HK1219895B - A method of increasing gipcr signalization in the cells of a scoliotic subject - Google Patents

Description

A method for increasing GIPCR signaling in cells from subjects with scoliosis

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is PCT application serial number PCT/CA2014/*, filed on June 17, 2014, and published in English under PCT Article 21(2), which claims the benefit of U.S. Provisional Application Serial No. 61/835,926, filed on June 17, 2013. All of the above documents are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

not applicable.

Field of the Invention

The present invention relates to a method of increasing GiPCR signaling in cells of a subject with scoliosis.

Reference to a sequence listing

Pursuant to 37 C.F.R. 1.821(c), the Sequence Listing is hereby submitted as an ASCII-compatible text file named 14033_121_ST25.txt, which was created on June 17, 2014 and has a size of 49 kilobytes. The contents of the above-mentioned file are hereby incorporated by reference in their entirety.

Background of the Invention

Idiopathic scoliosis is an unexplained spinal deformity that is generally defined as a sideways curve greater than 10 degrees with associated spinal rotation. ¹ Adolescent idiopathic scoliosis (AIS) is one of the most frequent childhood deformities worldwide, characterized by an unexplained 3D spinal deformity and representing both an immediate medical challenge and a chronic condition that affects individuals throughout their lives. It is the most common orthopedic condition requiring surgery in adolescents, affecting 4% of this population. The condition is most commonly diagnosed between the ages of 9 and 13 years. ^2,3,4 The diagnosis is primarily one of exclusion and is made only after other causes of spinal deformity, such as vertebral deformities, neuromuscular disorders, or syndromic conditions have been ruled out. Traditionally, trunk asymmetry is revealed during a physical examination using the Adams flexion test and measured with a scoliometer. The diagnosis can then be confirmed using the Cobb method by radiographic observation of the curve and angle measurement.

Once diagnosed, a physician's primary concern in managing children with scoliosis is whether the curve will progress. Indeed, curve progression is often unpredictable and is observed more frequently among girls than among boys. If untreated, the curve can progress rapidly, resulting in significant physical deformities and even cardiopulmonary problems. When the curve exceeds 70 degrees, these manifestations become life-threatening. Current treatment options for preventing or halting curve progression include braces and surgery. Generally, braces are recommended for curves between 25 and 40 degrees, while surgery is reserved for curves greater than 45 degrees or those that do not respond to braces. Currently in the United States, approximately one million children between the ages of 10 and 16 have some degree of idiopathic scoliosis. Approximately 10% of children diagnosed with idiopathic scoliosis have a curve progression that requires corrective surgery. Approximately 29,000 scoliosis surgeries are performed annually in North America, resulting in significant psychological and physical morbidity. (Goldberg MS, Mayo NE, Poitras B, et al. The Ste-Justine Adolescent IdiopathicScoliosis Cohort Study.Part I: Description of the study. Spine 1994; 19:1551-61; Poitras B, Mayo NE, Goldberg MS, et al. The Ste-Justine Adolescent IdiopathicScoliosis Cohort Study. Part IV: Surgical correction and back pain. Spine 1994;19:1582-8).

Currently, there are no reliable methods or tests available to identify subjects at risk of developing IS to predict which affected individuals require treatment to prevent or halt the progression of the disease, allowing for early provision of appropriate treatment and preventing surgical complications and cardiac and/or respiratory problems (Weinstein SL, Dolan LA, Cheng JC et al. Adolescent idiopathic scoliosis. Lancet 2008;371:1527-37).

Therefore, the application of current treatments such as braces or surgical correction is delayed until significant deformity is detected or until significant progression is clearly demonstrated, resulting in delayed, less than optimal treatment and often severe psychological sequelae (Society SR. Morbidity & Mortality Committee annual Report 1997).

Currently, in order to detect the deformity, diagnosed children are subjected to multiple radiographs over several years, usually until they reach skeletal maturity. It is estimated that a typical patient with scoliosis will have approximately 22 radiographic examinations over a 3-year period. Multiple radiographic examinations present potential risks. Also for this reason, an alternative method that would allow for the prognosis of idiopathic scoliosis is highly desirable.

A major limitation in developing diagnostic tests that can aid in treatment selection for patients is the heterogeneous nature of AIS. At the clinical level, the heterogeneity of AIS is clearly demonstrated by variability in curve morphology, localization, and curve amplitude, even in families with multiple affected members.

In the absence of a reliable phenotype for AIS, a better understanding of the molecular changes associated with disease onset and progression of spinal deformity is needed. Molecular definitions of disease are rapidly replacing traditional pathology-based descriptions of the disease, in part due to their utility in identifying the optimal treatment options for patients.

To this end, differential melatonin signaling dysfunction has been reported in AIS patients, leading to their stratification into three functional groups or biological endophenotypes (Moreau et al., 2004); (Azeddine et al., 2007); (Letellier et al., 2008) and Moreau WO2003/073102. More specifically, AIS patients were stratified into three functional groups (FG1, FG2, and FG3) representing distinct biological endophenotypes. According to this stratification, scoliotic patients and children at increased risk of developing scoliosis were less responsive to Gi protein stimulation compared to healthy control subjects, and the classification was based on the percentage reduction relative to the control group. The classification ranges were fixed at approximately 10% to 40% reduction in response for FG3, approximately 40% to 60% reduction in response for FG2, and approximately 60% to 90% reduction in response for FG1 relative to the control group.

Recently, using cellular dielectric spectroscopy (CDS), a label-free method for functional evaluation of G proteins and endogenous receptors coupled to these proteins (Verdonk et al., 2006), it was found that the cellular response after stimulation of the melatonin receptor by melatonin was primarily G-dependent in normal osteoblasts and was reduced to varying degrees in osteoblasts derived from AIS patients (Akoume et al., 2010). Approximately 33% of asymptomatic children diagnosed with defective G protein function develop scoliosis many years later (Akoume et al., 2010).

Early detection/prognosis of scoliosis is not only crucial for successful and less invasive clinical outcomes, but also expands the range of treatment options available to clinicians. Indeed, improving patient stratification and disease staging represents a key step in selecting AIS patients for minimally invasive surgery before their spinal deformity becomes too advanced. OPN (a multifunctional cytokine) has been identified as a potential key pathophysiological contributing factor in the development of idiopathic scoliosis. Specifically, increased plasma OPN levels in patients with idiopathic scoliosis and in bipedal mice (a well-established animal model of the disease) are associated with the disease (see WO 2008/119170 by Moreau).

It is generally believed that the development of scoliosis is affected by posture mechanisms. The bipedal pathology naturally present in humans or experimentally induced in animals seems to play an important role in the manifestation of scoliotic deformity (Machida et al., 1999). Importantly, it is reported that when mice with C57Bl/6 or C3HHe backgrounds obtain a bipedal posture for 40 weeks after the amputation of their forelimbs and tails, the mice develop a scoliosis that is closely similar to human idiopathic scoliosis (Machida et al., 2006); (Oyama et al., 2006). Therefore, in order to solve the problem of whether OPN contributes to the development of idiopathic scoliosis, we took advantage of the availability of OPN-deficient mice on C57Bl/6 background.

This specification refers to various documents, the contents of which are incorporated herein by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention shows that OPN reduces in vitro signaling primarily via _{α5β1 integrin} engagement. Independent of this hypothesis, the mechanism by which OPN interferes with GiPCR signaling is believed to be through depletion of functional Gi proteins necessary for these receptors by sequestering a portion of Gi proteins via the integrin _β1 subunit and inactivation of the remaining Gi proteins after phosphorylation by _different kinases in the signaling cascade by engaging the _α5β1 integrin. Furthermore, OPN's inhibitory effect on GiPCR signaling can be modulated by CD44 levels and/or bioavailability in conjunction with OPN. The present invention shows that CD44 depletion exacerbates OPN's inhibitory effect and that hyaluronic acid (HA), a known CD44 ligand, further enhances the effect of OPN in a dose-dependent manner. HA exhibits a higher binding affinity for CD44 than OPN. Thus, HA competes with OPN for binding to CD44; thereby increasing the bioavailability of OPN _to bind to _α5β1 integrin and inhibit GiPCR signaling. Finally, the present invention shows that the presence of mutations in CD44 further reduces GiPCR signaling in osteoblasts of scoliosis subjects.

Therefore, the present invention relates to a method of increasing GiPCR signaling in cells of a subject in need thereof, comprising administering to a subject in need thereof an effective amount of: (a) an inhibitor of OPN expression and/or activity; (b) an inhibitor of _{integrin α5β1} ; or (c) a combination of (a) and (b), thereby increasing GiPCR signaling in cells of a subject in need thereof.

According to a related aspect, the present invention relates to a method of treating or preventing scoliosis in a subject in need thereof, the method comprising reducing α5β1 expression or activity (e.g., binding to OPN) in cells of the subject. In one embodiment, the method comprises administering to the subject an effective amount of an inhibitor of integrin α5β1 expression or activity.

In one embodiment, the inhibitor of OPN activity is an OPN antibody. In another embodiment, _the inhibitor of OPN expression is an siRNA specific for OPN. In another embodiment, the inhibitor of _α5β1 is (a) an antibody that specifically binds to the integrin subunit _α5 ; (b) an antibody that specifically binds to the integrin subunit _β1 ; or (c) a combination of (a) and (b). In one embodiment, the inhibitor _{of α5β1} is an siRNA specific for _α5 or _β1 .

According to another aspect of the present invention, a method for selecting an agent as a potential candidate for reducing or preventing GiPCR signaling inhibition induced by OPN is provided, the method comprising contacting the candidate agent with a cell expressing (i) _OPN , (ii) integrin _α5β1 and/or (iii) CD44/sCD44, wherein the candidate agent is selected (a) when OPN expression or activity is decreased in the presence of the candidate agent compared to in the absence of the candidate agent; (b) when _{integrin α5β1} expression or activity (e.g., binding to OPN) is decreased in the presence of the candidate agent compared to in the absence of the candidate agent; (c) when sCD44 and/or CD44 expression (level) or binding to OPN is increased in the presence of the candidate agent compared to in the absence of the candidate agent; or (d) any combination of at least two of (a), (b) and (c).

According to another aspect of the present invention, a method for selecting an agent as a potential candidate for reducing or preventing GiPCR signaling is provided, the method comprising contacting the candidate agent with a cell expressing (a) OPN and/or (b) integrin _α5β1 , wherein the candidate agent is _selected when (a) OPN expression or activity is increased in the presence of the candidate agent compared to in the absence of the candidate agent; and/or (b) integrin α5 _{/ β1} is increased in the presence of the candidate agent compared to in the absence of the candidate agent.

According to another aspect of the present invention, there is provided (i) an inhibitor of OPN expression or activity; ii) an inhibitor of _{integrin α5β1} expression or activity; iii) a stimulator of sCD44 or sCD44/CD44 expression; or iv) any combination of at least two of (i) to (iii) for use in increasing GiPCR signaling in cells of a subject in need thereof.

According to another aspect of the present invention, there is provided the use of (i) an inhibitor of OPN expression or activity; ii) an inhibitor of _{integrin α5β1} expression or activity; iii) a stimulator of sCD44 or sCD44/CD44 expression; or iv) any combination of at least two of (i) to (iii) for the preparation of a medicament for increasing GiPCR signaling in cells of a subject in need thereof.

According to another aspect of the present invention, a composition for increasing GiPCR signaling in cells of a subject in need thereof is provided, the composition comprising (i) an inhibitor of OPN expression or activity; (ii) an inhibitor of _α5β1 expression or activity; (iii) a stimulator of sCD44 _or sCD44/CD44 expression; or (iv) any combination of at least two of (i) to (iii).

In a specific embodiment, the subject in need thereof is a subject diagnosed with or at risk of developing scoliosis.

According to another aspect of the present invention, a method of increasing GiPCR signaling in cells of a subject in need thereof is provided, the method comprising administering to the subject an effective amount of an inhibitor _of integrin _α5β1 expression and/or activity, thereby increasing GiPCR signaling in cells of the subject.

In one embodiment, the method further comprises administering to the subject an effective amount of an inhibitor of OPN expression and/or activity. In another embodiment, the inhibitor of OPN activity is an OPN antibody. In another embodiment, the inhibitor of OPN expression is an siRNA specific for OPN. In another embodiment, the inhibitor of _α5β1 activity is (a ₎ an antibody that specifically binds to the integrin subunit _α5 ; (b) an antibody that specifically binds to the integrin subunit _β1 ; (c) an antibody that specifically binds to the _integrin subunit _α5β1 ; (d) a molecule that specifically blocks the binding of OPN to the _{α5β1 integrin} ; or (e) any combination of at least two of (a) to (d). In another embodiment, the molecule that specifically blocks the binding of OPN to the _{α5β1 integrin} is an RGD peptide or a derivative thereof. In another embodiment, the molecule that specifically blocks the binding of OPN to the _{α5β1 integrin} is a peptide fragment of OPN that contains the RGD motif. In another specific embodiment, the peptide fragment of OPN comprises the amino acid sequence GRGDSVVYG LRS (SEQ ID NO: 18 ₎ . In another specific embodiment, the inhibitor of _α5β1 expression is an siRNA specific for _α5 or _β1 . In one specific embodiment, the method further comprises administering to the subject i) an effective amount of sCD44 or a fragment thereof that specifically binds to OPN; or ii) a stimulator of sCD44 and/or CD44 expression.

