Patent application title: METHOD OF DETERMINING THE RISK OF SCOLIOSIS
Inventors:
Alain Moreau (Montreal, CA)
Alain Moreau (Montreal, CA)
IPC8 Class: AC12Q168FI
USPC Class:
435 6
Class name: Chemistry: molecular biology and microbiology measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving nucleic acid
Publication date: 2010-03-25
Patent application number: 20100075333
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Patent application title: METHOD OF DETERMINING THE RISK OF SCOLIOSIS
Inventors:
Alain Moreau
Agents:
KLARQUIST SPARKMAN, LLP
Assignees:
Origin: PORTLAND, OR US
IPC8 Class: AC12Q168FI
USPC Class:
435 6
Patent application number: 20100075333
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.Claims:
1. A method for determining the risk for developing a scoliosis
comprising(a) monitoring osteopontin (OPN) expression in a sample from a
subject over time; or(b) measuring osteopontin (OPN) expression in a
sample from a subject;wherein an OPN expression that increases in the
subject sample over time or 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.
2. The method of claim 1, wherein the monitoring begins when the subject is about three years old.
3. The method of claim 1, wherein the monitoring is performed by measuring OPN expression at a frequency of at least about once per month.
4. The method of claim 1, wherein the monitoring is performed by measuring OPN expression at a frequency of at least about once per six month.
5. The method of claim 1, wherein the monitoring OPN 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. A method of stratifying a subject having a scoliosis comprisingmeasuring osteopontin (OPN) expression in a sample from the subject;whereby the measuring step enables the stratification of the subject into a scoliosis subgroup.
12. A method for assessing the efficacy of a brace on a subject having a scoliosis comprisingmeasuring 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.
13. The method of claim 12, wherein the determining the OPN expression after the bracing is performed at least one month after the bracing.
14. The method of claim 12, wherein the determining the OPN expression after bracing the subject is performed at least two months hours after the bracing.
15. The method of claim 12, wherein the determining the OPN expression after bracing the subject is performed at least three months after the bracing.
16. The method of claim 12, wherein the determining the OPN expression after bracing the subject is performed at least six months after the bracing.
17. The method of claim 1, wherein the method further comprises measuring soluble CD44 receptor (sCD44) expression in the sample from the subject.
18. The method of claim 1, wherein the sample from the subject is a biological fluid from the subject.
19. The method of claim 18, 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. The method of claim 1, wherein the OPN expression is OPN protein.
22. The method of claim 21, wherein the determining of the OPN expression is performed with an antibody that specifically binds to OPN.
23. The method of claim 22, wherein the measuring OPN 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 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 expression that is higher than 800 nanograms per milliliter of plasma is indicative that the subject is at risk for developing a scoliosis.
26. The method of claim 1, wherein the OPN expression is OPN RNA.
27. The method of claim 1, wherein the sample from the subject is a paraspinal muscle biopsy and the OPN expression is OPN RNA.
28. A method of selecting an agent as a potential candidate for the reduction or prevention of scoliosis comprising: (a) 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; (b) contacting a candidate agent with a cell expressing sCD44, and detecting the expression of sCD44, wherein when the expression of sCD44 is higher in the presence of the candidate agent as compared to in the absence thereof, the candidate agent is selected; or (c) 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.
29. (canceled)
30. The method of claim 28, wherein the cell is a cell derived from a scoliotic patient.
31. (canceled)
32. A method of preventing or reducing scoliosis comprising administering to a subject having scoliosis a therapeutically effective amount of (a) an osteopontin inhibitor (OPN) or a selenium rich diet; (b) a CD44 inhibitor; or (c) a sCD44 stimulator, whereby scoliosis is thereby prevented or treated.
33-34. (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.
38-47. (canceled)
48. 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.
49. The kit of claim 48, further comprising a ligand specific to soluble CD44 (sCD44).
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001]This application claims priority, under 35 U.S.C. §119(e), 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 documents above are incorporated herein in their entirety by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002]N/A.
FIELD OF THE INVENTION
[0003]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.
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.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.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 C57BI/6j mice after surgery, and in the right panel presents typical x-rays of scoliotic deformities observed in bipedal C57BI/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 (45°) 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 r2=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≧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°; n=14) and in those without significant change in curve (no change in Cobb's angle, decrease, or increase smaller than 3°; 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--001040058 (SEQ ID NO: 1); transcript variant 2, mRNA NM--000582 (SEQ ID NO: 2); transcript variant 3, mRNA NM--001040060 (SEQ ID NO: 3) and the amino acid sequences of the three human OPN isoforms (isoform a NP--001035147 (SEQ ID NO: 4); isoform b NP--000573 (SEQ ID NO: 5); and isoform c NP--001035149 (SEQ ID NO: 6));
[0061]FIG. 21 presents the nucleotide sequences (mRNA) of six isoforms of human CD44 (NM--000610 transcript variant 1 (SEQ ID NO: 7); NM--001001389 transcript variant 2 (SEQ ID NO: 8); NM--001001390 transcript variant 3 (SEQ ID NO: 9); NM--001001391 transcript variant 4 (SEQ ID NO: 10); NM--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--000601 isoform 1 precursor (SEQ ID NO: 13); NP--001001389 isoform 2 precursor (SEQ ID NO: 14); NP 001001390 isoform 3 precursor (SEQ ID NO: 15); NP 001001391 isoform 4 precursor (SEQ ID NO: 16); NP--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 C57BI/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° (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° (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 C57BI/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.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 conditions30-33.
[0067]OPN signaling action: 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.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 isoforms18,17 and such structural heterogeneity is responsible of the ligand repertoire of CD44, which includes fibronectin18, chondroitin sulphate19, osteopontin20, at least two heparin binding growth hormones and hyaluronan.21,22 Soluble variant isoforms of sCD44 (sCD44var) have been associated with several pathological conditions.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.
[0068]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.
[0069]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.
[0070]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.
[0071]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°), orthopaedic devices (when Cobb's angle is around 25-30°), and surgery (over 45°). 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).
[0072]As used herein, the terms "severe AIS" refers to a scoliosis characterized by Cobb's angle of 45° or more.
[0073]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° to 50°, or from 18° to 25°) is a "development" of scoliosis.
[0074]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.
[0075]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.
[0076]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.
[0077]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.
[0078]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.
[0079]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.
[0080]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.
[0081]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.
[0082]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.
[0083]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".
[0084]The terms "such as" are used herein to mean, and is used interchangeably with, the phrase "such as but not limited to".
[0085]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.
[0086]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).
[0087]Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and α-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.
[0088]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.
[0089]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.
[0090]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.
[0091]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.
[0092]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.
[0093]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.
[0094]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.
[0095]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
[0096]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 Kit 900-142 0.11 ng/ml American Research Osteopontin, human kit 17158 ? Products, Inc. R&D Systems Human Osteopontin (OPN) DOST00 0.024 ng/mL ELISA Kit Promokine Human Osteopontin ELISA PK-EL-KA4231 3.6 ng/ml Uscnlife Human Osteopontin, OPN E0899h ? ELISA Kit
[0097]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.
[0098]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')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, VH regions (VH, VH-VH), anticalins, PepBodies®, 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.
[0099]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.
[0100]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, SOCl2, or R1N═C═NR, where R and R1 are different alkyl groups.
[0101]Animals may be immunized against the antigen, immunogenic conjugates, or derivatives by combining the antigen or conjugate (e.g., 100 μg for rabbits or 5 μg 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.
[0102]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).
[0103]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.
[0104]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.
[0105]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.
[0106]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.
[0107]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.
[0108]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.
[0109]The present invention also encompasses the monitoring of the biomarkers disclosed herein 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 . . . ).
[0110]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 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.
[0111]In a particular embodiment, it includes cells of any cell type derived from a scoliotic patient. (whole cell assay). 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.
[0112]Pharmaceutical compositions can also be administered by routes such as nasally, intravenously, intramuscularly, subcutaneously, sublingually, intrathecally, or intradermally. The route of administration can depend on a variety of factors, such as the environment and therapeutic goals.
Dosage
[0113]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".
[0114]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.
[0115]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.
[0116]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.
[0117]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.
[0118]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.).
[0119]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.
[0120]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.
[0121]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.
[0122]The present invention is illustrated in further details by the following non-limiting examples.
EXAMPLE 1
Material and Methods
[0123]GENERATION OF BIPEDAL C57BL/6J OPN-NULL AND CD44-NULL MICE. Experiments in mice were conducted according to protocols approved by The Step-Justine Hospital's Animal Health Care Review Committee. Breeding pairs of C57BI/6 devoid of either OPN(OPN-null mice) or CD44 receptor (CD44-null mice) backcrossed for more than 10 generations in C57BI/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 C57BI/6j mice served as wild-type control mice (Charles-River, Wilmington, Mass., USA). The C57BI6/6j mouse strain was used because it is naturally deficient in melatonin.sup.(26), 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® 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° or higher was retained as a significant scoliotic condition.
[0124]IMMUNODETECTION OF MOUSE OPN 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° 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® Expert-96 microplate reader (Biochrom, Cambridge, UK). Although serum was used in mice herein, the present invention also encompasses measuring OPN in mice plasma.
[0125]GENERATION OF PINEALECTOMIZED CHICKENS. 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).
[0126]EXPRESSION ANALYSIS AND IMMUNODETECTION OF CHICKEN OPN. 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® 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 MgCl2, 10 picomolars each primer, and 1U 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° C./45 seconds, 66° C./45 seconds, 72° C./1 minute. For quantitative analysis, all amplifications were normalized against that of the housekeeping gene β-actin; chicken β-actin (460 by PCR product) 5'-GGAAATCGTGCGTGACAT-3' (SEQ ID NO: 21) (forward), 5'-TCATGATGGAGTTGAATGTAGTT-3' (SEQ ID NO: 22) (reverse) 32 cycles: 94° C./45 seconds, 55° C./45 seconds, 72° 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).
[0127]HUMAN POPULATIONS 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.
[0128]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.
[0129]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. †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.
[0130]OSTEOPONTIN, SCD44 AND HA ENZYME-LINKED IMMUNOSORBENT ASSAYS 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° 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® 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.
[0131]STATISTICAL ANALYSIS 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 (www.r-project.org).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
[0132]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 C57BI/6j Mice
[0133]Bipedal C57BI/6j mice were generated and their OPN level was determined as described in Example 1 above. Bipedal ambulation for 8 weeks in C57BI/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 C57BI/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
[0134]FIG. 2 shows that the OPN protein level strongly increases after bipedal surgery (i.e. during scoliosis development) in scoliotic C57BI/6j mice.
EXAMPLE 4
Observation of Effect of Absence of OPN or CD44 Bipedal C57BI/6j Mice on Scoliosis
[0135]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 C57BI/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 C57BI/6j OPN-null (n=54) and C57BI/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.
[0136]In parallel, melatonin circulating levels were measured in wild-type and OPN-KO mice to exclude the possibility that absence of scoliosis in bipedal C57BI/6 OPN-KO mice was due to an increased production of melatonin.
[0137]FIG. 3 shows a two-fold decrease in circulating melatonin level of bipedal C57BI/6j OPN KO mice when compared to wild-type ones (C57BI/6j, C57BI/6j and FVB).
[0138]As indicated above, C57BI/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 (C57BI6/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
[0139]Two compounds suspected of having an effect on OPN transcription or synthesis were injected intraperitonealy at a dosage of 500 μg/kg of body weight/day to chicken 24-48 h prior pinealectomy.
[0140]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
[0141]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°-44° vs. ≧15°) 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 30°), a greater proportion of girls in the AIS groups (86% and 84% in the 10°-44° and ≧15° subgroups, respectively were observed compared to the control groups (54% and 64% in healthy and at risk control groups, respectively, p≦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≧45° compared to those with 10-44° 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).
[0142]The plasma OPN levels in patients with AIS exhibiting a severe deformity (Cobb's angle≧45°), low to moderate curve (Cobb's angle between 10° and 44°) 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≧45°) (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 Mean Male Female + Male biomarker Mean Mean level biomarker biomarker Subject Type N (ng/ml) Range N level (ng/ml) Range N level (ng/ml) Range P-value† OPN Healthy controls 19 580 ± 150 318-882 16 558 ± 168 308-856 35 570 ± 156 308-882 -- At risk controls 45 829 ± 419 208-1834 25 877 ± 378 391-1629 70 846 ± 402 208-1834 <0.001 AIS < 45° 162 774 ± 268 373-1585 27 948 ± 335 445-1668 189 799 ± 284 373-1668 <0.001 AIS ≧ 45° 53 913 ± 398 201-1821 10 1238 ± 409 575-1872 63 965 ± 414 201-1872 <0.001 sCD44 Healthy controls 19 522 ± 99 373-829 16 575 ± 92 404-800 35 546 ± 98 373-829 -- At risk controls 45 508 ± 96 316-760 25 533 ± 98 304-510 70 517 ± 97 304-760 >0.5 AIS < 45° 162 503 ± 161 194-1253 27 527 ± 110 364-793 189 506 ± 155 194-1253 >0.5 AIS ≧ 45° 53 436 ± 251 87-882 10 402 ± 216 147-962 63 431 ± 245 87-962 0.066 HA Healthy controls 19 128 ± 38 72-236 16 132 ± 49 80-255 35 130 ± 43 72-255 -- At risk controls 45 119 ± 51 36-257 25 117 ± 52 33-226 70 118 ± 51 33-257 >0.5 AIS < 45° 162 112 ± 60 18-356 27 124 ± 60 27-283 189 114 ± 60 18-356 >0.5 AIS ≧ 45° 53 93 ± 40 32-222 10 128 ± 71 41-25435 63 98 ± 48 32-254 0.140 *SD is standard deviation †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).
