Met Arg Glu Ala Asn Tyr Ile
Gly Ser Asp Lys Tyr Phe His 35 40 45Ala Arg Gly Asn Tyr Asp Ala Ala
Lys Arg Gly Pro Gly Gly Leu Gly 50 55 608369DNAhomo sapiens
8atgaagcttc tcacgggcct ggttttctgc tccttggtcc tgagtgtcag cagccgaagc
60ttcttttcgt tccttggcga ggcttttgat ggggctcggg acatgtggag agcctactct
120gacatgagag aagccaatta catcggctca gacaaatact tccatgctcg
ggggaactat 180gatgctgcca aaaggggacc tgggggtgcc tgggccgcag
aagtgatcag caatgccaga 240gagaatatcc agagactcac aggccatggt
gcggaggact cgctggccga tcaggctgcc 300aataaatggg gcaggagtgg
cagagacccc aatcacttcc gacctgctgg cctgcctgag 360aaatactga
3699122PRThomo sapiens 9Met Lys Leu Leu Thr Gly Leu Val Phe Cys Ser
Leu Val Leu Ser Val1 5 10 15Ser Ser Arg Ser Phe Phe Ser Phe Leu Gly
Glu Ala Phe Asp Gly Ala 20 25 30Arg Asp Met Trp Arg Ala Tyr Ser Asp
Met Arg Glu Ala Asn Tyr Ile 35 40 45Gly Ser Asp Lys Tyr Phe His Ala
Arg Gly Asn Tyr Asp Ala Ala Lys 50 55 60Arg Gly Pro Gly Gly Ala Trp
Ala Ala Glu Val Ile Ser Asn Ala Arg65 70 75 80Glu Asn Ile Gln Arg
Leu Thr Gly His Gly Ala Glu Asp Ser Leu Ala 85 90 95Asp Gln Ala Ala
Asn Lys Trp Gly Arg Ser Gly Arg Asp Pro Asn His 100 105 110Phe Arg
Pro Ala Gly Leu Pro Glu Lys Tyr 115 12010369DNAhomo sapiens
10atgaagcttc tcacgggcct ggttttctgc tccttggtcc tgagtgtcag cagccgaagc
60ttcttttcgt tccttggcga ggcttttgat ggggctcggg acatgtggag agcctactct
120gacatgagag aagccaatta catcggctca gacaaatact tccatgctcg
ggggaactat 180gatgctgcca aaaggggacc tgggggtgcc tgggccgcag
aagtgatcag caatgccaga 240gagaatatcc agagactcac aggccgtggt
gcggaggact cgctggccga tcaggctgcc 300aataaatggg gcaggagtgg
cagagacccc aatcacttcc gacctgctgg cctgcctgag 360aaatactga
36911122PRThomo sapiens 11Met Lys Leu Leu Thr Gly Leu Val Phe Cys
Ser Leu Val Leu Ser Val1 5 10 15Ser Ser Arg Ser Phe Phe Ser Phe Leu
Gly Glu Ala Phe Asp Gly Ala 20 25 30Arg Asp Met Trp Arg Ala Tyr Ser
Asp Met Arg Glu Ala Asn Tyr Ile 35 40 45Gly Ser Asp Lys Tyr Phe His
Ala Arg Gly Asn Tyr Asp Ala Ala Lys 50 55 60Arg Gly Pro Gly Gly Ala
Trp Ala Ala Glu Val Ile Ser Asn Ala Arg65 70 75 80Glu Asn Ile Gln
Arg Leu Thr Gly Arg Gly Ala Glu Asp Ser Leu Ala 85 90 95Asp Gln Ala
Ala Asn Lys Trp Gly Arg Ser Gly Arg Asp Pro Asn His 100 105 110Phe
Arg Pro Ala Gly Leu Pro Glu Lys Tyr 115 1201210001DNAhomo sapiens
12gggtggatca cgaggtcagg agatcgagac catcttggct aacatggtga aaccccgtct
60ctactaaaaa tacaaaaaaa ttagccgggc gtcatggtgg gcgcctgtag tcccagctac
120tcgggaggct gaggcaggag aatggtgtga acccgggagg cagaacttgc
agtgagccta 180gatcgcgcca ctgcactcca gcctggggga caaaacgaga
ctctgtctca aaaaaaaaaa 240aaaaaattcc cacattagag ttggggaaat
gggcagtcct ggtggaagtt agggaacaga 300tctgggacac gttatagcca
gctggactac aggaggccat aagctcaatt cttccttgac 360tctgaaacct
tccactggtc ctaatgccta gtaattccag gcctttccca gttgtgccag
420gcttggaggt gaacacatct atgtgccaag aaggaaaggt atgccaagca
ggggcttaag 480tcatccttat cctcagtctg tctatgagtg gtatgtaccc
ctgttcccct tgcaagatct 540gctgggctta ggtctcctgg ctgtgagttc
cccatacctg ggcataaatg tagtgagcct 600gagctcccaa ataaggttgg
gggctccaga gaggtggaga gccctgtgtc tgggaagtgt 660gcccacccag
caggtctgac caggaagata cactgctagg gttatggaaa aagactatgt
720gtcaaggtct cttgattctc catctaggca gagaatcatc tttaattaat
gggaaactgg 780aaggcaaatt acttggacct gaaattactt tttgtttatt
gaaccactgt gttgtaaatc 840acatctctct gaaggcaaga gaaatcaggg