According to another aspect of the present invention, _there is provided use of an inhibitor of integrin _α5β1 expression and/or activity in the preparation of a medicament for inhibiting GiPCR signaling in cells of a subject in need thereof.

According to another aspect of the present invention, _there is provided a use of an inhibitor of integrin _α5β1 expression and/or activity for inhibiting GiPCR signaling in cells of a subject in need thereof.

According to another aspect of the present invention, there is provided an inhibitor _of integrin _α5β1 expression and/or activity for use in inhibiting GiPCR signaling in cells of a subject in need thereof.

In one embodiment of the use or the inhibitor, the inhibitor _{of α5β1} activity is (a) an antibody that specifically binds to the integrin subunit _α5 ; (b) an antibody that specifically binds to the integrin subunit _β1 ; (c) an antibody that specifically binds to the _integrin subunit _α5β1 ; (d) a molecule that specifically blocks the binding of OPN to the _{α5β1 integrin} ; or (e) any combination of at least two of (a) to (d). In another embodiment of the use or the inhibitor, the molecule that specifically blocks the binding of OPN to the _{α5β1 integrin} is an RGD peptide or a derivative thereof. In another embodiment, the molecule that specifically blocks the binding of OPN to the _{α5β1 integrin} is a peptide fragment of OPN that contains an RGD motif. In another embodiment, the peptide fragment of OPN contains the amino acid sequence GRGDSVVYGLRS (SEQ ID NO: 18). In another specific embodiment, the inhibitor _of integrin _α5β1 expression is an siRNA specific for _α5 or _β1 .

According to another aspect of the present invention, a composition is provided, comprising the inhibitor of the present invention and a drug carrier.

In another specific embodiment, the composition is for increasing GiPCR signaling in a cell of a subject in need thereof.

In a specific embodiment of the methods, uses, inhibitors or compositions of the invention, the subject is a subject diagnosed with or at risk of developing scoliosis. In another specific embodiment, the scoliosis is idiopathic scoliosis. In another specific embodiment, the scoliosis is adolescent idiopathic scoliosis (AIS).

According to another aspect of the present invention, a method for determining the risk of developing scoliosis in a subject is provided, the method comprising: (i) providing a cell sample isolated from the subject; (ii) detecting the presence of at least one copy of a CD44 risk allele or a marker in linkage disequilibrium therewith in the cell sample from the subject, wherein the CD44 risk allele introduces a mutation at amino acid position 230 of CD44; and (iii) determining that the subject is at risk of developing scoliosis when at least one copy of the risk allele or a marker in linkage disequilibrium therewith is detected in the cell sample from the subject.

In a specific embodiment, the risk allele comprises SNP rs1467558 and the mutation changes isoleucine at position 230 of CD44 to threonine.

According to another aspect of the present invention, a method for stratifying subjects having scoliosis (e.g., idiopathic scoliosis, such as adolescent idiopathic scoliosis (AIS)) is provided, the method comprising: (i) providing a cell sample isolated from the subject; (ii) detecting the presence of at least one copy of a CD44 risk allele, or a marker in linkage disequilibrium therewith, in the cell sample from the subject, wherein the CD44 risk allele introduces a mutation at amino acid position 230 of CD44; and (iii) (a) stratifying the subject into a first AIS subclass when at least one copy of the CD44 risk allele, or a marker in linkage disequilibrium therewith, is detected in the cell sample from the subject; or (b) stratifying the subject into a second AIS subclass when the CD44 risk allele is not detected in the cell sample from the subject.

In one embodiment, the mutation changes the isoleucine at position 230 of CD44 to a threonine. In another embodiment, the at least one copy of the CD44 risk allele consists of two copies of the CD44 risk allele, and the subject is homozygous for the mutation. In another embodiment, the sample is a nucleic acid sample. In another embodiment, the sample is a protein sample.

According to another aspect of the present invention, the present invention provides a kit for performing the method as defined in any one of claims 24 to 30, the kit comprising: (i) a nucleic acid probe or primer for detecting a CD44 risk allele, the CD44 risk allele comprising a mutation in the codon encoding amino acid 230 of CD44; and/or (ii) a protein ligand that specifically detects a mutation at amino acid 230 of CD44.

In one embodiment, the nucleic acid probe or primer comprises a nucleic acid sequence that specifically hybridizes to a nucleic acid encoding a threonine at amino acid position 230 of CD44. In another embodiment, the protein ligand is an antibody specific for a CD44 protein comprising a threonine at amino acid position 230.

Other objects, advantages and features of the present invention will become more apparent upon reading the following non-restrictive description of specific embodiments of the invention, given by way of example only with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the attached figure:

Figure 1 shows that genetic deletion of OPN protects bipedal C57BL6 from scoliosis by improving Gi protein-mediated receptor signal transduction. (AD) Female C57Bl/6 wild-type (WT) and OPN knockout (OPN ^-/- ) mice were amputated of their forelimbs and tails at 1 month of age and subjected to bipedal walking for 36 weeks to induce scoliosis. X-ray images of the spine as taken at the end of the experimental period. ^OPN-/- mice do not develop scoliosis in quadrupedal or even bipedal mice, while WT develop scoliosis in bipedal mice. (E) Plasma OPN was detected in C57Bl/6 (WT) quadrupedal and bipedal mice, and bipedal mice showed higher plasma OPN than quadrupedal WT mice. (FI) GiPCR signaling in osteoblasts from WT and OPN-/- mice was examined using DAMGO, somatostatin, oxymethazolin, and apelin. The response was greater in OPN ^-/- osteoblasts than in WT osteoblasts. On the other hand, bipedal WT mice showed a lower response than quadrupedal WT. (JM) Osteoblasts from WT and OPN ^-/- mice pretreated with PTX responded to each of the previous agonists blocking the coupling of Gi to its cognate receptor in these cells. This indicates that these compounds elicited a typical CDS response profile of GiPCR in WT and OPN ^-/- osteoblasts. (NO) Isoproterenol and desmopressin were used to activate Gs, while bradykinin and endothelin-1 were used to activate Gq via its cognate receptor. The response of osteoblasts from OPN ^-/- mice to Gs stimulation was smaller than that of osteoblasts from WT mice, while there was no difference in the Gq stimulation elicited in WT and OPN ^-/- osteoblasts.

Figure 2 shows that extracellular OPN causes Gi protein-coupled receptor signaling dysfunction. (A) OPN was knocked down by siRNA in the MC3T3-E1 osteoblast cell line and the ability of GiPCR ligands to induce cell signaling was measured by CDS. The cellular response to DAMGO and oxymetazoline was significantly greater than that of cells depleted of OPN. (B) OPN blocked by an OPN-specific antibody in the MC3T3-E1 osteoblast cell line gave the same results as in (A). (C-D) MC3T3-E1 osteoblasts were treated with exogenous recombinant OPN (rOPN) before DAMGO and oxymetazoline stimulation. In each case, rOPN caused a decrease in the overall response in a concentration-dependent manner, which was prevented by the OPN antibody.

Figure 3 shows that CD44 is not involved in the inhibition of GiPCR signaling caused by extracellular OPN but can regulate the effect of OPN. (A) Blocking CD44 by CD44-specific antibodies in the MC3T3-E1 osteoblast cell line and treating cells with rOPN 0.5 μg/ml further aggravated the GiPCR signaling defect induced by OPN without reducing the GiPCR signaling defect caused by OPN. DAMGO and oxymetazoline were used as agonists of GiPCR. (B-C) To verify the activity of anti-CD44 antibodies, cells pretreated with IgG control or CD44 antibodies were stimulated with increasing concentrations of hyaluronic acid (HA), a high-affinity ligand for CD44. The comprehensive reaction induced by HA was completely eliminated by pretreatment with CD44 antibodies. In addition, the effect of CD44 antibodies on the response to HA stimulation was concentration-dependent. (D-E) CD44 was knocked down by siRNA in the MC3T3-E1 osteoblast cell line. The efficiency of siRNA transfection and inhibition of CD44 expression are demonstrated by qPCR (D) and Western blot analysis (E). (F) Knockdown of CD44 resulted in similar results to (A), and CD44 knockdown did not reduce the GiPCR signaling defect caused by OPN, but rather further aggravated the defect. (G-H) rOPN treatment caused a concentration-dependent decrease in the response to both DAMGO and oxymetazoline in osteoblasts from bipedal CD44 knockout (CD44-/-) mice. (I-J) Osteoblasts from bipedal (CD44-/-) mice were less responsive to DAMG or oxymetazoline when compared to osteoblasts from quadrupedal (CD44-/-) mice. (K-P) CD44 inhibition and HA potentiated the effect of OPN on GiPCR signaling in a dose-dependent manner. (K) In response to LPA stimulation of GiPCR signaling, CD44-/- osteoblasts responded less and OPN-/- osteoblasts responded more in a dose-dependent manner compared to wild-type cells. (L) Treatment with HA, a natural agonist of the CD44 receptor, exacerbated the GiPCR signaling defect in wild-type osteoblasts compared to controls. Inhibition of CD44 using HA or antibody (CD44fab) in the presence of recombinant OPN (rOPN) also exacerbated the signaling defect. The response was most reduced when CD44 was inhibited in both ways (HA+CD44Fab). (M) The same experiment using CD44-/- osteoblasts did not show any sensitivity to HA or anti-CD44 antibodies. (N, O, and P). Inhibition of the CD44 receptor by depleting CD44 using CD44-/- osteoblasts or by treating wild-type cells with HA in the presence of OPN had the same effect on the GiPCR signaling response.

FIG4 illustrates the RGD-dependent integrin-mediated inhibitory effect of OPN on GiPCR signaling. (AB and C) OPN binds to integrins via the RGD motif, and this is evident when bio1211 inhibits the SVVYGLR binding motif, which selectively inhibits _{α4β1 integrin} (the only SVVYGLR-containing integrin present in osteoblasts). Bio1211 does not affect OPN signaling, while co-incubation of cells with rOPN and RDG peptide completely prevents the inhibitory effect of rOPN on responses to DAMGO and oxymetazoline. (DE) Osteoblasts from WT mice incubated with high concentrations of RGD exhibit a significant increase in responses to DAMGO or oxymetazoline, whereas no changes were observed in osteoblasts from OPN ^−/− mice.

FIG5 illustrates the identification of integrins involved in the inhibition of GiPCR signaling by OPN. (A, B) Different integrins were blocked using specific antibodies and (C) these integrins were knocked down in MC3T3-E1 osteoblasts. Only the _α5 and _β1 integrins in the inhibitory cells reversed the inhibitory effect of OPN on GiPCR signaling. (D) Knockdown of _α5 and _β1 integrins in C57B1/6 bipedal WT and CD44 ^−/− mice reversed the inhibitory effect of rOPN on the response to both DAMGO and oxymetazoline.

Figure 6 shows that OPN reduces the availability of Gi proteins to their cognate receptors. (A, B, C) MC3T3-E1 osteoblasts were treated with PBS or rOPN. Cell lysates were analyzed by Western blot for the expression of three different GPCRs: μ-opioid (MO) receptor, lysophosphatidic acid type 1 (LPA1) receptor, and melatonin type 2 (MT2) receptor (A), while _Gi1 , _Gi2 , and _Gi3 protein isoforms were detected by Western blot (B) and qPCR (C). rOPN treatment had no significant effect on the amount of Gi proteins or mRNA (B, C) or GiPCR proteins (A). (D, F) Cell lysates were immunoprecipitated with antibodies against MO, MT2, or β1 integrin receptors, and the presence of Gi proteins in each precipitate was examined by Western blot using antibodies specific for _Gi1 , _Gi2 , or _Gi3 isoforms. The amount of each of these Gi protein isoforms was elevated in the β1 integrin precipitate following rOPN treatment.

FIG7 illustrates that OPN enhances the phosphorylation of Gi proteins. (A) MC3T3-E1 cells were treated with rOPN or pretreated with antibodies against _{α5β1 integrin} before rOPN treatment, and then cell lysates were immunoprecipitated (IP) using antibodies against _Gi1 , _Gi2 , or _Gi3 protein isoforms, and the presence of phosphorylation was revealed by Western blotting using anti-phosphoserine/threonine or anti-tyrosine antibodies. (B) Osteoblast lysates from scoliotic bipedal WT or CD44 ^−/− mice were immunoprecipitated with antibodies against _Gi1 , _Gi2 , and _Gi3 proteins, followed by Western blotting using antibodies against anti-phosphoserine/threonine or anti-phosphotyrosine. (CE) Cell extracts prepared from MC3T3-E1 cells were treated with rOPN in the presence or absence of kinase inhibitors, followed by immunoprecipitation with antibodies against Gi ₁ , Gi ₂ , or Gi ₃ protein isoforms and Western blotting using anti-phosphoserine/threonine or anti-phosphotyrosine antibodies.

Figure 8 shows the effect of OPN on GsPCR protein. (A) MC3T3-E1 cells were treated with rOPN or PBS (A) stimulated by isoproterenol or desmopressin; (B) immunoprecipitated with antibodies against Gs, followed by immunoblotting with antibodies against phosphoserine/threonine; or (C-D) immunoprecipitated with antibodies against MOR or MT2R, followed by Western blotting with antibodies against Gs. (E) MC3T3-E1 cells were treated with PTX to simulate the destructive effects of rOPN and compared to cells treated with rOPN alone or in combination with a Src inhibitor (PP2) to prevent tyrosine phosphorylation.