[0143]Receiver-operating characteristics (ROC) curves analyzes of plasma OPN comparing the patients with AIS more severely affected (Cobb's angle≧45°) 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).
[0144]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
[0145]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).
[0146]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).
[0147]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
[0148]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 45°) had also the lowest mean plasma sCD44 level when compared to the other three groups (p=0.066).
[0149]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°) 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
[0150]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.
[0151]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° and in those without significant change in curve (no change in Cobb's angle, decrease, or increase smaller than 3°; 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).
[0152]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;
[0153]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
[0154]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.
[0155]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° 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 ± 8.32 645.84 148.32 ± 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. †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°. Patient Timepoint Cobb's Angle Curve ID Date of Birth Gender Age Collection Date (mths) Pre-op Type 102 1991-09-12 F 13.8 2005-06-10 T0 18 rT 14.3 2006-01-13 T7 16 rT 15.8 2007-06-01 T12 16 rT 16.2 2007-11-30 T29 17 rT 103 1991-09-04 M 13.8 2005-06-10 T0 13 lT 104 1992-01-29 F 13.4 2005-06-10 T0 21-22 rTlL 106 1992-08-10 F 14.8 2007-06-05 T0 25-24 rTlL 15.2 2007-10-05 T4 22-18 rTlL 107 1991-09-09 F 13.8 2005-06-20 T0 31-32 rTlL 113 1995-11-21 F 9.7 2005-07-22 T0 10 rT 11.5 2007-05-18 T22 15 rT 118 1991-06-04 F 16.6 2008-01-18 T0 22-22 rTlTL 123 1993-09-23 F 12.1 2005-11-04 T0 28 rTL 14.3 2008-01-18 T26 19-31 lTrTL 124 1990-12-09 F 14.9 2005-11-04 T0 33-32 rTlTL 127 1992-01-18 F 13.9 2005-12-02 T0 33-19 rTrT 128 1997-03-18 F 8.8 2005-12-02 T0 10 lTL 130 1991-06-05 F 14.5 2005-12-09 T0 19 rTL 131 1992-11-09 F 13.1 2005-12-09 T0 32-24 rTlL 15.0 2007-11-12 T23 32-24 rTlL 136 1989-10-10 F 16.3 2006-01-13 T0 14 lTL 138 1993-06-04 F 12.7 2006-02-17 T0 24-26 rTlL 14.3 2007-10-24 T20 22-25 rTlL 14.7 2008-02-04 T24 23-26 rTlTL 139 1993-12-06 F 12.2 2006-02-24 T0 12.-14 rTlL 14.2 2008-02-08 T24 12.-6 rTlL 141 1992-07-20 F 13.7 2006-03-10 T0 20-18 rTlL 15.5 2008-01-22 T22 9.-13 rTlTL 142 1992-12-19 F 13.2 2006-03-10 T0 31 lTL 15.1 2008-01-23 T22 25 lTL 146 1990-05-13 F 16.0 2006-05-26 T0 32-22 rTlL 148 1993-08-12 F 14.3 2007-12-07 T0 11 lTL 149 1988-09-28 M 17.7 2006-06-02 T0 31-26 rTlL 150 1992-10-16 F 13.6 2006-06-02 T0 25 rT 151 1993-04-11 F 14.7 2007-12-03 T0 28-20 rTlL 152 1990-10-04 F 15.7 2006-06-02 T0 34 lL 154 1989-11-24 F 16.6 2006-06-08 T0 40 lTL 18.1 2007-12-07 T18 38 lTL 155 1991-01-01 F 15.4 2006-06-08 T0 26 lTL 159 1998-03-04 F 9.7 2007-11-06 T0 3 lTL 161 1994-04-27 F 13.6 2007-11-30 T0 15 lTL 165 1995-08-30 F 12.3 2007-12-03 T0 34-20 rTlL 168 1992-04-24 F 14.2 2006-06-26 T0 16-18 rTlL 14.6 2006-11-21 T5 17-16 rTlL 15.5 2007-10-01 T16 14-16 rTlTL 176 1992-10-24 F 13.8 2006-07-03 T0 29 rT 14.2 2007-01-15 T6 27 rT 183 1991-09-13 M 14.8 2006-05-07 T0 17 rL 15.4 2007-06-02 T13 7.-19 rTlL 200 1992-07-29 M 15.2 2007-10-30 T0 23-24 rTlL 201 1992-11-27 F 13.7 2006-07-12 T0 10-17. rTlL 225 1994-05-09 F 12.2 2006-07-24 T0 15-19 lTrTL 12.8 2007-02-27 T7 13-18 lTrL 234 1990-07-16 M 16.2 2006-10-13 T0 26 rT 235 1991-10-29 M 15 2006-10-13 T0 20 lTL 16 2007-10-11 T12 18 lTL 240 1993-10-04 F 13.2 2006-12-11 T0 17-23 rTlL 242 1989-09-12 F 17.3 2007-01-12 T0 6 lTL 244 1990-10-20 F 16.2 2007-01-19 T0 27-29 rTlL 17.3 2008-02-13 T13 NA NA 245 1992-01-27 F 15.0 2007-01-22 T0 31-35 rTlL 15.8 2007-11-14 T10 28-35 rTlL 247 1994-12-18 F 12.1 2007-01-26 T0 9 rTL 12.8 2007-10-09 T9 6 rTL 248 1997-06-16 F 9.6 2007-01-26 T0 9 rL 10.3 2007-10-09 T9 3 lTL 249 1991-03-25 F 15.9 2007-02-02 T0 31 lTL 16.4 2007-08-03 T6 NA lTL 16.9 2008-02-01 T12 36 lTL 250 1992-05-08 F 14.7 2007-02-02 T0 32 lTL 15.4 2007-10-15 T8 21 lTL 251 1991-09-05 F 15.4 2007-02-02 T0 40-30 rTlL 253 1992-10-18 M 14.3 2007-02-27 T0 31 rT 254 1991-12-11 F 15.2 2007-03-09 T0 28 lTL 15.9 2007-11-12 T8 15 lTL 256 1996-03-19 F 11.0 2007-03-09 T0 11 lTL 257 1995-04-15 F 11.9 2007-03-09 T0 6 rTL 12.5 2007-10-16 T7 NA NA 258 1990-06-24 M 16.8 2007-03-09 T0 14 rT 17.3 2007-10-02 T8 NA NA 259 1994-07-07 F 12.7 2007-03-16 T0 8 lTL 13.5 2007-10-15 T7 11 lTL 260 1994-07-07 M 12.7 2007-03-16 T0 6 rTL 13.5 2007-10-05 T7 4 lTL 261 1997-06-19 F 9.7 2007-03-16 T0 21 lL 10.3 2007-10-17 T7 10 lTL 10.4 2008-02-06 T11 5 lTL 263 1994-10-13 F 12.4 2007-03-20 T0 7.-12 rTlL 264 1992-05-24 F 14.8 2007-03-20 T0 23-30 rTlL 265 1993-05-04 F 13.9 2007-03-20 T0 23 lL 14.5 2007-11-13 T8 11-14. rTlL 266 1991-01-25 F 16.2 2007-04-02 T0 34 rTL 16.8 2007-11-15 T7 34 rTL 267 1994-05-14 F 12.9 2007-04-02 T0 5 rTL 13.5 2007-11-15 T7 5 rTL 268 1994-08-17 F 12.6 2007-04-04 T0 12.-4 rTlL 271 1994-11-17 F 12.4 2007-04-13 T0 23 rTL 12.9 2007-10-15 T6 24 rTL 272 1994-04-14 F 13.0 2007-04-13 T0 22-24 rTlL 13.6 2007-12-05 T8 14-15 rTlL 273 1991-06-30 F 15.8 2007-04-13 T0 25 rTL 274 1990-02-28 F 17.1 2007-04-17 T0 11.-22 rTlL 275 1996-04-08 F 11.0 2007-04-19 T0 27-1. rTlL 11.5 2007-10-15 T6 26-19 rTlTL 276 1994-09-26 F 13.1 2007-10-15 T0 19-19 rTlL 277 1994-11-02 F 12.4 2007-04-19 T0 12 lL 13.0 2007-11-14 T7 15-13 rTlL 278 1992-06-08 M 14.9 2007-05-04 T0 22-14 rTlL 15.3 2007-10-23 T5 26-28 rTlL 279 1998-09-22 F 8.7 2007-05-30 T0 19 rT 9.2 2007-10-05 T5 8 rT 280 1992-12-18 F 14.4 2007-05-30 T0 19 rT 14.9 2007-11-02 T6 24 rTL 281 1994-10-17 F 12.6 2007-06-01 T0 11 rT 13.1 2007-11-09 T5 9 lTL 282 1997-09-30 F 9.7 2007-06-13 T0 20 rT 10.3 2008-01-30 T7 NA NA 286 1994-06-01 F 13.3 2007-09-17 T0 28 lTL 287 1991-11-15 F 15.8 2007-09-18 T0 11 rTL 288 1996-05-13 M 11.3 2007-09-18 T0 20 lL 289 1992-10-23 F 14.9 2007-09-18 T0 18 rT 290 1993-10-02 F 14.0 2007-09-18 T0 22 rTL 291 1992-07-10 F 20.9 2007-09-18 T0 25-31 rTlL 292 1994-01-23 F 13.7 2007-09-21 T0 20 lTL 293 1993-04-03 F 14.5 2007-09-21 T0 16 rT 295 1991-08-09 M 16.1 2007-09-26 T0 11.-8 rTlL 296 1992-04-04 F 15.5 2007-09-28 T0 15-18 lTrL 297 1997-07-13 M 10.2 2007-09-28 T0 20 lT 298 1994-11-09 F 12.9 2007-09-28 T0 18-21 rTlL 299 1990-03-21 F 17.5 2007-10-03 T0 33-43 rTlL 301 1995-02-06 F 12.7 2007-10-09 T0 13 lT 302 1993-05-07 F 14.4 2007-10-09 T0 14.