U.S. patent application number 13/775069 was filed with the patent office on 2013-10-10 for method of determining risk of scoliosis.
This patent application is currently assigned to CHU SAINTE-JUSTINE. The applicant listed for this patent is Alain Moreau. Invention is credited to Alain Moreau.
Application Number | 20130266966 13/775069 |
Document ID | / |
Family ID | 39807753 |
Filed Date | 2013-10-10 |
United States Patent
Application |
20130266966 |
Kind Code |
A1 |
Moreau; Alain |
October 10, 2013 |
METHOD OF DETERMINING RISK OF SCOLIOSIS
Abstract
A method for determining the risk for developing a scoliosis
comprising monitoring osteopontin (OPN) expression in a sample from
a subject over time; wherein an OPN expression that increases in
the subject sample over time is indicative that the subject is at
risk for developing a scoliosis.
Inventors: |
Moreau; Alain; (Montreal,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Moreau; Alain |
Montreal |
|
CA |
|
|
Assignee: |
CHU SAINTE-JUSTINE
Montreal
CA
|
Family ID: |
39807753 |
Appl. No.: |
13/775069 |
Filed: |
February 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12594181 |
Sep 30, 2009 |
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PCT/CA2008/000595 |
Mar 31, 2008 |
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13775069 |
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60909408 |
Mar 30, 2007 |
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61025571 |
Feb 1, 2008 |
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Current U.S.
Class: |
435/7.24 ;
435/7.92; 436/501 |
Current CPC
Class: |
A61F 5/0102 20130101;
G01N 33/6893 20130101; A61P 43/00 20180101; G01N 33/6872 20130101;
A61P 19/08 20180101; G01N 2800/50 20130101; A61K 33/04 20130101;
A23V 2002/00 20130101; G01N 2800/108 20130101; A23L 33/30
20160801 |
Class at
Publication: |
435/7.24 ;
436/501; 435/7.92 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Claims
1. A method for determining the risk for developing a scoliosis,
comprising: (a) measuring osteopontin (OPN) protein expression in a
biological fluid sample from a subject over time; or (b) measuring
osteopontin (OPN) protein expression in a biological fluid sample
from a subject and comparing the OPN protein expression to an OPN
protein expression in a control biological fluid sample; wherein an
OPN protein expression that increases in the subject sample over
time or wherein an OPN protein expression that is higher in the
subject sample than that in the control sample, is indicative that
the subject is at risk for developing a scoliosis.
2. The method of claim 1, wherein the measuring OPN protein
expression begins when the subject is about three years old.
3. The method of claim 1, wherein the measuring OPN protein
expression is performed at a frequency of at least about once per
month.
4. The method of claim 1, wherein the measuring OPN protein
expression is performed at a frequency of at least about once per
six months.
5. The method of claim 1, wherein the measuring OPN protein
expression is performed using an enzyme-linked immunosorbent assay
(ELISA) or radioimmunoassay (RIA).
6. (canceled)
7. The method of claim 1, wherein the subject is a likely candidate
for developing a scoliosis.
8. The method of claim 1, wherein the subject is a likely candidate
for developing adolescent idiopathic scoliosis.
9. The method of claim 1, wherein the subject is pre-diagnosed as
having a scoliosis.
10. The method of claim 1, wherein the subject is pre-diagnosed
with adolescent idiopathic scoliosis.
11-16. (canceled)
17. The method of claim 1, wherein the method further comprises (a)
measuring soluble CD44 receptor (sCD44) expression in the
biological fluid sample from the subject over time; or (b)
measuring sCD44 expression in the biological fluid sample from a
subject and comparing the sCD44 expression to a sCD44 expression in
the control biological fluid sample; Wherein a sCD44 expression
that decreases in the subject sample over time or wherein a sCD44
expression that is lower in the subject sample than that in the
control sample, is a further indication that the subject is at risk
for developing a scoliosis.
18. (canceled)
19. The method of claim 1, wherein the biological fluid is selected
from the group consisting of blood, urine, tear and saliva.
20. The method of claim 19, wherein the biological fluid is
plasma.
21. (canceled)
22. The method of claim 1, wherein the measuring of the OPN protein
expression is performed with an antibody that specifically binds to
OPN.
23. The method of claim 22, wherein the measuring of the OPN
protein expression is performed using an enzyme-linked
immunosorbent assay (ELISA).
24. The method of claim 23, wherein the sample is a plasma sample
and an OPN protein expression that is higher than 700 nanograms per
milliliter of plasma is indicative that the subject is at risk for
developing a scoliosis.
25. The method of claim 23, wherein the sample is a plasma sample
and an OPN protein expression that is higher than 800 nanograms per
milliliter of plasma is indicative that the subject is at risk for
developing a scoliosis.
26-34. (canceled)
35. The method of claim 1 wherein the subject is human.
36. The method of claim 1 wherein the subject is human female.
37. The method of claim 1 wherein the subject is human male.
38-49. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/594,181, filed Sep. 30, 2009, which is a National Entry
Application of PCT application no. PCT/CA2008/000595 filed on Mar.
31, 2008 and published in English under PCT Article 21(2), which
itself claims the benefit of U.S. provisional application Ser. No.
60/909,408, filed on Mar. 30, 2007 and on U.S. provisional
application Ser. No. 61/025,571, filed on Feb. 1, 2008. All of
these applications are incorporated herein in their entirety by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to methods of determining the
risk of developing scoliosis, methods of stratifying a subject
having a scoliosis, methods for assessing the efficacy of a brace
on a subject having a scoliosis, and kits therefor.
SEQUENCE LISTING
[0003] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. 1.822. Only one strand of each
nucleic acid sequence is shown, but the complementary strand is
understood as included by any reference to the displayed strand.
The Sequence Listing is submitted as an ASCII text file named
Sequences.txt, created on Feb. 22, 2013, .about.80 KB, which is
incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0004] Spinal deformities and scoliosis in particular, represent
the most prevalent type of orthopedic deformities in children and
adolescents, while adolescent idiopathic scoliosis (AIS) represents
the most common form of scoliosis.
[0005] The etiology of adolescent idiopathic scoliosis (AIS)
remains poorly understood resulting in the traditional paradigm
that AIS is a multi-factorial disease with a genetic
predisposition..sup.(1-7) The occurrence of a melatonin signaling
dysfunction in cells derived from biopsies obtained
intraoperatively from affected AIS patients has been
reported..sup.8
[0006] Unfortunately, there is no proven method or test available
to identify children or adolescents at risk of developing AIS or to
identify, which of the affected individuals may require treatment
due to the risk of progression. Consequently, the application of
current treatments, such as bracing or surgical correction, is
delayed until a significant deformity is detected or until a
significant progression is clearly demonstrated, resulting in a
delayed and less optimal treatment..sup.29
[0007] The present description refers to a number of documents, the
content of which is herein incorporated by reference in their
entirety.
SUMMARY OF THE INVENTION
[0008] More specifically, in accordance with the present invention,
there is provided a method for determining the risk for developing
a scoliosis comprising monitoring osteopontin (OPN) expression in a
sample from a subject over time; wherein an OPN expression that
increases in the subject sample over time is indicative that the
subject is at risk for developing a scoliosis.
[0009] In a specific embodiment, the monitoring begins when the
subject is about three years old. In another specific embodiment,
the monitoring is performed by measuring OPN expression at a
frequency of at least about once per month. In another specific
embodiment, the monitoring is performed by measuring OPN expression
at a frequency of at least about once per six month. In another
specific embodiment, the method further comprises measuring sCD44
expression in a sample from the subject. In another specific
embodiment, the monitoring OPN expression is performed using an
enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay
(RIA).
[0010] In accordance with the present invention, there is provided
a method for determining the risk for developing a scoliosis
comprising measuring osteopontin (OPN) expression in a sample from
a subject; wherein an OPN expression that is higher in the subject
sample than that in a control sample is indicative that the subject
is at risk for developing a scoliosis.
[0011] In another specific embodiment, the subject is a likely
candidate for developing a scoliosis. In another specific
embodiment, the subject is a likely candidate for developing
adolescent idiopathic scoliosis. In another specific embodiment,
the subject is pre-diagnosed as having a scoliosis.
[0012] In another specific embodiment, the subject is pre-diagnosed
with adolescent idiopathic scoliosis.
[0013] In accordance with another aspect of the present invention,
there is provided a method of stratifying a subject having a
scoliosis comprising measuring osteopontin (OPN) expression in a
sample from the subject; whereby the measuring step enables the
stratification of the subject into a scoliosis subgroup.
[0014] In accordance with another aspect of the present invention,
there is provided a method for assessing the efficacy of a brace on
a subject having a scoliosis comprising measuring osteopontin (OPN)
expression in a sample from the subject prior to and at least once
after bracing the subject, wherein an increase in the OPN
expression after as compared to prior to bracing the subject is
indicative that the brace is ineffective.
[0015] In a specific embodiment, the determining the OPN expression
after the bracing is performed at least one month after the
bracing. In another specific embodiment, the determining the OPN
expression after bracing the subject is performed at least 2 months
hours after the bracing. In another specific embodiment, the
determining the OPN expression after bracing the subject is
performed at least three months after the bracing. In another
specific embodiment, the determining the OPN expression after
bracing the subject is performed at least six months after the
bracing.
[0016] In another specific embodiment, the method further comprises
measuring soluble CD44 receptor (sCD44) expression in the sample
from the subject.
[0017] In another specific embodiment, the sample from the subject
is a biological fluid from the subject. In another specific
embodiment, the biological fluid is selected from the group
consisting of blood, urine, tear and saliva. In another specific
embodiment, the biological fluid is plasma.
[0018] In another specific embodiment, the OPN expression is OPN
protein. In another specific embodiment, the determining of the OPN
expression is performed with an antibody that specifically binds to
OPN. In another specific embodiment, the measuring OPN expression
is performed using an enzyme-linked immunosorbent assay (ELISA). In
another specific embodiment, the sample is a plasma sample and an
OPN expression that is higher than 700 nanograms per milliliter of
plasma is indicative that the subject is at risk for developing a
scoliosis. In another specific embodiment, the sample is a plasma
sample and an OPN expression that is higher than 800 nanograms per
milliliter of plasma is indicative that the subject is at risk for
developing a scoliosis.
[0019] In another specific embodiment, the OPN expression is OPN
RNA. In another specific embodiment, the sample from the subject is
a paraspinal muscle biopsy and the OPN expression is OPN RNA.
[0020] In accordance with another aspect of the present invention,
there is provided a method of selecting an agent as a potential
candidate for the reduction or prevention of scoliosis comprising
contacting a candidate agent with a cell expressing osteopontin
(OPN), and detecting the expression of OPN, wherein when the
expression of OPN is lower in the presence of the candidate agent
as compared to in the absence thereof, the candidate agent is
selected.
[0021] In accordance with another aspect of the present invention,
there is provided a method of selecting an agent as a potential
candidate for the reduction or prevention of scoliosis comprising
contacting a candidate agent with a cell expressing sCD44, and
detecting the expression of sCD44, wherein when the expression of
OPN is higher in the presence of the candidate agent as compared to
in the absence thereof, the candidate agent is selected.
[0022] In another specific embodiment, the cell is a cell derived
from a scoliotic patient.
[0023] In accordance with another aspect of the present invention,
there is provided a method of selecting an agent as a potential
candidate for the prevention or reduction of scoliosis comprising
administering a candidate agent to a scoliosis model animal before
scoliosis has developed in the animal, whereby the candidate is
selected when the scoliosis is prevented or reduced in the model
animal as compared to in a control animal who was not administered
the candidate agent.
[0024] In accordance with another aspect of the present invention,
there is provided a method of preventing or reducing scoliosis
comprising administering to a subject having scoliosis a
therapeutically effective amount of an osteopontin inhibitor (OPN)
or a selenium rich diet, whereby scoliosis is thereby prevented or
treated.
[0025] In accordance with another aspect of the present invention,
there is provided a method of preventing or reducing scoliosis
comprising administering to a subject having scoliosis a
therapeutically effective amount of a CD44 inhibitor, whereby
scoliosis is thereby prevented or treated.
[0026] In accordance with another aspect of the present invention,
there is provided a method of preventing or reducing scoliosis
comprising administering to a subject having scoliosis a
therapeutically effective amount of a sCD44 stimulator, whereby
scoliosis is thereby prevented or treated.
[0027] In a specific embodiment of the methods of the present
invention, the subject is human. In another specific embodiment of
the methods of the present invention, the subject is human female.
In another specific embodiment of the methods of the present
invention, the subject is human male.
[0028] In accordance with another aspect of the present invention,
there is provided an osteopontin inhibitor for use in the treatment
or prevention of scoliosis.
[0029] In accordance with another aspect of the present invention,
there is provided a CD44 inhibitor for use in the treatment or
prevention of scoliosis.
[0030] In accordance with another aspect of the present invention,
there is provided a sCD44 stimulator for use in the treatment or
prevention of scoliosis.
[0031] In accordance with another aspect of the present invention,
there is provided a use of an osteopontin inhibitor in the
manufacture of a medicament for the prevention or the treatment of
scoliosis.
[0032] In accordance with another aspect of the present invention,
there is provided a use of an osteopontin inhibitor for the
prevention or the treatment of scoliosis.
[0033] In accordance with another aspect of the present invention,
there is provided a use of a CD44 inhibitor in the manufacture of a
medicament for the prevention or the treatment of scoliosis.
[0034] In accordance with another aspect of the present invention,
there is provided a use of a CD44 inhibitor for the prevention or
the treatment of scoliosis.
[0035] In accordance with another aspect of the present invention,
there is provided a use of a sCD44 stimulator in the manufacture of
a medicament for the prevention or the treatment of scoliosis.
[0036] In accordance with another aspect of the present invention,
there is provided a use of a sCD44 stimulator for the prevention or
the treatment of scoliosis.
[0037] In a specific embodiment of the uses of the present
invention, the scoliosis is adolescent idiopathic scoliosis.
[0038] In accordance with another aspect of the present invention,
there is provided a kit for predicting the risk of developing a
scoliosis comprising a ligand specific to osteopontin (OPN) and
instructions to use the kit for predicting the risk of developing a
scoliosis. In a specific embodiment, the kit further comprises a
ligand specific to soluble CD44 (sCD44).
[0039] Other objects, advantages and features of the present
invention will become more apparent upon reading of the following
non-restrictive description of specific embodiments thereof, given
by way of example only with reference to the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] In the appended drawings:
[0041] FIG. 1 presents OPN detection in pinealectomized chicken and
corresponding scoliosis. Upper and lower panels illustrates the up
regulation of OPN expression detected in paraspinal muscles of
pinealectomized chicken developing a scoliosis (S) vs. those
remaining unaffected (NS) at the mRNA and protein levels
respectively.
[0042] FIG. 2 graphically presents in the left panel the dynamic
variation of circulating OPN levels in scoliotic bipedal C57Bl/6j
mice after surgery, and in the right panel presents typical x-rays
of scoliotic deformities observed in bipedal C57Bl/6j mice, where
females (708) are more severely affected than males (907);
[0043] FIG. 3 shows a variation in plasma melatonin concentrations
in different mouse strains. S=scoliotic; NS=non-scoliotic;
[0044] FIG. 4 shows the effect of the pharmacological inhibition of
OPN transcription on scoliotic pinealectomized chicken;
[0045] FIG. 5 graphically presents the sensitivity and specificity
of plasma osteopontin in healthy control subjects, AIS patients and
at risk asymptomatic subjects. In Panel A, an analysis that
included 33 healthy control subjects and 32 AIS patients with
severe Cobb's Angle (.gtoreq.45.degree. revealed an area under the
curve (AUC) of 0.94 with a standard error of 0.03 (95 percent
confidence interval [CI], 0.88 to 1.000). In Panel B, the use of a
cut-off value of 700 nanograms per ml of osteopontin showed a high
sensitivity (90.6%) and a very good specificity (81.8%) for the
early detection of AIS and for detecting the risk of scoliosis
progression. In Panel C, the use of a cut-off value of 800
nanograms/ml of osteopontin also showed a high sensitivity (84.9%)
and a higher specificity (90.9%) for the early detection of AIS and
for detecting the risk of scoliosis progression. In Panel D, a
clear correlation between the levels of plasma osteopontin and the
Cobb's angle is demonstrated using all AIS patients, yielding a
p-value <0.001 and r.sup.2=0.26;
[0046] FIG. 6 presents graphs showing the distribution of age in
the different groups for male and female combined (control, at
risk, AIS<45 and AIS.gtoreq.45) (Panel A), and separated by sex
female (Panel B) and male (Panel C);
[0047] FIG. 7 shows profiles of change in OPN levels, sCD44 levels,
and Cobb's angle over follow up time in 4 selected AIS female
patients (not under brace treatment) aged 12 (red), 14 (green and
blue), and 17 (yellow) at baseline visit;
[0048] FIG. 8 shows the distribution of total change in OPN (left
panel) and sCD44 (left panel) levels over follow-up time in AIS
patients with worsened curve deformity (total increase in Cobb's
angle greater than 3.degree.; n=14) and in those without
significant change in curve (no change in Cobb's angle, decrease,
or increase smaller than 3.degree.; n=36);
[0049] FIG. 9 presents graphs showing OPN progression correlated
with Cobb's angle progression in AIS patients;
[0050] FIG. 10 presents graphs showing OPN regression or
stabilization correlated with Cobb's angle regression or
stabilization in AIS patients;
[0051] FIG. 11 shows profiles of change in OPN and sCD44 levels
over follow up time in 4 selected at risk subjects without
scoliosis: one male aged 13 (green), and 3 female aged 5 (gold), 11
(blue), and 9 (red) at baseline visit;
[0052] FIG. 12 compares OPN, sCD44 and HA levels in non AIS
scoliotic patients (NAIS) (OPN (n=28), sCD44 (n=18), HA (n=24)),
healthy controls (n=35) and AIS patients (n=252);
[0053] FIG. 13 presents a histogram comparison of circulating
levels of OPN change in function of spine biomechanics in
pre-operated AIS patients (n=79) vs. post-operated AIS patients
(n=28);
[0054] FIG. 14 presents a histogram comparison of circulating
levels of OPN and sCD44 of in pre-operated AIS female (OPN (n=10);
sCD44 (n=15)) vs. post-operated AIS female (OPN (n=10); sCD44
(n=12));
[0055] FIG. 15 presents charts distributing AIS patients across the
predefined cut-off zones pre-operation (Panel A) and post-operation
(Panel B);
[0056] FIG. 16 presents charts distributing AIS patients across the
predefined cut-off zones prior to being treated with bracing (Panel
A) and after bracing (Panel B);
[0057] FIG. 17 illustrates a hypothetic molecular concept
underlying spinal deformity progression in AIS;
[0058] FIG. 18 presents a graph that correlates selenium levels in
AIS patients with OPN levels;
[0059] FIG. 19 presents a histogram comparing selenium levels in
three categories of subjects: controls, low OPN producers and high
OPN producers;
[0060] FIG. 20 presents the nucleotide sequences of the three human
OPN isoforms (transcript variant 1, mRNA NM.sub.--001040058 (SEQ ID
NO: 1); transcript variant 2, mRNA NM.sub.--000582 (SEQ ID NO: 2);
transcript variant 3, mRNA NM.sub.--001040060 (SEQ ID NO: 3) and
the amino acid sequences of the three human OPN isoforms (isoform a
NP.sub.--001035147 (SEQ ID NO: 4); isoform b NP.sub.--000573 (SEQ
ID NO: 5); and isoform c NP.sub.--001035149 (SEQ ID NO: 6));
[0061] FIG. 21 presents the nucleotide sequences (mRNA) of six
isoforms of human CD44 (NM.sub.--000610 transcript variant 1 (SEQ
ID NO: 7); NM.sub.--001001389 transcript variant 2 (SEQ ID NO: 8);
NM.sub.--001001390 transcript variant 3 (SEQ ID NO: 9);
NM.sub.--001001391 transcript variant 4 (SEQ ID NO: 10);
NM.sub.--001001392 transcript variant 5 (SEQ ID NO: 11); X62739
Isoform identified in tumour cells (SEQ ID NO: 12)) and amino acid
sequences of six isoforms of human sCD44 (NP.sub.--000601 isoform 1
precursor (SEQ ID NO: 13); NP.sub.--001001389 isoform 2 precursor
(SEQ ID NO: 14); NP.sub.--001001390 isoform 3 precursor (SEQ ID NO:
15); NP.sub.--001001391 isoform 4 precursor (SEQ ID NO: 16);
NP.sub.--001001392 isoform 5 precursor (SEQ ID NO: 17); and
CAA44602 Isoform identified in tumour cells (SEQ ID NO: 18));
and
[0062] FIG. 22 shows the structure of sCD44 (Panel A), the origin
of the various CD44 isoforms (Panel B) and the cleavage site in one
sCD44 isoform (SEQ ID NO: 23).
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0063] The involvement of osteopontin (OPN) (also called secreted
phosphoprotein 1, bone sialoprotein I, early T-lymphocyte
activation 1), a multifunctional cytokine, was investigated in
adolescent idiopathic scoliosis (AIS) and plasma OPN concentrations
were determined in three populations: patients with AIS, healthy
controls without any family antecedent for scoliosis and
asymptomatic offspring, born from at least one scoliotic parent,
who are considered as at risk ("children at risk").
[0064] A group of 252 consecutive patients with AIS were compared
with 35 healthy control subjects without any family history of
scoliosis and 70 asymptomatic at risk subjects. All subjects were
Caucasians and demographic characteristics are shown in Table 2
below. Plasma OPN, soluble CD44 receptor (sCD44), and hyaluronan
(HA) levels were measured by enzyme-linked immunosorbent assays.
Pinealectomized chicken and genetically modified bipedal C57Bl/6j
mice devoid of either OPN or CD44 receptor, a known OPN receptor,
were also studied.
[0065] Mean plasma OPN concentration in patients with AIS were
significantly higher (p-value <0.001) in patients with AIS
having a Cobb's angle >45.degree. (965.+-.414 nanograms per
milliliter) than that in healthy controls (570.+-.156 nanograms per
milliliter) and than that in AIS patients with a Cobb's angle
<45.degree. (799.+-.284 nanograms per milliliter). Diagnostic
sensitivity and specificity of OPN for AIS was 84.4 percent and
90.6 percent respectively (cut-off value 800 nanograms per
milliliter). Subgroup analysis showed that 47.9 percent of children
at risk had OPN values higher than 800 nanograms per milliliter as
opposed to only 8.6 percent for the controls indicating that
elevated plasma OPN levels precede scoliosis formation. There were
no significant differences in mean plasma sCD44 levels and HA
levels between all groups. In respect to pathophysiology of
scoliosis, the bipedal C57Bl/6j mouse model demonstrated that the
development of scoliosis requires OPN interactions with CD44
receptors since none of the genetically modified bipedal mice
developed a scoliosis. Cut-off values for OPN disclosed herein were
calculated using the commercial Elisa kit specific to human OPN
from IBL. They may vary when a OPN expression (mRNA or protein) is
measured differently (e.g. measuring OPN expression in a different
biological sample through OPN RNA or OPN protein but using a
different antibody).
[0066] OPN (also called secreted phosphoprotein-1, minopontin, or
Eta-1) is a phosphorylated glycoprotein containing an
arginine-glycine-aspartate (RGD) sequence present in mineralized
tissues such as extracellular matrices. This multifunctional
cytokine is involved in many pathological conditions..sup.9,10 The
presence of OPN transcripts and proteins in postural control
centers such as the cerebellum, skeletal muscle proprioceptive
sensory organs, and inner ear structures that control of
equilibrium.sup.(11) is of interest, since AIS patients also
exhibit defects in postural control, proprioception and
equilibrium..sup.(12; 13) High plasma OPN levels have been found in
different adult cancers and inflammatory conditions.sup.30-33.
[0067] OPN Signaling Action:
[0068] The OPN signaling pathways are not well understood, although
it is known that aside from interacting with integrins, OPN can
interact with CD44 receptor at the cell surface..sup.14,15 Although
CD44 is a major receptor for hyaluronan (HA), it also acts as a
receptor for OPN and has multiple RGD binding sites. All human
isoforms of the CD44 family of adhesion molecules are encoded by a
single gene. Alternate splicing of 12 of the 19 exons in the human
CD44 gene leads to the production of multiple variant
isoforms.sup.16,17 and such structural heterogeneity is responsible
of the ligand repertoire of CD44, which includes
fibronectin.sup.18, chondroitin sulphate.sup.19,
osteopontin.sup.20, at least two heparin binding growth hormones
and hyaluronan..sup.21,22 Soluble variant isoforms of sCD44
(sCD44var) have been associated with several pathological
conditions..sup.16,18,23,24 It has been proposed that sCD44
isoforms are either generated through proteolytic cleavage of cell
surface CD44 or by de novo synthesis due to alternative splicing.
Functional diversity among CD44 molecules, unrelated to variant
exon usage, is demonstrated by observations that CD44H, or any
particular splice-variant, can be active for hyaluronan (HA)
binding when expressed in some cell types but inactive in others.
Many CD44 isoforms are tissue specific, but the full range of
soluble variant isoform(s) of sCD44 has been associated with some
pathological conditions. Indeed, circulating levels of total sCD44
and specific soluble CD44 isoforms have been shown to correlate
with tumor metastasis in some malignancies, including non-Hodgkin's
lymphoma and breast, gastric, and colon carcinomas. The level of
soluble CD44 is also known to be higher in the body fluids of
subjects with particular inflammatory conditions, such as
rheumatoid arthritis, pouchitis and colitis, and bronchitis.
Hyaluronan (HA), also called hyaluronate or hyaluronic acid, is a
mucopolysaccharide widely distributed throughout the body and
produced by a variety of cells including fibroblasts and other
specialized connective tissue cells.
[0069] As used herein the term "subject" is meant to refer to any
mammal including human, mice, rat, dog, cat, pig, monkey, horse,
etc. In a particular embodiment, it refers to a human.
[0070] As used herein the term "brace" is meant to include dental
and orthopedic brace and "bracing" thus refers to the action of
placing the braces on the subject. In a specific embodiment, it is
meant to refer to braces for scoliotic subjects.
[0071] As used herein the terminology "spinal disorders and
disorders causing scoliosis" refers to disorders that may involve
development of a scoliosis. Without so limited, it includes AIS,
congenital scoliosis, congenital cyphose scoliosis, neurological
scoliosis, dysplasic scoliosis, neurofibromatosis, cerebral palsy,
muscular dystrophies, neuromuscular scoliosis, spondylolesthesis
and Noonan syndrome. Scoliosis that may be stratified or predicted
excludes those caused by an accident and certain congenital
malformations.
[0072] As used herein the terms "likely candidate for developing
adolescent idiopathic scoliosis" include children of which a least
one parent has adolescent idiopathic scoliosis. Among other
factors, age (adolescence), gender and heredity (i.e. born from a
mother or father having a scoliosis) are factors that are known to
contribute to the risk of developing a scoliosis and are used to a
certain degree to assess the risk of developing AIS. In certain
subjects, scoliosis develops rapidly over a short period of time to
the point of requiring a corrective surgery. Current courses of
action available from the moment AIS is diagnosed (when scoliosis
is apparent) include observation (when Cobb's angle is around
10-25.degree.), orthopaedic devices (when Cobb's angle is around
25-30.degree.), and surgery (over 45.degree.). The more reliable
methods of determining the risk of progression and of monitoring
treatment efficiency in accordance of the present invention may
assist in 1) selecting an appropriate diet to remove certain food
products identified as contributors to scoliosis; 2) selecting the
best therapeutic agent; 3) selecting the least invasive preventive
action and/or available treatment such as postural exercises,
orthopaedic device, and/or less invasive surgeries or surgeries
without fusions (a surgery that does not fuse vertebra and
preserves column mobility).
[0073] As used herein, the terms "severe AIS" refers to a scoliosis
characterized by Cobb's angle of 45.degree. or more.
[0074] As used herein the terms "risk of developing scoliosis"
refer to a genetic or metabolic predisposition of a subject to
develop a scoliosis (i.e. spinal deformity) and/or to develop a
more severe scoliosis at a future time. For instance, an increase
of the Cobb's angle of a subject (e.g. from 40.degree. to
50.degree., or from 18.degree. to 25.degree.) is a "development" of
scoliosis.
[0075] As used herein the terminology "biological sample" refers to
any solid or liquid sample isolated from a living being. In a
particular embodiment, it refers to any solid or liquid sample
isolated from a human. Without being so limited it includes a
biopsy material, blood, tears (48), saliva, maternal milk, synovial
fluid, urine, ear fluid, amniotic fluid and cerebrospinal fluid. In
a specific embodiment it refers to a blood sample.
[0076] As used herein the terminology "blood sample" is meant to
refer to blood, plasma or serum. In a preferred embodiment, plasma
is used. In a more specific embodiment it refers to a plasma
sample.
[0077] As used herein the terminology "control sample" is meant to
refer to a sample that does not come from a subject known to have
scoliosis or known to be a likely candidate for developing a
scoliosis. In methods for determining the risk of developing
scoliosis in a subject that is pre-diagnosed with scoliosis, the
sample may however also come from the subject under scrutiny at an
earlier stage of the disease or disorder.
[0078] As used herein the term "treating" or "treatment" in
reference to scoliosis is meant to refer to at least one of a
reduction of Cobb's angle in a preexisting spinal deformity,
improvement of column mobility, preservation/maintenance of column
mobility, improvement of equilibrium and balance in a specific
plan; maintenance/preservation of equilibrium and balance in a
specific plan; improvement of functionality in a specific plan,
preservation/maintenance of functionality in a specific plan,
cosmetic improvement, and combination of any of the above.
[0079] As used herein the term "preventing" or "prevention" in
reference to scoliosis is meant to refer to a at least one of a
reduction in the progression of a Cobb's angle in a patient having
a scoliosis or in an asymptomatic patient, a complete prevention of
apparition of a spinal deformity, including changes affecting the
rib cage and pelvis in 3D, and a combination of any of the
above.
[0080] As used herein the term "osteopontin inhibitor" refers to an
agent able to reduce or block expression (transcription or
translation) of OPN (gene called sspi1), an agent able to reduce or
block OPN secretion or an agent able to reduce or block OPN binding
to its receptor CD44. Without being so limited, the agent can be
natural or synthetic and can be a protein such as but not limited
to an antibody that specifically binds to OPN, a peptide, a small
molecule, a nucleotide such as but not limited to an antisense or a
siRNA specific to OPN.
[0081] As used herein the term "CD44 inhibitor" refers to an agent
able to reduce expression (transcription or translation) of CD44,
or an agent able to reduce CD44 localization at the cellular
membrane. Without being so limited, the agent can be natural or
synthetic and can be a protein such as but not limited to an
antibody that specifically binds to CD44, a peptide, a small
molecule, a nucleotide such as but not limited to an antisense or a
siRNA specific to CD44.
[0082] As used herein the term "sCD44 stimulator" refers to an
agent able to increase expression (transcription or translation) of
sCD44, an agent able to increase sCD44 secretion or an agent able
to increase sCD44 affinity toward OPN. Without being so limited,
the agent can be a protein, a peptide, a small molecule or a
nucleotide.
[0083] The articles "a," an and "the" are used herein to refer to
one or to more than one (i.e., to at least one) of the grammatical
object of the article.
[0084] The term "including" and "comprising" are used herein to
mean, and re used interchangeably with, the phrases "including but
not limited to" and "comprising but not limited to".
[0085] The terms "such as" are used herein to mean, and is used
interchangeably with, the phrase "such as but not limited to".
[0086] The present invention also relates to methods for the
determination of the level of expression (i.e. transcript or
translation product) of OPN, HA or sCD44. The present invention
therefore encompasses any known method for such determination
including Elisa (Enzyme Linked Immunosorbent Assay), RIA
(Radioimmunoassay), real time PCR and competitive PCR, Northern
blots, nuclease protection, plaque hybridization and slot
blots.
