Identification Of Predisposition Genes Associated With Adolescent Idiopathic Scoliosis And Its Imaging Study

Chapter 2(section 1)Genome-wide association study identifies new susceptibility loci for adolescent idiopathic scoliosis in Chinese populationObjective To illustrate the genetic architecture of adolescent idiopathic scoliosis(AIS)using genome-wide association study(GWAS)in Chines Han populationMethods 5201 AIS patients were enrolled in the study from Nanjing,Hong Kong,Hangzhou and Changchun since 2009 to 2016.6787 normal subjects were enrolled as controls.Blood samples were collected with informed consent signed.Affymetrix Genome-Wide Human SNP Array 6.0 chips were used for genotyping.Quality control was performed using EIGENSTRAT.Principal component analysis(PCA)was used to investigate potential population stratification.PLINK software package was used in case-control association analysis to determine SNPs significantly associated with AIS.A p value of 5 x 10-8 was considered as genome significance.Results The PCA analysis showed that all the subjects were clustered in the Chinese Han population.The genome inflation factor was 1.13,indicating that there was no significant population stratification.After quality control,435183 SNPs were included in the case-control association analysis.We conducted a 3-stage GWAS study.Overall,we identified 3 novel susceptibility loci at 2p14 near MEIS1(rs7593846,Pcombined = 4.33 x 10-12),3p14.1 near MAGI1(rs7633294,Pcombined?3.41×10-11),3q26.2 near TNIK(rs9810566,Pcombined = 1.26 × 10-10).We also confirmed a recently reported region associated with AIS at 20p 11.22(rs6047663,Pcombined = 2.12 x 10-14).Conclusion New AIS susceptible genes were identified in the study with remarkable genome significance,which may serve as an important work for future study on the etiology of AIS in Chinese population.Chapter 2(section 2)The Wnt/?-catenin pathway:a novel pathway underlying adolescent idiopathic scoliosisObjective Our previous GWAS studies identified several AIS susceptible genes in Chinese population such as LBX1,PAX3,TNIK and MEIS1,which were associated with the Wnt/?-catenin signaling pathway.However,the underlying mechanism of these genes in AIS patients remains unknown.The purpose of this study was to investigate the role of these downstream genes of Wnt/?-catenin signaling pathway in the onset and development of AIS.Methods Paravertebral muscles were collected from 124 AIS female patients,20 Chiari-associated scoliosis female patients and 24 lumbar disc herniation(LDH)female patients.Total RNA and protein were extracted from muscles.The expression level of p-catenin,TNIK,TCF4,PAX3,LBX1,MyoD,MEIS1 and PAX1 were compared between concave and convex sides among three groups.The correlation analysis was calculated among different genes.Skeletal muscle fiber typing was performed using myofibrillar adenosine triphosphatase methods.Immunofluorescence was used to detect muscle fiber ?/? and MyoD protein.The proportion of muscle fibers was compared between concave and convex sides of AIS patients.The correlation analysis were calculated between concave/convex ratio of fiber type I and expression of MyoD.Results The three groups were age-matched.The AIS group was Cobb angle-matched with Chiari group.The mRNA expression level of each gene was lower in AIS group than control group.Also,the mRNA expression level was lower in concave than convex side in AIS group.There were no significant differences between concave and convex side in control groups.Most genes in the pathway were significantly correlated with each other.The protein expression level of ?-catenin and LBX1 were lower in concave than convex side,also lower in AIS than controls.The rate of Type ?fiber was significantly lower in the concave side than in the convex side(49.5%±18.0%vs.61.5%±7.7%,p=0.002).In addition,the concave/convex ratio of type I fiber was significantly correlated with the concave/convex ratio of MyoD in the paravertebral muscles of AIS patients(r=0.67,p=0.02).Conclusion We observed significantly asymmetric expression of Wnt/?-catenin pathway in the bilateral paraspinal muscle of AIS patients,including ?-catenin,TNIK and LBX1,etc.This is the first study that unveils the potential role of Wnt/p-catenin pathway in the development of AIS,which may shed new light on the etiopathogenesis of AIS.Chapter 2(section 3)Genetic variant of GPR126 gene is functionally associated with adolescent idiopathic scoliosis in Chinese populationObjective To investigate whether rs9403380 of GPR126 gene is susceptible locus of AIS and to further determine the functional variants regulating gene expression in tissues of AIS.Methods Rs9403380 were genotyped in 975 AIS patients and 1049 controls.The differences of genotype and allele distributions between patients and controls were calculated using Chi-square test.Paravertebral muscles were collected from 48 AIS and 10 lumbar disc herniation(LDH)patients.AIS patients were classified into three groups according to the genotypes of the SNP,and One-way ANOVA test was used to compare GPR126 expression