-12 rTlL 303 1991-03-29 F 16.5 2007-10-15 T0 14 lTL 304 1991-10-25 F 16.0 2007-10-16 T0 25 lT 305 1992-02-24 F 15.7 2007-10-19 T0 23 lTL 306 1994-09-22 F 13.1 2007-10-19 T0 13-18 rTlL 307 1994-01-25 M 13.7 2007-10-24 T0 8-11-11. lTrTlL 308 1997-05-22 F 10.4 2007-10-26 T0 8 rTL 309 1996-04-10 F 11.5 2007-10-26 T0 10 lTL 311 1993-05-07 F 14.5 2007-10-26 T0 17 lTL 313 1993-06-04 F 14.4 2007-10-26 T0 20-18 rTlL 314 1993-03-11 F 14.6 2007-10-29 T0 24 rL 315 1993-12-16 F 13.9 2007-10-31 T0 14 lTL 316 1992-10-07 M 15.1 2007-10-31 T0 28 rT 318 1997-05-25 F 10.4 2007-10-15 T0 11 rTL 319 1993-06-28 F 14.4 2007-11-06 T0 22 lTL 320 1993-09-24 F 14.1 2007-11-09 T0 15 rT 321 1992-07-04 F 15.3 2007-11-09 T0 16 rTL 322 1996-06-01 F 11.4 2007-11-09 T0 4 lTL 324 1991-04-20 F 16.6 2007-11-09 T0 19-19 rTlL 325 1994-03-26 F 13.6 2007-11-09 T0 21 rTL 326 1994-02-02 M 13.8 2007-11-13 T0 13 lTL 328 1994-09-24 F 12.8 2007-11-14 T0 11 lTL 329 1996-05-29 F 11.5 2007-11-14 T0 6 rTL 330 1994-02-05 F 13.8 2007-11-16 T0 12 lTL 332 1992-01-26 M 15.8 2007-11-23 T0 24 lTL 333 1993-10-21 F 14.1 2007-11-23 T0 30 lTL 334 1993-08-07 F 14.3 2007-11-23 T0 29-27 rTlL 335 1996-01-16 F 11.9 2007-11-23 T0 28-27 rTlL 337 1991-09-04 M 16.2 2007-11-28 T0 24 lL 338 1994-12-31 F 12.9 2007-11-30 T0 10 lTL 339 1992-03-17 F 15.7 2007-11-30 T0 25 lTL 340 1995-05-21 F 12.5 2007-11-30 T0 30 lTL 341 1996-02-11 F 11.8 2007-11-30 T0 15-14 rTlL 342 1993-12-01 F 14.0 2007-12-07 T0 16 rTL 343 1993-06-29 M 14.4 2007-12-07 T0 15 rTL 344 1996-03-26 F 11.7 2007-12-07 T0 10 rTL 345 1993-04-12 F 14.6 2007-12-07 T0 30 lTL 346 1996-10-11 F 11.2 2007-12-07 T0 18-17 rTlTL 347 1997-04-07 F 10.7 2007-12-11 T0 5-6. rTlL 348 1995-06-10 M 12.5 2007-12-11 T0 10 rTL 350 1995-02-22 F 12.8 2007-12-13 T0 25 rTL 351 1992-05-19 F 15.6 2007-12-13 T0 14 rTL 352 1996-04-13 M 11.7 2007-12-13 T0 14 rTL 353 1993-08-12 M 14.3 2007-12-13 T0 24 rT 354 1994-06-07 F 13.5 2007-12-13 T0 8 lT 355 1993-08-08 F 14.3 2007-12-13 T0 27 lTL 356 1995-05-17 F 12.6 2007-12-13 T0 19 lTL 358 1997-02-27 F 10.9 2008-01-11 T0 18 rTL 359 1995-11-08 F 13.0 2008-01-15 T0 14 rTL 360 1992-05-24 F 15.6 2008-01-15 T0 14 lTL 361 1996-06-29 F 11.5 2008-01-15 T0 23 rTL 362 1997-08-21 F 10.4 2008-01-16 T0 11 lTL 363 1993-05-24 F 14.6 2008-01-16 T0 20-24-19 lTrTlTL 364 1995-03-24 F 12.8 2008-01-16 T0 10 lTL 365 1999-07-26 F 9.3 2008-01-16 T0 5 rTL 368 1996-07-12 F 11.5 2008-01-18 T0 14 rTL 369 1992-05-21 F 15.7 2008-01-18 T0 25 rTL 370 1994-12-01 F 13.1 2008-01-18 T0 18-15 rTlL 371 1992-02-04 F 16.0 2008-01-18 T0 26-20 rTlTL 372 1991-06-21 F 16.6 2008-01-21 T0 23-21 rTlL 374 1992-05-26 F 15.7 2008-01-21 T0 25 lL 375 1992-10-21 F 15.3 2008-01-22 T0 31-55 rTlTL 376 1993-05-18 F 14.7 2008-01-22 T0 16 rTL 377 1995-01-31 F 13.0 2008-01-22 T0 27 lTL 379 1996-09-14 F 11.4 2008-01-25 T0 5.-5 lTrTL 381 1992-01-11 M 16.0 2008-01-25 T0 24 rT 382 1993-10-21 F 14.2 2008-01-25 T0 28-25 rTlTL 383 1994-11-20 F 13.2 2008-01-25 T0 30-27 rTlTL 384 1992-02-09 M 16.0 2008-01-29 T0 25-19 rTlL 386 1994-09-02 F 13.4 2008-02-01 T0 25-14 lTrT 387 1994-04-11 F 13.8 2008-02-01 T0 14-15 rTlTL 388 1995-11-24 F 12.2 2008-02-01 T0 34 rT 389 1997-04-13 F 10.8 2008-02-04 T0 14 lTL 390 1994-04-28 F 13.8 2008-02-04 T0 28-26 rTlL 391 1994-07-01 F 13.6 2008-02-05 T0 37 rTL 392 1998-11-25 F 9.2 2008-02-05 T0 16 rTL 393 1993-09-30 M 14.3 2008-02-05 T0 26 rTL 395 1995-05-24 F 12.7 2008-02-08 T0 11 rT 397 1999-02-20 F 9.0 2008-02-08 T0 10 rTL 398 1997-09-16 F 10.4 2008-02-08 T0 16 rTL 399 2000-09-28 M 7.4 2008-02-08 T0 22-20 rTlTL 400 1994-05-25 F 13.7 2008-02-08 T0 12 rTL 401 1994-02-17 F 14.0 2008-02-18 T0 28-21 rTlTL 402 1991-07-15 F 16.6 2008-02-14 T0 19-12 rTlL 403 1995-02-21 F 13.0 2008-02-14 T0 13-13 rTlTL 1264 1997-09-22 F 15.2 2005-04-18 T0 40 rTL 1276 1997-09-23 F 15.2 2005-05-16 T0 42 lT 1364 1997-09-24 M 14.9 2006-04-24 T0 44 lTL 1365 1990-05-11 F 15.9 2006-04-26 T0 23-53 lTrL 1366 1993-04-06 F 13.1 2006-05-01 T0 36 NA 1373 1991-10-07 F 14.6 2006-05-17 T0 41-48 rTlL 1380 1989-10-09 F 16.7 2006-06-26 T0 35 rL 1384 1991-01-17 F 15.5 2006-07-03 T0 41 lTL 15.8 2006-11-15 T4 9-4 1385 1990-06-12 F 16.1 2006-07-04 T0 42-23 rTlL 1387 1991-07-15 F 15.0 2006-07-17 T0 29-37-35 rTlL 1388 1991-12-13 F 14.6 2006-07-19 T0 38 rTL 1409 1993-02-11 F 13.6 2006-09-26 T0 40 rT 1433 1992-07-03 F 14.5 2007-01-10 T0 44 rT 1451 1995-01-13 F 12.2 2007-03-14 T0 42 rT 1478 1990-08-06 F 16.8 2007-06-11 T0 41 rTL 1481 1990-08-15 F 16.8 2007-06-18 T0 40 rT 1483 1989-06-26 F 18.0 2007-06-19 T0 37-25 rTlL 1487 1990-05-30 F 17.1 2007-07-03 T0 35-58-35 lCrTlL Patient Date of [sCD44] ID surgery Family history [OPN] (ng/ml) (ng/ml) [HA] (ng/ml) 102 -- Cousin 1265.10 375.56 132.06 ± 39.35 766.80 408.06 388.93 ± 23.42
933.77 ± 13.23 437.55 71.91 ± 4.23 591.72 ± 66.49 311.40 27.92 ± 1.72 103 -- Father (cyphose) 1338.32 792.62 207.12 104 -- -- 1221.83 742.48 132.24 106 -- -- 972.87 ± 16.73 488.72 86.78 ± 6.34 485.82 ± 34.70 475.13 293.05 ± 40.93 107 -- Mother 739.61 1253.3 109.39 ± 26.70 113 -- -- 670.49 ± 5.45 695.21 41.10 ± 8.51 688.49 ± 23.78 613.79 49.16 ± 9.14 118 -- Both parents 372.79 ± 10.86 273.31 70.42 ± 4.85 123 -- Both parents 1466.97 931.05 128.78 ± 4.22 779.90 ± 16.68 410.10 179.52 ± 21.17 124 -- Cousins 625.97 816.60 96.08 127 -- -- 786.71 755.60 131.36 ± 22.43 128 -- -- 837.64 628.74 118.73 ± 10.43 130 -- -- 559.85 552.78 75.09 ± 7.11 131 -- -- 568.01 578.96 101.00 ± 11.04 450.45 ± 9.36 505.94 100.03 ± 9.66 136 -- -- 411.02 670.31 84.81 ± 2.56 138 -- Cousin 577.78 293.51 63.86 ± 4.11 379.04 ± 18.07 388.16 86.23 ± 11.26 529.70 ± 4.86 378.03 227.26 ± 0.94 139 -- -- 847.98 868.95 136.19 ± 7.63 1192.61 ± 10.71 444.33 73.88 ± 19.39 141 -- Grand-mother, 658.28 735.50 90.51 cousins, uncle 172.67 ± 8.59 433.6 37.31 ± 7.61 142 -- Mother, cousin 776.43 907.96 122.73 ± 7.61 542.85 ± 1.41 511.4 146.43 ± 63.23 146 -- -- 1501.42 475.91 75.68 ± 10.22 148 -- Mother 1416.91 ± 41.50 550.4 37.79 ± 6.19 149 -- -- 472.61 559.97 138.95 ± 7.42 150 -- Sister 805.88 543.22 71.24 ± 1.52 151 -- -- 732.19 ± 2.30 403.51 20.80 ± 3.30 152 -- Father 655.10 551.24 122.69 ± 0.10 154 -- Cousin 541.07 639.52 104.09 ± 13.96 1101.07 ± 38.84 342.17 35.08 ± 5.40 155 -- Aunt 738.59 796.06 121.33 ± 17.72 159 -- Mother 769.50 ± 21.57 831.18 107.5 ± 1.08 161 -- -- 487.11 ± 29.43 355.79 23.63 ± 0.53 165 -- -- 1148.04 ± 47.51 607.43 42.39 ± 7.68 168 -- -- 810.21 ± 28.48 244.4 103.10 ± 10.39 582.52 ± 23.29 338.03 99.20 ± 18.18 441.81 ± 7.29 333.4 126.96 ± 1.45 176 -- -- 503.88 ± 35.81 331.65 91.50 ± 21.99 675.38 ± 44.20 305.92 193.26 ± 2.38 183 -- -- 733.99 ± 17.33 550.24 72.91 ± 10.68 781.03 ± 3.27 531.96 69.83 ± 7.07 200 -- -- 972.10 ± 4.92 401.94 88.41 ± 10.08 201 -- Sister 782.77 ± 2.63 498.93 142.57 ± 44.69 225 -- -- 406.67 ± 3.40 617.37 248.10 ± 24.21 651.89 ± 21.69 524.9 47.95 ± 3.60 234 -- -- 840.88 ± 1.98 491.26 89.04 ± 5.66 235 -- -- 586.25 ± 0.32 403.8 181.655 ± 48.71 523.39 ± 9.76 428.29 188.63 ± 6.83 240 -- Mother, brother, 525.88 ± 7.74 428.83 71.91 ± 4.23 cousin 242 -- Sister 590.13 ± 6.00 435.59 80.24 ± 3.69 244 -- -- 735.26 ± 4.42 510.44 73.81 ± 6.20 1293.68 ± 36.92 449.1 44.51 ± 4.81 245 -- -- 496.26 ± 3.54 333.97 70.41 ± 0.88 363.60 ± 2.97 562.52 54.98 ± 5.08 247 -- Mother, sister 1148.31 ± 2.17 371.29 164.68 ± 23.99 806.91 ± 16.69 393.27 141.16 ± 2.62 248 -- Mother, sister 1010.38 ± 5.14 443.83 142.95 ± 4.69 841.24 ± 18.47 490.2 158.10 ± 33.95 249 -- -- 534.09 ± 7.74 459.52 74.98 ± 0.08 340.44 ± 12.89 499.97 132.91 ± 37.20 579.65 ± 8.62 413.67 98.93 ± 19.98 250 -- Uncle 688.35 ± 9.46 587.17 74.40 ± 3.75 612.19 ± 22.36 540.29 150.73 251 -- -- 1146.66 ± 7.34 437.25 80.50 ± 5.24 253 -- -- 634.83 ± 0.90 486.03 184.50 ± 20.76 254 -- -- 701.23 ± 1.92 362.22 72.85 ± 2.66 548.26 ± 25.55 538.63 83.17 ± 0.07 256 -- -- 575.73 ± 5.49 530.67 97.73 ± 3.00 257 -- Mother 995.77 ± 8.22 468.59 94.49 ± 8.02 879.54 ± 20.53 421.24 102.11 ± 5.69 258 -- -- 876.44 ± 9.21 564.15 89.36 ± 4.66 520.58 ± 8.52 483.28 175.81 ± 53.68 259 -- -- 1095.11 ± 7.88 397.45 85.33 ± 4.07 1050.58 ± 5.08 466.58 139.86 ± 15.48 260 -- -- 1084.13 ± 1.82 480.1 127.84 ± 8.13 494.25 ± 22.05 401.01 188.45 ± 31.29 261 -- -- 745.79 ± 22.70 568.33 122.95 ± 2.89 1150.38 ± 5.64 506.72 206.45 ± 14.75 852.44 ± 31.69 432.45 142.46 ± 27.89 263 -- -- 989.52 ± 4.54 617.16 74.05 ± 5.38 264 -- Uncle 579.22 ± 9.53 580.38 100.39 ± 2.76 265 -- -- 696.52 ± 8.57 491.96 105.88 ± 7.86 848.34 ± 8.38 531.14 106.80 ± 1.16 266 -- -- 728.63 ± 5.47 462.66 78.08 ± 1.06 392.63 ± 9.28 349.34 73.67 ± 3.30 267 -- -- 809.78 ± 2.39 579.14 70.57 ± 2.92 925.13 ± 23.50 827.31 59.18 ± 8.22 268 -- Mother 750.67 ± 17.49 385.93 107.96 ± 12.28 271 -- -- 925.40 ± 10.01 482.89 87.43 ± 12.34 1087.79 ± 22.62 423.61 186.49 ± 10.22 272 -- Aunt 634.87 ± 15.77 531.54 86.12 ± 1.03 515.84 ± 13.88 594.47 30.80 ± 7.99 273 -- -- 455.86 ± 7.52 548.8 91.21 ± 10.34 274 -- -- 856.81 ± 23.09 461.61 103.50 ± 8.99 275 -- -- 943.57 ± 8.27 469.65 66.73 ± 5.64 339.71 ± 8.66 513.42 159.78 ± 30.24 276 -- -- 430.84 ± 16.02 431.09 234.52 ± 26.95 277 -- -- 724.67 ± 0.64 394.65 96.43 ± 0.04 634.03 ± 28.77 659.6 127.07 ± 4.00 278 -- Mother 1045.58 ± 1.10 364.31 106.88 ± 8.57 1118.55 ± 3.48 457.48 234.68 ± 24.37 279 -- -- 978.20 ± 17.94 442.08 85.62 ± 0.14 851.57 ± 67.60 573.28 64.64 280 -- Grand-parents 839.91 ± 4.88 415.23 82.19 ± 6.30 930.08 ± 11.55 468.35 63.88 ± 1.83 281 -- -- 991.09 ± 2.95 522.65 151.89 ± 1.15 655.22 ± 54.74 505.44 112.65 ± 14.80 282 -- -- 732.03 ± 19.20 547.53 138.06 ± 12.04 1196.46 ± 21.91 487.63 129.70 ± 7.80 286 -- -- 499.69 ± 1.97 400.19 130.85 ± 3.82 287 -- -- 602.68 ± 0.65 418.92 190.43 288 -- -- 927.74 ± 4.10 533.37 55.21 ± 10.16 289 -- -- 509.91 ± 5.91 362.72 81.33 ± 11.16 290 -- Aunts 498.69 ± 46.68 507.71 127.53 ± 8.29 291 -- -- 637.03 ± 7.11 467.8 154.54 ± 1.72 292 -- Grand-Mother 691.71 ± 37.30 581.43 76.54 ± 1.66 293 -- -- 494.81 ± 7.56 359.46 166.11 295 -- -- 838.72 ± 39.67 405.48 159.20 ± 22.89 296 -- -- 761.74 ± 25.61 494.27 237.77 297 -- Uncle 768.08 ± 6.70 515.45 100.00 ± 9.41 298 -- -- 750.91 ± 16.94 348.87 290.06 ± 38.15 299 -- -- 625.36 ± 6.80 306.11 135.94 ± 1.36 301 -- Grand-mother 948.83 ± 11.23 578.58 150.57 ± 4.40 302 -- -- 873.77 ± 2.17 373.31 230.66 ± 10.50 303 -- -- 767.96 ± 29.04 458.27 192.45 ± 10.19 304 -- Brother, father, all 493.39 ± 34.21 446.06 185.69 ± 12.07 paternal family 305 -- Mother 533.91 ± 18.09 364.52 123.23 ± 15.87 306 -- Mother 1016.54 ± 23.75 623.32 216.02 ± 19.04 307 -- -- 1328.92 ± 1.50 569.35 165.08 ± 16.63 308 -- Aunts 430.39 ± 5.44 519.72 133.63 ± 11.13 309 -- Mother, cousins 536.77 ± 9.30 485.45 285.92 ± 25.08 311 -- -- 493.18 ± 23.85 546.9 110.66 ± 9.59 313 -- Cousin 536.22 ± 4.65 379.49 99.52 ± 2.41 314 -- Mother 939.67 ± 37.16 549.66 78.11 ± 7.22 315 -- -- 537.59 ± 1.16 481.91 142.26 ± 23.98 316 -- -- 636.17 ± 2.31 576.05 94.21 ±
5.42 318 -- Mother 1151.62 ± 33.64 634.57 112.13 ± 23.16 319 -- Cousin 518.10 ± 27.77 667.02 79.46 ± 6.89 320 -- -- 452.54 ± 10.01 765.38 134.09 ± 21.38 321 -- -- 470.02 ± 16.75 377.13 110.37 ± 12.77 322 -- -- 565.20 ± 48.73 492.94 95.12 ± 7.44 324 -- -- 659.93 ± 14.39 562.52 98.61 ± 6.25 325 -- Mother, grand- 761.48 ± 3.82 846.66 89.91 ± 12.48 parents 326 -- -- 1451.37 ± 77.12 617.35 240.72 ± 27.74 328 -- -- 580.55 ± 24.91 876.97 174.59 329 -- Mother 877.16 ± 27.08 953.41 269.12 ± 4.88 330 -- -- 1403.38 ± 20.98 465.43 279.56 332 -- -- 864.14 ± 43.84 699.27 175.34 ± 30.44 333 -- Cousin 564.09 ± 7.37 762.16 143.10 ± 30.54 334 -- -- 896.91 ± 29.60 727.33 155.95 ± 38.28 335 -- -- 1192.08 ± 14.98 839.56 162.32 ± 0.67 337 -- Sister 914.93 ± 10.71 788.28 114.15 ± 25.71 338 -- Aunt 539.94 ± 1.35 301.42 38.44 ± 5.53 339 -- Grand-father 747.48 ± 9.20 444.12 253.92 340 -- -- 746.48 ± 45.11 498.56 259.46 341 -- Cousin 947.50 ± 31.38 662.73 75.40 ± 1.41 342 -- -- 993.33 ± 55.93 376.73 19.57 ± 5.63 343 -- Grand-mother 996.61 ± 25.86 541.76 43.48 ± 2.96 344 -- -- 637.78 ± 7.73 702.48 26.94 ± 5.89 345 -- Cousin 722.43 ± 18.56 429.44 31.74 ± 1.77 346 -- -- 576.26 ± 24.83 436.35 29.25 ± 2.56 347 -- Sister 1272.11 ± 18.19 425.98 41.20 ± 4.60 348 -- Sister 776.87 ± 50.77 384.51 27.13 ± 1.84 350 -- -- 1020.59 ± 46.63 488.19 32.35 ± 2.16 351 -- Father 557.14 ± 25.67 475.23 20.16 ± 2.76 352 -- Father 1339.62 ± 39.88 566.82 97.02 353 -- -- 1569.33 ± 43.27 607.43 105.59 ± 95.83 354 -- -- 608.88 ± 6.80 431.16 69.78 ± 40.24 355 -- -- 691.05 ± 37.53 378.46 24.41 ± 12.43 356 -- -- 824.89 ± 1.39 467.45 43.63 358 -- -- 554.86 ± 8.43 387.21 116.04 ± 22.53 359 -- -- 709.63 ± 3.85 485.94 195.32 ± 34.14 360 -- Mother 466.35 ± 12.61 335.02 157.17 ± 7.22 361 -- Aunt 899.31 ± 10.09 441.72 81.52 ± 1.47 362 -- Grand-mother 471.73 ± 21.57 437.35 110.36 ± 7.42 363 -- Mother, grand- 743.10 ± 15.01 353.53 161.77 ± 25.40 mother, aunt 364 -- Mother, grand- 767.06 ± 11.17 460.75 160.24 ± 26.97 mother, aunt 365 -- Mother, grand- 883.48 ± 2.32 403.41 127.81 ± 23.58 mother, aunt 368 -- -- 1206.06 ± 43.70 415.24 136.62 ± 28.94 369 -- -- 454.71 ± 13.34 431.44 132.25 ± 19.69 370 -- -- 855.36 ± 10.35 395.7 140.53 ± 2.77 371 -- Aunt, cousin 740.05 ± 5.38 487.74 112.07 ± 3.13 372 -- -- 436.58 ± 40.88 395.61 170.65 ± 13.44 374 -- -- 496.50 ± 28.07 401.4 77.69 ± 6.60 375 -- -- 475.88 ± 0.00 385.69 130.95 ± 3.80 376 -- -- 554.83 ± 44.65 387.61 73.78 ± 0.15 377 -- -- 739.47 ± 8.03 384.16 79.40 ± 1.15 379 -- -- 1404.12 ± 66.84 659.32 78.73 ± 2.62 381 -- -- 782.27 ± 1.42 505.65 283.01 ± 26.97 382 -- -- 998.95 ± 9.12 327.82 77.64 ± 12.98 383 -- -- 900.32 ± 24.08 401.79 83.98 ± 7.31 384 -- -- 479.70 ± 36.72 444.82 134.93 ± 7.83 386 -- -- 732.99 ± 28.62 637.86 129.78 ± 2.15 387 -- Cousin 853.05 ± 70.97 373.81 146.21 ± 6.37 388 -- -- 963.01 ± 40.86 465.02 66.49 ± 7.43 389 -- Father 689.25 ± 35.56 435.9 67.38 ± 15.52 390 -- Father 930.28 ± 18.25 368.83 56.32 ± 0.12 391 -- -- 540.38 ± 9.17 501.81 49.99 ± 7.23 392 -- Brother 661.55 ± 38.23 412.14 77.84 ± 23.22 393 -- Brother 1235.01 ± 29.98 488.02 106.86 ± 17.43 395 -- Mother 716.48 ± 30.93 496.45 82.74 ± 2.92 397 -- Mother, grand- 751.57 ± 2.34 543.59 85.71 ± 21.81 mother 398 -- Mother, grand- 872.92 ± 6.46 526.34 98.45 ± 6.33 mother 399 -- -- 444.55 ± 43.23 481.5 74.45 ± 10.16 400 -- Mother, aunt 1492.58 ± 30.46 477.59 135.22 ± 2.80 401 -- -- 691.24 ± 23.14 316.38 50.01 ± 1.95 402 -- -- 423.93 ± 1.08 314.48 36.64 ± 2.04 403 -- Sister 1216.81 ± 131.72 354.37 52.43 ± 15.76 1264 2005-04-18 -- 616.12 578.96 65.92 1276 2005-05-16 -- 817.56 450.13 107.62 ± 12.96 1364 2006-04-24 Sister, aunt 1668.06 407.4 80.85 ± 6.90 1365 2006-04-26 -- 947.35 642.66 63.18 ± 5.41 1366 2006-05-01 -- 1317.97 323.04 89.70 ± 20.57 1373 2006-05-17 -- 1584.54 583.14 80.12 ± 18.75 1380 2006-06-26 -- 1289.98 602.35 139.38 1384 2006-07-03 -- 1502.51 ± 18.63 194.3 121.65 ± 44.94 1258.85 ± 16.20 448.68 162.01 ± 11.64 1385 2006-07-04 -- 1098.75 523.52 102.35 1387 2006-07-17 Mother 1017.47 689.52 78.42 1388 2006-07-19 -- 1080.53 811.37 87.57 1409 2006-09-26 -- 499.41 ± 67.54 389.14 113.56 ± 15.03 1433 2007-01-10 Uncle 459.61 ± 17.79 287.42 263.55 ± 34.89 1451 2007-03-14 Grand-mother 1099.93 ± 48.11 290.5 158.45 ± 3.94 1478 2007-06-11 Father 619.94 ± 46.51 251.56 190.25 ± 18.46 1481 2007-06-18 -- 748.36 ± 9.30 250.14 95.34 ± 6.52 1483 2007-06-19 -- 489.30 ± 93.18 396.39 167.02 ± 28.62 1487 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 †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° 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 †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, grand- 1650.21 ± 13.90 416.99 199.56 ± 55.60 9.2 2007-04-19 T6 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, grand- 493.98 ± 7.26 463.68 43.99 ± 3.74 6.5 2007-12-12 T8 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-mother 730.44 ± 33.95 397.12 41.87 ± 4.55 13.6 2007-05-02 T7 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-mother 1082.23 ± 65.01 445.66 81.35 ± 14.77 14.9 2007-04-11 T5 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-mother 1573.79 ± 0.72 576.5 142.70 ± 0.57 3.1 2007-07-04 T7 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 40 (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. †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
[0156]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 †Plus-minus values are means ± standard deviations *Other scoliosis types include one neuromuscular scoliosis and one dysplasic scoliosis.
