U.S. patent application number 12/038673 was filed with the patent office on 2009-02-05 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 ALCON, INC.. Invention is credited to Abbot F. CLARK, Loretta MCNATT, Wan-Heng WANG.
Application Number | 20090036371 12/038673 |
Document ID | / |
Family ID | 34710163 |
Filed Date | 2009-02-05 |
United States Patent
Application |
20090036371 |
Kind Code |
A1 |
CLARK; Abbot F. ; et
al. |
February 5, 2009 |
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; (Hurst, TX) |
Correspondence
Address: |
ALCON
IP LEGAL, TB4-8, 6201 SOUTH FREEWAY
FORT WORTH
TX
76134
US
|
Assignee: |
ALCON, INC.
FORT WORTH
TX
|
Family ID: |
34710163 |
Appl. No.: |
12/038673 |
Filed: |
February 27, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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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: |
514/8.8 ;
514/545; 514/571 |
Current CPC
Class: |
A61P 27/02 20180101;
G01N 2800/168 20130101; A61K 38/046 20130101; G01N 2333/4709
20130101; G01N 33/5023 20130101; A61K 31/385 20130101; A61P 27/06
20180101; A61P 27/00 20180101; G01N 2500/02 20130101 |
Class at
Publication: |
514/12 ; 514/545;
514/571 |
International
Class: |
A61K 38/00 20060101
A61K038/00; A61K 31/216 20060101 A61K031/216; A61K 31/192 20060101
A61K031/192; A61P 27/00 20060101 A61P027/00 |
Claims
1. (canceled)
2. (canceled)
3. A method for treating glaucoma, said method comprising
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.
4. The method of claim 3, wherein said agent is a peroxisome
proliferator-activated receptor .alpha. (PPAR.alpha.) agonists,
tachykinin peptides and their non-peptide analogs or .alpha.-lipoic
acid.
5. The method of claim 4, wherein the agent is 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.
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] 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.
[0003] 2. Description of the Related Art
[0004] 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)).
[0005] 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.
[0006] 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).
[0007] 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.
[0008] 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.
[0009] 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
[0010] 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.
[0011] 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.
[0012] 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.
[0013] In yet another embodiment, the present invention provides a
method for diagnosing glaucoma, by the following steps: [0014] a)
obtaining a biological sample from a patient; and [0015] 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; wherein the
aberrantly high level, aberrantly high bioactivity or mutations of
the SAA genes or the gene products indicates a diagnosis of
glaucoma.
[0016] 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.
[0017] Alternatively, the present invention provides a method for
diagnosing glaucoma in a patient, by the steps: [0018] a)
collecting cells from a patient; [0019] b) isolating nucleic acid
from the cells; [0020] 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 [0021] d) detecting the amplification product;
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.
[0022] The present invention also provides a method for identifying
agents potentially useful for treating glaucoma, by the steps:
[0023] 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; [0024] b) admixing a candidate
substance with the cells; and [0025] 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; 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.
[0026] In another aspect, the present invention provides a method
for identifying an agent potentially useful for treating glaucoma,
by the steps: [0027] a) forming a reaction mixture comprising:
[0028] (i) an SAA protein or a cell expressing SAA or a reporter
gene driven by an SAA promoter; [0029] (ii) an SAA protein binding
partner; and [0030] (iii) a test compound; and [0031] 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;
[0032] 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.
[0033] In another aspect, the present invention provides a method
for identifying an agent potentially useful for treating glaucoma,
by the steps: [0034] a) forming a reaction mixture comprising:
[0035] (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 [0036] (ii) a test compound; and
[0037] 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.
[0038] In preferred aspects, the cells containing the SAA protein
or expression vectors will be HL-60 cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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).
[0045] FIG. 5. IL-8 secretion by HL-60 cells in response to
increasing concentrations of rhSAA.
DETAILED DESCRIPTION PREFERRED EMBODIMENTS
[0046] 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.
[0047] 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.
[0048] 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. SAA1 (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 SAA1 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, SAA2.alpha.
(SEQ ID NO:9), encoded by SEQ ID NO:8, and SAA2.beta. (SEQ ID
NO:1), encoded by SEQ ID NO:10, which differ by only one amino
acid. SAA1 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).
[0049] 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).
[0050] 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).
[0051] 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
[0052] 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.
[0053] 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.
[0054] 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).
[0055] 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).
[0056] 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).
[0057] 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.
[0058] 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
hybridication 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.
[0059] 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).
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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).
[0067] 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).
[0068] 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).
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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 Tobe et al. (1996), describe such techniques that are
frequently used.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] The Compounds can also be used in combination with other
agents for treating glaucoma, such as, but not limited to,
.beta.-blockers, prostaglandins, carbonic anhydrase inhibitors,
.alpha..sub.2 agonists, miotics, and neuroprotectants.
[0078] 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
[0079] 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.
[0080] 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
[0081] 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 ingredient NOC hydroxypropyl 0.5% W/V %
viscosity modifier methylcellulose (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, 0.01% W/V % preservative
nf 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
[0082] 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
[0083] 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; is 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
[0084] 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.
[0085] 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
[0086] 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.
[0087] United States Patents
[0088] Books
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Hosakawa, T., Ideda, Y., Woo, P., Sipe, J.D., and Hashimoto, K.,
Transcriptional regulation of Serum Amyloid A1 gene expression in
human aortic smooth muscle cells involves CCAAT/enhancer binding
proteins (C/EBP) and is distinct from HepG2 cells, SCAND. J.
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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 Ala20 25 30Arg Asp Met
Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile35 40 45Gly Ser
Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Lys50 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 Ala85
90 95Asp Gln Ala Ala Asn Glu Trp Gly Arg Ser Gly Lys Asp Pro Asn
His100 105 110Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr115
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 Ala20 25 30Arg Asp Met
Trp Arg Ala Tyr Ser Asp Met Arg Glu Ala Asn Tyr Ile35 40 45Gly Ser
Asp Lys Tyr Phe His Ala Arg Gly Asn Tyr Asp Ala Ala Lys50 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 Ala85
90 95Asp Gln Ala Ala Asn Lys Trp Gly Arg Ser Gly Arg Asp Pro Asn
His100 105 110Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr115
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 Tyr20 25 30Ser Asp Met Arg Glu Ala Asn Tyr Ile
Gly Ser Asp Lys Tyr Phe His35 40 45Ala Arg Gly Asn Tyr Asp Ala Ala
Lys Arg Gly Pro Gly Gly Leu Gly50 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 Ala20 25 30Arg Asp Met Trp Arg Ala Tyr Ser Asp
Met Arg Glu Ala Asn Tyr Ile35 40 45Gly Ser Asp Lys Tyr Phe His Ala
Arg Gly Asn Tyr Asp Ala Ala Lys50 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 Ala85 90 95Asp Gln Ala Ala
Asn Lys Trp Gly Arg Ser Gly Arg Asp Pro Asn His100 105 110Phe Arg
Pro Ala Gly Leu Pro Glu Lys Tyr115 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 Ala20 25 30Arg Asp Met Trp Arg Ala Tyr Ser
Asp Met Arg Glu Ala Asn Tyr Ile35 40 45Gly Ser Asp Lys Tyr Phe His
Ala Arg Gly Asn Tyr Asp Ala Ala Lys50 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 Ala85 90 95Asp Gln Ala
Ala Asn Lys Trp Gly Arg Ser Gly Arg Asp Pro Asn His100 105 110Phe
Arg Pro Ala Gly Leu Pro Glu Lys Tyr115 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