[0087] The present invention also concerns isolated nucleic acid
molecules including probes and primers to detect OPN, sCD44 or
CD44. In specific embodiments, the isolated nucleic acid molecules
have no more than 300, or no more than 200, or no more than 100, or
no more than 90, or no more than 80, or no more than 70, or no more
than 60, or no more than 50, or no more than 40 or no more than 30
nucleotides. In specific embodiments, the isolated nucleic acid
molecules have at least 17, or at least 18, or at least 19, or at
least 20, or at least 30, or at least 40 nucleotides. In other
specific embodiments, the isolated nucleic acid molecules have at
least 20 and no more than 300 nucleotides. In other specific
embodiments, the isolated nucleic acid molecules have at least 20
and no more than 200 nucleotides. In other specific embodiments,
the isolated nucleic acid molecules have at least 20 and no more
than 100 nucleotides. In other specific embodiments, the isolated
nucleic acid molecules have at least 20 and no more than 90
nucleotides. In other specific embodiments, the isolated nucleic
acid molecules have at least 20 and no more than 80 nucleotides. In
other specific embodiments, the isolated nucleic acid molecules
have at least 20 and no more than 70 nucleotides. In other specific
embodiments, the isolated nucleic acid molecules have at least 20
and no more than 60 nucleotides. In other specific embodiments, the
isolated nucleic acid molecules have at least 20 and no more than
50 nucleotides. In other specific embodiments, the isolated nucleic
acid molecules have at least 20 and no more than 40 nucleotides. In
other specific embodiments, the isolated nucleic acid molecules
have at least 17 and no more than 40 nucleotides. In other specific
embodiments, the isolated nucleic acid molecules have at least 20
and no more than 30 nucleotides. In other specific embodiments, the
isolated nucleic acid molecules have at least 17 and no more than
30 nucleotides. In other specific embodiments, the isolated nucleic
acid molecules have at least 30 and no more than 300 nucleotides.
In other specific embodiments, the isolated nucleic acid molecules
have at least 30 and no more than 200 nucleotides. In other
specific embodiments, the isolated nucleic acid molecules have at
least 30 and no more than 100 nucleotides. In other specific
embodiments, the isolated nucleic acid molecules have at least 30
and no more than 90 nucleotides. In other specific embodiments, the
isolated nucleic acid molecules have at least 30 and no more than
80 nucleotides. In other specific embodiments, the isolated nucleic
acid molecules have at least 30 and no more than 70 nucleotides. In
other specific embodiments, the isolated nucleic acid molecules
have at least 30 and no more than 60 nucleotides. In other specific
embodiments, the isolated nucleic acid molecules have at least 30
and no more than 50 nucleotides. In other specific embodiments, the
isolated nucleic acid molecules have at least 30 and no more than
40 nucleotides. It should be understood that in real-time PCR,
primers also constitute probe without the traditional meaning of
this term. Primers or probes appropriate to detect OPN sCD44 and
CD44 in the methods of the present invention can be designed with
known methods using sequences distributed across their respective
nucleotide sequence (49).
[0088] Probes of the invention can be utilized with naturally
occurring sugar-phosphate backbones as well as modified backbones
including phosphorothioates, dithionates, alkyl phosphonates and
.alpha.-nucleotides and the like. Modified sugar-phosphate
backbones are generally known. Probes of the invention can be
constructed of either ribonucleic acid (RNA) or deoxyribonucleic
acid (DNA), and preferably of DNA.
[0089] The types of detection methods in which probes can be used
include Southern blots (DNA detection), dot or slot blots (DNA,
RNA), and Northern blots (RNA detection). Although less preferred,
labeled proteins could also be used to detect a particular nucleic
acid sequence to which it binds. Other detection methods include
kits containing probes on a dipstick setup and the like.
[0090] As used herein the terms "detectably labeled" refer to a
marking of a probe or an antibody in accordance with the presence
invention that will allow the detection of OPN, HA and/or sCD44 in
accordance with the present invention. Although the present
invention is not specifically dependent on the use of a label for
the detection of a particular nucleic acid sequence, such a label
might be beneficial, by increasing the sensitivity of the
detection. Furthermore, it enables automation. Probes can be
labeled according to numerous well known methods. Non-limiting
examples of labels include 3H, 14C, 32P, and 35S, Non-limiting
examples of detectable markers include ligands, fluorophores,
chemiluminescent agents, enzymes, and antibodies. Other detectable
markers for use with probes, which can enable an increase in
sensitivity of the method of the invention, include biotin and
radionucleotides. It will become evident to the person of ordinary
skill that the choice of a particular label dictates the manner in
which it is bound to the probe.
[0091] As commonly known, radioactive nucleotides can be
incorporated into probes of the invention by several methods.
Non-limiting examples thereof include kinasing the 5' ends of the
probes using gamma 32P ATP and polynucleotide kinase, using the
Klenow fragment of Pol I of E. coli in the presence of radioactive
dNTP (e.g. uniformly labeled DNA probe using random oligonucleotide
primers in low-melt gels), using the SP6/T7 system to transcribe a
DNA segment in the presence of one or more radioactive NTP, and the
like.
[0092] The present invention also relates to methods of selecting
compounds. As used herein the term "compound" is meant to encompass
natural, synthetic or semi-synthetic compounds, including without
being so limited chemicals, macromolecules, cell or tissue extracts
(from plants or animals), nucleic acid molecules, peptides,
antibodies and proteins.
[0093] The present invention also relates to arrays. As used
herein, an "array" is an intentionally created collection of
molecules which can be prepared either synthetically or
biosynthetically. The molecules in the array can be identical or
different from each other. The array can assume a variety of
formats, e.g., libraries of soluble molecules; libraries of
compounds tethered to resin beads, silica chips, or other solid
supports.
[0094] As used herein "array of nucleic acid molecules" is an
intentionally created collection of nucleic acids which can be
prepared either synthetically or biosynthetically in a variety of
different formats (e.g., libraries of soluble molecules; and
libraries of oligonucleotides tethered to resin beads, silica
chips, or other solid supports). Additionally, the term "array" is
meant to include those libraries of nucleic acids which can be
prepared by spotting nucleic acids of essentially any length (e.g.,
from 1 to about 1000 nucleotide monomers in length) onto a
substrate. The term "nucleic acid" as used herein refers to a
polymeric form of nucleotides of any length, either
ribonucleotides, deoxyribonucleotides or peptide nucleic acids
(PNAs), that comprise purine and pyrimidine bases, or other
natural, chemically or biochemically modified, non-natural, or
derivatized nucleotide bases. The backbone of the polynucleotide
can comprise sugars and phosphate groups, as may typically be found
in RNA or DNA, or modified or substituted sugar or phosphate
groups. A polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and nucleotide analogs. The sequence of
nucleotides may be interrupted by non-nucleotide components. Thus
the terms nucleoside, nucleotide, deoxynucleoside and
deoxynucleotide generally include analogs such as those described
herein. These analogs are those molecules having some structural
features in common with a naturally occurring nucleoside or
nucleotide such that when incorporated into a nucleic acid or
oligonucleotide sequence, they allow hybridization with a naturally
occurring nucleic acid sequence in solution. Typically, these
analogs are derived from naturally occurring nucleosides and
nucleotides by replacing and/or modifying the base, the ribose or
the phosphodiester moiety. The changes can be tailor made to
stabilize or destabilize hybrid formation or enhance the
specificity of hybridization with a complementary nucleic acid
sequence as desired.
[0095] As used herein "solid support", "support", and "substrate"
are used interchangeably and refer to a material or group of
materials having a rigid or semi-rigid surface or surfaces. In many
embodiments, at least one surface of the solid support will be
substantially flat, although in some embodiments it may be
desirable to physically separate synthesis regions for different
compounds with, for example, wells, raised regions, pins, etched
trenches, or the like. According to other embodiments, the solid
support(s) will take the form of beads, resins, gels, microspheres,
or other geometric configurations.
[0096] Any known nucleic acid arrays can be used in accordance with
the present invention. For instance, such arrays include those
based on short or longer oligonucleotide probes as well as cDNAs or
polymerase chain reaction (PCR) products. Other methods include
serial analysis of gene expression (SAGE), differential display, as
well as subtractive hybridization methods, differential screening
(DS), RNA arbitrarily primer (RAP)-PCR, restriction endonucleolytic
analysis of differentially expressed sequences (READS), amplified
restriction fragment-length polymorphisms (AFLP).
Antibodies
[0097] The present invention encompasses using antibodies for
detecting or determining OPN, sCD44 or CD44 levels for instance in
the samples of a subject and for including in kits of the present
invention. Antibodies that specifically bind to these biological
markers can be produced routinely with methods further described
below. The present invention also encompasses using antibodies
commercially available. Without being so limited antibodies that
specifically bind to OPN include those listed in Table 1 below.
TABLE-US-00001 TABLE 1 commercially available human OPN Elisa kits.
Catalogue Company Kit name number Sensitivity IBL Hambourg Human
Osteopontin ELISA JP 171 58 3.33 ng/ml IBL America Human
Osteopontin N-Half 27258 3.90 pmol/L Assay Kit-IBL IBL-America
Human Osteopontin Assay 27158 3.33 ng/ml Kit-IBL Assay designs
Osteopontin (human) EIA 900-142 0.11 ng/ml Kit American
Osteopontin, human kit 17158 ? Research Products, Inc. R&D
Systems Human Osteopontin (OPN) DOST00 0.024 ng/mL ELISA Kit
Promokine Human Osteopontin ELISA PK-EL- 3.6 ng/ml KA4231 Uscnlife
Human Osteopontin, OPN E0899h ? ELISA Kit
[0098] Both monoclonal and polyclonal antibodies directed to OPN
are included within the scope of this invention as they can be
produced by well established procedures known to those of skill in
the art. Additionally, any secondary antibodies, either monoclonal
or polyclonal, directed to the first antibodies would also be
included within the scope of this invention.
[0099] As used herein, the term "anti-OPN antibody" or
"immunologically specific anti-OPN antibody" refers to an antibody
that specifically binds to (interacts with) an OPN protein and
displays no substantial binding to other naturally occurring
proteins other than the ones sharing the same antigenic
determinants as the OPN protein. The term antibody or
immunoglobulin is used in the broadest sense, and covers monoclonal
antibodies (including full length monoclonal antibodies),
polyclonal antibodies, multispecific antibodies, and antibody
fragments so long as they exhibit the desired biological activity.
Antibody fragments comprise a portion of a full length antibody,
generally an antigen binding or variable region thereof. Examples
of antibody fragments include Fab, Fab', F(ab').sub.2, and Fv
fragments, diabodies, linear antibodies, single-chain antibody
molecules, single domain antibodies (e.g., from camelids), shark
NAR single domain antibodies, and multispecific antibodies formed
from antibody fragments. Antibody fragments can also refer to
binding moieties comprising CDRs or antigen binding domains
including, but not limited to, V.sub.H regions (V.sub.H,
V.sub.H-V.sub.H), anticalins, PepBodies.TM., antibody-T-cell
epitope fusions (Troybodies) or Peptibodies. Additionally, any
secondary antibodies, either monoclonal or polyclonal, directed to
the first antibodies would also be included within the scope of
this invention.
[0100] In general, techniques for preparing antibodies (including
monoclonal antibodies and hybridomas) and for detecting antigens
using antibodies are well known in the art (Campbell, 1984, In
"Monoclonal Antibody Technology: Laboratory Techniques in
Biochemistry and Molecular Biology", Elsevier Science Publisher,
Amsterdam, The Netherlands) and in Harlow et al., 1988 (in:
Antibody A Laboratory Manual, CSH Laboratories). The term antibody
encompasses herein polyclonal, monoclonal antibodies and antibody
variants such as single-chain antibodies, humanized antibodies,
chimeric antibodies and immunologically active fragments of
antibodies (e.g. Fab and Fab' fragments) which inhibit or
neutralize their respective interaction domains in Hyphen and/or
are specific thereto.
[0101] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc), intravenous (iv) or intraperitoneal
(ip) injections of the relevant antigen with or without an
adjuvant. It may be useful to conjugate the relevant antigen to a
protein that is immunogenic in the species to be immunized, e.g.,
keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or
soybean trypsin inhibitor using a bifunctional or derivatizing
agent, for example, maleimidobenzoyl sulfosuccinimide ester
(conjugation through cysteine residues), N-hydroxysuccinimide
(through lysine residues), glutaraldehyde, succinic anhydride,
SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R and R.sup.1 are
different alkyl groups.
[0102] Animals may be immunized against the antigen, immunogenic
conjugates, or derivatives by combining the antigen or conjugate
(e.g., 100 .mu.g for rabbits or 5 .mu.g for mice) with 3 volumes of
Freund's complete adjuvant and injecting the solution intradermally
at multiple sites. One month later the animals are boosted with the
antigen or conjugate (e.g., with 1/5 to 1/10 of the original amount
used to immunize) in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later the animals are
bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Preferably, for conjugate
immunizations, the animal is boosted with the conjugate of the same
antigen, but conjugated to a different protein and/or through a
different cross-linking reagent. Conjugates also can be made in
recombinant cell culture as protein fusions. Also, aggregating
agents such as alum are suitably used to enhance the immune
response.
[0103] Monoclonal antibodies may be made using the hybridoma method
first described by Kohler et al., Nature, 256: 495 (1975), or may
be made by recombinant DNA methods (e.g., U.S. Pat. No. 6,204,023).
Monoclonal antibodies may also be made using the techniques
described in U.S. Pat. Nos. 6,025,155 and 6,077,677 as well as U.S.
Patent Application Publication Nos. 2002/0160970 and 2003/0083293
(see also, e.g., Lindenbaum et al., 2004).
[0104] In the hybridoma method, a mouse or other appropriate host
animal, such as a rat, hamster or monkey, is immunized (e.g., as
hereinabove described) to elicit lymphocytes that produce or are
capable of producing antibodies that will specifically bind to the
antigen used for immunization. Alternatively, lymphocytes may be
immunized in vitro. Lymphocytes then are fused with myeloma cells
using a suitable fusing agent, such as polyethylene glycol, to form
a hybridoma cell.
[0105] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0106] As used herein, the term "purified" in the expression
"purified antibody" is simply meant to distinguish man-made
antibody from an antibody that may naturally be produced by an
animal against its own antigens. Hence, raw serum and hybridoma
culture medium containing anti-OPN antibody are "purified
antibodies" within the meaning of the present invention.
[0107] The present invention also encompasses arrays to detect
and/or quantify the translation products of OPN, HA or sCD44. Such
arrays include protein micro- or macroarrays, gel technologies
including high-resolution 2D-gel methodologies, possibly coupled
with mass spectrometry imaging system at the cellular level such as
microscopy combined with a fluorescent labeling system.
[0108] The present invention also encompasses methods for
identifying specific mutation(s) directly or indirectly affecting
the transcription, translation, post-translational modification or
activity of OPN. Without being so limited, mutations of interest
include any mutation affecting the interactions between OPN and any
soluble or non soluble isoform of CD44 or the binding of HA to any
soluble or non soluble isoform of CD44.
[0109] The present invention also encompasses the monitoring of the
biomarkers disclosed herein to assess the efficacy of numerous
approaches to prevent scoliosis and curve progression such as any
physical therapies (e.g. postural exercises, physiotherapies,
biomechanical stimulations by manipulation or using specific
devices e.g. vibrant plates); the monitoring of bracing efficacy or
development of novel braces; the monitoring of new surgical devices
with or without fusion of vertebras, and the monitoring of the
efficacy of specific diet, nutraceutical and/or pharmacological
treatments. Without being so limited, the first measure after the
braces have been applied could be performed 1 month later to
determine for instance whether the braces are well adjusted and
determine whether the patient is compliant to the treatment.
Thereafter, the monitoring could be performed every three to six
months depending on whether high OPN levels are detected or not.
This method of the present invention may advantageously reduces the
requirement for x-rays. X-rays could be performed for instance only
at visits where OPN levels detected are too high.
[0110] The present invention also encompasses the monitoring of the
biomarkers disclosed herein to identify patients having a risk of
progression for early bracing or for less-invasive surgeries with
novel fusionless devices, for pharmacological treatments and to
monitor responses to treatment in patients with AIS. Of note,
fusionless devices are particularly useful for patients still
possessing a growth potential so that identification of the risk of
developing a scoliosis as early as possible in the life of the
subject is beneficial. In a specific embodiment, monitoring begins
when the subject is about 5 years old or less in subjects having a
scoliosis family antecedent/history. The frequency of the testing
could typically be every six months. In case where OPN values are
above the cut-off value (i.e. >800 ng/ml when the OPN IBL ELISA
kit code No. 27158 is used), the frequency would be advantageously
significantly increased (e.g. every month, every two months, every
three months . . . ).
[0111] The present invention also encompasses methods to
screen/select for potential useful therapeutic agents using whole
cells assays, the therapeutic compound being able to repress the
transcription and/or synthesis of OPN (encoded by ssp1 gene),
and/or able to increase the production of sCD44 which could
sequester circulating OPN, and/or able to interfere with OPN
liaison with the CD44 receptor, and/or able to block the CD44
receptor. Cells for use in such methods includes cells of any
source (including in house or commercially available cell lines)
and type (any tissue). In house cell lines could be made for
instance by immortalizing cells from AIS subjects. In specific
embodiments, methods of screening of the invention seek to identify
agents that inhibit OPN expression (transcription and/or
translation) and agents that increase sCD44 expression
(transcription and/or translation). Useful cell lines for these
embodiments include those producing high levels of OPN and/or low
levels of sCD44. Such useful cell lines are described in references
43-56.
[0112] In a particular embodiment, it includes cells of any cell
type derived from a scoliotic patient. In specific embodiments, it
includes osteoblasts, chondrocytes, myoblasts or blood cells
including lymphocytes. As used herein, the term "cell derived from
a scoliotic patient" refers to cells isolated directly from
scoliotic patients, or immortalized cell lines originating from
cells isolated directly from scoliotic patients. In specific
embodiments, the cells are paraspinal muscle cells. Such cells may
be isolated by a subject through needle biopsies for instance.
[0113] Pharmaceutical compositions can also be administered by
routes such as nasal, intravenous, intramuscular, subcutaneous,
sublingual, intrathecal, or intradermal route. The route of
administration can depend on a variety of factors, such as the
environment and therapeutic goals.
Dosage
[0114] Any amount of a pharmaceutical and/or nutraceutical and/or
dietary supplement compositions can be administered to a subject.
The dosages will depend on many factors including the mode of
administration. Typically, the amount of anti-scoliosis composition
(e.g. osteopontin inhibitor or selenium compound) contained within
a single dose will be an amount that effectively prevents, delays
or reduces scoliosis without inducing significant toxicity
"therapeutically effective amount".
[0115] In some embodiments, the therapeutically effective amount of
the neutraceutical anti-scoliosis composition (e.g. selenium
supplement) can be altered. Useful effective amount concentrations
include amounts ranging from about 0.01% to about 10% of a total
diet on a weight by weight basis, from about 1% to about 6% of a
total diet on a weight by weight basis, or from about 02% to about
6% of a total diet on a weight by weight basis.
[0116] The effective amount of the osteopontin inhibitor or
selenium compound may also be measured directly. The effective
amount may be given daily or weekly or fractions thereof.
Typically, a pharmaceutical and/or nutraceutical and/or dietary
supplement composition of the invention can be administered in an
amount from about 0.001 mg up to about 500 mg per kg of body weight
per day (e.g., 10 mg, 50 mg, 100 mg, or 250 mg). Dosages may be
provided in either a single or multiple dosage regimen. For
example, in some embodiments the effective amount is a dose that
ranges from about 1 mg to about 25 grams of the anti-scoliose
preparation per day, about 50 mg to about 10 grams of the
anti-scoliose preparation per day, from about 100 mg to about 5
grams of the anti-scoliose preparation per day, about 1 gram of the
anti-scoliose preparation per day, about 1 mg to about 25 grams of
the anti-scoliose preparation per week, about 50 mg to about 10
grams of the anti-scoliose preparation per week, about 100 mg to
about 5 grams of the anti-scoliose preparation every other day, and
about 1 gram of the anti-scoliose preparation once a week.
[0117] By way of example, a pharmaceutical (e.g. containing an
osteopontin inhibitor) and/or nutraceutical (e.g. containing
selenium) and/or dietary supplement (e.g. containing selenium)
composition of the invention can be in the form of a liquid,
solution, suspension, pill, capsule, tablet, gelcap, powder, gel,
ointment, cream, nebulae, mist, atomized vapor, aerosol, or
phytosome. For oral administration, tablets or capsules can be
prepared by conventional means with at least one pharmaceutically
acceptable excipient such as binding agents, fillers, lubricants,
disintegrants, or wetting agents. The tablets can be coated by
methods known in the art. Liquid preparations for oral
administration can take the form of, for example, solutions,
syrups, or suspension, or they can be presented as a dry product
for constitution with saline or other suitable liquid vehicle
before use. Dietary supplements of the invention also can contain
pharmaceutically acceptable additives such as suspending agents,
emulsifying agents, non-aqueous vehicles, preservatives, buffer
salts, flavoring, coloring, and sweetening agents as appropriate.
Preparations for oral administration also can be suitably
formulated to give controlled release of the active
ingredients.
[0118] In addition, a pharmaceutical (e.g. containing an
osteopontin inhibitor) and/or nutraceutical (e.g. containing
selenium) and/or dietary supplement (e.g. containing selenium)
composition of the invention can contain a pharmaceutically
acceptable carrier for administration to a mammal, including,
without limitation, sterile aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents
include, without limitation, propylene glycol, polyethylene glycol,
vegetable oils, and injectable organic esters. Aqueous carriers
include, without limitation, water, alcohol, saline, and buffered
solutions. Pharmaceutically acceptable carriers also can include
physiologically acceptable aqueous vehicles (e.g., physiological
saline) or other known carriers appropriate to specific routes of
administration.
[0119] An osteopontin inhibitor or selenium may be incorporated
into dosage forms in conjunction with any of the vehicles which are
commonly employed in pharmaceutical preparations, e.g. talc, gum
arabic, lactose, starch, magnesium searate, cocoa butter, aqueous
or non-aqueous solvents, oils, paraffin derivatives or glycols.
Emulsions such as those described in U.S. Pat. No. 5,434,183, may
also be used in which vegetable oil (e.g., soybean oil or safflower
oil), emulsifying agent (e.g., egg yolk phospholipid) and water are
combined with glycerol. Methods for preparing appropriate
formulations are well known in the art (see e.g., Remington's
Pharmaceutical Sciences, 16th Ed., 1980, A. Oslo Ed., Easton,
Pa.).
[0120] In cases where parenteral administration is elected as the
route of administration, preparations containing osteopontin
inhibitor or selenium may be provided to patients in combination
with pharmaceutically acceptable sterile aqueous or non-aqueous
solvents, suspensions or emulsions. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oil,
fish oil, and injectable organic esters. Aqueous carriers include
water, water-alcohol solutions, emulsions or suspensions, including
saline and buffered medical parenteral vehicles including sodium
chloride solution, Ringer's dextrose solution, dextrose plus sodium
chloride solution, Ringer's solution containing lactose, or fixed
oils. Intravenous vehicles may include fluid and nutrient
replenishers, electrolyte replenishers, such as those based upon
Ringer's dextrose, and the like.
[0121] These are simply guidelines since the actual dose must be
carefully selected and titrated by the attending physician based
upon clinical factors unique to each patient or by a nutritionist.
The optimal daily dose will be determined by methods known in the
art and will be influenced by factors such as the age of the
patient and other clinically relevant factors. In addition,
patients may be taking medications for other diseases or
conditions. The other medications may be continued during the time
that the osteopontin inhibitor or selenium compound is given to the
patient, but it is particularly advisable in such cases to begin
with low doses to determine if adverse side effects are
experienced.
[0122] The present invention also relates to kits. Without being so
limited, it relates to kits for stratifying scoliotic subjects
and/or predicting whether a subject is at risk of developing a
scoliosis comprising an isolated nucleic acid, a protein or a
ligand such as an antibody in accordance with the present invention
as described above. For example, a compartmentalized kit in
accordance with the present invention includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers or strips of
plastic or paper. Such containers allow the efficient transfer of
reagents from one compartment to another compartment such that the
samples and reagents are not cross-contaminated and the agents or
solutions of each container can be added in a quantitative fashion
from one compartment to another. Such containers will include a
container which will accept the subject sample (DNA genomic nucleic
acid, cell sample or blood samples), a container which contains in
some kits of the present invention, the probes used in the methods
of the present invention, containers which contain enzymes,
containers which contain wash reagents, and containers which
contain the reagents used to detect the extension products. Kits of
the present invention may also contain instructions to use these
probes and or antibodies to stratify scoliotic subjects or predict
whether a subject is at risk of developing a scoliosis.
[0123] The present invention is illustrated in further details by
the following non-limiting examples.
Example 1
Material and methods
[0124] Generation of Bipedal C57Bl/6J OPN-Null and CD44-Null
Mice.
[0125] Experiments in mice were conducted according to protocols
approved by The Ste-Justine Hospital's Animal Health Care Review
Committee. Breeding pairs of C57Bl/6 devoid of either OPN(OPN-null
mice) or CD44 receptor (CD44-null mice) backcrossed for more than
10 generations in C57Bl/6j mice were graciously obtained from Dr.
Susan Rittling, (Rutger University, NJ, USA) and Dr. Tak Mak
(University of Toronto, ON, Canada), respectively, to establish new
colonies, while C57Bl/6j mice served as wild-type control mice
(Charles-River, Wilmington, Mass., USA). The C57Bl6/6j mouse strain
was used because it is naturally deficient in melatonin.sup.(28),
exhibits high circulating OPN levels.sup.(27) and develops
scoliosis when they are maintained in a bipedal state..sup.(28) It
is a well known scoliosis animal model. Bipedal surgeries were
performed after weaning by amputation of the forelimbs and tail
under anesthesia as reported previously..sup.(28) All mice
underwent complete radiographic examination under anesthesia using
a Faxitron.TM. X-rays apparatus (Faxitron X-rays Corp. Wheeling,
Ill., USA) every two weeks starting at the age of six weeks.
Anteroposterior X-rays were taken and each digital image was
evaluated subsequently for the presence of scoliosis. Cobb's angle
threshold value of 10.degree. or higher was retained as a
significant scoliotic condition.
[0126] Immunodetection of Mouse OPN
[0127] Mouse serum was obtained from peripheral blood samples for
the determination of serum levels of OPN and were collected in
serum separator tubes containing silica gel (BD Microtainer, BD New
Jersey, USA) and then centrifuged. Derived serum samples were
aliquoted and kept frozen at -80.degree. C. until thawed and
analyzed. Serum concentrations of OPN were measured by capture
enzyme-linked immunosorbent assays (ELISA) according to the
protocol provided by the manufacturer (IBL, Hamburg, Germany). The
OPN ELISA kit measured total concentration of both phosphorylated
and non-phosphorylated of all isoforms of OPN in serum. ELISA tests
were performed in duplicate and the optical density was measured at
450 nm using an AsysHiTech.TM. Expert-96 microplate reader
(Biochrom, Cambridge, UK). Although serum was used in mice herein,
the present invention also encompasses measuring OPN in mice
plasma.
[0128] Generation of Pinealectomized Chickens.
[0129] A percentage of pinealectomized chickens develop a scoliosis
and they are thus used as a scoliosis model. For this study, 145
newly hatched chickens (Mountain Hubbard) were purchased at a local
hatchery and pinealectomy were performed as previously
described.sup.(25).
[0130] Expression Analysis and Immunodetection of Chicken OPN.
[0131] Total cellular RNA was prepared from paraspinal muscles of
pinealectomized chickens by phenol/chloroform extraction. For
RT-PCR, 1 microgramme total RNA was reversed transcribed using
ThermoScript.TM. reverse transcriptase (Invitrogen), and the
equivalent of 0.1 microgramme of reverse-transcribed RNA used for
PCR reactions. These were carried out in a final volume of 50
microliters containing 200 micromolars dNTPs, 1.5 millimolars
MgCl.sub.2, 10 picomolars each primer, and 1 U Pfu DNA-polymerase
(Stratagene, LaJolla, Calif., USA). PCR reactions were performed
using the following primers and conditions: chicken OPN (420 by PCR
product): 5'-ACACTTTCACTCCAATCGTCC-3' (SEQ ID NO: 19) (forward),
5'-TGCCCTTTCCGTTGTTGTCC-3' (SEQ ID NO: 20) (reverse) 35 cycles:
95.degree. C./45 seconds, 66.degree. C./45 seconds, 72.degree. C./1
minute. For quantitative analysis, all amplifications were
normalized against that of the housekeeping gene .beta.-actin;
chicken .beta.-actin (460 bp PCR product) 5'-GGAAATCGTGCGTGACAT-3'
(SEQ ID NO: 21) (forward), 5'-TCATGATGGAGTTGAATGTAGTT-3' (SEQ ID
NO: 22) (reverse) 32 cycles: 94.degree. C./45 seconds, 55.degree.
C./45 seconds, 72.degree. C./1 minute. PCR amplified products were
analyzed on 1.5% agarose gel containing ethidium bromide. Total
protein extracts of paraspinal muscles were used to detect chicken
OPN by Western blot using anti-human OPN antibodies cross-reacting
with chicken OPN (clone 8E5, Kamiya Biomedial, WA, USA).
[0132] Human Populations
[0133] The institutional review boards of The Sainte-Justine
Hospital, The Montreal Children's Hospital, The Shriners Hospital
for Children in Montreal, McGill University and The Affluent School
Board, approved the study. Parents or legal guardians of all
participants gave written informed consent, and minors gave their
assent.
[0134] All patients with AIS were examined by one of six orthopedic
surgeons. A person was deemed to be affected if history and
physical examination were consistent with the diagnosis of
idiopathic scoliosis and a minimum of a ten degree curvature in the
coronal plane with vertebral rotation was found on a standing
radiograph of the spine. Healthy controls were recruited in
elementary schools of Montreal. Each subject was examined by the
same orthopedic surgeon using Adam's forward bending-test with a
scoliometer.
[0135] Three populations were investigated: patients with AIS,
healthy controls without any family antecedent/history for
scoliosis and asymptomatic offspring, born from at least one
scoliotic parent, who are considered as at risk of developing a
scoliosis. A group of 252 consecutive patients with AIS, 35 healthy
control subjects and 70 asymptomatic children at risk of developing
a scoliosis were recruited. All subjects were Caucasians and
demographic characteristics are shown in Table 2 below).
TABLE-US-00002 TABLE 2 Demographic and clinical characteristics of
patients with AIS, healthy control and at risk control subjects.
Subject Type Healthy At Risk AIS Control Subjects Control Subjects
Characteristics Female Male Female Male Female Male Number 215 37
19 16 45 25 Mean Age (Years) 14.1 .+-. 2.1 14.8 .+-. 2.2 10.6 .+-.
0.6 10.9 .+-. 0.6 9.8 .+-. 3.7 10.0 .+-. 2.9 Patient percentage
& Mean Cobb's Angle Thoracolumbar 35.8% 22.5 .+-. 15.2 29.7%
28.3 .+-. 22.8 -- -- -- -- Thoracic 20.5% 39.7 .+-. 20.4 29.7% 34.1
.+-. 22.3 -- -- -- -- Double Scoliosis 30.2% 24.3% -- -- -- --
(Thoracic + Lumbar) Thoracic Curvature 34.8 .+-. 19.0 38.9 .+-.
21.2 Lumbar Curvature 31.0 .+-. 17.3 33.0 .+-. 18.7 Lumbar 4.7%
25.4 .+-. 10.7 8.1% 20.3 .+-. 3.5 -- -- -- -- Double Scoliosis 6.0%
5.4% -- -- -- -- (Thoracic + Thoracolumbar) Thoracic Curvature 25.4
.+-. 13.5 36.0 .+-. 19.8 Lumbar Curvature 25.2 .+-. 15.5 41.0 .+-.
29.7 Triple Scoliosis 1.9% 36.8 .+-. 18.5 2.7% 8.0 -- -- -- -- 41.0
.+-. 14.3 11.0 30.5 .+-. 7.7 11.0 Double Scoliosis 0.9% -- -- -- --
-- (Thoracic + Thoracic) 29.0 .+-. 5.7 -- 16.5 .+-. 3.5 -- Heredity
36.3% 37.8% 0.0% 0.0% 100.0% 100.0% * Plus-minus values are means
.+-. standard deviations. .dagger. Mean Cobb's Angles for double
scoliosis are represented by the curvatures on the thoracic and
lumbar levels separately. .dagger-dbl. Mean Cobb's Angle for the
triple scoliosis represents two thoracic curvatures and one lumbar
curvature.