FIG9 shows that the CT SNP in CD44 exacerbates the inhibitory effect of OPN on GiPCR signaling. (A) Osteoblasts from subjects with AIS harboring the CT SNP and from healthy subjects were treated with OPN and assessed for the effect on GiPCR signaling after stimulation with LPA. (B) Same as in (A), except that the cell samples were treated with increasing concentrations of OPN. The presence of the CT SNP reduced GiPCR signaling by approximately 50%.

Figure 10 shows a schematic illustration of the proposed mechanism by which OPN reduces GiPCR signaling and contributes to the development of spinal deformities. Following OPN binding, _{α5β1 integrin} recruits a portion of Gi proteins from the common pool, which then promotes different signaling pathways, leading to the activation of different kinases, which directly or indirectly phosphorylate a portion of the remaining Gi proteins in the common pool, resulting in reduced GiPCR signaling.

Description of Illustrative Embodiments

As used herein, the term "risk of developing scoliosis" refers to a subject's genetic or metabolic predisposition to developing scoliosis (i.e., spinal deformity) and/or developing more severe scoliosis (i.e., progression of the curve) in the future. For example, an increase in a subject's Cobb's angle (e.g., from 40° to 50° or from 18° to 25°) is "development" of scoliosis.

In one embodiment, the subject is a possible candidate for developing scoliosis, such as idiopathic scoliosis (e.g., infantile idiopathic scoliosis, juvenile idiopathic scoliosis, or adolescent idiopathic scoliosis (AIS)). As used herein, the term "possible candidate for developing scoliosis" includes subjects (e.g., children) whose parents have at least one scoliosis (e.g., adolescent idiopathic scoliosis). Among other factors, age (puberty), gender, and other family antecedents are known factors that contribute to the risk of developing scoliosis and are used to assess the risk of developing scoliosis to a certain extent. In some subjects, scoliosis progresses rapidly over a short period of time to the point where corrective surgery is required (often when the deformity reaches a Cobb angle of ≥50°). Current practices available from the time a scoliosis, such as AIS, is diagnosed (when the scoliosis is evident) include observation (when the Cobb angle is about 10°-25°), orthopedic devices (when the Cobb angle is about 25°-30°), and surgery (over 45°). A more reliable determination of the risk of progression can enable 1) selection of an appropriate diet to eliminate certain foods identified as contributing factors to scoliosis; 2) selection of the best therapeutic agent; and/or 3) selection of the least invasive available treatment, such as postural exercises, orthotic devices, or less invasive surgery or fusion-free surgery (a surgery that does not fuse the spine and preserves spinal mobility). The present invention encompasses selecting the most effective and least invasive known preventive measure or treatment given the determined risk of developing scoliosis.

As used herein, the term "subject" refers to any mammal, including humans, mice, rats, dogs, chickens, cats, pigs, monkeys, horses, etc. In a specific embodiment, the subject is a human.

In the context of the present invention, a "subject in need thereof" or "patient" is intended to include any subject who would benefit or may benefit from modulating (increasing or decreasing) the effect of OPN on GiPCR signaling. In one embodiment, a subject in need thereof is a subject who would benefit or may benefit from an increase in GiPCR signaling and, therefore, would benefit from (a) an antagonist (inhibitor) of OPN expression and/or activity; (b) an antagonist (inhibitor) of _α5 (e.g., Gene ID 3678, NP_002196) and/or _β1 expression and/or activity; or (c) an increase in sCD44 or CD44 expression. In a specific embodiment, a subject in need thereof is a subject with high OPN levels compared to a control subject. In a specific embodiment, high OPN levels correspond to OPN levels of ≥ to about 600 ng/ml, ≥ to about 700 ng/ml, ≥ to about 800 ng/ml, ≥ to about 900 ng/ml, or ≥ to about 1000 ng/ml, as measured in a blood sample from the subject. In one embodiment, a subject in need thereof is a subject diagnosed with scoliosis (eg, AIS). In another embodiment, a subject in need thereof is a subject at risk of developing scoliosis (eg, AIS).

As used herein, the term "biological sample" refers to any solid or liquid sample isolated from an organism. In a specific embodiment, a biological sample refers to any solid or liquid sample isolated from a human. Without being so limited, biological samples include biopsy material, blood, tears, saliva, breast milk, synovial fluid, urine, ear fluid, amniotic fluid, and cerebrospinal fluid. In a specific embodiment, a biological sample is a blood sample.

As used herein, the term "blood sample" means blood, plasma, or serum.

As used herein, the term "control sample" means a sample that is not from a subject known to have scoliosis or known to be a possible candidate for developing scoliosis. In methods for determining the risk of developing scoliosis in a subject previously diagnosed with scoliosis, the sample may also be from a subject who is under close observation at an early stage of the disease or condition. In a specific embodiment, the control sample may be from another subject diagnosed with scoliosis and belonging to the same functional group (e.g., FG1, FG2, or FG3) at an early stage (or late stage) of the disease or condition.

As used herein, the term "control" is meant to encompass a "control sample." In certain embodiments, the term "control" also refers to the mean or median value obtained after assaying GiPCR signaling in multiple samples (e.g., samples obtained from several subjects who are not known to have scoliosis and are not known to be possible candidates for developing scoliosis).

As used herein, the terms "treating" or "treatment" with respect to scoliosis means at least one of: reduction of the Cobb angle of a pre-existing spinal deformity; improvement of spinal mobility; preservation/maintenance of spinal mobility; improvement of equilibrium and balance in a specific plan; maintenance/preservation of equilibrium/balance in a specific plan; improvement of functionality in a specific plan; preservation/maintenance of functionality in a specific plan; cosmetic improvement; and any combination of the above.

As used herein, the terms "preventing" or "prevention" with respect to scoliosis means at least one of: a reduction in the progression of the Cobb angle in a patient with scoliosis or in an asymptomatic patient; complete prevention of the development of spinal deformity, including changes affecting the rib cage and pelvis in 3D; and any combination of the foregoing.

The terms "inhibitor," "inhibitor," and "antagonist" are well known in the art and are used interchangeably herein. They include intracellular as well as extracellular inhibitors.

The term "OPN inhibitor" or "OPN antagonist" includes any compound that is capable of adversely affecting (i.e., completely or partially reducing) the expression and/or activity of OPN (e.g., Gene ID 6696, NP_001035147.1 (SEQ ID NO: 25) and NM_001040058 (SEQ ID NO: 26)) in a cell. In a specific embodiment, the activity of OPN in a cell is a reduction in GiPCR signaling. Similarly, the terms "inhibitor of _α5β1 ,"_" inhibitor of _α5 ," or "inhibitor of _β1, " and the like include any compound that can adversely affect the expression and/or activity of _α5 (e.g., Gene ID 3678, NP_002196 (SEQ ID NO: 23) and NM_002205.2 (SEQ ID NO: 24)) and/or _β1 (Gene ID 3688, NP_002202 (SEQ ID NO: 21) and NM_002211.3 (SEQ ID NO: 22)) in a cell. In a specific embodiment, the "activity" of _α5 and/or _β1 in a cell is the transduction of a signal that results in OPN-dependent inhibition of GiPCR signaling.

The term "inhibitor of OPN expression" or "inhibitor of _α5β1 expression" includes any _compound that can adversely affect the expression of OPN, _α5 and/or _β1 (i.e., at the transcription and/or translation level), i.e., the level of OPN/ _α5 or _β1 mRNA and/or protein, or the stability of the protein. Without being so limited, such inhibitors include RNA interfering agents (siRNA, shRNA, miRNA), antisense molecules, and ribozymes. Such RNA interfering agents are designed to specifically hybridize with their target nucleic acid under suitable conditions and are therefore substantially complementary to their target nucleic acid.

The term "inhibitor of OPN activity" or "inhibitor of _α5β1 activity" refers to any molecule _that can reduce or block the effect of OPN or _α5β1 on Gi-mediated signaling. These molecules increase GiPCR signaling in cells by completely or partially blocking/ _reducing the inhibitory effect induced _by OPN and/or _α5β1 activity. Non-limiting examples of inhibitors of OPN activity include proteins (e.g., dominant negative, inactive variants), peptides, small molecules, anti-OPN antibodies (neutralizing antibodies), antibody fragments, inactive fragments of _α5 and/or _β1 integrins, and the like. Non-limiting examples of inhibitors _{of α5β1} activity include proteins (e.g., dominant negative, inactive variants), peptides (RGD peptides or RGD peptide derivatives), small molecules, anti- _α5 and/or _β1 antibodies (e.g., neutralizing antibodies, such as Volociximab ^™ M200), antibody fragments, and the like. In one embodiment, the RGD peptide is a peptide fragment of OPN comprising an RGD motif comprising the amino acid sequence GRGDSVVYGLRS (SEQ ID NO: 18) corresponding to amino acids 158 to 169 of OPN. In one embodiment, the OPN fragment comprising the RGD motif comprises amino acids 158 to 162, 158 to 165, 158 to 167, 158 to 170, 158 to 175, 158 to 180, 158 to 185, 158 to 190, 158 to 195, or 158 to 200 of OPN (e.g., SEQ ID NO: 25). In one embodiment, the peptide fragment of OPN comprising the RGD motif comprises amino acids 158 to 161, 156 to 161, 154 to 161, 152 to 162, 150 to 162, 148 to 162, 146 to 162, 144 to 162, 140 to 162, 159 to 163, 159 to 164, 159 to 162, 159 to 166, 159 to 167, or 159 to 169 of OPN (e.g., SEQ ID NO: 25).

In one embodiment, an "inhibitor of OPN activity" is a neutralizing antibody directed against (or specifically binding to) a human OPN polypeptide, wherein the neutralizing antibody inhibits the binding of OPN to _α5β1 (i.e., binding to _α5 and/ _{or β1} integrins). In one embodiment, an "inhibitor of _α5β1 activity" is a neutralizing antibody directed against ( _or specifically binding to) _a human _α5β1 ( _α5 and/or _β1 ) polypeptide, wherein the neutralizing antibody inhibits the binding of OPN to _α5β1 (i.e., binding to _α5 and/or β1 _integrins ). In one embodiment, the antibody binds to the RGD domain of OPN. In one embodiment, the antibody is directed against amino acids 159 to 162, 158 to 162, 158 to 165, 158 to 167, 158 to 170, 158 to 175, 158 to 180, 158 to 185, 158 to 190, 158 to 195, or 158 to 200 of OPN (e.g., SEQ ID NO: 25). In one embodiment, the antibody is directed against amino acids 158 to 161, 156 to 161, 154 to 161, 152 to 162, 150 to 162, 148 to 162, 146 to 162, 144 to 162, 140 to 162, 159 to 163, 159 to 164, 159 to 162, 159 to 166, 159 to 167, or 159 to 169 of OPN (e.g., SEQ ID NO: 25).

The term "stimulator" or "enhancer" of sCD44/CD44 expression (e.g., Gene ID 960, NM_000610 (SEQ ID NO: 19), NP_000601.3 (SEQ ID NO: 20)) refers to an agent that increases the level or expression of CD44, or an agent that increases CD44 secretion. In one embodiment, the stimulator of sCD44/CD44 is an agent that increases the affinity of CD44 for OPN. Without being so limited, the agent can be a protein, peptide, small molecule, or nucleotide.

One possible way to reduce the effect of OPN on GiPCR signaling is to reduce the interaction of OPN with _{α5β1 integrin} by, for example, increasing or promoting the binding of OPN to CD44. Hyaluronic acid (HA) is known to bind CD44. As shown herein, HA may enhance the effect of OPN by increasing the bioavailability of OPN to _α5β1 (e.g., by chelating CD44 _that is no longer available for binding to OPN). Therefore, subjects who would benefit from an increase in OPN inhibition and GiPCR signaling should not take HA supplements and/or should reduce their food HA intake by, for example, avoiding or reducing his/her consumption of foods that are particularly rich in HA or known to increase HA synthesis. Non-limiting examples of such foods include meat and meat organs (e.g., veal, lamb, beef and gizzards, liver, heart, and kidney), fish, poultry (including meat, fish, and poultry broth), soy (including soy milk), starchy root vegetables (including potatoes and sweet potatoes). Sweet potatoes that are particularly rich in HA include satoimo and imoji (two Japanese sweet potatoes).

The present invention also relates to methods for determining the level of expression (i.e., transcript (RNA) or translation product (protein)) _of OPN, _{α5β1 integrin} , and/or CD44. As used herein, the term _α5β1 is used herein to refer to _{α5β1 integrin} . In specific embodiments, the present invention also includes a method comprising determining the expression level of one or more other scoliosis markers (see, for example, WO 2008/119170 to Moreau). The present invention thus encompasses any known method for such determination, including ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), immunofluorescence, real-time and competitive PCR, Northern blotting, nuclease protection, plaque hybridization, and slot blotting.

