METHOD OF DETERMINING THE RISK OF SCOLIOSIS

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority, under 35 U.S.C. §119(e), of U.S. provisional application Ser. No. 60/909,408, filed on Mar. 30, 2007 and on U.S. provisional application Ser. No. 61/025,571, filed on Feb. 1, 2008. All documents above are incorporated herein in their entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N/A.

FIELD OF THE INVENTION

The present invention relates to methods of determining the risk of developing scoliosis, methods of stratifying a subject having a scoliosis, methods for assessing the efficacy of a brace on a subject having a scoliosis, and kits therefor.

BACKGROUND OF THE INVENTION

Spinal deformities and scoliosis in particular, represent the most prevalent type of orthopedic deformities in children and adolescents, while adolescent idiopathic scoliosis (AIS) represents the most common form of scoliosis.

The etiology of adolescent idiopathic scoliosis (AIS) remains poorly understood resulting in the traditional paradigm that AIS is a multi-factorial disease with a genetic predisposition.^(1-7) The occurrence of a melatonin signaling dysfunction in cells derived from biopsies obtained intraoperatively from affected AIS patients has been reported.⁸

Unfortunately, there is no proven method or test available to identify children or adolescents at risk of developing AIS or to identify, which of the affected individuals may require treatment due to the risk of progression. Consequently, the application of current treatments, such as bracing or surgical correction, is delayed until a significant deformity is detected or until a significant progression is clearly demonstrated, resulting in a delayed and less optimal treatment.²⁹

The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

More specifically, in accordance with the present invention, there is provided a method for determining the risk for developing a scoliosis comprising monitoring osteopontin (OPN) expression in a sample from a subject over time; wherein an OPN expression that increases in the subject sample over time is indicative that the subject is at risk for developing a scoliosis.

In a specific embodiment, the monitoring begins when the subject is about three years old. In another specific embodiment, the monitoring is performed by measuring OPN expression at a frequency of at least about once per month. In another specific embodiment, the monitoring is performed by measuring OPN expression at a frequency of at least about once per six month. In another specific embodiment, the method further comprises measuring sCD44 expression in a sample from the subject. In another specific embodiment, the monitoring OPN expression is performed using an enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA).

In accordance with the present invention, there is provided a method for determining the risk for developing a scoliosis comprising measuring osteopontin (OPN) expression in a sample from a subject; wherein an OPN expression that is higher in the subject sample than that in a control sample is indicative that the subject is at risk for developing a scoliosis.

In another specific embodiment, the subject is a likely candidate for developing a scoliosis. In another specific embodiment, the subject is a likely candidate for developing adolescent idiopathic scoliosis. In another specific embodiment, the subject is pre-diagnosed as having a scoliosis.

In another specific embodiment, the subject is pre-diagnosed with adolescent idiopathic scoliosis.

In accordance with another aspect of the present invention, there is provided a method of stratifying a subject having a scoliosis comprising measuring osteopontin (OPN) expression in a sample from the subject; whereby the measuring step enables the stratification of the subject into a scoliosis subgroup.

In accordance with another aspect of the present invention, there is provided a method for assessing the efficacy of a brace on a subject having a scoliosis comprising measuring osteopontin (OPN) expression in a sample from the subject prior to and at least once after bracing the subject, wherein an increase in the OPN expression after as compared to prior to bracing the subject is indicative that the brace is ineffective.

In a specific embodiment, the determining the OPN expression after the bracing is performed at least one month after the bracing. In another specific embodiment, the determining the OPN expression after bracing the subject is performed at least 2 months hours after the bracing. In another specific embodiment, the determining the OPN expression after bracing the subject is performed at least three months after the bracing. In another specific embodiment, the determining the OPN expression after bracing the subject is performed at least six months after the bracing.

In another specific embodiment, the method further comprises measuring soluble CD44 receptor (sCD44) expression in the sample from the subject.

In another specific embodiment, the sample from the subject is a biological fluid from the subject. In another specific embodiment, the biological fluid is selected from the group consisting of blood, urine, tear and saliva. In another specific embodiment, the biological fluid is plasma.

In another specific embodiment, the OPN expression is OPN protein. In another specific embodiment, the determining of the OPN expression is performed with an antibody that specifically binds to OPN. In another specific embodiment, the measuring OPN expression is performed using an enzyme-linked immunosorbent assay (ELISA). In another specific embodiment, the sample is a plasma sample and an OPN expression that is higher than 700 nanograms per milliliter of plasma is indicative that the subject is at risk for developing a scoliosis. In another specific embodiment, the sample is a plasma sample and an OPN expression that is higher than 800 nanograms per milliliter of plasma is indicative that the subject is at risk for developing a scoliosis.

In another specific embodiment, the OPN expression is OPN RNA. In another specific embodiment, the sample from the subject is a paraspinal muscle biopsy and the OPN expression is OPN RNA.

In accordance with another aspect of the present invention, there is provided a method of selecting an agent as a potential candidate for the reduction or prevention of scoliosis comprising contacting a candidate agent with a cell expressing osteopontin (OPN), and detecting the expression of OPN, wherein when the expression of OPN is lower in the presence of the candidate agent as compared to in the absence thereof, the candidate agent is selected.

In accordance with another aspect of the present invention, there is provided a method of selecting an agent as a potential candidate for the reduction or prevention of scoliosis comprising contacting a candidate agent with a cell expressing sCD44, and detecting the expression of sCD44, wherein when the expression of OPN is higher in the presence of the candidate agent as compared to in the absence thereof, the candidate agent is selected.

In another specific embodiment, the cell is a cell derived from a scoliotic patient.

In accordance with another aspect of the present invention, there is provided a method of selecting an agent as a potential candidate for the prevention or reduction of scoliosis comprising administering a candidate agent to a scoliosis model animal before scoliosis has developed in the animal, whereby the candidate is selected when the scoliosis is prevented or reduced in the model animal as compared to in a control animal who was not administered the candidate agent.

In accordance with another aspect of the present invention, there is provided a method of preventing or reducing scoliosis comprising administering to a subject having scoliosis a therapeutically effective amount of an osteopontin inhibitor (OPN) or a selenium rich diet, whereby scoliosis is thereby prevented or treated.

In accordance with another aspect of the present invention, there is provided a method of preventing or reducing scoliosis comprising administering to a subject having scoliosis a therapeutically effective amount of a CD44 inhibitor, whereby scoliosis is thereby prevented or treated.

In accordance with another aspect of the present invention, there is provided a method of preventing or reducing scoliosis comprising administering to a subject having scoliosis a therapeutically effective amount of a sCD44 stimulator, whereby scoliosis is thereby prevented or treated.

In a specific embodiment of the methods of the present invention, the subject is human. In another specific embodiment of the methods of the present invention, the subject is human female. In another specific embodiment of the methods of the present invention, the subject is human male.

In accordance with another aspect of the present invention, there is provided an osteopontin inhibitor for use in the treatment or prevention of scoliosis.

In accordance with another aspect of the present invention, there is provided a CD44 inhibitor for use in the treatment or prevention of scoliosis.

In accordance with another aspect of the present invention, there is provided a sCD44 stimulator for use in the treatment or prevention of scoliosis.

In accordance with another aspect of the present invention, there is provided a use of an osteopontin inhibitor in the manufacture of a medicament for the prevention or the treatment of scoliosis.

In accordance with another aspect of the present invention, there is provided a use of an osteopontin inhibitor for the prevention or the treatment of scoliosis.

In accordance with another aspect of the present invention, there is provided a use of a CD44 inhibitor in the manufacture of a medicament for the prevention or the treatment of scoliosis.

In accordance with another aspect of the present invention, there is provided a use of a CD44 inhibitor for the prevention or the treatment of scoliosis.

In accordance with another aspect of the present invention, there is provided a use of a sCD44 stimulator in the manufacture of a medicament for the prevention or the treatment of scoliosis.

In accordance with another aspect of the present invention, there is provided a use of a sCD44 stimulator for the prevention or the treatment of scoliosis.

In a specific embodiment of the uses of the present invention, the scoliosis is adolescent idiopathic scoliosis.

In accordance with another aspect of the present invention, there is provided a kit for predicting the risk of developing a scoliosis comprising a ligand specific to osteopontin (OPN) and instructions to use the kit for predicting the risk of developing a scoliosis. In a specific embodiment, the kit further comprises a ligand specific to soluble CD44 (sCD44).

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

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 presents OPN detection in pinealectomized chicken and corresponding scoliosis. Upper and lower panels illustrates the up regulation of OPN expression detected in paraspinal muscles of pinealectomized chicken developing a scoliosis (S) vs. those remaining unaffected (NS) at the mRNA and protein levels respectively;

FIG. 2 graphically presents in the left panel the dynamic variation of circulating OPN levels in scoliotic bipedal C57BI/6j mice after surgery, and in the right panel presents typical x-rays of scoliotic deformities observed in bipedal C57BI/6j mice, where females (708) are more severely affected than males (907);

FIG. 3 shows a variation in plasma melatonin concentrations in different mouse strains. S=scoliotic; NS=non-scoliotic;

FIG. 4 shows the effect of the pharmacological inhibition of OPN transcription on scoliotic pinealectomized chicken;

FIG. 5 graphically presents the sensitivity and specificity of plasma osteopontin in healthy control subjects, AIS patients and at risk asymptomatic subjects. In Panel A, an analysis that included 33 healthy control subjects and 32 AIS patients with severe Cobb's Angle (45°) revealed an area under the curve (AUC) of 0.94 with a standard error of 0.03 (95 percent confidence interval [CI], 0.88 to 1.000). In Panel B, the use of a cut-off value of 700 nanograms per ml of osteopontin showed a high sensitivity (90.6%) and a very good specificity (81.8%) for the early detection of AIS and for detecting the risk of scoliosis progression. In Panel C, the use of a cut-off value of 800 nanograms/ml of osteopontin also showed a high sensitivity (84.9%) and a higher specificity (90.9%) for the early detection of AIS and for detecting the risk of scoliosis progression. In Panel D, a clear correlation between the levels of plasma osteopontin and the Cobb's angle is demonstrated using all AIS patients, yielding a p-value<0.001 and r²=0.26;

FIG. 6 presents graphs showing the distribution of age in the different groups for male and female combined (control, at risk, AIS<45 and AIS≧45) (Panel A), and separated by sex female (Panel B) and male (Panel C);

FIG. 7 shows profiles of change in OPN levels, sCD44 levels, and Cobb's angle over follow up time in 4 selected AIS female patients (not under brace treatment) aged 12 (red), 14 (green and blue), and 17 (yellow) at baseline visit;

FIG. 8 shows the distribution of total change in OPN (left panel) and sCD44 (left panel) levels over follow-up time in AIS patients with worsened curve deformity (total increase in Cobb's angle greater than 3°; n=14) and in those without significant change in curve (no change in Cobb's angle, decrease, or increase smaller than 3°; n=36);

FIG. 9 presents graphs showing OPN progression correlated with Cobb's angle progression in AIS patients;

FIG. 10 presents graphs showing OPN regression or stabilization correlated with Cobb's angle regression or stabilization in AIS patients;

FIG. 11 shows profiles of change in OPN and sCD44 levels over follow up time in 4 selected at risk subjects without scoliosis: one male aged 13 (green), and 3 female aged 5 (gold), 11 (blue), and 9 (red) at baseline visit;

FIG. 12 compares OPN, sCD44 and HA levels in non AIS scoliotic patients (NAIS) (OPN (n=28), sCD44 (n=18), HA (n=24)), healthy controls (n=35) and AIS patients (n=252);

FIG. 13 presents a histogram comparison of circulating levels of OPN change in function of spine biomechanics in pre-operated AIS patients (n=79) vs. post-operated AIS patients (n=28);

FIG. 14 presents a histogram comparison of circulating levels of OPN and sCD44 of in pre-operated AIS female (OPN (n=10); sCD44 (n=15)) vs. post-operated AIS female (OPN (n=10); sCD44 (n=12));

FIG. 15 presents charts distributing AIS patients across the predefined cut-off zones pre-operation (Panel A) and post-operation (Panel B);

FIG. 16 presents charts distributing AIS patients across the predefined cut-off zones prior to being treated with bracing (Panel A) and after bracing (Panel B);

FIG. 17 illustrates a hypothetic molecular concept underlying spinal deformity progression in AIS;

FIG. 18 presents a graph that correlates selenium levels in AIS patients with OPN levels;

FIG. 19 presents a histogram comparing selenium levels in three categories of subjects: controls, low OPN producers and high OPN producers;

FIG. 20 presents the nucleotide sequences of the three human OPN isoforms (transcript variant 1, mRNA NM_—001040058 (SEQ ID NO: 1); transcript variant 2, mRNA NM_—000582 (SEQ ID NO: 2); transcript variant 3, mRNA NM_—001040060 (SEQ ID NO: 3) and the amino acid sequences of the three human OPN isoforms (isoform a NP_—001035147 (SEQ ID NO: 4); isoform b NP_—000573 (SEQ ID NO: 5); and isoform c NP_—001035149 (SEQ ID NO: 6));

FIG. 21 presents the nucleotide sequences (mRNA) of six isoforms of human CD44 (NM_—000610 transcript variant 1 (SEQ ID NO: 7); NM_—001001389 transcript variant 2 (SEQ ID NO: 8); NM_—001001390 transcript variant 3 (SEQ ID NO: 9); NM_—001001391 transcript variant 4 (SEQ ID NO: 10); NM_—001001392 transcript variant 5 (SEQ ID NO: 11); X62739 Isoform identified in tumour cells (SEQ ID NO: 12)) and amino acid sequences of six isoforms of human sCD44 (NP_—000601 isoform 1 precursor (SEQ ID NO: 13); NP_—001001389 isoform 2 precursor (SEQ ID NO: 14); NP 001001390 isoform 3 precursor (SEQ ID NO: 15); NP 001001391 isoform 4 precursor (SEQ ID NO: 16); NP_—001001392 isoform 5 precursor (SEQ ID NO: 17); and CAA44602 Isoform identified in tumour cells (SEQ ID NO: 18)); and

FIG. 22 shows the structure of sCD44 (Panel A), the origin of the various CD44 isoforms (Panel B) and the cleavage site in one sCD44 isoform (SEQ ID NO: 23).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The involvement of osteopontin (OPN) (also called secreted phosphoprotein 1, bone sialoprotein I, early T-lymphocyte activation 1), a multifunctional cytokine, was investigated in adolescent idiopathic scoliosis (AIS) and plasma OPN concentrations were determined in three populations: patients with AIS, healthy controls without any family antecedent for scoliosis and asymptomatic offspring, born from at least one scoliotic parent, who are considered as at risk (“children at risk”).

A group of 252 consecutive patients with AIS were compared with 35 healthy control subjects without any family history of scoliosis and 70 asymptomatic at risk subjects. All subjects were Caucasians and demographic characteristics are shown in Table 2 below. Plasma OPN, soluble CD44 receptor (sCD44), and hyaluronan (HA) levels were measured by enzyme-linked immunosorbent assays. Pinealectomized chicken and genetically modified bipedal C57BI/6j mice devoid of either OPN or CD44 receptor, a known OPN receptor, were also studied.

Mean plasma OPN concentration in patients with AIS were significantly higher (p-value<0.001) in patients with AIS having a Cobb's angle>45° (965±414 nanograms per milliliter) than that in healthy controls (570±156 nanograms per milliliter) and than that in AIS patients with a Cobb's angle<45° (799±284 nanograms per milliliter). Diagnostic sensitivity and specificity of OPN for AIS was 84.4 percent and 90.6 percent respectively (cut-off value≧800 nanograms per milliliter). Subgroup analysis showed that 47.9 percent of children at risk had OPN values higher than 800 nanograms per milliliter as opposed to only 8.6 percent for the controls indicating that elevated plasma OPN levels precede scoliosis formation. There were no significant differences in mean plasma sCD44 levels and HA levels between all groups. In respect to pathophysiology of scoliosis, the bipedal C57BI/6j mouse model demonstrated that the development of scoliosis requires OPN interactions with CD44 receptors since none of the genetically modified bipedal mice developed a scoliosis. Cut-off values for OPN disclosed herein were calculated using the commercial Elisa kit specific to human OPN from IBL. They may vary when a OPN expression (mRNA or protein) is measured differently (e.g. measuring OPN expression in a different biological sample through OPN RNA or OPN protein but using a different antibody).

OPN (also called secreted phosphoprotein-1, minopontin, or Eta-1) is a phosphorylated glycoprotein containing an arginine-glycine-aspartate (RGD) sequence present in mineralized tissues such as extracellular matrices. This multifunctional cytokine is involved in many pathological conditions.^9,10 The presence of OPN transcripts and proteins in postural control centers such as the cerebellum, skeletal muscle proprioceptive sensory organs, and inner ear structures that control of equilibrium⁽¹¹⁾ is of interest, since AIS patients also exhibit defects in postural control, proprioception and equilibrium.^(12,13) High plasma OPN levels have been found in different adult cancers and inflammatory conditions^30-33.

OPN signaling action: The OPN signaling pathways are not well understood, although it is known that aside from interacting with integrins, OPN can interact with CD44 receptor at the cell surface.^14,15 Although CD44 is a major receptor for hyaluronan (HA), it also acts as a receptor for OPN and has multiple RGD binding sites. All human isoforms of the CD44 family of adhesion molecules are encoded by a single gene. Alternate splicing of 12 of the 19 exons in the human CD44 gene leads to the production of multiple variant isoforms^18,17 and such structural heterogeneity is responsible of the ligand repertoire of CD44, which includes fibronectin¹⁸, chondroitin sulphate¹⁹, osteopontin²⁰, at least two heparin binding growth hormones and hyaluronan.^21,22 Soluble variant isoforms of sCD44 (sCD44var) have been associated with several pathological conditions.^16,18,23,24 It has been proposed that sCD44 isoforms are either generated through proteolytic cleavage of cell surface CD44 or by de novo synthesis due to alternative splicing. Functional diversity among CD44 molecules, unrelated to variant exon usage, is demonstrated by observations that CD44H, or any particular splice-variant, can be active for hyaluronan (HA) binding when expressed in some cell types but inactive in others. Many CD44 isoforms are tissue specific, but the full range of soluble variant isoform(s) of sCD44 has been associated with some pathological conditions. Indeed, circulating levels of total sCD44 and specific soluble CD44 isoforms have been shown to correlate with tumor metastasis in some malignancies, including non-Hodgkin's lymphoma and breast, gastric, and colon carcinomas. The level of soluble CD44 is also known to be higher in the body fluids of subjects with particular inflammatory conditions, such as rheumatoid arthritis, pouchitis and colitis, and bronchitis. Hyaluronan (HA), also called hyaluronate or hyaluronic acid, is a mucopolysaccharide widely distributed throughout the body and produced by a variety of cells including fibroblasts and other specialized connective tissue cells.