[0136] Osteopontin, sCD44 and Ha Enzyme-Linked Immunosorbent
Assays
[0137] Peripheral blood samples for AIS patients, asymptomatic
children and control groups were collected in EDTA-containing tubes
and then centrifuged. Derived plasma samples were aliquoted and
kept frozen at -80.degree. C. until thawed and analyzed. Plasma
concentrations of OPN and sCD44 were measured by capture
enzyme-linked immunosorbent assays (ELISA) according to protocols
provided by the manufacturer (IBL, Hamburg, Germany). The sCD44
Elisa kit (sCD44std) measured all circulating (soluble) CD44
isoforms comprising the standard protein sequences but not the rare
isoforms associated with alternative splicing between exons V2 and
V10 (50) (see also FIG. 22). The OPN IBL ELISA kit (code No. 27158)
measures total concentration of both phosphorylated and
non-phosphorylated of all isoforms of OPN in plasma. Circulating
levels of HA were measured in all plasma samples using an ELISA kit
(HA-Elisa (K-1200), Echelon Biosciences, Salt Lake City, Utah). All
ELISA tests were performed in duplicate and the optical density was
measured at 450 nm (for OPN and sCD44) and 405 nm (for HA) using an
AsysHiTech Expert-96.TM. microplate reader (Biochrom, Cambridge,
UK). Other Elisa kits available commercially or house made can be
used in methods of the present invention. The cut-off value that
statistically distinguishes non scoliotic subjects from scoliotic
subjects that will help predict the risk of scoliosis progression
as determined with these other kits will likely differ from that
calculated with the kit used herein. It may however be calculated
for each new antibody used as described herein.
[0138] Statistical Analysis
[0139] Age and gender differences among the different AIS and
control groups were assessed using Pearson's Chi-square and
Student's t tests, respectively. Multiple linear regression models
were used to test for association between groups and levels of OPN,
sCD44, and HA. Values were adjusted for age, gender, and age-gender
interaction when these potential confounders were associated with
the biomarker levels at p<0.1. Interactions between group and
gender were also investigated. It was first tested for an overall
group effect using a global F test comparing models with and
without group effects. Were then tested specific differences
between groups, applying a Bonferroni correction for multiple
testing. Receiver-operating characteristics (ROC) curves were used
to evaluate the diagnostic value of OPN, and to identify the
optimal threshold values. The sensitivity (proportion of
true-positive results when the assay was applied to patients known
to have AIS) and specificity (proportion of true-negative results
when the assay was applied to healthy controls) of OPN were
profiled by curves. The area under ROC curve (AUC) and associated
95% confidence interval were calculated. The test of the hypothesis
that the theoretical AUC is 0.5 was based on the confidence
interval. Statistical analysis was performed with the SAS software,
version 9.1, with the exception of the ROC curve analysis, which
was performed with the ROCR package for R.sup.(51,52). In all
analyses except when otherwise mentioned a p-value <0.05 was
considered statistically significant.
Example 2
mRNA and Protein OPN Levels Pinealectomized Chicken
[0140] Expression analysis and immunodetection analysis of OPN in
pinealectomized chicken were performed as described in Example 1
above. OPN at the mRNA and protein levels occurring in
pinealectomized chicken were measured. FIG. 1 shows a strong
increase of OPN at the mRNA and protein levels only in
pinealectomized chicken that developed a scoliosis.
Example 3
OPN Protein Levels in C57Bl/6j Mice
[0141] Bipedal C57Bl/6j mice were generated and their OPN level was
determined as described in Example 1 above. Bipedal ambulation for
8 weeks in C57Bl/6j mice induced scoliosis at a rate of 46 percent
in females and 24 percent in males which correlated well with
higher plasma OPN levels found in females (Table 3 below). The
relevance of this animal model is strengthened by the fact that
scoliosis are more frequently seen in number and severity in
bipedal C57Bl/6j females (46%) when compared to bipedal males (24%)
as is also observed in humans.
TABLE-US-00003 TABLE 3 Scoliosis frequency in naturally melatonin
deficient mouse strain C57BI/6j mice and genetically modified C57BI
mice devoid of OPN or CD44. Mean period of n % of scoliosis
follow-up C57BI/6j 21 24% 57 weeks +/- 3 28 46% 57 weeks +/- 3
C57BI/6j 30 0% 54 weeks +/- 2 OPN-null 24 0% 54 weeks +/- 2
C57BI/6j 29 0% 52 weeks +/- 2 CD44-null 31 0% 52 weeks +/- 2
[0142] FIG. 2 shows that the OPN protein level strongly increases
after bipedal surgery (i.e. during scoliosis development) in
scoliotic C57Bl/6j mice.
Example 4
Observation of Effect of Absence of OPN or CD44 Bipedal C57Bl/6j
Mice on Scoliosis
[0143] The contribution of OPN and CD44 receptor as an integral
part of the pathophysiology cascade in scoliosis formation and
curve progression was also examined by studying genetically
modified bipedal C57Bl/6j mice by conducting experiments as
described in Example 1 above. As shown in Table 3 above, it was
found that none of the bipedal C57Bl/6j OPN-null (n=54) and
C57Bl/6j CD44-null mice (n=60) respectively, developed a scoliosis
even if their analysis was extended over 52 weeks. Scoliosis
development is detected 8 weeks after the surgery. A longer
follow-up was performed to demonstrate that scoliosis development
was not simply delayed in OPN-null and CD44-null mice.
[0144] In parallel, melatonin circulating levels were measured in
wild-type and OPN-KO mice to exclude the possibility that absence
of scoliosis in bipedal C57Bl/6 OPN-KO mice was due to an increased
production of melatonin.
[0145] FIG. 3 shows a two-fold decrease in circulating melatonin
level of bipedal C57Bl/6j OPN KO mice when compared to wild-type
ones (C57Bl/6j, C57Bl/6j and FVB).
[0146] As indicated above, C57Bl/6j mice are melatonin deficient
and may develop a scoliosis (S) in contrast to the FVB strain,
which produces high melatonin levels. OPN-knockout mice do not
develop a scoliosis (NS) even if they are in the same genomic
background (C57Bl6/j), although melatonin is markedly decreased,
suggesting that melatonin negatively regulates OPN expression and
synthesis in vivo. Without being bound by this hypothesis, it is
also suggested that in absence of OPN in genetically modified mice,
the melatonin level will be further decreased accordingly as an
adaptive physiological response to enhance OPN expression and
synthesis.
Example 5
Effect of OPN Inhibitors on Scoliosis Prevention
[0147] Two compounds suspected of having an effect on OPN
transcription or synthesis were injected intraperitonealy at a
dosage of 500 .mu.g/kg of body weight/day to chicken 24-48 h prior
pinealectomy.
[0148] As is apparent in FIG. 4, fewer pinealectomized chicken
pre-treated with the drugs developed scoliosis (a reduction of 50%)
than untreated pinealectomized chickens.
Example 6
Comparing the Level of Circulating OPN in AIS Patients Classified
in Two Groups and Healthy Controls
[0149] A group of 252 patients with AIS and 35 healthy control
subjects were tested as described in Example 1 above. Patients with
AIS were divided into two subgroups according to their spinal curve
severity (10.degree.-44.degree. vs. .gtoreq.45.degree.) In the most
severely affected AIS subgroup, none of the patients had corrective
surgery at the time of the tests. Consistent with literature
reporting increased AIS prevalence in teenage girls when compared
to boys for moderate curves (ratio 10:1 for curve with a Cobb's
angle .gtoreq.30.degree.), a greater proportion of girls in the AIS
groups (86% and 84% in the 10.degree.-44.degree. and 45.degree.
subgroups, respectively were observed compared to the control
groups (54% and 64% in healthy and at risk control groups,
respectively, p.ltoreq.0.0001 when comparing the control groups).
There was no significant gender difference between the two AIS
subgroups (p=0.76) or between the two control groups (p=0.32). Mean
age was significantly higher in AIS patients with Cobb's angle
.gtoreq.45.degree. compared to those with 10-40.degree. angle
(15.2.+-.1.8 vs. 13.8.+-.2.1, p<0.0001). Both AIS groups had
higher mean age compared to control groups (10.7.+-.0.6 for the
healthy and 9.9.+-.3.4 for the at risk group, p<0.0001 when
comparing to either AIS group).
[0150] The plasma OPN levels in patients with AIS exhibiting a
severe deformity (Cobb's angle .gtoreq.45.degree.), low to moderate
curve (Cobb's angle between 10.degree. and 44.degree.) and healthy
controls are summarized in Table 4 below according to various
clinical parameters. The mean plasma OPN levels were significantly
higher in both AIS groups when compared to healthy control group
although plasma OPN levels were more elevated in patients with the
most severe deformities (Cobb's angle .gtoreq.45.degree.)
(Bonferroni-corrected p<0.001 after adjustment for age, gender,
and age-gender interaction). Plasma OPN levels in AIS patients were
correlated with the severity of curve deformity (FIG. 5D) in girls
and boys (Partial Pearson correlation coefficient adjusted for
age=0.29, p<0.001, and 0.33, p=0.04, respectively). Mean plasma
OPN levels in the group at risk of developing scoliosis (846.+-.402
ng/ml) differed significantly (Bonferroni-corrected p<0.001)
from the healthy controls (570.+-.156 ng/ml).
TABLE-US-00004 TABLE 4 Mean biochemical values of patients with
AIS, healthy control subjects and asymptomatic at risk control
subjects*. Female Male Female + Male Mean Mean Mean biomarker
biomarker biomarker Subject Type N level (ng/ml) Range N level
(ng/ml) Range N level (ng/ml) Range P-value.dagger. OPN Healthy 19
580 .+-. 150 318-882 16 558 .+-. 168 308-856 35 570 .+-. 156
308-882 -- controls At risk controls 45 829 .+-. 419 208-1834 25
877 .+-. 378 391-1629 70 846 .+-. 402 208-1834 <0.001 AIS <
45.degree. 162 774 .+-. 268 373-1585 27 948 .+-. 335 445-1668 189
799 .+-. 284 373-1668 <0.001 AIS .gtoreq. 45.degree. 53 913 .+-.
398 201-1821 10 1238 .+-. 409 575-1872 63 965 .+-. 414 201-1872
<0.001 sCD44 Healthy 19 522 .+-. 99 373-829 16 575 .+-. 92
404-800 35 546 .+-. 98 373-829 -- controls At risk controls 45 508
.+-. 96 316-760 25 533 .+-. 98 304-510 70 517 .+-. 97 304-760
>0.5 AIS < 45.degree. 162 503 .+-. 161 194-1253 27 527 .+-.
110 364-793 189 506 .+-. 155 194-1253 >0.5 AIS .gtoreq.
45.degree. 53 436 .+-. 251 87-882 10 402 .+-. 216 147-962 63 431
.+-. 245 87-962 0.066 HA Healthy 19 128 .+-. 38 72-236 16 132 .+-.
49 80-255 35 130 .+-. 43 72-255 -- controls At risk controls 45 119
.+-. 51 36-257 25 117 .+-. 52 33-226 70 118 .+-. 51 33-257 >0.5
AIS < 45.degree. 162 112 .+-. 60 18-356 27 124 .+-. 60 27-283
189 114 .+-. 60 18-356 >0.5 AIS .gtoreq. 45.degree. 53 93 .+-.
40 32-222 10 128 .+-. 71 41-25435 63 98 .+-. 48 32-254 0.140 *SD is
standard deviation .dagger.P-value is from the comparison with
healthy control group in all subjects after Bonferroni correction
and adjustment for age, gender, and age-gender interaction (OPN and
HA) or age (sCD44). After the same adjustments, overall F test
p-values for association between group and biomarker levels were
<0.001 (OPN), 0.035 (sCD44), and 0.163 (HA).
[0151] Receiver-operating characteristics (ROC) curves analyzes of
plasma OPN comparing the patients with AIS more severely affected
(Cobb's angle .gtoreq.45.degree.) with healthy controls showed an
AUC of 0.94 with a standard error of 0.03 (95 percent confidence
interval 0.88 to 0.99) (see FIG. 5A). A cut-off value >700
nanograms per milliliter gave a sensitivity of 90.6 percent and a
specificity of 81.8 percent with (see FIG. 5B). A cut-off value
>800 nanograms per milliliter had the highest accuracy with a
sensitivity of 84.4 percent and specificity of 90.6 percent for
confirming scoliosis (minimal false negative and false positive
results) (see FIG. 5C).
[0152] Although as indicated above, high levels of OPN are found in
other adult diseases, high plasma OPN levels found in patients with
scoliosis are unique in the pediatric population. The detection of
OPN level can thus be used to identify within asymptomatic children
those who are at risk of developing a scoliosis (AIS or other
spinal disorders and disorders causing scoliosis) and identify
among scoliotic subjects, those or are at risk of experiencing a
progression of scoliosis. Moreover, plasma OPN levels found in AIS
patients were often higher than those measured in adult diseases.
OPN levels can also be used to predict the risk in adults (e.g.
degenerative scoliosis and idiopathic scoliosis that progress
through adulthood). Certain mutations have already been associated
with other disorders that may lead to scoliosis. In a particular
embodiment, the OPN levels could be used in combination with the
detection of these mutations.
Example 7
Comparing the Level of Circulating OPN in Asymptomatic Children at
Risk and Healthy Controls
[0153] A group of 70 asymptomatic children at risk of developing a
scoliosis and 35 healthy control subjects were tested as described
in Example 1 above. The mean plasma OPN levels in the group at risk
of developing a scoliosis (846.30.+-.402 nanograms per milliliter)
differed significantly (p=0.001) from the healthy controls
(570.+-.156 nanograms per milliliter) and both groups were age- and
gender-matched. No significant gender difference was observed (see
Table 4 above).
[0154] Using a cut-off value of 800 nanograms per milliliter, it
was observed that 47.9 percent of asymptomatic children in that
group were above this plasma OPN value while only 8.6 percent of
healthy controls were above this value. These results are in
agreement with previous reports showing that the offspring of at
least one affected parent develops more often a scoliosis than ones
born from unaffected parents (34, 35).
[0155] An enzyme-linked immunosorbent assay (ELISA) or RIA for OPN
for instance can thus be used for early identification of subjects
at risk of developing a scoliosis for purposes of prognosis and/or
scoliotic patients stratification for early bracing and
less-invasive surgeries with novel fusionless devices, for
pharmacological treatments and to monitor responses to treatment in
patients with AIS.
Example 8
Comparing the Level of Circulating sCD44 in AIS Patients Classified
Two Groups and Healthy Controls
[0156] Experiments were conducted as described in Example 1 above.
The plasma sCD44 and HA levels in healthy controls, both AIS groups
and asymptomatic at risk children are displayed in Table 4 above.
Comparison among all groups showed no significant change in mean
plasma sCD44 and HA values. However, AIS patients exhibiting the
most severe spinal deformities (.ltoreq.45.degree.) had also the
lowest mean plasma sCD44 level when compared to the other three
groups (p=0.066).
[0157] CD44 and sCD44 can act as a receptor and decoy receptor for
OPN respectively. In spite that no significant changes were
measured among all groups tested, the most severely affected AIS
patients 45.degree.) showed the lowest mean sCD44 value among all
groups tested. Interestingly, decreased plasma sCD44 levels were
found in immunodeficiency and autoimmune diseases.sup.(35-37), but
none of these conditions normally lead to scoliosis in absence of
high plasma OPN levels, suggesting that sCD44 could play a role in
AIS as disease-modifying factor by interfering with the action of
OPN (see FIG. 17).
Example 9
Profiles of Change in OPN Levels, sCD44 Levels, and Cobb's Angle of
AIS Patients Over Time
[0158] The progression of biomarkers (OPN and sCD44 levels) and
Cobb's angle was measured over follow up time in AIS patients. FIG.
7 presents these progression in 4 selected AIS female patients (not
under brace treatment) aged 12 (red), 14 (green and blue), and 17
(yellow) at baseline visit.
[0159] FIG. 8 presents the distribution of total change in OPN
(left panel) and sCD44 (right panel) levels over follow-up time in
AIS patients with worsened curve deformity (total increase in
Cobb's angle greater than 3.degree.) and in those without
significant change in curve (no change in Cobb's angle, decrease,
or increase smaller than 3.degree.; also presents for all Average
change in OPN levels was significantly higher in the group with
worsened curve deformity (Wilcoxon rank sum test p<0.01). No
significant difference was detected for sCD44 (p>0.5). Length of
follow-up time was similar between the 2 groups (p>0.5).
[0160] FIG. 9 shows OPN progression correlated with Cobb's angle
progression in a group of AIS patients while FIG. 10 shows OPN
regression or stabilization correlated with Cobb's angle regression
or stabilization in other AIS patients;
[0161] OPN level can be used to identify among pre-diagnosed
patients those in which scoliosis will progress.
Example 10
Profiles of Change in OPN Levels, sCD44 Levels, and Cobb's Angle of
Asymptomatic at Risk Patients Over Time
[0162] FIG. 11 shows profiles of change in OPN and sCD44 levels
angle in 4 selected at risk subjects without scoliosis: one male
aged 13 (green), and 3 female aged 5 (gold), 11 (blue), and 9 (red)
at baseline visit. Significant inter-subject variability was
observed in the baseline levels of biomarkers and change over time
among at risk subjects (especially for OPN), indicating the
potential of using this biomarker as a tool to monitor onset of
scoliosis in at risk subjects.
[0163] Tables 5 to 8 below present the clinical and biochemical
profiles in detail for each of the healthy control subjects (Table
5), of the AIS patients with Cobb's angles of less than 45 degrees
(Table 6), of the AIS patients with Cobb's angles 45.degree. or
more (Table 7), and of the asymptomatic at risk children (Table
8).
TABLE-US-00005 TABLE 5 Clinical and biochemical profile of healthy
control subjects. Date of Collection Timepoint [sCD44] Random Birth
Gender Age Date (months) [OPN] (ng/ml) (ng/ml) [HA] (ng/ml) 1
1996-03-21 M 11.2 2007-05-22 T0 663.92 .+-. 26.03 533.4 164.87 .+-.
6.05 2 1996-06-26 M 10.9 2007-05-22 T0 418.23 .+-. 12.49 504.38
120.49 .+-. 2.06 11.6 2008-01-16 T8 593.64 .+-. 28.77 555.88 150.02
.+-. 15.74 3 1996-05-28 F 11.0 2007-05-22 T0 629.52 .+-. 0.64
829.35 140.89 .+-. 3.90 11.7 2008-01-16 T8 892.76 .+-. 1.54 507.54
146.71 .+-. 24.69 4 1996-06-22 M 10.9 2007-05-22 T0 458.68 .+-.
11.40 799.57 100.98 .+-. 6.89 5 1996-10-13 F 10.6 2007-05-22 T0
459.33 .+-. 2.90 525.76 139.84 .+-. 2.89 11.3 2008-01-16 T8 464.46
.+-. 2.29 476.43 157.36 .+-. 20.10 7 1996-08-08 F 10.8 2007-05-22
T0 691.18 .+-. 2.50 664.38 120.69 .+-. 2.79 11.5 2008-01-16 T8
825.38 .+-. 1.16 545.85 180.39 .+-. 42.55 8 1996-02-01 M 11.3
2007-05-22 T0 498.86 .+-. 0.66 643.38 99.24 .+-. 2.35 12.0
2008-01-16 T8 469.87 .+-. 11.47 440.44 154.20 .+-. 2.53 9
1997-06-28 M 9.9 2007-05-22 T0 517.11 .+-. 53.44 582.66 134.43 .+-.
6.42 10 1997-07-23 F 9.8 2007-05-22 T0 756.24 .+-. 23.61 499.03
131.04 .+-. 1.98 10.5 2008-01-16 T8 1039.80 .+-. 3.10 337.33 167.84
.+-. 2.48 11 1996-02-22 M 11.3 2007-06-06 T0 653.09 .+-. 15.14
581.14 191.13 .+-. 17.98 11.8 2007-12-04 T6 521.00 .+-. 5.82 861.46
265.54 .+-. 6.97 12 1996-02-09 F 11.3 2007-06-06 T0 449.97 .+-.
11.21 490.25 112.71 .+-. 17.95 11.8 2007-12-04 T6 923.12 .+-. 1.03
476.09 188.80 .+-. 15.17 13 1996-05-17 F 11.1 2007-06-06 T0 488.30
.+-. 0.80 428.77 168.61 .+-. 9.49 11.6 2007-12-04 T6 659.35 .+-.
1.68 584.96 182.09 .+-. 13.74 14 1995-10-20 M 11.6 2007-06-06 T0
610.77 .+-. 8.93 573.88 128.40 .+-. 6.58 12.1 2007-12-04 T6 469.87
.+-. 19.12 527.07 167.16 .+-. 44.48 16 1997-03-07 F 10.2 2007-06-06
T0 544.82 .+-. 7.91 516.6 132.83 .+-. 2.07 10.7 2007-12-04 T6
723.88 .+-. 8.56 503.74 65.43 .+-. 9.60 17 1996-05-09 M 11.1
2007-06-06 T0 450.87 .+-. 6.41 553.26 255.19 .+-. 14.61 11.6
2007-12-04 T6 530.37 .+-. 16.78 267.86 42.33 .+-. 7.47 18
1997-09-02 F 9.8 2007-06-06 T0 555.41 .+-. 32.17 498.65 127.24 .+-.
10.65 19 1996-11-04 M 10.6 2007-06-06 T0 314.85 .+-. 9.93 682.71
175.92 .+-. 16.20 20 1997-05-30 F 10.0 2007-06-06 T0 381.57 .+-.
4.61 373.01 87.65 .+-. 3.71 10.5 2007-12-04 T6 434.48 .+-. 5.73
497.7 142.61 .+-. 8.42 21 1997-01-07 F 10.4 2007-06-06 T0 318.19
.+-. 6.62 474.59 235.76 .+-. 3.68 10.9 2007-12-04 T6 393.98 .+-.
3.87 571.14 209.26 .+-. 2.40 22 1997-02-09 F 10.3 2007-06-06 T0
882.15 .+-. 18.31 542.95 131.86 .+-. 1.13 10.8 2007-12-04 T6 804.46
593.61 120.43 .+-. 14.60 23 1997-03-02 M 10.3 2007-06-06 T0 307.71
.+-. 4.88 621.23 157.12 .+-. 2.29 24 1997-06-19 F 10.0 2007-06-06
T0 423.06 .+-. 13.90 561.28 149.88 .+-. 5.65 25 1997-04-12 F 10.1
2007-06-06 T0 758.88 .+-. 5.74 478.79 169.32 .+-. 8.25 26
1997-12-02 M 9.5 2007-06-06 T0 441.36 .+-. 8.3 2 645.84 148.32 .+-.
16.36 27 1996-04-03 F 11.2 2007-06-06 T0 794.21 .+-. 5.50 545.62
77.58 .+-. 8.87 11.7 2007-12-04 T6 748.79 .+-. 7.61 575.46 228.08
.+-. 27.64 28 1995-09-30 F 11.7 2007-06-12 T0 503.25 .+-. 8.16
451.68 71.91 .+-. 4.23 29 1996-09-15 M 10.7 2007-06-12 T0 576.62
.+-. 5.29 554.79 80.24 .+-. 3.69 11.2 2007-12-04 T6 552.15 598.79
108.09 .+-. 16.44 30 1996-01-18 F 11.4 2007-06-12 T0 578.62 .+-.
0.24 634.22 126.21 .+-. 4.18 11.9 2007-12-04 T6 498.67 .+-. 8.60
606.57 192.18 .+-. 31.90 31 1996-08-24 F 10.8 2007-06-12 T0 531.91
.+-. 4.36 432.2 132.19 .+-. 5.06 11.3 2007-12-04 T6 455.46 .+-.
4.85 660.14 244.46 .+-. 3.49 32 1997-04-19 F 10.1 2007-06-12 T0
611.32 .+-. 6.46 481.47 92.69 .+-. 2.87 10.6 2007-12-04 T6 406.38
.+-. 19.28 415.61 142.80 .+-. 25.25 33 1997-04-21 M 10.1 2007-06-12
T0 543.15 .+-. 7.32 403.56 91.82 .+-. 4.49 10.6 2007-12-04 T6
360.77 .+-. 9.93 544.36 81.68 .+-. 23.85 34 1995-11-15 M 11.6
2007-06-12 T0 856.07 .+-. 3.82 501.71 96.37 .+-. 4.15 12.1
2007-12-04 T6 922.12 .+-. 20.68 535.71 56.34 .+-. 1.86 35
1996-04-22 F 11.1 2007-06-12 T0 659.81 .+-. 5.54 502.09 87.90 .+-.
4.85 11.6 2007-12-04 T6 596.77 .+-. 10.14 378.46 242.42 .+-. 36.30
36 1995-12-09 M 11.5 2007-06-12 T0 816.64 .+-. 14.56 502.85 83.26
.+-. 0.12 37 1995-10-07 M 11.7 2007-06-12 T0 805.92 .+-. 14.01
511.63 80.24 .+-. 3.69 12.2 2007-12-04 T6 304.61 .+-. 14.94 489.06
141.51 .+-. 21.50 * Plus-minus values are means .+-. standard
deviations. .dagger. Healthy control subjects have no family
history of scoliosis and are examined before sample collection by
an orthopaedic surgeon.
TABLE-US-00006 TABLE 6 Clinical and biochemical profiles of AIS
patients with Cobb's angles less than 45.degree.. Cobb's Angle
Patient ID Date of Birth Gender Age Collection Date Timepoint
(mths) Pre-op Curve Type Date of surgery Family history [OPN]
(ng/ml) [sCD44] (ng/ml) [HA] (ng/ml) 102 1991-09-12 F 13.8
2005-06-10 T0 18 rT -- Cousin 1265.10 375.56 132.06 .+-. 39.35 14.3
2006-01-13 T7 16 rT 766.80 408.06 388.93 .+-. 23.42 15.8 2007-06-01
T12 16 rT 933.77 .+-. 13.23 437.55 71.91 .+-. 4.23 16.2 2007-11-30
T29 17 rT 591.72 .+-. 66.49 311.40 27.92 .+-. 1.72 103 1991-09-04 M
13.8 2005-06-10 T0 13 lT -- Father (cyphose) 1338.32 792.62 207.12
104 1992-01-29 F 13.4 2005-06-10 T0 21-22 rTlL -- -- 1221.83 742.48
132.24 106 1992-08-10 F 14.8 2007-06-05 T0 25-24 rTlL -- -- 972.87
.+-. 16.73 488.72 86.78 .+-. 6.34 15.2 2007-10-05 T4 22-18 rTlL
485.82 .+-. 34.70 475.13 293.05 .+-. 40.93 107 1991-09-09 F 13.8
2005-06-20 T0 31-32 rTlL -- Mother 739.61 1253.3 109.39 .+-. 26.70
113 1995-11-21 F 9.7 2005-07-22 T0 10 rT -- -- 670.49 .+-. 5.45
695.21 41.10 .+-. 8.51 11.5 2007-05-18 T22 15 rT 688.49 .+-. 23.78
613.79 49.16 .+-. 9.14 118 1991-06-04 F 16.6 2008-01-18 T0 22-22
rTlTL -- Both parents 372.79 .+-. 10.86 273.31 70.42 .+-. 4.85 123
1993-09-23 F 12.1 2005-11-04 T0 28 rTL -- Both parents 1466.97
931.05 128.78 .+-. 4.22 14.3 2008-01-18 T26 19-31 lTrTL 779.90 .+-.
16.68 410.10 179.52 .+-. 21.17 124 1990-12-09 F 14.9 2005-11-04 T0
33-32 rTlTL -- Cousins 625.97 816.60 96.08 127 1992-01-18 F 13.9
2005-12-02 T0 33-19 rTrT -- -- 786.71 755.60 131.36 .+-. 22.43 128
1997-03-18 F 8.8 2005-12-02 T0 10 lTL -- -- 837.64 628.74 118.73
.+-. 10.43 130 1991-06-05 F 14.5 2005-12-09 T0 19 rTL -- -- 559.85
552.78 75.09 .+-. 7.11 131 1992-11-09 F 13.1 2005-12-09 T0 32-24
rTlL -- -- 568.01 578.96 101.00 .+-. 11.04 15.0 2007-11-12 T23
32-24 rTlL 450.45 .+-. 9.36 505.94 100.03 .+-. 9.66 136 1989-10-10
F 16.3 2006-01-13 T0 14 lTL -- -- 411.02 670.31 84.81 .+-. 2.56 138
1993-06-04 F 12.7 2006-02-17 T0 24-26 rTlL -- Cousin 577.78 293.51
63.86 .+-. 4.11 14.3 2007-10-24 T20 22-25 rTlL 379.04 .+-. 18.07
388.16 86.23 .+-. 11.26 14.7 2008-02-04 T24 23-26 rTlTL 529.70 .+-.
4.86 378.03 227.26 .+-. 0.94 139 1993-12-06 F 12.2 2006-02-24 T0
12.-14 rTlL -- -- 847.98 868.95 136.19 .+-. 7.63 14.2 2008-02-08
T24 12.-6 rTlL 1192.61 .+-. 10.71 444.33 73.88 .+-. 19.39 141
1992-07-20 F 13.7 2006-03-10 T0 20-18 rTlL -- Grand-mother, 658.28
735.50 90.51 15.5 2008-01-22 T22 9.-13 rTlTL cousins, uncle 172.67
.+-. 8.59 433.6 37.31 .+-. 7.61 142 1992-12-19 F 13.2 2006-03-10 T0
31 lTL -- Mother, cousin 776.43 907.96 122.73 .+-. 7.61 15.1
2008-01-23 T22 25 lTL 542.85 .+-. 1.41 511.4 146.43 .+-. 63.23 146
1990-05-13 F 16.0 2006-05-26 T0 32-22 rTlL -- -- 1501.42 475.91
75.68 .+-. 10.22 148 1993-08-12 F 14.3 2007-12-07 T0 11 lTL --
Mother 1416.91 .+-. 41.50 550.4 37.79 .+-. 6.19 149 1988-09-28 M
17.7 2006-06-02 T0 31-26 rTlL -- -- 472.61 559.97 138.95 .+-. 7.42
150 1992-10-16 F 13.6 2006-06-02 T0 25 rT -- Sister 805.88 543.22
71.24 .+-. 1.52 151 1993-04-11 F 14.7 2007-12-03 T0 28-20 rTlL --
-- 732.19 .+-. 2.30 403.51 20.80 .+-. 3.30 152 1990-10-04 F 15.7
2006-06-02 T0 34 lL -- Father 655.10 551.24 122.69 .+-. 0.10 154
1989-11-24 F 16.6 2006-06-08 T0 40 lTL -- Cousin 541.07 639.52
104.09 .+-. 13.96 18.1 2007-12-07 T18 38 lTL 1101.07 .+-. 38.84
342.17 35.08 .+-. 5.40 155 1991-01-01 F 15.4 2006-06-08 T0 26 lTL
-- Aunt 738.59 796.06 121.33 .+-. 17.72 159 1998-03-04 F 9.7
2007-11-06 T0 3 lTL -- Mother 769.50 .+-. 21.57 831.18 107.5 .+-.
1.08 161 1994-04-27 F 13.6 2007-11-30 T0 15 lTL -- -- 487.11 .+-.
29.43 355.79 23.63 .+-. 0.53 165 1995-08-30 F 12.3 2007-12-03 T0
34-20 rTlL -- -- 1148.04 .+-. 47.51 607.43 42.39 .+-. 7.68 168
1992-04-24 F 14.2 2006-06-26 T0 16-18 rTlL -- -- 810.21 .+-. 28.48
244.4 103.10 .+-. 10.39 14.6 2006-11-21 T5 17-16 rTlL 582.52 .+-.
23.29 338.03 99.20 .+-. 18.18 15.5 2007-10-01 T16 14-16 rTlTL
441.81 .+-. 7.29 333.4 126.96 .+-. 1.45 176 1992-10-24 F 13.8
2006-07-03 T0 29 rT -- -- 503.88 .+-. 35.81 331.65 91.50 .+-. 21.99
14.2 2007-01-15 T6 27 rT 675.38 .+-. 44.20 305.92 193.26 .+-. 2.38
183 1991-09-13 M 14.8 2006-05-07 T0 17 rL -- -- 733.99 .+-. 17.33
550.24 72.91 .+-. 10.68 15.4 2007-06-02 T13 7.-19 rTlL 781.03 .+-.
3.27 531.96 69.83 .+-. 7.07 200 1992-07-29 M 15.2 2007-10-30 T0
23-24 rTlL -- -- 972.10 .+-. 4.92 401.94 88.41 .+-. 10.08 201
1992-11-27 F 13.7 2006-07-12 T0 10-17. rTlL -- Sister 782.77 .+-.