agttacaaaa tgtttaggag aactaaacag 900gactccctgt tttgctaact
aatcagattg agacaggctc tctggtaaat ctacaaattt 960gatgttgttc
aaccataagc agtaaatttc ctatgctgga ttttcctgac aatgaatgta
1020aaaggaaaag gagtcttttt gacaaaatat tttattgttc atctaaactg
aaaaacttct 1080ctatttttca aaattgctat acgtgtttaa agatgtagat
atttgaatag cctaactggt 1140acagaaggtt taatgatgat tcctaagaca
tacctataaa ttacttgaaa ttgaaacgaa 1200atttaagaag aattattgga
attttcccct tctcaaatga gttcttagtt tcataaatac 1260tatacaagtc
cataagagat ttggggtttt gagatgtctt tttttttttt tttttttcag
1320acggagtttc actgttgttg cctaggctgg agtgcaatgg cgtgacctca
gctcactaca 1380acctccacct cccaggttca agcgattttc ctgcctcagc
ctcccaagta gctgggatta 1440cagggacctg ccacaacgcc aagctaatgt
tttgtatttt tagtagagat ggggttcacc 1500atgttggcca ggcttgtctg
gaactcctga cctcaggtga tccacccgcc tataatttat 1560tactcccttt
tgcaaatgtt tgaaaaggaa taaagtgcaa tatttttaaa cagaatgcag
1620agttctgttg tcctttggca ataccagttt cagactctga gagtggctct
tgctgttgcc 1680gacagtgggc tgatgaccaa atcccaacat gcccccgctg
cgagtccttc ataacctgat 1740tcagtcatca cttagaggcc agcaggcttc
agggaggcgt gagcctcagc caacaaccta 1800taggggaaga gacgcagaac
tcaatgcaga caggtttgga ttctggtgcc tagagaatgc 1860aacttggaaa
ctctgagcca ggagaaaagg gttctctctc catgagagag tgtgggcttt
1920gtgagaagcg acacacagca aacacaatta agagtccacc cctcagcggg
gcgcaggggc 1980tcacgcctgt aatcccagca ctttgggagg ccgaggcggg
tggatcacga ggtcaggaga 2040tcaagaccat cctggctaac acagtgaaac
cctgtctcta ctaaaaatac aaaaaaatta 2100gccgggcgtg gtggcgggcg
cctgtggtcc cagctactcg ggaggctgag gcaggagaat 2160ggtgtgaacc
cgggaggtgg agcttgcagt gagccgagat cgcgccactg cactccagcc
2220tgggcgacag agcgagactc catctcaaaa aaaaaaagaa aaagaaaaag
aaaaagagtc 2280cgcccctgaa ttaaatagtt ggtccttttg tgttcctggt
gattcacttg ctaagtggaa 2340gaaacaggag ggaatctttt ctcctgccct
cctggtaatc catagcccat ggcctggctt 2400tacttctgta aagtggcagg
agaccttttg acagctgagc catttcttat tttatttatt 2460ttaataagag
atggtaggaa tgagcaatga tattagtacc tggggactgt tgttcttaag
2520gagaaacaat cttagaatga ttagtgatac cccttgcttt ctcttttctt
tcattatact 2580ttttgtacac atatttttcc catttattta ttggaatctt
actgatttat tataagtata 2640agctttatgt ctacacatgt ataatcattt
ttccccaagt ataagtctct ttttcatgga 2700ggcacagcct agacctggtt
agccgccatc tcccctcatt gtatgcccaa tatctattgt 2760agtatctgct
gcatagaagg cactcgatgc gtgaatggat aatgactgat gatgaatcaa
2820taaataaatg gacatgtcat tgtaaaaaat tctaaaaatc tagaataaca
caagctgttg 2880gcactaccta gaaacacaga tgtaaaactt cctaggttgt
gtttcaccat gggaacatgt 2940ctttgaacaa aaatgggatc atattctatt
gcactctttc ccttaagaga tacttctcca 3000ggtcattaag tgctcttcca
caatatcagt atatggcaga ggcaaggtca taccaggtct 3060gtctgaaacc
agggcttggc tcttaacttg cagccatact gcctccaagt ctaggtggct
3120gggttttagg atctgtaatg ggaactcagt gtcacaacct ctactgggaa
ggtattctgg 3180tgttgcataa caggactttc tgttagagat aaccatggca
aaatggaata gagacaaagt 3240tcaggtttct gctgccagga gctgagattg
ctgtgaccaa tggcattctc ccaaaccaaa 3300taatccaacc tggaattacc
ataaaccact cctcatcttt tcaaggggtg tccaagttcc 3360cagaaaagaa
catttgttaa gggatggagg caaggaggtg gagaagaaag agcactggcc
3420aaggtatcat gagtgtcctg ggttctggtc cttgaataag ccatttatct
tctctgcagc 3480ttctccatct gataggagtt tggaggcaga gttttttctt
aatgagcaaa agacagtcgt 3540gcctaggaga tgtggtgtac atgttagaaa
gaagggactg gctgtgactc tataaaagat 3600gaattcatac aaaaacaaat
