Proteome Analysis Of Cartilage Endplate In Adolescent Idiopathic Scoliosis

BackgroundAdolescent idiopathic scoliosis(AIS) is the most common form of spinal deformity of teenagers.The incidence of AIS among adolescents is approximately 3% as identified by a Cobb angle≥10°.AIS falls within the subcategory of "late-onset" idiopathic scoliosis which is defined as diagnosis at age≥10 years of age as opposed to "early onset" which can include infantile and juvenile cases.It is the most common form of idiopathic scoliosis accounting for between 80-85%of cases.Diagnostic criteria include:age≥10 years,Cobb angle of≥10°and exclusion of neuromuscular or congenital etiologies.Clinically AIS can be screened for using the Adams forward bend test and quantifying the degree of trunk rotation using a scoliometer,while the radiographic determination of the Cobb angle remains the gold standard.AIS may progress to result in pain,cardiopulmonary dysfunction,and dissatisfaction with appearance.More severe curves and progressive curves may require treatment including bracing or surgery.AIS has no validated etiology and occurs in otherwise healthy adolescents. Several hypotheses have been brought forward to explain the etiology of AIS, including genetic,biochemical,mechanical,neurological,muscular,hormonal,and even environmental factors.Twin studies have shown a much higher incidence of disease among monozygotic twins compared to dizygotic twins,lending credibility to a genetic component to the disease. Controversy,however,exists as to the exact mode of inheritance.Recent research has mapped genetic loci of AIS to chromosomes 9,17 and 19,but the inheritance pattern is not clear.Other research has failed to demonstrate a role for the growth hormone receptor gene as a predisposing gene or disease modifier gene in AIS.Asymmetrical interverteberal disc formation and growth is the structural foundation of deformity and disease progression in AIS patients.It has been shown that the convex side of interverteberal discs are found well developed compared to the concave side,both on radiographs and during anterior correction surgery procedures.The difference in histological grades and cellular activity between the convex and concave side indicated that the growth of AIS patients have different growth kinetics in endplate cartilage which may affect the curve progression. Ultimately,the differential growth in the endplates must result from some origin factors leading to differential expression of proteins.New advances in proteomics bio-technology now make it possible to directly analyze those proteins from harvested cartilage tissues.The pathogenesis of AIS is still unknown yet.Many different theories exist for the etiology.This study is the first major proteomic investigation of interverteberal disc cartilage.The proteins found with significant asymmetry in expression patterns may be implicated as causative of deformity,or may be the result of differences in the biomechanical environment within the disc,and secondary epiphenomena.Objective:To identify protein expression in asymmetrically developed intervertebral discs of adolescent idiopathic scoliosis(AIS) patients and try to find out factors that may originate or strengthen the situation.Methods:The apex intervertebral discs were harvested from 6 AIS patients during anterior correction surgery.Protein extraction were performed and we used shotgun strategy (2D-LC-MS/MS analysis) to achieve proteomics investigation.MS and tandem mass spectra were extracted from the XCalibur data system format(.RAW) into DTA format by Extact_msn.Tandem mass spectra were interpreted by SEQUEST against human ref protein database.CILP and HSP27 were performed Western Blot procedure to testify the mass spectra results.Results:The consistency of the extracted protein was tested through Bradford procedure[23], and the average concentration is 3.02mg/ml(2.20-3.97mg/ml).SDS-PAGE was performed to collect major complexion of the protein samples.Both quantity and concentration of the proteins from convex side were found to be higher than that from the opposite side.One hundred and three proteins were identified from samples of the convex side and fifty-five from the concave side,forty-five proteins were found both in convex side and concave side,while 58 and 10 proteins were found only in the convex side versus the concave side respectively.Conclusions:This is the first proteomic characterization of endplate cartilage phenotypes.Different protein expression levels between the convex and concave sides of the intervertebral discs endplate cartilage in AIS patients were indentified by shot-gun proteomics strategy.Though the result is preliminary,and it is still not clear that these differences are original factors of AIS or secondary cellular responses to mechanical compression and tension on the intervertebral disc at the apex of the curve. The study sheds new light on the genetic pathogenesis of adolescent idiopathic scoliosis and other disc related spinal diseases.The future directions identified appear promising.(For example in vitro mutation analysis studies could help in determining how these proteins affect cartilage tissue and AIS progress) Molecular and genetic mediations could help on finding how these proteins affect cartilage tissue and AIS progress.