As used herein the term “subject” is meant to refer to any mammal including human, mice, rat, dog, cat, pig, monkey, horse, etc. In a particular embodiment, it refers to a human.

As used herein the term “brace” is meant to include dental and orthopedic brace and “bracing” thus refers to the action of placing the braces on the subject. In a specific embodiment, it is meant to refer to braces for scoliotic subjects.

As used herein the terminology “spinal disorders and disorders causing scoliosis” refers to disorders that may involve development of a scoliosis. Without so limited, it includes AIS, congenital scoliosis, congenital cyphose scoliosis, neurological scoliosis, dysplasic scoliosis, neurofibromatosis, cerebral palsy, muscular dystrophies, neuromuscular scoliosis, spondylolesthesis and Noonan syndrome. Scoliosis that may be stratified or predicted excludes those caused by an accident and certain congenital malformations.

As used herein the terms “likely candidate for developing adolescent idiopathic scoliosis” include children of which a least one parent has adolescent idiopathic scoliosis. Among other factors, age (adolescence), gender and heredity (i.e. born from a mother or father having a scoliosis) are factors that are known to contribute to the risk of developing a scoliosis and are used to a certain degree to assess the risk of developing AIS. In certain subjects, scoliosis develops rapidly over a short period of time to the point of requiring a corrective surgery. Current courses of action available from the moment AIS is diagnosed (when scoliosis is apparent) include observation (when Cobb's angle is around 10-25°), orthopaedic devices (when Cobb's angle is around 25-30°), and surgery (over 45°). The more reliable methods of determining the risk of progression and of monitoring treatment efficiency in accordance of the present invention may assist in 1) selecting an appropriate diet to remove certain food products identified as contributors to scoliosis; 2) selecting the best therapeutic agent; 3) selecting the least invasive preventive action and/or available treatment such as postural exercises, orthopaedic device, and/or less invasive surgeries or surgeries without fusions (a surgery that does not fuse vertebra and preserves column mobility).

As used herein, the terms “severe AIS” refers to a scoliosis characterized by Cobb's angle of 45° or more.

As used herein the terms “risk of developing scoliosis” refer to a genetic or metabolic predisposition of a subject to develop a scoliosis (i.e. spinal deformity) and/or to develop a more severe scoliosis at a future time. For instance, an increase of the Cobb's angle of a subject (e.g. from 40° to 50°, or from 18° to 25°) is a “development” of scoliosis.

As used herein the terminology “biological sample” refers to any solid or liquid sample isolated from a living being. In a particular embodiment, it refers to any solid or liquid sample isolated from a human. Without being so limited it includes a biopsy material, blood, tears (48), saliva, maternal milk, synovial fluid, urine, ear fluid, amniotic fluid and cerebrospinal fluid. In a specific embodiment it refers to a blood sample.

As used herein the terminology “blood sample” is meant to refer to blood, plasma or serum. In a preferred embodiment, plasma is used. In a more specific embodiment it refers to a plasma sample.

As used herein the terminology “control sample” is meant to refer to a sample that does not come from a subject known to have scoliosis or known to be a likely candidate for developing a scoliosis. In methods for determining the risk of developing scoliosis in a subject that is pre-diagnosed with scoliosis, the sample may however also come from the subject under scrutiny at an earlier stage of the disease or disorder.

As used herein the term “treating” or “treatment” in reference to scoliosis is meant to refer to at least one of a reduction of Cobb's angle in a preexisting spinal deformity, improvement of column mobility, preservation/maintenance of column mobility, improvement of equilibrium and balance in a specific plan; maintenance/preservation of equilibrium and balance in a specific plan; improvement of functionality in a specific plan, preservation/maintenance of functionality in a specific plan, cosmetic improvement, and combination of any of the above.

As used herein the term “preventing” or “prevention” in reference to scoliosis is meant to refer to a at least one of a reduction in the progression of a Cobb's angle in a patient having a scoliosis or in an asymptomatic patient, a complete prevention of apparition of a spinal deformity, including changes affecting the rib cage and pelvis in 3D, and a combination of any of the above.

As used herein the term “osteopontin inhibitor” refers to an agent able to reduce or block expression (transcription or translation) of OPN (gene called sspi1), an agent able to reduce or block OPN secretion or an agent able to reduce or block OPN binding to its receptor CD44. Without being so limited, the agent can be natural or synthetic and can be a protein such as but not limited to an antibody that specifically binds to OPN, a peptide, a small molecule, a nucleotide such as but not limited to an antisense or a siRNA specific to OPN.

As used herein the term “CD44 inhibitor” refers to an agent able to reduce expression (transcription or translation) of CD44, or an agent able to reduce CD44 localization at the cellular membrane. Without being so limited, the agent can be natural or synthetic and can be a protein such as but not limited to an antibody that specifically binds to CD44, a peptide, a small molecule, a nucleotide such as but not limited to an antisense or a siRNA specific to CD44.

As used herein the term “sCD44 stimulator” refers to an agent able to increase expression (transcription or translation) of sCD44, an agent able to increase sCD44 secretion or an agent able to increase sCD44 affinity toward OPN. Without being so limited, the agent can be a protein, a peptide, a small molecule or a nucleotide.

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

The term “including” and “comprising” are used herein to mean, and re used interchangeably with, the phrases “including but not limited to” and “comprising but not limited to”.

The terms “such as” are used herein to mean, and is used interchangeably with, the phrase “such as but not limited to”.

The present invention also relates to methods for the determination of the level of expression (i.e. transcript or translation product) of OPN, HA or sCD44. The present invention therefore encompasses any known method for such determination including Elisa (Enzyme Linked Immunosorbent Assay), RIA (Radioimmunoassay), real time PCR and competitive PCR, Northern blots, nuclease protection, plaque hybridization and slot blots.

The present invention also concerns isolated nucleic acid molecules including probes and primers to detect OPN, sCD44 or CD44. In specific embodiments, the isolated nucleic acid molecules have no more than 300, or no more than 200, or no more than 100, or no more than 90, or no more than 80, or no more than 70, or no more than 60, or no more than 50, or no more than 40 or no more than 30 nucleotides. In specific embodiments, the isolated nucleic acid molecules have at least 17, or at least 18, or at least 19, or at least 20, or at least 30, or at least 40 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 300 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 200 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 100 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 90 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 80 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 70 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 60 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 50 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 40 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 17 and no more than 40 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 20 and no more than 30 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 17 and no more than 30 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 300 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 200 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 100 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 90 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 80 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 70 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 60 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 50 nucleotides. In other specific embodiments, the isolated nucleic acid molecules have at least 30 and no more than 40 nucleotides. It should be understood that in real-time PCR, primers also constitute probe without the traditional meaning of this term. Primers or probes appropriate to detect OPN sCD44 and CD44 in the methods of the present invention can be designed with known methods using sequences distributed across their respective nucleotide sequence (49).

Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and α-nucleotides and the like. Modified sugar-phosphate backbones are generally known. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.

The types of detection methods in which probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection). Although less preferred, labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds. Other detection methods include kits containing probes on a dipstick setup and the like.

As used herein the terms “detectably labeled” refer to a marking of a probe or an antibody in accordance with the presence invention that will allow the detection of OPN, HA and/or sCD44 in accordance with the present invention. Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labeled according to numerous well known methods. Non-limiting examples of labels include ³H, ¹⁴C, ³²P, and ³⁵S, Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radionucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.

As commonly known, radioactive nucleotides can be incorporated into probes of the invention by several methods. Non-limiting examples thereof include kinasing the 5′ ends of the probes using gamma ³²P ATP and polynucleotide kinase, using the Klenow fragment of Pol I of E. coli in the presence of radioactive dNTP (e.g. uniformly labeled DNA probe using random oligonucleotide primers in low-melt gels), using the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like.

The present invention also relates to methods of selecting compounds. As used herein the term “compound” is meant to encompass natural, synthetic or semi-synthetic compounds, including without being so limited chemicals, macromolecules, cell or tissue extracts (from plants or animals), nucleic acid molecules, peptides, antibodies and proteins.

The present invention also relates to arrays. As used herein, an “array” is an intentionally created collection of molecules which can be prepared either synthetically or biosynthetically. The molecules in the array can be identical or different from each other. The array can assume a variety of formats, e.g., libraries of soluble molecules; libraries of compounds tethered to resin beads, silica chips, or other solid supports.

As used herein “array of nucleic acid molecules” is an intentionally created collection of nucleic acids which can be prepared either synthetically or biosynthetically in a variety of different formats (e.g., libraries of soluble molecules; and libraries of oligonucleotides tethered to resin beads, silica chips, or other solid supports). Additionally, the term “array” is meant to include those libraries of nucleic acids which can be prepared by spotting nucleic acids of essentially any length (e.g., from 1 to about 1000 nucleotide monomers in length) onto a substrate. The term “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNAs), that comprise purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups, as may typically be found in RNA or DNA, or modified or substituted sugar or phosphate groups. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. Thus the terms nucleoside, nucleotide, deoxynucleoside and deoxynucleotide generally include analogs such as those described herein. These analogs are those molecules having some structural features in common with a naturally occurring nucleoside or nucleotide such that when incorporated into a nucleic acid or oligonucleotide sequence, they allow hybridization with a naturally occurring nucleic acid sequence in solution. Typically, these analogs are derived from naturally occurring nucleosides and nucleotides by replacing and/or modifying the base, the ribose or the phosphodiester moiety. The changes can be tailor made to stabilize or destabilize hybrid formation or enhance the specificity of hybridization with a complementary nucleic acid sequence as desired.

As used herein “solid support”, “support”, and “substrate” are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. In many embodiments, at least one surface of the solid support will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like. According to other embodiments, the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations.

Any known nucleic acid arrays can be used in accordance with the present invention. For instance, such arrays include those based on short or longer oligonucleotide probes as well as cDNAs or polymerase chain reaction (PCR) products. Other methods include serial analysis of gene expression (SAGE), differential display, as well as subtractive hybridization methods, differential screening (DS), RNA arbitrarily primer (RAP)-PCR, restriction endonucleolytic analysis of differentially expressed sequences (READS), amplified restriction fragment-length polymorphisms (AFLP).

Antibodies

The present invention encompasses using antibodies for detecting or determining OPN, sCD44 or CD44 levels for instance in the samples of a subject and for including in kits of the present invention. Antibodies that specifically bind to these biological markers can be produced routinely with methods further described below. The present invention also encompasses using antibodies commercially available. Without being so limited antibodies that specifically bind to OPN include those listed in Table 1 below.

TABLE 1
commercially available human OPN Elisa kits.

		Catalogue
Company	Kit name	number	Sensitivity

IBL Hambourg	Human Osteopontin ELISA	JP 171 58	3.33	ng/ml
IBL America	Human Osteopontin N-Half	27258	3.90	pmol/L
	Assay Kit-IBL
IBL-America	Human Osteopontin Assay	27158	3.33	ng/ml
	Kit-IBL
Assay designs	Osteopontin (human) EIA Kit	900-142	0.11	ng/ml

American Research	Osteopontin, human kit	17158	?
Products, Inc.

R&D Systems	Human Osteopontin (OPN)	DOST00	0.024	ng/mL
	ELISA Kit
Promokine	Human Osteopontin ELISA	PK-EL-KA4231	3.6	ng/ml

Uscnlife	Human Osteopontin, OPN	E0899h	?
	ELISA Kit

Both monoclonal and polyclonal antibodies directed to OPN are included within the scope of this invention as they can be produced by well established procedures known to those of skill in the art. Additionally, any secondary antibodies, either monoclonal or polyclonal, directed to the first antibodies would also be included within the scope of this invention.

As used herein, the term “anti-OPN antibody” or “immunologically specific anti-OPN antibody” refers to an antibody that specifically binds to (interacts with) an OPN protein and displays no substantial binding to other naturally occurring proteins other than the ones sharing the same antigenic determinants as the OPN protein. The term antibody or immunoglobulin is used in the broadest sense, and covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, and antibody fragments so long as they exhibit the desired biological activity. Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (V_H, V_H-V_H), anticalins, PepBodies™, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. Additionally, any secondary antibodies, either monoclonal or polyclonal, directed to the first antibodies would also be included within the scope of this invention.

In general, techniques for preparing antibodies (including monoclonal antibodies and hybridomas) and for detecting antigens using antibodies are well known in the art (Campbell, 1984, In “Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology”, Elsevier Science Publisher, Amsterdam, The Netherlands) and in Harlow et al., 1988 (in: Antibody A Laboratory Manual, CSH Laboratories). The term antibody encompasses herein polyclonal, monoclonal antibodies and antibody variants such as single-chain antibodies, humanized antibodies, chimeric antibodies and immunologically active fragments of antibodies (e.g. Fab and Fab′ fragments) which inhibit or neutralize their respective interaction domains in Hyphen and/or are specific thereto.

Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc), intravenous (iv) or intraperitoneal (ip) injections of the relevant antigen with or without an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, where R and R¹ are different alkyl groups.

Animals may be immunized against the antigen, immunogenic conjugates, or derivatives by combining the antigen or conjugate (e.g., 100 μg for rabbits or 5 μg for mice) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with the antigen or conjugate (e.g., with ⅕ to 1/10 of the original amount used to immunize) in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, for conjugate immunizations, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.

Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or may be made by recombinant DNA methods (e.g., U.S. Pat. No. 6,204,023). Monoclonal antibodies may also be made using the techniques described in U.S. Pat. Nos. 6,025,155 and 6,077,677 as well as U.S. Patent Application Publication Nos. 2002/0160970 and 2003/0083293 (see also, e.g., Lindenbaum et al., 2004).

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

The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

As used herein, the term “purified” in the expression “purified antibody” is simply meant to distinguish man-made antibody from an antibody that may naturally be produced by an animal against its own antigens. Hence, raw serum and hybridoma culture medium containing anti-OPN antibody are “purified antibodies” within the meaning of the present invention.

The present invention also encompasses arrays to detect and/or quantify the translation products of OPN, HA or sCD44. Such arrays include protein micro- or macroarrays, gel technologies including high-resolution 2D-gel methodologies, possibly coupled with mass spectrometry imaging system at the cellular level such as microscopy combined with a fluorescent labeling system.

The present invention also encompasses methods for identifying specific mutation(s) directly or indirectly affecting the transcription, translation, post-translational modification or activity of OPN. Without being so limited, mutations of interest include any mutation affecting the interactions between OPN and any soluble or non soluble isoform of CD44 or the binding of HA to any soluble or non soluble isoform of CD44.

The present invention also encompasses the monitoring of the biomarkers disclosed herein to assess the efficacy of numerous approaches to prevent scoliosis and curve progression such as any physical therapies (e.g. postural exercises, physiotherapies, biomechanical stimulations by manipulation or using specific devices e.g. vibrant plates); the monitoring of bracing efficacy or development of novel braces; the monitoring of new surgical devices with or without fusion of vertebras, and the monitoring of the efficacy of specific diet, nutraceutical and/or pharmacological treatments. Without being so limited, the first measure after the braces have been applied could be performed 1 month later to determine for instance whether the braces are well adjusted and determine whether the patient is compliant to the treatment. Thereafter, the monitoring could be performed every three to six months depending on whether high OPN levels are detected or not. This method of the present invention may advantageously reduces the requirement for x-rays. X-rays could be performed for instance only at visits where OPN levels detected are too high.

The present invention also encompasses the monitoring of the biomarkers disclosed herein identify patients having a risk of progression for early bracing or for less-invasive surgeries with novel fusionless devices, for pharmacological treatments and to monitor responses to treatment in patients with AIS. Of note, fusionless devices are particularly useful for patients still possessing a growth potential so that identification of the risk of developing a scoliosis as early as possible in the life of the subject is beneficial. In a specific embodiment, monitoring begins when the subject is about 5 years old or less in subjects having a scoliosis family antecedent/history. The frequency of the testing could typically be every six months. In case where OPN values are above the cut-off value (i.e. >800 ng/ml when the OPN IBL ELISA kit code No. 27158 is used), the frequency would be advantageously significantly increased (e.g. every month, every two months, every three months . . . ).

The present invention also encompasses methods to screen/select for potential useful therapeutic agents using whole cells assays, the therapeutic compound being able to repress the transcription and/or synthesis of OPN (encoded by ssp1 gene), and/or able to increase the production of sCD44 which could sequester circulating OPN, and/or able to interfere with OPN liaison with the CD44 receptor, and/or able to block CD44 receptor. Cells for use in such methods includes cells of any source (including in house or commercially available cell lines) and type (any tissue). In house cell lines could be made for instance by immortalizing cells from AIS subjects. In specific embodiments, methods of screening of the invention seek to identify agents that inhibit OPN expression (transcription and/or translation) and agents that increase sCD44 expression (transcription and/or translation). Useful cell lines for these embodiments include those producing high levels of OPN and/or low levels of sCD44. Such useful cell lines are described in references 43-56.

In a particular embodiment, it includes cells of any cell type derived from a scoliotic patient. (whole cell assay). In specific embodiments, it includes osteoblasts, chondrocytes, myoblasts or blood cells including lymphocytes. As used herein, the term “cell derived from a scoliotic patient” refers to cells isolated directly from scoliotic patients, or immortalized cell lines originating from cells isolated directly from scoliotic patients. In specific embodiments, the cells are paraspinal muscle cells. Such cells may be isolated by a subject through needle biopsies for instance.

Pharmaceutical compositions can also be administered by routes such as nasally, intravenously, intramuscularly, subcutaneously, sublingually, intrathecally, or intradermally. The route of administration can depend on a variety of factors, such as the environment and therapeutic goals.

Dosage

Any amount of a pharmaceutical and/or nutraceutical and/or dietary supplement compositions can be administered to a subject. The dosages will depend on many factors including the mode of administration. Typically, the amount of anti-scoliosis composition (e.g. osteopontin inhibitor or selenium compound) contained within a single dose will be an amount that effectively prevents, delays or reduces scoliosis without inducing significant toxicity “therapeutically effective amount”.

In some embodiments, the therapeutically effective amount of the neutraceutical anti-scoliosis composition (e.g. selenium supplement) can be altered. Useful effective amount concentrations include amounts ranging from about 0.01% to about 10% of a total diet on a weight by weight basis, from about 1% to about 6% of a total diet on a weight by weight basis, or from about 02% to about 6% of a total diet on a weight by weight basis.

