MOLECULAR CLASSIFICATION OF AIS PATIENTS: TOWARD NEW EMERGING CONCEPTS IN MOLECULAR MEDICINE TO TREAT ADOLESCENT IDIOPATHIC SCOLIOSIS B. Azeddine, H. Labelle, B. Poitras, CH. Rivard, and A. Moreau. Bone Molecular Genetics and Musculoskeletal Malformations Laboratory, Research Centre, Hôpital Sainte-Justine POES, 06 mai 2005

Adolescent idiopathic scoliosis Definition: «  Idiopathic scoliosis is a three-dimensional deformity of the spine with lateral curvature combined with vertebral rotation, which affect mostly girls. » It’s etiology is still unknown

Tissues & systems targeted by melatonin action and affected in AIS Postural control defect (CNS) AIS patient Equilibrium & vestibular dysfonction Proprioception dysfonction Platelet abnomalities Increased calmodulin level Osteopenia Reduced muscle spindles Muscle fibers abnormalities Abnormal muscular tone GH secretion NOVEL HYPOTHESIS Melatonin signaling dysfunction in AIS Mel   i GTP AC MT1-2 ATP Gi GDP AMPc First unifying concept supporting the multiplicity of disorders associated with AIS

Aims The goals of this study were to assess the possibility to establish a molecular classification of AIS patients and to demonstrate the feasibility to correct this melatonin signaling defect using therapeutic compounds

Materials and methods AIS CTRL Patients number Total 62 22 7 8 55 14 Clinical data of scoliotic patients and control subjects AIS CTRL Patients number Total 62 22 7 8 55 14 Mean age (year) boys girls 16,4 15 14,7 16 Mean Cobb’s angle thoracic double thoracolumbar lumbar 50,9° 53°-48° 49,9° 47,1° 51,9° 60°-30° 53° no scoliosis 76,5°

Percentage of cAMP inhibition Melatonin signaling dysfunction in osteoblasts isolated from AIS patients 300 250 200 150 Percentage of cAMP inhibition per 100,000 cells GR 1 (n=13) 100 GR 2 (n=25) GR 3 (n=16) 50 CTRL (n=4) F -11 -9 -7 -5 Melatonin concentration (M) Moreau et al., Spine 2004; 29:1772-1781

Phosphorylation of Gi proteins is increased in osteoblasts derived from AIS patients Melatonin Vanadate Genistein Control Inactive form 60kDa Ctrl Active form 43kDa 60kDa 1003 43kDa 60kDa 1007 43kDa

Search for regulators involved in Gi protein phosphorylation TKs Rack1 P TPs Y PKCδ activation P Gi S inactivation

Abnormal interactions between MT2 melatonin receptor subtype and PKCδ occur in AIS cells Patients Gender Age (years) Curve pattern Cobb's angle Pain Group # 1056 F 18 right thoracic 34 Yes 2 # 1084 F 12 right thoracic 70 No 3 # 1138 F 14 right thoracic 58 No 1

Rack1 interactions with PKCδ are affected in AIS cells Patients Gender Age (years) Curve pattern Cobb's angle Pain Group # 1056 F 18 right thoracic 34 Yes 2 # 1084 F 12 right thoracic 70 No 3 # 1138 F 14 right thoracic 58 No 1

Screening for therapeutic compounds that could rescue melatonin signaling dysfunction in AIS 120 100 80 Control % cAMP 60 Compound B 40 20 Compound A F -11 -9 -7 -5 melatonin concentration [M] Patient #1004

Summary Increased phosphorylation of Gi proteins could be at the origin of the melatonin signaling defect in AIS although such dysfunction may involve more than one gene. Functional assays offer the mean to identify for the first time children at risk of progression without the knowledge of specific mutations. Such dysfunction could be rescued in some patients using therapeutic compounds and may offer innovative approaches to prevent or stop spinal deformities.

X The future of scoliosis treatments Scoliosis Early detection 25˚- 40˚ Identification of patients with a risk of progression 45˚+, progressive Surgery Prior the clinical manifestations 1. Goal: To prevent scoliosis using pharmacotherapies and/or braces Functional & molecular classification of patients already diagnosed 2. Goal: To stop the progression of scoliotic deformities Development of new generation of braces Use of a tailored pharmacotherapy (selection of specific drugs) Identification of biochemical/molecular markers

Bone Molecular Genetics and Musculoskeletal Malformations Laboratory Acknowledgements Bone Molecular Genetics and Musculoskeletal Malformations Laboratory Collaborators University of Milan, Italy Dr Debora Angeloni Prof Franco Fraschini Hôpital Sainte-Justine Dr Hubert Labelle Dr Benoît Poitras Dr Charles-Hilaire Rivard Dr Guy Grimard Dr Gilles Chabot Julie Joncas Dr Florina Moldovan Dr. Moreau’s team Dr Alain Moreau Hugo Boulanger Catherine Chassaing Ginette Lacroix Virginie Landreville Kareen Letellier Isabelle Turgeon Dr Da Shen Wang Montreal’s Children Hospital Dr Jean Ouellette Dr Vincent Arlet