[0157]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. Cobb's Patient Collection Angle Curve ID Date of Birth Gender Age Date Diagnosis Pre-op Type 1208 1990-01-19 M 17.8 2007-10-03 Congenital cyphose scoliosis 72 lT 1256 1992-03-27 M 13.0 2005-05-09 Congenital scoliosis 44-65 rTlL 1278 1998-07-22 F 6.8 2005-05-30 Congenital neurological 60 lT scoliosis 1281 1985-05-21 M 20.1 2005-06-01 Spondylolisthesis 16 -- 1286 1990-05-08 M 15.1 2005-06-15 Dysplasic scoliosis 62-66 rTlL 1356 1993-02-22 F 13.2 2006-04-03 Congenital scoliosis 75 rT 1358 2003-11-09 M 2.4 2006-04-04 Congenital scoliosis 33-35 rTlL 1367 1993-12-12 F 12.4 2006-02-01 Neurological scoliosis 90 lTL 1368 1990-06-21 F 15.9 2006-05-02 Neurological cyphosis 50 lTL 1370 1995-09-15 M 10.7 2006-05-09 Neurological scoliosis 65 rT 1375 1992-09-13 F 13.7 2006-05-30 Congenital scoliosis 53 rTL 1407 1990-12-22 M 16.8 2007-10-31 Spondylolisthesis 9 lL 1431 1987-11-23 M 19.2 2007-01-08 Neurological scoliosis 90-90 rTlT 1432 1992-08-08 M 14.4 2007-01-09 Neurological scoliosis 64 rT 1434 1994-08-07 F 12.4 2007-01-10 Congenital scoliosis 79-77 rTlL 1436 1993-02-16 F 13.9 2007-01-22 Cyphose scoliosis 120 -- 1437 1992-11-06 M 14.2 2007-02-05 Neurological scoliosis 100 NA 1455 1996-12-14 F 10.3 2007-04-03 Congenital cyphose scoliosis 61 lTL 1456 1990-10-03 F 16.5 2007-04-17 Neuromuscular scoliosis 87 rTL 1462 1997-10-22 F 9.5 2007-04-23 Neurological scoliosis 76 lTL 1463 1989-03-19 F 18.1 2007-04-24 Scoliosis + Spondylolisthesis 33 rT 1466 1997-08-24 F 9.8 2007-05-08 Congenital scoliosis 39 rL 1475 1993-05-25 M 14.1 2007-06-05 Cyphose scoliosis 98 -- 1479 1996-01-24 F 11.4 2007-06-05 Neurological scoliosis 90 rTlL 1480 2003-06-13 F 4.0 2007-06-18 Congenital scoliosis 56 lT 1482 1989-03-30 F 18.2 2007-06-19 Spondylolisthesis gr 1 -- NA 1486 1993-01-15 M 14.4 2007-06-27 Spondylolisthesis gr 2 -- NA 357 1996-07-08 F 11.4 2007-12-18 Congenital scoliosis 30-31 rTlT Patient Date of Age at Family [sCD44] [HA] ID Surgery Surgery History [OPN] (ng/ml) (ng/ml) (ng/ml) 1208 2004-11- 14.8 -- 1101.06 ± 31.26 444.81 82.89 ± 15.11 08 1256 2005-03- 13.0 -- 1490.59 NA 127.74 ± 9.29 29 1278 2005-05- 6.8 -- 1401.88 NA 75.65 ± 5.16 30 1281 2005-06- 20.1 -- 985.85 NA 150.30 ± 7.93 01 1286 2005-06- 15.1 -- 549.60 ± 5.06 NA NA 15 1356 2006-04- 13.2 -- 1181.85 NA 111.51 ± 2.30 03 1358 2006-04- 2.4 -- 1530.6 NA 284.60 ± 69.00 04 1367 2006-05- 12.4 -- 1525.13 NA 350.01 ± 36.55 01 1368 2006-05- 15.9 -- 1079.23 NA 126.44 ± 3.63 02 1370 2006-05- 10.7 -- 1318.58 NA 104.06 ± 5.18 09 1375 2006-05- 13.7 Cousin 380.08 ± 12.95 NA NA 30 1407 2006-09- 15.8 -- 818.17 ± 1.52 441.73 116.09 ± 3.88 25 1431 2007-01- 19.2 -- 450.78 ± 101.56 275.62 130.30 ± 23.92 08 1432 2007-01- 14.4 -- 558.47 ± 4.70 145.15 98.99 ± 13.92 09 1434 2007-01- 12.4 -- 631.59 ± 7.42 325.95 44.79 ± 5.73 10 1436 2007-01- 13.9 -- 220.32 ± 2.94 322.03 44.34 ± 6.37 22 1437 2007-02- 14.2 -- 388.01 ± 8.22 225.71 76.96 ± 4.53 05 1455 2007-04- 10.3 -- 1090.51 ± 5.57 323.24 34.79 ± 0.32 03 1456 2007-04- 16.5 -- 622.46 ± 7.15 240.22 NA 17 1462 2007-04- 9.5 -- 1118.25 ± 1.32 607.1 55.90 ± 1.82 23 1463 2007-04- 18.1 -- 751.54 ± 8.69 284.71 21.56 ± 4.58 24 1466 2007-05- 9.8 -- 1110.01 ± 2.38 510.18 47.07 ± 1.48 08 1475 2007-06- 14.1 -- 1123.49 ± 5.56 319.93 166.63 ± 34.63 04 1479 2007-06- 11.4 -- 1098.54 ± 131.44 119.17 NA 05 1480 2007-06- 4.0 -- 809.8 468.03 120.72 ± 40.73 18 1482 2007-06- 18.2 -- 678.49 ± 18.32 187.48 46.07 ± 5.27 19 1486 2007-06- 14.4 -- 924.40 ± 17.16 628.78 47.06 ± 6.84 27 357 -- -- -- 996.58 ± 8.51 423.72 127.33 ± 3.13 *Plus-minus values are means ± standard deviations. †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
[0158]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).
[0159]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.
[0160]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.
[0161]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
[0162]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.
[0163]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
[0164]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).
[0165]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.
[0166]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.
REFERENCES
[0167](1) Brodner W, Krepler P, Nicolakis M et al. Melatonin and adolescent idiopathic scoliosis. J Bone Joint Surg Br 2000; 82(3):399-403. [0168](2) Lowe T G, Edgar M, Margulies J Y et al. Etiology of idiopathic scoliosis: current trends in research. J Bone Joint Surg Am 2000; 82-A(8):1157-1168. [0169](3) Veldhuizen A G, Wever D J, Webb P J. The aetiology of idiopathic scoliosis: biomechanical and neuromuscular factors. Eur Spine J 2000; 9(3):178-184. [0170](4) Miller N H. Cause and natural history of adolescent idiopathic scoliosis. Orthop Clin North Am 1999; 30(3):343-52, vii. [0171](5) Miller N H. Genetics of familial idiopathic scoliosis. Clin Orthop 2002; (401):60-64. [0172](6) Miller N H, Schwab D L, Sponseller P D, Manolio T A, Pugh E W, Wilson A P. Characterization of idiopathic scoliosis in a clinically well-defined population. Clin Orthop 2001; (392):349-357. [0173](7) Wise C A, Barnes R, Gillum J, Herring J A, Bowcock A M, Lovett M. Localization of susceptibility to familial idiopathic scoliosis. Spine 2000; 25(18):2372-2380. [0174](8) Moreau A, Wang D S, Forget S et al. Melatonin Signaling Dysfunction in Adolescent Idiopathic Scoliosis. Spine 2004. [0175](9) Denhardt D T, Noda M, O'Regan A W, Pavlin D, Berman J S. Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival. J Clin Invest 2001; 107(9):1055-1061. [0176](10) Mazzali M, Kipari T, Ophascharoensuk V, Wesson J A, Johnson R, Hughes J. Osteopontin--a molecule for all seasons. QJM 2002; 95(1):3-13. [0177](11) Lopez C A, Olson E S, Adams J C, Mou K, Denhardt D T, Davis R L. Osteopontin expression detected in adult cochleae and inner ear fluids. Hear Res 1995; 85(1-2):210-222. [0178](12) Simoneau M, Richer N, Mercier P, Allard P, Teasdale N. Sensory deprivation and balance control in idiopathic scoliosis adolescent. Exp Brain Res 2006; 170(4):576-582. [0179](13) Guo X, Chau W W, Hui-Chan C W, Cheung C S, Tsang W W, Cheng J C. Balance control in adolescents with idiopathic scoliosis and disturbed somatosensory function. Spine 2006; 31(14):E437-E440. [0180](14) Weber B, Rosel M, Arch R, Moller P, Zoller M. Transient expression of CD44 variant isoforms in the ontogeny of the rat: ectoderm-, endoderm- and mesoderm-derived cells express different exon combinations. Differentiation 1996; 60(1):17-29. [0181](15) Panda D, Kundu G C, Lee B I et al. Potential roles of osteopontin and alphaVbeta3 integrin in the development of coronary artery restenosis after angioplasty. Proc Natl Acad Sci USA 1997; 94(17):9308-9313. [0182](16) Ruiz P, Schwarzler C, Gunthert U. CD44 isoforms during differentiation and development. Bioessays 1995; 17(1):17-24. [0183](17) Katagiri Y U, Sleeman J, Fujii H et al. CD44 variants but not CD44s cooperate with beta1-containing integrins to permit cells to bind to osteopontin independently of arginine-glycine-aspartic acid, thereby stimulating cell motility and chemotaxis. Cancer Res 1999; 59(1):219-226. [0184](18) Jalkanen S, Jalkanen M. Lymphocyte CD44 binds the COOH-terminal heparin-binding domain of fibronectin. J Cell Biol 1992; 116(3):817-825. [0185](19) Naujokas M F, Morin M, Anderson M S, Peterson M, Miller J. The chondroitin sulfate form of invariant chain can enhance stimulation of T cell responses through interaction with CD44. Cell 1993; 74(2):257-268. [0186](20) Weber G F, Ashkar S, Glimcher M J, Cantor H. Receptor-ligand interaction between CD44 and osteopontin (Eta-1). Science 1996; 271(5248):509-512. [0187](21) Bennett K L, Modrell B, Greenfield B et al. Regulation of CD44 binding to hyaluronan by glycosylation of variably spliced exons. J Cell Biol 1995; 131(6 Pt 1):1623-1633. [0188](22) Stamenkovic I, Aruffo A, Amiot M, Seed B. The hematopoietic and epithelial forms of CD44 are distinct polypeptides with different adhesion potentials for hyaluronate-bearing cells. EMBO J. 1991; 10(2):343-348. [0189](23) Komura K, Sato S, Fujimoto M, Hasegawa M, Takehara K. Elevated levels of circulating CD44 in patients with systemic sclerosis: association with a milder subset. Rheumatology (Oxford) 2002; 41(10):1149-1154. [0190](24) Scott D A, Stapleton J A, Palmer R M et al. Plasma concentrations of reputed tumor-associated soluble CD44 isoforms (v5 and v6) in smokers are dose related and decline on smoking cessation. Cancer Epidemiol Biomarkers Prey 2000; 9(11):1211-1214. [0191](25) Wang X, Jiang H, Raso J et al. Characterization of the scoliosis that develops after pinealectomy in the chicken and comparison with adolescent idiopathic scoliosis in humans. Spine 1997; 22(22):2626-2635. [0192](26) von Gall C, Lewy A, Schomerus C et al. Transcription factor dynamics and neuroendocrine