The effective amount of the osteopontin inhibitor or selenium compound may also be measured directly. The effective amount may be given daily or weekly or fractions thereof. Typically, a pharmaceutical and/or nutraceutical and/or dietary supplement composition of the invention can be administered in an amount from about 0.001 mg up to about 500 mg per kg of body weight per day (e.g., 10 mg, 50 mg, 100 mg, or 250 mg). Dosages may be provided in either a single or multiple dosage regimen. For example, in some embodiments the effective amount is a dose that ranges from about 1 mg to about 25 grams of the anti-scoliose preparation per day, about 50 mg to about 10 grams of the anti-scoliose preparation per day, from about 100 mg to about 5 grams of the anti-scoliose preparation per day, about 1 gram of the anti-scoliose preparation per day, about 1 mg to about 25 grams of the anti-scoliose preparation per week, about 50 mg to about 10 grams of the anti-scoliose preparation per week, about 100 mg to about 5 grams of the anti-scoliose preparation every other day, and about 1 gram of the anti-scoliose preparation once a week.

By way of example, a pharmaceutical (e.g. containing an osteopontin inhibitor) and/or nutraceutical (e.g. containing selenium) and/or dietary supplement (e.g. containing selenium) composition of the invention can be in the form of a liquid, solution, suspension, pill, capsule, tablet, gelcap, powder, gel, ointment, cream, nebulae, mist, atomized vapor, aerosol, or phytosome. For oral administration, tablets or capsules can be prepared by conventional means with at least one pharmaceutically acceptable excipient such as binding agents, fillers, lubricants, disintegrants, or wetting agents. The tablets can be coated by methods known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspension, or they can be presented as a dry product for constitution with saline or other suitable liquid vehicle before use. Dietary supplements of the invention also can contain pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles, preservatives, buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration also can be suitably formulated to give controlled release of the active ingredients.

In addition, a pharmaceutical (e.g. containing an osteopontin inhibitor) and/or nutraceutical (e.g. containing selenium) and/or dietary supplement (e.g. containing selenium) composition of the invention can contain a pharmaceutically acceptable carrier for administration to a mammal, including, without limitation, sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include, without limitation, propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters. Aqueous carriers include, without limitation, water, alcohol, saline, and buffered solutions. Pharmaceutically acceptable carriers also can include physiologically acceptable aqueous vehicles (e.g., physiological saline) or other known carriers appropriate to specific routes of administration.

An osteopontin inhibitor or selenium may be incorporated into dosage forms in conjunction with any of the vehicles which are commonly employed in pharmaceutical preparations, e.g. talc, gum arabic, lactose, starch, magnesium searate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives or glycols. Emulsions such as those described in U.S. Pat. No. 5,434,183, may also be used in which vegetable oil (e.g., soybean oil or safflower oil), emulsifying agent (e.g., egg yolk phospholipid) and water are combined with glycerol. Methods for preparing appropriate formulations are well known in the art (see e.g., Remington's Pharmaceutical Sciences, 16th Ed., 1980, A. Oslo Ed., Easton, Pa.).

In cases where parenteral administration is elected as the route of administration, preparations containing osteopontin inhibitor or selenium may be provided to patients in combination with pharmaceutically acceptable sterile aqueous or non-aqueous solvents, suspensions or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters. Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions, including saline and buffered medical parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose, or fixed oils. Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers, such as those based upon Ringer's dextrose, and the like.

These are simply guidelines since the actual dose must be carefully selected and titrated by the attending physician based upon clinical factors unique to each patient or by a nutritionist. The optimal daily dose will be determined by methods known in the art and will be influenced by factors such as the age of the patient and other clinically relevant factors. In addition, patients may be taking medications for other diseases or conditions. The other medications may be continued during the time that the osteopontin inhibitor or selenium compound is given to the patient, but it is particularly advisable in such cases to begin with low doses to determine if adverse side effects are experienced.

The present invention also relates to kits. Without being so limited, it relates to kits for stratifying scoliotic subjects and/or predicting whether a subject is at risk of developing a scoliosis comprising an isolated nucleic acid, a protein or a ligand such as an antibody in accordance with the present invention as described above. For example, a compartmentalized kit in accordance with the present invention includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the subject sample (DNA genomic nucleic acid, cell sample or blood samples), a container which contains in some kits of the present invention, the probes used in the methods of the present invention, containers which contain enzymes, containers which contain wash reagents, and containers which contain the reagents used to detect the extension products. Kits of the present invention may also contain instructions to use these probes and or antibodies to stratify scoliotic subjects or predict whether a subject is at risk of developing a scoliosis.

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

EXAMPLE 1

Material and Methods

GENERATION OF BIPEDAL C57BL/6J OPN-NULL AND CD44-NULL MICE. Experiments in mice were conducted according to protocols approved by The Step-Justine Hospital's Animal Health Care Review Committee. Breeding pairs of C57BI/6 devoid of either OPN(OPN-null mice) or CD44 receptor (CD44-null mice) backcrossed for more than 10 generations in C57BI/6j mice were graciously obtained from Dr. Susan Rittling, (Rutger University, NJ, USA) and Dr. Tak Mak (University of Toronto, ON, Canada), respectively, to establish new colonies, while C57BI/6j mice served as wild-type control mice (Charles-River, Wilmington, Mass., USA). The C57BI6/6j mouse strain was used because it is naturally deficient in melatonin⁽²⁶⁾, exhibits high circulating OPN levels⁽²⁷⁾ and develops scoliosis when they are maintained in a bipedal state.⁽²⁸⁾ It is a well known scoliosis animal model. Bipedal surgeries were performed after weaning by amputation of the forelimbs and tail under anesthesia as reported previously.⁽²⁸⁾ All mice underwent complete radiographic examination under anesthesia using a Faxitron™ X-rays apparatus (Faxitron X-rays Corp. Wheeling, Ill., USA) every two weeks starting at the age of six weeks. Anteroposterior X-rays were taken and each digital image was evaluated subsequently for the presence of scoliosis. Cobb's angle threshold value of 10° or higher was retained as a significant scoliotic condition.

IMMUNODETECTION OF MOUSE OPN Mouse serum was obtained from peripheral blood samples for the determination of serum levels of OPN and were collected in serum separator tubes containing silica gel (BD Microtainer, BD New Jersey, USA) and then centrifuged. Derived serum samples were aliquoted and kept frozen at −80° C. until thawed and analyzed. Serum concentrations of OPN were measured by capture enzyme-linked immunosorbent assays (ELISA) according to the protocol provided by the manufacturer (IBL, Hamburg, Germany). The OPN ELISA kit measured total concentration of both phosphorylated and non-phosphorylated of all isoforms of OPN in serum. ELISA tests were performed in duplicate and the optical density was measured at 450 nm using an AsysHiTech™ Expert-96 microplate reader (Biochrom, Cambridge, UK). Although serum was used in mice herein, the present invention also encompasses measuring OPN in mice plasma.

GENERATION OF PINEALECTOMIZED CHICKENS. A percentage of pinealectomized chickens develop a scoliosis and they are thus used as a scoliosis model. For this study, 145 newly hatched chickens (Mountain Hubbard) were purchased at a local hatchery and pinealectomy were performed as previously described⁽²⁵⁾.

EXPRESSION ANALYSIS AND IMMUNODETECTION OF CHICKEN OPN. Total cellular RNA was prepared from paraspinal muscles of pinealectomized chickens by phenol/chloroform extraction. For RT-PCR, 1 microgramme total RNA was reversed transcribed using ThermoScript™ reverse transcriptase (Invitrogen), and the equivalent of 0.1 microgramme of reverse-transcribed RNA used for PCR reactions. These were carried out in a final volume of 50 microliters containing 200 micromolars dNTPs, 1.5 millimolars MgCl₂, 10 picomolars each primer, and 1U Pfu DNA-polymerase (Stratagene, LaJolla, Calif., USA). PCR reactions were performed using the following primers and conditions: chicken OPN (420 by PCR product): 5′-ACACTTTCACTCCAATCGTCC-3′ (SEQ ID NO: 19)(forward), 5′-TGCCCTTTCCGTTGTTGTCC-3′ (SEQ ID NO: 20) (reverse) 35 cycles: 95° C./45 seconds, 66° C./45 seconds, 72° C./1 minute. For quantitative analysis, all amplifications were normalized against that of the housekeeping gene β-actin; chicken β-actin (460 by PCR product) 5′-GGAAATCGTGCGTGACAT-3′ (SEQ ID NO: 21) (forward), 5′-TCATGATGGAGTTGAATGTAGTT-3′ (SEQ ID NO: 22) (reverse) 32 cycles: 94° C./45 seconds, 55° C./45 seconds, 72° C./1 minute. PCR amplified products were analyzed on 1.5% agarose gel containing ethidium bromide. Total protein extracts of paraspinal muscles were used to detect chicken OPN by Western blot using anti-human OPN antibodies cross-reacting with chicken OPN (clone 8E5, Kamiya Biomedial, WA, USA).

HUMAN POPULATIONS The institutional review boards of The Sainte-Justine Hospital, The Montreal Children's Hospital, The Shriners Hospital for Children in Montreal, McGill University and The Affluent School Board, approved the study. Parents or legal guardians of all participants gave written informed consent, and minors gave their assent.

All patients with AIS were examined by one of six orthopedic surgeons. A person was deemed to be affected if history and physical examination were consistent with the diagnosis of idiopathic scoliosis and a minimum of a ten degree curvature in the coronal plane with vertebral rotation was found on a standing radiograph of the spine. Healthy controls were recruited in elementary schools of Montreal. Each subject was examined by the same orthopedic surgeon using Adam's forward bending-test with a scoliometer.

Three populations were investigated: patients with AIS, healthy controls without any family antecedent/history for scoliosis and asymptomatic offspring, born from at least one scoliotic parent, who are considered as at risk of developing a scoliosis. A group of 252 consecutive patients with AIS, 35 healthy control subjects and 70 asymptomatic children at risk of developing a scoliosis were recruited. All subjects were Caucasians and demographic characteristics are shown in Table 2 below).

TABLE 2
Demographic and clinical characteristics of patients with AIS, healthy control and at risk control subjects.

Subject Type

		Healthy	At Risk
	AIS	Control Subjects	Control Subjects

Characteristics	Female	Male	Female	Male	Female	Male
Number	215	37	19	16	45	25
Mean Age (Years)	14.1 ± 2.1	14.8 ± 2.2	10.6 ± 0.6	10.9 ± 0.6	9.8 ± 3.7	10.0 ± 2.9

Patient percentage & Mean Cobb's Angle
Thoracolumbar	35.8%	22.5 ± 15.2	29.7%	28.3 ± 22.8	—	—	—	—
Thoracic	20.5%	39.7 ± 20.4	29.7%	34.1 ± 22.3	—	—	—	—
Double Scoliosis	30.2%		24.3%		—	—	—	—
(Thoracic + Lumbar)
Thoracic Curvature		34.8 ± 19.0		38.9 ± 21.2
Lumbar Curvature		31.0 ± 17.3		33.0 ± 18.7
Lumbar	4.7%	25.4 ± 10.7	8.1%	20.3 ± 3.5	—	—	—	—
Double Scoliosis	6.0%		5.4%		—	—	—	—
(Thoracic + Thoracolumbar)
Thoracic Curvature		25.4 ± 13.5		36.0 ± 19.8
Lumbar Curvature		25.2 ± 15.5		41.0 ± 29.7
Triple Scoliosis	1.9%	36.8 ± 18.5	2.7%	8.0	—	—	—	—
		41.0 ± 14.3		11.0
		30.5 ± 7.7		11.0
Double Scoliosis	0.9%		—		—	—	—	—
(Thoracic + Thoracic)
		29.0 ± 5.7		—
		16.5 ± 3.5		—
Heredity	36.3%		37.8%		0.0%	0.0%	100.0%	100.0%
*Plus-minus values are means ± standard deviations.
†Mean Cobb's Angles for double scoliosis are represented by the curvatures on the thoracic and lumbar levels separately.
‡Mean Cobb's Angle for the triple scoliosis represents two thoracic curvatures and one lumbar curvature.

OSTEOPONTIN, SCD44 AND HA ENZYME-LINKED IMMUNOSORBENT ASSAYS Peripheral blood samples for AIS patients, asymptomatic children and control groups were collected in EDTA-containing tubes and then centrifuged. Derived plasma samples were aliquoted and kept frozen at −80° C. until thawed and analyzed. Plasma concentrations of OPN and sCD44 were measured by capture enzyme-linked immunosorbent assays (ELISA) according to protocols provided by the manufacturer (IBL, Hamburg, Germany). The sCD44 Elisa kit (sCD44std) measured all circulating (soluble) CD44 isoforms comprising the standard protein sequences but not the rare isoforms associated with alternative splicing between exons V2 and V10 (50) (see also FIG. 22). The OPN IBL ELISA kit (code No. 27158) measures total concentration of both phosphorylated and non-phosphorylated of all isoforms of OPN in plasma. Circulating levels of HA were measured in all plasma samples using an ELISA kit (HA-Elisa (K-1200), Echelon Biosciences, Salt Lake City, Utah). All ELISA tests were performed in duplicate and the optical density was measured at 450 nm (for OPN and sCD44) and 405 nm (for HA) using an AsysHiTech Expert-96™ microplate reader (Biochrom, Cambridge, UK). Other Elisa kits available commercially or house made can be used in methods of the present invention. The cut-off value that statistically distinguishes non scoliotic subjects from scoliotic subjects that will help predict the risk of scoliosis progression as determined with these other kits will likely differ from that calculated with the kit used herein. It may however be calculated for each new antibody used as described herein.

STATISTICAL ANALYSIS Age and gender differences among the different AIS and control groups were assessed using Pearson's Chi-square and Student's t tests, respectively. Multiple linear regression models were used to test for association between groups and levels of OPN, sCD44, and HA. Values were adjusted for age, gender, and age-gender interaction when these potential confounders were associated with the biomarker levels at p<0.1. Interactions between group and gender were also investigated. It was first tested for an overall group effect using a global F test comparing models with and without group effects. Were then tested specific differences between groups, applying a Bonferroni correction for multiple testing. Receiver-operating characteristics (ROC) curves were used to evaluate the diagnostic value of OPN, and to identify the optimal threshold values. The sensitivity (proportion of true-positive results when the assay was applied to patients known to have AIS) and specificity (proportion of true-negative results when the assay was applied to healthy controls) of OPN were profiled by curves. The area under ROC curve (AUC) and associated 95% confidence interval were calculated. The test of the hypothesis that the theoretical AUC is 0.5 was based on the confidence interval. Statistical analysis was performed with the SAS software, version 9.1, with the exception of the ROC curve analysis, which was performed with the ROCR package for R (www.r-project.org)^(51,52). In all analyses except when otherwise mentioned a p-value<0.05 was considered statistically significant.

EXAMPLE 2

mRNA and Protein OPN Levels Pinealectomized Chicken

Expression analysis and immunodetection analysis of OPN in pinealectomized chicken were performed as described in Example 1 above. OPN at the mRNA and protein levels occurring in pinealectomized chicken were measured. FIG. 1 shows a strong increase of OPN at the mRNA and protein levels only in pinealectomized chicken that developed a scoliosis.

EXAMPLE 3

OPN Protein Levels in C57BI/6j Mice

Bipedal C57BI/6j mice were generated and their OPN level was determined as described in Example 1 above. Bipedal ambulation for 8 weeks in C57BI/6j mice induced scoliosis at a rate of 46 percent in females and 24 percent in males which correlated well with higher plasma OPN levels found in females (Table 3 below). The relevance of this animal model is strengthened by the fact that scoliosis are more frequently seen in number and severity in bipedal C57BI/6j females (46%) when compared to bipedal males (24%) as is also observed in humans.

TABLE 3
Scoliosis frequency in naturally melatonin deficient mouse
strain C57BI/6j mice and genetically modified C57BI mice
devoid of OPN or CD44.

			Mean period of
	n	% of scoliosis	follow-up

	C57BI/6j	♂	21	24%	57 weeks +/− 3
		♀	28	46%	57 weeks +/− 3
	C57BI/6j	♂	30	0%	54 weeks +/− 2
	OPN-null	♀	24	0%	54 weeks +/− 2
	C57BI/6j	♂	29	0%	52 weeks +/− 2
	CD44-null	♀	31	0%	52 weeks +/− 2

FIG. 2 shows that the OPN protein level strongly increases after bipedal surgery (i.e. during scoliosis development) in scoliotic C57BI/6j mice.

EXAMPLE 4

Observation of Effect of Absence of OPN or CD44 Bipedal C57BI/6j Mice on Scoliosis

The contribution of OPN and CD44 receptor as an integral part of the pathophysiology cascade in scoliosis formation and curve progression was also examined by studying genetically modified bipedal C57BI/6j mice by conducting experiments as described in Example 1 above. As shown in Table 3 above, it was found that none of the bipedal C57BI/6j OPN-null (n=54) and C57BI/6j CD44-null mice (n=60) respectively, developed a scoliosis even if their analysis was extended over 52 weeks. Scoliosis development is detected 8 weeks after the surgery. A longer follow-up was performed to demonstrate that scoliosis development was not simply delayed in OPN-null and CD44-null mice.

In parallel, melatonin circulating levels were measured in wild-type and OPN-KO mice to exclude the possibility that absence of scoliosis in bipedal C57BI/6 OPN-KO mice was due to an increased production of melatonin.

FIG. 3 shows a two-fold decrease in circulating melatonin level of bipedal C57BI/6j OPN KO mice when compared to wild-type ones (C57BI/6j, C57BI/6j and FVB).

As indicated above, C57BI/6j mice are melatonin deficient and may develop a scoliosis (S) in contrast to the FVB strain, which produces high melatonin levels. OPN-knockout mice do not develop a scoliosis (NS) even if they are in the same genomic background (C57BI6/j), although melatonin is markedly decreased, suggesting that melatonin negatively regulates OPN expression and synthesis in vivo. Without being bound by this hypothesis, it is also suggested that in absence of OPN in genetically modified mice, the melatonin level will be further decreased accordingly as an adaptive physiological response to enhance OPN expression and synthesis.

EXAMPLE 5

Effect of OPN Inhibitors on Scoliosis Prevention

Two compounds suspected of having an effect on OPN transcription or synthesis were injected intraperitonealy at a dosage of 500 μg/kg of body weight/day to chicken 24-48 h prior pinealectomy.

As is apparent in FIG. 4, fewer pinealectomized chicken pre-treated with the drugs developed scoliosis (a reduction of 50%) than untreated pinealectomized chickens.