among different groups and genotypes.Results Rs9403380 were found significantly associated with AIS.Allele C of the SNP can significantly add to the risk of AIS with an odds ratio of 1.16.AIS patients were found to have significantly higher GPR126 expression than controls.Moreover,patients with rs9403380 genotype CC have a significantly increased expression of GPR126 than those with TT.Conclusion Rs9403380 could be a functional variant regulating the expression of GPR126 in the paraspinal muscles of AIS,which might be a potential biomarker for the early diagnosis of AIS.Chapter 3(section 1)Anterior spinal overgrowth in patients with adolescent idiopathic scoliosis:primary or secondary to the spinal deformity?Objective Relative anterior spinal overgrowth has been reported in morphological studies and is believed to be a potential driver for both AIS initiation and progression.However,contradictory findings have been reported on whether the altered vertebral morphology is primary or secondary?No study has compared the AIS with the non-idiopathic scoliosis on the shape of the vertebral bodies.This study was designed to compare vertebral morphology among AIS,age-,sex-,curve pattern-matched Chiari I malformation(CMI)-associated scoliosis and normal controls by using 3D CT scans,aiming to investigate whether the phenomenon "'overgrowth of anterior column" is primary or secondary to the deformity.Methods A total of 142 girls were enrolled into the study,included 60 Lenke type 1 AIS patients and 52 age-,gender-,curve pattern-matched CMI patients as well as 30 age-,gender-matched normal controls.All had whole spine CT images.The anterior and posterior heights of the vertebral body(VBHa and VBHp),the height of the pedicles(PH)and vertebral central width(VCW)for each level were measured on CT scans.The ratios of VBHa and PH,VBHp and PH were calculated and compared among three groups respectively.Results Compared with control group,the VBHa and VBHp of most vertebral bodies were consistently longer in both AIS and CMI group between T1 and T12,but no significant difference was found between AIS and CMI group.Also,the PHs of most vertebral bodies were consistently shorter in both AIS and CMI group,but no significant difference was found between AIS and CMI group.There was no significant difference of VCW among three groups.The ratios for differential growth between the anterior and posterior elements of each thoracic vertebra in both AIS and CMI group were significantly larger than that in the controls,but the ratios were not significantly different between AIS and CMI group.Conclusion The faster growth of the anterior spinal column was confirmed by the longer vertebral bodies and shorter pedicles in both AIS and scoliosis secondary to CMI patients.The relative anterior spinal overgrowth of the thoracic spine is not involved in the initiation of AIS.The altered vertebral growth in AIS might be a secondary change to scoliosis.Chapter 3(section 2)Selecting the last "substantially"touching vertebra as lowest instrumented vertebra in Lenke type 1A curve:radiographic outcomes with a minimum of 2-year follow-upObjective Previous studies have documented good outcomes when last touching vertebra(LTV)was selected as LIV.However,it is sometimes confusing to determine the proper LTV when central sacral vertical line(CSVL)slightly touches the vertebra.The purpose of this study is to compare the long-term outcomes of correction surgery for Lenke 1A scoliosis patients among those with non-Substantially Touched Vertebra(nSTV),nSTV+1 or Substantially Touched Vertebra(STV)selected as lowest instrumented vertebra(LIV).Methods 104 patients were included in the study with a minimum of 2-year follow-up after selective posterior thoracic instrumentation.STV was defined as the LTV where CSVL was between the pedicles or touching the pedicle.nSTV was defined as the LTV where CSVL was touching the corner of the vertebra lateral to the pedicle border.Patients with nSTV,nSTV+1,or STV selected as LIV were assigned to three groups with clinical outcomes compared among them.Factors associated with the incidence of adding-on were analyzed.Results Distal adding-on was observed in 23 patients(22.1%).The incidence of distal adding-on was significantly higher in nSTV group than STV group or nSTV+1 group.Several risk factors significantly associated with adding-on were identified,including the distance between LIV and STV/nSTV+1,preoperative proximal thoracic curve and sagittal vertical axis,postoperative lumbar lordosis,apical translation,trunk shift and radiographical shoulder height.Logistic regression analysis showed that the distance between LIV and STV/nSTV+1(LIV-STV<0 or LIV-(nSTVH-1)<0)was the only independent factor associated with the incidence of adding-on(OR=27.1,95%CI=2.3-311.2,P=0.002).Conclusion Differentiating STV from nSTV properly can facilitate the determination of optimal LIV and decrease the incidence of distal adding-on.Selecting STV or nSTV+1 as LIV could yield a promising outcome for Lenke 1A scoliosis patients undergoing selective posterior thoracic fusion.