signalling in the mouse pineal gland: a comparative analysis of melatonin-deficient C57BL mice and melatonin-proficient C3H mice. Eur J Neurosci 2000; 12(3):964-972. [0193](27) Aherrahrou Z, Axtner S B, Kaczmarek P M et al. A locus on chromosome 7 determines dramatic up-regulation of osteopontin in dystrophic cardiac calcification in mice. Am J Pathol 2004; 164(4):1379-1387. [0194](28) Machida M, Dubousset J, Yamada T et al. Experimental scoliosis in melatonin-deficient C57BL/6J mice without pinealectomy. J Pineal Res 2006; 41(1):1-7. [0195](29) Scoliosis Research Society. Morbidity & Mortality Committee annual report 1997. [0196](30) Mishima R, Takeshima F, Sawai T et al. High plasma osteopontin levels in patients with inflammatory bowel disease. J Clin Gastroenterol 2007; 41(2):167-172. [0197](31) Ang C, Chambers A F, Tuck A B, Winquist E, Izawa J I. Plasma osteopontin levels are predictive of disease stage in patients with transitional cell carcinoma of the bladder. BJU Int 2005; 96(6):803-805. [0198](32) Wong C K, Lit L C, Tam L S, Li E K, Lam C W. Elevation of plasma osteopontin concentration is correlated with disease activity in patients with systemic lupus erythematosus. Rheumatology (Oxford) 2005; 44(5):602-606. [0199](33) Kim J, Ki S S, Lee S D et al. Elevated plasma osteopontin levels in patients with hepatocellular carcinoma. Am J Gastroenterol 2006; 101(9):2051-2059. [0200](34) Wynne-Davies R. Familial (idiopathic) scoliosis. A family survey. J Bone Joint Surg Br 1968; 50(1):24-30. [0201](35) De George F V, Fisher R L. Idiopathic scoliosis: genetic and environmental aspects. J Med Genet. 1967; 4(4):251-257. [0202](36) Lein M, Jung K, Weiss S, Schnorr D, Loening S A. Soluble CD44 variants in the serum of patients with urological malignancies. Oncology 1997; 54(3):226-230. [0203](37) Karjalainen J M, Tammi R H, Tammi M I et al. Reduced level of CD44 and hyaluronan associated with unfavorable prognosis in clinical stage I cutaneous melanoma. Am J Pathol 2000; 157(3):957-965. [0204](38) Schlosser W, Gansauge F, Schlosser S, Gansauge S, Beger H G. Low serum levels of CD44, CD44v6, and neopterin indicate immune dysfunction in chronic pancreatitis. Pancreas 2001; 23(4):335-340. [0205](39) Sjoberg S, Fogelstrand L, Hulthe J, Fagerberg B, Krettek A. Circulating soluble CD44 is higher among women than men and is not associated with cardiovascular risk factors or subclinical atherosclerosis. Metabolism 2005; 54(2):139-141. [0206](40) Jenkins R H, Thomas G J, Williams J D, Steadman R. Myofibroblastic differentiation leads to hyaluronan accumulation through reduced hyaluronan turnover. J Biol Chem 2004; 279(40):41453-41460. [0207](41) Lien Y H, Fu J, Rucker R B, Scheck M, Abbott U, Stern R. Collagen, proteoglycan and hyaluronidase activity in cultures from normal and scoliotic chicken fibroblasts. Biochim Biophys Acta 1990; 1034(3):318-325. [0208](42) Dastych M, Cienciala J. Idiopathic scoliosis and concentrations of zinc, copper, and selenium in blood plasma. Biol Trace Elem Res 2002; 89(2):105-110. [0209](43) El-Bayoumy K, Sinha R. Molecular chemoprevention by selenium: a genomic approach. Mutat Res 2005; 591(1-2):224-236. [0210](44) Unni E, Kittrell F S, Singh U, Sinha R. Osteopontin is a potential target gene in mouse mammary cancer chemoprevention by Se-methylselenocysteine. Breast Cancer Res 2004; 6(5):R586-R592. [0211](45) He Y T, Liu D W, Ding L Y, Li Q, Xiao Y H. Therapeutic effects and molecular mechanisms of anti-fibrosis herbs and selenium on rats with hepatic fibrosis. World J Gastroenterol 2004; 10(5):703-706. [0212](46) Sheehan T M, Gao M. Simplified fluorometric assay of total selenium in plasma and urine. Clin Chem 1990; 36(12):2124-2126. [0213](47) Ando M, Takizawa M, Suwabe S, Yamato S, Shimada K. Determination of selenium in human serum by liquid chromatography/electron capture atmospheric pressure chemical ionization mass spectrometry after acid digestion and derivatization using 2,3-diaminonaphthalene. Eur J Mass Spectrom (Chichester, Eng) 2003; 9(6):619-622. [0214](48) Uchio E, Matsuura N, Kadonosono K, Ohno S, Uede T. Tear osteopontin levels in patients with allergic conjunctival diseases. Graefes ArCh Clin Exp Opthalmol, 2002; 240(11): 924-8. [0215](49) Buck et al. Design Strategies and Performance of Custom DNA Sequencing primers. Biotechniques 1999; 27:528-536. [0216](50) Ponta, H, Sherman L, Herrlich, P A. CD44: from Adhesion molecules to signalling regulators. Nature Reviews. 2004; 4:33-45. [0217](51) Garrett, K. A., P. D. Esker, and A. H. Sparks. 2007. Introduction to the R Programming Environment. The Plant Health Instructor. DOI:10.1094/PHI-A-2007-1226-02. [0218](52) Ihaka R, Gentleman R. A language for data analysis and graphics. Journal of Computational and Graphical Statistics1996, 5(3):299-314. [0219](53) Goodison S, and Tarin D. Clinical Implications Of Anomalous Cd44 Gene Expression In Neoplasia. Frontiers in Bioscience 1998, 3, e89-109. [0220](54) Ito T, Hashimoto Y, Tanaka E, Kan T, Tsunoda S, Sato F, Higashiyama M, Okumura T, Shimada Y. An Inducible Short-Hairpin RNAVector against Osteopontin Reduces Metastatic Potential of Human Esophageal Squamous Cell Carcinoma In vitro and In vivo Clin Cancer Res 2006; 12(4) 1308-1316. [0221](55) Kadkol S S, Lin A Y, Barak V, Kalickman I, Leach L, Valyi-Nagy K, Majumdar D, Setty S, Maniotis A J, Folberg R, Pe'er J. Osteopontin Expression and Serum Levels in Metastatic Uveal Melanoma A Pilot Study Invest Opthalmol V is Sci. 2006; 47(3): 802-806. [0222](56) Guarino V, Faviana P, Salvatore G, Castellone M D, Cirafici A, De Falco V, Celetti A, Giannini R, Basolo F, Melillo R M, Santoro M. Osteopontin Is Overexpressed in Human Papillary Thyroid Carcinomas and Enhances Thyroid Carcinoma Cell Invasiveness. The Journal of Clinical Endocrinology & Metabolism. 2005 90(9):5270-5278. [0223](57) Ponta et al, Nat Rev Mol Cell Biol. 2003 January; 4(1):33-45. Review.
Sequence CWU
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 Ala1 5 10
15Ile Pro Val Lys Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys Gln Leu
20 25 30Tyr Asn Lys Tyr Pro Asp Ala
Val Ala Thr Trp Leu Asn Pro Asp Pro 35 40
45Ser Gln Lys Gln Asn Leu Leu Ala Pro Gln Asn Ala Val Ser Ser
Glu 50 55 60Glu Thr Asn Asp Phe Lys
Gln Glu Thr Leu Pro Ser Lys Ser Asn Glu65 70
75 80Ser His Asp His Met Asp Asp Met Asp Asp Glu
Asp Asp Asp Asp His 85 90
95Val Asp Ser Gln Asp Ser Ile Asp Ser Asn Asp Ser Asp Asp Val Asp
100 105 110Asp Thr Asp Asp Ser His
Gln Ser Asp Glu Ser His His Ser Asp Glu 115 120
125Ser Asp Glu Leu Val Thr Asp Phe Pro Thr Asp Leu Pro Ala
Thr Glu 130 135 140Val Phe Thr Pro Val
Val Pro Thr Val Asp Thr Tyr Asp Gly Arg Gly145 150
155 160Asp Ser Val Val Tyr Gly Leu Arg Ser Lys
Ser Lys Lys Phe Arg Arg 165 170
175Pro Asp Ile Gln Tyr Pro Asp Ala Thr Asp Glu Asp Ile Thr Ser His
180 185 190Met Glu Ser Glu Glu
Leu Asn Gly Ala Tyr Lys Ala Ile Pro Val Ala 195
200 205Gln Asp Leu Asn Ala Pro Ser Asp Trp Asp Ser Arg
Gly Lys Asp Ser 210 215 220Tyr Glu Thr
Ser Gln Leu Asp Asp Gln Ser Ala Glu Thr His Ser His225
230 235 240Lys Gln Ser Arg Leu Tyr Lys
Arg Lys Ala Asn Asp Glu Ser Asn Glu 245
250 255His Ser Asp Val Ile Asp Ser Gln Glu Leu Ser Lys
Val Ser Arg Glu 260 265 270Phe
His Ser His Glu Phe His Ser His Glu Asp Met Leu Val Val Asp 275
280 285Pro Lys Ser Lys Glu Glu Asp Lys His
Leu Lys Phe Arg Ile Ser His 290 295
300Glu Leu Asp Ser Ala Ser Ser Glu Val Asn305
3105300PRTHomo sapiens 5Met Arg Ile Ala Val Ile Cys Phe Cys Leu Leu Gly
Ile Thr Cys Ala1 5 10
15Ile Pro Val Lys Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys Gln Leu
20 25 30Tyr Asn Lys Tyr Pro Asp Ala
Val Ala Thr Trp Leu Asn Pro Asp Pro 35 40
45Ser Gln Lys Gln Asn Leu Leu Ala Pro Gln Thr Leu Pro Ser Lys
Ser 50 55 60Asn Glu Ser His Asp His
Met Asp Asp Met Asp Asp Glu Asp Asp Asp65 70
75 80Asp His Val Asp Ser Gln Asp Ser Ile Asp Ser
Asn Asp Ser Asp Asp 85 90
95Val Asp Asp Thr Asp Asp Ser His Gln Ser Asp Glu Ser His His Ser
100 105 110Asp Glu Ser Asp Glu Leu
Val Thr Asp Phe Pro Thr Asp Leu Pro Ala 115 120
125Thr Glu Val Phe Thr Pro Val Val Pro Thr Val Asp Thr Tyr
Asp Gly 130 135 140Arg Gly Asp Ser Val
Val Tyr Gly Leu Arg Ser Lys Ser Lys Lys Phe145 150
155 160Arg Arg Pro Asp Ile Gln Tyr Pro Asp Ala
Thr Asp Glu Asp Ile Thr 165 170
175Ser His Met Glu Ser Glu Glu Leu Asn Gly Ala Tyr Lys Ala Ile Pro
180 185 190Val Ala Gln Asp Leu
Asn Ala Pro Ser Asp Trp Asp Ser Arg Gly Lys 195
200 205Asp Ser Tyr Glu Thr Ser Gln Leu Asp Asp Gln Ser
Ala Glu Thr His 210 215 220Ser His Lys
Gln Ser Arg Leu Tyr Lys Arg Lys Ala Asn Asp Glu Ser225
230 235 240Asn Glu His Ser Asp Val Ile
Asp Ser Gln Glu Leu Ser Lys Val Ser 245
250 255Arg Glu Phe His Ser His Glu Phe His Ser His Glu
Asp Met Leu Val 260 265 270Val
Asp Pro Lys Ser Lys Glu Glu Asp Lys His Leu Lys Phe Arg Ile 275
280 285Ser His Glu Leu Asp Ser Ala Ser Ser
Glu Val Asn 290 295 3006287PRTHomo