EXAMPLE 6

Comparing the Level of Circulating OPN in AIS Patients Classified in Two Groups and Healthy Controls

A group of 252 patients with AIS and 35 healthy control subjects were tested as described in Example 1 above. Patients with AIS were divided into two subgroups according to their spinal curve severity (10°-44° vs. ≧15°) In the most severely affected AIS subgroup, none of the patients had corrective surgery at the time of the tests. Consistent with literature reporting increased AIS prevalence in teenage girls when compared to boys for moderate curves (ratio 10:1 for curve with a Cobb's angle 30°), a greater proportion of girls in the AIS groups (86% and 84% in the 10°-44° and ≧15° subgroups, respectively were observed compared to the control groups (54% and 64% in healthy and at risk control groups, respectively, p≦0.0001 when comparing the control groups). There was no significant gender difference between the two AIS subgroups (p=0.76) or between the two control groups (p=0.32). Mean age was significantly higher in AIS patients with Cobb's angle≧45° compared to those with 10-44° angle (15.2±1.8 vs. 13.8±2.1, p<0.0001). Both AIS groups had higher mean age compared to control groups (10.7±0.6 for the healthy and 9.9±3.4 for the at risk group, p<0.0001 when comparing to either AIS group).

The plasma OPN levels in patients with AIS exhibiting a severe deformity (Cobb's angle≧45°), low to moderate curve (Cobb's angle between 10° and 44°) and healthy controls are summarized in Table 4 below according to various clinical parameters. The mean plasma OPN levels were significantly higher in both AIS groups when compared to healthy control group although plasma OPN levels were more elevated in patients with the most severe deformities (Cobb's angle≧45°) (Bonferroni-corrected p<0.001 after adjustment for age, gender, and age-gender interaction). Plasma OPN levels in AIS patients were correlated with the severity of curve deformity (FIG. 5D) in girls and boys (Partial Pearson correlation coefficient adjusted for age=0.29, p<0.001, and 0.33, p=0.04, respectively). Mean plasma OPN levels in the group at risk of developing scoliosis (846±402 ng/ml) differed significantly (Bonferroni-corrected p<0.001) from the healthy controls (570±156 ng/ml).

TABLE 4
Mean biochemical values of patients with AIS, healthy control subjects and asymptomatic at risk control subjects*.

Female

Mean

Male

Female + Male

			biomarker			Mean			Mean
			level			biomarker			biomarker
	Subject Type	N	(ng/ml)	Range	N	level (ng/ml)	Range	N	level (ng/ml)	Range	P-value†

OPN	Healthy controls	19	580 ± 150	318-882	16	558 ± 168	308-856	35	570 ± 156	308-882	—
	At risk controls	45	829 ± 419	208-1834	25	877 ± 378	391-1629	70	846 ± 402	208-1834	<0.001
	AIS < 45°	162	774 ± 268	373-1585	27	948 ± 335	445-1668	189	799 ± 284	373-1668	<0.001
	AIS ≧ 45°	53	913 ± 398	201-1821	10	1238 ± 409	575-1872	63	965 ± 414	201-1872	<0.001
sCD44	Healthy controls	19	522 ± 99	373-829	16	575 ± 92	404-800	35	546 ± 98	373-829	—
	At risk controls	45	508 ± 96	316-760	25	533 ± 98	304-510	70	517 ± 97	304-760	>0.5
	AIS < 45°	162	503 ± 161	194-1253	27	527 ± 110	364-793	189	506 ± 155	194-1253	>0.5
	AIS ≧ 45°	53	436 ± 251	87-882	10	402 ± 216	147-962	63	431 ± 245	87-962	0.066
HA	Healthy controls	19	128 ± 38	72-236	16	132 ± 49	80-255	35	130 ± 43	72-255	—
	At risk controls	45	119 ± 51	36-257	25	117 ± 52	33-226	70	118 ± 51	33-257	>0.5
	AIS < 45°	162	112 ± 60	18-356	27	124 ± 60	27-283	189	114 ± 60	18-356	>0.5
	AIS ≧ 45°	53	93 ± 40	32-222	10	128 ± 71	41-25435	63	98 ± 48	32-254	0.140
*SD is standard deviation
†P-value is from the comparison with healthy control group in all subjects after Bonferroni correction and adjustment for age, gender, and age-gender interaction (OPN and HA) or age (sCD44). After the same adjustments, overall F test p-values for association between group and biomarker levels were <0.001 (OPN), 0.035 (sCD44), and 0.163 (HA).

Receiver-operating characteristics (ROC) curves analyzes of plasma OPN comparing the patients with AIS more severely affected (Cobb's angle≧45°) with healthy controls showed an AUC of 0.94 with a standard error of 0.03 (95 percent confidence interval 0.88 to 0.99) (see FIG. 5A). A cut-off value>700 nanograms per milliliter gave a sensitivity of 90.6 percent and a specificity of 81.8 percent with (see FIG. 5B). A cut-off value>800 nanograms per milliliter had the highest accuracy with a sensitivity of 84.4 percent and specificity of 90.6 percent for confirming scoliosis (minimal false negative and false positive results) (see FIG. 5C).

Although as indicated above, high levels of OPN are found in other adult diseases, high plasma OPN levels found in patients with scoliosis are unique in the pediatric population. The detection of OPN level can thus be used to identify within asymptomatic children those who are at risk of developing a scoliosis (AIS or other spinal disorders and disorders causing scoliosis) and identify among scoliotic subjects, those or are at risk of experiencing a progression of scoliosis. Moreover, plasma OPN levels found in AIS patients were often higher than those measured in adult diseases. OPN levels can also be used to predict the risk in adults (e.g. degenerative scoliosis and idiopathic scoliosis that progress through adulthood). Certain mutations have already been associated with other disorders that may lead to scoliosis. In a particular embodiment, the OPN levels could be used in combination with the detection of these mutations.

EXAMPLE 7

Comparing the Level of Circulating OPN in Asymptomatic Children at Risk and Healthy Controls

A group of 70 asymptomatic children at risk of developing a scoliosis and 35 healthy control subjects were tested as described in Example 1 above. The mean plasma OPN levels in the group at risk of developing a scoliosis (846.30±402 nanograms per milliliter) differed significantly (p=0.001) from the healthy controls (570±156 nanograms per milliliter) and both groups were age- and gender-matched. No significant gender difference was observed (see Table 4 above).

Using a cut-off value of 800 nanograms per milliliter, it was observed that 47.9 percent of asymptomatic children in that group were above this plasma OPN value while only 8.6 percent of healthy controls were above this value. These results are in agreement with previous reports showing that the offspring of at least one affected parent develops more often a scoliosis than ones born from unaffected parents (34, 35).

An enzyme-linked immunosorbent assay (ELISA) or RIA for OPN for instance can thus be used for early identification of subjects at risk of developing a scoliosis for purposes of prognosis and/or scoliotic patients stratification for early bracing and less-invasive surgeries with novel fusionless devices, for pharmacological treatments and to monitor responses to treatment in patients with AIS.

EXAMPLE 8

Comparing the Level of Circulating sCD44 in AIS Patients Classified Two Groups and Healthy Controls

Experiments were conducted as described in Example 1 above. The plasma sCD44 and HA levels in healthy controls, both AIS groups and asymptomatic at risk children are displayed in Table 4 above. Comparison among all groups showed no significant change in mean plasma sCD44 and HA values. However, AIS patients exhibiting the most severe spinal deformities 45°) had also the lowest mean plasma sCD44 level when compared to the other three groups (p=0.066).

CD44 and sCD44 can act as a receptor and decoy receptor for OPN respectively. In spite that no significant changes were measured among all groups tested, the most severely affected AIS patients (≧45°) showed the lowest mean sCD44 value among all groups tested, Interestingly, decreased plasma sCD44 levels were found in immunodeficiency and autoimmune diseases^(35-37), but none of these conditions normally lead to scoliosis in absence of high plasma OPN levels, suggesting that sCD44 could play a role in AIS as disease-modifying factor by interfering with the action of OPN (see FIG. 17).

EXAMPLE 9

Profiles of Change in OPN Levels, sCD44 Levels, and Cobb's Angle of AIS Patients Over Time

The progression of biomarkers (OPN and sCD44 levels) and Cobb's angle was measured over follow up time in AIS patients. FIG. 7 presents these progression in 4 selected AIS female patients (not under brace treatment) aged 12 (red), 14 (green and blue), and 17 (yellow) at baseline visit.

FIG. 8 presents the distribution of total change in OPN (left panel) and sCD44 (right panel) levels over follow-up time in AIS patients with worsened curve deformity (total increase in Cobb's angle greater than)₃° and in those without significant change in curve (no change in Cobb's angle, decrease, or increase smaller than 3°; also presents for all Average change in OPN levels was significantly higher in the group with worsened curve deformity (Wilcoxon rank sum test p<0.01). No significant difference was detected for sCD44 (p>0.5). Length of follow-up time was similar between the 2 groups (p>0.5).

FIG. 9 shows OPN progression correlated with Cobb's angle progression in a group of AIS patients while FIG. 10 shows OPN regression or stabilization correlated with Cobb's angle regression or stabilization in other AIS patients;

OPN level can be used to identify among pre-diagnosed patients those in which scoliosis will progress.

EXAMPLE 10

Profiles of Change in OPN Levels, sCD44 Levels, and Cobb's Angle of Asymptomatic at Risk Patients Over Time

FIG. 11 shows profiles of change in OPN and sCD44 levels angle in 4 selected at risk subjects without scoliosis: one male aged 13 (green), and 3 female aged 5 (gold), 11 (blue), and 9 (red) at baseline visit. Significant inter-subject variability was observed in the baseline levels of biomarkers and change over time among at risk subjects (especially for OPN), indicating the potential of using this biomarker as a tool to monitor onset of scoliosis in at risk subjects.

Tables 5 to 8 below present the clinical and biochemical profiles in detail for each of the healthy control subjects (Table 5), of the AIS patients with Cobb's angles of less than 45 degrees (Table 6), of the AIS patients with Cobb's angles 45° or more (Table 7), and of the asymptomatic at risk children (Table 8).

TABLE 5
Clinical and biochemical profile of healthy control subjects.

	Date of			Collection	Timepoint		[sCD44]
Random	Birth	Gender	Age	Date	(months)	[OPN] (ng/ml)	(ng/ml)	[HA] (ng/ml)

1	1996-03-21	M	11.2	2007-05-22	T0	663.92 ± 26.03	533.4	164.87 ± 6.05
2	1996-06-26	M	10.9	2007-05-22	T0	418.23 ± 12.49	504.38	120.49 ± 2.06
			11.6	2008-01-16	T8	593.64 ± 28.77	555.88	150.02 ± 15.74
3	1996-05-28	F	11.0	2007-05-22	T0	629.52 ± 0.64	829.35	140.89 ± 3.90
			11.7	2008-01-16	T8	892.76 ± 1.54	507.54	146.71 ± 24.69
4	1996-06-22	M	10.9	2007-05-22	T0	458.68 ± 11.40	799.57	100.98 ± 6.89
5	1996-10-13	F	10.6	2007-05-22	T0	459.33 ± 2.90	525.76	139.84 ± 2.89
			11.3	2008-01-16	T8	464.46 ± 2.29	476.43	157.36 ± 20.10
7	1996-08-08	F	10.8	2007-05-22	T0	691.18 ± 2.50	664.38	120.69 ± 2.79
			11.5	2008-01-16	T8	825.38 ± 1.16	545.85	180.39 ± 42.55
8	1996-02-01	M	11.3	2007-05-22	T0	498.86 ± 0.66	643.38	99.24 ± 2.35
			12.0	2008-01-16	T8	469.87 ± 11.47	440.44	154.20 ± 2.53
9	1997-06-28	M	9.9	2007-05-22	T0	517.11 ± 53.44	582.66	134.43 ± 6.42
10	1997-07-23	F	9.8	2007-05-22	T0	756.24 ± 23.61	499.03	131.04 ± 1.98
			10.5	2008-01-16	T8	1039.80 ± 3.10	337.33	167.84 ± 2.48
11	1996-02-22	M	11.3	2007-06-06	T0	653.09 ± 15.14	581.14	191.13 ± 17.98
			11.8	2007-12-04	T6	521.00 ± 5.82	861.46	265.54 ± 6.97
12	1996-02-09	F	11.3	2007-06-06	T0	449.97 ± 11.21	490.25	112.71 ± 17.95
			11.8	2007-12-04	T6	923.12 ± 1.03	476.09	188.80 ± 15.17
13	1996-05-17	F	11.1	2007-06-06	T0	488.30 ± 0.80	428.77	168.61 ± 9.49
			11.6	2007-12-04	T6	659.35 ± 1.68	584.96	182.09 ± 13.74
14	1995-10-20	M	11.6	2007-06-06	T0	610.77 ± 8.93	573.88	128.40 ± 6.58
			12.1	2007-12-04	T6	469.87 ± 19.12	527.07	167.16 ± 44.48
16	1997-03-07	F	10.2	2007-06-06	T0	544.82 ± 7.91	516.6	132.83 ± 2.07
			10.7	2007-12-04	T6	723.88 ± 8.56	503.74	65.43 ± 9.60
17	1996-05-09	M	11.1	2007-06-06	T0	450.87 ± 6.41	553.26	255.19 ± 14.61
			11.6	2007-12-04	T6	530.37 ± 16.78	267.86	42.33 ± 7.47
18	1997-09-02	F	9.8	2007-06-06	T0	555.41 ± 32.17	498.65	127.24 ± 10.65
19	1996-11-04	M	10.6	2007-06-06	T0	314.85 ± 9.93	682.71	175.92 ± 16.20
20	1997-05-30	F	10.0	2007-06-06	T0	381.57 ± 4.61	373.01	87.65 ± 3.71
			10.5	2007-12-04	T6	434.48 ± 5.73	497.7	142.61 ± 8.42
21	1997-01-07	F	10.4	2007-06-06	T0	318.19 ± 6.62	474.59	235.76 ± 3.68
			10.9	2007-12-04	T6	393.98 ± 3.87	571.14	209.26 ± 2.40
22	1997-02-09	F	10.3	2007-06-06	T0	882.15 ± 18.31	542.95	131.86 ± 1.13
			10.8	2007-12-04	T6	804.46	593.61	120.43 ± 14.60
23	1997-03-02	M	10.3	2007-06-06	T0	307.71 ± 4.88	621.23	157.12 ± 2.29
24	1997-06-19	F	10.0	2007-06-06	T0	423.06 ± 13.90	561.28	149.88 ± 5.65
25	1997-04-12	F	10.1	2007-06-06	T0	758.88 ± 5.74	478.79	169.32 ± 8.25
26	1997-12-02	M	9.5	2007-06-06	T0	441.36 ± 8.32	645.84	148.32 ± 16.36
27	1996-04-03	F	11.2	2007-06-06	T0	794.21 ± 5.50	545.62	77.58 ± 8.87
			11.7	2007-12-04	T6	748.79 ± 7.61	575.46	228.08 ± 27.64
28	1995-09-30	F	11.7	2007-06-12	T0	503.25 ± 8.16	451.68	71.91 ± 4.23
29	1996-09-15	M	10.7	2007-06-12	T0	576.62 ± 5.29	554.79	80.24 ± 3.69
			11.2	2007-12-04	T6	552.15	598.79	108.09 ± 16.44
30	1996-01-18	F	11.4	2007-06-12	T0	578.62 ± 0.24	634.22	126.21 ± 4.18
			11.9	2007-12-04	T6	498.67 ± 8.60	606.57	192.18 ± 31.90
31	1996-08-24	F	10.8	2007-06-12	T0	531.91 ± 4.36	432.2	132.19 ± 5.06
			11.3	2007-12-04	T6	455.46 ± 4.85	660.14	244.46 ± 3.49
32	1997-04-19	F	10.1	2007-06-12	T0	611.32 ± 6.46	481.47	92.69 ± 2.87
			10.6	2007-12-04	T6	406.38 ± 19.28	415.61	142.80 ± 25.25
33	1997-04-21	M	10.1	2007-06-12	T0	543.15 ± 7.32	403.56	91.82 ± 4.49
			10.6	2007-12-04	T6	360.77 ± 9.93	544.36	81.68 ± 23.85
34	1995-11-15	M	11.6	2007-06-12	T0	856.07 ± 3.82	501.71	96.37 ± 4.15
			12.1	2007-12-04	T6	922.12 ± 20.68	535.71	56.34 ± 1.86
35	1996-04-22	F	11.1	2007-06-12	T0	659.81 ± 5.54	502.09	87.90 ± 4.85
			11.6	2007-12-04	T6	596.77 ± 10.14	378.46	242.42 ± 36.30
36	1995-12-09	M	11.5	2007-06-12	T0	816.64 ± 14.56	502.85	83.26 ± 0.12
37	1995-10-07	M	11.7	2007-06-12	T0	805.92 ± 14.01	511.63	80.24 ± 3.69
			12.2	2007-12-04	T6	304.61 ± 14.94	489.06	141.51 ± 21.50
*Plus-minus values are means ± standard deviations.
†Healthy control subjects have no family history of scoliosis and are examined before sample collection by an orthopaedic surgeon.

TABLE 6
Clinical and biochemical profiles of AIS patients with Cobb's angles less than 45°.