taccctttcc cagggagaaa gtttggatcc agtaattaga 3660gatctcaaaa
agtagaagac ctgccctgtg aggcctgtgg cctccaagtt tgaatgctgt
3720gtgtcagctt taaaaactag tttcttgctg ataaatgttt catattaagc
atgtgttgag 3780agtactcctt gcctaccttc actagccact gtttccttcc
cctcctccct tgtcccttca 3840ttctctccag aactttctgc taacttccat
tctcttcagg acttcagcat ggttgggaga 3900agatcagaaa ggcatcctca
ctgtttttat tttagtccac ttgacctttg gggagtagtt 3960ccactggctc
ataagtatca gccccccata gcacagcacc ccacactgag cccggaagca
4020ataaagaatc ccaatctgct gtcactaacc agcacgctca actgccatgc
cctttactct 4080tctcatctcc ctgctttcac gtcacaccaa ctaatttctc
tatgagtcag cctcaactct 4140cccaacactc tgcccaccct tcttctacta
ccttccagtg agctcctcga aagaagggtc 4200tgcggtgagg atgccccttt
atctctgcct atttccttcc cattacaaaa acttgaaacc 4260tgcctttccc
atgttgattt cactttattc tcatctttac ccatggggta tgcctcctgc
4320aattcctcct agacaataga atgagaaaga ggggtcctcg tcctctttgc
tttccatgac 4380catttctcca ttcttcacct ctgtgatgtg tcctctttga
agtccctgat aaattcatta 4440ccaccttctc tccagtctta ctaatgttat
ctgcacaagt gatttccaaa caggaagatt 4500ttcaaacact gattcctgaa
gatcaccccc aactcgctga actgagacca agacctccaa 4560gattatggct
taggaatctg catttttttt ttttttttga gacaagagtc tcgctctgtt
4620gccaggctag agtgcaatgg tggaatcata gctcattgta acctcaaact
cctgggctca 4680agtgatcttc ctgcctcagc ctcccaagta gtgaggacaa
caggagtgtg ccaccatgcc 4740cagctaattg ttaatttttt gtagaaatgg
agtctcacta tgttgctcgg gctggtctca 4800aactcctgac cttaacccat
cctccgcctc cgcccccaaa agtgttggga ttacaggtgt 4860gagccaccgt
gcccagccta gaaataccca ctagaagctt ctgtgtagac aatctgctta
4920gtgatgtttg gagacaaagt acctctttat tgtattcatt gacaaaactc
tccagtcctc 4980tcccatcttc atggaaaatt ttcacagttc atttacggcc
ctctttccaa cacattcact 5040gccaatactc ttattgacaa taactgtatt
gttgaacctt
ccagtatcct gcattcccgg 5100atcaaggccc cctcaaagcc ctgatatgca
aatatctggg aaaagaatgt tccagaggaa 5160aggaacagct aatccgaggc
ccctagggta agatgtgcct gggggtttgg agaccagtgt 5220ggccagagca
aaatgagcag gaggagagaa ttggatgatg aggtacgaga ggaaggagtt
5280aggacagttt gagtaaagtt tgaaaaccat tataagggct ttgacttcaa
ctatgagtgg 5340aagtggaatc ctccggagag ttttgaatgg agagtgatag
aagttgtctt gtgttgtaac 5400agtctggctg ctatactgaa aagagactag
ttggcggcaa agggggaaat gtggaagcca 5460gttaagaagc catcataacc
cagaaggtga tgcctaataa catctctctg ggagcagcgg 5520agagatgata
agggtttgcc ttctgaatat gttttttgac aattaatgta aacatttcaa
5580gtaggctgag attttattgc atattaacaa tgtccatgtt cactcgcggc
agccgccccc 5640ttctgcgcgg tcatgccgag ccagcacctg ggcctggaac
tgggccgcag cccccagctt 5700cacccaccac ctccctacca tggacccctg
caaagtgaac gagcttcggg cctttgtgaa 5760aatgtgtaag caggatccga
gcgttctgca caccgaggaa atgcgcttcc tgagagagtg 5820ggtggagagc
atgggaggta aagtaccacc tgctactcag aaggctaaat cagaagaaaa
5880taccaaggaa gaaaaacctg atagtaagaa ggtggaggaa gacttaaagg
cagacgaacc 5940atcaactgag gaaagtgatc tagaaattga taaagaaggt
gtgattgaac cagacactga 6000tgctcctcaa gaaatgggag atgaaaatgt
ggagataacg gaggagatga tggatcaggc 6060aaatgataaa aaagtggctg
ctattgaagt cctaaatgat ggtgaactcc agaaagccat 6120tgacttattc
acagatgcca tcaagctgaa tcctcgcttg gccattttgt atgcaaagag
6180ggccagtgtc ttcgtcaaat tacagaagcc aaatgctgcc atccaagact
gtgacagagc 6240cattgaaata aatcctgatt cagctcagcc ttacaagtgg
cgggggaaag cacacagact 6300tctaggccac tgggaagaag cagcccatga