The present invention also relates to isolated _nucleic acid molecules, including probes and primers for detecting OPN, _α5β1 and sCD44/CD44 (and optionally other scoliosis markers). 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 molecule has at least 20 and no more than 80 nucleotides. In other specific embodiments, the isolated nucleic acid molecule has at least 20 and no more than 70 nucleotides. In other specific embodiments, the isolated nucleic acid molecule has at least 20 and no more than 60 nucleotides. In other specific embodiments, the isolated nucleic acid molecule has at least 20 and no more than 50 nucleotides. In other specific embodiments, the isolated nucleic acid molecule has at least 20 and no more than 40 nucleotides. In other specific embodiments, the isolated nucleic acid molecule has at least 17 and no more than 40 nucleotides. In other specific embodiments, the isolated nucleic acid molecule has at least 20 and no more than 30 nucleotides. In other specific embodiments, the isolated nucleic acid molecule has at least 17 and no more than 30 nucleotides. In other specific embodiments, the isolated nucleic acid molecule has at least 30 and no more than 300 nucleotides. In other specific embodiments, the isolated nucleic acid molecule has at least 30 and no more than 200 nucleotides. In other specific embodiments, the isolated nucleic acid molecule has at least 30 and no more than 100 nucleotides. In other specific embodiments, the nucleic acid molecule of separation has at least 30 and is no more than 90 nucleotides. In other specific embodiments, the nucleic acid molecule of separation has at least 30 and is no more than 80 nucleotides. In other specific embodiments, the nucleic acid molecule of separation has at least 30 and is no more than 70 nucleotides. In other specific embodiments, the nucleic acid molecule of separation has at least 30 and is no more than 60 nucleotides. In other specific embodiments, the nucleic acid molecule of separation has at least 30 and is no more than 50 nucleotides. In other specific embodiments, the nucleic acid molecule of separation has at least 30 and is no more than 40 nucleotides. It should be understood that in real-time PCR, primers also constitute probes, rather than the traditional meaning of the term. Primers or probes suitable for detecting OPN and/or α ₅ β ₁ in the method of the present invention can be designed using sequences distributed on their corresponding nucleotide sequences using known methods. The probes and/or primers of the present invention are designed to specifically hybridize to their target nucleic acids ( _α5 (SEQ ID NO: 24), _β1 (SEQ ID NO: 22), CD44 wild type (SEQ ID NO: 19) or CD44 risk allele (e.g., comprising the 230I→T (SNP (CT)) variant) and/or OPN (SEQ ID NO: 26)). In one embodiment, the primers and probes of the present invention are substantially complementary to their target nucleic acids.

Substantially complementary nucleic acids are nucleic acids in which the complementary sequence of one molecule is substantially identical to the other molecule. In optimal alignment, two nucleic acid or protein sequences are considered to be substantially identical if they share at least about 70% sequence identity. In alternative embodiments, the sequence identity can be, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 98%, or at least 99%. Optimal alignment of sequences for identity comparison can be performed using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2:482; the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443; the similarity search method of Pearson and Lipman; and computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, U.S.A.). Sequence identity can also be determined using the BLAST algorithm described in Altschul et al. 1990 (using the disclosed default settings). Software for performing BLAST analysis is available through the National Center for Biotechnology Information (via the Internet at http://www.ncbi.nlm.nih.gov/). The BLAST algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with a word of the same length in a database sequence, either match or satisfy some positive-valued threshold score T. T is referred to as the neighborhood word score threshold. Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is stopped when: the cumulative alignment score decreases by the amount X from its maximum value reached; the cumulative score drops to zero or below due to the accumulation of negative values for one or more residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. One measure of statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

The alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under medium stringency conditions or preferably under stringent conditions.Sequence hybridization combined with filter membrane under medium stringency conditions can be, for example, carried out at 65 ℃ in 0.5M NaHPO4, 7% sodium lauryl sulfate (SDS), 1mM EDTA, and washed at 42 ℃ in 0.2x SSC/0.1% SDS. Alternatively, sequence hybridization combined with filter membrane under stringent conditions can be, for example, carried out at 65 ℃ in 0.5M NaHPO4, 7% SDS, 1mM EDTA, and washed at 68 ℃ in 0.1x SSC/0.1% SDS. Hybridization conditions can be modified according to known methods depending on the target sequence. In general, stringent conditions are selected to be about 5 ℃ lower than the thermal melting point for the specific sequence under limited ionic strength and pH.

The probes of the present invention can be used with naturally occurring sugar-phosphate backbones as well as modified backbones (including phosphorothioates, dithionates, alkylphosphonates, and α-nucleotides, etc.). Modified sugar-phosphate backbones are generally known. The probes of the present invention can be constructed from ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and are preferably constructed from DNA.

Types of detection methods that can use probes include Southern blotting (DNA detection), dot or slot blotting (DNA, RNA), and Northern blotting (RNA detection). Although less preferred, labeled proteins can also be used to detect the specific nucleic acid sequence to which they bind. Other detection methods include kits containing probes on a dipstick device, etc.

As used herein, the term "detectably labeled" refers to labeling _a probe or antibody according to the present invention, which will allow detection of OPN and/or _α5β1 according to the present invention. Although the present invention does not specifically rely on the use of a label for detecting a specific nucleic acid sequence, such labeling may be beneficial by increasing the sensitivity of the detection. In addition, the labeling can be automated. The probe can be labeled according to numerous well-known methods. Non-limiting examples of labels include ³ H, ¹⁴ C, ³² P, and ³⁵ S. Non-limiting examples of detectable labels include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable labels for use with the probe that can achieve increased sensitivity of the method of the present invention include biotin and radionucleotides. It will become apparent to those skilled in the art that the choice of a specific label determines how it binds to the probe.

As is generally known, radionucleotides can be incorporated into the probes of the present invention by several methods. Non-limiting examples include kinasing the 5' end of the probe using γ ³² P ATP and polynucleotide kinase, using the Klenow fragment of Escherichia coli Pol I in the presence of radioactive dNTPs (e.g., using DNA probes uniformly labeled with random oligonucleotide primers in a low melting point gel), transcribing DNA segments using the SP6/T7 system in the presence of one or more radioactive NTPs, and the like.

The present invention also relates to methods for selecting compounds. As used herein, the term "compound" is intended to encompass natural, synthetic or semi-synthetic compounds, including but not limited to chemicals, macromolecules, cell or tissue extracts (from plants or animals), nucleic acid molecules, peptides, antibodies and proteins.

The present invention also relates to arrays. As used herein, an "array" is a collection of intentionally produced molecules that can be prepared synthetically or biosynthetically. The molecules in the array can be identical or different from one another. The array can take a variety of forms, such as a library of soluble molecules; a library of compounds attached to resin beads, a silica chip, or other solid support.

As used herein, an "array of nucleic acid molecules" is a collection of intentionally produced nucleic acids that can be synthesized or biosynthetically prepared in a variety of different ways (e.g., a library of soluble molecules; and a library of oligonucleotides attached to resin beads, silica chips, or other solid supports). In addition, the term "array" is intended to include libraries of nucleic acids that can be prepared by spotting nucleic acids of essentially any length (e.g., 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 (ribonucleotides, deoxyribonucleotides, or peptide nucleic acids (PNAs)) of any length that contain purine and pyrimidine bases or other natural, synthetic, or biosynthetically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide may contain sugar and phosphate groups (as commonly found in RNA or DNA) or modified or substituted sugar or phosphate groups. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. The term "nucleoside" or "nucleotide" generally refers to a nucleoside or nucleotide sequence having a plurality of common structural features. These analogs are molecules having some common structural features with naturally occurring nucleoside or nucleotide, so that when incorporated into nucleic acid or oligonucleotide sequences, the molecules allow hybridization with the naturally occurring nucleic acid sequences in the solution. Typically, these analogs are derived from naturally occurring nucleoside and nucleotide sequences by substituting and/or modifying base, ribose or phosphodiester moieties. The variation can be customized to allow hybrids to form stable or unstable or enhanced specificity with the hybridization of complementary nucleic acid sequences as required.

As used herein, "solid support," "support," and "substrate" are used interchangeably and refer to a material or group of materials having one or more rigid or semi-rigid surfaces. In many embodiments, at least one surface of the solid support will be substantially flat, but in some embodiments, it may be desirable to physically separate synthesis areas for different compounds using, for example, wells, raised areas, pins, etched grooves, etc. According to other embodiments, the solid support will take the form of beads, resins, gels, microspheres, or other geometric configurations.

Any known nucleic acid array can be used in accordance with the present invention. For example, such arrays include those based on short or long oligonucleotide probes and cDNA or polymerase chain reaction (PCR) products. Other methods include serial analysis of gene expression (SAGE), differential display and subtractive hybridization methods, differential screening (DS), RNA arbitrary primer (RAP)-PCR, restriction endonucleoside analysis of differentially expressed sequences (READS), and amplified restriction fragment length polymorphism (AFLP).

Antibody

The present invention encompasses the use of antibodies for detecting or determining the levels _of OPN and/or _α5β1 in a sample from, for example, a subject and for inclusion in the kits of the present invention. Antibodies that specifically bind to these biomarkers can be routinely produced using methods described further below. The present invention also encompasses the use of commercially available antibodies. Without being so limited, antibodies that specifically bind to OPN and/or _α5β1 include those listed in Table ₁ below.

Table 1: Non-limiting examples of commercially available human OPN Elisa kits

Table 2: Non-limiting examples of commercially available antibodies against OPN (human, unconjugated)

Table 4: Non-limiting examples of commercially available ELISA kits for Integrin alpha ₅ (ITGA5, human)

Table 5: Non-limiting examples of commercially available antibodies against _α5 (ITGA5, human)

Table 6: Non-limiting examples of commercially available ELISA kits for β1 (ITGB1, human)

Company Name Catalog Number scope Sensitivity antibodies-online ABIN833710 Not analyzed Not analyzed Merck Millipore ECM470 Not analyzed Not analyzed DLdevelop DL-ITGb1-Hu 1.56-100 ng/mL Not analyzed Biomatik E91042Hu 1.56-100 ng/mL 0.64 ng/mL

Table 7: Non-limiting examples of commercially available antibodies against β ₁ ITGB1 (human, unconjugated)

Both monoclonal and polyclonal antibodies to OPN, α ₅ and/or β ₁ are included within the scope of the present invention, as they can be produced by well-established procedures known to those skilled in the art. In addition, any secondary antibody (monoclonal or polyclonal) to the first antibody is also included within the scope of the present invention.

As used herein, the terms "anti-OPN antibody,""anti- _α5 antibody,""anti- _β1 antibody," and "anti _{- α5β1} antibody," or "immunospecific anti-OPN antibody,""immunospecific anti- _α5 antibody,""immunospecific anti- _β1 antibody," or "immunospecific _anti - _α5β1 antibody" refer to antibodies _that specifically bind (interact) with OPN, _α5 , _β1 , or _α5β1 protein, respectively, and do not exhibit substantial binding to other naturally occurring proteins other than proteins that share the same antigenic determinants with OPN, _α5 , _β1 , or _α5β1 protein _, respectively. The terms antibody or immunoglobulin are used in the broadest sense and encompass 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, typically the 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 may also refer to binding portions 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. In addition, any secondary antibody (monoclonal or polyclonal) directed against the primary antibody is also within the scope of the present invention.

In general, the techniques for preparing antibodies (including monoclonal antibodies and hybridomas) and using antibodies to detect antigens are well known in the art (Campbell, 1984, In "Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology", Elsevier Science Publishers, Amsterdam, The Netherlands) and Harlow et al., 1988 (published in: Antibody A Laboratory Manual, CSH Laboratories). The term antibody herein encompasses polyclonal antibodies, 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) that inhibit or neutralize the corresponding interacting domain in its hybridoma and/or are specific for it.

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 bifunctional or derivatizing agents such as maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues ⁾ , glutaraldehyde, succinic anhydride, SOCl ₂ or R ¹ N═C═NR (wherein R and R 1 are different alkyl groups).

Animals can be immunized against antigens, immunogenic conjugates or derivatives by mixing the antigen or conjugate (e.g., 100 μg for rabbits or 5 μg for mice) with 3 volumes of complete Freund's adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with the antigen or conjugate in complete Freund's adjuvant (e.g., 1/5 to 1/10 of the original amount used for immunization) by subcutaneous injection at multiple sites. After 7 to 14 days, the animals are bled and the serum is assayed for antibody titers. Animals are boosted until the titer reaches a stable level. Preferably, for conjugate immunization, animals are boosted with the same antigen but conjugated to different proteins and/or conjugated by different cross-linking reagents. Conjugates can also be prepared as protein fusions in recombinant cell culture. In addition, aggregating agents such as alum are also suitable for enhancing immune responses.

Monoclonal antibodies can be prepared using the hybridoma method originally described by Kohler et al., Nature, 256:495 (1975) or can be prepared by recombinant DNA methods (e.g., U.S. Patent No. 6,204,023). Monoclonal antibodies can also be prepared using the technology described in U.S. Patent Nos. 6,025,155 and 6,077,677 and U.S. Patent Application Publication Nos. 2002/0160970 and 2003/0083293.

In the hybridoma method, a mouse or other appropriate host animal, such as a rat, hamster, or monkey, is immunized (e.g., as described above) to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent (e.g., polyethylene glycol) to form hybridoma cells.

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 phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

As used herein, the term "purified" in the expression "purified antibodies" is intended only to distinguish artificial antibodies from antibodies naturally produced by animals against their own antigens. Thus, the original serum and hybridoma culture medium containing anti-OPN antibodies are "purified antibodies" within the meaning of the present invention.

The present invention also encompasses arrays for detecting and/or quantifying _the translation products of OPN and _α5β1 . Such arrays include protein microarrays or macroarrays, gel technology including high-resolution 2D-gel methods, and possibly coupled to mass spectrometry imaging systems at the cellular level, such as microscopes combined with fluorescent labeling systems.