2.63 498.93 142.57 .+-. 44.69 225 1994-05-09 F 12.2 2006-07-24 T0
15-19 lTrTL -- -- 406.67 .+-. 3.40 617.37 248.10 .+-. 24.21 12.8
2007-02-27 T7 13-18 lTrL 651.89 .+-. 21.69 524.9 47.95 .+-. 3.60
234 1990-07-16 M 16.2 2006-10-13 T0 26 rT -- -- 840.88 .+-. 1.98
491.26 89.04 .+-. 5.66 235 1991-10-29 M 15 2006-10-13 T0 20 lTL --
-- 586.25 .+-. 0.32 403.8 181.655 .+-. 48.71 16 2007-10-11 T12 18
lTL 523.39 .+-. 9.76 428.29 188.63 .+-. 6.83 240 1993-10-04 F 13.2
2006-12-11 T0 17-23 rTlL -- Mother, brother, 525.88 .+-. 7.74
428.83 71.91 .+-. 4.23 cousin 242 1989-09-12 F 17.3 2007-01-12 T0 6
lTL -- Sister 590.13 .+-. 6.00 435.59 80.24 .+-. 3.69 244
1990-10-20 F 16.2 2007-01-19 T0 27-29 rTlL -- -- 735.26 .+-. 4.42
510.44 73.81 .+-. 6.20 17.3 2008-02-13 T13 NA NA 1293.68 .+-. 36.92
449.1 44.51 .+-. 4.81 245 1992-01-27 F 15.0 2007-01-22 T0 31-35
rTlL -- -- 496.26 .+-. 3.54 333.97 70.41 .+-. 0.88 15.8 2007-11-14
T10 28-35 rTlL 363.60 .+-. 2.97 562.52 54.98 .+-. 5.08 247
1994-12-18 F 12.1 2007-01-26 T0 9 rTL -- Mother, sister 1148.31
.+-. 2.17 371.29 164.68 .+-. 23.99 12.8 2007-10-09 T9 6 rTL 806.91
.+-. 16.69 393.27 141.16 .+-. 2.62 248 1997-06-16 F 9.6 2007-01-26
T0 9 rL -- Mother, sister 1010.38 .+-. 5.14 443.83 142.95 .+-. 4.69
10.3 2007-10-09 T9 3 lTL 841.24 .+-. 18.47 490.2 158.10 .+-. 33.95
249 1991-03-25 F 15.9 2007-02-02 T0 31 lTL -- -- 534.09 .+-. 7.74
459.52 74.98 .+-. 0.08 16.4 2007-08-03 T6 NA lTL 340.44 .+-. 12.89
499.97 132.91 .+-. 37.20 16.9 2008-02-01 T12 36 lTL 579.65 .+-.
8.62 413.67 98.93 .+-. 19.98 250 1992-05-08 F 14.7 2007-02-02 T0 32
lTL -- Uncle 688.35 .+-. 9.46 587.17 74.40 .+-. 3.75 15.4
2007-10-15 T8 21 lTL 612.19 .+-. 22.36 540.29 150.73 251 1991-09-05
F 15.4 2007-02-02 T0 40-30 rTlL -- -- 1146.66 .+-. 7.34 437.25
80.50 .+-. 5.24 253 1992-10-18 M 14.3 2007-02-27 T0 31 rT -- --
634.83 .+-. 0.90 486.03 184.50 .+-. 20.76 254 1991-12-11 F 15.2
2007-03-09 T0 28 lTL -- -- 701.23 .+-. 1.92 362.22 72.85 .+-. 2.66
15.9 2007-11-12 T8 15 lTL 548.26 .+-. 25.55 538.63 83.17 .+-. 0.07
256 1996-03-19 F 11.0 2007-03-09 T0 11 lTL -- -- 575.73 .+-. 5.49
530.67 97.73 .+-. 3.00 257 1995-04-15 F 11.9 2007-03-09 T0 6 rTL --
Mother 995.77 .+-. 8.22 468.59 94.49 .+-. 8.02 12.5 2007-10-16 T7
NA NA 879.54 .+-. 20.53 421.24 102.11 .+-. 5.69 258 1990-06-24 M
16.8 2007-03-09 T0 14 rT -- -- 876.44 .+-. 9.21 564.15 89.36 .+-.
4.66 17.3 2007-10-02 T8 NA NA 520.58 .+-. 8.52 483.28 175.81 .+-.
53.68 259 1994-07-07 F 12.7 2007-03-16 T0 8 lTL -- -- 1095.11 .+-.
7.88 397.45 85.33 .+-. 4.07 13.5 2007-10-15 T7 11 lTL 1050.58 .+-.
5.08 466.58 139.86 .+-. 15.48 260 1994-07-07 M 12.7 2007-03-16 T0 6
rTL -- -- 1084.13 .+-. 1.82 480.1 127.84 .+-. 8.13 13.5 2007-10-05
T7 4 lTL 494.25 .+-. 22.05 401.01 188.45 .+-. 31.29 261 1997-06-19
F 9.7 2007-03-16 T0 21 lL -- -- 745.79 .+-. 22.70 568.33 122.95
.+-. 2.89 10.3 2007-10-17 T7 10 lTL 1150.38 .+-. 5.64 506.72 206.45
.+-. 14.75 10.4 2008-02-06 T11 5 lTL 852.44 .+-. 31.69 432.45
142.46 .+-. 27.89 263 1994-10-13 F 12.4 2007-03-20 T0 7.-12 rTlL --
-- 989.52 .+-. 4.54 617.16 74.05 .+-. 5.38 264 1992-05-24 F 14.8
2007-03-20 T0 23-30 rTlL -- Uncle 579.22 .+-. 9.53 580.38 100.39
.+-. 2.76 265 1993-05-04 F 13.9 2007-03-20 T0 23 lL -- -- 696.52
.+-. 8.57 491.96 105.88 .+-. 7.86 14.5 2007-11-13 T8 11-14. rTlL
848.34 .+-. 8.38 531.14 106.80 .+-. 1.16 266 1991-01-25 F 16.2
2007-04-02 T0 34 rTL -- -- 728.63 .+-. 5.47 462.66 78.08 .+-. 1.06
16.8 2007-11-15 T7 34 rTL 392.63 .+-. 9.28 349.34 73.67 .+-. 3.30
267 1994-05-14 F 12.9 2007-04-02 T0 5 rTL -- -- 809.78 .+-. 2.39
579.14 70.57 .+-. 2.92 13.5 2007-11-15 T7 5 rTL 925.13 .+-. 23.50
827.31 59.18 .+-. 8.22 268 1994-08-17 F 12.6 2007-04-04 T0 12.-4
rTlL -- Mother 750.67 .+-. 17.49 385.93 107.96 .+-. 12.28 271
1994-11-17 F 12.4 2007-04-13 T0 23 rTL -- -- 925.40 .+-. 10.01
482.89 87.43 .+-. 12.34 12.9 2007-10-15 T6 24 rTL 1087.79 .+-.
22.62 423.61 186.49 .+-. 10.22 272 1994-04-14 F 13.0 2007-04-13 T0
22-24 rTlL -- Aunt 634.87 .+-. 15.77 531.54 86.12 .+-. 1.03 13.6
2007-12-05 T8 14-15 rTlL 515.84 .+-. 13.88 594.47 30.80 .+-. 7.99
273 1991-06-30 F 15.8 2007-04-13 T0 25 rTL -- -- 455.86 .+-. 7.52
548.8 91.21 .+-. 10.34 274 1990-02-28 F 17.1 2007-04-17 T0 11.-22
rTlL -- -- 856.81 .+-. 23.09 461.61 103.50 .+-. 8.99 275 1996-04-08
F 11.0 2007-04-19 T0 27-1. rTlL -- -- 943.57 .+-. 8.27 469.65 66.73
.+-. 5.64 11.5 2007-10-15 T6 26-19 rTlTL 339.71 .+-. 8.66 513.42
159.78 .+-. 30.24 276 1994-09-26 F 13.1 2007-10-15 T0 19-19 rTlL --
-- 430.84 .+-. 16.02 431.09 234.52 .+-. 26.95 277 1994-11-02 F 12.4
2007-04-19 T0 12 lL -- -- 724.67 .+-. 0.64 394.65 96.43 .+-. 0.04
13.0 2007-11-14 T7 15-13 rTlL 634.03 .+-. 28.77 659.6 127.07 .+-.
4.00 278 1992-06-08 M 14.9 2007-05-04 T0 22-14 rTlL -- Mother
1045.58 .+-. 1.10 364.31 106.88 .+-. 8.57 15.3 2007-10-23 T5 26-28
rTlL 1118.55 .+-. 3.48 457.48 234.68 .+-. 24.37 279 1998-09-22 F
8.7 2007-05-30 T0 19 rT -- -- 978.20 .+-. 17.94 442.08 85.62 .+-.
0.14 9.2 2007-10-05 T5 8 rT 851.57 .+-. 67.60 573.28 64.64 280
1992-12-18 F 14.4 2007-05-30 T0 19 rT -- Grand-parents 839.91 .+-.
4.88 415.23 82.19 .+-. 6.30 14.9 2007-11-02 T6 24 rTL 930.08 .+-.
11.55 468.35 63.88 .+-. 1.83 281 1994-10-17 F 12.6 2007-06-01 T0 11
rT -- -- 991.09 .+-. 2.95 522.65 151.89 .+-. 1.15 13.1 2007-11-09
T5 9 lTL 655.22 .+-. 54.74 505.44 112.65 .+-. 14.80 282 1997-09-30
F 9.7 2007-06-13 T0 20 rT -- -- 732.03 .+-. 19.20 547.53 138.06
.+-. 12.04 10.3 2008-01-30 T7 NA NA 1196.46 .+-. 21.91 487.63
129.70 .+-. 7.80 286 1994-06-01 F 13.3 2007-09-17 T0 28 lTL -- --
499.69 .+-. 1.97 400.19 130.85 .+-. 3.82 287 1991-11-15 F 15.8
2007-09-18 T0 11 rTL -- -- 602.68 .+-. 0.65 418.92 190.43 288
1996-05-13 M 11.3 2007-09-18 T0 20 lL -- -- 927.74 .+-. 4.10 533.37
55.21 .+-. 10.16 289 1992-10-23 F 14.9 2007-09-18 T0 18 rT -- --
509.91 .+-. 5.91 362.72 81.33 .+-. 11.16 290 1993-10-02 F 14.0
2007-09-18 T0 22 rTL -- Aunts 498.69 .+-. 46.68 507.71 127.53 .+-.
8.29 291 1992-07-10 F 20.9 2007-09-18 T0 25-31 rTlL -- -- 637.03
.+-. 7.11 467.8 154.54 .+-. 1.72 292 1994-01-23 F 13.7 2007-09-21
T0 20 lTL -- Grand-mother 691.71 .+-. 37.30 581.43 76.54 .+-. 1.66
293 1993-04-03 F 14.5 2007-09-21 T0 16 rT -- -- 494.81 .+-. 7.56
359.46 166.11 295 1991-08-09 M 16.1 2007-09-26 T0 11.-8 rTlL -- --
838.72 .+-. 39.67 405.48 159.20 .+-. 22.89 296 1992-04-04 F 15.5
2007-09-28 T0 15-18 lTrL -- -- 761.74 .+-. 25.61 494.27 237.77 297
1997-07-13 M 10.2 2007-09-28 T0 20 lT -- Uncle 768.08 .+-. 6.70
515.45 100.00 .+-. 9.41 298 1994-11-09 F 12.9 2007-09-28 T0 18-21
rTlL -- -- 750.91 .+-. 16.94 348.87 290.06 .+-. 38.15 299
1990-03-21 F 17.5 2007-10-03 T0 33-43 rTlL -- -- 625.36 .+-. 6.80
306.11 135.94 .+-. 1.36 301 1995-02-06 F 12.7 2007-10-09 T0 13 lT
-- Grand-mother 948.83 .+-.
11.23 578.58 150.57 .+-. 4.40 302 1993-05-07 F 14.4 2007-10-09 T0
14.-12 rTlL -- -- 873.77 .+-. 2.17 373.31 230.66 .+-. 10.50 303
1991-03-29 F 16.5 2007-10-15 T0 14 lTL -- -- 767.96 .+-. 29.04
458.27 192.45 .+-. 10.19 304 1991-10-25 F 16.0 2007-10-16 T0 25 lT
-- Brother, father, all 493.39 .+-. 34.21 446.06 185.69 .+-. 12.07
paternal family 305 1992-02-24 F 15.7 2007-10-19 T0 23 lTL --
Mother 533.91 .+-. 18.09 364.52 123.23 .+-. 15.87 306 1994-09-22 F
13.1 2007-10-19 T0 13-18 rTlL -- Mother 1016.54 .+-. 23.75 623.32
216.02 .+-. 19.04 307 1994-01-25 M 13.7 2007-10-24 T0 8-11-11.
lTrTlL -- -- 1328.92 .+-. 1.50 569.35 165.08 .+-. 16.63 308
1997-05-22 F 10.4 2007-10-26 T0 8 rTL -- Aunts 430.39 .+-. 5.44
519.72 133.63 .+-. 11.13 309 1996-04-10 F 11.5 2007-10-26 T0 10 lTL
-- Mother, cousins 536.77 .+-. 9.30 485.45 285.92 .+-. 25.08 311
1993-05-07 F 14.5 2007-10-26 T0 17 lTL -- -- 493.18 .+-. 23.85
546.9 110.66 .+-. 9.59 313 1993-06-04 F 14.4 2007-10-26 T0 20-18
rTlL -- Cousin 536.22 .+-. 4.65 379.49 99.52 .+-. 2.41 314
1993-03-11 F 14.6 2007-10-29 T0 24 rL -- Mother 939.67 .+-. 37.16
549.66 78.11 .+-. 7.22 315 1993-12-16 F 13.9 2007-10-31 T0 14 lTL
-- -- 537.59 .+-. 1.16 481.91 142.26 .+-. 23.98 316 1992-10-07 M
15.1 2007-10-31 T0 28 rT -- -- 636.17 .+-. 2.31 576.05 94.21 .+-.
5.42 318 1997-05-25 F 10.4 2007-10-15 T0 11 rTL -- Mother 1151.62
.+-. 33.64 634.57 112.13 .+-. 23.16 319 1993-06-28 F 14.4
2007-11-06 T0 22 lTL -- Cousin 518.10 .+-. 27.77 667.02 79.46 .+-.
6.89 320 1993-09-24 F 14.1 2007-11-09 T0 15 rT -- -- 452.54 .+-.
10.01 765.38 134.09 .+-. 21.38 321 1992-07-04 F 15.3 2007-11-09 T0
16 rTL -- -- 470.02 .+-. 16.75 377.13 110.37 .+-. 12.77 322
1996-06-01 F 11.4 2007-11-09 T0 4 lTL -- -- 565.20 .+-. 48.73
492.94 95.12 .+-. 7.44 324 1991-04-20 F 16.6 2007-11-09 T0 19-19
rTlL -- -- 659.93 .+-. 14.39 562.52 98.61 .+-. 6.25 325 1994-03-26
F 13.6 2007-11-09 T0 21 rTL -- Mother, grand- 761.48 .+-. 3.82
846.66 89.91 .+-. 12.48 parents 326 1994-02-02 M 13.8 2007-11-13 T0
13 lTL -- -- 1451.37 .+-. 77.12 617.35 240.72 .+-. 27.74 328
1994-09-24 F 12.8 2007-11-14 T0 11 lTL -- -- 580.55 .+-. 24.91
876.97 174.59 329 1996-05-29 F 11.5 2007-11-14 T0 6 rTL -- Mother
877.16 .+-. 27.08 953.41 269.12 .+-. 4.88 330 1994-02-05 F 13.8
2007-11-16 T0 12 lTL -- -- 1403.38 .+-. 20.98 465.43 279.56 332
1992-01-26 M 15.8 2007-11-23 T0 24 lTL -- -- 864.14 .+-. 43.84
699.27 175.34 .+-. 30.44 333 1993-10-21 F 14.1 2007-11-23 T0 30 lTL
-- Cousin 564.09 .+-. 7.37 762.16 143.10 .+-. 30.54 334 1993-08-07
F 14.3 2007-11-23 T0 29-27 rTlL -- -- 896.91 .+-. 29.60 727.33
155.95 .+-. 38.28 335 1996-01-16 F 11.9 2007-11-23 T0 28-27 rTlL --
-- 1192.08 .+-. 14.98 839.56 162.32 .+-. 0.67 337 1991-09-04 M 16.2
2007-11-28 T0 24 lL -- Sister 914.93 .+-. 10.71 788.28 114.15 .+-.
25.71 338 1994-12-31 F 12.9 2007-11-30 T0 10 lTL -- Aunt 539.94
.+-. 1.35 301.42 38.44 .+-. 5.53 339 1992-03-17 F 15.7 2007-11-30
T0 25 lTL -- Grand-father 747.48 .+-. 9.20 444.12 253.92 340
1995-05-21 F 12.5 2007-11-30 T0 30 lTL -- -- 746.48 .+-. 45.11
498.56 259.46 341 1996-02-11 F 11.8 2007-11-30 T0 15-14 rTlL --
Cousin 947.50 .+-. 31.38 662.73 75.40 .+-. 1.41 342 1993-12-01 F
14.0 2007-12-07 T0 16 rTL -- -- 993.33 .+-. 55.93 376.73 19.57 .+-.
5.63 343 1993-06-29 M 14.4 2007-12-07 T0 15 rTL -- Grand-mother
996.61 .+-. 25.86 541.76 43.48 .+-. 2.96 344 1996-03-26 F 11.7
2007-12-07 T0 10 rTL -- -- 637.78 .+-. 7.73 702.48 26.94 .+-. 5.89
345 1993-04-12 F 14.6 2007-12-07 T0 30 lTL -- Cousin 722.43 .+-.
18.56 429.44 31.74 .+-. 1.77 346 1996-10-11 F 11.2 2007-12-07 T0
18-17 rTlTL -- -- 576.26 .+-. 24.83 436.35 29.25 .+-. 2.56 347
1997-04-07 F 10.7 2007-12-11 T0 5-6. rTlL -- Sister 1272.11 .+-.
18.19 425.98 41.20 .+-. 4.60 348 1995-06-10 M 12.5 2007-12-11 T0 10
rTL -- Sister 776.87 .+-. 50.77 384.51 27.13 .+-. 1.84 350
1995-02-22 F 12.8 2007-12-13 T0 25 rTL -- -- 1020.59 .+-. 46.63
488.19 32.35 .+-. 2.16 351 1992-05-19 F 15.6 2007-12-13 T0 14 rTL
-- Father 557.14 .+-. 25.67 475.23 20.16 .+-. 2.76 352 1996-04-13 M
11.7 2007-12-13 T0 14 rTL -- Father 1339.62 .+-. 39.88 566.82 97.02
353 1993-08-12 M 14.3 2007-12-13 T0 24 rT -- -- 1569.33 .+-. 43.27
607.43 105.59 .+-. 95.83 354 1994-06-07 F 13.5 2007-12-13 T0 8 lT
-- -- 608.88 .+-. 6.80 431.16 69.78 .+-. 40.24 355 1993-08-08 F
14.3 2007-12-13 T0 27 lTL -- -- 691.05 .+-. 37.53 378.46 24.41 .+-.
12.43 356 1995-05-17 F 12.6 2007-12-13 T0 19 lTL -- -- 824.89 .+-.
1.39 467.45 43.63 358 1997-02-27 F 10.9 2008-01-11 T0 18 rTL -- --
554.86 .+-. 8.43 387.21 116.04 .+-. 22.53 359 1995-11-08 F 13.0
2008-01-15 T0 14 rTL -- -- 709.63 .+-. 3.85 485.94 195.32 .+-.
34.14 360 1992-05-24 F 15.6 2008-01-15 T0 14 lTL -- Mother 466.35
.+-. 12.61 335.02 157.17 .+-. 7.22 361 1996-06-29 F 11.5 2008-01-15
T0 23 rTL -- Aunt 899.31 .+-. 10.09 441.72 81.52 .+-. 1.47 362
1997-08-21 F 10.4 2008-01-16 T0 11 lTL -- Grand-mother 471.73 .+-.
21.57 437.35 110.36 .+-. 7.42 363 1993-05-24 F 14.6 2008-01-16 T0
20-24-19 lTrTlTL -- Mother, grand- 743.10 .+-. 15.01 353.53 161.77
.+-. 25.40 mother, aunt 364 1995-03-24 F 12.8 2008-01-16 T0 10 lTL
-- Mother, grand- 767.06 .+-. 11.17 460.75 160.24 .+-. 26.97
mother, aunt 365 1999-07-26 F 9.3 2008-01-16 T0 5 rTL -- Mother,
grand- 883.48 .+-. 2.32 403.41 127.81 .+-. 23.58 mother, aunt 368
1996-07-12 F 11.5 2008-01-18 T0 14 rTL -- -- 1206.06 .+-. 43.70
415.24 136.62 .+-. 28.94 369 1992-05-21 F 15.7 2008-01-18 T0 25 rTL
-- -- 454.71 .+-. 13.34 431.44 132.25 .+-. 19.69 370 1994-12-01 F
13.1 2008-01-18 T0 18-15 rTlL -- -- 855.36 .+-. 10.35 395.7 140.53
.+-. 2.77 371 1992-02-04 F 16.0 2008-01-18 T0 26-20 rTlTL -- Aunt,
cousin 740.05 .+-. 5.38 487.74 112.07 .+-. 3.13 372 1991-06-21 F
16.6 2008-01-21 T0 23-21 rTlL -- -- 436.58 .+-. 40.88 395.61 170.65
.+-. 13.44 374 1992-05-26 F 15.7 2008-01-21 T0 25 lL -- -- 496.50
.+-. 28.07 401.4 77.69 .+-. 6.60 375 1992-10-21 F 15.3 2008-01-22
T0 31-55 rTlTL -- -- 475.88 .+-. 0.00 385.69 130.95 .+-. 3.80 376
1993-05-18 F 14.7 2008-01-22 T0 16 rTL -- -- 554.83 .+-. 44.65
387.61 73.78 .+-. 0.15 377 1995-01-31 F 13.0 2008-01-22 T0 27 lTL
-- -- 739.47 .+-. 8.03 384.16 79.40 .+-. 1.15 379 1996-09-14 F 11.4
2008-01-25 T0 5.-5 lTrTL -- -- 1404.12 .+-. 66.84 659.32 78.73 .+-.
2.62 381 1992-01-11 M 16.0 2008-01-25 T0 24 rT -- -- 782.27 .+-.
1.42 505.65 283.01 .+-. 26.97 382 1993-10-21 F 14.2 2008-01-25 T0
28-25 rTlTL -- -- 998.95 .+-. 9.12 327.82 77.64 .+-. 12.98 383
1994-11-20 F 13.2 2008-01-25 T0 30-27 rTlTL -- -- 900.32 .+-. 24.08
401.79 83.98 .+-. 7.31 384 1992-02-09 M 16.0 2008-01-29 T0 25-19
rTlL -- -- 479.70 .+-. 36.72 444.82 134.93 .+-. 7.83 386 1994-09-02
F 13.4 2008-02-01 T0 25-14 lTrT -- -- 732.99 .+-. 28.62 637.86
129.78 .+-. 2.15 387 1994-04-11 F 13.8 2008-02-01 T0 14-15 rTlTL --
Cousin 853.05 .+-. 70.97 373.81 146.21 .+-. 6.37 388 1995-11-24 F
12.2 2008-02-01 T0 34 rT -- -- 963.01 .+-. 40.86 465.02 66.49 .+-.
7.43 389 1997-04-13 F 10.8 2008-02-04 T0 14 lTL -- Father 689.25
.+-. 35.56 435.9 67.38 .+-. 15.52 390 1994-04-28 F 13.8 2008-02-04
T0 28-26 rTlL -- Father 930.28 .+-. 18.25 368.83 56.32 .+-. 0.12
391 1994-07-01 F 13.6 2008-02-05 T0 37 rTL -- -- 540.38 .+-. 9.17
501.81 49.99 .+-. 7.23 392 1998-11-25 F 9.2 2008-02-05 T0 16 rTL --
Brother 661.55 .+-. 38.23 412.14 77.84 .+-. 23.22 393 1993-09-30 M
14.3 2008-02-05 T0 26 rTL -- Brother 1235.01 .+-. 29.98 488.02
106.86 .+-. 17.43 395 1995-05-24 F 12.7 2008-02-08 T0 11 rT --
Mother 716.48 .+-. 30.93 496.45 82.74 .+-. 2.92 397 1999-02-20 F
9.0 2008-02-08 T0 10 rTL -- Mother, grand- 751.57 .+-. 2.34 543.59
85.71 .+-. 21.81 mother 398 1997-09-16 F 10.4 2008-02-08 T0 16 rTL
-- Mother, grand- 872.92 .+-. 6.46 526.34 98.45 .+-. 6.33 mother
399 2000-09-28 M 7.4 2008-02-08 T0 22-20 rTlTL -- -- 444.55 .+-.
43.23 481.5 74.45 .+-. 10.16 400 1994-05-25 F 13.7 2008-02-08 T0 12
rTL -- Mother, aunt 1492.58 .+-. 30.46 477.59 135.22 .+-. 2.80 401
1994-02-17 F 14.0 2008-02-18 T0 28-21 rTlTL -- -- 691.24 .+-. 23.14
316.38 50.01 .+-. 1.95 402 1991-07-15 F 16.6 2008-02-14 T0 19-12
rTlL -- -- 423.93 .+-. 1.08 314.48 36.64 .+-. 2.04 403 1995-02-21 F
13.0 2008-02-14 T0 13-13 rTlTL -- Sister 1216.81 .+-. 131.72 354.37
52.43 .+-. 15.76 1264 1997-09-22 F 15.2 2005-04-18 T0 40 rTL
2005-04-18 -- 616.12 578.96 65.92 1276 1997-09-23 F 15.2 2005-05-16
T0 42 lT 2005-05-16 -- 817.56 450.13 107.62 .+-. 12.96 1364
1997-09-24 M 14.9 2006-04-24 T0 44 lTL 2006-04-24 Sister, aunt
1668.06 407.4 80.85 .+-. 6.90 1365 1990-05-11 F 15.9 2006-04-26 T0
23-53 lTrL 2006-04-26 -- 947.35 642.66 63.18 .+-. 5.41 1366
1993-04-06 F 13.1 2006-05-01 T0 36 NA 2006-05-01 -- 1317.97 323.04
89.70 .+-. 20.57 1373 1991-10-07 F 14.6 2006-05-17 T0 41-48 rTlL
2006-05-17 -- 1584.54 583.14 80.12 .+-. 18.75 1380 1989-10-09 F
16.7 2006-06-26 T0 35 rL 2006-06-26 -- 1289.98 602.35 139.38 1384
1991-01-17 F 15.5 2006-07-03 T0 41 lTL 2006-07-03 -- 1502.51 .+-.
18.63 194.3 121.65 .+-. 44.94 15.8 2006-11-15 T4 9-4 1258.85 .+-.
16.20 448.68 162.01 .+-. 11.64 1385 1990-06-12 F 16.1 2006-07-04 T0
42-23 rTlL 2006-07-04 -- 1098.75 523.52 102.35 1387 1991-07-15 F
15.0 2006-07-17 T0 29-37-35 rTlL 2006-07-17 Mother 1017.47 689.52
78.42 1388 1991-12-13 F 14.6 2006-07-19 T0 38 rTL 2006-07-19 --
1080.53 811.37 87.57 1409 1993-02-11 F 13.6 2006-09-26 T0 40 rT
2006-09-26 -- 499.41 .+-. 67.54 389.14 113.56 .+-. 15.03 1433
1992-07-03 F 14.5 2007-01-10 T0 44 rT 2007-01-10 Uncle 459.61 .+-.
17.79 287.42 263.55 .+-. 34.89 1451 1995-01-13 F 12.2 2007-03-14 T0
42 rT 2007-03-14 Grand-mother 1099.93 .+-. 48.11 290.5 158.45 .+-.
3.94 1478 1990-08-06 F 16.8 2007-06-11 T0 41 rTL 2007-06-11 Father
619.94 .+-. 46.51 251.56 190.25 .+-. 18.46 1481 1990-08-15 F 16.8
2007-06-18 T0 40 rT 2007-06-18 -- 748.36 .+-. 9.30 250.14 95.34
.+-. 6.52 1483 1989-06-26 F 18.0 2007-06-19 T0 37-25 rTlL
2007-06-19 -- 489.30 .+-. 93.18 396.39 167.02 .+-. 28.62 1487
1990-05-30 F 17.1 2007-07-03 T0 35-58-35 lCrTlL 2007-07-03 Aunts
508.82 .+-. 50.08 281.48 17.75 .+-. 1.94 * Plus-minus values are
means .+-. standard deviations. ** All patients are diagnosed with
AIS .dagger. Curve type nomenclature: r, right/l, left/T,
Thoracic/L, Lumbar/TL, Thoracolumbar/C, Cervical. .dagger-dbl.
Certain clinical information may not have been available at the
time of the study, NA.
TABLE-US-00007 TABLE 7 Clinical and biochemical profiles of AIS
patients with Cobb's angles of 45.degree. or more. Cobb's Patient
Date of Collection Timepoint Angle Curve ID Birth Gender Age Date
(months) Pre-op Type 101 1988-05-22 F 17.1 2005-06-10 T0 47 rT 108
1989-08-29 F 15.9 2005-07-04 T0 45 lL 17.2 2006-11-21 T16 40 lL 135
1987-12-31 F 18.0 2006-01-13 T0 47-30 rTlL 145 1990-02-15 M 16.2
2006-04-21 T0 50-43 rTlTL 170 1991-07-08 F 14.9 2006-06-26 T0 53-22
rTlL 15.9 2007-04-18 T10 44-21 rTlL 1150 1992-04-18 F 12.1
2004-05-11 T0 84 rT 1169 1989-09-19 F 14.8 2004-06-22 T0 54-52 rTlL
1192 1990-10-16 F 13.9 2004-09-08 T0 59 rT 1212 1991-05-06 F 13.5
2004-11-22 T0 54 rT 1254 1991-07-23 F 13.7 2005-03-16 T0 52-49 rTlL
1267 1990-09-08 F 14.6 2005-04-25 T0 55 lT 1282 1988-12-29 F 16.5
2005-06-06 T0 49 rT 1310 1990-05-05 F 15.6 2005-11-09 T0 55-42 rTlL
1353 1989-08-08 F 16.6 2006-03-27 T0 46 lT 17.2 2006-10-06 T7 2 NA
1354 1991-11-18 F 14.3 2006-03-27 T0 45 rT 1355 1990-02-26 M 16.1
2006-03-28 T0 74-53 rTlL 1357 1990-08-23 F 14.8 2005-06-15 T0 47-50
rTlL 15.7 2006-04-04 T10 57-50 rTlL 1360 1996-05-09 F 9.9
2006-04-10 T0 53-46 rTlL 1361 1989-09-03 F 16.6 2006-04-10 T0 65-95
rTlL 1369 1992-02-19 F 14.2 2006-05-09 T0 88 rT 14.8 2006-11-24 T6
25 NA 1371 1991-01-30 F 15.3 2006-05-15 T0 72-59 rTlL 1372
1990-09-06 F 15.7 2006-05-16 T0 63-45- rTLlLC 33 1374 1989-10-05 F
16.6 2006-05-29 T0 45 lTL 1378 1992-12-14 M 13.5 2006-06-05 T0 70
lTL 1381 1990-10-03 F 15.7 2006-06-27 T0 66 lT 1389 1995-10-26 F
10.7 2006-07-24 T0 46-66 rTlTL 11.0 2006-10-02 T5 NA NA 1390
1990-12-12 F 15.6 2006-07-24 T0 53 lTL 1392 1993-05-25 F 13.2
2006-07-26 T0 48 rT 1393 1991-05-09 F 15.2 2006-07-26 T0 56 lTL
1395 1988-10-25 F 17.8 2006-08-08 T0 84 lTL 1396 1995-05-27 F 11.2
2006-08-14 T0 74-62 rTlL 11.3 2006-09-26 T1 NA NA 1397 1988-12-23 M
17.7 2006-08-29 T0 60-58 rTlL 17.9 2006-10-11 T2 34-23 NA 1406
1991-10-29 F 14.9 2006-09-20 T0 62-60 rTlL 1410 1993-01-04 F 13.7
2006-09-28 T0 56 rT 13.8 2006-11-21 T2 23 NA 1416 1991-07-10 F 15.4
2006-11-15 T0 56-30 rTlL 1420 1993-06-30 F 13.4 2006-11-29 T0 60-48
rTlL 1422 1994-06-27 F 12.4 2006-12-06 T0 60-50 rTlL 1430
1989-09-28 F 17.3 2007-01-03 T0 48 rT 1442 1994-08-21 F 12.5
2007-02-14 T0 60 rT 1446 1988-07-10 F 18.6 2007-02-26 T0 60 rT 1448
1992-12-07 F 14.3 2007-03-13 T0 49 lTL 1457 1993-05-30 F 13.9
2007-04-10 T0 50-43 rTlL 1458 1991-09-27 F 15.4 2007-04-11 T0 45 rT
1459 1990-03-28 F 17.1 2007-04-16 T0 72-36 rTlL 17.2 2007-05-18 T1
NA NA 1461 1990-05-17 F 16.9 2007-04-18 T0 48 rT 1464 1990-01-02 F
17.3 2007-04-25 T0 53 rT 1467 1990-11-18 F 16.5 2007-05-08 T0 60 rT
1468 1991-11-12 M 15.5 2007-05-14 T0 69 rTL 1471 1989-10-08 F 17.6
2007-05-29 T0 60 rTL 1474 1989-06-24 M 18.0 2007-06-04 T0 54-52
rTlL 1477 1992-10-17 F 14.6 2007-06-06 T0 62-65 rTlL 1484
1991-04-27 F 16.2 2007-06-26 T0 60 rT 1488 1992-02-17 M 15.4
2007-07-16 T0 87 rT 1489 1990-09-26 M 16.8 2007-07-17 T0 57 rT 1495
1992-03-19 F 15.5 2007-09-17 T0 67-39 rT 1498 1992-11-05 F 14.9
2007-09-18 T0 51-42 rTL 1501 1989-02-04 F 16.5 2005-07-22 T0 58 rTL
17.8 2006-11-21 T16 60 rTL 1502 1994-03-14 F 13.6 2007-10-15 T0
55-43 rTlL 13.8 2007-12-05 T2 NA NA 1506 1992-07-07 F 15.3
2007-11-06 T0 65 rT 1517 Nov. 20, 1990 M 17.2 2008-02-13 T0 50-62
rTlTL 1518 Dec. 8, 1991 F 16.2 2008-02-13 T0 62-62 rTlL 1519
1993-04-19 M 14.8 2008-02-08 T0 51 rT 1520 1993-06-26 F 14.6
2008-02-08 T0 54-42 rTlTL Patient Date of [sCD44] ID Surgery Family
History [OPN] (ng/ml) (ng/ml) [HA] (ng/ml) 101 -- -- 1047.64 728.42
221.97 .+-. 8.23 108 -- -- 774.45 704.05 86.15 .+-. 12.73 414.67
.+-. 55.62 361.83 172.00 .+-. 3.68 135 -- -- 657.01 839.02 117.48
.+-. 5.37 145 -- Brother 1178.85 961.85 120.52 .+-. 8.59 170
2007-08 Aunt 480.97 .+-. 29.49 317.2 33.76 .+-. 0.92 540.63 .+-.