sapiens 6Met Arg Ile Ala Val Ile Cys Phe Cys Leu Leu Gly Ile Thr Cys Ala1
5 10 15Ile Pro Val Lys
Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys Gln Asn 20
25 30Ala Val Ser Ser Glu Glu Thr Asn Asp Phe Lys
Gln Glu Thr Leu Pro 35 40 45Ser
Lys Ser Asn Glu Ser His Asp His Met Asp Asp Met Asp Asp Glu 50
55 60Asp Asp Asp Asp His Val Asp Ser Gln Asp
Ser Ile Asp Ser Asn Asp65 70 75
80Ser Asp Asp Val Asp Asp Thr Asp Asp Ser His Gln Ser Asp Glu
Ser 85 90 95His His Ser
Asp Glu Ser Asp Glu Leu Val Thr Asp Phe Pro Thr Asp 100
105 110Leu Pro Ala Thr Glu Val Phe Thr Pro Val
Val Pro Thr Val Asp Thr 115 120
125Tyr Asp Gly Arg Gly Asp Ser Val Val Tyr Gly Leu Arg Ser Lys Ser 130
135 140Lys Lys Phe Arg Arg Pro Asp Ile
Gln Tyr Pro Asp Ala Thr Asp Glu145 150
155 160Asp Ile Thr Ser His Met Glu Ser Glu Glu Leu Asn
Gly Ala Tyr Lys 165 170
175Ala Ile Pro Val Ala Gln Asp Leu Asn Ala Pro Ser Asp Trp Asp Ser
180 185 190Arg Gly Lys Asp Ser Tyr
Glu Thr Ser Gln Leu Asp Asp Gln Ser Ala 195 200
205Glu Thr His Ser His Lys Gln Ser Arg Leu Tyr Lys Arg Lys
Ala Asn 210 215 220Asp Glu Ser Asn Glu
His Ser Asp Val Ile Asp Ser Gln Glu Leu Ser225 230
235 240Lys Val Ser Arg Glu Phe His Ser His Glu
Phe His Ser His Glu Asp 245 250
255Met Leu Val Val Asp Pro Lys Ser Lys Glu Glu Asp Lys His Leu Lys
260 265 270Phe Arg Ile Ser His
Glu Leu Asp Ser Ala Ser Ser Glu Val Asn 275 280
28575748DNAHomo 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 Pro1 5
10 15Leu 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 45Ala Ala Asp Leu Cys Lys Ala Phe
Asn Ser Thr Leu Pro Thr Met Ala 50 55
60Gln Met Glu Lys Ala Leu Ser Ile Gly Phe Glu Thr Cys Arg Tyr Gly65
70 75 80Phe Ile Glu Gly His
Val Val Ile Pro Arg Ile His Pro Asn Ser Ile 85
90 95Cys Ala Ala Asn Asn Thr Gly Val Tyr Ile Leu
Thr Ser Asn Thr Ser 100 105
110Gln Tyr Asp Thr Tyr Cys Phe Asn Ala Ser Ala Pro Pro Glu Glu Asp
115 120 125Cys Thr Ser Val Thr Asp Leu
Pro Asn Ala Phe Asp Gly Pro Ile Thr 130 135
140Ile Thr Ile Val Asn Arg Asp Gly Thr Arg Tyr Val Gln Lys Gly
Glu145 150 155 160Tyr Arg
Thr Asn Pro Glu Asp Ile Tyr Pro Ser Asn Pro Thr Asp Asp
165 170 175Asp Val Ser Ser Gly Ser Ser
Ser Glu Arg Ser Ser Thr Ser Gly Gly 180 185
190Tyr Ile Phe Tyr Thr Phe Ser Thr Val His Pro Ile Pro Asp
Glu Asp 195 200 205Ser Pro Trp Ile
Thr Asp Ser Thr Asp Arg Ile Pro Ala Thr Thr Leu 210
215 220Met Ser Thr Ser Ala Thr Ala Thr Glu Thr Ala Thr
Lys Arg Gln Glu225 230 235
240Thr Trp Asp Trp Phe Ser Trp Leu Phe Leu Pro Ser Glu Ser Lys Asn
245 250 255His Leu His Thr Thr
Thr Gln Met Ala Gly Thr Ser Ser Asn Thr Ile 260
265 270Ser Ala Gly Trp Glu Pro Asn Glu Glu Asn Glu Asp
Glu Arg Asp Arg 275 280 285His Leu
Ser Phe Ser Gly Ser Gly Ile Asp Asp Asp Glu Asp Phe Ile 290
295 300Ser Ser Thr Ile Ser Thr Thr Pro Arg Ala Phe
Asp His Thr Lys Gln305 310 315
320Asn Gln Asp Trp Thr Gln Trp Asn Pro Ser His Ser Asn Pro Glu Val
325 330 335Leu Leu Gln Thr
Thr Thr Arg Met Thr Asp Val Asp Arg Asn Gly Thr 340
345 350Thr Ala Tyr Glu Gly Asn Trp Asn Pro Glu Ala
His Pro Pro Leu Ile 355 360 365His
His Glu His His Glu Glu Glu Glu Thr Pro His Ser Thr Ser Thr 370
375 380Ile Gln Ala Thr Pro Ser Ser Thr Thr Glu
Glu Thr Ala Thr Gln Lys385 390 395
400Glu Gln Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gln Thr
Pro 405 410 415Lys Glu Asp
Ser His Ser Thr Thr Gly Thr Ala Ala Ala Ser Ala His 420
425 430Thr Ser His Pro Met Gln Gly Arg Thr Thr
Pro Ser Pro Glu Asp Ser 435 440
445Ser Trp Thr Asp Phe Phe Asn Pro Ile Ser His Pro Met Gly Arg Gly 450
455 460His Gln Ala Gly Arg Arg Met Asp
Met Asp Ser Ser His Ser Ile Thr465 470
475 480Leu Gln Pro Thr Ala Asn Pro Asn Thr Gly Leu Val
Glu Asp Leu Asp 485 490
495Arg Thr Gly Pro Leu Ser Met Thr Thr Gln Gln Ser Asn Ser Gln Ser
500 505 510Phe Ser Thr Ser His Glu
Gly Leu Glu Glu Asp Lys Asp His Pro Thr 515 520
525Thr Ser Thr Leu Thr Ser Ser Asn Arg Asn Asp Val Thr Gly
Gly Arg 530 535 540Arg Asp Pro Asn His
Ser Glu Gly Ser Thr Thr Leu Leu Glu Gly Tyr545 550
555 560Thr Ser His Tyr Pro His Thr Lys Glu Ser
Arg Thr Phe Ile Pro Val 565 570
575Thr Ser Ala Lys Thr Gly Ser Phe Gly Val Thr Ala Val Thr Val Gly
580 585 590Asp Ser Asn Ser Asn
Val Asn Arg Ser Leu Ser Gly Asp Gln Asp Thr 595
600 605Phe His Pro Ser Gly Gly Ser His Thr Thr His Gly
Ser Glu Ser Asp 610 615 620Gly His Ser
His Gly Ser Gln Glu Gly Gly Ala Asn Thr Thr Ser Gly625
630 635 640Pro Ile Arg Thr Pro Gln Ile
Pro Glu Trp Leu Ile Ile Leu Ala Ser 645
650 655Leu Leu Ala Leu Ala Leu Ile Leu Ala Val Cys Ile
Ala Val Asn Ser 660 665 670Arg
Arg Arg Cys Gly Gln Lys Lys Lys Leu Val Ile Asn Ser Gly Asn 675
680 685Gly Ala Val Glu Asp Arg Lys Pro Ser
Gly Leu Asn Gly Glu Ala Ser 690 695
700Lys Ser Gln Glu Met Val His Leu Val Asn Lys Glu Ser Ser Glu Thr705
710 715 720Pro Asp Gln Phe
Met Thr Ala Asp Glu Thr Arg Asn Leu Gln Asn Val 725
730 735Asp Met Lys Ile Gly Val
74014699PRTHomo sapiens 14Met Asp Lys Phe Trp Trp His Ala Ala Trp Gly Leu
Cys Leu Val Pro1 5 10
15Leu Ser Leu Ala Gln Ile Asp Leu Asn Ile Thr Cys Arg Phe Ala Gly
20 25 30Val Phe His Val Glu Lys Asn
Gly Arg Tyr Ser Ile Ser Arg Thr Glu 35 40
45Ala Ala Asp Leu Cys Lys Ala Phe Asn Ser Thr Leu Pro Thr Met
Ala 50 55 60Gln Met Glu Lys Ala Leu
Ser Ile Gly Phe Glu Thr Cys Arg Tyr Gly65 70
75 80Phe Ile Glu Gly His Val Val Ile Pro Arg Ile
His Pro Asn Ser Ile 85 90
95Cys Ala Ala Asn Asn Thr Gly Val Tyr Ile Leu Thr Ser Asn Thr Ser
100 105 110Gln Tyr Asp Thr Tyr Cys
Phe Asn Ala Ser Ala Pro Pro Glu Glu Asp 115 120
125Cys Thr Ser Val Thr Asp Leu Pro Asn Ala Phe Asp Gly Pro
Ile Thr 130 135 140Ile Thr Ile Val Asn
Arg Asp Gly Thr Arg Tyr Val Gln Lys Gly Glu145 150
155 160Tyr Arg Thr Asn Pro Glu Asp Ile Tyr Pro
Ser Asn Pro Thr Asp Asp 165 170
175Asp Val Ser Ser Gly Ser Ser Ser Glu Arg Ser Ser Thr Ser Gly Gly
180 185 190Tyr Ile Phe Tyr Thr
Phe Ser Thr Val His Pro Ile Pro Asp Glu Asp 195
200 205Ser Pro Trp Ile Thr Asp Ser Thr Asp Arg Ile Pro
Ala Thr Ser Thr 210 215 220Ser Ser Asn
Thr Ile Ser Ala Gly Trp Glu Pro Asn Glu Glu Asn Glu225
230 235 240Asp Glu Arg Asp Arg His Leu
Ser Phe Ser Gly Ser Gly Ile Asp Asp 245
250 255Asp Glu Asp Phe Ile Ser Ser Thr Ile Ser Thr Thr
Pro Arg Ala Phe 260 265 270Asp
His Thr Lys Gln Asn Gln Asp Trp Thr Gln Trp Asn Pro Ser His 275
280 285Ser Asn Pro Glu Val Leu Leu Gln Thr
Thr Thr Arg Met Thr Asp Val 290 295
300Asp Arg Asn Gly Thr Thr Ala Tyr Glu Gly Asn Trp Asn Pro Glu Ala305
310 315 320His Pro Pro Leu
Ile His His Glu His His Glu Glu Glu Glu Thr Pro 325
330 335His Ser Thr Ser Thr Ile Gln Ala Thr Pro
Ser Ser Thr Thr Glu Glu 340 345
350Thr Ala Thr Gln Lys Glu Gln Trp Phe Gly Asn Arg Trp His Glu Gly
355 360 365Tyr Arg Gln Thr Pro Lys Glu
Asp Ser His Ser Thr Thr Gly Thr Ala 370 375
380Ala Ala Ser Ala His Thr Ser His Pro Met Gln Gly Arg Thr Thr
Pro385 390 395 400Ser Pro
Glu Asp Ser Ser Trp Thr Asp Phe Phe Asn Pro Ile Ser His
405 410 415Pro Met Gly Arg Gly His Gln
Ala Gly Arg Arg Met Asp Met Asp Ser 420 425
430Ser His Ser Ile Thr Leu Gln Pro Thr Ala Asn Pro Asn Thr
Gly Leu 435 440 445Val Glu Asp Leu
Asp Arg Thr Gly Pro Leu Ser Met Thr Thr Gln Gln 450
455 460Ser Asn Ser Gln Ser Phe Ser Thr Ser His Glu Gly
Leu Glu Glu Asp465 470 475
480Lys Asp His Pro Thr Thr Ser Thr Leu Thr Ser Ser Asn Arg Asn Asp
485 490 495Val Thr Gly Gly Arg
Arg Asp Pro Asn His Ser Glu Gly Ser Thr Thr 500
505 510Leu Leu Glu Gly Tyr Thr Ser His Tyr Pro His Thr
Lys Glu Ser Arg 515 520 525Thr Phe
Ile Pro Val Thr Ser Ala Lys Thr Gly Ser Phe Gly Val Thr 530
535 540Ala Val Thr Val Gly Asp Ser Asn Ser Asn Val
Asn Arg Ser Leu Ser545 550 555
560Gly Asp Gln Asp Thr Phe His Pro Ser Gly Gly Ser His Thr Thr His
565 570 575Gly Ser Glu Ser
Asp Gly His Ser His Gly Ser Gln Glu Gly Gly Ala 580
585 590Asn Thr Thr Ser Gly Pro Ile Arg Thr Pro Gln
Ile Pro Glu Trp Leu 595 600 605Ile
Ile Leu Ala Ser Leu Leu Ala Leu Ala Leu Ile Leu Ala Val Cys 610
615 620Ile Ala Val Asn Ser Arg Arg Arg Cys Gly
Gln Lys Lys Lys Leu Val625 630 635
640Ile Asn Ser Gly Asn Gly Ala Val Glu Asp Arg Lys Pro Ser Gly
Leu 645 650 655Asn Gly Glu
Ala Ser Lys Ser Gln Glu Met Val His Leu Val Asn Lys 660
665 670Glu Ser Ser Glu Thr Pro Asp Gln Phe Met
Thr Ala Asp Glu Thr Arg 675 680
685Asn Leu Gln Asn Val Asp Met Lys Ile Gly Val 690
69515493PRTHomo sapiens 15Met Asp Lys Phe Trp Trp His Ala Ala Trp Gly Leu
Cys Leu Val Pro1 5 10
15Leu Ser Leu Ala Gln Ile Asp Leu Asn Ile Thr Cys Arg Phe Ala Gly
20 25 30Val Phe His Val Glu Lys Asn
Gly Arg Tyr Ser Ile Ser Arg Thr Glu 35 40
45Ala Ala Asp Leu Cys Lys Ala Phe Asn Ser Thr Leu Pro Thr Met
Ala 50 55 60Gln Met Glu Lys Ala Leu
Ser Ile Gly Phe Glu Thr Cys Arg Tyr Gly65 70
75 80Phe Ile Glu Gly His Val Val Ile Pro Arg Ile
His Pro Asn Ser Ile 85 90
95Cys Ala Ala Asn Asn Thr Gly Val Tyr Ile Leu Thr Ser Asn Thr Ser
100 105 110Gln Tyr Asp Thr Tyr Cys
Phe Asn Ala Ser Ala Pro Pro Glu Glu Asp 115 120
125Cys Thr Ser Val Thr Asp Leu Pro Asn Ala Phe Asp Gly Pro
Ile Thr 130 135 140Ile Thr Ile Val Asn
Arg Asp Gly Thr Arg Tyr Val Gln Lys Gly Glu145 150
155 160Tyr Arg Thr Asn Pro Glu Asp Ile Tyr Pro
Ser Asn Pro Thr Asp Asp 165 170