The present invention also encompasses methods for screening/selecting potentially useful therapeutic agents using whole cell assays that increase the transcription and/or synthesis of OPN and/or _α5 and/or _β1 , or reduce the transcription or synthesis of CD44/sCD44. Cells used in the methods include cells of any source (including in-house or commercially available cell lines) and type (of any tissue). In-house cell lines can be prepared, for example, by immortalizing cells from AIS subjects. In specific embodiments, the screening methods of the present invention seek to identify agents that inhibit the expression and/or activity _of OPN and/or _α5β1 . Cell lines useful for these embodiments include those that produce high levels of OPN and/ _{or α5β1} or low levels of CD44/sCD44. Non-limiting examples of useful cells include MC3T3-E1 cells and PBMCs.

In a specific embodiment, the cells include cells of any cell type derived from a scoliosis patient. (Whole cell assay). In a specific embodiment, the cells include osteoblasts, chondrocytes, myoblasts or blood cells, including PBMCs (including lymphocytes). As used herein, the term "cells derived from scoliosis patients" refers to cells directly isolated from scoliosis patients, or immortal cell lines derived from cells directly isolated from scoliosis patients. In a specific embodiment, the cells are paraspinal muscle cells. Such cells can be isolated from a subject by, for example, a puncture biopsy.

The present invention also relates to pharmaceutical compositions for regulating OPN-mediated GiPCR cell signaling and/or for treating or preventing scoliosis (e.g., idiopathic scoliosis or AIS) in a subject in need thereof. The compositions include agents for increasing or decreasing the inhibition of OPN-mediated GiPCR signaling in a subject in need _thereof . For example, the pharmaceutical compositions of the present invention may include OPN, _α5 , _β1 , _and /or _α5β1 antibodies (i.e., neutralizing antibodies), RGD peptides, or antisense/ _siRNA to reduce _OPN and/or _α5β1 activity. In one embodiment, the drug mixture may include stimulators/enhancers for sCD44 or sCD44 _{/ CD44} expression. The pharmaceutical compositions may be administered by any suitable route, such as nasal, intravenous, intramuscular, subcutaneous, sublingual, intrathecal, or intradermal. The route of administration may depend on various factors, such as environment and therapeutic purpose.

dose

Any suitable amount of the pharmaceutical composition can be administered to a subject. The dosage will depend on many factors, including the mode of administration. Generally, the amount of the anti-scoliosis composition (e.g., an agent that reduces OPN and/or _α5β1 ) contained in a _single dose will be an amount that is effective in preventing, delaying, or alleviating scoliosis without inducing significant toxicity, i.e., a "therapeutically effective amount."

The effective dose of the medicament that can also directly measure and reduce OPN and/or α ₅ β _1.Can give described effective dose or its fraction every day or every week.Usually, medicine of the present invention and/or nutraceutical and/or dietary supplement composition can be pressed every day every kg body weight about 0.001mg to the amount of about 500mg (for example, 10mg, 50mg, 100mg or 250mg) at most and use.Dosage can be provided by single or multiple dose scheme.For example, in some embodiments, effective dose is the dosage within the following scope: every day about 1mg to about 25 grams of antiscoliosis preparations, every day about 50mg to about 10 grams of antiscoliosis preparations, every day about 100mg to about 5 grams of antiscoliosis preparations, every day about 1 gram of antiscoliosis preparations, every day about 1mg to about 25 grams of antiscoliosis preparations, every week about 50mg to about 10 grams of antiscoliosis preparations, every other day about 100mg to about 5 grams of antiscoliosis preparations and once a week about 1 gram of antiscoliosis preparation.

By way of example, pharmaceutical compositions of the invention (e.g., containing an agent _that reduces OPN and/or _α5β1 ) may be in the form of liquids, solutions, suspensions, pills, capsules, tablets, soft capsules, powders, gels, ointments, creams, sprays, mists, atomized steam, aerosols, or phytosomes. For oral administration, tablets or capsules may be prepared by conventional methods using at least one pharmaceutically acceptable excipient such as a binder, filler, lubricant, disintegrant, or wetting agent. Tablets may be coated by methods known in the art. Liquid formulations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or they may be presented as dry products for reconstitution with saline or other suitable liquid vehicles prior to use. Formulations for oral administration may also be suitably formulated to give controlled release of the active ingredient.

In addition, the pharmaceutical compositions of the present invention (e.g., containing an agent _that reduces OPN and/or _α5β1 ) may contain a pharmaceutically acceptable carrier for administration to a mammal, including but not limited to sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include but are not limited to propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters. Aqueous carriers include but are not limited to water, alcohol, saline, and buffered solutions. Pharmaceutically acceptable carriers may also include physiologically acceptable aqueous vehicles (e.g., physiological saline) or other known carriers that are appropriate for a particular route of administration.

Agents that reduce the expression or activity of OPN and/or _α5 and/or _β1 or agents that increase sCD44/CD44 levels (e.g., binding to OPN) can be incorporated into dosage forms in combination with any vehicle commonly used in pharmaceutical formulations, such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives, or ethylene glycol. Emulsions, such as those described in U.S. Patent No. 5,434,183, in which a vegetable oil (e.g., soybean oil or safflower oil), an emulsifier (e.g., egg yolk phospholipids), and water are combined with glycerol, can also be used. Methods for preparing appropriate formulations are well known in the art (see, for example, Remington's Pharmaceutical Sciences, 16th edition, 1980, A. Oslo Ed., Easton, Pa.).

In the case of selecting parenteral administration as route of administration, the preparation containing the medicament that reduces OPN and/or α ₅ β ₁ can be provided to the patient in combination with pharmaceutically acceptable sterile aqueous or non-aqueous solvent, suspension or emulsion. The example of non-aqueous solvent is propylene glycol, polyethylene glycol, vegetable oil, fish oil and injectable organic ester. Aqueous carrier comprises water, water-alcohol solution, emulsion or suspension, comprises saline or buffered medical parenteral vehicle, comprises sodium chloride solution, Ringer's dextrose solution, glucose plus sodium chloride solution, Ringer's solution or the fixed oil containing lactose. Intravenous vehicle can comprise fluid and nutrient supplement, electrolyte supplement, as those supplements based on Ringer's dextrose etc.

Since the actual dosage must be carefully selected and titrated by the attending physician based on the unique clinical factors of each patient or by a nutritionist, these are merely simple guidelines. The optimal daily dose will be determined by methods known in the art and will be affected by factors such as the patient's age and other clinically relevant factors. In addition, _the patient may be taking medications for other diseases or conditions. Other medications can be continued while the agent that reduces OPN and/or _α5β1 is being administered to the patient, but in such cases it is particularly recommended to start with a low dose to determine whether adverse side effects are experienced.

The present invention also relates to detecting CD44 risk alleles (e.g., comprising one or more SNPs) in subjects in need thereof. According to the present invention, the CD44 risk allele comprises a SNP located in the coding region of CD44 and is therefore detectable at the genomic, mRNA or protein level. SNP genotyping is the measurement of genetic variation of single nucleotide polymorphisms (SNPs) between members of a species (e.g., humans). SNPs are single base pair mutations at specific loci, typically consisting of two alleles (wherein the rare allele frequency is>1%). SNP genotyping can be performed using methods well known in the art, such as sequencing (e.g., microsequencing, pyrophosphate sequencing), hybridization-based methods (using probes complementary to the SNP sites, hybridized under high stringency conditions), enzyme-based methods, single-stranded conformational polymorphisms (SSCP), temperature gradient gel electrophoresis, denaturing high-performance liquid chromatography, high-resolution melting of the entire amplicon, using DNA mismatch-binding proteins or SNPlex™ platforms. Non-limiting examples of hybridization-based methods include dynamic allele-specific hybridization and the use of molecular beacons. Non-limiting examples of enzyme-based methods include restriction fragment length polymorphism (RFLP), PCR-based methods (e.g., ARMS), flanking endonucleases (e.g., Invader), primer extension, 5'-nuclease (TaqMan™ assay), oligonucleotide ligation assays.

In one embodiment, the methods of the invention include methods for: i) determining the risk of developing scoliosis; and ii) stratifying subjects with scoliosis, the method comprising determining the presence of a CD44 risk allele comprising SNP rs1467558 or a mutation/SNP/marker in linkage disequilibrium therewith.

In specific embodiments of the present invention, linkage disequilibrium (LD) is defined by specific quantitative cutoffs. As described in detail herein, linkage disequilibrium can be quantitatively determined by metrics such as ^r and |D'|. Therefore, certain embodiments of the present invention relate to replacing markers of linkage disequilibrium by metrics within a certain range specified by specific values of ^r and/or |D'|. In one embodiment, LD is characterized by a value greater than 0.1 for ^r . In another embodiment, LD is characterized by a value greater than 0.5 for ^r . In another embodiment, LD is characterized by a value greater than 0.8 for ^r . In another embodiment, LD is characterized by a value of 0.9 or greater for ^r .

linkage disequilibrium

The natural phenomenon of recombination, which occurs on average once per chromosome pair during each meiotic event, represents a way in which nature provides sequence (and therefore biological function) variation. It has been found that recombination does not occur randomly in the genome; rather, there is a large variation in the frequency of recombination rates, resulting in small regions of high recombination frequency (also referred to as recombination hotspots) and large regions of low recombination frequency, which are generally referred to as linkage disequilibrium (LD) blocks (Myers, S. et al., Biochem Soc Trans 34:526-530 (2006); Jeffreys, AJ. et al., Nature Genet 29:217-222 (2001); May, C.A. et al., Nature Genet 31:272-275 (2002)).

Linkage disequilibrium (LD) refers to the non-random distribution of two genetic elements.Therefore, the term " linkage disequilibrium " refers to the non-random genetic association between one or more alleles of two different polymorphic DNA sequences, and it is due to the physical proximity of two loci. When two DNA segments very close to each other on a given chromosome will tend to keep unseparated for several generations, there is linkage disequilibrium, wherein the allele of the DNA polymorphism (or marker) in a segment will show the allele non-random association with the different DNA polymorphisms (or markers) in another DNA segment located nearby. When studying two DNA polymorphisms very close to each other, this situation is encountered in whole human genome. Linkage disequilibrium and its use in genetic studies are well known in the art to which the present invention relates, as exemplified by publications such as Risch and Merikangas, Science 273:1516-1517 (1996); Maniatis, Methods Mol Biol. 376:109-21 (2007); and Borecki et al., Adv Genet 60:51-74 (2008).

For example, if a particular genetic element (e.g., an allele of a polypeptide marker, or a haplotype) occurs at a frequency of 0.25 (25%) in a population, and another element occurs at a frequency of 0.25 (25%), the predicted occurrence of a person with both elements is 0.125 (12.5%), assuming a random distribution of the elements. However, if the two elements are found to occur together at a frequency higher than 0.125, the elements are said to be in linkage disequilibrium because they tend to be inherited together at a ratio higher than that predicted for their individual frequencies of occurrence (e.g., allele or haplotype frequencies). Roughly speaking, LD is typically associated with the frequency of recombination events between the two elements. Allele or haplotype frequencies in the population can be determined by genotyping the individuals in the population and measuring the frequency of occurrence of each allele or haplotype in the population.

Many different metrics have been proposed for assessing the strength of linkage disequilibrium (LD). Most capture the strength of association between pairs of bi-allelic loci. Two important pairwise metrics of LD are ^r² (sometimes designated as Δ) and |D'|. Both metrics range from 0 (no imbalance) to 1 ("complete imbalance"), but their interpretations are slightly different. |D'| is defined in the following way: it equals 1 if only two to three of the possible haplotypes are present, and it <1 if all four possible haplotypes are present. Thus, a |D'| value <1 indicates that historical recombination may have occurred between the two sites (recurrent mutations can also cause |D'| <1, but for single nucleotide polymorphisms (SNPs), this is generally considered less likely than recombination). The measure ^r² represents the statistical correlation between the two sites and takes a value of 1 if only two haplotypes are present.

The r ² measure is arguably the most relevant measure for association mapping because there is a simple inverse correlation between r ² and the sample size required to detect association between a susceptibility locus and a SNP. These measures are defined for pairs of sites, but for some applications, one may wish to determine the extent of the strength of LD across a region encompassing many polypeptide sites (e.g., to test whether the strength of LD varies significantly among loci or within populations, or whether there is more or less LD in a region than predicted under a particular model).

In some embodiments, the LD of the present invention is measured by the r-value, which is a measure of the LD of the region. The LD of the region is measured by the r-value, which is a measure of the LD of the region. The LD of the region is measured by the r-value, which is a measure of the LD of the region. The LD of the region is measured by the r-value, which is a measure of the LD of the region. The LD of the region is measured by the r-value, which is a measure of the LD of the region. The LD of the region is measured by the r-value, which is a measure of the LD of the region. The LD of the region is measured by the r ^- value, which is a measure of the LD of the region. The LD of the region is measured by the r-value, which is a measure of the LD of the region. The LD of the region is measured by the r-value, which is a measure of the LD of the region. The LD of the region is measured by the r-value, which is a measure of the LD of the region. The LD of the region is measured by the r-value, which is a measure of the LD of the region. The LD of the region is measured by the r ^- value, which is a measure of the LD of the region. In one embodiment of the present invention, linkage disequilibrium is measured in the sample of one or more HapMap™ colonies (Caucasians, Africans, Japanese, Chinese). In one embodiment of the present invention, LD is measured in the sample of one or more HapMap ^™ colonies ( ^Caucasians , Africans, Japanese, Chinese). In one embodiment of the present invention, LD is measured in the CEU colony of HapMap ^™ III samples.