Patient					Timepoint	Cobb's Angle	Curve
ID	Date of Birth	Gender	Age	Collection Date	(mths)	Pre-op	Type
102	1991-09-12	F	13.8	2005-06-10	T0	18	rT
			14.3	2006-01-13	T7	16	rT
			15.8	2007-06-01	T12	16	rT
			16.2	2007-11-30	T29	17	rT
103	1991-09-04	M	13.8	2005-06-10	T0	13	lT
104	1992-01-29	F	13.4	2005-06-10	T0	21-22	rTlL
106	1992-08-10	F	14.8	2007-06-05	T0	25-24	rTlL
			15.2	2007-10-05	T4	22-18	rTlL
107	1991-09-09	F	13.8	2005-06-20	T0	31-32	rTlL
113	1995-11-21	F	9.7	2005-07-22	T0	10	rT
			11.5	2007-05-18	T22	15	rT
118	1991-06-04	F	16.6	2008-01-18	T0	22-22	rTlTL
123	1993-09-23	F	12.1	2005-11-04	T0	28	rTL
			14.3	2008-01-18	T26	19-31	lTrTL
124	1990-12-09	F	14.9	2005-11-04	T0	33-32	rTlTL
127	1992-01-18	F	13.9	2005-12-02	T0	33-19	rTrT
128	1997-03-18	F	8.8	2005-12-02	T0	10	lTL
130	1991-06-05	F	14.5	2005-12-09	T0	19	rTL
131	1992-11-09	F	13.1	2005-12-09	T0	32-24	rTlL
			15.0	2007-11-12	T23	32-24	rTlL
136	1989-10-10	F	16.3	2006-01-13	T0	14	lTL
138	1993-06-04	F	12.7	2006-02-17	T0	24-26	rTlL
			14.3	2007-10-24	T20	22-25	rTlL
			14.7	2008-02-04	T24	23-26	rTlTL
139	1993-12-06	F	12.2	2006-02-24	T0	12.-14	rTlL
			14.2	2008-02-08	T24	12.-6	rTlL
141	1992-07-20	F	13.7	2006-03-10	T0	20-18	rTlL
			15.5	2008-01-22	T22	9.-13	rTlTL
142	1992-12-19	F	13.2	2006-03-10	T0	31	lTL
			15.1	2008-01-23	T22	25	lTL
146	1990-05-13	F	16.0	2006-05-26	T0	32-22	rTlL
148	1993-08-12	F	14.3	2007-12-07	T0	11	lTL
149	1988-09-28	M	17.7	2006-06-02	T0	31-26	rTlL
150	1992-10-16	F	13.6	2006-06-02	T0	25	rT
151	1993-04-11	F	14.7	2007-12-03	T0	28-20	rTlL
152	1990-10-04	F	15.7	2006-06-02	T0	34	lL
154	1989-11-24	F	16.6	2006-06-08	T0	40	lTL
			18.1	2007-12-07	T18	38	lTL
155	1991-01-01	F	15.4	2006-06-08	T0	26	lTL
159	1998-03-04	F	9.7	2007-11-06	T0	3	lTL
161	1994-04-27	F	13.6	2007-11-30	T0	15	lTL
165	1995-08-30	F	12.3	2007-12-03	T0	34-20	rTlL
168	1992-04-24	F	14.2	2006-06-26	T0	16-18	rTlL
			14.6	2006-11-21	T5	17-16	rTlL
			15.5	2007-10-01	T16	14-16	rTlTL
176	1992-10-24	F	13.8	2006-07-03	T0	29	rT
			14.2	2007-01-15	T6	27	rT
183	1991-09-13	M	14.8	2006-05-07	T0	17	rL
			15.4	2007-06-02	T13	7.-19	rTlL
200	1992-07-29	M	15.2	2007-10-30	T0	23-24	rTlL
201	1992-11-27	F	13.7	2006-07-12	T0	10-17.	rTlL
225	1994-05-09	F	12.2	2006-07-24	T0	15-19	lTrTL
			12.8	2007-02-27	T7	13-18	lTrL
234	1990-07-16	M	16.2	2006-10-13	T0	26	rT
235	1991-10-29	M	15	2006-10-13	T0	20	lTL
			16	2007-10-11	T12	18	lTL
240	1993-10-04	F	13.2	2006-12-11	T0	17-23	rTlL
242	1989-09-12	F	17.3	2007-01-12	T0	6	lTL
244	1990-10-20	F	16.2	2007-01-19	T0	27-29	rTlL
			17.3	2008-02-13	T13	NA	NA
245	1992-01-27	F	15.0	2007-01-22	T0	31-35	rTlL
			15.8	2007-11-14	T10	28-35	rTlL
247	1994-12-18	F	12.1	2007-01-26	T0	9	rTL
			12.8	2007-10-09	T9	6	rTL
248	1997-06-16	F	9.6	2007-01-26	T0	9	rL
			10.3	2007-10-09	T9	3	lTL
249	1991-03-25	F	15.9	2007-02-02	T0	31	lTL
			16.4	2007-08-03	T6	NA	lTL
			16.9	2008-02-01	T12	36	lTL
250	1992-05-08	F	14.7	2007-02-02	T0	32	lTL
			15.4	2007-10-15	T8	21	lTL
251	1991-09-05	F	15.4	2007-02-02	T0	40-30	rTlL
253	1992-10-18	M	14.3	2007-02-27	T0	31	rT
254	1991-12-11	F	15.2	2007-03-09	T0	28	lTL
			15.9	2007-11-12	T8	15	lTL
256	1996-03-19	F	11.0	2007-03-09	T0	11	lTL
257	1995-04-15	F	11.9	2007-03-09	T0	6	rTL
			12.5	2007-10-16	T7	NA	NA
258	1990-06-24	M	16.8	2007-03-09	T0	14	rT
			17.3	2007-10-02	T8	NA	NA
259	1994-07-07	F	12.7	2007-03-16	T0	8	lTL
			13.5	2007-10-15	T7	11	lTL
260	1994-07-07	M	12.7	2007-03-16	T0	6	rTL
			13.5	2007-10-05	T7	4	lTL
261	1997-06-19	F	9.7	2007-03-16	T0	21	lL
			10.3	2007-10-17	T7	10	lTL
			10.4	2008-02-06	T11	5	lTL
263	1994-10-13	F	12.4	2007-03-20	T0	7.-12	rTlL
264	1992-05-24	F	14.8	2007-03-20	T0	23-30	rTlL
265	1993-05-04	F	13.9	2007-03-20	T0	23	lL
			14.5	2007-11-13	T8	11-14.	rTlL
266	1991-01-25	F	16.2	2007-04-02	T0	34	rTL
			16.8	2007-11-15	T7	34	rTL
267	1994-05-14	F	12.9	2007-04-02	T0	5	rTL
			13.5	2007-11-15	T7	5	rTL
268	1994-08-17	F	12.6	2007-04-04	T0	12.-4	rTlL
271	1994-11-17	F	12.4	2007-04-13	T0	23	rTL
			12.9	2007-10-15	T6	24	rTL
272	1994-04-14	F	13.0	2007-04-13	T0	22-24	rTlL
			13.6	2007-12-05	T8	14-15	rTlL
273	1991-06-30	F	15.8	2007-04-13	T0	25	rTL
274	1990-02-28	F	17.1	2007-04-17	T0	11.-22	rTlL
275	1996-04-08	F	11.0	2007-04-19	T0	27-1.	rTlL
			11.5	2007-10-15	T6	26-19	rTlTL
276	1994-09-26	F	13.1	2007-10-15	T0	19-19	rTlL
277	1994-11-02	F	12.4	2007-04-19	T0	12	lL
			13.0	2007-11-14	T7	15-13	rTlL
278	1992-06-08	M	14.9	2007-05-04	T0	22-14	rTlL
			15.3	2007-10-23	T5	26-28	rTlL
279	1998-09-22	F	8.7	2007-05-30	T0	19	rT
			9.2	2007-10-05	T5	8	rT
280	1992-12-18	F	14.4	2007-05-30	T0	19	rT
			14.9	2007-11-02	T6	24	rTL
281	1994-10-17	F	12.6	2007-06-01	T0	11	rT
			13.1	2007-11-09	T5	9	lTL
282	1997-09-30	F	9.7	2007-06-13	T0	20	rT
			10.3	2008-01-30	T7	NA	NA
286	1994-06-01	F	13.3	2007-09-17	T0	28	lTL
287	1991-11-15	F	15.8	2007-09-18	T0	11	rTL
288	1996-05-13	M	11.3	2007-09-18	T0	20	lL
289	1992-10-23	F	14.9	2007-09-18	T0	18	rT
290	1993-10-02	F	14.0	2007-09-18	T0	22	rTL
291	1992-07-10	F	20.9	2007-09-18	T0	25-31	rTlL
292	1994-01-23	F	13.7	2007-09-21	T0	20	lTL
293	1993-04-03	F	14.5	2007-09-21	T0	16	rT
295	1991-08-09	M	16.1	2007-09-26	T0	11.-8	rTlL
296	1992-04-04	F	15.5	2007-09-28	T0	15-18	lTrL
297	1997-07-13	M	10.2	2007-09-28	T0	20	lT
298	1994-11-09	F	12.9	2007-09-28	T0	18-21	rTlL
299	1990-03-21	F	17.5	2007-10-03	T0	33-43	rTlL
301	1995-02-06	F	12.7	2007-10-09	T0	13	lT
302	1993-05-07	F	14.4	2007-10-09	T0	14.-12	rTlL
303	1991-03-29	F	16.5	2007-10-15	T0	14	lTL
304	1991-10-25	F	16.0	2007-10-16	T0	25	lT
305	1992-02-24	F	15.7	2007-10-19	T0	23	lTL
306	1994-09-22	F	13.1	2007-10-19	T0	13-18	rTlL
307	1994-01-25	M	13.7	2007-10-24	T0	8-11-11.	lTrTlL
308	1997-05-22	F	10.4	2007-10-26	T0	8	rTL
309	1996-04-10	F	11.5	2007-10-26	T0	10	lTL
311	1993-05-07	F	14.5	2007-10-26	T0	17	lTL
313	1993-06-04	F	14.4	2007-10-26	T0	20-18	rTlL
314	1993-03-11	F	14.6	2007-10-29	T0	24	rL
315	1993-12-16	F	13.9	2007-10-31	T0	14	lTL
316	1992-10-07	M	15.1	2007-10-31	T0	28	rT
318	1997-05-25	F	10.4	2007-10-15	T0	11	rTL
319	1993-06-28	F	14.4	2007-11-06	T0	22	lTL
320	1993-09-24	F	14.1	2007-11-09	T0	15	rT
321	1992-07-04	F	15.3	2007-11-09	T0	16	rTL
322	1996-06-01	F	11.4	2007-11-09	T0	4	lTL
324	1991-04-20	F	16.6	2007-11-09	T0	19-19	rTlL
325	1994-03-26	F	13.6	2007-11-09	T0	21	rTL
326	1994-02-02	M	13.8	2007-11-13	T0	13	lTL
328	1994-09-24	F	12.8	2007-11-14	T0	11	lTL
329	1996-05-29	F	11.5	2007-11-14	T0	6	rTL
330	1994-02-05	F	13.8	2007-11-16	T0	12	lTL
332	1992-01-26	M	15.8	2007-11-23	T0	24	lTL
333	1993-10-21	F	14.1	2007-11-23	T0	30	lTL
334	1993-08-07	F	14.3	2007-11-23	T0	29-27	rTlL
335	1996-01-16	F	11.9	2007-11-23	T0	28-27	rTlL
337	1991-09-04	M	16.2	2007-11-28	T0	24	lL
338	1994-12-31	F	12.9	2007-11-30	T0	10	lTL
339	1992-03-17	F	15.7	2007-11-30	T0	25	lTL
340	1995-05-21	F	12.5	2007-11-30	T0	30	lTL
341	1996-02-11	F	11.8	2007-11-30	T0	15-14	rTlL
342	1993-12-01	F	14.0	2007-12-07	T0	16	rTL
343	1993-06-29	M	14.4	2007-12-07	T0	15	rTL
344	1996-03-26	F	11.7	2007-12-07	T0	10	rTL
345	1993-04-12	F	14.6	2007-12-07	T0	30	lTL
346	1996-10-11	F	11.2	2007-12-07	T0	18-17	rTlTL
347	1997-04-07	F	10.7	2007-12-11	T0	5-6.	rTlL
348	1995-06-10	M	12.5	2007-12-11	T0	10	rTL
350	1995-02-22	F	12.8	2007-12-13	T0	25	rTL
351	1992-05-19	F	15.6	2007-12-13	T0	14	rTL
352	1996-04-13	M	11.7	2007-12-13	T0	14	rTL
353	1993-08-12	M	14.3	2007-12-13	T0	24	rT
354	1994-06-07	F	13.5	2007-12-13	T0	8	lT
355	1993-08-08	F	14.3	2007-12-13	T0	27	lTL
356	1995-05-17	F	12.6	2007-12-13	T0	19	lTL
358	1997-02-27	F	10.9	2008-01-11	T0	18	rTL
359	1995-11-08	F	13.0	2008-01-15	T0	14	rTL
360	1992-05-24	F	15.6	2008-01-15	T0	14	lTL
361	1996-06-29	F	11.5	2008-01-15	T0	23	rTL
362	1997-08-21	F	10.4	2008-01-16	T0	11	lTL
363	1993-05-24	F	14.6	2008-01-16	T0	20-24-19	lTrTlTL
364	1995-03-24	F	12.8	2008-01-16	T0	10	lTL
365	1999-07-26	F	9.3	2008-01-16	T0	5	rTL
368	1996-07-12	F	11.5	2008-01-18	T0	14	rTL
369	1992-05-21	F	15.7	2008-01-18	T0	25	rTL
370	1994-12-01	F	13.1	2008-01-18	T0	18-15	rTlL
371	1992-02-04	F	16.0	2008-01-18	T0	26-20	rTlTL
372	1991-06-21	F	16.6	2008-01-21	T0	23-21	rTlL
374	1992-05-26	F	15.7	2008-01-21	T0	25	lL
375	1992-10-21	F	15.3	2008-01-22	T0	31-55	rTlTL
376	1993-05-18	F	14.7	2008-01-22	T0	16	rTL
377	1995-01-31	F	13.0	2008-01-22	T0	27	lTL
379	1996-09-14	F	11.4	2008-01-25	T0	5.-5	lTrTL
381	1992-01-11	M	16.0	2008-01-25	T0	24	rT
382	1993-10-21	F	14.2	2008-01-25	T0	28-25	rTlTL
383	1994-11-20	F	13.2	2008-01-25	T0	30-27	rTlTL
384	1992-02-09	M	16.0	2008-01-29	T0	25-19	rTlL
386	1994-09-02	F	13.4	2008-02-01	T0	25-14	lTrT
387	1994-04-11	F	13.8	2008-02-01	T0	14-15	rTlTL
388	1995-11-24	F	12.2	2008-02-01	T0	34	rT
389	1997-04-13	F	10.8	2008-02-04	T0	14	lTL
390	1994-04-28	F	13.8	2008-02-04	T0	28-26	rTlL
391	1994-07-01	F	13.6	2008-02-05	T0	37	rTL
392	1998-11-25	F	9.2	2008-02-05	T0	16	rTL
393	1993-09-30	M	14.3	2008-02-05	T0	26	rTL
395	1995-05-24	F	12.7	2008-02-08	T0	11	rT
397	1999-02-20	F	9.0	2008-02-08	T0	10	rTL
398	1997-09-16	F	10.4	2008-02-08	T0	16	rTL
399	2000-09-28	M	7.4	2008-02-08	T0	22-20	rTlTL
400	1994-05-25	F	13.7	2008-02-08	T0	12	rTL
401	1994-02-17	F	14.0	2008-02-18	T0	28-21	rTlTL
402	1991-07-15	F	16.6	2008-02-14	T0	19-12	rTlL
403	1995-02-21	F	13.0	2008-02-14	T0	13-13	rTlTL
1264	1997-09-22	F	15.2	2005-04-18	T0	40	rTL
1276	1997-09-23	F	15.2	2005-05-16	T0	42	lT
1364	1997-09-24	M	14.9	2006-04-24	T0	44	lTL
1365	1990-05-11	F	15.9	2006-04-26	T0	23-53	lTrL
1366	1993-04-06	F	13.1	2006-05-01	T0	36	NA
1373	1991-10-07	F	14.6	2006-05-17	T0	41-48	rTlL
1380	1989-10-09	F	16.7	2006-06-26	T0	35	rL
1384	1991-01-17	F	15.5	2006-07-03	T0	41	lTL
			15.8	2006-11-15	T4	9-4
1385	1990-06-12	F	16.1	2006-07-04	T0	42-23	rTlL
1387	1991-07-15	F	15.0	2006-07-17	T0	29-37-35	rTlL
1388	1991-12-13	F	14.6	2006-07-19	T0	38	rTL
1409	1993-02-11	F	13.6	2006-09-26	T0	40	rT
1433	1992-07-03	F	14.5	2007-01-10	T0	44	rT
1451	1995-01-13	F	12.2	2007-03-14	T0	42	rT
1478	1990-08-06	F	16.8	2007-06-11	T0	41	rTL
1481	1990-08-15	F	16.8	2007-06-18	T0	40	rT
1483	1989-06-26	F	18.0	2007-06-19	T0	37-25	rTlL
1487	1990-05-30	F	17.1	2007-07-03	T0	35-58-35	lCrTlL