tcttgccttt gcctgtaaat tggattatga 6360tgaagatgct agtgcaatgc
tgaaagaagt tcaacctagg gcacagaaaa ttgcagaaca 6420ttggagaaag
tatgagcgaa aacatgaaga gcgagagatc aaagaaagaa tagaacgagt
6480taagaaggct caagaagagc aggagagagc ccagagggag gaagaagcca
gacgacagtc 6540aggagctcac tatggccctt ttccaggtgg ctttcctggt
ggaatgcctg gtaattttcc 6600cggaggaatg cctggaatgg gaggggacat
gcctggaatg gccggaatgc ctggactcaa 6660tgaaattctt agtgatccag
aggctcttgc agccatgcag gatccagaag ttatggtggc 6720cttccaggat
gtggctcaga acccagcaaa tatgtcaaaa taccagagca acccaaaggt
6780tatgaatctc atcagtaaat tgtcagccaa atttggaggt caagcataat
gcccttctga 6840taaataaagc cctgctgaag gaaaagcaac ctagatcacc
ttatggatgt cgcaataata 6900caaaccaacg tacctctgac cttctcatca
agagagctgg ggtgctttga agataatccc 6960tacccctctc ccccaaatgc
agctgaagca ttttacagtg gtttgccatt agggtattca 7020ttcagataat
gttttcctac taggaattac aaactttaaa cactttttaa atcttcaaat
7080atttaaaaca aatttaaagg gtctgttaat tcttatattt ttctttacta
atcattgtgg 7140atttttcctt aaattattgg gcagggaata tacttattta
tggaagatta ctgctctaat 7200ttgagtgaaa taaaagttat tagtgcgagg
caaacataaa aaaaaaaagt ccatgttcat 7260ctctaaatga catcattgtt
ccaaagcttt tccattcttc ttaaccttcc acctgtcaat 7320ctataggaga
tgacttctcc tacttcactc atgcattgac tccttcaatc aataaaagtg
7380actaagaacc tgctacaggt gaggtgctgt gtttggtgtt aaagtgacaa
cagttatctg 7440tcaataagcc tgacaaggtt cctatccctg tgttttgtgc
actctgggtc aaactcagaa 7500atgcaaacag gtggagagcg atgagttcta
tgactggtaa agaaaagggc ctgctggttt 7560ccctcaggat ctctgtcctt
catctcaaaa tgcatcttcc ttgttatcgt tcctctcctt 7620cctgtctcag
aggaagacct gctcctgcta cactctgggc aaccttgtcc ccgtggccct
7680gtggcccctt ggttgttgaa gtctatgtta tgccctatct tttaccctca
gtcactctct 7740ctgttaacat tctccctgtg ccctgtaacc ctccctcatc
tttaaataaa tcctcctcct 7800ttgaccttcg catgtattca gtcatgcaac
tcaacaagca tttattgcac agtgatattc 7860aatttgccac ttgctaaaag
tctgaacctt ggcagctgaa tgtgatcaga aaaaaagcac 7920gactgctatg
actagtctca ctttaaattc atggtcgttg accaagagct accatacaat
7980ccactacctt tctcaagttc agtcacattc ttcctttcct agatgtctgc
tttctacttc 8040tcttctcttc tgaaacttcc cacaactcct cgttcattct
cttctcagtt gacaactttg 8100cttcctattt cactgaaaaa tagaagcaat
cagatatgaa cttctggctg ggcatggtag 8160ctcatgccta taatctcagc
actttgggag gccaaggcag gaggactgca ggttaggaat 8220ttgagaccag
cctgggcaac atggtgaaac tcccactgta ctaaaaattt taaaaattac
8280tcaaacatat tggcaaacaa ctgcagtccc agctacttgg gaggttgaga
tgcaaggatc 8340acttaaacct gggaggctga ggctgcagtg agccatgatt
gcaccactgc actccagctc 8400aggcaacaga gcaagaccct gtcttgagag
gagaggagaa gagaggaggg gaggggaggg 8460caggggaggg gaggggaggg
gaagggagag gggaggggag aggggaggag agaggggagg 8520ggaggggagg
ggaggggagg ggaggagagg aggatcaggt gaggagtatg ccaaggagtg
8580tttttaagac ttactgtttt ctctttccca acaagattgt catttccttt
aaaaagtagt 8640tatcctgagg cctatattca tagcattctg aaagaaagaa
aagaaaagag gaaagaaaga 8700gagaggaagg aaggaaggag aaagagagag
gaaggaagga gaaagagaga ggaaggaagg 8760gaggaagaga agaagggagg
aagaaaagaa ggaaggaagg agggagggag ggaagggagg 8820gagggaaaga
ggaagaaagg agggaaagaa ggaaggaaga gagagaggaa ggaaggagga
8880agagagaaga aggaaggagg aagacagaga gggagtaagg aaggaaggaa
ggagaaagag 8940agaggaagga agaaatgaag gaaggaagga aagaaagaaa
aaataaaaga gtgaaaacgg 9000actggagaag aagaaaccac agttgctgct
atatccacca gcctctctgc atgtcctggc 9060ctcagccctg ctgggctctg
