Marker for the classification, diagnosis and treatment of scoliosis

Abstract

A method of classifying a subject having idiopathic scoliosis (IS) or at risk of developing IS comprising: determining the cellular response to Gi stimulation in a cell sample from the subject in the presence of OPN; determining the cellular response to Gi stimulation in a cell sample from the subject in the absence of OPN; and comparing the cellular response obtained in the presence of OPN with the cellular response obtained in the absence of OPN, whereby the comparing step enables the classification of the subject into one IS functional group. Also provided is the use of the foregoing method to classify borderline subjects and kits for applying the methods.

1 . A method of classifying a subject having idiopathic scoliosis (IS) or at risk of developing IS or a method of determining the risk of developing IS in a subject comprising:
 (A)   a) contacting a cell sample from the subject with i) an agonist of cluster I or cluster II GiPCR; and ii) Pertussis toxin (PXT);   b) determining the cellular response to Gi stimulation; and   c) classifying said subject into a IS functional group according to the subject's cellular response profile; or   (B)   a) contacting a cell sample from the subject with an agonist of a cluster II GiPCR;   b) determining the cellular response to Gi protein stimulation by cellular impedance; and   c) classifying the subject into a IS functional group according to the subject's cellular impedance profile.   
     
     
         2 . The method of  claim 1 :
 i.  1 (A), wherein step (c) comprises (i) classifying said subject into a first functional group when the cellular response is above the cellular response of a control sample; or (ii) classifying said subject into a second functional group when the cellular response is substantially comparable to the cellular response of a control sample; or   ii. 1(B), wherein step (c) comprises: (i) classifying the subject into a first IS functional group when said cellular impedance profile comprises a phase of negative impedance followed by a phase of positive impedance; or (ii) classifying the subject into a second IS functional group when the cellular impedance profile does not comprise a phase of negative impedance.   
     
     
         3 . The method of  claim 1 , wherein said cluster I GiPCR is lysophosphatidic acid receptor (LPAR), melatonin receptor (MT1 or MT2), mu-opioid receptor (OR) or adenosine receptor (AR) or wherein said cluster II GiPCR is cannabinoid receptor (CB2R) or a α2-Adrenoreceptor (α2AR). 
     
     
         4 . The method of  claim 3 , wherein said agonist of cluster I GiPCR is melatonin, iodomelatonin, phenylmelatonin, LPA, DAMGO or NECA or wherein said agonist of cluster II GiPCR is CB65 or UK14304. 
     
     
         5 . The method of claim  1 (A), wherein the cellular response to Gi stimulation is determined by measuring cellular impedance, preferably by cellular dielectric spectroscopy. 
     
     
         6 . The method of  claim 1 , further comprising classifying borderline subjects into one IS functional group by using an alternative classification method, and wherein said alternative classification method is preferably performed prior to step (a). 
     
     
         7 . The method of  claim 6 , wherein the alternative classification method comprises:
 (i) determining the difference between cellular responses to Gi and Gs protein stimulations in the cell sample from the subject;   (ii) determining the magnitude of a cellular response to Gi stimulation in the cell sample relative to a control sample;   (iii) measuring cAMP concentration;   (iv) determining the phosphorylation state of Giα proteins in the cell sample; or   (v) determining cellular proliferation of the cell sample relative to a control sample.   
     
     
         8 . The method of  claim 7 , wherein (i) determining the difference between cellular responses to Gi and Gs protein stimulations comprises (i a) measuring cAMP cellular concentration produced by each of Gi and Gs protein stimulations; (i b) measuring cellular impedance, wherein said cellular impedance is preferably measured by cellular dielectric spectroscopy (CDS); (i c) determining the ΔG in the sample from the subject; or (i d) by determining a ratio between cellular responses to Gi and Gs protein stimulations (Gi/Gs). 
     
     
         9 . The method of  claim 8 , wherein:
 in (i c) the ΔG is determined by measuring cellular impedance and the cellular impedance is measured by CDS, and wherein i) a ΔG below −10 is indicative that the subject belongs to IS functional group FG1; ii) a ΔG above −10 and below +10 is indicative that the subject belongs to IS functional group FG2; and iii) a ΔG above +10 is indicative that the subject belongs to IS functional group FG3; or   in (i d) i) a ratio below about 0.5 is indicative that the subject belongs to IS functional group FG1; ii) a ratio between about 0.5 and 1.5, is indicative that the subject belongs to IS functional group FG2; and iii) a ratio above 1.5 is indicative that the subject belongs to IS functional group FG3.   
     
     
         10 . The method of  claim 7 , wherein:
 in (ii):(ii a) a reduction of between about 60-90% of the cellular response to Gi stimulation relative to control is indicative that the subject belongs to IS functional group FG1; (ii b) a reduction of between about 40-60% of the cellular response to Gi stimulation relative to control is indicative that the subject belongs to IS functional group FG2; and (ii c) a reduction between about 10-40% of the cellular response to Gi stimulation relative to control is indicative that the subject belongs to IS functional group FG3;   in (iii):(iii a) an increase in cAMP concentration relative to baseline is indicative that the subject belongs to IS functional group FG1, (iii b) no significant or weak reduction in cAMP concentration relative to baseline is indicative that the subject belongs to IS functional group FG2; and (iii c) a reduction in cAMP concentration relative to baseline is indicative that the subject belongs to IS functional group FG3;   in (iv): (iv a) an increase in the level of serine phosphorylated Giα1, Giα2 and Giα3 proteins as compared to a control sample is indicative that the subject belongs to IS functional group FG1; (iv b) an absence of serine phosphorylation in Giα3 proteins or a level of serine phosphorylation on Giα3 proteins comparable or lower to the level of serine phosphorylation in Giα3 proteins in a control sample is indicative that the subject belongs to IS functional group FG2; and (iv c) an absence of serine phosphorylation in Giα1 proteins or a level of serine phosphorylation on Giα1 proteins comparable to the level of serine phosphorylation in Giα1 proteins in a control sample is indicative that the subject belongs to IS functional group FG3; or   in (v): (v a) a reduction in cellular proliferation of about 50% or more relative to control is indicative that the subject belongs to IS functional group FG1; (v b) a reduction in cellular proliferation between about 25% and 45% relative to control is indicative that the subject belongs to IS functional group FG2; and (v c) a reduction in cellular proliferation of about 25% or less relative to control is indicative that the subject belongs to IS functional group FG3.   
     