If all polymorphisms in the genome are identical under the colony level, each of the polymorphisms will need to be studied in an association study. However, due to the linkage disequilibrium between the polymorphisms, the tightly linked polymorphisms are strongly correlated, and their reduction requires the number of polymorphisms to be studied in an association study to observe significant associations. Genome LD maps have been produced within the entire genome, and this type of LD map has been proposed to serve as a framework (Risch, N and Merkiangas, K, Science 273: 1516-1517 (1996) for drawing the map of disease genes; Maniatis, N. et al., Proc Natl Acad Sci USA 99: 2228-2233 (2002); Reich, DE et al., Nature 411: 199-204 (2001)).

It is now established that many parts of the human genome can be resolved into a series of discrete haplotype blocks containing some common haplotypes; for these blocks, linkage disequilibrium data provide little evidence indicative of recombination (see, e.g., Wall., J.D. and Pritchard, J.K., Nature Reviews Genetics 4:587-597 (2003); Daly, M. et al., Nature Genet. 29:229-232 (2001); Gabriel, S.B. et al., Science 296:2225-2229 (2002); Patil, N. et al., Science 294:1719-1723 (2001); Dawson, E. et al., Nature 4JS:544-548 (2002); Phillips, M.S. et al., Nature Genet 33:382-387 (2003)).

There are two main approaches for defining these haplotype blocks: blocks can be defined as regions of DNA with limited haplotype diversity (see, e.g., Daly, M. et al., Nature Genet. 29:229-232 (2001); Patil, N. et al., Science 294:1719-1723 (2001); Dawson, E. et al., Nature 418:544-548 (2002); Zhang, K. et al., Proc. Natl. Acad. Sci. USA 99:7335-7339 (2002)), or as regions between translated regions with extensive historical recombination identified using linkage disequilibrium (see, e.g., Gabriel, S.B. et al., Science 296:2225-2229 (2002); Phillips, M.S. et al., Nature Genet.33:382-387 (2003); Wang, N. et al., Am.J.Hum.Genet.7:1227-1234 (2002); Stumpf, M.P. and Goldstein, D.B., Curr.Biol.13:1-8 (2003)). Recently, a fine-scale map of recombination rates and corresponding hotspots throughout the human genome has been generated (Myers, S. et al., Science 310:321-32324 (2005); Myers, S. et al., Biochem Soc Trans 34:526530 (2006)). The map reveals a huge variation in recombination within the genome, with recombination rates as high as 10-60 cM/Mb in hotspots and closer to 0 in intercalated regions, which therefore represent regions of limited haplotype diversity and high LD. The map can therefore be used to define haplotype blocks/LD blocks as regions flanked by recombination hotspots. As used herein, the term "haplotype block" or "LD block" includes blocks defined by any of the above characteristics or other alternative methods used by those skilled in the art to define such regions.

Haplotype blocks can be used to map the association between phenotypes and haplotype states, using either a single marker or haplotypes encompassing multiple markers.

The major haplotype can be identified in each haplotype block, and then a set of "marked" SNPs or markers can be identified (the minimum set of SNPs or markers is required to distinguish between the haplotypes). These marked SNPs or markers can then be used to assess samples from multiple groups of individuals to identify the association between phenotypes and haplotypes. If desired, adjacent haplotype blocks can be assessed simultaneously because linkage disequilibrium may also exist in the haplotype blocks. Thus, a skilled artisan can easily identify SNPs/variants/markers that are disequilibrium with the CD44 risk alleles identified herein, and can use markers of such linkage disequilibrium to stratify subjects and determine the risk of developing scoliosis.

When the SNP is translated at the protein level, the mutation/variation can be detected using well-known protein detection methods such as ELISA, immunofluorescence, Western blotting, sequencing, electrophoresis, HPLC, MS, etc.

The present invention also relates to kits. Without being so limited, the present invention relates to kits for: i) increasing GiPCR signaling in cells; ii) stratifying subjects with scoliosis; and/or iii) predicting whether a subject is at risk for developing scoliosis, the kit comprising an isolated nucleic acid (e.g., as described above, for a protein or ligand according to the present invention, such as an antibody (for _α5 , _β1 , CD44 (wild type or SNP risk allele (e.g., 230I→T variant) and/or OPN)) having specific primers or probes (the primers or probes are substantially complementary to or hybridize with _α5 (SEQ ID NO: 24), _β1 (SEQ ID NO: 22), CD44 (wild type (SEQ ID NO: 19) or SNP risk allele (e.g., 230I→T (CT) variant) and/or OPN (SEQ ID NO: 26)). For example, a compartmentalized kit according to the present invention Kit) includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, or plastic or paper strips. Such containers allow reagents to be effectively transferred from one compartment to another so that the sample and reagents will not cross-contaminate, and the reagents or solutions in each container can be added from one compartment to another in a quantitative manner. Such containers will include containers that will receive subject samples (DNA genomic nucleic acid, cell samples, or blood samples), containers containing (in some kits of the present invention) probes used in the methods of the present invention, containers containing enzymes, containers containing washing reagents, and containers containing reagents for detecting extension products. The kit of the present invention may also include instructions for using these probes and or antibodies to stratify scoliosis subjects or predict whether a subject is at risk of developing scoliosis.

The articles "a," "an," and "the" are used herein to refer to one or to more than one of the grammatical object of the article (ie, to "at least one").

The terms "including" and "comprising" are used herein to mean and are interchangeable with the phrases "including but not limited to" and "including but not limited to."

The term "such as" is used herein to mean, and is used interchangeably with, the phrase "such as, but not limited to."

The present invention is further illustrated in detail by the following non-limiting examples.

Example 1

Materials and methods

Experimental animal models

The Review Committee for the Care and Handling of Animals Used in the Study (CHU Sainte-Justine) approved the protocol in accordance with the guidelines of the Canadian Council of Animal Care.

Breeding pairs of C57Bl/6j mice without OPN (OPN-deficient mice) or CD44 (CD44-deficient mice) were obtained from Dr. Susan Rittling (Rutger University, NJ, USA) and backcrossed for more than 10 generations in the C57Bl/6j background to establish our own colony, while C57Bl/6j mice were used as wild-type control mice (Charles-River, Wilmington, MA, USA). The C57Bl6/6j mouse strain was used because it naturally lacks melatonin (Von Gall et al., 2000) and exhibits high circulating OPN levels (Aherrahrou et al., 2004). These mice develop scoliosis when they are maintained in the bipedal stage. (Machida et al., 2006). As previously reported, bipedal surgery was performed by amputating the forelimbs and tail under anesthesia after weaning (5 weeks after birth). (Machida et al., 2006). Similarly, bipedal C57Bl/6jCD44-deficient mice were generated.

Derivation of primary osteoblast cultures

Bone specimens from mice were obtained from the spine after euthanasia. Bone fragments were reduced to smaller fragments using a cutter under sterile conditions. The smaller bone fragments were incubated in αMEM culture medium containing 10% fetal bovine serum (FBS; guaranteed qualified FBS, Invitrogen, Burlington, ON, Canada) and 1% penicillin/streptomycin (Invitrogen) in a 10- ^cm2 culture _dish at 37°C in 5% CO2. After one month, the osteoblasts that emerged from the bone fragments were separated from the remaining bone fragments by trypsinization. RNA was extracted from the osteoblasts using the TRIzol ^™ method (Invitrogen). Expression profiles were studied by qPCR. Transcript expression was assessed using Stratagene ^™ Mx3000P (Agilent Technologies, La Jolla, CA).

Functional evaluation of G protein

Functional evaluation of Gi, Gs, and Gq proteins was assessed by CDS assay using osteoblasts derived from C57Bl/6j WT mice and C57Bl/6j OPN ^−/− mice (bipedal and quadrupedal) using the CellKey ^™ instrument as previously described (Akoume et al., 2010 and Moreau et al., WO 2010/040234).

Bone bridge ELISA

Peripheral blood samples were collected in EDTA-treated tubes and then centrifuged. Plasma samples were obtained, and then the plasma samples were divided into aliquots and stored frozen at -80 ° C until thawed and analyzed. As described in the protocol provided by manufacturer (IBL, Hamburg, Germany), the concentration of plasma OPN was measured by enzyme-linked immunosorbent assay (ELISA). This OPN ELISA kit measures the total concentration of both the phosphorylated form and the non-phosphorylated form of OPN in plasma. All ELISA tests were performed in duplicate and the optical density at 450 nm was measured using a DTX880 microplate reader (Beckman Coulter, USA).

Quantitative reverse transcription-polymerase chain reaction (qPCR)

Thermo-Script ^™ reverse transcriptase (Invitrogen) was used to reverse transcribe mRNA into cDNA (1 mg total concentration). Several dilutions were tested to select the concentration that produced the most efficient amplification. The human primers used were as follows:

β-actin forward 5'-GGAAATCGTGCGTGACAT-3' (SEQ ID NO: 1),

β-actin reverse 5'-TCATGATGGAGTTGAAGGTAGTT-3' (SEQ ID NO: 2),

CD44 forward 5'-AGCATCGGATTTGAGACCTG-3' (SEQ ID NO: 3),

CD44 reverse 5'-TGAGTCCACTTGGCTTTCTG-3' (SEQ ID NO: 4),

β1 integrin forward 5'-ATGTGTCAGACCTGCCTTG-3' (SEQ ID NO: 5),

β1 integrin reverse 5'-TTGTCCCGACTTTCTACCTTG-3' (SEQ ID NO: 6),

αv integrin forward 5'-GTCCCCACAGTAGACACATATG-3' (SEQ ID NO: 7),

αv Integrin Reverse 5'-TCAACTCCTCGCTTTCCATG-3' (SEQ ID NO: 8).

Each amplification was performed in duplicate using 5 ml of diluted cDNA, 7.5 ml of 3 mM primer solution, and 12.5 ml of 2X QuantiTect ^™ SYBR Green PCR Master Mix (QIAGEN Inc, Ontario, Canada). All reaction mixtures were run on an Mx3000P ^™ system from Stratagene (Agilent Technologies Company, La Jolla, CA) and analyzed using MxPro ^™ QPCR software, also from Stratagene. Relative quantification was calculated using the ΔCT method using β-actin as an endogenous control.

Cell lines and siRNA transfection

MC3T3-E1 cells were used to examine the effects of knockdown of OPN and its receptors. MC3T3-E1 osteoblasts were transiently transfected in serum-free medium using Lipofectamine ^™ RNAiMAX reagent (Invitrogen) according to the manufacturer's instructions, and functional experiments were performed 48 hours after transfection. The sequences of the RNA oligonucleotides used to knock down different genes were: ssp1 (encoding OPN) (CCA CAG CCA CAA GCA GUC CAG AUU A (SEQ ID NO:9)), integrin β1 (CCUAAG UCA GCA GUA GGA ACA UUA U (SEQ ID NO:10)), integrin β3 (CCU CCA GCU CAU UGUUGA UGC UUA U (SEQ ID NO:11)), integrin α5 (CCG AGU ACC UGA UCA ACC UGG UUC A (SEQ ID NO:12)), integrin α8 (GAG AUG AAA CUG AAU UCC GAG AUA A (SEQ ID NO:13)), integrin αv (GAC UGU UGA GUA UGC UCC AUG UAG A (SEQ ID NO:14)), and CD44 (GAA CAA GGA GUCGUC AGA AAC UCC A (SEQ ID NO:15)). NO: 15)). Two other siRNAs against integrin α5 (UCC ACCAUG UCU AUG AGC UCA UCA A (SEQ ID NO: 16) and UCA GGA GCA GAU UGC AGA AUC UUA U (SEQ ID NO: 17)) were tested. Comparable results were obtained for all siRNAs.

Evaluation of the effects of OPN on GPCR and Gi protein expression

The expression levels of Gi protein isoforms were determined using Western blotting. MC3T3-E1 cells were treated with PBS or rOPN 0.5 μg/ml overnight (R&D systems Inc., Minneapolis, USA). The cells were washed once with cold PBS and then dissolved in RIPA buffer (25 mM Tris.Hcl pH 7.4, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with 5 mM NaVO ₄ and a protease inhibitor cocktail (Roche molecular Biochemicals, Mannheim, Germany). Immunoblotting was performed with primary antibodies specific for _Gi1 , _Gi2 , Gi3 _, Gs, phosphoserine, integrin β1 (Santa Cruz Biotechnology, Santa Cruz, CA), μ opioid receptor (MOR) (abcam Toronto, ON, Canada), lysophosphatidic acid receptor 1 (LPAR1) (assaybiotech Inc., USA), melatonin receptor 2 (MT2), and peroxidase-conjugated secondary antibodies. Bands were then visualized using SuperSignal ^™ chemiluminescent substrate (Pierce, Rockford, IL).