gtactgacca cttccttcct tcctaatttc ctaattgact 9120aggccagctg
agcagggctt ttctgtgctg aggaggtaaa tctctggata tctagactga
9180ggggtggaag gagccttcca gggcacacat gagacatggc aggggtaggc
tgctagtttt 9240attttgtttt cttttagaca cagggtcttg ctctgttaac
caggctggag tgcagtggcg 9300tgattatagc tcactgcagc cttgacctcc
tgggtctccc acaatccttc cgcttcagcc 9360tcttgagtag ctgggactgc
aggtgcacac taccacaccc ggtccattta tttttatatt 9420tcgtagagac
aagatcttac agttttgcac agagtgatct taaactcttg accccaagtg
9480atcctcctgc cttggcctcc aaaagcattg ggattatagg agtgagccac
tgtgctggac 9540ctagtctgtc agctttgaag ctttagatat gaactcagag
ggacttcatt tcagaggcat 9600ctgccatgtg gcccagcaga gcccatcctg
aggaaatgac tggtagagtc aggagctggc 9660ttcaaagctg ccctcacttc
acaccttcca gcagcccagg tgccgccatc acggggctcc 9720cactctcaac
tccgcagcct cagccccctc aatgctgagg agcagagctg gtctcctgcc
9780ctgacagctg ccaggcacat cttgttccct caggttgcac aactgggata
aatgacccgg 9840gatgaagaaa ccactggcat ccaggaactt gtcttagacc
gttttgtagg ggaaatgacc 9900tgcagggact ttccccaggg accacatcca
gcttttcttc gctcccaaga aaccagcagg 9960gaaggctcag tataaatagc
agccaccgct ccctggcagg c 100011310001DNAhomo sapiens 13gtctgccagg
gagaggtggc tgctatttat agtgagcctt gctggtctct tgggagggaa 60gaaaagctgg
atgtggtccc tggggaaagt ccctgcaggt catttcccct acaaactggt
120ctaagacaag ttcctggatg ccggtggttt cttcatcccg ggtcatttat
cccagttgtg 180taacctatgg gaacaagaga ggtttgctgt gccttggcaa
tggacagggt gctagatcag 240ctctgctcct cagcattggg ggaagtgcag
ctgcagagat gccagtggga gccccgtgat 300ggcggcacct gggctgctgg
aaggtgtgga gtgagggcag ctcttcagcc agctcctgac 360tataccggtc
atttcctcag gatgggccct gctgggccac atggcagatg accctgactg
420aaatccctgt gagttcatgt ctaaagcttt aagctttaaa acggacagcc
tacccctgcc 480acatctcatg tgtgccctgg aagcctcctt ccacccctct
ggatgtcctg atatttctca 540gcacagaaaa tctctgctcc gctggcttag
ccaatttgga aatgcttttt ctaagttggc 600tcctgagcca aggacaatgt
agagaggggg actttctgct gccccagcct agtcctggag 660ccccaccttg
ggagaatgag agtgtggtgc gttaaatagg cagcccagct ggggacgtgc
720ccagcatcca ggcagggaag ggtgggagag ctcttggtct gctgtattat
cacggagggg 780tgcagggggc atgcagatca ctctctcatg agaacatcaa
cagggtcaga ttagctctgc 840agaggcttat ggaggagcat ggtggccaga
gatgggtcag taccagagcc caggggggct 900gaggccagga catgcagaga
ggctggtgga catagcagca actctggttt cttcttctcc 960agtccatgtt
cataccctga gggctaggca tttgtaataa caaacaaaca agcaatttag
1020aaatgggcca ggcatggtgg catgtgccta tagtcccagc tacttgggag
gccaaggcag 1080gaggcctgct tgaacccaga aatttgaggc cagcctgggc
aacacagcaa gattatctta 1140aaaaattttt tttaatctct gagaaatggg
tagggccagg aagtaaagga tggccaaata 1200ctccataagc agcaaatgcg
tggctccaat gtgaacaatg atattataga ctctgttctg 1260agacctatgc
attgacacct ccacctcccc cactacatct tgccacctta aaaccactga
1320gagtggtacc tgctggaatg ggtccacaca cacagtcaca catattttag
gcagggtagt 1380tgacatcccc agggaaaaag agctcacaga gagaggctga
atgtttccaa ctgggtagca 1440gtaatagtac atcatgctgt acatggtaca
gcacagatca ggtgaaaata atagcacatc 1500gtgattaacc agggcttatt
ccagggagtc aagaagagtt tcatatcaga aaaatctatc 1560tttgtaattc
actataccag taatcaaaga aaaggattgt acatttattt tactagatgc
1620agaaaatgaa tttcataatt gtcaacatct actgatgata aggaaaatgt
ataacaaaat 1680aaagagacca tttctgactt gagaaaggat aaataccaat
atgttatagc aacagttctc 1740aaactgtttt ccagggaacc ctaagaatcc
ctccttaggg aggctttgat ctcaaaatta 1800tttttagaat agtgctaaca