10.65 410.66 70.69 .+-. 4.67 1150 2004-05-11 Mother, grand- 884.02
874.59 97.74 mother 1169 2004-06-22 -- 776.13 868.43 101.22 .+-.
9.41 1192 2004-09-08 -- 1140.09 596.41 66.97 1212 2004-11-22
Great-aunt 834.47 796.56 75.57 1254 2005-03-16 -- 1091.92 882.29
82.8 1267 2005-04-25 -- 509.48 596.41 76.87 1282 2005-06-06 --
718.45 788.41 53.95 .+-. 16.65 1310 2005-11-09 -- 1042.25 789.32
132.89 1353 2006-03-27 -- 1078.92 .+-. 33.32 262.59 90.88 .+-. 1.59
44.35 .+-. 0.50 342.48 157.74 .+-. 37.90 1354 2006-03-27 --
1378.360 725.138 61.016 1355 2006-03-28 -- 1871.67 467.38 253.56
.+-. 6.84 1357 2006-04-04 Brother 705.92 .+-. 16.09 415.22 174.61
.+-. 74.40 1788.1 374.7 76.86 .+-. 4.78 1360 2006-04-10 Father,
aunt 1820.95 444.42 80.45 .+-. 29.61 1361 2006-04-10 -- 1512.16
599.64 67.13 .+-. 10.66 1369 2006-05-09 -- 1498.66 262.58 91.42
.+-. 8.52 541.43 .+-. 10.31 317.72 166.79 .+-. 35.56 1371
2006-05-15 -- 1723.91 224.15 89.53 .+-. 18.60 1372 2006-05-16 Aunt
1016.66 597.2 65.24 .+-. 5.40 1374 2006-05-29 -- 1698.01 544.71
70.32 .+-. 16.24 1378 2006-06-05 -- 1531.64 394.74 249.97 1381
2006-06-27 -- 1032.61 626.25 89.25 1389 2006-07-24 -- 899.76 .+-.
20.49 359.31 187.61 .+-. 62.69 770.91 .+-. 13.31 533.42 82.67 .+-.
1.55 1390 2006-07-24 -- 1269.89 839.02 78.42 1392 2006-07-26
Grand-mother, 1341.80 .+-. 15.38 87.13 105.48 .+-. 0.34 aunts 1393
2006-07-26 -- 969.63 821.21 81.59 1395 2006-08-08 Aunt 1205.3
450.13 41.8 1396 2006-08-14 -- 1624.64 .+-. 5.10 166.83 172.75 .+-.
26.23 773.40 .+-. 16.42 342.29 218.18 .+-. 2.83 1397 2006-08-29
Uncle 1581.40 .+-. 11.23 440.95 106.21 .+-. 10.20 1191.01 .+-.
14.64 546.18 158.77 .+-. 21.05 1406 2006-09-20 -- 628.36 .+-. 45.23
304.04 52.88 .+-. 0.66 1410 2006-09-28 Mother, aunt 1287.16 .+-.
3.12 133.56 119.48 .+-. 24.22 903.57 .+-. 52.88 328.75 141.76 .+-.
12.56 1416 2006-11-15 -- 514.30 .+-. 15.49 233.55 121.42 .+-. 28.69
1420 2006-11-29 Sister, aunt 661.35 .+-. 21.22 314.01 127.14 .+-.
1.06 1422 2006-12-06 Sister 530.56 .+-. 6.57 190.55 61.30 .+-.
14.49 1430 2007-01-03 -- 533.56 .+-. 24.89 228.54 51.29 .+-. 7.00
1442 2007-02-14 -- 512.99 .+-. 44.58 163.01 162.44 .+-. 3.03 1446
2007-02-26 -- 537.87 .+-. 4.70 332.42 66.44 .+-. 20.48 1448
2007-03-13 -- 588.73 .+-. 25.88 110.3 138.81 .+-. 10.07 1457
2007-04-10 -- 1073.67 .+-. 69.04 401.79 83.21 .+-. 0.17 1458
2007-04-11 -- 401.08 .+-. 22.88 212.16 66.48 .+-. 0.55 1459
2007-04-16 -- 761.78 .+-. 11.69 104.61 42.08 .+-. 5.99 744.34 .+-.
10.91 340.71 1461 2007-04-18 Sister 200.53 .+-. 3.68 371.51 112.29
.+-. 27.44 1464 2007-04-25 -- 778.26 .+-. 19.40 163.01 133.86 .+-.
4.16 1467 2007-05-08 -- 453.32 .+-. 17.32 236.23 48.59 .+-. 6.73
1468 2007-05-14 Cousin 574.80 .+-. 42.46 283.37 116.85 .+-. 14.54
1471 2007-05-29 -- 907.06 .+-. 34.13 332.42 66.91 .+-. 28.51 1474
2007-06-04 -- 1254.39 .+-. 4.53 334.72 71.72 .+-. 16.08 1477
2007-06-06 Mother, brother 829.32 .+-. 15.89 355.03 150.57 .+-.
28.87 1484 2007-06-26 -- 489.15 .+-. 20.09 216.67 88.54 .+-. 422
1488 2007-07-16 Mother 1358.23 .+-. 56.62 304.83 120.78 .+-. 13.25
1489 2007-07-17 -- 1417.61 .+-. 0.00 146.93 135.42 .+-. 2.53 1495
2007-09-17 -- 437.55 .+-. 14.74 227.82 32.06 .+-. 0.29 1498
2007-09-18 -- 557.43 .+-. 50.58 152.3 62.63 .+-. 12.90 1501 -- --
939.53 711.38 144.30 .+-. 16.14 580.11 .+-. 7.56 503.43 107.24 .+-.
7.29 1502 2007-10-15 -- 856.14 .+-. 4.95 386.19 152.27 .+-. 5.09
1089.57 .+-. 22.51 349.14 55.91 .+-. 10.45 1506 2007-11-06 --
675.53 .+-. 13.63 241.98 85.64 .+-. 24.87 1517 -- -- 666.49 .+-.
65.68 328.96 41.3 .+-. 8.74 1518 -- -- 672.59 .+-. 35.53 440.55
67.71 .+-. 6.81 1519 -- -- 945.23 .+-. 53.53 360.02 66.48 .+-. 1.10
1520 -- -- 752.87 .+-. 23.12 288.35 87.08 .+-. 0.36 * Plus-minus
values are means .+-. standard deviations. ** All patients are
diagnosed with AIS .dagger. Curve type nomenclature: r, right/l,
left/T, Thoracic/L, Lumbar/TL, Thoracolumbar/C, Cervical.
.dagger-dbl. Certain clinical information may not have been
available at the time of the study, NA.
TABLE-US-00008 TABLE 8 Clinical and biochemical profiles of
asymptomatic at risk children. Collection Timepoint [sCD44] Family
Id Date of Birth Gender Age Date (months) Family History [OPN]
(ng/ml) (ng/ml) [HA] (ng/ml) 1 1997-09-02 M 8.8 2006-07-10 T0
Mother 439.72 .+-. 12.32 561.46 118.71 .+-. 8.74 1 1995-09-06 F
10.8 2006-07-10 T0 Mother 207.88 .+-. 0.93 315.67 180.71 .+-. 19.91
2 1998-02-08 F 8.7 2006-10-03 T0 Mother, uncle, 1650.21 .+-. 13.90
416.99 199.56 .+-. 55.60 9.2 2007-04-19 T6 grand-father 1966.98
.+-. 1.96 459.89 207.57 .+-. 39.18 9.8 2007-12-12 T14 1816.83 .+-.
24.08 387.1 209.86 .+-. 21.38 2 2001-06-18 M 5.8 2007-04-19 T0
Mother, uncle, 493.98 .+-. 7.26 463.68 43.99 .+-. 3.74 6.5
2007-12-12 T8 grand-father 684.54 .+-. 10.06 438.94 102.21 .+-.
61.17 3 1994-08-24 F 12.2 2006-10-19 T0 Sister 690.58 .+-. 2.92
418.18 220.8 12.6 2007-05-02 T7 727.27 .+-. 17.36 467.79 196.82
.+-. 18.74 13.2 2007-12-12 T14 1212.32 .+-. 0.48 311.06 279.74 .+-.
30.33 4 2003-10-17 F 3.0 2006-10-19 T0 Mother 1530.90 .+-. 28.42
478.58 225.02 .+-. 20.51 3.5 2007-04-11 T6 1021.07 .+-. 7.22 464.63
122.36 .+-. 15.35 4.2 2007-12-12 T14 1594.42 .+-. 23.36 470.05
332.11 5 2003-07-17 M 3.2 2006-10-19 T0 Mother 905.58 .+-. 30.14
563.44 58.88 .+-. 3.86 3.7 2007-04-19 T6 1865.13 .+-. 7.35 434.93
128.14 .+-. 4.00 4.4 2007-12-09 T14 960.14 .+-. 26.22 631.93 32.64
.+-. 5.81 6 1998-07-26 F 8.2 2006-10-19 T0 Mother 505.03 .+-. 8.92
564.17 81.86 .+-. 13.18 7 1995-06-16 F 11.3 2006-10-24 T0 Mother
548.59 .+-. 6.61 512.92 80.39 .+-. 31.53 11.8 2007-04-11 T6 766.85
.+-. 5.73 396.69 103.31 .+-. 22.50 12.3 2007-10-17 T12 596.91 .+-.
35.50 465.36 122.40 .+-. 8.97 8 1996-04-10 F 10.5 2006-10-26 T0
Mother 1109.78 .+-. 47.61 401.66 77.16 .+-. 9.72 11 2007-04-11 T6
875.81 .+-. 14.01 366.36 176.96 .+-. 4.68 9 1995-05-09 F 11.4
2006-10-26 T0 Mother 1657.97 440.3 112.58 .+-. 0.45 11.9 2007-04-11
T6 782.29 .+-. 1.47 429.56 86.57 .+-. 1.46 12.8 2008-02-13 T16
885.10 .+-. 35.98 255.6 63.42 .+-. 7.99 10 2002-08-03 F 4.2
2006-10-26 T0 Mother 901.66 .+-. 12.01 398.27 158.65 .+-. 60.85 4.7
2007-04-11 T6 929.42 .+-. 3.07 356.88 167.19 .+-. 0.13 11
1992-09-07 F 14.1 2006-10-26 T0 Mother 528.00 .+-. 8.83 469.78
69.05 .+-. 4.37 14.8 2007-07-11 T9 714.79 .+-. 14.44 383.1 37.97
.+-. 3.99 15.3 2008-01-23 T15 443.30 .+-. 0.58 472.69 80.27 .+-.
11.45 12 1991-12-15 F 14.8 2006-10-26 T0 Mother 818.88 .+-. 0.94
518.03 134.08 .+-. 84.67 15.3 2007-04-11 T6 648.15 487.38 140.02
.+-. 50.63 15.9 2007-11-14 T13 398.28 .+-. 19.81 521.44 191.07 .+-.
8.20 12 1996-02-23 M 10.7 2006-10-26 T0 Mother 1203.88 .+-. 55.29
681.23 85.30 .+-. 36.75 11.2 2007-04-11 T6 1930.95 .+-. 1.96 633.37
107.10 .+-. 15.99 11.8 2007-11-14 T13 1341.78 .+-. 31.57 687.61
170.54 .+-. 25.46 13 1993-10-09 F 13.0 2006-10-26 T0 Mother, grand-
730.44 .+-. 33.95 397.12 41.87 .+-. 4.55 13.6 2007-05-02 T7 mother
420.91 .+-. 23.59 412.49 216.75 .+-. 27.71 14.1 2007-11-14 T13
943.64 .+-. 1.96 698.95 124.28 .+-. 15.03 14 2001-09-07 F 5.2
2006-11-16 T0 Father 919.94 .+-. 11.91 510.08 45.28 .+-. 10.89 15
1997-02-18 M 9.8 2006-11-16 T0 Mother 1629.22 .+-. 12.49 611.25
129.80 .+-. 30.80 10.2 2007-04-11 T5 1030.34 .+-. 6.55 690.56
146.19 .+-. 2.58 10.7 2007-10-10 T11 929.36 .+-. 11.23 590.8 135.89
.+-. 18.75 16 2002-02-21 F 4.8 2006-11-16 T0 Mother 1834.30 .+-.
4.16 628.94 149.05 .+-. 19.17 5.2 2007-04-11 T5 909.22 .+-. 6.67
661.18 125.31 5.9 2007-12-12 T13 877.48 .+-. 23.75 466.59 70.10
.+-. 33.68 17 2000-03-30 F 6.7 2006-11-16 T0 Mother 482.76 .+-.
10.64 678.55 95.92 .+-. 18.21 18 2000-08-01 F 6.2 2006-11-16 T0
Mother 870.73 .+-. 21.30 644.62 146.12 .+-. 36.68 18 1997-05-05 M
9.5 2006-11-16 T0 Mother 1123.32 .+-. 7.06 401.66 112.68 .+-. 11.34
20 1998-09-27 F 8.2 2006-11-22 T0 Father 506.21 .+-. 10.03 456.42
59.40 .+-. 30.21 8.8 2007-07-11 T8 677.71 .+-. 13.95 416.28 37.11
.+-. 6.95 21 (015) 1998-11-17 F 8.0 2006-11-22 T0 Sister 482.63
.+-. 7.58 458.02 99.16 .+-. 5.46 8.5 2007-05-23 T6 511.46 488.33
151.08 9.0 2007-11-14 T12 760.00 .+-. 3.99 589.62 190.77 .+-. 5.64
21 (016) 1991-08-13 F 15.2 2006-11-22 T0 Sister 617.06 .+-. 7.65
511.71 110.15 .+-. 12.37 15.7 2007-05-23 T6 619.60 .+-. 17.63 519.3
93.16 .+-. 0.39 16.2 2007-11-14 T12 685.18 .+-. 0.80 529.63 218.26
.+-. 27.22 22 1992-05-15 M 14.5 2006-11-22 T0 Mother, grand-
1082.23 .+-. 65.01 445.66 81.35 .+-. 14.77 14.9 2007-04-11 T5
mother 1044.90 .+-. 3.21 432.72 152.54 .+-. 10.62 15.6 2008-01-23
T14 1010.18 .+-. 60.70 384.16 106.42 .+-. 10.80 23 (334) 1994-09-24
F 12.2 2006-11-29 T0 Sister 1365.94 .+-. 1.71 346.45 150.14 .+-.
2.53 12.6 2007-04-19 T5 1856.82 .+-. 12.74 501.92 167.91 .+-. 17.19
13.1 2007-10-10 T11 947.97 .+-. 16.31 489.38 271.36 .+-. 20.40 24
1994-11-24 M 12.0 2006-11-29 T0 Mother, aunt 775.28 .+-. 20.77
427.49 84.54 .+-. 0.14 12.5 2007-05-02 T6 610.29 .+-. 10.86 436.82
130.53 .+-. 2.30 13.1 2007-12-12 T13 718.55 .+-. 5.97 355.99 127.92
.+-. 3.93 24 1994-11-24 F 12 2006-11-29 T0 Mother, aunt 815.81 .+-.
22.25 473.76 160.63 .+-. 8.36 12.5 2007-05-02 T6 673.56 .+-. 16.29
445.36 127.40 .+-. 37.13 13.1 2007-12-12 T13 1299.89 .+-. 28.77
662.73 276.97 25 1998-06-05 F 8.4 2006-11-29 T0 Mother, father
1245.41 .+-. 13.75 441.4 108.75 .+-. 18.90 8.8 2007-04-19 T5
1766.40 .+-. 2.69 500.34 197.20 .+-. 31.62 9.3 2007-10-10 T11
944.99 .+-. 25.37 476.76 115.66 .+-. 10.09 25 2001-06-04 M 5.4
2006-11-29 T0 Mother, father 1181.70 .+-. 50.65 303.75 157.81 .+-.
11.99 5.8 2007-04-19 T5 1707.51 .+-. 30.62 319.63 113.24 .+-. 2.45
6.3 2007-10-10 T11 867.79 .+-. 25.36 364.76 114.76 .+-. 33.42 26
1994-03-18 F 12.7 2006-11-29 T0 Mother 676.95 .+-. 9.57 432.08
86.09 27 1987-12-13 F 19 2006-12-19 T0 Father 287.27 .+-. 8.96
572.38 101.88 .+-. 13.89 28 2003-05-23 F 3.6 2006-12-19 T0 Mother
612.92 .+-. 3.03 760.08 45.57 .+-. 3.40 29 1990-10-17 M 16.2
2006-12-19 T0 Mother 459.54 .+-. 29.16 488.33 99.03 .+-. 54.21 17.0
2007-10-10 T10 505.24 .+-. 39.04 441.73 121.53 .+-. 15.54 29 (652)
1999-05-11 F 7.6 2006-12-19 T0 Mother 576.64 .+-. 20.73 656.77
114.39 8.4 2007-10-10 T10 972.66 .+-. 7.97 636.32 138.53 .+-. 16.69
29 (160) 1996-12-02 F 10.0 2006-12-19 T0 Mother 583.62 .+-. 19.18
600.16 136.79 .+-. 10.66 10.8 2007-10-10 T10 874.79 .+-. 2.17
535.48 112.73 .+-. 7.74 30 1995-03-09 M 11.8 2006-12-19 T0 Mother
1608.98 .+-. 8.37 607.15 115.19 .+-. 6.27 12.3 2007-07-04 T7
1107.95 .+-. 0.53 504.15 40.04 .+-. 11.63 12.8 2008-01-23 T13
1578.17 .+-. 18.50 469.62 93.33 .+-. 3.68 30 1997-06-08 F 9.5
2006-12-19 T0 Mother 1211.80 .+-. 5.47 586.43 172.18 .+-. 4.00 10.1
2007-07-04 T7 774.18 .+-. 21.15 534.59 40.03 .+-. 11.95 10.6
2008-01-23 T13 697.49 .+-. 12.25 473.45 95.89 .+-. 6.16 31
1998-03-18 F 8.8 2006-12-19 T0 Mother, aunt, grand- 467.80 .+-.
1.39 574.23 106.48 .+-. 29.19 father 31 1999-11-03 M 7.1 2006-12-19
T0 Mother, aunt, grand- 745.53 .+-. 40.56 552.66 98.22 .+-. 1.18
father 32 2004-06-20 F 2.5 2006-12-19 T0 Mother, grand- 1573.79
.+-. 0.72 576.5 142.70 .+-. 0.57 3.1 2007-07-04 T7 mother 1034.97
.+-. 25.55 494.82 52.38 .+-. 5.01 3.6 2008-01-23 T13 1237.94 .+-.
48.60 374.2 152.27 .+-. 0.32 33 1996-05-17 M 10.7 2007-01-10 T0
Mother 623.78 .+-. 2.66 649.44 166.16 .+-. 32.22 11.5 2007-11-07
T10 671.14 .+-. 0.27 634.5 36.87 .+-. 2.05 33 1996-06-25 F 11.2
2007-01-10 T0 Mother 893.13 .+-. 34.21 436.86 92.74 .+-. 2.45 11.7
2007-07-11 T6 716.31 .+-. 27.52 543.59 37.95 .+-. 5.33 34
1996-08-14 F 10.3 2006-12-21 T0 Mother 1135.80 .+-. 18.20 508.95
256.64 .+-. 37.18 10.8 2007-06-13 T6 594.41 .+-. 0.37 490.61 96.56
.+-. 2.45 11.4 2008-01-23 T13 978.10 .+-. 49.46 450.46 103.67 .+-.
10.95 34 1994-06-21 M 12.5 2006-12-21 T0 Mother 1010.70 .+-. 22.34
416.71 172.33 .+-. 50.68 13.0 2007-06-13 T6 739.31 .+-. 3.43 499.04
93.55 .+-. 6.90 13.6 2008-01-23 T13 777.22 .+-. 39.78 448.93 92.70
.+-. 21.91 35 (605) 1995-03-31 M 11.8 2006-12-21 T0 Mother 1126.22
.+-. 46.08 552.37 163.66 .+-. 0.79 35 (604) 1995-03-31 M 11.8
2006-12-21 T0 Mother 933.16 .+-. 14.20 437.43 118.57 .+-. 6.65 35
1993-05-12 F 13.6 2006-12-21 T0 Mother 1679.45 436.58 128.45 .+-.
17.60 36 1998-09-06 M 8.3 2007-01-10 T0 Mother 1520.81 .+-. 20.48
485.39 225.68 .+-. 85.59 9.2 2007-11-14 T10 1103.50 .+-. 27.07
899.87 114.96 .+-. 0.11 37 2001-07-11 F 5.5 2007-01-17 T0 Mother
419.51 .+-. 10.21 524.02 35.52 .+-. 0.52 6.0 2007-07-04 T6 606.10
.+-. 14.32 490.91 209.23 38 1995-01-19 M 12.0 2007-01-17 T0 Mother
435.87 .+-. 7.38 600.34 164.49 .+-. 10.01 38 1992-08-02 F 14.4
2007-01-17 T0 Mother 328.67 .+-. 25.67 564.58 166.19 .+-. 2.53 39
1996-06-08 M 10.6 2007-01-24 T0 Mother 437.90 .+-. 23.91 529.14
215.53 .+-. 70.15 11.1 2007-07-18 T6 617.26 .+-. 5.45 445.15 146.08
.+-. 8.82 39 1997-08-08 F 9.4 2007-01-24 T0 Mother 399.82 .+-.
14.71 452.38 71.339 .+-. 22.51 9.9 2007-07-18 T6 648.28 .+-. 6.30
462.01 188.78 .+-. 12.79 40 1996-05-05 F 10.9 2007-04-05 T0 Mother
986.26 .+-. 9.88 478.27 99.9 40 1999-04-23 M 8.0 2007-04-05 T0
Mother 851.99 .+-. 4.04 710.05 52.81 .+-. 12.17 41 1995-03-29 F
12.2 2007-05-30 T0 Father 500.68 .+-. 20.08 416.56 71.27 .+-. 0.30
42 1996-07-03 M 10.8 2007-05-02 T0 Father 391.38 .+-. 30.03 620.65
32.83 11.3 2007-11-14 T6 393.23 .+-. 4.22 445.78 167.25 .+-. 27.97
42 1992-04-14 F 15.1 2007-05-02 T0 Father 452.43 .+-. 1.68 519.81
38.46 .+-. 16.02 15.6 2007-11-14 T6 658.95 .+-. 1.62 938.89 232.91
.+-. 2.00 43 2001-11-20 F 5.5 2007-05-23 T0 Mother 892.70 .+-.
21.23 484.89 97.65 .+-. 30.81 44 1995-09-11 M 11.8 2007-06-13 T0
Mother 1058.59 .+-. 6.11 547.8 41.15 .+-. 11.08 12.2 2007-12-12 T6
1160.10 .+-. 16.16 456.22 145.61 .+-. 51.30 45 1994-05-10 F 13.2
2007-08-29 T0 Mother 714.66 .+-. 6.88 482.12 120.00 .+-. 13.64 13.8
2008-02-13 T6 801.53 .+-. 42.46 358.64 134.84 .+-. 16.18 46
1999-11-04 M 7.8 2007-09-12 T0 Mother 603.75 .+-. 10.96 569.62
111.95 .+-. 5.86 46 (980) 1996-04-15 F 11.4 2007-09-13 T0 Mother
504.38 .+-. 35.85 540.29 118.25 .+-. 9.11 46 (982) 2004-01-24 F 3.7
2007-09-12 T0 Mother 718.72 .+-. 78.98 510.97 153.13 .+-. 4.50 47
1996-12-07 F 10.8 2007-10-17 T0 Mother 1010.10 .+-. 17.02 494.12
147.00 .+-. 87.36 47 1999-04-03 M 8.5 2007-10-17 T0 Mother 844.83
.+-. 30.84 456.7 156.33 .+-. 50.36 C6 1997-02-06 F 10.3 2007-05-22
T0 Mother 669.60 .+-. 4.19 755.65 133.68 .+-. 4.10 11.0 2008-01-16
T8 733.30 .+-. 11.16 620.67 250.52 .+-. 38.11 C15 1997-05-27 M 10.0
2007-06-06 T0 Brother 441.81 .+-. 0.64 640.33 106.53 .+-. 1.88 10.5
2007-12-04 T6 444.69 .+-. 3.82 958.24 151.86 .+-. 17.41 *
Plus-minus values are means .+-. standard deviations. .dagger. All
subjects are examined before sample collection by an orthopedic
surgeon to monitor possible scoliosis development.
Example 11
OPN, sCD44 and HA Levels in Non AIS Scoliotic Patients
[0164] OPN levels were measured in non AIS scoliotic patients (NAIS
patients). Results are summarized in Table 9 below. A comparison of
OPN, sCD44 and HA levels in healthy, AIS and NAIS patients is also
provided in FIG. 12.
TABLE-US-00009 TABLE 9 Biomarkers Comparison of non-AIS scoliotic
Patients. Characteristics Mean OPN Mean sCD44 Mean HA Mean Age Mean
Cobb Concentration Concentration Concentration Type of Scoliosis
Number (Years) Angle (ng/ml) (ng/ml) (ng/ml) Neurological 8 12.3
.+-. 3.7 79.4 .+-. 15.1 982 .+-. 452 274 .+-. 196 127 .+-. 101
Scoliosis Congenital Scoliosis 8 10.0 .+-. 4.4 51.8 .+-. 18.1 1016
.+-. 400 432 .+-. 79 123 .+-. 80 Spondylolisthesis 5 17.5 .+-. 2.1
21.0 .+-. 17.0 832 .+-. 125 386 .+-. 193 76 .+-. 54 Kyphosis
Scoliosis 5 14.4 .+-. 2.8 80.2 .+-. 28.5 923 .+-. 393 352 .+-. 62
91 .+-. 56 Other* 2 15.1 74.5 .+-. 17.7 586 .+-. 52 240 NA .dagger.
Plus-minus values are means .+-. standard deviations *Other
scoliosis types include one neuromuscular scoliosis and one
dysplasic scoliosis.
[0165] Table 10 below presents in detail biomarkers levels for non
AIS scoliotic patients.
TABLE-US-00010 TABLE 10 Clinical and biochemical profiles of non
AIS scoliotic patients. Collection Cobb's Angle Curve Date of Age
at Family [sCD44] [HA] Patient ID Date of Birth Gender Age Date
Diagnosis Pre-op Type Surgery Surgery History [OPN] (ng/ml) (ng/ml)
(ng/ml) 1208 1990-01-19 M 17.8 2007-10-03 Congenital cyphose
scoliosis 72 lT 2004-11- 14.8 -- 1101.06 .+-. 31.26 444.81 82.89
.+-. 15.11 08 1256 1992-03-27 M 13.0 2005-05-09 Congenital
scoliosis 44-65 rTlL 2005-03- 13.0 -- 1490.59 NA 127.74 .+-. 9.29
29 1278 1998-07-22 F 6.8 2005-05-30 Congenital neurological 60 lT
2005-05- 6.8 -- 1401.88 NA 75.65 .+-. 5.16 scoliosis 30 1281
1985-05-21 M 20.1 2005-06-01 Spondylolisthesis 16 -- 2005-06- 20.1
-- 985.85 NA 150.30 .+-. 7.93 01 1286 1990-05-08 M 15.1 2005-06-15
Dysplasic scoliosis 62-66 rTlL 2005-06- 15.1 -- 549.60 .+-. 5.06 NA
NA 15 1356 1993-02-22 F 13.2 2006-04-03 Congenital scoliosis 75 rT
2006-04- 13.2 -- 1181.85 NA 111.51 .+-. 2.30 03 1358 2003-11-09 M
2.4 2006-04-04 Congenital scoliosis 33-35 rTlL 2006-04- 2.4 --
1530.6 NA 284.60 .+-. 69.00 04 1367 1993-12-12 F 12.4 2006-02-01
Neurological scoliosis 90 lTL 2006-05- 12.4 -- 1525.13 NA 350.01
.+-. 36.55 01 1368 1990-06-21 F 15.9 2006-05-02 Neurological
cyphosis 50 lTL 2006-05- 15.9 -- 1079.23 NA 126.44 .+-. 3.63 02
1370 1995-09-15 M 10.7 2006-05-09 Neurological scoliosis 65 rT
2006-05- 10.7 -- 1318.58 NA 104.06 .+-. 5.18 09 1375 1992-09-13 F
13.7 2006-05-30 Congenital scoliosis 53 rTL 2006-05- 13.7 Cousin
380.08 .+-. 12.95 NA NA 30 1407 1990-12-22 M 16.8 2007-10-31
Spondylolisthesis 9 lL 2006-09- 15.8 -- 818.17 .+-. 1.52 441.73
116.09 .+-. 3.88 25 1431 1987-11-23 M 19.2 2007-01-08 Neurological
scoliosis 90-90 rTlT 2007-01- 19.2 -- 450.78 .+-. 101.56 275.62
130.30 .+-. 23.92 08 1432 1992-08-08 M 14.4 2007-01-09 Neurological
scoliosis 64 rT 2007-01- 14.4 -- 558.47 .+-. 4.70 145.15 98.99 .+-.
13.92 09 1434 1994-08-07 F 12.4 2007-01-10 Congenital scoliosis
79-77 rTlL 2007-01- 12.4 -- 631.59 .+-. 7.42 325.95 44.79 .+-. 5.73
10 1436 1993-02-16 F 13.9 2007-01-22 Cyphose scoliosis 120 --
2007-01- 13.9 -- 220.32 .+-. 2.94 322.03 44.34 .+-. 6.37 22 1437
1992-11-06 M 14.2 2007-02-05 Neurological scoliosis 100 NA 2007-02-
14.2 -- 388.01 .+-. 8.22 225.71 76.96 .+-. 4.53 05 1455 1996-12-14
F 10.3 2007-04-03 Congenital cyphose scoliosis 61 lTL 2007-04- 10.3
-- 1090.51 .+-. 5.57 323.24 34.79 .+-. 0.32 03 1456 1990-10-03 F
16.5 2007-04-17 Neuromuscular scoliosis 87 rTL 2007-04- 16.5 --
622.46 .+-. 7.15 240.22 NA 17 1462 1997-10-22 F 9.5 2007-04-23
Neurological scoliosis 76 lTL 2007-04- 9.5 -- 1118.25 .+-. 1.32
607.1 55.90 .+-. 1.82 23 1463 1989-03-19 F 18.1 2007-04-24
Scoliosis + Spondylolisthesis 33 rT 2007-04- 18.1 -- 751.54 .+-.