Patient	Date of			[sCD44]
ID	surgery	Family history	[OPN] (ng/ml)	(ng/ml)	[HA] (ng/ml)
102	—	Cousin	1265.10	375.56	132.06 ± 39.35
			766.80	408.06	388.93 ± 23.42
			933.77 ± 13.23	437.55	71.91 ± 4.23
			591.72 ± 66.49	311.40	27.92 ± 1.72
103	—	Father (cyphose)	1338.32	792.62	207.12
104	—	—	1221.83	742.48	132.24
106	—	—	972.87 ± 16.73	488.72	86.78 ± 6.34
			485.82 ± 34.70	475.13	293.05 ± 40.93
107	—	Mother	739.61	1253.3	109.39 ± 26.70
113	—	—	670.49 ± 5.45	695.21	41.10 ± 8.51
			688.49 ± 23.78	613.79	49.16 ± 9.14
118	—	Both parents	372.79 ± 10.86	273.31	70.42 ± 4.85
123	—	Both parents	1466.97	931.05	128.78 ± 4.22
			779.90 ± 16.68	410.10	179.52 ± 21.17
124	—	Cousins	625.97	816.60	96.08
127	—	—	786.71	755.60	131.36 ± 22.43
128	—	—	837.64	628.74	118.73 ± 10.43
130	—	—	559.85	552.78	75.09 ± 7.11
131	—	—	568.01	578.96	101.00 ± 11.04
			450.45 ± 9.36	505.94	100.03 ± 9.66
136	—	—	411.02	670.31	84.81 ± 2.56
138	—	Cousin	577.78	293.51	63.86 ± 4.11
			379.04 ± 18.07	388.16	86.23 ± 11.26
			529.70 ± 4.86	378.03	227.26 ± 0.94
139	—	—	847.98	868.95	136.19 ± 7.63
			1192.61 ± 10.71	444.33	73.88 ± 19.39
141	—	Grand-mother,	658.28	735.50	90.51
		cousins, uncle	172.67 ± 8.59	433.6	37.31 ± 7.61
142	—	Mother, cousin	776.43	907.96	122.73 ± 7.61
			542.85 ± 1.41	511.4	146.43 ± 63.23
146	—	—	1501.42	475.91	75.68 ± 10.22
148	—	Mother	1416.91 ± 41.50	550.4	37.79 ± 6.19
149	—	—	472.61	559.97	138.95 ± 7.42
150	—	Sister	805.88	543.22	71.24 ± 1.52
151	—	—	732.19 ± 2.30	403.51	20.80 ± 3.30
152	—	Father	655.10	551.24	122.69 ± 0.10
154	—	Cousin	541.07	639.52	104.09 ± 13.96
			1101.07 ± 38.84	342.17	35.08 ± 5.40
155	—	Aunt	738.59	796.06	121.33 ± 17.72
159	—	Mother	769.50 ± 21.57	831.18	107.5 ± 1.08
161	—	—	487.11 ± 29.43	355.79	23.63 ± 0.53
165	—	—	1148.04 ± 47.51	607.43	42.39 ± 7.68
168	—	—	810.21 ± 28.48	244.4	103.10 ± 10.39
			582.52 ± 23.29	338.03	99.20 ± 18.18
			441.81 ± 7.29	333.4	126.96 ± 1.45
176	—	—	503.88 ± 35.81	331.65	91.50 ± 21.99
			675.38 ± 44.20	305.92	193.26 ± 2.38
183	—	—	733.99 ± 17.33	550.24	72.91 ± 10.68
			781.03 ± 3.27	531.96	69.83 ± 7.07
200	—	—	972.10 ± 4.92	401.94	88.41 ± 10.08
201	—	Sister	782.77 ± 2.63	498.93	142.57 ± 44.69
225	—	—	406.67 ± 3.40	617.37	248.10 ± 24.21
			651.89 ± 21.69	524.9	47.95 ± 3.60
234	—	—	840.88 ± 1.98	491.26	89.04 ± 5.66
235	—	—	586.25 ± 0.32	403.8	181.655 ± 48.71
			523.39 ± 9.76	428.29	188.63 ± 6.83
240	—	Mother, brother,	525.88 ± 7.74	428.83	71.91 ± 4.23
		cousin
242	—	Sister	590.13 ± 6.00	435.59	80.24 ± 3.69
244	—	—	735.26 ± 4.42	510.44	73.81 ± 6.20
			1293.68 ± 36.92	449.1	44.51 ± 4.81
245	—	—	496.26 ± 3.54	333.97	70.41 ± 0.88
			363.60 ± 2.97	562.52	54.98 ± 5.08
247	—	Mother, sister	1148.31 ± 2.17	371.29	164.68 ± 23.99
			806.91 ± 16.69	393.27	141.16 ± 2.62
248	—	Mother, sister	1010.38 ± 5.14	443.83	142.95 ± 4.69
			841.24 ± 18.47	490.2	158.10 ± 33.95
249	—	—	534.09 ± 7.74	459.52	74.98 ± 0.08
			340.44 ± 12.89	499.97	132.91 ± 37.20
			579.65 ± 8.62	413.67	98.93 ± 19.98
250	—	Uncle	688.35 ± 9.46	587.17	74.40 ± 3.75
			612.19 ± 22.36	540.29	150.73
251	—	—	1146.66 ± 7.34	437.25	80.50 ± 5.24
253	—	—	634.83 ± 0.90	486.03	184.50 ± 20.76
254	—	—	701.23 ± 1.92	362.22	72.85 ± 2.66
			548.26 ± 25.55	538.63	83.17 ± 0.07
256	—	—	575.73 ± 5.49	530.67	97.73 ± 3.00
257	—	Mother	995.77 ± 8.22	468.59	94.49 ± 8.02
			879.54 ± 20.53	421.24	102.11 ± 5.69
258	—	—	876.44 ± 9.21	564.15	89.36 ± 4.66
			520.58 ± 8.52	483.28	175.81 ± 53.68
259	—	—	1095.11 ± 7.88	397.45	85.33 ± 4.07
			1050.58 ± 5.08	466.58	139.86 ± 15.48
260	—	—	1084.13 ± 1.82	480.1	127.84 ± 8.13
			494.25 ± 22.05	401.01	188.45 ± 31.29
261	—	—	745.79 ± 22.70	568.33	122.95 ± 2.89
			1150.38 ± 5.64	506.72	206.45 ± 14.75
			852.44 ± 31.69	432.45	142.46 ± 27.89
263	—	—	989.52 ± 4.54	617.16	74.05 ± 5.38
264	—	Uncle	579.22 ± 9.53	580.38	100.39 ± 2.76
265	—	—	696.52 ± 8.57	491.96	105.88 ± 7.86
			848.34 ± 8.38	531.14	106.80 ± 1.16
266	—	—	728.63 ± 5.47	462.66	78.08 ± 1.06
			392.63 ± 9.28	349.34	73.67 ± 3.30
267	—	—	809.78 ± 2.39	579.14	70.57 ± 2.92
			925.13 ± 23.50	827.31	59.18 ± 8.22
268	—	Mother	750.67 ± 17.49	385.93	107.96 ± 12.28
271	—	—	925.40 ± 10.01	482.89	87.43 ± 12.34
			1087.79 ± 22.62	423.61	186.49 ± 10.22
272	—	Aunt	634.87 ± 15.77	531.54	86.12 ± 1.03
			515.84 ± 13.88	594.47	30.80 ± 7.99
273	—	—	455.86 ± 7.52	548.8	91.21 ± 10.34
274	—	—	856.81 ± 23.09	461.61	103.50 ± 8.99
275	—	—	943.57 ± 8.27	469.65	66.73 ± 5.64
			339.71 ± 8.66	513.42	159.78 ± 30.24
276	—	—	430.84 ± 16.02	431.09	234.52 ± 26.95
277	—	—	724.67 ± 0.64	394.65	96.43 ± 0.04
			634.03 ± 28.77	659.6	127.07 ± 4.00
278	—	Mother	1045.58 ± 1.10	364.31	106.88 ± 8.57
			1118.55 ± 3.48	457.48	234.68 ± 24.37
279	—	—	978.20 ± 17.94	442.08	85.62 ± 0.14
			851.57 ± 67.60	573.28	64.64
280	—	Grand-parents	839.91 ± 4.88	415.23	82.19 ± 6.30
			930.08 ± 11.55	468.35	63.88 ± 1.83
281	—	—	991.09 ± 2.95	522.65	151.89 ± 1.15
			655.22 ± 54.74	505.44	112.65 ± 14.80
282	—	—	732.03 ± 19.20	547.53	138.06 ± 12.04
			1196.46 ± 21.91	487.63	129.70 ± 7.80
286	—	—	499.69 ± 1.97	400.19	130.85 ± 3.82
287	—	—	602.68 ± 0.65	418.92	190.43
288	—	—	927.74 ± 4.10	533.37	55.21 ± 10.16
289	—	—	509.91 ± 5.91	362.72	81.33 ± 11.16
290	—	Aunts	498.69 ± 46.68	507.71	127.53 ± 8.29
291	—	—	637.03 ± 7.11	467.8	154.54 ± 1.72
292	—	Grand-Mother	691.71 ± 37.30	581.43	76.54 ± 1.66
293	—	—	494.81 ± 7.56	359.46	166.11
295	—	—	838.72 ± 39.67	405.48	159.20 ± 22.89
296	—	—	761.74 ± 25.61	494.27	237.77
297	—	Uncle	768.08 ± 6.70	515.45	100.00 ± 9.41
298	—	—	750.91 ± 16.94	348.87	290.06 ± 38.15
299	—	—	625.36 ± 6.80	306.11	135.94 ± 1.36
301	—	Grand-mother	948.83 ± 11.23	578.58	150.57 ± 4.40
302	—	—	873.77 ± 2.17	373.31	230.66 ± 10.50
303	—	—	767.96 ± 29.04	458.27	192.45 ± 10.19
304	—	Brother, father, all	493.39 ± 34.21	446.06	185.69 ± 12.07
		paternal family
305	—	Mother	533.91 ± 18.09	364.52	123.23 ± 15.87
306	—	Mother	1016.54 ± 23.75	623.32	216.02 ± 19.04
307	—	—	1328.92 ± 1.50	569.35	165.08 ± 16.63
308	—	Aunts	430.39 ± 5.44	519.72	133.63 ± 11.13
309	—	Mother, cousins	536.77 ± 9.30	485.45	285.92 ± 25.08
311	—	—	493.18 ± 23.85	546.9	110.66 ± 9.59
313	—	Cousin	536.22 ± 4.65	379.49	99.52 ± 2.41
314	—	Mother	939.67 ± 37.16	549.66	78.11 ± 7.22
315	—	—	537.59 ± 1.16	481.91	142.26 ± 23.98
316	—	—	636.17 ± 2.31	576.05	94.21 ± 5.42
318	—	Mother	1151.62 ± 33.64	634.57	112.13 ± 23.16
319	—	Cousin	518.10 ± 27.77	667.02	79.46 ± 6.89
320	—	—	452.54 ± 10.01	765.38	134.09 ± 21.38
321	—	—	470.02 ± 16.75	377.13	110.37 ± 12.77
322	—	—	565.20 ± 48.73	492.94	95.12 ± 7.44
324	—	—	659.93 ± 14.39	562.52	98.61 ± 6.25
325	—	Mother, grand-	761.48 ± 3.82	846.66	89.91 ± 12.48
		parents
326	—	—	1451.37 ± 77.12	617.35	240.72 ± 27.74
328	—	—	580.55 ± 24.91	876.97	174.59
329	—	Mother	877.16 ± 27.08	953.41	269.12 ± 4.88
330	—	—	1403.38 ± 20.98	465.43	279.56
332	—	—	864.14 ± 43.84	699.27	175.34 ± 30.44
333	—	Cousin	564.09 ± 7.37	762.16	143.10 ± 30.54
334	—	—	896.91 ± 29.60	727.33	155.95 ± 38.28
335	—	—	1192.08 ± 14.98	839.56	162.32 ± 0.67
337	—	Sister	914.93 ± 10.71	788.28	114.15 ± 25.71
338	—	Aunt	539.94 ± 1.35	301.42	38.44 ± 5.53
339	—	Grand-father	747.48 ± 9.20	444.12	253.92
340	—	—	746.48 ± 45.11	498.56	259.46
341	—	Cousin	947.50 ± 31.38	662.73	75.40 ± 1.41
342	—	—	993.33 ± 55.93	376.73	19.57 ± 5.63
343	—	Grand-mother	996.61 ± 25.86	541.76	43.48 ± 2.96
344	—	—	637.78 ± 7.73	702.48	26.94 ± 5.89
345	—	Cousin	722.43 ± 18.56	429.44	31.74 ± 1.77
346	—	—	576.26 ± 24.83	436.35	29.25 ± 2.56
347	—	Sister	1272.11 ± 18.19	425.98	41.20 ± 4.60
348	—	Sister	776.87 ± 50.77	384.51	27.13 ± 1.84
350	—	—	1020.59 ± 46.63	488.19	32.35 ± 2.16
351	—	Father	557.14 ± 25.67	475.23	20.16 ± 2.76
352	—	Father	1339.62 ± 39.88	566.82	97.02
353	—	—	1569.33 ± 43.27	607.43	105.59 ± 95.83
354	—	—	608.88 ± 6.80	431.16	69.78 ± 40.24
355	—	—	691.05 ± 37.53	378.46	24.41 ± 12.43
356	—	—	824.89 ± 1.39	467.45	43.63
358	—	—	554.86 ± 8.43	387.21	116.04 ± 22.53
359	—	—	709.63 ± 3.85	485.94	195.32 ± 34.14
360	—	Mother	466.35 ± 12.61	335.02	157.17 ± 7.22
361	—	Aunt	899.31 ± 10.09	441.72	81.52 ± 1.47
362	—	Grand-mother	471.73 ± 21.57	437.35	110.36 ± 7.42
363	—	Mother, grand-	743.10 ± 15.01	353.53	161.77 ± 25.40
		mother, aunt
364	—	Mother, grand-	767.06 ± 11.17	460.75	160.24 ± 26.97
		mother, aunt
365	—	Mother, grand-	883.48 ± 2.32	403.41	127.81 ± 23.58
		mother, aunt
368	—	—	1206.06 ± 43.70	415.24	136.62 ± 28.94
369	—	—	454.71 ± 13.34	431.44	132.25 ± 19.69
370	—	—	855.36 ± 10.35	395.7	140.53 ± 2.77
371	—	Aunt, cousin	740.05 ± 5.38	487.74	112.07 ± 3.13
372	—	—	436.58 ± 40.88	395.61	170.65 ± 13.44
374	—	—	496.50 ± 28.07	401.4	77.69 ± 6.60
375	—	—	475.88 ± 0.00	385.69	130.95 ± 3.80
376	—	—	554.83 ± 44.65	387.61	73.78 ± 0.15
377	—	—	739.47 ± 8.03	384.16	79.40 ± 1.15
379	—	—	1404.12 ± 66.84	659.32	78.73 ± 2.62
381	—	—	782.27 ± 1.42	505.65	283.01 ± 26.97
382	—	—	998.95 ± 9.12	327.82	77.64 ± 12.98
383	—	—	900.32 ± 24.08	401.79	83.98 ± 7.31
384	—	—	479.70 ± 36.72	444.82	134.93 ± 7.83
386	—	—	732.99 ± 28.62	637.86	129.78 ± 2.15
387	—	Cousin	853.05 ± 70.97	373.81	146.21 ± 6.37
388	—	—	963.01 ± 40.86	465.02	66.49 ± 7.43
389	—	Father	689.25 ± 35.56	435.9	67.38 ± 15.52
390	—	Father	930.28 ± 18.25	368.83	56.32 ± 0.12
391	—	—	540.38 ± 9.17	501.81	49.99 ± 7.23
392	—	Brother	661.55 ± 38.23	412.14	77.84 ± 23.22
393	—	Brother	1235.01 ± 29.98	488.02	106.86 ± 17.43
395	—	Mother	716.48 ± 30.93	496.45	82.74 ± 2.92
397	—	Mother, grand-	751.57 ± 2.34	543.59	85.71 ± 21.81
		mother
398	—	Mother, grand-	872.92 ± 6.46	526.34	98.45 ± 6.33
		mother
399	—	—	444.55 ± 43.23	481.5	74.45 ± 10.16
400	—	Mother, aunt	1492.58 ± 30.46	477.59	135.22 ± 2.80
401	—	—	691.24 ± 23.14	316.38	50.01 ± 1.95
402	—	—	423.93 ± 1.08	314.48	36.64 ± 2.04
403	—	Sister	1216.81 ± 131.72	354.37	52.43 ± 15.76
1264	2005-04-18	—	616.12	578.96	65.92
1276	2005-05-16	—	817.56	450.13	107.62 ± 12.96
1364	2006-04-24	Sister, aunt	1668.06	407.4	80.85 ± 6.90
1365	2006-04-26	—	947.35	642.66	63.18 ± 5.41
1366	2006-05-01	—	1317.97	323.04	89.70 ± 20.57
1373	2006-05-17	—	1584.54	583.14	80.12 ± 18.75
1380	2006-06-26	—	1289.98	602.35	139.38
1384	2006-07-03	—	1502.51 ± 18.63	194.3	121.65 ± 44.94
			1258.85 ± 16.20	448.68	162.01 ± 11.64
1385	2006-07-04	—	1098.75	523.52	102.35
1387	2006-07-17	Mother	1017.47	689.52	78.42
1388	2006-07-19	—	1080.53	811.37	87.57
1409	2006-09-26	—	499.41 ± 67.54	389.14	113.56 ± 15.03
1433	2007-01-10	Uncle	459.61 ± 17.79	287.42	263.55 ± 34.89
1451	2007-03-14	Grand-mother	1099.93 ± 48.11	290.5	158.45 ± 3.94
1478	2007-06-11	Father	619.94 ± 46.51	251.56	190.25 ± 18.46
1481	2007-06-18	—	748.36 ± 9.30	250.14	95.34 ± 6.52
1483	2007-06-19	—	489.30 ± 93.18	396.39	167.02 ± 28.62
1487	2007-07-03	Aunts	508.82 ± 50.08	281.48	17.75 ± 1.94
*Plus-minus values are means ± standard deviations.
**All patients are diagnosed with AIS
†Curve type nomenclature: r, right/l, left/T, Thoracic/L, Lumbar/TL, Thoracolumbar/C, Cervical.
‡Certain clinical information may not have been available at the time of the study, NA.

TABLE 7
Clinical and biochemical profiles of AIS patients with Cobb's angles of 45° or more.

						Cobb's
Patient	Date of			Collection	Timepoint	Angle	Curve
ID	Birth	Gender	Age	Date	(months)	Pre-op	Type
101	1988-05-22	F	17.1	2005-06-10	T0	47	rT
108	1989-08-29	F	15.9	2005-07-04	T0	45	lL
			17.2	2006-11-21	T16	40	lL
135	1987-12-31	F	18.0	2006-01-13	T0	47-30	rTlL
145	1990-02-15	M	16.2	2006-04-21	T0	50-43	rTlTL
170	1991-07-08	F	14.9	2006-06-26	T0	53-22	rTlL
			15.9	2007-04-18	T10	44-21	rTlL
1150	1992-04-18	F	12.1	2004-05-11	T0	84	rT
1169	1989-09-19	F	14.8	2004-06-22	T0	54-52	rTlL
1192	1990-10-16	F	13.9	2004-09-08	T0	59	rT
1212	1991-05-06	F	13.5	2004-11-22	T0	54	rT
1254	1991-07-23	F	13.7	2005-03-16	T0	52-49	rTlL
1267	1990-09-08	F	14.6	2005-04-25	T0	55	lT
1282	1988-12-29	F	16.5	2005-06-06	T0	49	rT
1310	1990-05-05	F	15.6	2005-11-09	T0	55-42	rTlL
1353	1989-08-08	F	16.6	2006-03-27	T0	46	lT
			17.2	2006-10-06	T7	2	NA
1354	1991-11-18	F	14.3	2006-03-27	T0	45	rT
1355	1990-02-26	M	16.1	2006-03-28	T0	74-53	rTlL
1357	1990-08-23	F	14.8	2005-06-15	T0	47-50	rTlL
			15.7	2006-04-04	T10	57-50	rTlL
1360	1996-05-09	F	9.9	2006-04-10	T0	53-46	rTlL
1361	1989-09-03	F	16.6	2006-04-10	T0	65-95	rTlL
1369	1992-02-19	F	14.2	2006-05-09	T0	88	rT
			14.8	2006-11-24	T6	25	NA
1371	1991-01-30	F	15.3	2006-05-15	T0	72-59	rTlL
1372	1990-09-06	F	15.7	2006-05-16	T0	63-45-	rTLlLC
						33
1374	1989-10-05	F	16.6	2006-05-29	T0	45	lTL
1378	1992-12-14	M	13.5	2006-06-05	T0	70	lTL
1381	1990-10-03	F	15.7	2006-06-27	T0	66	lT
1389	1995-10-26	F	10.7	2006-07-24	T0	46-66	rTlTL
			11.0	2006-10-02	T5	NA	NA
1390	1990-12-12	F	15.6	2006-07-24	T0	53	lTL
1392	1993-05-25	F	13.2	2006-07-26	T0	48	rT
1393	1991-05-09	F	15.2	2006-07-26	T0	56	lTL
1395	1988-10-25	F	17.8	2006-08-08	T0	84	lTL
1396	1995-05-27	F	11.2	2006-08-14	T0	74-62	rTlL
			11.3	2006-09-26	T1	NA	NA
1397	1988-12-23	M	17.7	2006-08-29	T0	60-58	rTlL
			17.9	2006-10-11	T2	34-23	NA
1406	1991-10-29	F	14.9	2006-09-20	T0	62-60	rTlL
1410	1993-01-04	F	13.7	2006-09-28	T0	56	rT
			13.8	2006-11-21	T2	23	NA
1416	1991-07-10	F	15.4	2006-11-15	T0	56-30	rTlL
1420	1993-06-30	F	13.4	2006-11-29	T0	60-48	rTlL
1422	1994-06-27	F	12.4	2006-12-06	T0	60-50	rTlL
1430	1989-09-28	F	17.3	2007-01-03	T0	48	rT
1442	1994-08-21	F	12.5	2007-02-14	T0	60	rT
1446	1988-07-10	F	18.6	2007-02-26	T0	60	rT
1448	1992-12-07	F	14.3	2007-03-13	T0	49	lTL
1457	1993-05-30	F	13.9	2007-04-10	T0	50-43	rTlL
1458	1991-09-27	F	15.4	2007-04-11	T0	45	rT
1459	1990-03-28	F	17.1	2007-04-16	T0	72-36	rTlL
			17.2	2007-05-18	T1	NA	NA
1461	1990-05-17	F	16.9	2007-04-18	T0	48	rT
1464	1990-01-02	F	17.3	2007-04-25	T0	53	rT
1467	1990-11-18	F	16.5	2007-05-08	T0	60	rT
1468	1991-11-12	M	15.5	2007-05-14	T0	69	rTL
1471	1989-10-08	F	17.6	2007-05-29	T0	60	rTL
1474	1989-06-24	M	18.0	2007-06-04	T0	54-52	rTlL
1477	1992-10-17	F	14.6	2007-06-06	T0	62-65	rTlL
1484	1991-04-27	F	16.2	2007-06-26	T0	60	rT
1488	1992-02-17	M	15.4	2007-07-16	T0	87	rT
1489	1990-09-26	M	16.8	2007-07-17	T0	57	rT
1495	1992-03-19	F	15.5	2007-09-17	T0	67-39	rT
1498	1992-11-05	F	14.9	2007-09-18	T0	51-42	rTL
1501	1989-02-04	F	16.5	2005-07-22	T0	58	rTL
			17.8	2006-11-21	T16	60	rTL
1502	1994-03-14	F	13.6	2007-10-15	T0	55-43	rTlL
			13.8	2007-12-05	T2	NA	NA
1506	1992-07-07	F	15.3	2007-11-06	T0	65	rT
1517	Nov. 20, 1990	M	17.2	2008-02-13	T0	50-62	rTlTL
1518	Dec. 8, 1991	F	16.2	2008-02-13	T0	62-62	rTlL
1519	1993-04-19	M	14.8	2008-02-08	T0	51	rT
1520	1993-06-26	F	14.6	2008-02-08	T0	54-42	rTlTL