cactattttc atgtttcagt ctcattttct catgagtaca 1860cacaatatga
caagttagtt gatatgagtg tggatttcca catggtaact gacttttcag
1920aagctaccac ttgttgagtt tggtataata tagaatagcc acaattatct
aaaaatacca 1980ttaaaataca ctcccccatt tcaactatat atctgtgtga
ggctgaattt tcttcatata 2040ctccaaccta aataacatat taaaacaggt
tggatgatga atcagatagg aaaatccagc 2100tatgaaaaaa aaatcagaca
tgaaaaattt tcaaaagggt aaaaccatag tactcttctt 2160actttttttc
ttttggaaga tggttatttt tcataaaaat atattattta tgttaacata
2220tagaagatgg ataatttttt gaagaattga taaatgttta aattttttct
ttctattatg 2280gtaaatactg atgaatagag tccccataaa taaaagttct
ttggggtatt caataatttt 2340taatagtgta atgggatcct gagaccaaaa
ggtttgagaa tcattgctct acagcaaaca 2400ttatgtgtaa ttaagacact
tcaggtgcat tctcaagaag accaataaag aggccacaat 2460ggcaggcgtg
gtggctcaca cttgtaatcc aagaacttag agaggacgag gcaggtggat
2520cactggaggt caggaattct caaccagcct ggccaacatg gtgaaaccct
gtctctacta 2580aaagtacaaa aattagtcgg gtgtagtggc aggtacctgt
aatcccaagt acttgggggg 2640ttgaggcagg agaatcactt gaagccggga
ggtggaggct gcagtgagcc gagatcgtgc 2700cactgcactc cagcctgggc
aacggagtga gacttcatca tggaaaaaaa aacaaagagg 2760ccaggatgtc
tggttgttac tgccactgtt tcacatatcc ctgaaggacc tgcccaatgc
2820taaagaaaca caaggaaggt aagaggtgaa agagaagaaa tgaaactatc
attgtttgaa 2880gatgacacca tcttttacat agaaaacctg ttagaatcaa
atggcaagct attagaacta 2940ctaagagaat tcagtgaggc tgctgtattc
atggcaaaat tttaacaatt gatagcattt 3000ctctgcaaca ttccttaata
gttataaaat acagcacaaa gtagtaccaa aaatattaac 3060tatctaggaa
ataacctctt acagagaaaa tttagtctgt taaaggataa acagtggcaa
3120tgtacgtcat gtccacagag attatatttt agcttagcaa agataccaat
tctcccaaat 3180ttatttataa attaaatgca atgtgaatca aaatttccca
ctggaatttt tatcaggaag 3240gcaacaaatt ctttctttct ttctttcttt
ctttctttat ttatttattt atttatttat 3300ttatttattt ccttccttcc
ttccttcctt ccttccttcc tttctttctt tctttctttc 3360tttctttctt
tctttctctc tctctttctc tctccccccc tctctctctc tctgtctctc
3420tctctctctc tttctttctt tctttctttc tttcttttta agacaaagtc
tggctctgtc 3480acccaggctg cagtgcagtg atacaatctc agctcactga
aacctcaacc tctccggcat 3540caggtgaacc tcccacctca gccccccgag
tagctgggac tacaggtgca caccactggg 3600cctagataac tttttgtatt
tattgtaaat aaacacaaaa aataaatatt ttgctcaggt 3660tggtctggaa
ctcctgggct caagcaatcc gcctgccttg gcctcccaaa gtgctagaat
3720tacagttgtg agccaccaca cccagccaat aaattaattc tttatgatga
ataagttatc 3780tatgaaaatt aagtcagctg ggtgcggtgg ctcacgcctg
taatcccagc actttgccgg 3840gctgaagcag gtggatcacc tgaggttggg
agttcaagac cagccggacc aacatagaga 3900aaacccgtct ctactaaaaa
tgcaaaatta gctgggtgtg gtggcatatg cctgtaatcc 3960cagatactta
ggaggctgag gcaggagaat tgcttgaacc cgggcggtgg aggttgcggt
4020gagccaagat tgcaccattg cactccagcc tgggccacaa gagcgaaact
ccatctcaaa 4080aaaaaaaaaa gagaagttaa gtcaatgaaa agttaagtca
attaaaaaag taagagctgt 4140agtgtttaga tatatacaca cacacatata
tatatattta tctttatata tgtatatata 4200tcttttcctt tttttgagac
cgagtctgtt tttgttgccc aggctggaat gcagtggcgc 4260gatctctgct
tactgcaacc tctgcctccc aggttcaagc gattctcgtg cctcagcctc
4320ccgagtagct gggattacag gtgcctgccc ccatgcccgg ctaatttttg
catttttagt 4380agagacgggg tttcaccatg ttggccaggc tggtctcaaa
ctcctgacct caggtgatcc 4440accggcctca gcctcccaaa gtgctgggat
tacaggtgtg agccaccgcg cccagccata 4500tattttgctt ttcatctgca