175Asp Val Ser Ser Gly Ser Ser Ser Glu Arg Ser Ser Thr Ser Gly Gly
180 185 190Tyr Ile Phe Tyr Thr
Phe Ser Thr Val His Pro Ile Pro Asp Glu Asp 195
200 205Ser Pro Trp Ile Thr Asp Ser Thr Asp Arg Ile Pro
Ala Thr Asn Met 210 215 220Asp Ser Ser
His Ser Ile Thr Leu Gln Pro Thr Ala Asn Pro Asn Thr225
230 235 240Gly Leu Val Glu Asp Leu Asp
Arg Thr Gly Pro Leu Ser Met Thr Thr 245
250 255Gln Gln Ser Asn Ser Gln Ser Phe Ser Thr Ser His
Glu Gly Leu Glu 260 265 270Glu
Asp Lys Asp His Pro Thr Thr Ser Thr Leu Thr Ser Ser Asn Arg 275
280 285Asn Asp Val Thr Gly Gly Arg Arg Asp
Pro Asn His Ser Glu Gly Ser 290 295
300Thr Thr Leu Leu Glu Gly Tyr Thr Ser His Tyr Pro His Thr Lys Glu305
310 315 320Ser Arg Thr Phe
Ile Pro Val Thr Ser Ala Lys Thr Gly Ser Phe Gly 325
330 335Val Thr Ala Val Thr Val Gly Asp Ser Asn
Ser Asn Val Asn Arg Ser 340 345
350Leu Ser Gly Asp Gln Asp Thr Phe His Pro Ser Gly Gly Ser His Thr
355 360 365Thr His Gly Ser Glu Ser Asp
Gly His Ser His Gly Ser Gln Glu Gly 370 375
380Gly Ala Asn Thr Thr Ser Gly Pro Ile Arg Thr Pro Gln Ile Pro
Glu385 390 395 400Trp Leu
Ile Ile Leu Ala Ser Leu Leu Ala Leu Ala Leu Ile Leu Ala
405 410 415Val Cys Ile Ala Val Asn Ser
Arg Arg Arg Cys Gly Gln Lys Lys Lys 420 425
430Leu Val Ile Asn Ser Gly Asn Gly Ala Val Glu Asp Arg Lys
Pro Ser 435 440 445Gly Leu Asn Gly
Glu Ala Ser Lys Ser Gln Glu Met Val His Leu Val 450
455 460Asn Lys Glu Ser Ser Glu Thr Pro Asp Gln Phe Met
Thr Ala Asp Glu465 470 475
480Thr Arg Asn Leu Gln Asn Val Asp Met Lys Ile Gly Val
485 49016361PRTHomo sapiens 16 Met Asp Lys Phe Trp Trp
His Ala Ala Trp Gly Leu Cys Leu Val Pro1 5
10 15Leu Ser Leu Ala Gln Ile Asp Leu Asn Ile Thr Cys
Arg Phe Ala Gly 20 25 30Val
Phe His Val Glu Lys Asn Gly Arg Tyr Ser Ile Ser Arg Thr Glu 35
40 45Ala Ala Asp Leu Cys Lys Ala Phe Asn
Ser Thr Leu Pro Thr Met Ala 50 55
60Gln Met Glu Lys Ala Leu Ser Ile Gly Phe Glu Thr Cys Arg Tyr Gly65
70 75 80Phe Ile Glu Gly His
Val Val Ile Pro Arg Ile His Pro Asn Ser Ile 85
90 95Cys Ala Ala Asn Asn Thr Gly Val Tyr Ile Leu
Thr Ser Asn Thr Ser 100 105
110Gln Tyr Asp Thr Tyr Cys Phe Asn Ala Ser Ala Pro Pro Glu Glu Asp
115 120 125Cys Thr Ser Val Thr Asp Leu
Pro Asn Ala Phe Asp Gly Pro Ile Thr 130 135
140Ile Thr Ile Val Asn Arg Asp Gly Thr Arg Tyr Val Gln Lys Gly
Glu145 150 155 160Tyr Arg
Thr Asn Pro Glu Asp Ile Tyr Pro Ser Asn Pro Thr Asp Asp
165 170 175Asp Val Ser Ser Gly Ser Ser
Ser Glu Arg Ser Ser Thr Ser Gly Gly 180 185
190Tyr Ile Phe Tyr Thr Phe Ser Thr Val His Pro Ile Pro Asp
Glu Asp 195 200 205Ser Pro Trp Ile
Thr Asp Ser Thr Asp Arg Ile Pro Ala Thr Arg Asp 210
215 220Gln Asp Thr Phe His Pro Ser Gly Gly Ser His Thr
Thr His Gly Ser225 230 235
240Glu Ser Asp Gly His Ser His Gly Ser Gln Glu Gly Gly Ala Asn Thr
245 250 255Thr Ser Gly Pro Ile
Arg Thr Pro Gln Ile Pro Glu Trp Leu Ile Ile 260
265 270Leu Ala Ser Leu Leu Ala Leu Ala Leu Ile Leu Ala
Val Cys Ile Ala 275 280 285Val Asn
Ser Arg Arg Arg Cys Gly Gln Lys Lys Lys Leu Val Ile Asn 290
295 300Ser Gly Asn Gly Ala Val Glu Asp Arg Lys Pro
Ser Gly Leu Asn Gly305 310 315
320Glu Ala Ser Lys Ser Gln Glu Met Val His Leu Val Asn Lys Glu Ser
325 330 335Ser Glu Thr Pro
Asp Gln Phe Met Thr Ala Asp Glu Thr Arg Asn Leu 340
345 350Gln Asn Val Asp Met Lys Ile Gly Val
355 36017139PRTHomo sapiens 17Met Asp Lys Phe Trp Trp His
Ala Ala Trp Gly Leu Cys Leu Val Pro1 5 10
15Leu Ser Leu Ala Gln Ile Asp Leu Asn Ile Thr Cys Arg
Phe Ala Gly 20 25 30Val Phe
His Val Glu Lys Asn Gly Arg Tyr Ser Ile Ser Arg Thr Glu 35
40 45Ala Ala Asp Leu Cys Lys Ala Phe Asn Ser
Thr Leu Pro Thr Met Ala 50 55 60Gln
Met Glu Lys Ala Leu Ser Ile Gly Phe Glu Thr Cys Ser Leu His65
70 75 80Cys Ser Gln Gln Ser Lys
Lys Val Trp Ala Glu Glu Lys Ala Ser Asp 85
90 95Gln Gln Trp Gln Trp Ser Cys Gly Gly Gln Lys Ala
Lys Trp Thr Gln 100 105 110Arg
Arg Gly Gln Gln Val Ser Gly Asn Gly Ala Phe Gly Glu Gln Gly 115
120 125Val Val Arg Asn Ser Arg Pro Val Tyr
Asp Ser 130 13518337PRTHomo sapiens 18Thr Ser Ser Asn
Thr Ile Ser Ala Gly Trp Glu Pro Asn Glu Glu Asn1 5
10 15Glu Asp Glu Arg Asp Arg His Leu Ser Phe
Ser Gly Ser Gly Ile Asp 20 25
30Asp Asp Glu Asp Phe Ile Ser Ser Thr Ile Ser Thr Thr Pro Arg Ala
35 40 45Phe Asp His Thr Lys Gln Asn Gln
Asp Trp Thr Gln Trp Asn Pro Ser 50 55
60His Ser Asn Pro Glu Val Leu Leu Gln Thr Thr Thr Arg Met Thr Asp65
70 75 80Val Asp Arg Asn Gly
Thr Thr Ala Tyr Glu Gly Asn Trp Asn Pro Glu 85
90 95Ala His Pro Pro Leu Ile His His Glu His His
Glu Glu Glu Glu Thr 100 105
110Pro His Ser Thr Ser Thr Ile Gln Ala Thr Pro Ser Ser Thr Thr Glu
115 120 125Glu Thr Ala Thr Gln Lys Glu
Gln Trp Phe Gly Asn Arg Trp His Glu 130 135
140Gly Tyr Arg Gln Thr Pro Arg Glu Asp Ser His Ser Thr Thr Gly
Thr145 150 155 160Ala Ala
Ala Ser Ala His Thr Ser His Pro Met Gln Gly Arg Thr Thr
165 170 175Pro Ser Pro Glu Asp Ser Ser
Trp Thr Asp Phe Phe Asn Pro Ile Ser 180 185
190His Pro Met Gly Arg Gly His Gln Ala Gly Arg Arg Met Asp
Met Asp 195 200 205Ser Ser His Ser
Thr Thr Leu Gln Pro Thr Ala Asn Pro Asn Thr Gly 210
215 220Leu Val Glu Asp Leu Asp Arg Thr Gly Pro Leu Ser
Met Thr Thr Gln225 230 235
240Gln Ser Asn Ser Gln Ser Phe Ser Thr Ser His Glu Gly Leu Glu Glu
245 250 255Asp Lys Asp His Pro
Thr Thr Ser Thr Leu Thr Ser Ser Asn Arg Asn 260
265 270Asp Val Thr Gly Gly Arg Arg Asp Pro Asn His Ser
Glu Gly Ser Thr 275 280 285Thr Leu
Leu Glu Gly Tyr Thr Ser His Tyr Pro His Thr Lys Glu Ser 290
295 300Arg Thr Phe Ile Pro Val Thr Ser Ala Lys Thr
Gly Ser Phe Gly Val305 310 315
320Thr Ala Val Thr Val Gly Asp Ser Asn Ser Asn Val Asn Arg Ser Leu
325 330
335Ser1921DNAArtificial 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 Lys1
5 10 15Asn Gly Arg Tyr Ser Ile
Ser Arg Thr Glu Ala Ala Asp Leu Cys Lys 20 25
30Ala Phe Asn Ser Thr Leu Pro Thr Met Ala Gln Met Glu
Lys Ala Leu 35 40 45Ser Ile Gly
Phe Glu Thr Cys Arg Tyr Gly Phe Ile Glu Gly His Val 50
55 60Val Ile Pro Arg Ile His Pro Asn Ser Ile Cys Ala
Ala Asn Asn Thr65 70 75
80Gly Val Tyr Ile Leu Thr Ser Asn Thr Ser Gln Tyr Asp Thr Tyr Cys
85 90 95Phe Asn Ala Ser Ala Pro
Pro Glu Glu Asp Cys Thr Ser Val Thr Asp 100
105 110Leu Pro Asn Ala Phe Asp Gly Pro Ile Thr Ile Thr
Ile Val Asn Arg 115 120 125Asp Gly
Thr Arg Tyr Val Gln Lys Gly Glu Tyr Arg Thr Asn Pro Glu 130
135 140Asp Ile Tyr Pro Ser Asn Pro Thr Asp Asp Asp
Val Ser Ser Gly Ser145 150 155
160Ser Ser Glu Arg Ser Ser Thr Ser Gly Gly Tyr Ile Phe Tyr Thr Phe
165 170 175Ser Thr Val His
Pro Ile Pro Asp Glu Asp Ser Pro Trp Ile Thr Asp 180
185 190Ser Thr Asp Arg Ile Pro Ala Thr Thr Leu Met
Ser Thr Ser Ala Thr 195 200 205Ala
Thr Glu Thr Ala Thr Lys Arg Gln Glu Thr Trp Asp Trp Phe Ser 210
215 220Trp Leu Phe Leu Pro Ser Glu Ser Lys Asn
His Leu His Thr Thr Thr225 230 235
240Gln Met Ala Gly Thr Ser Ser Asn Thr Ile Ser Ala Gly Trp Glu
Pro 245 250 255Asn Glu Glu
Asn Glu Asp Glu Arg Asp Arg His Leu Ser Phe Ser Gly 260
265 270Ser Gly Ile Asp Asp Asp Glu Asp Phe Ile
Ser Ser Thr Ile Ser Thr 275 280
285Thr Pro Arg Ala Phe Asp His Thr Lys Gln Asn Gln Asp Trp Thr Gln 290
295 300Trp Asn Pro Ser His Ser Asn Pro
Glu Val Leu Leu Gln Thr Thr Thr305 310
315 320Arg Met Thr Asp Val Asp Arg Asn Gly Thr Thr Ala
Tyr Glu Gly Asn 325 330
335Trp Asn Pro Glu Ala His Pro Pro Leu Ile His His Glu His His Glu
340 345 350Glu Glu Glu Thr Pro His
Ser Thr Ser Thr Ile Gln Ala Thr Pro Ser 355 360
365Ser Thr Thr Glu Glu Thr Ala Thr Gln Lys Glu Gln Trp Phe
Gly Asn 370 375 380Arg Trp His Glu Gly
Tyr Arg Gln Thr Pro Lys Glu Asp Ser His Ser385 390
395 400Thr Thr Gly Thr Ala Ala Ala Ser Ala His
Thr Ser His Pro Met Gln 405 410
415Gly Arg Thr Thr Pro Ser Pro Glu Asp Ser Ser Trp Thr Asp Phe Phe
420 425 430Asn Pro Ile Ser His
Pro Met Gly Arg Gly His Gln Ala Gly Arg Arg 435
440 445Met Asp Met Asp Ser Ser His Ser Ile Thr Leu Gln
Pro Thr Ala Asn 450 455 460Pro Asn Thr
Gly Leu Val Glu Asp Leu Asp Arg Thr Gly Pro Leu Ser465
470 475 480Met Thr Thr Gln Gln Ser Asn
Ser Gln Ser Phe Ser Thr Ser His Glu 485
490 495Gly Leu Glu Glu Asp Lys Asp His Pro Thr Thr Ser
Thr Leu Thr Ser 500 505 510Ser
Asn Arg Asn Asp Val Thr Gly Gly Arg Arg Asp Pro Asn His Ser 515
520 525Glu Gly Ser Thr Thr Leu Leu Glu Gly
Tyr Thr Ser His Tyr Pro His 530 535
540Thr Lys Glu Ser Arg Thr Phe Ile Pro Val Thr Ser Ala Lys Thr Gly545
550 555 560Ser Phe Gly Val
Thr Ala Val Thr Val Gly Asp Ser Asn Ser Asn Val 565
570 575Asn Arg Ser Leu Ser Gly Asp Gln Asp Thr
Phe His Pro Ser Gly Gly 580 585
590Ser His Thr Thr His Gly Ser Glu Ser Asp Gly His Ser His Gly Ser
595 600 605Gln Glu Gly Gly Ala Asn Thr
Thr Ser Gly Pro Ile Arg Thr Pro Gln 610 615
620Ile Pro Glu Trp Leu Ile Ile Leu Ala Ser Leu Leu Ala Leu Ala
Leu625 630 635 640Ile Leu
Ala Val Cys Ile Ala Val Asn Ser Arg Arg Arg Cys Gly Gln
645 650 655Lys Lys Lys Leu Val Ile Asn
Ser Gly Asn Gly Ala Val Glu Asp Arg 660 665
670Lys Pro Ser Gly Leu Asn Gly Glu Ala Ser Lys Ser Gln Glu
Met Val 675 680 685His Leu Val Asn
Lys Glu Ser Ser Glu Thr Pro Asp Gln Phe Met Thr 690
695 700Ala Asp Glu Thr Arg Asn Leu Gln Asn Val Asp Met
Lys Ile Gly Val705 710 715
720
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