Effects of OPN on the interaction of Gi proteins with GPCRs and β1 integrin

MC3T3-E1 cells were treated with PBS or rOPN (0.5 μg/ml) and then whole cell protein (1 mg) was incubated overnight at 4°C with protein G beads for immunoprecipitation (IP) with anti-MOR (Abcam Toronto, ON, Canada), anti-MT2 or anti-β ₁ integrin (Santa Cruz Biotechnology). After immunoprecipitation, the purified protein was loaded on a 10% gel and then processed for gel transfer to a PVDF membrane and blocked with 5% BSA. The membrane was exposed to antibodies specific for Gi ₁ , Gi ₂ , Gi ₃ , MOR, MT2 and β ₁ integrin overnight at 4°C and subsequently treated with secondary antibodies for 1 hour at room temperature. SuperSignal ^™ chemiluminescence (Pierce, Rockford, IL) was used to visualize the bands and quantified by optical density scanning.

Whole-exome sequencing identification of SNPs associated with increased risk of spinal deformity progression in AIS subjects

Whole exome sequencing (WES) studies were performed using Agilent targeted exon capture and Life technologies SOLiD 5500™ for sequencing. GEMINI1™ was used for SNV (single nucleotide variant) annotation. A SNP (rs1467558) was identified in AIS subjects with more severe AIS that changes the amino acid isoleucine 230 in the CD44 coding sequence to threonine.

Statistical analysis

Data are presented as mean ± SE and analyzed by ANOVA or Student's t-test using GraphPad ^™ Prism 4.0 software. Multiple comparisons of means were performed using one-way ANOVA followed by Dunnett's post hoc test. Only P values < 0.05 were considered significant.

Example 2

Genetic deletion of OPN protects bipedal C57Bl/6 from scoliosis

Bipedal C57Bl/6 mice are widely used as animal models to study the pathophysiological events that lead to idiopathic scoliosis. These mice rapidly develop spinal deformities after 40 weeks of bipedal walking (Oyama et al., 2006). In order to determine whether OPN is involved in the development of idiopathic scoliosis, female wild-type (WT) and OPN knockout (OPN ^-/- ) C57Bl/6 mice were amputated of their forelimbs and tails at 1 month of age and subjected to bipedal walking for 36 weeks to induce scoliosis. Representative radiographs of the spine obtained at the end of the experimental period are shown in Figures 1A-1D. As expected, scoliosis did not develop in any one of the WT or OPN ^-/- quadrupedal mice. However, scoliosis was obvious in all bipedal WT mice. The convexity of the curve was directed to either side, without consistent bias. In contrast, analysis of radiographs did not produce evidence of scoliosis in bipedal OPN ^-/- mice. All bipedal OPN ^-/- mice had straight spines that were indistinguishable from quadrupedal mice. These data strongly emphasize that genetic OPN deletion protects bipedal C57Bl/6 mice from scoliosis.

To provide further evidence for this hypothesis, OPN was measured in the plasma of bipedal mice 12, 24, and 36 weeks after surgery (Figure 1E) and compared with OPN levels in age-matched quadrupedal mice. Although OPN could not be detected in quadrupedal or bipedal OPN ^-/- mice (data not shown), bipedal WT showed significantly higher plasma OPN than quadrupedal WT mice at all indicated time points. A maximum was observed at thirty-six weeks post-surgery.

Example 3

OPN deficiency improves Gi protein-mediated receptor signaling

Next determine whether the lack of OPN can affect the signal transduction mediated by Gi protein.For this purpose, the osteoblasts from WT and OPN ^-/- mice are screened for their response to DAMGO, somatostatin, oxymetazoline and apelin respectively activated opioid, somatostatin, α2-adrenergic receptor and APJ receptor.These receptors are well known to mediate signal transduction by Gi protein. The results shown in Figure 1F-1I show that all four compounds cause the concentration-dependent reaction in the osteoblasts from WT and OPN ^-/- mice to increase. However, in each case, the reaction amplitude is larger in OPN ^-/- osteoblasts than in WT osteoblasts, which shows that the activation of Gi protein is promoted in OPN ^-/- osteoblasts. In addition, although the reaction amplitude is similar in the osteoblasts from quadruped and bipedal OPN ^-/- mice, significant differences (data not shown) are observed between the reactions from quadruped and bipedal WT mice. The degree of reaction is much lower in the osteoblasts from bipedal WT mice.

To confirm these results with the function of Gi proteins, osteoblasts were treated with pertussis toxin (PTX), which ADP-ribosylates Gi proteins and disrupts their interaction with receptors (Gilman, 1987); (Moss et al., 1984). The results shown in Figures 1G-1M show that the cellular response to each of the four tested agonists was largely reduced by PTX pretreatment in osteoblasts from either phenotype. However, the extent of the reduction was higher in OPN ^-/- osteoblasts. It is worth noting that the difference in response amplitude between WT and OPN ^-/- osteoblasts was completely eliminated by PTX treatment. Overall, these data indicate that OPN deficiency enhances receptor signal transduction mediated by Gi proteins in osteoblasts.

To determine whether loss of OPN specifically enhances Gi protein signaling, we examined the effects of OPN on signaling initiated by other G proteins, such as Gs and Gq. We used isoproterenol and desmopressin to activate Gs through the β-adrenergic and vasopressin receptors, respectively, and bradykinin and endothelin-1 to activate Gq through its cognate receptors. The results in Figures 1N and 1O show that osteoblasts from OPN ^−/− mice responded less to Gs stimulation than osteoblasts from WT mice, whereas Gq stimulation elicited in WT and OPN ^−/− osteoblasts was of comparable magnitude. These findings strongly indicate that OPN deficiency exclusively improves Gi protein-mediated signaling.

Example 4

Extracellular OPN disrupts Gi protein-mediated receptor signaling

In order to further study the effect of OPN in Gi protein-mediated receptor signaling, several cell line experiments were carried out using the MC3T3-E1 osteoblast cell line. Because these cells express OPN, first check whether OPN affects the ability of GiPCR ligand-induced cell signaling by siRNA elimination, as measured by CDS in MC3T3-E1 cells. The results shown in Figure 2A show that the comprehensive response to DAMGO is significantly larger in the cells that eliminate OPN. When stimulating cells with oxymetazoline, similar results are obtained. Interestingly, by exposing cells to the OPN that blocks secretion in neutralization with OPN antibodies, the reaction to both compounds is also increased (Fig. 2B). These results show that extracellular OPN destroys GiPCR signaling. In order to confirm this hypothesis, cells were treated with exogenous recombinant OPN (rOPN) before DAMGO and oxymetazoline stimulation. In each case, rOPN causes a reduction in comprehensive response in a concentration-dependent manner, which is prevented by OPN antibodies (Fig. 2C-2D).

Example 5

CD44 is not involved in the inhibition of GiPCR signaling caused by extracellular OPN

OPN is known to work by interacting with a variety of cell surface receptors, including CD44 and many integrins (Weber et al., 1996); (Rangaswami et al., 2006). Because CD44 is considered a key receptor for OPN, it was first explored whether CD44 is responsible for the inhibitory effect of OPN on GiPCR signaling. For this purpose, a CD44 antibody was used to interfere with the interaction between OPN and CD44. As shown in Figure 3A, OPN reduced the overall response to DAMGO in cells pretreated with IgG control or CD44 antibody. Similar results were obtained when cells were stimulated with oxymetazoline (Figure 3A), indicating that blocking CD44 does not prevent the reduction of GiPCR signaling induced by OPN and even enhances its effect. In order to exclude that the lack of OPN inhibition by CD44 antibody is due to its ineffectiveness, cells pretreated with IgG control or CD44 antibody were stimulated with increasing concentrations of hyaluronic acid (HA), a high-affinity ligand for CD44. As shown in Figure 3B, the overall response induced by HA was completely eliminated by pretreatment with CD44 antibody. Furthermore, the effect of the CD44 antibody on the response to HA stimulation was concentration-dependent ( FIG3C ). These data clearly indicate that the CD44 antibody is active.

Small interfering RNA (siRNA) method is further used to knock down the expression of CD44, and by quantitative real-time PCR and Western blot analysis, the efficiency (Fig. 3 D and 3E) of siRNA transfection is demonstrated. CD44 can not be prevented from the inhibitory effect (Fig. 3 F) of OPN for the reaction to DAMGO or oxymetazoline by siRNA deletion CD44. Consistent with these findings, rOPN treatment causes the concentration-dependent reduction (Fig. 3 G and H) of the reaction to DAMGO and oxymetazoline in the osteoblasts from bipedal CD44 knockout (CD44 ^-/- ) mice. In addition, when compared with the osteoblasts from quadrupedal (CD44 ^-/- ) mice, the reaction of these osteoblasts to DAMG or oxymetazoline is lower (Fig. 3 I and 3J).

Further experiments in wild-type and CD44-/- cells in the presence or absence of HA confirmed that the inhibitory effect of OPN was exacerbated in the absence of CD44 and enhanced in the presence of HA (a known CD44 ligand) (Figures 3K to 3P). CD44 was not the cause of the GiPCR signaling defect, but rather the effect was due to OPN. However, because CD44 binds to OPN, inhibiting CD44 or its effects (via HA, CD44 antibodies, anti-CD44 siRNA, or using CD44-/- cells) exacerbated the destructive effects of OPN.

Collectively, these data clearly indicate that CD44 is not involved in the inhibition of GiPCR signaling by OPN and that its presence reduces the effects of OPN on GiPCR signaling.

Example 6

RGD-dependent integrin mediates the inhibitory effect of OPN on GiPCR signaling

Next, the possible requirement for integrins was examined. Given that OPN contains an arginine-glycine-aspartic acid (RGD) motif that engages a subset of cell surface integrins, as well as a domain containing serine-valine-valine-tyrosine-glutamate-leucine-arginine (SVVYGLR) that interacts with other integrins, it was interesting to examine whether specific domain-dependent integrins are required for OPN action. To this end, small synthetic peptides were used to compete with integrins for binding to either the _RGD or SVVYLRG sequences in OPN. BIO1211 was used to selectively inhibit _α4β1 integrin, the only integrin containing SVVYLRG present in osteoblasts. As shown in Figure 4A, the effect of OPN on GiPCR signaling was not significantly affected by BIO1211 in MC3T3-E1 cells. The response to DAMGO or oxymetazoline was reduced by rOPN in the presence or absence of BIO1211, indicating that integrins with SVVYLRG recognition specificity are not required in this process. In contrast, co-incubation of cells with rOPN and RDG peptide completely prevented the inhibitory effect of rOPN for the reaction to DAMGO and oxymetazoline. Figures 4 B and 4C illustrate that this preventive effect of RDG is concentration-dependent. Increasing the concentration of RGD causes the gradual attenuation of the inhibitory effect of OPN, thereby reversing the effect of OPN when the concentration exceeds 10 μg/ml. Similarly, incubation of osteoblasts from WT mice with high concentrations of RGD showed a significant increase (Figure 4 D) in the reaction to DAMGO or oxymetazoline, thereby indicating that RGD effectively inhibits the effect of secreted OPN. Consistent with this view, RGD had no effect (Figure 4 E) when using osteoblasts from OPN ^-/- mice, thereby indicating that the effect of RGD strictly depends on the presence of OPN.

Collectively, these data suggest that the inhibitory effect of OPN on GiPCR signaling is mediated through one or more RGD-dependent integrins expressed in osteoblasts.

Example 7

Identification of integrins involved in the inhibition of GiPCR signaling caused by OPN

Given the potential role of RGD-dependent integrins in the inhibitory effect of OPN on GiPCR signaling in osteoblasts, specific integrins were examined to determine whether they could mediate this effect. To this end, antibodies were used to selectively neutralize subunits _{of αvβ1} , _αvβ3 , _αvβ5 , _{α5β1 ,} and _α8β1 , RGD-dependent integrins that have been shown to be expressed in _osteoblasts ( _Hughes et al., 1997); (Gronthos et al. _, 1997); (Grzesik and Robey, 1994); (Clover et al., 1992); (Moursi et al., 1997); (Pistone et al., 1996). Figures 5A and 5B show that the inhibitory effect of rOPN on DAMGO and oxymetazoline was attenuated to varying degrees by _β3 , _β5 , _αv , and _α8 antibodies, but not abolished. In contrast, antibodies against _α5 or _β1 reversed the inhibitory effect of rOPN on the responses to both DAMGO and oxymetazoline. Cells pretreated with these antibodies showed an increase in the amplitude of the overall response compared to untreated cells in the presence of rOPN, which was in stark contrast to the decrease induced by rOPN in cells pretreated with IgG. These results indicate that _α5 and _β1 integrins are the primary mediators of the inhibitory effect of OPN on GiPCR signaling.

To further support this hypothesis, siRNA methods were used. When compared with untreated cells, MC3T3-E1 transfected with scrambled siRNA showed a lower response, while cells transfected with _α5 or _β1 integrin siRNA showed a high response in the presence of rOPN. Cells transfected with both _α5 and _β1 integrin siRNA showed a further increase (Fig. 5C). In addition, the silencing of two integrin subunits simultaneously resulted in an increase in the response to DAMGO and oxymetazoline in osteoblasts from bipedal WT and CD44 ^-/- cells (Fig. 5D and 5E). The effective silencing of integrin expression was supported by q-RT-PCR (Fig. 5F). In all cases, cells transfected with other integrins resulted in a partial attenuation of the inhibitory effect of OPN on GiPCR signal transduction (data not shown). In short, these results are convincing evidence _{that α5β1} is the main integrin dimer involved in mediating the inhibitory effect of OPN on GiPCR signal transduction in osteoblasts.