gctcctggat cctaactcct tgttatattg ttgggcactt 4560taggcctcag
taaacagaat ctctgtctat gaccttctcc tgtccttctt ccacctgccc
4620aaagcaggac tctaatttga ttgtgggtca aaagactctc attccagaaa
gggccttgcc 4680tcatacccta gaggaaggaa tgctgcacag aaacgccaag
tctgaacaga caagccttgc 4740tgggtttata ccatatgctt tttgtccaat
cacatttctt catggttgcc aatcatgcct 4800atgtaatgaa gcctccataa
gaacccagaa ggacagggtt cagagagttt ccacatagct 4860gaacactatc
tggagagtga acacttccta gagagtggca cacccagaga gatcatgaaa
4920gctccacgcc cctttcccct tacctcgccc tccacatctc ttcatctgta
tctttcataa 4980tatcctttat aaataaacca gcaaatgtgt ttccctgagt
tatgtgagtc actctagcaa 5040attaatcgaa cccaaagagg gggtcatggg
aaccccaact tgaagccagt cagtcagaag 5100ttccagaggc ccagacttgc
aactggggag aaagaggggg aggtcttggg gactgagccc 5160ccaacctgtg
ggatctgaca ctgtctccag gtaggtagtg ttggaactgc attggaggac
5220actcctggtg tctgctgctt ggtgtgtggg gggaaaaacc cacacctttg
gttacggagg 5280tcttctgtgt tgacgatcat tgctgtttga gggcagaggg
aatacacggt ttgagagagt 5340ttttccctga catgagcgaa caggggacat
gtactggtct ctgagatggg ggatcatggg 5400atctgccaca agtggggaga
ccactgtgac ccctgccaca gtctttgggg cagagggtgt 5460ctcgggggca
gaagaagcga gagttgtttg cagtagcagt tatgtccaaa gtgggcgcca
5520ggaaagtagg gctgcccagc tttgaagagc ctccttactc ccagcctgaa
tgaaaccatt 5580tcctgtaaag cgctaagcat aaagtttgcc aatggtgatc
cacggagaag tgagtgtacc 5640ccaccccgcc atcccacagg gaatgtcgga
gtgatgttga tctgcaccta gggaaggaat 5700ggttcatgag atgtggtgga
gatgctgagg gcccgtggac atcagatcct accctacctg 5760tgccaggaca
agccatgcgc atgtgcttca gaccaccagg caacaggagt gttgcatgag
5820gtgtgaagca ggcacctggg aaagaggagt gtgaacagca gatgggacac
actgggggca 5880gtcataggaa tgaaatgtcc caggatggat gcaggcaggt
tatggaggac ttagtgagga 5940ctgctctcct ggtgggaatt gtggagtggg
agactggatg gagactggag gtgttttaag 6000tagggaagcc aacttgcaag
ggtgaccagg gaaactatgt cggccaaggg tgagacatgc 6060actggcaaga
ctctcagaca gcctggctta tctaagcaga atgcttgagc catgccaacg
6120gtgcctcgca agttgtatta atcatgtcct ttcattttgt gtttttggtg
cttggcatct 6180gggcccttgc tgaccctaag ggaccatttc tctcagagct
agtcaagtcc tagacacagt 6240aaatgactct cctgggagca tgccttccat
gtgcagacca accaatcaag agtccacact 6300cccacccacc tcctttatcg
agctctcaca tcctggggca ccatccacct gccctaatca 6360ctcaaggacc
acgtcccaaa caactaggga cagcctccat gcccctgcac ccattgaaat
6420tattcatgct agccaatcct aaacctgtgt atgctgccac accattcctt
cctgcagaaa 6480cacagtaagg actcttccta cacctcccct acttcctctg
ctccctgact tacccactta 6540cttcctggtg cagtcccctg tggcatagtt
cactctcttc ttttgggaac tgtgaggcta 6600tcttctcaat ggcagtcatc
tcctgagctg ttggccttgc catacctaac taataataaa 6660atctatattc
taaggtaaaa acaaaacaga tagggtctca ctctgttgcc caggctggag
6720tacagtggtg tgatcatgac tcactgcagc ctcaaactcc tgggctcaag
cagttctctc 6780atctcaacct cccgagtagc tgggactaca ggcacacacc
accatgcctg gctagttttc 6840ttattttttt tgtagataca gggtcttgtt
atgttgccaa ggctggtctt gaactcctgg 6900gctcaagtga tcctcctgcc
ttggcctccc aaactgctgc aattacaggc atgagccacc 6960atgcccagat
cagaaatctt actaaaaata tttcaaggag aagagaaagc caaagatgtt
7020gaatatatat atatgtgtgt gtgtgtgtgt gtatatatat gtatatatgt
gtatatatgt 7080gtgtatatat atatgtatat atgtatatat atatgtatat
atgtatatat atatgtatat 7140tggggcaggc gtggtggctc atgcctgtgg
tcctaactac ttgagagtct gaggtgggag 7200gattgcttga gcctgggaga
tcgaggctgc tgtgagctga gactacacca ctgcactcca 7260gcttgggtga