8.69 284.71 21.56 .+-. 4.58 24 1466 1997-08-24 F 9.8 2007-05-08
Congenital scoliosis 39 rL 2007-05- 9.8 -- 1110.01 .+-. 2.38 510.18
47.07 .+-. 1.48 08 1475 1993-05-25 M 14.1 2007-06-05 Cyphose
scoliosis 98 -- 2007-06- 14.1 -- 1123.49 .+-. 5.56 319.93 166.63
.+-. 34.63 04 1479 1996-01-24 F 11.4 2007-06-05 Neurological
scoliosis 90 rTlL 2007-06- 11.4 -- 1098.54 .+-. 131.44 119.17 NA 05
1480 2003-06-13 F 4.0 2007-06-18 Congenital scoliosis 56 lT
2007-06- 4.0 -- 809.8 468.03 120.72 .+-. 40.73 18 1482 1989-03-30 F
18.2 2007-06-19 Spondylolisthesis gr 1 -- NA 2007-06- 18.2 --
678.49 .+-. 18.32 187.48 46.07 .+-. 5.27 19 1486 1993-01-15 M 14.4
2007-06-27 Spondylolisthesis gr 2 -- NA 2007-06- 14.4 -- 924.40
.+-. 17.16 628.78 47.06 .+-. 6.84 27 357 1996-07-08 F 11.4
2007-12-18 Congenital scoliosis 30-31 rTlT -- -- -- 996.58 .+-.
8.51 423.72 127.33 .+-. 3.13 * Plus-minus values are means .+-.
standard deviations. .dagger. Curve type nomenclature: r, right/l,
left/T, Thoracic/L, Lumbar/TL, Thoracolumbar/C, Cervical
Example 12
OPN and sCD44 Levels in AIS Patients Pre and Post Operations
[0166] OPN levels were measured in AIS patients pre (n=79) and post
(N=28) operations. Interestingly, comparison of AIS patients in
pre-operation vs. post operation showed a reduction in circulating
OPN levels, which further support the role of OPN at the cellular
level as mechanosensor (FIG. 13).
[0167] OPN were measured in AIS female patients pre (n=10) and post
(N=10) treatment with braces. Similarly, sCD44 levels were measured
in AIS female patients pre (n=15) and post (N=12) operations.
Results are presented in FIG. 14.
[0168] A distribution of 12 AIS patients was also performed across
the predefined cut-off zones pre-operation and post-operation. FIG.
15 shows 92% of the surgically treated patients had pre-operation
OPN levels in the red-zone (>800 ng/mL of plasma OPN level),
while the remaining 8% were in the yellow zone (700-800 ng/mL). No
patients were in the green zone representing plasma OPN levels
<700 ng/mL. This also shows a strong correlation between high
OPN concentrations and the progression of scoliotic curves.
[0169] Panel B of FIG. 15 show that red zone patients who were
treated surgically experienced a decline in OPN concentrations in
the blood. 75% of the surgically treated patients fell into the
green and yellow zones (800 ng/mL or less).
Example 13
OPN Levels in AIS Patients with Various Types of Braces
[0170] OPN levels were also measured in AIS patients prior to being
treated with brace (n=79) and after brace (N=28). Table 11 below
also shows the effect of braces on biomarkers.
TABLE-US-00011 TABLE 11 Possible effects of brace treatment on
biomarker concentrations. Characteristics Mean Mean Brace Mean Mean
OPN Mean sCD44 Mean HA Age Wear Cobb's Concentration Concentration
Concentration Treatment No. (Years) (Months) Angle (ng/ml) (ng/ml)
(ng/ml) Without Brace Female 193 14.2 .+-. 2.1 -- 30.9 .+-. 19.3
809 .+-. 376 474 .+-. 179 108 .+-. 58 Male 36 14.8 .+-. 2.2 -- 32.2
.+-. 21.1 1034 .+-. 376 492 .+-. 155 126 .+-. 62 With Brace (All
Female) All Braces 21 14.0 .+-. 1.8 12.0 21.2 .+-. 8.3 664 .+-. 282
483 .+-. 112 118 .+-. 60 Combined Boston 5 13.0 .+-. 1.4 10.6 25.8
.+-. 4.4 735 .+-. 358 568 .+-. 184 150 .+-. 57 SpineCor 14 14.5
.+-. 1.6 12.7 20.6 .+-. 8.7 626 .+-. 279 451 .+-. 81 108 .+-. 62
Charleston 1 15.4 10.0 7.0 781 532 70 Providence 1 9.7 1.0 20.0 732
547 138 Night Brace P-value.dagger-dbl. 0.018 0.879 0.608 *
Plus-minus values are means .+-. standard deviations. .dagger-dbl.
Statistical analysis to compare patients with or without brace was
done by bilateral unpaired Student's T-test with equal variance. A
difference was considered statistically significant with a p-value
<0.05.
[0171] A distribution of AIS patients across the predefined cut-off
zones was also performed prior to being treated with bracing and
after bracing. Eight patients were tested a certain number of
months after bracing, namely for each of patients #1 to 8: 7, 7, 8,
22, 22, 22 and 26 months after bracing, respectively. FIG. 16 shows
that prior to being treated with bracing (Panel A), 63% of these
patients were in the red and yellow zones. A significant shift
towards the green zone (<700 ng/mL) was observed, which is
consistent with the trend observed in surgically treated patients,
as presented in FIGS. 13-15.
Example 14
Comparison of Selenium Levels in AIS Patients Vs. Healthy
Subjects
[0172] Selenium concentration was reported to be significantly
decreased in plasma of AIS patients (42). Selenium and more
specifically Se-methylselenocystein, an organoselenium naturally
occurring in diet, are used to prevent metastasis in breast cancer
as chemopreventive therapy by targeting OPN transcription
(43-45).
[0173] Plasma selenium concentration was thus measured in pediatric
populations (AIS vs. healthy controls) to determine whether or not
low selenium levels correlate with higher OPN concentrations in
AIS. Plasma selenium concentrations were determined by a
fluorometric method using 2,3-diaminonaphthalene (DAN) (46, 47).
Results presented in FIGS. 18 and 19 show a correlation between
high OPN levels and low selenium levels in scoliotic and
asymptomatic at risk children.
[0174] Although the present invention has been described
hereinabove by way of specific embodiments thereof, it can be
modified, without departing from the spirit and nature of the
subject invention as defined in the appended claims.
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Sequence CWU 1
1
2311641DNAHomo sapiens 1ctccctgtgt tggtggagga tgtctgcagc agcatttaaa
ttctgggagg gcttggttgt 60cagcagcagc aggaggaggc agagcacagc atcgtcggga
ccagactcgt ctcaggccag 120ttgcagcctt ctcagccaaa cgccgaccaa
ggaaaactca ctaccatgag aattgcagtg 180atttgctttt gcctcctagg
catcacctgt gccataccag ttaaacaggc tgattctgga 240agttctgagg
aaaagcagct ttacaacaaa tacccagatg ctgtggccac atggctaaac
300cctgacccat ctcagaagca gaatctccta gccccacaga atgctgtgtc
ctctgaagaa 360accaatgact ttaaacaaga gacccttcca agtaagtcca
acgaaagcca tgaccacatg 420gatgatatgg atgatgaaga tgatgatgac
catgtggaca gccaggactc cattgactcg 480aacgactctg atgatgtaga
tgacactgat gattctcacc agtctgatga gtctcaccat 540tctgatgaat
ctgatgaact ggtcactgat tttcccacgg acctgccagc aaccgaagtt
600ttcactccag ttgtccccac agtagacaca tatgatggcc gaggtgatag
tgtggtttat 660ggactgaggt caaaatctaa gaagtttcgc agacctgaca
tccagtaccc tgatgctaca 720gacgaggaca tcacctcaca catggaaagc
gaggagttga atggtgcata caaggccatc 780cccgttgccc aggacctgaa
cgcgccttct gattgggaca gccgtgggaa ggacagttat 840gaaacgagtc
agctggatga ccagagtgct gaaacccaca gccacaagca gtccagatta
900tataagcgga aagccaatga tgagagcaat gagcattccg atgtgattga
tagtcaggaa 960ctttccaaag tcagccgtga attccacagc catgaatttc
acagccatga agatatgctg 1020gttgtagacc ccaaaagtaa ggaagaagat
aaacacctga aatttcgtat ttctcatgaa 1080ttagatagtg catcttctga
ggtcaattaa aaggagaaaa aatacaattt ctcactttgc 1140atttagtcaa
aagaaaaaat gctttatagc aaaatgaaag agaacatgaa atgcttcttt
1200ctcagtttat tggttgaatg tgtatctatt tgagtctgga aataactaat
gtgtttgata 1260attagtttag tttgtggctt catggaaact ccctgtaaac
taaaagcttc agggttatgt 1320ctatgttcat tctatagaag aaatgcaaac
tatcactgta ttttaatatt tgttattctc 1380tcatgaatag aaatttatgt
agaagcaaac aaaatacttt tacccactta aaaagagaat 1440ataacatttt
atgtcactat aatcttttgt tttttaagtt agtgtatatt ttgttgtgat
1500tatctttttg tggtgtgaat aaatctttta tcttgaatgt aataagaatt
tggtggtgtc 1560aattgcttat ttgttttccc acggttgtcc agcaattaat
aaaacataac cttttttact 1620gcctaaaaaa aaaaaaaaaa a 164121616DNAHomo
sapiens 2ctccctgtgt tggtggagga tgtctgcagc agcatttaaa ttctgggagg
gcttggttgt 60cagcagcagc aggaggaggc agagcacagc atcgtcggga ccagactcgt
ctcaggccag 120ttgcagcctt ctcagccaaa cgccgaccaa ggaaaactca
ctaccatgag aattgcagtg 180atttgctttt gcctcctagg catcacctgt
gccataccag ttaaacaggc tgattctgga 240agttctgagg aaaagcagct
ttacaacaaa tacccagatg ctgtggccac atggctaaac 300cctgacccat
ctcagaagca gaatctccta gccccacaga cccttccaag taagtccaac
360gaaagccatg accacatgga tgatatggat gatgaagatg atgatgacca
tgtggacagc 420caggactcca ttgactcgaa cgactctgat gatgtagatg
acactgatga ttctcaccag 480tctgatgagt ctcaccattc tgatgaatct
gatgaactgg tcactgattt tcccacggac 540ctgccagcaa ccgaagtttt
cactccagtt gtccccacag tagacacata tgatggccga 600ggtgatagtg
tggtttatgg actgaggtca aaatctaaga agtttcgcag acctgacatc
660cagtaccctg atgctacaga cgaggacatc acctcacaca tggaaagcga
ggagttgaat 720ggtgcataca aggccatccc cgttgcccag gacctgaacg
cgccttctga ttgggacagc 780cgtgggaagg acagttatga aacgagtcag
ctggatgacc agagtgctga aacccacagc 840cacaagcagt ccagattata
taagcggaaa gccaatgatg agagcaatga gcattccgat 900gtgattgata
gtcaggaact ttccaaagtc agccgtgaat tccacagcca tgaatttcac
960agccatgaag atatgctggt tgtagacccc aaaagtaagg aagaagataa
acacctgaaa 1020tttcgtattt ctcatgaatt agatagtgca tcttctgagg
tcaattaaaa ggagaaaaaa 1080tacaatttct cactttgcat ttagtcaaaa
gaaaaaatgc tttatagcaa aatgaaagag 1140aacatgaaat gcttctttct
cagtttattg gttgaatgtg tatctatttg agtctggaaa 1200taactaatgt
gtttgataat tagtttagtt tgtggcttca tggaaactcc ctgtaaacta
1260aaagcttcag ggttatgtct atgttcattc tatagaagaa atgcaaacta
tcactgtatt 1320ttaatatttg ttattctctc atgaatagaa atttatgtag
aagcaaacaa aatactttta 1380cccacttaaa aagagaatat aacattttat
gtcactataa tcttttgttt tttaagttag 1440tgtatatttt gttgtgatta
tctttttgtg gtgtgaataa atcttttatc ttgaatgtaa 1500taagaatttg
gtggtgtcaa ttgcttattt gttttcccac ggttgtccag caattaataa
1560aacataacct tttttactgc ctaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa
161631560DNAHomo sapiens 3ctccctgtgt tggtggagga tgtctgcagc
agcatttaaa ttctgggagg gcttggttgt 60cagcagcagc aggaggaggc agagcacagc
atcgtcggga ccagactcgt ctcaggccag 120ttgcagcctt ctcagccaaa
cgccgaccaa ggaaaactca ctaccatgag aattgcagtg 180atttgctttt
gcctcctagg catcacctgt gccataccag ttaaacaggc tgattctgga
240agttctgagg aaaagcagaa tgctgtgtcc tctgaagaaa ccaatgactt
taaacaagag 300acccttccaa gtaagtccaa cgaaagccat gaccacatgg
atgatatgga tgatgaagat 360gatgatgacc atgtggacag ccaggactcc
attgactcga acgactctga tgatgtagat 420gacactgatg attctcacca
gtctgatgag tctcaccatt ctgatgaatc tgatgaactg 480gtcactgatt
ttcccacgga cctgccagca accgaagttt tcactccagt tgtccccaca
540gtagacacat atgatggccg aggtgatagt gtggtttatg gactgaggtc
aaaatctaag 600aagtttcgca gacctgacat ccagtaccct gatgctacag
acgaggacat cacctcacac 660atggaaagcg aggagttgaa tggtgcatac
aaggccatcc ccgttgccca ggacctgaac 720gcgccttctg attgggacag
ccgtgggaag gacagttatg aaacgagtca gctggatgac 780cagagtgctg
aaacccacag ccacaagcag tccagattat ataagcggaa agccaatgat
840gagagcaatg agcattccga tgtgattgat agtcaggaac tttccaaagt
cagccgtgaa 900ttccacagcc atgaatttca cagccatgaa gatatgctgg
ttgtagaccc caaaagtaag 960gaagaagata aacacctgaa atttcgtatt
tctcatgaat tagatagtgc atcttctgag 1020gtcaattaaa aggagaaaaa
atacaatttc tcactttgca tttagtcaaa agaaaaaatg 1080ctttatagca
aaatgaaaga gaacatgaaa tgcttctttc tcagtttatt ggttgaatgt
1140gtatctattt gagtctggaa ataactaatg tgtttgataa ttagtttagt
ttgtggcttc 1200atggaaactc cctgtaaact aaaagcttca gggttatgtc
tatgttcatt ctatagaaga 1260aatgcaaact atcactgtat tttaatattt
gttattctct catgaataga aatttatgta 1320gaagcaaaca aaatactttt
acccacttaa aaagagaata taacatttta tgtcactata 1380atcttttgtt
ttttaagtta gtgtatattt tgttgtgatt atctttttgt ggtgtgaata
1440aatcttttat cttgaatgta ataagaattt ggtggtgtca attgcttatt
tgttttccca 1500cggttgtcca gcaattaata aaacataacc ttttttactg
cctaaaaaaa aaaaaaaaaa 15604314PRTHomo sapiens 4Met Arg Ile Ala Val
Ile Cys Phe Cys Leu Leu Gly Ile Thr Cys Ala 1 5 10 15 Ile Pro Val
Lys Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys Gln Leu 20 25 30 Tyr
Asn Lys Tyr Pro Asp Ala Val Ala Thr Trp Leu Asn Pro Asp Pro 35 40
45 Ser Gln Lys Gln Asn Leu Leu Ala Pro Gln Asn Ala Val Ser Ser Glu
50 55 60 Glu Thr Asn Asp Phe Lys Gln Glu Thr Leu Pro Ser Lys Ser
Asn Glu 65 70 75 80 Ser His Asp His Met Asp Asp Met Asp Asp Glu Asp
Asp Asp Asp His 85 90 95 Val Asp Ser Gln Asp Ser Ile Asp Ser Asn
Asp Ser Asp Asp Val Asp 100 105 110 Asp Thr Asp Asp Ser His Gln Ser
Asp Glu Ser His His Ser Asp Glu 115 120 125 Ser Asp Glu Leu Val Thr
Asp Phe Pro Thr Asp Leu Pro Ala Thr Glu 130 135 140 Val Phe Thr Pro
Val Val Pro Thr Val Asp Thr Tyr Asp Gly Arg Gly 145 150 155 160 Asp
Ser Val Val Tyr Gly Leu Arg Ser Lys Ser Lys Lys Phe Arg Arg 165 170
175 Pro Asp Ile Gln Tyr Pro Asp Ala Thr Asp Glu Asp Ile Thr Ser His
180 185 190 Met Glu Ser Glu Glu Leu Asn Gly Ala Tyr Lys Ala Ile Pro
Val Ala 195 200 205 Gln Asp Leu Asn Ala Pro Ser Asp Trp Asp Ser Arg
Gly Lys Asp Ser 210 215 220 Tyr Glu Thr Ser Gln Leu Asp Asp Gln Ser
Ala Glu Thr His Ser His 225 230 235 240 Lys Gln Ser Arg Leu Tyr Lys
Arg Lys Ala Asn Asp Glu Ser Asn Glu 245 250 255 His Ser Asp Val Ile
Asp Ser Gln Glu Leu Ser Lys Val Ser Arg Glu 260 265 270 Phe His Ser
His Glu Phe His Ser His Glu Asp Met Leu Val Val Asp 275 280 285 Pro
Lys Ser Lys Glu Glu Asp Lys His Leu Lys Phe Arg Ile Ser His 290 295
300 Glu Leu Asp Ser Ala Ser Ser Glu Val Asn 305 310 5300PRTHomo
sapiens 5Met Arg Ile Ala Val Ile Cys Phe Cys Leu Leu Gly Ile Thr
Cys Ala 1 5 10 15 Ile Pro Val Lys Gln Ala Asp Ser Gly Ser Ser Glu
Glu Lys Gln Leu 20 25 30 Tyr Asn Lys Tyr Pro Asp Ala Val Ala Thr
Trp Leu Asn Pro Asp Pro 35 40 45 Ser Gln Lys Gln Asn Leu Leu Ala
Pro Gln Thr Leu Pro Ser Lys Ser 50 55 60 Asn Glu Ser His Asp His
Met Asp Asp Met Asp Asp Glu Asp Asp Asp 65 70 75 80 Asp His Val Asp
Ser Gln Asp Ser Ile Asp Ser Asn Asp Ser Asp Asp 85 90 95 Val Asp
Asp Thr Asp Asp Ser His Gln Ser Asp Glu Ser His His Ser 100 105 110
Asp Glu Ser Asp Glu Leu Val Thr Asp Phe Pro Thr Asp Leu Pro Ala 115
120 125 Thr Glu Val Phe Thr Pro Val Val Pro Thr Val Asp Thr Tyr Asp
Gly 130 135 140 Arg Gly Asp Ser Val Val Tyr Gly Leu Arg Ser Lys Ser
Lys Lys Phe 145 150 155 160 Arg Arg Pro Asp Ile Gln Tyr Pro Asp Ala
Thr Asp Glu Asp Ile Thr 165 170 175 Ser His Met Glu Ser Glu Glu Leu
Asn Gly Ala Tyr Lys Ala Ile Pro 180 185 190 Val Ala Gln Asp Leu Asn
Ala Pro Ser Asp Trp Asp Ser Arg Gly Lys 195 200 205 Asp Ser Tyr Glu
Thr Ser Gln Leu Asp Asp Gln Ser Ala Glu Thr His 210 215 220 Ser His
Lys Gln Ser Arg Leu Tyr Lys Arg Lys Ala Asn Asp Glu Ser 225 230 235
240 Asn Glu His Ser Asp Val Ile Asp Ser Gln Glu Leu Ser Lys Val Ser
245 250 255 Arg Glu Phe His Ser His Glu Phe His Ser His Glu Asp Met
Leu Val 260 265 270 Val Asp Pro Lys Ser Lys Glu Glu Asp Lys His Leu
Lys Phe Arg Ile 275 280 285 Ser His Glu Leu Asp Ser Ala Ser Ser Glu
Val Asn 290 295 300 6287PRTHomo sapiens 6Met Arg Ile Ala Val Ile
Cys Phe Cys Leu Leu Gly Ile Thr Cys Ala 1 5 10 15 Ile Pro Val Lys
Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys Gln Asn 20 25 30 Ala Val
Ser Ser Glu Glu Thr Asn Asp Phe Lys Gln Glu Thr Leu Pro 35 40 45
Ser Lys Ser Asn Glu Ser His Asp His Met Asp Asp Met Asp Asp Glu 50
55 60 Asp Asp Asp Asp His Val Asp Ser Gln Asp Ser Ile Asp Ser Asn
Asp 65 70 75 80 Ser Asp Asp Val Asp Asp Thr Asp Asp Ser His Gln Ser
Asp Glu Ser 85 90 95 His His Ser Asp Glu Ser Asp Glu Leu Val Thr
Asp Phe Pro Thr Asp 100 105 110 Leu Pro Ala Thr Glu Val Phe Thr Pro
Val Val Pro Thr Val Asp Thr 115 120 125 Tyr Asp Gly Arg Gly Asp Ser
Val Val Tyr Gly Leu Arg Ser Lys Ser 130 135 140 Lys Lys Phe Arg Arg
Pro Asp Ile Gln Tyr Pro Asp Ala Thr Asp Glu 145 150 155 160 Asp Ile
Thr Ser His Met Glu Ser Glu Glu Leu Asn Gly Ala Tyr Lys 165 170 175
Ala Ile Pro Val Ala Gln Asp Leu Asn Ala Pro Ser Asp Trp Asp Ser 180
185 190 Arg Gly Lys Asp Ser Tyr Glu Thr Ser Gln Leu Asp Asp Gln Ser
Ala 195 200 205 Glu Thr His Ser His Lys Gln Ser Arg Leu Tyr Lys Arg
Lys Ala Asn 210 215 220 Asp Glu Ser Asn Glu His Ser Asp Val Ile Asp
Ser Gln Glu Leu Ser 225 230 235 240 Lys Val Ser Arg Glu Phe His Ser
His Glu Phe His Ser His Glu Asp 245 250 255 Met Leu Val Val Asp Pro
Lys Ser Lys Glu Glu Asp Lys His Leu Lys 260 265 270 Phe Arg Ile Ser
His Glu Leu Asp Ser Ala Ser Ser Glu Val Asn 275 280 285
75748DNAHomo sapiens 7gagaagaaag ccagtgcgtc tctgggcgca ggggccagtg
gggctcggag gcacaggcac 60cccgcgacac tccaggttcc ccgacccacg tccctggcag
ccccgattat ttacagcctc 120agcagagcac ggggcggggg cagaggggcc
cgcccgggag ggctgctact tcttaaaacc 180tctgcgggct gcttagtcac
agcccccctt gcttgggtgt gtccttcgct cgctccctcc 240ctccgtctta
ggtcactgtt ttcaacctcg aataaaaact gcagccaact tccgaggcag
300cctcattgcc cagcggaccc cagcctctgc caggttcggt ccgccatcct
cgtcccgtcc 360tccgccggcc cctgccccgc gcccagggat cctccagctc
ctttcgcccg cgccctccgt 420tcgctccgga caccatggac aagttttggt
ggcacgcagc ctggggactc tgcctcgtgc 480cgctgagcct ggcgcagatc
gatttgaata taacctgccg ctttgcaggt gtattccacg 540tggagaaaaa
tggtcgctac agcatctctc ggacggaggc cgctgacctc tgcaaggctt
600tcaatagcac cttgcccaca atggcccaga tggagaaagc tctgagcatc
ggatttgaga 660cctgcaggta tgggttcata gaagggcacg tggtgattcc
ccggatccac cccaactcca 720tctgtgcagc aaacaacaca ggggtgtaca
tcctcacatc caacacctcc cagtatgaca 780catattgctt caatgcttca
gctccacctg aagaagattg tacatcagtc acagacctgc 840ccaatgcctt
tgatggacca attaccataa ctattgttaa ccgtgatggc acccgctatg
900tccagaaagg agaatacaga acgaatcctg aagacatcta ccccagcaac
cctactgatg 960atgacgtgag cagcggctcc tccagtgaaa ggagcagcac
ttcaggaggt tacatctttt 1020acaccttttc tactgtacac cccatcccag
acgaagacag tccctggatc accgacagca 1080cagacagaat ccctgctacc
actttgatga gcactagtgc tacagcaact gagacagcaa 1140ccaagaggca
agaaacctgg gattggtttt catggttgtt tctaccatca gagtcaaaga
1200atcatcttca cacaacaaca caaatggctg gtacgtcttc aaataccatc
tcagcaggct 1260gggagccaaa tgaagaaaat gaagatgaaa gagacagaca
cctcagtttt tctggatcag 1320gcattgatga tgatgaagat tttatctcca
gcaccatttc aaccacacca cgggcttttg 1380accacacaaa acagaaccag
gactggaccc agtggaaccc aagccattca aatccggaag 1440tgctacttca
gacaaccaca aggatgactg atgtagacag aaatggcacc actgcttatg
1500aaggaaactg gaacccagaa gcacaccctc ccctcattca ccatgagcat
catgaggaag 1560aagagacccc acattctaca agcacaatcc aggcaactcc
tagtagtaca acggaagaaa 1620cagctaccca gaaggaacag tggtttggca
acagatggca tgagggatat cgccaaacac 1680ccaaagaaga ctcccattcg
acaacaggga cagctgcagc ctcagctcat accagccatc 1740caatgcaagg
aaggacaaca ccaagcccag aggacagttc ctggactgat ttcttcaacc
1800caatctcaca ccccatggga cgaggtcatc aagcaggaag aaggatggat
atggactcca 1860gtcatagtat aacgcttcag cctactgcaa atccaaacac
aggtttggtg gaagatttgg 1920acaggacagg acctctttca atgacaacgc
agcagagtaa ttctcagagc ttctctacat 1980cacatgaagg cttggaagaa
gataaagacc atccaacaac ttctactctg acatcaagca 2040ataggaatga
tgtcacaggt ggaagaagag acccaaatca ttctgaaggc tcaactactt
2100tactggaagg ttatacctct cattacccac acacgaagga aagcaggacc
ttcatcccag 2160tgacctcagc taagactggg tcctttggag ttactgcagt
tactgttgga gattccaact 2220ctaatgtcaa tcgttcctta tcaggagacc
aagacacatt ccaccccagt ggggggtccc 2280ataccactca tggatctgaa
tcagatggac actcacatgg gagtcaagaa ggtggagcaa 2340acacaacctc
tggtcctata aggacacccc aaattccaga atggctgatc atcttggcat
2400ccctcttggc cttggctttg attcttgcag tttgcattgc agtcaacagt
cgaagaaggt 2460gtgggcagaa gaaaaagcta gtgatcaaca gtggcaatgg
agctgtggag gacagaaagc 2520caagtggact caacggagag gccagcaagt
ctcaggaaat ggtgcatttg gtgaacaagg 2580agtcgtcaga aactccagac
cagtttatga cagctgatga gacaaggaac ctgcagaatg 2640tggacatgaa
gattggggtg taacacctac accattatct tggaaagaaa caaccgttgg
2700aaacataacc attacaggga gctgggacac ttaacagatg caatgtgcta
ctgattgttt 2760cattgcgaat cttttttagc ataaaatttt ctactctttt
tgttttttgt gttttgttct 2820ttaaagtcag gtccaatttg taaaaacagc
attgctttct gaaattaggg cccaattaat 2880aatcagcaag aatttgatcg
ttccagttcc cacttggagg cctttcatcc ctcgggtgtg 2940ctatggatgg
cttctaacaa aaactacaca tatgtattcc tgatcgccaa cctttccccc
3000accagctaag gacatttccc agggttaata gggcctggtc cctgggagga
aatttgaatg 3060ggtccatttt gcccttccat agcctaatcc ctgggcattg
ctttccactg aggttggggg 3120ttggggtgta ctagttacac atcttcaaca
gaccccctct agaaattttt cagatgcttc 3180tgggagacac ccaaagggtg
aagctattta tctgtagtaa actatttatc tgtgtttttg 3240aaatattaaa
ccctggatca gtcctttgat cagtataatt ttttaaagtt actttgtcag
3300aggcacaaaa gggtttaaac tgattcataa taaatatctg tacttcttcg
atcttcacct 3360tttgtgctgt gattcttcag tttctaaacc agcactgtct
gggtccctac aatgtatcag 3420gaagagctga gaatggtaag gagactcttc
taagtcttca tctcagagac cctgagttcc 3480cactcagacc cactcagcca
aatctcatgg aagaccaagg agggcagcac tgtttttgtt 3540ttttgttttt
tgtttttttt ttttgacact gtccaaaggt tttccatcct gtcctggaat
3600cagagttgga agctgaggag cttcagcctc ttttatggtt taatggccac
ctgttctctc 3660ctgtgaaagg ctttgcaaag tcacattaag tttgcatgac
ctgttatccc tggggcccta 3720tttcatagag gctggcccta ttagtgattt
ccaaaaacaa tatggaagtg ccttttgatg 3780tcttacaata agagaagaag
ccaatggaaa tgaaagagat tggcaaaggg gaaggatgat 3840gccatgtaga
tcctgtttga catttttatg gctgtatttg taaacttaaa cacaccagtg
3900tctgttcttg atgcagttgc tatttaggat gagttaagtg cctggggagt
ccctcaaaag 3960gttaaaggga ttcccatcat tggaatctta tcaccagata
ggcaagttta tgaccaaaca 4020agagagtact ggctttatcc tctaacctca
tattttctcc cacttggcaa gtcctttgtg 4080gcatttattc atcagtcagg
gtgtccgatt ggtcctagaa cttccaaagg ctgcttgtca 4140tagaagccat
tgcatctata aagcaacggc tcctgttaaa
tggtatctcc tttctgaggc 4200tcctactaaa agtcatttgt tacctaaact
tatgtgctta acaggcaatg cttctcagac 4260cacaaagcag aaagaagaag
aaaagctcct gactaaatca gggctgggct tagacagagt 4320tgatctgtag
aatatcttta aaggagagat gtcaactttc tgcactattc ccagcctctg
4380ctcctccctg tctaccctct cccctccctc tctccctcca cttcacccca
caatcttgaa 4440aaacttcctt tctcttctgt gaacatcatt ggccagatcc
attttcagtg gtctggattt 4500ctttttattt tcttttcaac ttgaaagaaa
ctggacatta ggccactatg tgttgttact 4560gccactagtg ttcaagtgcc
tcttgttttc ccagagattt cctgggtctg ccagaggccc 4620agacaggctc
actcaagctc tttaactgaa aagcaacaag ccactccagg acaaggttca