Patient	Date of			[sCD44]
ID	Surgery	Family History	[OPN] (ng/ml)	(ng/ml)	[HA] (ng/ml)
101	—	—	1047.64	728.42	221.97 ± 8.23
108	—	—	774.45	704.05	86.15 ± 12.73
			414.67 ± 55.62	361.83	172.00 ± 3.68
135	—	—	657.01	839.02	117.48 ± 5.37
145	—	Brother	1178.85	961.85	120.52 ± 8.59
170	2007-08	Aunt	480.97 ± 29.49	317.2	33.76 ± 0.92
			540.63 ± 10.65	410.66	70.69 ± 4.67
1150	2004-05-11	Mother, grand-	884.02	874.59	97.74
		mother
1169	2004-06-22	—	776.13	868.43	101.22 ± 9.41
1192	2004-09-08	—	1140.09	596.41	66.97
1212	2004-11-22	Great-aunt	834.47	796.56	75.57
1254	2005-03-16	—	1091.92	882.29	82.8
1267	2005-04-25	—	509.48	596.41	76.87
1282	2005-06-06	—	718.45	788.41	53.95 ± 16.65
1310	2005-11-09	—	1042.25	789.32	132.89
1353	2006-03-27	—	1078.92 ± 33.32	262.59	90.88 ± 1.59
			44.35 ± 0.50	342.48	157.74 ± 37.90
1354	2006-03-27	—	1378.360	725.138	61.016
1355	2006-03-28	—	1871.67	467.38	253.56 ± 6.84
1357	2006-04-04	Brother	705.92 ± 16.09	415.22	174.61 ± 74.40
			1788.1	374.7	76.86 ± 4.78
1360	2006-04-10	Father, aunt	1820.95	444.42	80.45 ± 29.61
1361	2006-04-10	—	1512.16	599.64	67.13 ± 10.66
1369	2006-05-09	—	1498.66	262.58	91.42 ± 8.52
			541.43 ± 10.31	317.72	166.79 ± 35.56
1371	2006-05-15	—	1723.91	224.15	89.53 ± 18.60
1372	2006-05-16	Aunt	1016.66	597.2	65.24 ± 5.40
1374	2006-05-29	—	1698.01	544.71	70.32 ± 16.24
1378	2006-06-05	—	1531.64	394.74	249.97
1381	2006-06-27	—	1032.61	626.25	89.25
1389	2006-07-24	—	899.76 ± 20.49	359.31	187.61 ± 62.69
			770.91 ± 13.31	533.42	82.67 ± 1.55
1390	2006-07-24	—	1269.89	839.02	78.42
1392	2006-07-26	Grand-mother,	1341.80 ± 15.38	87.13	105.48 ± 0.34
		aunts
1393	2006-07-26	—	969.63	821.21	81.59
1395	2006-08-08	Aunt	1205.3	450.13	41.8
1396	2006-08-14	—	1624.64 ± 5.10	166.83	172.75 ± 26.23
			773.40 ± 16.42	342.29	218.18 ± 2.83
1397	2006-08-29	Uncle	1581.40 ± 11.23	440.95	106.21 ± 10.20
			1191.01 ± 14.64	546.18	158.77 ± 21.05
1406	2006-09-20	—	628.36 ± 45.23	304.04	52.88 ± 0.66
1410	2006-09-28	Mother, aunt	1287.16 ± 3.12	133.56	119.48 ± 24.22
			903.57 ± 52.88	328.75	141.76 ± 12.56
1416	2006-11-15	—	514.30 ± 15.49	233.55	121.42 ± 28.69
1420	2006-11-29	Sister, aunt	661.35 ± 21.22	314.01	127.14 ± 1.06
1422	2006-12-06	Sister	530.56 ± 6.57	190.55	61.30 ± 14.49
1430	2007-01-03	—	533.56 ± 24.89	228.54	51.29 ± 7.00
1442	2007-02-14	—	512.99 ± 44.58	163.01	162.44 ± 3.03
1446	2007-02-26	—	537.87 ± 4.70	332.42	66.44 ± 20.48
1448	2007-03-13	—	588.73 ± 25.88	110.3	138.81 ± 10.07
1457	2007-04-10	—	1073.67 ± 69.04	401.79	83.21 ± 0.17
1458	2007-04-11	—	401.08 ± 22.88	212.16	66.48 ± 0.55
1459	2007-04-16	—	761.78 ± 11.69	104.61	42.08 ± 5.99
			744.34 ± 10.91	340.71
1461	2007-04-18	Sister	200.53 ± 3.68	371.51	112.29 ± 27.44
1464	2007-04-25	—	778.26 ± 19.40	163.01	133.86 ± 4.16
1467	2007-05-08	—	453.32 ± 17.32	236.23	48.59 ± 6.73
1468	2007-05-14	Cousin	574.80 ± 42.46	283.37	116.85 ± 14.54
1471	2007-05-29	—	907.06 ± 34.13	332.42	66.91 ± 28.51
1474	2007-06-04	—	1254.39 ± 4.53	334.72	71.72 ± 16.08
1477	2007-06-06	Mother, brother	829.32 ± 15.89	355.03	150.57 ± 28.87
1484	2007-06-26	—	489.15 ± 20.09	216.67	88.54 ± 422
1488	2007-07-16	Mother	1358.23 ± 56.62	304.83	120.78 ± 13.25
1489	2007-07-17	—	1417.61 ± 0.00	146.93	135.42 ± 2.53
1495	2007-09-17	—	437.55 ± 14.74	227.82	32.06 ± 0.29
1498	2007-09-18	—	557.43 ± 50.58	152.3	62.63 ± 12.90
1501	—	—	939.53	711.38	144.30 ± 16.14
			580.11 ± 7.56	503.43	107.24 ± 7.29
1502	2007-10-15	—	856.14 ± 4.95	386.19	152.27 ± 5.09
			1089.57 ± 22.51	349.14	55.91 ± 10.45
1506	2007-11-06	—	675.53 ± 13.63	241.98	85.64 ± 24.87
1517	—	—	666.49 ± 65.68	328.96	41.3 ± 8.74
1518	—	—	672.59 ± 35.53	440.55	67.71 ± 6.81
1519	—	—	945.23 ± 53.53	360.02	66.48 ± 1.10
1520	—	—	752.87 ± 23.12	288.35	87.08 ± 0.36
*Plus-minus values are means ± standard deviations.
**All patients are diagnosed with AIS
†Curve type nomenclature: r, right/l, left/T, Thoracic/L, Lumbar/TL, Thoracolumbar/C, Cervical.
‡Certain clinical information may not have been available at the time of the study, NA.

TABLE 8
Clinical and biochemical profiles of asymptomatic at risk children.

				Collection	Timepoint			[sCD44]
Family Id	Date of Birth	Gender	Age	Date	(months)	Family History	[OPN] (ng/ml)	(ng/ml)	[HA] (ng/ml)

1	1997-09-02	M	8.8	2006-07-10	T0	Mother	439.72 ± 12.32	561.46	118.71 ± 8.74
1	1995-09-06	F	10.8	2006-07-10	T0	Mother	207.88 ± 0.93	315.67	180.71 ± 19.91
2	1998-02-08	F	8.7	2006-10-03	T0	Mother, uncle, grand-	1650.21 ± 13.90	416.99	199.56 ± 55.60
			9.2	2007-04-19	T6	father	1966.98 ± 1.96	459.89	207.57 ± 39.18
			9.8	2007-12-12	T14		1816.83 ± 24.08	387.1	209.86 ± 21.38
2	2001-06-18	M	5.8	2007-04-19	T0	Mother, uncle, grand-	493.98 ± 7.26	463.68	43.99 ± 3.74
			6.5	2007-12-12	T8	father	684.54 ± 10.06	438.94	102.21 ± 61.17
3	1994-08-24	F	12.2	2006-10-19	T0	Sister	690.58 ± 2.92	418.18	220.8
			12.6	2007-05-02	T7		727.27 ± 17.36	467.79	196.82 ± 18.74
			13.2	2007-12-12	T14		1212.32 ± 0.48	311.06	279.74 ± 30.33
4	2003-10-17	F	3.0	2006-10-19	T0	Mother	1530.90 ± 28.42	478.58	225.02 ± 20.51
			3.5	2007-04-11	T6		1021.07 ± 7.22	464.63	122.36 ± 15.35
			4.2	2007-12-12	T14		1594.42 ± 23.36	470.05	332.11
5	2003-07-17	M	3.2	2006-10-19	T0	Mother	905.58 ± 30.14	563.44	58.88 ± 3.86
			3.7	2007-04-19	T6		1865.13 ± 7.35	434.93	128.14 ± 4.00
			4.4	2007-12-09	T14		960.14 ± 26.22	631.93	32.64 ± 5.81
6	1998-07-26	F	8.2	2006-10-19	T0	Mother	505.03 ± 8.92	564.17	81.86 ± 13.18
7	1995-06-16	F	11.3	2006-10-24	T0	Mother	548.59 ± 6.61	512.92	80.39 ± 31.53
			11.8	2007-04-11	T6		766.85 ± 5.73	396.69	103.31 ± 22.50
			12.3	2007-10-17	T12		596.91 ± 35.50	465.36	122.40 ± 8.97
8	1996-04-10	F	10.5	2006-10-26	T0	Mother	1109.78 ± 47.61	401.66	77.16 ± 9.72
			11	2007-04-11	T6		875.81 ± 14.01	366.36	176.96 ± 4.68
9	1995-05-09	F	11.4	2006-10-26	T0	Mother	1657.97	440.3	112.58 ± 0.45
			11.9	2007-04-11	T6		782.29 ± 1.47	429.56	86.57 ± 1.46
			12.8	2008-02-13	T16		885.10 ± 35.98	255.6	63.42 ± 7.99
10	2002-08-03	F	4.2	2006-10-26	T0	Mother	901.66 ± 12.01	398.27	158.65 ± 60.85
			4.7	2007-04-11	T6		929.42 ± 3.07	356.88	167.19 ± 0.13
11	1992-09-07	F	14.1	2006-10-26	T0	Mother	528.00 ± 8.83	469.78	69.05 ± 4.37
			14.8	2007-07-11	T9		714.79 ± 14.44	383.1	37.97 ± 3.99
			15.3	2008-01-23	T15		443.30 ± 0.58	472.69	80.27 ± 11.45
12	1991-12-15	F	14.8	2006-10-26	T0	Mother	818.88 ± 0.94	518.03	134.08 ± 84.67
			15.3	2007-04-11	T6		648.15	487.38	140.02 ± 50.63
			15.9	2007-11-14	T13		398.28 ± 19.81	521.44	191.07 ± 8.20
12	1996-02-23	M	10.7	2006-10-26	T0	Mother	1203.88 ± 55.29	681.23	85.30 ± 36.75
			11.2	2007-04-11	T6		1930.95 ± 1.96	633.37	107.10 ± 15.99
			11.8	2007-11-14	T13		1341.78 ± 31.57	687.61	170.54 ± 25.46
13	1993-10-09	F	13.0	2006-10-26	T0	Mother, grand-mother	730.44 ± 33.95	397.12	41.87 ± 4.55
			13.6	2007-05-02	T7		420.91 ± 23.59	412.49	216.75 ± 27.71
			14.1	2007-11-14	T13		943.64 ± 1.96	698.95	124.28 ± 15.03
14	2001-09-07	F	5.2	2006-11-16	T0	Father	919.94 ± 11.91	510.08	45.28 ± 10.89
15	1997-02-18	M	9.8	2006-11-16	T0	Mother	1629.22 ± 12.49	611.25	129.80 ± 30.80
			10.2	2007-04-11	T5		1030.34 ± 6.55	690.56	146.19 ± 2.58
			10.7	2007-10-10	T11		929.36 ± 11.23	590.8	135.89 ± 18.75
16	2002-02-21	F	4.8	2006-11-16	T0	Mother	1834.30 ± 4.16	628.94	149.05 ± 19.17
			5.2	2007-04-11	T5		909.22 ± 6.67	661.18	125.31
			5.9	2007-12-12	T13		877.48 ± 23.75	466.59	70.10 ± 33.68
17	2000-03-30	F	6.7	2006-11-16	T0	Mother	482.76 ± 10.64	678.55	95.92 ± 18.21
18	2000-08-01	F	6.2	2006-11-16	T0	Mother	870.73 ± 21.30	644.62	146.12 ± 36.68
18	1997-05-05	M	9.5	2006-11-16	T0	Mother	1123.32 ± 7.06	401.66	112.68 ± 11.34
20	1998-09-27	F	8.2	2006-11-22	T0	Father	506.21 ± 10.03	456.42	59.40 ± 30.21
			8.8	2007-07-11	T8		677.71 ± 13.95	416.28	37.11 ± 6.95
21 (015)	1998-11-17	F	8.0	2006-11-22	T0	Sister	482.63 ± 7.58	458.02	99.16 ± 5.46
			8.5	2007-05-23	T6		511.46	488.33	151.08
			9.0	2007-11-14	T12		760.00 ± 3.99	589.62	190.77 ± 5.64
21 (016)	1991-08-13	F	15.2	2006-11-22	T0	Sister	617.06 ± 7.65	511.71	110.15 ± 12.37
			15.7	2007-05-23	T6		619.60 ± 17.63	519.3	93.16 ± 0.39
			16.2	2007-11-14	T12		685.18 ± 0.80	529.63	218.26 ± 27.22
22	1992-05-15	M	14.5	2006-11-22	T0	Mother, grand-mother	1082.23 ± 65.01	445.66	81.35 ± 14.77
			14.9	2007-04-11	T5		1044.90 ± 3.21	432.72	152.54 ± 10.62
			15.6	2008-01-23	T14		1010.18 ± 60.70	384.16	106.42 ± 10.80
23 (334)	1994-09-24	F	12.2	2006-11-29	T0	Sister	1365.94 ± 1.71	346.45	150.14 ± 2.53
			12.6	2007-04-19	T5		1856.82 ± 12.74	501.92	167.91 ± 17.19
			13.1	2007-10-10	T11		947.97 ± 16.31	489.38	271.36 ± 20.40
24	1994-11-24	M	12.0	2006-11-29	T0	Mother, aunt	775.28 ± 20.77	427.49	84.54 ± 0.14
			12.5	2007-05-02	T6		610.29 ± 10.86	436.82	130.53 ± 2.30
			13.1	2007-12-12	T13		718.55 ± 5.97	355.99	127.92 ± 3.93
24	1994-11-24	F	12	2006-11-29	T0	Mother, aunt	815.81 ± 22.25	473.76	160.63 ± 8.36
			12.5	2007-05-02	T6		673.56 ± 16.29	445.36	127.40 ± 37.13
			13.1	2007-12-12	T13		1299.89 ± 28.77	662.73	276.97
25	1998-06-05	F	8.4	2006-11-29	T0	Mother, father	1245.41 ± 13.75	441.4	108.75 ± 18.90
			8.8	2007-04-19	T5		1766.40 ± 2.69	500.34	197.20 ± 31.62
			9.3	2007-10-10	T11		944.99 ± 25.37	476.76	115.66 ± 10.09
25	2001-06-04	M	5.4	2006-11-29	T0	Mother, father	1181.70 ± 50.65	303.75	157.81 ± 11.99
			5.8	2007-04-19	T5		1707.51 ± 30.62	319.63	113.24 ± 2.45
			6.3	2007-10-10	T11		867.79 ± 25.36	364.76	114.76 ± 33.42
26	1994-03-18	F	12.7	2006-11-29	T0	Mother	676.95 ± 9.57	432.08	86.09
27	1987-12-13	F	19	2006-12-19	T0	Father	287.27 ± 8.96	572.38	101.88 ± 13.89
28	2003-05-23	F	3.6	2006-12-19	T0	Mother	612.92 ± 3.03	760.08	45.57 ± 3.40
29	1990-10-17	M	16.2	2006-12-19	T0	Mother	459.54 ± 29.16	488.33	99.03 ± 54.21
			17.0	2007-10-10	T10		505.24 ± 39.04	441.73	121.53 ± 15.54
29 (652)	1999-05-11	F	7.6	2006-12-19	T0	Mother	576.64 ± 20.73	656.77	114.39
			8.4	2007-10-10	T10		972.66 ± 7.97	636.32	138.53 ± 16.69
29 (160)	1996-12-02	F	10.0	2006-12-19	T0	Mother	583.62 ± 19.18	600.16	136.79 ± 10.66
			10.8	2007-10-10	T10		874.79 ± 2.17	535.48	112.73 ± 7.74
30	1995-03-09	M	11.8	2006-12-19	T0	Mother	1608.98 ± 8.37	607.15	115.19 ± 6.27
			12.3	2007-07-04	T7		1107.95 ± 0.53	504.15	40.04 ± 11.63
			12.8	2008-01-23	T13		1578.17 ± 18.50	469.62	93.33 ± 3.68
30	1997-06-08	F	9.5	2006-12-19	T0	Mother	1211.80 ± 5.47	586.43	172.18 ± 4.00
			10.1	2007-07-04	T7		774.18 ± 21.15	534.59	40.03 ± 11.95
			10.6	2008-01-23	T13		697.49 ± 12.25	473.45	95.89 ± 6.16
31	1998-03-18	F	8.8	2006-12-19	T0	Mother, aunt, grand-	467.80 ± 1.39	574.23	106.48 ± 29.19
						father
31	1999-11-03	M	7.1	2006-12-19	T0	Mother, aunt, grand-	745.53 ± 40.56	552.66	98.22 ± 1.18
						father
32	2004-06-20	F	2.5	2006-12-19	T0	Mother, grand-mother	1573.79 ± 0.72	576.5	142.70 ± 0.57
			3.1	2007-07-04	T7		1034.97 ± 25.55	494.82	52.38 ± 5.01
			3.6	2008-01-23	T13		1237.94 ± 48.60	374.2	152.27 ± 0.32
33	1996-05-17	M	10.7	2007-01-10	T0	Mother	623.78 ± 2.66	649.44	166.16 ± 32.22
			11.5	2007-11-07	T10		671.14 ± 0.27	634.5	36.87 ± 2.05
33	1996-06-25	F	11.2	2007-01-10	T0	Mother	893.13 ± 34.21	436.86	92.74 ± 2.45
			11.7	2007-07-11	T6		716.31 ± 27.52	543.59	37.95 ± 5.33
34	1996-08-14	F	10.3	2006-12-21	T0	Mother	1135.80 ± 18.20	508.95	256.64 ± 37.18
			10.8	2007-06-13	T6		594.41 ± 0.37	490.61	96.56 ± 2.45
			11.4	2008-01-23	T13		978.10 ± 49.46	450.46	103.67 ± 10.95
34	1994-06-21	M	12.5	2006-12-21	T0	Mother	1010.70 ± 22.34	416.71	172.33 ± 50.68
			13.0	2007-06-13	T6		739.31 ± 3.43	499.04	93.55 ± 6.90
			13.6	2008-01-23	T13		777.22 ± 39.78	448.93	92.70 ± 21.91
35 (605)	1995-03-31	M	11.8	2006-12-21	T0	Mother	1126.22 ± 46.08	552.37	163.66 ± 0.79
35 (604)	1995-03-31	M	11.8	2006-12-21	T0	Mother	933.16 ± 14.20	437.43	118.57 ± 6.65
35	1993-05-12	F	13.6	2006-12-21	T0	Mother	1679.45	436.58	128.45 ± 17.60
36	1998-09-06	M	8.3	2007-01-10	T0	Mother	1520.81 ± 20.48	485.39	225.68 ± 85.59
			9.2	2007-11-14	T10		1103.50 ± 27.07	899.87	114.96 ± 0.11
37	2001-07-11	F	5.5	2007-01-17	T0	Mother	419.51 ± 10.21	524.02	35.52 ± 0.52
			6.0	2007-07-04	T6		606.10 ± 14.32	490.91	209.23
38	1995-01-19	M	12.0	2007-01-17	T0	Mother	435.87 ± 7.38	600.34	164.49 ± 10.01
38	1992-08-02	F	14.4	2007-01-17	T0	Mother	328.67 ± 25.67	564.58	166.19 ± 2.53
39	1996-06-08	M	10.6	2007-01-24	T0	Mother	437.90 ± 23.91	529.14	215.53 ± 70.15
			11.1	2007-07-18	T6		617.26 ± 5.45	445.15	146.08 ± 8.82
39	1997-08-08	F	9.4	2007-01-24	T0	Mother	399.82 ± 14.71	452.38	71.339 ± 22.51
			9.9	2007-07-18	T6		648.28 ± 6.30	462.01	188.78 ± 12.79
40	1996-05-05	F	10.9	2007-04-05	T0	Mother	986.26 ± 9.88	478.27	99.9
40	1999-04-23	M	8.0	2007-04-05	T0	Mother	851.99 ± 4.04	710.05	52.81 ± 12.17
41	1995-03-29	F	12.2	2007-05-30	T0	Father	500.68 ± 20.08	416.56	71.27 ± 0.30
42	1996-07-03	M	10.8	2007-05-02	T0	Father	391.38 ± 30.03	620.65	32.83
			11.3	2007-11-14	T6		393.23 ± 4.22	445.78	167.25 ± 27.97
42	1992-04-14	F	15.1	2007-05-02	T0	Father	452.43 ± 1.68	519.81	38.46 ± 16.02
			15.6	2007-11-14	T6		658.95 ± 1.62	938.89	232.91 ± 2.00
43	2001-11-20	F	5.5	2007-05-23	T0	Mother	892.70 ± 21.23	484.89	97.65 ± 30.81
44	1995-09-11	M	11.8	2007-06-13	T0	Mother	1058.59 ± 6.11	547.8	41.15 ± 11.08
			12.2	2007-12-12	T6		1160.10 ± 16.16	456.22	145.61 ± 51.30
45	1994-05-10	F	13.2	2007-08-29	T0	Mother	714.66 ± 6.88	482.12	120.00 ± 13.64
			13.8	2008-02-13	T6		801.53 ± 42.46	358.64	134.84 ± 16.18
46	1999-11-04	M	7.8	2007-09-12	T0	Mother	603.75 ± 10.96	569.62	111.95 ± 5.86
40 (980)	1996-04-15	F	11.4	2007-09-13	T0	Mother	504.38 ± 35.85	540.29	118.25 ± 9.11
46 (982)	2004-01-24	F	3.7	2007-09-12	T0	Mother	718.72 ± 78.98	510.97	153.13 ± 4.50
47	1996-12-07	F	10.8	2007-10-17	T0	Mother	1010.10 ± 17.02	494.12	147.00 ± 87.36
47	1999-04-03	M	8.5	2007-10-17	T0	Mother	844.83 ± 30.84	456.7	156.33 ± 50.36
C6	1997-02-06	F	10.3	2007-05-22	T0	Mother	669.60 ± 4.19	755.65	133.68 ± 4.10
			11.0	2008-01-16	T8		733.30 ± 11.16	620.67	250.52 ± 38.11
C15	1997-05-27	M	10.0	2007-06-06	T0	Brother	441.81 ± 0.64	640.33	106.53 ± 1.88
			10.5	2007-12-04	T6		444.69 ± 3.82	958.24	151.86 ± 17.41
*Plus-minus values are means ± standard deviations.
†All subjects are examined before sample collection by an orthopedic surgeon to monitor possible scoliosis development.