cagagtgaga ccctgtctcc aaaaaaacaa aaagaaaaag aaaaaaagat
7320ggaaaaagac atgaaaaaac aacaacagaa atacccacac atcatcaatg
ggagggaagc 7380atcttgaggc agcaaagcgg gagtgctagt agagaggcag
atagggcgtt ggacctgagg 7440cattaaggaa agtcaggatt tggagcttac
aagtctctca ttggagatgg gatggggttg 7500gaatgaatgt ctgagcaaac
acaaagcatt tccttcccta atgactcccc accagtctaa 7560agaatcccac
attaggtcga acacggtggc tcacgcctgt aatcccagca ctttgggagg
7620ccaaggcggg tggatcacga ggtcaggaga tcgagaccat cttggctaac
atggtgaaac 7680cccgtctcta ctaaaaatac aaaaaaatta gccgggcgtc
atggtgggcg cctgtagtcc 7740cagctactcg ggaggctgag gcaggagaat
ggtgtgaacc cgggaggcag aacttgcagt 7800gagcctagat cgcgccactg
cactccagcc tgggggacaa aacgagactc tgtctcaaaa 7860aaaaaaaaaa
aaattcccac attagagttg gggaaatggg cagtcctggt ggaagttagg
7920gaacagatct gggacacgtt atagccagct ggactacagg aggccataag
ctcaattctt 7980ccttgactct gaaaccttcc actggtccta atgcctagta
attccaggcc tttcccagtt 8040gtgccaggct tggaggtgaa cacatctatg
tgccaagaag gaaaggtatg ccaagcaggg 8100gcttaagtca tccttatcct
cagtctgtct atgagtggta tgtacccctg ttccccttgc 8160aagatctgct
gggcttaggt ctcctggctg tgagttcccc atacctgggc ataaatgtag
8220tgagcctgag ctcccaaata aggttggggg ctccagagag gtggagagcc
ctgtgtctgg 8280gaagtgtgcc cacccagcag gtctgaccag gaagatacac
tgctagggtt atggaaaaag 8340actatgtgtc aaggtctctt gattctccat
ctaggcagag aatcatcttt aattaatggg 8400aaactggaag gcaaattact
tggacctgaa attacttttt gtttattgaa ccactgtgtt 8460gtaaatcaca
tctctctgaa ggcaagagaa atcagggagt tacaaaatgt ttaggagaac
8520taaacaggac tccctgtttt gctaactaat cagattgaga caggctctct
ggtaaatcta 8580caaatttgat gttgttcaac cataagcagt aaatttccta
tgctggattt tcctgacaat 8640gaatgtaaaa ggaaaaggag tctttttgac
aaaatatttt attgttcatc taaactgaaa 8700aacttctcta tttttcaaaa
ttgctatacg tgtttaaaga tgtagatatt tgaatagcct 8760aactggtaca
gaaggtttaa tgatgattcc taagacatac ctataaatta cttgaaattg
8820aaacgaaatt taagaagaat tattggaatt ttccccttct caaatgagtt
cttagtttca 8880taaatactat acaagtccat aagagatttg gggttttgag
atgtcttttt tttttttttt 8940ttttcagacg gagtttcact gttgttgcct
aggctggagt gcaatggcgt gacctcagct 9000cactacaacc tccacctccc
aggttcaagc gattttcctg cctcagcctc ccaagtagct 9060gggattacag
ggacctgcca caacgccaag ctaatgtttt gtatttttag tagagatggg
9120gttcaccatg ttggccaggc ttgtctggaa ctcctgacct caggtgatcc
acccgcctat 9180aatttattac tcccttttgc aaatgtttga aaaggaataa
agtgcaatat ttttaaacag 9240aatgcagagt tctgttgtcc tttggcaata
ccagtttcag actctgagag tggctcttgc 9300tgttgccgac agtgggctga
tgaccaaatc ccaacatgcc cccgctgcga gtccttcata 9360acctgattca
gtcatcactt agaggccagc aggcttcagg gaggcgtgag cctcagccaa
9420caacctatag gggaagagac gcagaactca atgcagacag gtttggattc
tggtgcctag 9480agaatgcaac ttggaaactc tgagccagga gaaaagggtt
ctctctccat gagagagtgt 9540gggctttgtg agaagcgaca cacagcaaac
acaattaaga gtccacccct cagcggggcg 9600caggggctca cgcctgtaat
cccagcactt tgggaggccg aggcgggtgg atcacgaggt 9660caggagatca
agaccatcct ggctaacaca gtgaaaccct gtctctacta aaaatacaaa
9720aaaattagcc gggcgtggtg gcgggcgcct gtggtcccag ctactcggga
ggctgaggca 9780ggagaatggt gtgaacccgg gaggtggagc ttgcagtgag
ccgagatcgc gccactgcac 9840tccagcctgg gcgacagagc gagactccat
ctcaaaaaaa aaaagaaaaa gaaaaagaaa 9900aagagtccgc ccctgaatta
aatagttggt ccttttgtgt tcctggtgat tcacttgcta 9960agtggaagaa
acaggaggga atcttttctc ctgccctcct g 10001
* * * * *
References