4680aaatggttac aacagcctct acctgtcgcc ccagggagaa aggggtagtg
atacaagtct 4740catagccaga gatggttttc cactccttct agatattccc
aaaaagaggc tgagacagga 4800ggttattttc aattttattt tggaattaaa
tacttttttc cctttattac tgttgtagtc 4860cctcacttgg atatacctct
gttttcacga tagaaataag ggaggtctag agcttctatt 4920ccttggccat
tgtcaacgga gagctggcca agtcttcaca aacccttgca acattgcctg
4980aagtttatgg aataagatgt attctcactc ccttgatctc aagggcgtaa
ctctggaagc 5040acagcttgac tacacgtcat ttttaccaat gattttcagg
tgacctgggc taagtcattt 5100aaactgggtc tttataaaag taaaaggcca
acatttaatt attttgcaaa gcaacctaag 5160agctaaagat gtaatttttc
ttgcaattgt aaatcttttg tgtctcctga agacttccct 5220taaaattagc
tctgagtgaa aaatcaaaag agacaaaaga catcttcgaa tccatatttc
5280aagcctggta gaattggctt ttctagcaga acctttccaa aagttttata
ttgagattca 5340taacaacacc aagaattgat tttgtagcca acattcattc
aatactgtta tatcagagga 5400gtaggagaga ggaaacattt gacttatctg
gaaaagcaaa atgtacttaa gaataagaat 5460aacatggtcc attcaccttt
atgttataga tatgtctttg tgtaaatcat ttgttttgag 5520ttttcaaaga
atagcccatt gttcattctt gtgctgtaca atgaccactg ttattgttac
5580tttgactttt cagagcacac ccttcctctg gtttttgtat atttattgat
ggatcaataa 5640taatgaggaa agcatgatat gtatattgct gagttgaaag
cacttattgg aaaatattaa 5700aaggctaaca ttaaaagact aaaggaaaca
gaaaaaaaaa aaaaaaaa 574885619DNAHomo sapiens 8gagaagaaag ccagtgcgtc
tctgggcgca ggggccagtg gggctcggag gcacaggcac 60cccgcgacac tccaggttcc
ccgacccacg tccctggcag ccccgattat ttacagcctc 120agcagagcac
ggggcggggg cagaggggcc cgcccgggag ggctgctact tcttaaaacc
180tctgcgggct gcttagtcac agcccccctt gcttgggtgt gtccttcgct
cgctccctcc 240ctccgtctta ggtcactgtt ttcaacctcg aataaaaact
gcagccaact tccgaggcag 300cctcattgcc cagcggaccc cagcctctgc
caggttcggt ccgccatcct cgtcccgtcc 360tccgccggcc cctgccccgc
gcccagggat cctccagctc ctttcgcccg cgccctccgt 420tcgctccgga
caccatggac aagttttggt ggcacgcagc ctggggactc tgcctcgtgc
480cgctgagcct ggcgcagatc gatttgaata taacctgccg ctttgcaggt
gtattccacg 540tggagaaaaa tggtcgctac agcatctctc ggacggaggc
cgctgacctc tgcaaggctt 600tcaatagcac cttgcccaca atggcccaga
tggagaaagc tctgagcatc ggatttgaga 660cctgcaggta tgggttcata
gaagggcacg tggtgattcc ccggatccac cccaactcca 720tctgtgcagc
aaacaacaca ggggtgtaca tcctcacatc caacacctcc cagtatgaca
780catattgctt caatgcttca gctccacctg aagaagattg tacatcagtc
acagacctgc 840ccaatgcctt tgatggacca attaccataa ctattgttaa
ccgtgatggc acccgctatg 900tccagaaagg agaatacaga acgaatcctg
aagacatcta ccccagcaac cctactgatg 960atgacgtgag cagcggctcc
tccagtgaaa ggagcagcac ttcaggaggt tacatctttt 1020acaccttttc
tactgtacac cccatcccag acgaagacag tccctggatc accgacagca
1080cagacagaat ccctgctacc agtacgtctt caaataccat ctcagcaggc
tgggagccaa 1140atgaagaaaa tgaagatgaa agagacagac acctcagttt
ttctggatca ggcattgatg 1200atgatgaaga ttttatctcc agcaccattt
caaccacacc acgggctttt gaccacacaa 1260aacagaacca ggactggacc
cagtggaacc caagccattc aaatccggaa gtgctacttc 1320agacaaccac
aaggatgact gatgtagaca gaaatggcac cactgcttat gaaggaaact
1380ggaacccaga agcacaccct cccctcattc accatgagca tcatgaggaa
gaagagaccc 1440cacattctac aagcacaatc caggcaactc ctagtagtac
aacggaagaa acagctaccc 1500agaaggaaca gtggtttggc aacagatggc
atgagggata tcgccaaaca cccaaagaag 1560actcccattc gacaacaggg
acagctgcag cctcagctca taccagccat ccaatgcaag 1620gaaggacaac
accaagccca gaggacagtt cctggactga tttcttcaac ccaatctcac
1680accccatggg acgaggtcat caagcaggaa gaaggatgga tatggactcc
agtcatagta 1740taacgcttca gcctactgca aatccaaaca caggtttggt
ggaagatttg gacaggacag 1800gacctctttc aatgacaacg cagcagagta
attctcagag cttctctaca tcacatgaag 1860gcttggaaga agataaagac
catccaacaa cttctactct gacatcaagc aataggaatg 1920atgtcacagg
tggaagaaga gacccaaatc attctgaagg ctcaactact ttactggaag
1980gttatacctc tcattaccca cacacgaagg aaagcaggac cttcatccca
gtgacctcag 2040ctaagactgg gtcctttgga gttactgcag ttactgttgg
agattccaac tctaatgtca 2100atcgttcctt atcaggagac caagacacat
tccaccccag tggggggtcc cataccactc 2160atggatctga atcagatgga
cactcacatg ggagtcaaga aggtggagca aacacaacct 2220ctggtcctat
aaggacaccc caaattccag aatggctgat catcttggca tccctcttgg
2280ccttggcttt gattcttgca gtttgcattg cagtcaacag tcgaagaagg
tgtgggcaga 2340agaaaaagct agtgatcaac agtggcaatg gagctgtgga
ggacagaaag ccaagtggac 2400tcaacggaga ggccagcaag tctcaggaaa
tggtgcattt ggtgaacaag gagtcgtcag 2460aaactccaga ccagtttatg
acagctgatg agacaaggaa cctgcagaat gtggacatga 2520agattggggt
gtaacaccta caccattatc ttggaaagaa acaaccgttg gaaacataac
2580cattacaggg agctgggaca cttaacagat gcaatgtgct actgattgtt
tcattgcgaa 2640tcttttttag cataaaattt tctactcttt ttgttttttg
tgttttgttc tttaaagtca 2700ggtccaattt gtaaaaacag cattgctttc
tgaaattagg gcccaattaa taatcagcaa 2760gaatttgatc gttccagttc
ccacttggag gcctttcatc cctcgggtgt gctatggatg 2820gcttctaaca
aaaactacac atatgtattc ctgatcgcca acctttcccc caccagctaa
2880ggacatttcc cagggttaat agggcctggt ccctgggagg aaatttgaat
gggtccattt 2940tgcccttcca tagcctaatc cctgggcatt gctttccact
gaggttgggg gttggggtgt 3000actagttaca catcttcaac agaccccctc
tagaaatttt tcagatgctt ctgggagaca 3060cccaaagggt gaagctattt
atctgtagta aactatttat ctgtgttttt gaaatattaa 3120accctggatc
agtcctttga tcagtataat tttttaaagt tactttgtca gaggcacaaa
3180agggtttaaa ctgattcata ataaatatct gtacttcttc gatcttcacc
ttttgtgctg 3240tgattcttca gtttctaaac cagcactgtc tgggtcccta
caatgtatca ggaagagctg 3300agaatggtaa ggagactctt ctaagtcttc
atctcagaga ccctgagttc ccactcagac 3360ccactcagcc aaatctcatg
gaagaccaag gagggcagca ctgtttttgt tttttgtttt 3420ttgttttttt
tttttgacac tgtccaaagg ttttccatcc tgtcctggaa tcagagttgg
3480aagctgagga gcttcagcct cttttatggt ttaatggcca cctgttctct
cctgtgaaag 3540gctttgcaaa gtcacattaa gtttgcatga cctgttatcc
ctggggccct atttcataga 3600ggctggccct attagtgatt tccaaaaaca
atatggaagt gccttttgat gtcttacaat 3660aagagaagaa gccaatggaa
atgaaagaga ttggcaaagg ggaaggatga tgccatgtag 3720atcctgtttg
acatttttat ggctgtattt gtaaacttaa acacaccagt gtctgttctt
3780gatgcagttg ctatttagga tgagttaagt gcctggggag tccctcaaaa
ggttaaaggg 3840attcccatca ttggaatctt atcaccagat aggcaagttt
atgaccaaac aagagagtac 3900tggctttatc ctctaacctc atattttctc
ccacttggca agtcctttgt ggcatttatt 3960catcagtcag ggtgtccgat
tggtcctaga acttccaaag gctgcttgtc atagaagcca 4020ttgcatctat
aaagcaacgg ctcctgttaa atggtatctc ctttctgagg ctcctactaa
4080aagtcatttg ttacctaaac ttatgtgctt aacaggcaat gcttctcaga
ccacaaagca 4140gaaagaagaa gaaaagctcc tgactaaatc agggctgggc
ttagacagag ttgatctgta 4200gaatatcttt aaaggagaga tgtcaacttt
ctgcactatt cccagcctct gctcctccct 4260gtctaccctc tcccctccct
ctctccctcc acttcacccc acaatcttga aaaacttcct 4320ttctcttctg
tgaacatcat tggccagatc cattttcagt ggtctggatt tctttttatt
4380ttcttttcaa cttgaaagaa actggacatt aggccactat gtgttgttac
tgccactagt 4440gttcaagtgc ctcttgtttt cccagagatt tcctgggtct
gccagaggcc cagacaggct 4500cactcaagct ctttaactga aaagcaacaa
gccactccag gacaaggttc aaaatggtta 4560caacagcctc tacctgtcgc
cccagggaga aaggggtagt gatacaagtc tcatagccag 4620agatggtttt
ccactccttc tagatattcc caaaaagagg ctgagacagg aggttatttt
4680caattttatt ttggaattaa atactttttt ccctttatta ctgttgtagt
ccctcacttg 4740gatatacctc tgttttcacg atagaaataa gggaggtcta
gagcttctat tccttggcca 4800ttgtcaacgg agagctggcc aagtcttcac
aaacccttgc aacattgcct gaagtttatg 4860gaataagatg tattctcact
cccttgatct caagggcgta actctggaag cacagcttga 4920ctacacgtca
tttttaccaa tgattttcag gtgacctggg ctaagtcatt taaactgggt
4980ctttataaaa gtaaaaggcc aacatttaat tattttgcaa agcaacctaa
gagctaaaga 5040tgtaattttt cttgcaattg taaatctttt gtgtctcctg
aagacttccc ttaaaattag 5100ctctgagtga aaaatcaaaa gagacaaaag
acatcttcga atccatattt caagcctggt 5160agaattggct tttctagcag
aacctttcca aaagttttat attgagattc ataacaacac 5220caagaattga
ttttgtagcc aacattcatt caatactgtt atatcagagg agtaggagag
5280aggaaacatt tgacttatct ggaaaagcaa aatgtactta agaataagaa
taacatggtc 5340cattcacctt tatgttatag atatgtcttt gtgtaaatca
tttgttttga gttttcaaag 5400aatagcccat tgttcattct tgtgctgtac
aatgaccact gttattgtta ctttgacttt 5460tcagagcaca cccttcctct
ggtttttgta tatttattga tggatcaata ataatgagga 5520aagcatgata
tgtatattgc tgagttgaaa gcacttattg gaaaatatta aaaggctaac
5580attaaaagac taaaggaaac agaaaaaaaa aaaaaaaaa 561995001DNAHomo
sapiens 9gagaagaaag ccagtgcgtc tctgggcgca ggggccagtg gggctcggag
gcacaggcac 60cccgcgacac tccaggttcc ccgacccacg tccctggcag ccccgattat
ttacagcctc 120agcagagcac ggggcggggg cagaggggcc cgcccgggag
ggctgctact tcttaaaacc 180tctgcgggct gcttagtcac agcccccctt
gcttgggtgt gtccttcgct cgctccctcc 240ctccgtctta ggtcactgtt
ttcaacctcg aataaaaact gcagccaact tccgaggcag 300cctcattgcc
cagcggaccc cagcctctgc caggttcggt ccgccatcct cgtcccgtcc
360tccgccggcc cctgccccgc gcccagggat cctccagctc ctttcgcccg
cgccctccgt 420tcgctccgga caccatggac aagttttggt ggcacgcagc
ctggggactc tgcctcgtgc 480cgctgagcct ggcgcagatc gatttgaata
taacctgccg ctttgcaggt gtattccacg 540tggagaaaaa tggtcgctac
agcatctctc ggacggaggc cgctgacctc tgcaaggctt 600tcaatagcac
cttgcccaca atggcccaga tggagaaagc tctgagcatc ggatttgaga
660cctgcaggta tgggttcata gaagggcacg tggtgattcc ccggatccac
cccaactcca 720tctgtgcagc aaacaacaca ggggtgtaca tcctcacatc
caacacctcc cagtatgaca 780catattgctt caatgcttca gctccacctg
aagaagattg tacatcagtc acagacctgc 840ccaatgcctt tgatggacca
attaccataa ctattgttaa ccgtgatggc acccgctatg 900tccagaaagg
agaatacaga acgaatcctg aagacatcta ccccagcaac cctactgatg
960atgacgtgag cagcggctcc tccagtgaaa ggagcagcac ttcaggaggt
tacatctttt 1020acaccttttc tactgtacac cccatcccag acgaagacag
tccctggatc accgacagca 1080cagacagaat ccctgctacc aatatggact
ccagtcatag tataacgctt cagcctactg 1140caaatccaaa cacaggtttg
gtggaagatt tggacaggac aggacctctt tcaatgacaa 1200cgcagcagag
taattctcag agcttctcta catcacatga aggcttggaa gaagataaag
1260accatccaac aacttctact ctgacatcaa gcaataggaa tgatgtcaca
ggtggaagaa 1320gagacccaaa tcattctgaa ggctcaacta ctttactgga
aggttatacc tctcattacc 1380cacacacgaa ggaaagcagg accttcatcc
cagtgacctc agctaagact gggtcctttg 1440gagttactgc agttactgtt
ggagattcca actctaatgt caatcgttcc ttatcaggag 1500accaagacac
attccacccc agtggggggt cccataccac tcatggatct gaatcagatg
1560gacactcaca tgggagtcaa gaaggtggag caaacacaac ctctggtcct
ataaggacac 1620cccaaattcc agaatggctg atcatcttgg catccctctt
ggccttggct ttgattcttg 1680cagtttgcat tgcagtcaac agtcgaagaa
ggtgtgggca gaagaaaaag ctagtgatca 1740acagtggcaa tggagctgtg
gaggacagaa agccaagtgg actcaacgga gaggccagca 1800agtctcagga
aatggtgcat ttggtgaaca aggagtcgtc agaaactcca gaccagttta
1860tgacagctga tgagacaagg aacctgcaga atgtggacat gaagattggg
gtgtaacacc 1920tacaccatta tcttggaaag aaacaaccgt tggaaacata
accattacag ggagctggga 1980cacttaacag atgcaatgtg ctactgattg
tttcattgcg aatctttttt agcataaaat 2040tttctactct ttttgttttt
tgtgttttgt tctttaaagt caggtccaat ttgtaaaaac 2100agcattgctt
tctgaaatta gggcccaatt aataatcagc aagaatttga tcgttccagt
2160tcccacttgg aggcctttca tccctcgggt gtgctatgga tggcttctaa
caaaaactac 2220acatatgtat tcctgatcgc caacctttcc cccaccagct
aaggacattt cccagggtta 2280atagggcctg gtccctggga ggaaatttga
atgggtccat tttgcccttc catagcctaa 2340tccctgggca ttgctttcca
ctgaggttgg gggttggggt gtactagtta cacatcttca 2400acagaccccc
tctagaaatt tttcagatgc ttctgggaga cacccaaagg gtgaagctat
2460ttatctgtag taaactattt atctgtgttt ttgaaatatt aaaccctgga
tcagtccttt 2520gatcagtata attttttaaa gttactttgt cagaggcaca
aaagggttta aactgattca 2580taataaatat ctgtacttct tcgatcttca
ccttttgtgc tgtgattctt cagtttctaa 2640accagcactg tctgggtccc
tacaatgtat caggaagagc tgagaatggt aaggagactc 2700ttctaagtct
tcatctcaga gaccctgagt tcccactcag acccactcag ccaaatctca
2760tggaagacca aggagggcag cactgttttt gttttttgtt ttttgttttt
tttttttgac 2820actgtccaaa ggttttccat cctgtcctgg aatcagagtt
ggaagctgag gagcttcagc 2880ctcttttatg gtttaatggc cacctgttct
ctcctgtgaa aggctttgca aagtcacatt 2940aagtttgcat gacctgttat
ccctggggcc ctatttcata gaggctggcc ctattagtga 3000tttccaaaaa
caatatggaa gtgccttttg atgtcttaca ataagagaag aagccaatgg
3060aaatgaaaga gattggcaaa ggggaaggat gatgccatgt agatcctgtt
tgacattttt 3120atggctgtat ttgtaaactt aaacacacca gtgtctgttc
ttgatgcagt tgctatttag 3180gatgagttaa gtgcctgggg agtccctcaa
aaggttaaag ggattcccat cattggaatc 3240ttatcaccag ataggcaagt
ttatgaccaa acaagagagt actggcttta tcctctaacc 3300tcatattttc
tcccacttgg caagtccttt gtggcattta ttcatcagtc agggtgtccg
3360attggtccta gaacttccaa aggctgcttg tcatagaagc cattgcatct
ataaagcaac 3420ggctcctgtt aaatggtatc tcctttctga ggctcctact
aaaagtcatt tgttacctaa 3480acttatgtgc ttaacaggca atgcttctca
gaccacaaag cagaaagaag aagaaaagct 3540cctgactaaa tcagggctgg
gcttagacag agttgatctg tagaatatct ttaaaggaga 3600gatgtcaact
ttctgcacta ttcccagcct ctgctcctcc ctgtctaccc tctcccctcc
3660ctctctccct ccacttcacc ccacaatctt gaaaaacttc ctttctcttc
tgtgaacatc 3720attggccaga tccattttca gtggtctgga tttcttttta
ttttcttttc aacttgaaag 3780aaactggaca ttaggccact atgtgttgtt
actgccacta gtgttcaagt gcctcttgtt 3840ttcccagaga tttcctgggt
ctgccagagg cccagacagg ctcactcaag ctctttaact 3900gaaaagcaac
aagccactcc aggacaaggt tcaaaatggt tacaacagcc tctacctgtc
3960gccccaggga gaaaggggta gtgatacaag tctcatagcc agagatggtt
ttccactcct 4020tctagatatt cccaaaaaga ggctgagaca ggaggttatt
ttcaatttta ttttggaatt 4080aaatactttt ttccctttat tactgttgta
gtccctcact tggatatacc tctgttttca 4140cgatagaaat aagggaggtc
tagagcttct attccttggc cattgtcaac ggagagctgg 4200ccaagtcttc
acaaaccctt gcaacattgc ctgaagttta tggaataaga tgtattctca
4260ctcccttgat ctcaagggcg taactctgga agcacagctt gactacacgt
catttttacc 4320aatgattttc aggtgacctg ggctaagtca tttaaactgg
gtctttataa aagtaaaagg 4380ccaacattta attattttgc aaagcaacct
aagagctaaa gatgtaattt ttcttgcaat 4440tgtaaatctt ttgtgtctcc
tgaagacttc ccttaaaatt agctctgagt gaaaaatcaa 4500aagagacaaa
agacatcttc gaatccatat ttcaagcctg gtagaattgg cttttctagc
4560agaacctttc caaaagtttt atattgagat tcataacaac accaagaatt
gattttgtag 4620ccaacattca ttcaatactg ttatatcaga ggagtaggag
agaggaaaca tttgacttat 4680ctggaaaagc aaaatgtact taagaataag
aataacatgg tccattcacc tttatgttat 4740agatatgtct ttgtgtaaat
catttgtttt gagttttcaa agaatagccc attgttcatt 4800cttgtgctgt
acaatgacca ctgttattgt tactttgact tttcagagca cacccttcct
4860ctggtttttg tatatttatt gatggatcaa taataatgag gaaagcatga
tatgtatatt 4920gctgagttga aagcacttat tggaaaatat taaaaggcta
acattaaaag actaaaggaa 4980acagaaaaaa aaaaaaaaaa a 5001104605DNAHomo
sapiens 10gagaagaaag ccagtgcgtc tctgggcgca ggggccagtg gggctcggag
gcacaggcac 60cccgcgacac tccaggttcc ccgacccacg tccctggcag ccccgattat
ttacagcctc 120agcagagcac ggggcggggg cagaggggcc cgcccgggag
ggctgctact tcttaaaacc 180tctgcgggct gcttagtcac agcccccctt
gcttgggtgt gtccttcgct cgctccctcc 240ctccgtctta ggtcactgtt
ttcaacctcg aataaaaact gcagccaact tccgaggcag 300cctcattgcc
cagcggaccc cagcctctgc caggttcggt ccgccatcct cgtcccgtcc
360tccgccggcc cctgccccgc gcccagggat cctccagctc ctttcgcccg
cgccctccgt 420tcgctccgga caccatggac aagttttggt ggcacgcagc
ctggggactc tgcctcgtgc 480cgctgagcct ggcgcagatc gatttgaata
taacctgccg ctttgcaggt gtattccacg 540tggagaaaaa tggtcgctac
agcatctctc ggacggaggc cgctgacctc tgcaaggctt 600tcaatagcac
cttgcccaca atggcccaga tggagaaagc tctgagcatc ggatttgaga
660cctgcaggta tgggttcata gaagggcacg tggtgattcc ccggatccac
cccaactcca 720tctgtgcagc aaacaacaca ggggtgtaca tcctcacatc
caacacctcc cagtatgaca 780catattgctt caatgcttca gctccacctg
aagaagattg tacatcagtc acagacctgc 840ccaatgcctt tgatggacca
attaccataa ctattgttaa ccgtgatggc acccgctatg 900tccagaaagg
agaatacaga acgaatcctg aagacatcta ccccagcaac cctactgatg
960atgacgtgag cagcggctcc tccagtgaaa ggagcagcac ttcaggaggt
tacatctttt 1020acaccttttc tactgtacac cccatcccag acgaagacag
tccctggatc accgacagca 1080cagacagaat ccctgctacc agagaccaag
acacattcca ccccagtggg gggtcccata 1140ccactcatgg atctgaatca
gatggacact cacatgggag tcaagaaggt ggagcaaaca 1200caacctctgg
tcctataagg acaccccaaa ttccagaatg gctgatcatc ttggcatccc
1260tcttggcctt ggctttgatt cttgcagttt gcattgcagt caacagtcga
agaaggtgtg 1320ggcagaagaa aaagctagtg atcaacagtg gcaatggagc
tgtggaggac agaaagccaa 1380gtggactcaa cggagaggcc agcaagtctc
aggaaatggt gcatttggtg aacaaggagt 1440cgtcagaaac tccagaccag
tttatgacag ctgatgagac aaggaacctg cagaatgtgg 1500acatgaagat
tggggtgtaa cacctacacc attatcttgg aaagaaacaa ccgttggaaa
1560cataaccatt acagggagct gggacactta acagatgcaa tgtgctactg
attgtttcat 1620tgcgaatctt ttttagcata aaattttcta ctctttttgt
tttttgtgtt ttgttcttta 1680aagtcaggtc caatttgtaa aaacagcatt
gctttctgaa attagggccc aattaataat 1740cagcaagaat ttgatcgttc
cagttcccac ttggaggcct ttcatccctc gggtgtgcta 1800tggatggctt
ctaacaaaaa ctacacatat gtattcctga tcgccaacct ttcccccacc
1860agctaaggac atttcccagg gttaataggg cctggtccct gggaggaaat
ttgaatgggt 1920ccattttgcc cttccatagc ctaatccctg ggcattgctt
tccactgagg ttgggggttg 1980gggtgtacta gttacacatc ttcaacagac
cccctctaga aatttttcag atgcttctgg 2040gagacaccca aagggtgaag
ctatttatct gtagtaaact atttatctgt gtttttgaaa 2100tattaaaccc
tggatcagtc ctttgatcag tataattttt taaagttact ttgtcagagg
2160cacaaaaggg tttaaactga ttcataataa atatctgtac ttcttcgatc
ttcacctttt 2220gtgctgtgat tcttcagttt ctaaaccagc actgtctggg
tccctacaat gtatcaggaa 2280gagctgagaa tggtaaggag actcttctaa
gtcttcatct cagagaccct gagttcccac 2340tcagacccac tcagccaaat
ctcatggaag accaaggagg gcagcactgt ttttgttttt 2400tgttttttgt
tttttttttt tgacactgtc caaaggtttt ccatcctgtc ctggaatcag
2460agttggaagc tgaggagctt cagcctcttt tatggtttaa tggccacctg
ttctctcctg 2520tgaaaggctt tgcaaagtca cattaagttt gcatgacctg
ttatccctgg ggccctattt 2580catagaggct ggccctatta gtgatttcca
aaaacaatat ggaagtgcct tttgatgtct 2640tacaataaga gaagaagcca
atggaaatga aagagattgg caaaggggaa ggatgatgcc 2700atgtagatcc
tgtttgacat ttttatggct gtatttgtaa
acttaaacac accagtgtct 2760gttcttgatg cagttgctat ttaggatgag
ttaagtgcct ggggagtccc tcaaaaggtt 2820aaagggattc ccatcattgg
aatcttatca ccagataggc aagtttatga ccaaacaaga 2880gagtactggc
tttatcctct aacctcatat tttctcccac ttggcaagtc ctttgtggca
2940tttattcatc agtcagggtg tccgattggt cctagaactt ccaaaggctg
cttgtcatag 3000aagccattgc atctataaag caacggctcc tgttaaatgg
tatctccttt ctgaggctcc 3060tactaaaagt catttgttac ctaaacttat
gtgcttaaca ggcaatgctt ctcagaccac 3120aaagcagaaa gaagaagaaa
agctcctgac taaatcaggg ctgggcttag acagagttga 3180tctgtagaat
atctttaaag gagagatgtc aactttctgc actattccca gcctctgctc
3240ctccctgtct accctctccc ctccctctct ccctccactt caccccacaa
tcttgaaaaa 3300cttcctttct cttctgtgaa catcattggc cagatccatt
ttcagtggtc tggatttctt 3360tttattttct tttcaacttg aaagaaactg
gacattaggc cactatgtgt tgttactgcc 3420actagtgttc aagtgcctct
tgttttccca gagatttcct gggtctgcca gaggcccaga 3480caggctcact
caagctcttt aactgaaaag caacaagcca ctccaggaca aggttcaaaa
3540tggttacaac agcctctacc tgtcgcccca gggagaaagg ggtagtgata
caagtctcat 3600agccagagat ggttttccac tccttctaga tattcccaaa
aagaggctga gacaggaggt 3660tattttcaat tttattttgg aattaaatac
ttttttccct ttattactgt tgtagtccct 3720cacttggata tacctctgtt
ttcacgatag aaataaggga ggtctagagc ttctattcct 3780tggccattgt
caacggagag ctggccaagt cttcacaaac ccttgcaaca ttgcctgaag
3840tttatggaat aagatgtatt ctcactccct tgatctcaag ggcgtaactc
tggaagcaca 3900gcttgactac acgtcatttt taccaatgat tttcaggtga
cctgggctaa gtcatttaaa 3960ctgggtcttt ataaaagtaa aaggccaaca
tttaattatt ttgcaaagca acctaagagc 4020taaagatgta atttttcttg
caattgtaaa tcttttgtgt ctcctgaaga cttcccttaa 4080aattagctct
gagtgaaaaa tcaaaagaga caaaagacat cttcgaatcc atatttcaag
4140cctggtagaa ttggcttttc tagcagaacc tttccaaaag ttttatattg
agattcataa 4200caacaccaag aattgatttt gtagccaaca ttcattcaat
actgttatat cagaggagta 4260ggagagagga aacatttgac ttatctggaa
aagcaaaatg tacttaagaa taagaataac 4320atggtccatt cacctttatg
ttatagatat gtctttgtgt aaatcatttg ttttgagttt 4380tcaaagaata
gcccattgtt cattcttgtg ctgtacaatg accactgtta ttgttacttt
4440gacttttcag agcacaccct tcctctggtt tttgtatatt tattgatgga
tcaataataa 4500tgaggaaagc atgatatgta tattgctgag ttgaaagcac
ttattggaaa atattaaaag 4560gctaacatta aaagactaaa ggaaacagaa
aaaaaaaaaa aaaaa 4605113985DNAHomo sapiens 11gagaagaaag ccagtgcgtc
tctgggcgca ggggccagtg gggctcggag gcacaggcac 60cccgcgacac tccaggttcc
ccgacccacg tccctggcag ccccgattat ttacagcctc 120agcagagcac
ggggcggggg cagaggggcc cgcccgggag ggctgctact tcttaaaacc
180tctgcgggct gcttagtcac agcccccctt gcttgggtgt gtccttcgct
cgctccctcc 240ctccgtctta ggtcactgtt ttcaacctcg aataaaaact
gcagccaact tccgaggcag 300cctcattgcc cagcggaccc cagcctctgc
caggttcggt ccgccatcct cgtcccgtcc 360tccgccggcc cctgccccgc
gcccagggat cctccagctc ctttcgcccg cgccctccgt 420tcgctccgga
caccatggac aagttttggt ggcacgcagc ctggggactc tgcctcgtgc
480cgctgagcct ggcgcagatc gatttgaata taacctgccg ctttgcaggt
gtattccacg 540tggagaaaaa tggtcgctac agcatctctc ggacggaggc
cgctgacctc tgcaaggctt 600tcaatagcac cttgcccaca atggcccaga
tggagaaagc tctgagcatc ggatttgaga 660cctgcagttt gcattgcagt
caacagtcga agaaggtgtg ggcagaagaa aaagctagtg 720atcaacagtg
gcaatggagc tgtggaggac agaaagccaa gtggactcaa cggagaggcc
780agcaagtctc aggaaatggt gcatttggtg aacaaggagt cgtcagaaac
tccagaccag 840tttatgacag ctgatgagac aaggaacctg cagaatgtgg
acatgaagat tggggtgtaa 900cacctacacc attatcttgg aaagaaacaa
ccgttggaaa cataaccatt acagggagct 960gggacactta acagatgcaa
tgtgctactg attgtttcat tgcgaatctt ttttagcata 1020aaattttcta
ctctttttgt tttttgtgtt ttgttcttta aagtcaggtc caatttgtaa
1080aaacagcatt gctttctgaa attagggccc aattaataat cagcaagaat
ttgatcgttc 1140cagttcccac ttggaggcct ttcatccctc gggtgtgcta
tggatggctt ctaacaaaaa 1200ctacacatat gtattcctga tcgccaacct
ttcccccacc agctaaggac atttcccagg 1260gttaataggg cctggtccct
gggaggaaat ttgaatgggt ccattttgcc cttccatagc 1320ctaatccctg
ggcattgctt tccactgagg ttgggggttg gggtgtacta gttacacatc
1380ttcaacagac cccctctaga aatttttcag atgcttctgg gagacaccca
aagggtgaag 1440ctatttatct gtagtaaact atttatctgt gtttttgaaa
tattaaaccc tggatcagtc 1500ctttgatcag tataattttt taaagttact
ttgtcagagg cacaaaaggg tttaaactga 1560ttcataataa atatctgtac
ttcttcgatc ttcacctttt gtgctgtgat tcttcagttt 1620ctaaaccagc
actgtctggg tccctacaat gtatcaggaa gagctgagaa tggtaaggag