     
         11 . The method of  claim 10 , wherein in (ii), the magnitude of a cellular response to Gi stimulation is determined by measuring cellular impedance, preferably by cellular dielectric spectroscopy (CDS). 
     
     
         12 . A method of selecting a preventive measure, treatment or follow-up schedule for a subject suffering from IS or at risk of developing IS comprising classifying the subject using the method as defined in  claim 1 ; wherein
 a) when the subject is classified as belonging to the FG1 functional group:
 i) the level of OPN in said subject is increased; 
 ii) the level of HA in said subject is increased; 
 iii) the level of CD44 in said subject is decreased; and/or 
 iv) the frequency of assessment of scoliosis progression is decreased; 
   b) when the subject is classified as belonging to the FG2 functional group:
 i) the level of OPN is said subject is decreased; 
 ii) the level of HA in said subject is decreased; and/or 
 iii) the level of CD44 in said subject is increased; 
 iv) the frequency of assessment of scoliosis progression is increased; and/or 
 v) the subject is prescribed corrective surgery prior to reaching a scoliosis with a cobb angle of 45 degree; 
   c) when the subject is classified as belonging to the FG3 functional group:
 i) the level of OPN is said subject is decreased; 
 ii) the level of HA in said subject is decreased; 
 iii) the level of CD44 in said subject is increased; and/or 
   v) the frequency of assessment of scoliosis progression is decreased.   
     
     
         13 . The method of  claim 12 , wherein:
 (aa) increasing the level of OPN comprises administering OPN; applying pulsative compressive pressure to a body part of the subject or applying low intensity pulsed ultrasound to a body part of the subject;   (bb) increasing the level of HA comprises administering to the subject HA supplements or complying to a HA-rich diet and wherein decreasing the level of HA comprises complying to a HA-poor diet;   (cc) decreasing OPN level comprises brace treatment, accupoint heat sensitive moxibustion, heat therapy with pad, thermal bath, electroacupuncture, administering melatonin, administering selenium or administering PROTANDIM; or   (dd) decreasing the frequency of assessment of scoliosis progression comprises performing less than 22 radiological examinations within 3 years following classification.   
     
     
         14 . The method of  claim 1 , wherein (i) the subject is a subject pre-diagnosed with IS; (ii) the subject is asymptomatic; and/or (iii) the subject has at least one family member suffering from IS. 
     
     
         15 . The method of  claim 1 , wherein the cell sample comprises osteoblasts, chondrocytes, myoblasts and/or peripheral blood mononuclear cells (PBMCs), preferably fresh PBMCs comprising lymphocytes. 
     
     
         16 . The method of  claim 1 , wherein said Gi stimulation further comprises contacting the cell with GP Ant-2. 
     
     
         17 . The method of  claim 7 , wherein the Gs stimulation comprises contacting the cells with isoproterenol. 
     
     
         18 . A kit for classifying a subject having idiopathic scoliosis (IS) or at risk of developing IS or for predicting the risk of developing IS comprising:
 (A)   a) Pertussis toxin (PTX); and   b) a cluster I GiPCR and/or a cluster II receptor agonist; and optionally,   (B)   c) a ligand for Gs stimulation;   d) one or more antibodies for detecting Giα phosphorylation; and/or   e) instructions for classifying the subject or for predicting the risk of developing IS.   
     
     
         19 . The kit of  claim 18 , wherein the cluster I GiPCR is lysophosphatidic acid receptor (LPAR), mu-opioid receptor (OR) or adenosine receptor (AR) or melatonin receptor and the cluster II GiPCR is cannabinoid receptor (CB2R) or a α2-Adrenoreceptor (α2AR), and/or
 wherein the agonist is melatonin, iodomelatonin, phenylmelatonin, LPA, DAMGO, NECA, CB65 or UK14304. 
 
     
     
         20 . A composition for classifying a subject having idiopathic scoliosis (IS) or at risk of developing IS or for determining the risk of developing IS in a subject comprising:
 (A)   a) cell sample from the subject;   b) Pertussis toxin (PTX); and   c) a cluster I GiPCR and/or a cluster II GiPCR agonist; and optionally,   (B)   d) a ligand for Gs stimulation; and/or   e) one or more antibodies for detecting Giα phosphorylation.   
     
     
         21 . The composition of  claim 20 , wherein the cluster I GiPCR is LPAR, OR, A1R or melatonin receptor and the cluster II GiPCR is CB2R or α2AR, and/or
 wherein the agonist is melantonin, iodomelatonin, phenylmelatonin, LPA, DAMGO, NECA, CB65 or UK14304.