EXAMPLE 11

OPN, sCD44 and HA Levels in Non AIS Scoliotic Patients

OPN levels were measured in non AIS scoliotic patients (NAIS patients). Results are summarized in Table 9 below. A comparison of OPN, sCD44 and HA levels in healthy, AIS and NAIS patients is also provided in FIG. 12.

TABLE 9
Biomarkers Comparison of non-AIS scoliotic Patients.

Characteristics

				Mean OPN	Mean sCD44	Mean HA
		Mean Age	Mean Cobb	Concentration	Concentration	Concentration
Type of Scoliosis	Number	(Years)	Angle	(ng/ml)	(ng/ml)	(ng/ml)
Neurological	8	12.3 ± 3.7	79.4 ± 15.1	982 ± 452	274 ± 196	127 ± 101
Scoliosis
Congenital Scoliosis	8	10.0 ± 4.4	51.8 ± 18.1	1016 ± 400	432 ± 79	123 ± 80
Spondylolisthesis	5	17.5 ± 2.1	21.0 ± 17.0	832 ± 125	386 ± 193	76 ± 54
Kyphosis Scoliosis	5	14.4 ± 2.8	80.2 ± 28.5	923 ± 393	352 ± 62	91 ± 56
Other*	2	15.1	74.5 ± 17.7	586 ± 52	240	NA
†Plus-minus values are means ± standard deviations
*Other scoliosis types include one neuromuscular scoliosis and one dysplasic scoliosis.

Table 10 below presents in detail biomarkers levels for non AIS scoliotic patients.

TABLE 10
Clinical and biochemical profiles of non AIS scoliotic patients.

						Cobb's
Patient				Collection		Angle	Curve
ID	Date of Birth	Gender	Age	Date	Diagnosis	Pre-op	Type
1208	1990-01-19	M	17.8	2007-10-03	Congenital cyphose scoliosis	72	lT
1256	1992-03-27	M	13.0	2005-05-09	Congenital scoliosis	44-65	rTlL
1278	1998-07-22	F	6.8	2005-05-30	Congenital neurological	60	lT
					scoliosis
1281	1985-05-21	M	20.1	2005-06-01	Spondylolisthesis	16	—
1286	1990-05-08	M	15.1	2005-06-15	Dysplasic scoliosis	62-66	rTlL
1356	1993-02-22	F	13.2	2006-04-03	Congenital scoliosis	75	rT
1358	2003-11-09	M	2.4	2006-04-04	Congenital scoliosis	33-35	rTlL
1367	1993-12-12	F	12.4	2006-02-01	Neurological scoliosis	90	lTL
1368	1990-06-21	F	15.9	2006-05-02	Neurological cyphosis	50	lTL
1370	1995-09-15	M	10.7	2006-05-09	Neurological scoliosis	65	rT
1375	1992-09-13	F	13.7	2006-05-30	Congenital scoliosis	53	rTL
1407	1990-12-22	M	16.8	2007-10-31	Spondylolisthesis	9	lL
1431	1987-11-23	M	19.2	2007-01-08	Neurological scoliosis	90-90	rTlT
1432	1992-08-08	M	14.4	2007-01-09	Neurological scoliosis	64	rT
1434	1994-08-07	F	12.4	2007-01-10	Congenital scoliosis	79-77	rTlL
1436	1993-02-16	F	13.9	2007-01-22	Cyphose scoliosis	120	—
1437	1992-11-06	M	14.2	2007-02-05	Neurological scoliosis	100	NA
1455	1996-12-14	F	10.3	2007-04-03	Congenital cyphose scoliosis	61	lTL
1456	1990-10-03	F	16.5	2007-04-17	Neuromuscular scoliosis	87	rTL
1462	1997-10-22	F	9.5	2007-04-23	Neurological scoliosis	76	lTL
1463	1989-03-19	F	18.1	2007-04-24	Scoliosis + Spondylolisthesis	33	rT
1466	1997-08-24	F	9.8	2007-05-08	Congenital scoliosis	39	rL
1475	1993-05-25	M	14.1	2007-06-05	Cyphose scoliosis	98	—
1479	1996-01-24	F	11.4	2007-06-05	Neurological scoliosis	90	rTlL
1480	2003-06-13	F	4.0	2007-06-18	Congenital scoliosis	56	lT
1482	1989-03-30	F	18.2	2007-06-19	Spondylolisthesis gr 1	—	NA
1486	1993-01-15	M	14.4	2007-06-27	Spondylolisthesis gr 2	—	NA
357	1996-07-08	F	11.4	2007-12-18	Congenital scoliosis	30-31	rTlT

Patient	Date of	Age at	Family		[sCD44]	[HA]
ID	Surgery	Surgery	History	[OPN] (ng/ml)	(ng/ml)	(ng/ml)
1208	2004-11-	14.8	—	1101.06 ± 31.26	444.81	82.89 ± 15.11
	08
1256	2005-03-	13.0	—	1490.59	NA	127.74 ± 9.29
	29
1278	2005-05-	6.8	—	1401.88	NA	75.65 ± 5.16
	30
1281	2005-06-	20.1	—	985.85	NA	150.30 ± 7.93
	01
1286	2005-06-	15.1	—	549.60 ± 5.06	NA	NA
	15
1356	2006-04-	13.2	—	1181.85	NA	111.51 ± 2.30
	03
1358	2006-04-	2.4	—	1530.6	NA	284.60 ± 69.00
	04
1367	2006-05-	12.4	—	1525.13	NA	350.01 ± 36.55
	01
1368	2006-05-	15.9	—	1079.23	NA	126.44 ± 3.63
	02
1370	2006-05-	10.7	—	1318.58	NA	104.06 ± 5.18
	09
1375	2006-05-	13.7	Cousin	380.08 ± 12.95	NA	NA
	30
1407	2006-09-	15.8	—	818.17 ± 1.52	441.73	116.09 ± 3.88
	25
1431	2007-01-	19.2	—	450.78 ± 101.56	275.62	130.30 ± 23.92
	08
1432	2007-01-	14.4	—	558.47 ± 4.70	145.15	98.99 ± 13.92
	09
1434	2007-01-	12.4	—	631.59 ± 7.42	325.95	44.79 ± 5.73
	10
1436	2007-01-	13.9	—	220.32 ± 2.94	322.03	44.34 ± 6.37
	22
1437	2007-02-	14.2	—	388.01 ± 8.22	225.71	76.96 ± 4.53
	05
1455	2007-04-	10.3	—	1090.51 ± 5.57	323.24	34.79 ± 0.32
	03
1456	2007-04-	16.5	—	622.46 ± 7.15	240.22	NA
	17
1462	2007-04-	9.5	—	1118.25 ± 1.32	607.1	55.90 ± 1.82
	23
1463	2007-04-	18.1	—	751.54 ± 8.69	284.71	21.56 ± 4.58
	24
1466	2007-05-	9.8	—	1110.01 ± 2.38	510.18	47.07 ± 1.48
	08
1475	2007-06-	14.1	—	1123.49 ± 5.56	319.93	166.63 ± 34.63
	04
1479	2007-06-	11.4	—	1098.54 ± 131.44	119.17	NA
	05
1480	2007-06-	4.0	—	809.8	468.03	120.72 ± 40.73
	18
1482	2007-06-	18.2	—	678.49 ± 18.32	187.48	46.07 ± 5.27
	19
1486	2007-06-	14.4	—	924.40 ± 17.16	628.78	47.06 ± 6.84
	27
357	—	—	—	996.58 ± 8.51	423.72	127.33 ± 3.13
*Plus-minus values are means ± standard deviations.
†Curve type nomenclature: r, right/l, left/T, Thoracic/L, Lumbar/TL, Thoracolumbar/C, Cervical

EXAMPLE 12

OPN and sCD44 Levels in AIS Patients Pre and Post Operations

OPN levels were measured in AIS patients pre (n=79) and post (N=28) operations. Interestingly, comparison of AIS patients in pre-operation vs. post operation showed a reduction in circulating OPN levels, which further support the role of OPN at the cellular level as mechanosensor (FIG. 13).

OPN were measured in AIS female patients pre (n=10) and post (N=10) treatment with braces. Similarly, sCD44 levels were measured in AIS female patients pre (n=15) and post (N=12) operations. Results are presented in FIG. 14.

A distribution of 12 AIS patients was also performed across the predefined cut-off zones pre-operation and post-operation. FIG. 15 shows 92% of the surgically treated patients had pre-operation OPN levels in the red-zone (>800 ng/mL of plasma OPN level), while the remaining 8% were in the yellow zone (700-800 ng/mL). No patients were in the green zone representing plasma OPN levels<700 ng/mL. This also shows a strong correlation between high OPN concentrations and the progression of scoliotic curves.

Panel B of FIG. 15 show that red zone patients who were treated surgically experienced a decline in OPN concentrations in the blood. 75% of the surgically treated patients fell into the green and yellow zones (800 ng/mL or less).

EXAMPLE 13

OPN Levels in AIS Patients with Various Types of Braces

OPN levels were also measured in AIS patients prior to being treated with brace (n=79) and after brace (N=28). Table 11 below also shows the effect of braces on biomarkers.

TABLE 11
Possible effects of brace treatment on biomarker concentrations.

Characteristics

			Mean
		Mean	Brace	Mean	Mean OPN	Mean sCD44	Mean HA
		Age	Wear	Cobb's	Concentration	Concentration	Concentration
Treatment	No.	(Years)	(Months)	Angle	(ng/ml)	(ng/ml)	(ng/ml)

Without Brace
Female	193	14.2 ± 2.1	—	30.9 ± 19.3	809 ± 376	474 ± 179	108 ± 58
Male	36	14.8 ± 2.2	—	32.2 ± 21.1	1034 ± 376	492 ± 155	126 ± 62
With Brace
(All Female)
All Braces	21	14.0 ± 1.8	12.0	21.2 ± 8.3	664 ± 282	483 ± 112	118 ± 60
Combined
Boston	5	13.0 ± 1.4	10.6	25.8 ± 4.4	735 ± 358	568 ± 184	150 ± 57
SpineCor	14	14.5 ± 1.6	12.7	20.6 ± 8.7	626 ± 279	451 ± 81	108 ± 62
Charleston	1	15.4	10.0	7.0	781	532	70
Providence	1	9.7	1.0	20.0	732	547	138
Night Brace
P-value‡					0.018	0.879	0.608
*Plus-minus values are means ± standard deviations.
‡Statistical analysis to compare patients with or without brace was done by bilateral unpaired Student's T-test with equal variance. A difference was considered statistically significant with a p-value < 0.05.

A distribution of AIS patients across the predefined cut-off zones was also performed prior to being treated with bracing and after bracing. Eight patients were tested a certain number of months after bracing, namely for each of patients #1 to 8: 7, 7, 8, 22, 22, 22 and 26 months after bracing, respectively. FIG. 16 shows that prior to being treated with bracing (Panel A), 63% of these patients were in the red and yellow zones. A significant shift towards the green zone (<700 ng/mL) was observed, which is consistent with the trend observed in surgically treated patients, as presented in FIGS. 13-15.

EXAMPLE 14

Comparison of Selenium Levels in AIS Patients vs. Healthy Subjects

Selenium concentration was reported to be significantly decreased in plasma of AIS patients (42). Selenium and more specifically Se-methylselenocystein, an organoselenium naturally occurring in diet, are used to prevent metastasis in breast cancer as chemopreventive therapy by targeting OPN transcription (43-45).

Plasma selenium concentration was thus measured in pediatric populations (AIS vs. healthy controls) to determine whether or not low selenium levels correlate with higher OPN concentrations in AIS. Plasma selenium concentrations were determined by a fluorometric method using 2,3-diaminonaphthalene (DAN) (46, 47). Results presented in FIGS. 18 and 19 show a correlation between high OPN levels and low selenium levels in scoliotic and asymptomatic at risk children.

Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

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