1680actcttctaa gtcttcatct cagagaccct gagttcccac tcagacccac
tcagccaaat 1740ctcatggaag accaaggagg gcagcactgt ttttgttttt
tgttttttgt tttttttttt 1800tgacactgtc caaaggtttt ccatcctgtc
ctggaatcag agttggaagc tgaggagctt 1860cagcctcttt tatggtttaa
tggccacctg ttctctcctg tgaaaggctt tgcaaagtca 1920cattaagttt
gcatgacctg ttatccctgg ggccctattt catagaggct ggccctatta
1980gtgatttcca aaaacaatat ggaagtgcct tttgatgtct tacaataaga
gaagaagcca 2040atggaaatga aagagattgg caaaggggaa ggatgatgcc
atgtagatcc tgtttgacat 2100ttttatggct gtatttgtaa acttaaacac
accagtgtct gttcttgatg cagttgctat 2160ttaggatgag ttaagtgcct
ggggagtccc tcaaaaggtt aaagggattc ccatcattgg 2220aatcttatca
ccagataggc aagtttatga ccaaacaaga gagtactggc tttatcctct
2280aacctcatat tttctcccac ttggcaagtc ctttgtggca tttattcatc
agtcagggtg 2340tccgattggt cctagaactt ccaaaggctg cttgtcatag
aagccattgc atctataaag 2400caacggctcc tgttaaatgg tatctccttt
ctgaggctcc tactaaaagt catttgttac 2460ctaaacttat gtgcttaaca
ggcaatgctt ctcagaccac aaagcagaaa gaagaagaaa 2520agctcctgac
taaatcaggg ctgggcttag acagagttga tctgtagaat atctttaaag
2580gagagatgtc aactttctgc actattccca gcctctgctc ctccctgtct
accctctccc 2640ctccctctct ccctccactt caccccacaa tcttgaaaaa
cttcctttct cttctgtgaa 2700catcattggc cagatccatt ttcagtggtc
tggatttctt tttattttct tttcaacttg 2760aaagaaactg gacattaggc
cactatgtgt tgttactgcc actagtgttc aagtgcctct 2820tgttttccca
gagatttcct gggtctgcca gaggcccaga caggctcact caagctcttt
2880aactgaaaag caacaagcca ctccaggaca aggttcaaaa tggttacaac
agcctctacc 2940tgtcgcccca gggagaaagg ggtagtgata caagtctcat
agccagagat ggttttccac 3000tccttctaga tattcccaaa aagaggctga
gacaggaggt tattttcaat tttattttgg 3060aattaaatac ttttttccct
ttattactgt tgtagtccct cacttggata tacctctgtt 3120ttcacgatag
aaataaggga ggtctagagc ttctattcct tggccattgt caacggagag
3180ctggccaagt cttcacaaac ccttgcaaca ttgcctgaag tttatggaat
aagatgtatt 3240ctcactccct tgatctcaag ggcgtaactc tggaagcaca
gcttgactac acgtcatttt 3300taccaatgat tttcaggtga cctgggctaa
gtcatttaaa ctgggtcttt ataaaagtaa 3360aaggccaaca tttaattatt
ttgcaaagca acctaagagc taaagatgta atttttcttg 3420caattgtaaa
tcttttgtgt ctcctgaaga cttcccttaa aattagctct gagtgaaaaa
3480tcaaaagaga caaaagacat cttcgaatcc atatttcaag cctggtagaa
ttggcttttc 3540tagcagaacc tttccaaaag ttttatattg agattcataa
caacaccaag aattgatttt 3600gtagccaaca ttcattcaat actgttatat
cagaggagta ggagagagga aacatttgac 3660ttatctggaa aagcaaaatg
tacttaagaa taagaataac atggtccatt cacctttatg 3720ttatagatat
gtctttgtgt aaatcatttg ttttgagttt tcaaagaata gcccattgtt
3780cattcttgtg ctgtacaatg accactgtta ttgttacttt gacttttcag
agcacaccct 3840tcctctggtt tttgtatatt tattgatgga tcaataataa
tgaggaaagc atgatatgta 3900tattgctgag ttgaaagcac ttattggaaa
atattaaaag gctaacatta aaagactaaa 3960ggaaacagaa aaaaaaaaaa aaaaa
3985121014DNAHomo sapiens 12gtacgtcttc aaataccatc tcagcaggct
gggagccaaa tgaagaaaat gaagatgaaa 60gagacagaca cctcagtttt tctggatcag
gcattgatga tgatgaagat tttatctcca 120gcaccatttc aaccacacca
cgggcctttg accacacaaa acagaaccag gactggaccc 180agtggaaccc
aagccattca aatccggaag tgctacttca gacaaccaca aggatgactg
240atgtagacag aaatggcacc actgcttatg aaggaaactg gaacccagaa
gcacaccctc 300ccctcattca ccatgagcat catgaggaag aagagacccc
acattctaca agcacaatcc 360aggcaactcc tagtagtaca acggaagaaa
cagctaccca gaaggaacag tggtttggca 420acagatggca tgagggatat
cgccaaacac ccagagaaga ctcccattcg acaacaggga 480cagctgcagc
ctcagctcat accagccatc caatgcaagg aaggacaaca ccaagcccag
540aggacagttc ctggactgat ttcttcaacc caatctcaca ccccatggga
cgaggtcatc 600aagcaggaag aaggatggat atggactcca gtcatagtac
aacgcttcag cctactgcaa 660atccaaacac aggtttggtg gaagatttgg
acaggacagg acctctttca atgacaacgc 720agcagagtaa ttctcagagc
ttctctacat cacatgaagg cttggaagaa gataaagacc 780atccaacaac
ttctactctg acatcaagca ataggaatga tgtcacaggt ggaagaagag
840acccaaatca ttctgaaggc tcaactactt tactggaagg ttatacctct
cattacccac 900acacgaagga aagcaggacc ttcatcccag tgacctcagc
taagactggg tcctttggag 960ttactgcagt tactgttgga gattccaact
ctaatgtcaa tcgttcctta tcag 101413742PRTHomo sapiens 13Met Asp Lys
Phe Trp Trp His Ala Ala Trp Gly Leu Cys Leu Val Pro 1 5 10 15 Leu
Ser Leu Ala Gln Ile Asp Leu Asn Ile Thr Cys Arg Phe Ala Gly 20 25
30 Val Phe His Val Glu Lys Asn Gly Arg Tyr Ser Ile Ser Arg Thr Glu
35 40 45 Ala Ala Asp Leu Cys Lys Ala Phe Asn Ser Thr Leu Pro Thr
Met Ala 50 55 60 Gln Met Glu Lys Ala Leu Ser Ile Gly Phe Glu Thr
Cys Arg Tyr Gly 65 70 75 80 Phe Ile Glu Gly His Val Val Ile Pro Arg
Ile His Pro Asn Ser Ile 85 90 95 Cys Ala Ala Asn Asn Thr Gly Val
Tyr Ile Leu Thr Ser Asn Thr Ser 100 105 110 Gln Tyr Asp Thr Tyr Cys
Phe Asn Ala Ser Ala Pro Pro Glu Glu Asp 115 120 125 Cys Thr Ser Val
Thr Asp Leu Pro Asn Ala Phe Asp Gly Pro Ile Thr 130 135 140 Ile Thr
Ile Val Asn Arg Asp Gly Thr Arg Tyr Val Gln Lys Gly Glu 145 150 155
160 Tyr Arg Thr Asn Pro Glu Asp Ile Tyr Pro Ser Asn Pro Thr Asp Asp
165 170 175 Asp Val Ser Ser Gly Ser Ser Ser Glu Arg Ser Ser Thr Ser
Gly Gly 180 185 190 Tyr Ile Phe Tyr Thr Phe Ser Thr Val His Pro Ile
Pro Asp Glu Asp 195 200 205 Ser Pro Trp Ile Thr Asp Ser Thr Asp Arg
Ile Pro Ala Thr Thr Leu 210 215 220 Met Ser Thr Ser Ala Thr Ala Thr
Glu Thr Ala Thr Lys Arg Gln Glu 225 230 235 240 Thr Trp Asp Trp Phe
Ser Trp Leu Phe Leu Pro Ser Glu Ser Lys Asn 245 250 255 His Leu His
Thr Thr Thr Gln Met Ala Gly Thr Ser Ser Asn Thr Ile 260 265 270 Ser
Ala Gly Trp Glu Pro Asn Glu Glu Asn Glu Asp Glu Arg Asp Arg 275 280
285 His Leu Ser Phe Ser Gly Ser Gly Ile Asp Asp Asp Glu Asp Phe Ile
290 295 300 Ser Ser Thr Ile Ser Thr Thr Pro Arg Ala Phe Asp His Thr
Lys Gln 305 310 315 320 Asn Gln Asp Trp Thr Gln Trp Asn Pro Ser His
Ser Asn Pro Glu Val 325 330 335 Leu Leu Gln Thr Thr Thr Arg Met Thr
Asp Val Asp Arg Asn Gly Thr 340 345 350 Thr Ala Tyr Glu Gly Asn Trp
Asn Pro Glu Ala His Pro Pro Leu Ile 355 360 365 His His Glu His His
Glu Glu Glu Glu Thr Pro His Ser Thr Ser Thr 370 375 380 Ile Gln Ala
Thr Pro Ser Ser Thr Thr Glu Glu Thr Ala Thr Gln Lys 385 390 395 400
Glu Gln Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gln Thr Pro 405
410 415 Lys Glu Asp Ser His Ser Thr Thr Gly Thr Ala Ala Ala Ser Ala
His 420 425 430 Thr Ser His Pro Met Gln Gly Arg Thr Thr Pro Ser Pro
Glu Asp Ser 435 440 445 Ser Trp Thr Asp Phe Phe Asn Pro Ile Ser His
Pro Met Gly Arg Gly 450 455 460 His Gln Ala Gly Arg Arg Met Asp Met
Asp Ser Ser His Ser Ile Thr 465 470 475 480 Leu Gln Pro Thr Ala Asn
Pro Asn Thr Gly Leu Val Glu Asp Leu Asp 485 490 495 Arg Thr Gly Pro
Leu Ser Met Thr Thr Gln Gln Ser Asn Ser Gln Ser 500 505 510 Phe Ser
Thr Ser His Glu Gly Leu Glu Glu Asp Lys Asp His Pro Thr 515 520 525
Thr Ser Thr Leu Thr Ser Ser Asn Arg Asn Asp Val Thr Gly Gly Arg 530
535 540 Arg Asp Pro Asn His Ser Glu Gly Ser Thr Thr Leu Leu Glu Gly
Tyr 545 550 555 560 Thr Ser His Tyr Pro His Thr Lys Glu Ser Arg Thr
Phe Ile Pro Val 565 570 575 Thr Ser Ala Lys Thr Gly Ser Phe Gly Val
Thr Ala Val Thr Val Gly 580 585 590 Asp Ser Asn Ser Asn Val Asn Arg
Ser Leu Ser Gly Asp Gln Asp Thr 595 600 605 Phe His Pro Ser Gly Gly
Ser His Thr Thr His Gly Ser Glu Ser Asp 610 615 620 Gly His Ser His
Gly Ser Gln Glu Gly Gly Ala Asn Thr Thr Ser Gly 625 630 635 640 Pro
Ile Arg Thr Pro Gln Ile Pro Glu Trp Leu Ile Ile Leu Ala Ser 645 650
655 Leu Leu Ala Leu Ala Leu Ile Leu Ala Val Cys Ile Ala Val Asn Ser
660 665 670 Arg Arg Arg Cys Gly Gln Lys Lys Lys Leu Val Ile Asn Ser
Gly Asn 675 680 685 Gly Ala Val Glu Asp Arg Lys Pro Ser Gly Leu Asn
Gly Glu Ala Ser 690 695 700 Lys Ser Gln Glu Met Val His Leu Val Asn
Lys Glu Ser Ser Glu Thr 705 710 715 720 Pro Asp Gln Phe Met Thr Ala
Asp Glu Thr Arg Asn Leu Gln Asn Val 725 730 735 Asp Met Lys Ile Gly
Val 740 14699PRTHomo sapiens 14Met Asp Lys Phe Trp Trp His Ala Ala
Trp Gly Leu Cys Leu Val Pro 1 5 10 15 Leu Ser Leu Ala Gln Ile Asp
Leu Asn Ile Thr Cys Arg Phe Ala Gly 20 25 30 Val Phe His Val Glu
Lys Asn Gly Arg Tyr Ser Ile Ser Arg Thr Glu 35 40 45 Ala Ala Asp
Leu Cys Lys Ala Phe Asn Ser Thr Leu Pro Thr Met Ala 50 55 60 Gln
Met Glu Lys Ala Leu Ser Ile Gly Phe Glu Thr Cys Arg Tyr Gly 65 70
75 80 Phe Ile Glu Gly His Val Val Ile Pro Arg Ile His Pro Asn Ser
Ile 85 90 95 Cys Ala Ala Asn Asn Thr Gly Val Tyr Ile Leu Thr Ser
Asn Thr Ser 100 105 110 Gln Tyr Asp Thr Tyr Cys Phe Asn Ala Ser Ala
Pro Pro Glu Glu Asp 115 120 125 Cys Thr Ser Val Thr Asp Leu Pro Asn
Ala Phe Asp Gly Pro Ile Thr 130 135 140 Ile Thr Ile Val Asn Arg Asp
Gly Thr Arg Tyr Val Gln Lys Gly Glu 145 150 155 160 Tyr Arg Thr Asn
Pro Glu Asp Ile Tyr Pro Ser Asn Pro Thr Asp Asp 165 170 175 Asp Val
Ser Ser Gly Ser Ser Ser Glu Arg Ser Ser Thr Ser Gly Gly 180 185 190
Tyr Ile Phe Tyr Thr Phe Ser Thr Val His Pro Ile Pro Asp Glu Asp 195
200 205 Ser Pro Trp Ile Thr Asp Ser Thr Asp Arg Ile Pro Ala Thr Ser
Thr 210 215 220 Ser Ser Asn Thr Ile Ser Ala Gly Trp Glu Pro Asn Glu
Glu Asn Glu 225 230 235 240 Asp Glu Arg Asp Arg His Leu Ser Phe Ser
Gly Ser Gly Ile Asp Asp 245 250 255 Asp Glu Asp Phe Ile Ser Ser Thr
Ile Ser Thr Thr Pro Arg Ala Phe 260 265 270 Asp His Thr Lys Gln Asn
Gln Asp Trp Thr Gln Trp Asn Pro Ser His 275 280 285 Ser Asn Pro Glu
Val Leu Leu Gln Thr Thr Thr Arg Met Thr Asp Val 290 295 300 Asp Arg
Asn Gly Thr Thr Ala Tyr Glu Gly Asn Trp Asn Pro Glu Ala 305 310 315
320 His Pro Pro Leu Ile His His Glu His His Glu Glu Glu Glu Thr Pro
325 330 335 His Ser Thr Ser Thr Ile Gln Ala Thr Pro Ser Ser Thr Thr
Glu Glu 340 345 350 Thr Ala Thr Gln Lys Glu Gln Trp Phe Gly Asn Arg
Trp His Glu Gly 355 360 365 Tyr Arg Gln Thr Pro Lys Glu Asp Ser His
Ser Thr Thr Gly Thr Ala 370 375 380 Ala Ala Ser Ala His Thr Ser His
Pro Met Gln Gly Arg Thr Thr Pro 385 390 395 400 Ser Pro Glu Asp Ser
Ser Trp Thr Asp Phe Phe Asn Pro Ile Ser His 405 410 415 Pro Met Gly
Arg Gly His Gln Ala Gly Arg Arg Met Asp Met Asp Ser 420 425 430 Ser
His Ser Ile Thr Leu Gln Pro Thr Ala Asn Pro Asn Thr Gly Leu 435 440
445 Val Glu Asp Leu Asp Arg Thr Gly Pro Leu Ser Met Thr Thr Gln Gln
450 455 460 Ser Asn Ser Gln Ser Phe Ser Thr Ser His Glu Gly Leu Glu
Glu Asp 465 470 475 480 Lys Asp His Pro Thr Thr Ser Thr Leu Thr Ser
Ser Asn Arg Asn Asp 485
490 495 Val Thr Gly Gly Arg Arg Asp Pro Asn His Ser Glu Gly Ser Thr
Thr 500 505 510 Leu Leu Glu Gly Tyr Thr Ser His Tyr Pro His Thr Lys
Glu Ser Arg 515 520 525 Thr Phe Ile Pro Val Thr Ser Ala Lys Thr Gly
Ser Phe Gly Val Thr 530 535 540 Ala Val Thr Val Gly Asp Ser Asn Ser
Asn Val Asn Arg Ser Leu Ser 545 550 555 560 Gly Asp Gln Asp Thr Phe
His Pro Ser Gly Gly Ser His Thr Thr His 565 570 575 Gly Ser Glu Ser
Asp Gly His Ser His Gly Ser Gln Glu Gly Gly Ala 580 585 590 Asn Thr
Thr Ser Gly Pro Ile Arg Thr Pro Gln Ile Pro Glu Trp Leu 595 600 605
Ile Ile Leu Ala Ser Leu Leu Ala Leu Ala Leu Ile Leu Ala Val Cys 610
615 620 Ile Ala Val Asn Ser Arg Arg Arg Cys Gly Gln Lys Lys Lys Leu
Val 625 630 635 640 Ile Asn Ser Gly Asn Gly Ala Val Glu Asp Arg Lys
Pro Ser Gly Leu 645 650 655 Asn Gly Glu Ala Ser Lys Ser Gln Glu Met
Val His Leu Val Asn Lys 660 665 670 Glu Ser Ser Glu Thr Pro Asp Gln
Phe Met Thr Ala Asp Glu Thr Arg 675 680 685 Asn Leu Gln Asn Val Asp
Met Lys Ile Gly Val 690 695 15493PRTHomo sapiens 15Met Asp Lys Phe
Trp Trp His Ala Ala Trp Gly Leu Cys Leu Val Pro 1 5 10 15 Leu Ser
Leu Ala Gln Ile Asp Leu Asn Ile Thr Cys Arg Phe Ala Gly 20 25 30
Val Phe His Val Glu Lys Asn Gly Arg Tyr Ser Ile Ser Arg Thr Glu 35
40 45 Ala Ala Asp Leu Cys Lys Ala Phe Asn Ser Thr Leu Pro Thr Met
Ala 50 55 60 Gln Met Glu Lys Ala Leu Ser Ile Gly Phe Glu Thr Cys
Arg Tyr Gly 65 70 75 80 Phe Ile Glu Gly His Val Val Ile Pro Arg Ile
His Pro Asn Ser Ile 85 90 95 Cys Ala Ala Asn Asn Thr Gly Val Tyr
Ile Leu Thr Ser Asn Thr Ser 100 105 110 Gln Tyr Asp Thr Tyr Cys Phe
Asn Ala Ser Ala Pro Pro Glu Glu Asp 115 120 125 Cys Thr Ser Val Thr
Asp Leu Pro Asn Ala Phe Asp Gly Pro Ile Thr 130 135 140 Ile Thr Ile
Val Asn Arg Asp Gly Thr Arg Tyr Val Gln Lys Gly Glu 145 150 155 160
Tyr Arg Thr Asn Pro Glu Asp Ile Tyr Pro Ser Asn Pro Thr Asp Asp 165
170 175 Asp Val Ser Ser Gly Ser Ser Ser Glu Arg Ser Ser Thr Ser Gly
Gly 180 185 190 Tyr Ile Phe Tyr Thr Phe Ser Thr Val His Pro Ile Pro
Asp Glu Asp 195 200 205 Ser Pro Trp Ile Thr Asp Ser Thr Asp Arg Ile
Pro Ala Thr Asn Met 210 215 220 Asp Ser Ser His Ser Ile Thr Leu Gln
Pro Thr Ala Asn Pro Asn Thr 225 230 235 240 Gly Leu Val Glu Asp Leu
Asp Arg Thr Gly Pro Leu Ser Met Thr Thr 245 250 255 Gln Gln Ser Asn
Ser Gln Ser Phe Ser Thr Ser His Glu Gly Leu Glu 260 265 270 Glu Asp
Lys Asp His Pro Thr Thr Ser Thr Leu Thr Ser Ser Asn Arg 275 280 285
Asn Asp Val Thr Gly Gly Arg Arg Asp Pro Asn His Ser Glu Gly Ser 290
295 300 Thr Thr Leu Leu Glu Gly Tyr Thr Ser His Tyr Pro His Thr Lys
Glu 305 310 315 320 Ser Arg Thr Phe Ile Pro Val Thr Ser Ala Lys Thr
Gly Ser Phe Gly 325 330 335 Val Thr Ala Val Thr Val Gly Asp Ser Asn
Ser Asn Val Asn Arg Ser 340 345 350 Leu Ser Gly Asp Gln Asp Thr Phe
His Pro Ser Gly Gly Ser His Thr 355 360 365 Thr His Gly Ser Glu Ser
Asp Gly His Ser His Gly Ser Gln Glu Gly 370 375 380 Gly Ala Asn Thr
Thr Ser Gly Pro Ile Arg Thr Pro Gln Ile Pro Glu 385 390 395 400 Trp
Leu Ile Ile Leu Ala Ser Leu Leu Ala Leu Ala Leu Ile Leu Ala 405 410
415 Val Cys Ile Ala Val Asn Ser Arg Arg Arg Cys Gly Gln Lys Lys Lys
420 425 430 Leu Val Ile Asn Ser Gly Asn Gly Ala Val Glu Asp Arg Lys
Pro Ser 435 440 445 Gly Leu Asn Gly Glu Ala Ser Lys Ser Gln Glu Met
Val His Leu Val 450 455 460 Asn Lys Glu Ser Ser Glu Thr Pro Asp Gln
Phe Met Thr Ala Asp Glu 465 470 475 480 Thr Arg Asn Leu Gln Asn Val
Asp Met Lys Ile Gly Val 485 490 16361PRTHomo sapiens 16Met Asp Lys
Phe Trp Trp His Ala Ala Trp Gly Leu Cys Leu Val Pro 1 5 10 15 Leu
Ser Leu Ala Gln Ile Asp Leu Asn Ile Thr Cys Arg Phe Ala Gly 20 25
30 Val Phe His Val Glu Lys Asn Gly Arg Tyr Ser Ile Ser Arg Thr Glu
35 40 45 Ala Ala Asp Leu Cys Lys Ala Phe Asn Ser Thr Leu Pro Thr
Met Ala 50 55 60 Gln Met Glu Lys Ala Leu Ser Ile Gly Phe Glu Thr
Cys Arg Tyr Gly 65 70 75 80 Phe Ile Glu Gly His Val Val Ile Pro Arg
Ile His Pro Asn Ser Ile 85 90 95 Cys Ala Ala Asn Asn Thr Gly Val
Tyr Ile Leu Thr Ser Asn Thr Ser 100 105 110 Gln Tyr Asp Thr Tyr Cys
Phe Asn Ala Ser Ala Pro Pro Glu Glu Asp 115 120 125 Cys Thr Ser Val
Thr Asp Leu Pro Asn Ala Phe Asp Gly Pro Ile Thr 130 135 140 Ile Thr
Ile Val Asn Arg Asp Gly Thr Arg Tyr Val Gln Lys Gly Glu 145 150 155
160 Tyr Arg Thr Asn Pro Glu Asp Ile Tyr Pro Ser Asn Pro Thr Asp Asp
165 170 175 Asp Val Ser Ser Gly Ser Ser Ser Glu Arg Ser Ser Thr Ser
Gly Gly 180 185 190 Tyr Ile Phe Tyr Thr Phe Ser Thr Val His Pro Ile
Pro Asp Glu Asp 195 200 205 Ser Pro Trp Ile Thr Asp Ser Thr Asp Arg
Ile Pro Ala Thr Arg Asp 210 215 220 Gln Asp Thr Phe His Pro Ser Gly
Gly Ser His Thr Thr His Gly Ser 225 230 235 240 Glu Ser Asp Gly His
Ser His Gly Ser Gln Glu Gly Gly Ala Asn Thr 245 250 255 Thr Ser Gly
Pro Ile Arg Thr Pro Gln Ile Pro Glu Trp Leu Ile Ile 260 265 270 Leu
Ala Ser Leu Leu Ala Leu Ala Leu Ile Leu Ala Val Cys Ile Ala 275 280
285 Val Asn Ser Arg Arg Arg Cys Gly Gln Lys Lys Lys Leu Val Ile Asn
290 295 300 Ser Gly Asn Gly Ala Val Glu Asp Arg Lys Pro Ser Gly Leu
Asn Gly 305 310 315 320 Glu Ala Ser Lys Ser Gln Glu Met Val His Leu
Val Asn Lys Glu Ser 325 330 335 Ser Glu Thr Pro Asp Gln Phe Met Thr
Ala Asp Glu Thr Arg Asn Leu 340 345 350 Gln Asn Val Asp Met Lys Ile
Gly Val 355 360 17139PRTHomo sapiens 17Met Asp Lys Phe Trp Trp His
Ala Ala Trp Gly Leu Cys Leu Val Pro 1 5 10 15 Leu Ser Leu Ala Gln
Ile Asp Leu Asn Ile Thr Cys Arg Phe Ala Gly 20 25 30 Val Phe His
Val Glu Lys Asn Gly Arg Tyr Ser Ile Ser Arg Thr Glu 35 40 45 Ala
Ala Asp Leu Cys Lys Ala Phe Asn Ser Thr Leu Pro Thr Met Ala 50 55
60 Gln Met Glu Lys Ala Leu Ser Ile Gly Phe Glu Thr Cys Ser Leu His
65 70 75 80 Cys Ser Gln Gln Ser Lys Lys Val Trp Ala Glu Glu Lys Ala
Ser Asp 85 90 95 Gln Gln Trp Gln Trp Ser Cys Gly Gly Gln Lys Ala
Lys Trp Thr Gln 100 105 110 Arg Arg Gly Gln Gln Val Ser Gly Asn Gly
Ala Phe Gly Glu Gln Gly 115 120 125 Val Val Arg Asn Ser Arg Pro Val
Tyr Asp Ser 130 135 18337PRTHomo sapiens 18Thr Ser Ser Asn Thr Ile
Ser Ala Gly Trp Glu Pro Asn Glu Glu Asn 1 5 10 15 Glu Asp Glu Arg
Asp Arg His Leu Ser Phe Ser Gly Ser Gly Ile Asp 20 25 30 Asp Asp
Glu Asp Phe Ile Ser Ser Thr Ile Ser Thr Thr Pro Arg Ala 35 40 45
Phe Asp His Thr Lys Gln Asn Gln Asp Trp Thr Gln Trp Asn Pro Ser 50
55 60 His Ser Asn Pro Glu Val Leu Leu Gln Thr Thr Thr Arg Met Thr
Asp 65 70 75 80 Val Asp Arg Asn Gly Thr Thr Ala Tyr Glu Gly Asn Trp
Asn Pro Glu 85 90 95 Ala His Pro Pro Leu Ile His His Glu His His
Glu Glu Glu Glu Thr 100 105 110 Pro His Ser Thr Ser Thr Ile Gln Ala
Thr Pro Ser Ser Thr Thr Glu 115 120 125 Glu Thr Ala Thr Gln Lys Glu
Gln Trp Phe Gly Asn Arg Trp His Glu 130 135 140 Gly Tyr Arg Gln Thr
Pro Arg Glu Asp Ser His Ser Thr Thr Gly Thr 145 150 155 160 Ala Ala
Ala Ser Ala His Thr Ser His Pro Met Gln Gly Arg Thr Thr 165 170 175
Pro Ser Pro Glu Asp Ser Ser Trp Thr Asp Phe Phe Asn Pro Ile Ser 180
185 190 His Pro Met Gly Arg Gly His Gln Ala Gly Arg Arg Met Asp Met
Asp 195 200 205 Ser Ser His Ser Thr Thr Leu Gln Pro Thr Ala Asn Pro
Asn Thr Gly 210 215 220 Leu Val Glu Asp Leu Asp Arg Thr Gly Pro Leu
Ser Met Thr Thr Gln 225 230 235 240 Gln Ser Asn Ser Gln Ser Phe Ser
Thr Ser His Glu Gly Leu Glu Glu 245 250 255 Asp Lys Asp His Pro Thr
Thr Ser Thr Leu Thr Ser Ser Asn Arg Asn 260 265 270 Asp Val Thr Gly
Gly Arg Arg Asp Pro Asn His Ser Glu Gly Ser Thr 275 280 285 Thr Leu
Leu Glu Gly Tyr Thr Ser His Tyr Pro His Thr Lys Glu Ser 290 295 300
Arg Thr Phe Ile Pro Val Thr Ser Ala Lys Thr Gly Ser Phe Gly Val 305
310 315 320 Thr Ala Val Thr Val Gly Asp Ser Asn Ser Asn Val Asn Arg
Ser Leu 325 330 335 Ser 1921DNAArtificial sequenceOligonucleotide
primer 19acactttcac tccaatcgtc c 212020DNAArtificial
sequenceOligonucleotide primer 20tgccctttcc gttgttgtcc
202118DNAArtificial sequenceOligonucleotide primer 21ggaaatcgtg
cgtgacat 182223DNAArtificial sequenceOligonucleotide primer
22tcatgatgga gttgaatgta gtt 2323720PRTHomo
sapiensmisc_feature(1)..(1)Xaa can be any naturally occurring amino
acid 23Xaa Leu Asn Ile Thr Cys Arg Phe Ala Gly Val Phe His Val Glu
Lys 1 5 10 15 Asn Gly Arg Tyr Ser Ile Ser Arg Thr Glu Ala Ala Asp
Leu Cys Lys 20 25 30 Ala Phe Asn Ser Thr Leu Pro Thr Met Ala Gln
Met Glu Lys Ala Leu 35 40 45 Ser Ile Gly Phe Glu Thr Cys Arg Tyr
Gly Phe Ile Glu Gly His Val 50 55 60 Val Ile Pro Arg Ile His Pro
Asn Ser Ile Cys Ala Ala Asn Asn Thr 65 70 75 80 Gly Val Tyr Ile Leu
Thr Ser Asn Thr Ser Gln Tyr Asp Thr Tyr Cys 85 90 95 Phe Asn Ala
Ser Ala Pro Pro Glu Glu Asp Cys Thr Ser Val Thr Asp 100 105 110 Leu
Pro Asn Ala Phe Asp Gly Pro Ile Thr Ile Thr Ile Val Asn Arg 115 120
125 Asp Gly Thr Arg Tyr Val Gln Lys Gly Glu Tyr Arg Thr Asn Pro Glu
130 135 140 Asp Ile Tyr Pro Ser Asn Pro Thr Asp Asp Asp Val Ser Ser
Gly Ser 145 150 155 160 Ser Ser Glu Arg Ser Ser Thr Ser Gly Gly Tyr
Ile Phe Tyr Thr Phe 165 170 175 Ser Thr Val His Pro Ile Pro Asp Glu
Asp Ser Pro Trp Ile Thr Asp 180 185 190 Ser Thr Asp Arg Ile Pro Ala
Thr Thr Leu Met Ser Thr Ser Ala Thr 195 200 205 Ala Thr Glu Thr Ala
Thr Lys Arg Gln Glu Thr Trp Asp Trp Phe Ser 210 215 220 Trp Leu Phe
Leu Pro Ser Glu Ser Lys Asn His Leu His Thr Thr Thr 225 230 235 240
Gln Met Ala Gly Thr Ser Ser Asn Thr Ile Ser Ala Gly Trp Glu Pro 245
250 255 Asn Glu Glu Asn Glu Asp Glu Arg Asp Arg His Leu Ser Phe Ser
Gly 260 265 270 Ser Gly Ile Asp Asp Asp Glu Asp Phe Ile Ser Ser Thr
Ile Ser Thr 275 280 285 Thr Pro Arg Ala Phe Asp His Thr Lys Gln Asn
Gln Asp Trp Thr Gln 290 295 300 Trp Asn Pro Ser His Ser Asn Pro Glu
Val Leu Leu Gln Thr Thr Thr 305 310 315 320 Arg Met Thr Asp Val Asp
Arg Asn Gly Thr Thr Ala Tyr Glu Gly Asn 325 330 335 Trp Asn Pro Glu
Ala His Pro Pro Leu Ile His His Glu His His Glu 340 345 350 Glu Glu
Glu Thr Pro His Ser Thr Ser Thr Ile Gln Ala Thr Pro Ser 355 360 365
Ser Thr Thr Glu Glu Thr Ala Thr Gln Lys Glu Gln Trp Phe Gly Asn 370
375 380 Arg Trp His Glu Gly Tyr Arg Gln Thr Pro Lys Glu Asp Ser His
Ser 385 390 395 400 Thr Thr Gly Thr Ala Ala Ala Ser Ala His Thr Ser
His Pro Met Gln 405 410 415 Gly Arg Thr Thr Pro Ser Pro Glu Asp Ser
Ser Trp Thr Asp Phe Phe 420 425 430 Asn Pro Ile Ser His Pro Met Gly
Arg Gly His Gln Ala Gly Arg Arg 435 440 445 Met Asp Met Asp Ser Ser
His Ser Ile Thr Leu Gln Pro Thr Ala Asn 450 455 460 Pro Asn Thr Gly
Leu Val Glu Asp Leu Asp Arg Thr Gly Pro Leu Ser 465 470 475 480 Met
Thr Thr Gln Gln Ser Asn Ser Gln Ser Phe Ser Thr Ser His Glu 485 490
495 Gly Leu Glu Glu Asp Lys Asp His Pro Thr Thr Ser Thr Leu Thr Ser
500 505 510 Ser Asn Arg Asn Asp Val Thr Gly Gly Arg Arg Asp Pro Asn
His Ser 515 520 525 Glu Gly Ser Thr Thr Leu Leu Glu Gly Tyr Thr Ser
His Tyr Pro His 530 535 540 Thr Lys Glu Ser Arg Thr Phe Ile Pro Val
Thr Ser Ala Lys Thr Gly 545 550 555 560 Ser Phe Gly Val Thr Ala Val
Thr Val Gly Asp Ser Asn Ser Asn Val 565 570 575 Asn Arg Ser Leu Ser
Gly Asp Gln Asp Thr Phe His Pro Ser Gly Gly 580 585 590 Ser His Thr
Thr His Gly Ser Glu Ser Asp Gly His Ser His Gly Ser 595 600 605 Gln
Glu Gly Gly Ala Asn Thr Thr Ser Gly Pro Ile Arg Thr Pro Gln 610 615
620 Ile Pro Glu Trp Leu Ile Ile Leu Ala Ser Leu Leu Ala Leu Ala Leu
625 630 635 640 Ile Leu Ala Val Cys Ile Ala Val Asn Ser Arg Arg Arg
Cys Gly Gln 645 650 655 Lys Lys Lys Leu Val Ile Asn Ser Gly Asn Gly
Ala Val Glu Asp Arg 660 665 670 Lys Pro Ser Gly Leu Asn Gly Glu Ala
Ser Lys Ser Gln Glu Met Val
675 680 685 His Leu Val Asn Lys Glu Ser Ser Glu Thr Pro Asp Gln Phe
Met Thr 690 695 700 Ala Asp Glu Thr Arg Asn Leu Gln Asn Val Asp Met
Lys Ile Gly Val 705 710 715 720
* * * * *