Example 8

OPN has no effect on the expression of Gi proteins and their cognate receptors

In view of the fact that the level of GPCR is key for its signal transduction, it is interesting to check whether the defective GiPCR signal transduction induced by OPN is the result of the quantitative reduction of these receptors. For this purpose, MC3T3-E1 cells are processed with PBS or rOPN and analyzed by Western blotting for the expression of three different GPCRs to cell lysates; Including μ-opioid receptor (MOR), lysophosphatidic acid type 1 receptor (LPA1R) and melatonin type 2 receptor (MT2R). As shown in Figure 6 A, in MC3T3-E1 cells, all three receptors are detected by immunohistochemistry and the level of every kind of receptor is not changed by rOPN processing. Therefore, the effect of rOPN on the expression of Gi protein has been checked. Relative to the cell processed by PBS, rOPN processing does not have a significant effect on the amount of Gi protein, as measured by immunoblotting (Fig. 6 B) using antibodies for Gi ₁ , Gi ₂ and Gi ₃ isotypes. qPCR analysis also revealed no significant difference in mRNA expression of any of these isoforms between PBS-treated and rOPN-treated cells (Figure 6C).These results exclude the possibility that rOPN reduces expression of the receptor or Gi protein.

Example 9

OPN reduces the availability of Gi proteins to their cognate receptors

Gi proteins have been shown to be recruited by _β1 integrin via adapter molecules and mediate integrin signaling (Green et al., 1999). It is worth noting that when different receptors transmit signals through the same G protein subfamily, the receptors share the same limited G protein pool. Therefore, if OPN enhances the recruitment of Gi proteins in the _β1 integrin complex, then a reduction in the amount of Gi proteins in the GiPCR complex will be expected. To evaluate this possibility, cell lysates were immunoprecipitated with antibodies against MO, MT2, or _β1 integrin receptors, and the presence of Gi proteins in each precipitate was examined by Western blotting using antibodies specific for _Gi1 , _Gi2 , or _Gi3 isoforms. Figures 6D, 6E, and 6F show that the relative amount of _Gi1 isoforms immunoprecipitated with MO or MT2 receptors decreased in rOPN-treated cells compared to PBS-treated cells. Similar observations were noted for _Gi2 and _Gi3 isoforms. In contrast, the amount of each of these Gi protein isoforms was elevated in the _β1 integrin precipitate after rOPN treatment.These results suggest that OPN causes the kidnapping of Gi proteins and reduces their availability to GiPCR.

Example 10

OPN enhances the phosphorylation of Gi proteins

In view of the defective GiPCR signal conduction relevant to idiopathic scoliosis has increased in the cause of disease with the phosphorylation of Gi protein (Moreau etc., 2004), it is interesting to check whether OPN affects the phosphorylation state of Gi protein. Therefore, MC3T3-E1 cells are processed with rOPN, then with the antibody immunoprecipitation cell lysate for Gi ₁ , Gi ₂ or Gi ₃ protein isotype, and use anti-phosphoserine/threonine antibody or anti-tyrosine antibody to check phosphorylation level by western blotting. The result discloses that there is phosphorylated serine and tyrosine residue in the Gi ₁ precipitate obtained from the cell processed with PBS or rOPN. However, the band density is higher in the cell processed by OPN. Similar observations (Fig. 7 A) are noted in Gi ₂ and Gi ₃ precipitates. These results show that OPN enhances the phosphorylation of Gi protein at serine and tyrosine residues.

To support the involvement of _{α5β1 integrin} in the process, cells were pretreated with antibodies against _{α5β1 integrin} before rOPN treatment. Western blot analysis revealed that Gi phosphorylation induced by rOPN treatment was attenuated by _{α5β1 integrin} blocking antibodies (Figure 7A).

To correlate these findings with reduced GiPCR signaling in scoliosis, Gi proteins were directly examined to determine whether they undergo increased phosphorylation in osteoblasts from scoliotic mice. Immunoprecipitations of _Gi1 , _Gi2 , and _Gi3 proteins were probed with anti-phospho-serine/threonine or anti-phospho-tyrosine specific antibodies and it was observed that the phosphorylation level of each Gi isoform was significantly higher in osteoblasts from scoliotic bipedal WT or CD44 ^-/- mice compared to osteoblasts from quadrupedal control mice (Figure 7B). Importantly, phosphorylation levels were attenuated by _anti - _α5β1 integrin blocking antibodies (data not shown). These results provide convincing evidence that GiPCR signaling in scoliotic mice is associated with Gi protein phosphorylation induced by OPN engagement via _{α5β1 integrin} .

Example 11

OPN-induced phosphorylation of Gi proteins involves various kinases

In order to identify which kinases participate in the Gi protein phosphorylation induced by OPN, FAK, MEK, ERK1/2, P38, JNK, PI3K, Src, PKC and CaMKII are pharmacologically inhibited. Cell extracts prepared from MC3T3-E1 processed with rOPN in the presence or absence of a specific inhibitor for each kinase were subjected to immunoprecipitation and western blot analysis. The results in Fig. 7 C-7E show that the inhibitor of PKC attenuates the serine phosphorylation level in the Gi protein precipitate, but has no effect on tyrosine phosphorylation level. However, both serine and tyrosine phosphorylation are attenuated by FAK (FAK inhibitor-14), Scr (PP2), CaMKII (KN93), PI3K (wortmannin-data not shown), MEK (PD98059), ERK1/2 (FR180204), JNK (SP60125) and P38 (SB203580). These results indicate that various kinases are directly or indirectly involved in the phosphorylation of Gi proteins induced by OPN.

Example 12

The effect of OPN on Gi and Gs proteins facilitates GsPCR signaling

Some reports indicate that inhibition of Gi proteins disrupts signals from GiPCR and enhances GsPCR signaling (Itoh et al., 1984); (Katada et al., 1985); (Wesslau and Smith, 1992). On the other hand, it has been shown that Gs proteins also enhance GsPCR signaling through tyrosine phosphorylation of Src, possibly by increasing the activity of Gs proteins and their association with receptors (Hausdorff et al., 1992); (Chakrabarti and Gintzler, 2007).

Taking these factors into account and having observed that the genetic deletion of OPN affects the reaction to Gi and Gs stimulation (Fig. 1), it is interesting to check the function and phosphorylation state of OPN to Gs protein and the effect on its interaction with receptor.For this purpose, cells were subjected to isoprenaline and desmopressin in the presence of rOPN or PBS. As expected, the reaction to isoprenaline in the cells processed by rOPN was higher than that in the cells processed by PBS. When stimulating cells with desmopressin, similar results were obtained, thereby indicating that OPN increases the reaction to GsPCR stimulation (Fig. 8 A). Interestingly, the immunoprecipitate of the Gs protein detected with anti-tyrosine antibody showed an intensity higher than that of the cells processed by PBS in the cells processed by rOPN (Fig. 8 B). More interestingly, rOPN processes the presence (Fig. 8 C-8D) of the immunoprecipitate enhancing Gs protein in MT2 and MO receptors.

Collectively, these results demonstrate that OPN not only enhances GsPCR signaling but also enhances tyrosine phosphorylation of the Gs protein and its association with the receptor.

It is assumed that the inhibition of Gi protein and the phosphorylation of Gs protein are responsible for the increase in GsPCR signal transduction associated with rOPN, and the following experimental purpose is to determine the contribution of each parameter. For this purpose, cells were treated with PTX to simulate the destructive effect of rOPN and compared with cells treated with rOPN alone or with a Src inhibitor (PP2) for preventing tyrosine phosphorylation. The results in Figure 8 E show that the cells treated with rOPN had 30% more responses to isoproterenol stimulation than those treated with PTX. This moderate difference was eliminated by the Src inhibitor (PP2). When cells were stimulated with desmopressin, similar observations were noted. These results indicate that Gs phosphorylation only contributes to the part of the stimulating effect of rOPN on GsPCR signal transduction and destroys the main reason that the inhibitory signal from GiPCR represents this phenomenon.

Example 13:

Identification of CT SNPs associated with increased risk of spinal deformity progression in AIS subjects and their effect on GiPCR signaling

Whole exome sequencing (WES) studies were performed on cell samples from AIS subjects and identified a SNP (rs1467558) that changes the amino acid isoleucine 230 in the CD44 coding sequence to threonine in AIS subjects with severe AIS.

Next, the effect of CT SNP on GiPCR signaling was assessed. Osteoblasts carrying the CT SNP from severe AIS patients who underwent surgery and osteoblasts with wild-type CD44 (CC) from healthy controls were stimulated with LPA in the presence of recombinant OPN (rOPN). The GiPCR-deficient signaling effect in response to the same rOPN treatment was reduced by more than 50% in mutant CD44 (CT) osteoblasts compared to wild-type CD44 (CC) osteoblasts (Figure 9A). Comparable results have been obtained using different concentrations of OPN. The CT mutation in CD44 therefore increases the sensitivity of scoliosis patients to the disruptive effects of OPN on GiPCR signaling, thereby contributing to the more severe phenotype observed in these subjects.

Based on the findings presented herein and available evidence, and without limitation, the hypothetical scenario outlined in FIG9 explains the mechanism by which OPN reduces GiPCR signaling and contributes to the development of spinal deformities. According to the proposed mechanism, after OPN binding, _{α5β1 integrin} recruits a portion of Gi proteins from a common pool, which then promotes different signaling pathways, leading to the activation of different kinases, which directly or indirectly phosphorylate a portion of the remaining Gi proteins in the common pool. These events simultaneously cause depletion of the pool and inactivation of different isoforms, resulting in a reduction in the amount of functional Gi proteins required for effective GiPCR signaling. This will reduce the inhibitory control of Gi proteins and favor GsPCR signaling, which is exacerbated by phosphorylation of Gs proteins by Src. The increased GsPCR signaling increases bone formation (Hsiao et al., 2008) and reduces myoblast proliferation (Marchal et al., 1995), leading to an imbalance between bone mass and muscle strength around the spine. As a result, the spine will lose balance and scoliosis will occur. Because OPN mRNA and protein levels are enhanced by Gs protein signaling (Nagao et al., 2011), increased GsPCR signaling will maintain high OPN production and amplify the events that drive the development of spinal deformities. Although additional mechanisms may be operative, the contribution of GsPCR signaling is further supported by the addition of spinal deformities to the constellation of orthopedic problems commonly associated with fibrous dysplasia of bone, an uncommon disease caused by congenital mutations in the Gs protein (Leet et al., 2004).

The results presented herein demonstrate that the absence of CD44 expression enhances the effect of OPN and further reduces the GiPCR signaling in cells (Fig. 3). In addition, HA shows enhanced effects. CD44 is known to bind to OPN and HA. Finally, in AIS subjects with severe scoliosis (not limited to any specific theory), CD44 can act by chelating a portion of the OPN reservoir available for interaction with integrin receptors. In the absence of CD44 available for binding to OPN (e.g., when the expression level of CD44 is reduced, when the affinity of OPN to CD44 is reduced, or when the binding to OPN is blocked (e.g., by an antibody specific for CD44 or by another ligand such as HA)), the bioavailability of OPN to α ₅ β ₁ increases and the GiPCR signaling is further reduced. On the contrary, increasing CD44/sCD44 expression (in conjunction with the availability of OPN) can promote the reduction of OPN binding to integrin and increase the GiPCR signaling in cells.

The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description overall.

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Claims (8)

1. Use of reagents for detecting the presence of at least one copy of the CD44 risk allele in a cell sample in the preparation of a kit for determining the risk of developing scoliosis in a subject, wherein the determination of risk includes: (i) Provide cell samples isolated from the subject; (ii) Detecting the presence of at least one copy of the CD44 risk allele of SNP rs1467558 in the cell sample from the subject using the reagent; and (iii) When at least one copy of the risk allele is detected in the cell sample from the subject, the subject is determined to be at risk of developing scoliosis. 2. Use of reagents for detecting the presence of at least one copy of the CD44 risk allele in a cell sample in the preparation of a kit for stratifying subjects with scoliosis, the stratification comprising: (i) Provide cell samples isolated from the subject; (ii) Detecting the presence of at least one copy of the CD44 risk allele of SNP rs1467558 in the cell sample from the subject using the reagent; and (iii)(a) When at least one copy of the CD44 risk allele is detected in the cell sample from the subject, the subject is stratified into a first AIS subclass; or (b) When the CD44 risk allele is not detected in the cell sample from the subject, the subject is stratified into a second AIS subclass. 3. The use as described in claim 1 or 2, wherein the at least one copy of the CD44 risk allele consists of two copies of the CD44 risk allele, and the subject is homozygous for the mutant SNP rs1467558. 4. The use as described in claim 1 or 2, wherein the sample is a nucleic acid sample. 5. The use as described in claim 1 or 2, wherein the sample is a protein sample. 6. The use as described in claim 1 or 2, wherein the kit comprises: (i) nucleic acid probes or primers for detecting the CD44 risk allele containing SNP rs1467558; and/or (ii) Specific detection of protein ligands of threonine encoded by mutation of SNP rs1467558 in CD44 protein. 7. The use as claimed in claim 6, wherein the nucleic acid probe or primer comprises a nucleic acid sequence that specifically hybridizes with a nucleic acid containing SNP rs1467558. 8. The use as claimed in claim 6, wherein the protein ligand is an antibody specific to CD44 protein containing a threonine residue encoded by a mutation of SNP rs1467558.