The Association Study Of CALM1 Gene Polymorphisms With Susceptibility To Adolescent Idiopathic Scoliosis

BackgroundIdiopathic scoliosis is the most common pediatric spinal deformity affecting 1%to 3%of the population,and adolescent idiopathic scoliosis(AIS) accounts for approximately 80%of these cases.The etiology of AIS is still uncertain,but with the rapid development of genetics and molecular biology in recent years,genetics researches of AIS are brought into focus gradually.Nowadays,AIS is widely believed a multifactorial inheritance disease.Recent linkage studies on familial idiopathic scoliosis revealed multiple putative predisposition loci.As for investigating complicated multigenetic disease,candidate gene analysis, association analysis and haplotype analysis are advanced and exact methods,and they are direct and effective means of elucidating the mechanism of disease-related gene.In addition,AIS has complex clinical phenotypes,such as PUMC classification classification,yet there are no report about the association of genetic mechanism with different clinical classifications.Objects1.To identify the relationship between CALM1 gene and AIS,and to investigate the major effect of CALM1 Gene in AIS patients,2.To identify the relationship between the genotypes of SNPs and the clinical phenotypes of AIS.3.To explore possible etiologic hypothesis contributed to the development of AIS and the different clinical phenotypes of AIS at gene level.MethodsA hospital-based case-ontrol design was applied in this study.A total of 146 patients(24 boys,122 girls,mean age 14.89 y/o) diagnosed with adolescent idiopathic scoliosis admitted in Peking Union Medical College(PUMC) hospital were enrolled in this study according to inclusion and exclusion criteria between October 2005 and February 2008.The control group also comprised 146 patients(24 boys,122 girls,mean age 15.18 y/o) and consisted of infection(64),inflammatory disease(53),trauma(11),hypersensitive disease(6)and others(12) at PUMC hospital during the same period.All the control subjects were frequency-matched to the cases on age(±3 years),gender and the Han nationality. PUMC classification,curve pattern,cobb's angle of main curve of AIS patients were recorded.Genomic DNA was extracted from peripheral blood leukocytes of each subject who had signed informed consent with QIAamp DNA Blood Mini Kit.Based on genotype data from the international HapMap project,14 single nucleotide polymorphisms(SNPs) in calmodulin 1(CALM1) gene were initially choosed by Haploview 4.0 software.Genotyping of all selected SNPs was done by SNPstream technolofy(Beckman Coulter SNPstream).However,2 SNPs failed in composition of premier and were abandoned.All the data of 12 SNPs with polymorphism were analysed by the association analysis based on alleles and phenotypes of SNPs.Odds ratio(ORs) and 95%confidence intervals(CIs) were computed by the unconditional logistic regression to estimate the relative risk for the single locus genotypes by online software—SNPstats.Haplotype frequencies were estimated and difference in haplotype distributions between cases and controls were assessed by Haploview 4.0 software. Results1.Epidemiologic data analysis:there were no significant difference of age distribution and sex proportion between case group and control group.2.We failed in composing premier of two SNPs(rs12885713,rs5871) and abandoned them in this study.The remained 12 SNPs were genotyped and all polymorphisms were in Hardy-Weinberg equilibrium between case group and contrl group.3.SNP Allele-AIS:the allele frequency distributions of rs2300496,rs2300500 and rs3213718 were statistically different between case group and control group(P=0.0079,0.0079,0.0257 respectively).4.SNP genotype-AIS(1)The genotypes distributions of rs2300496,rs2300500 and rs3213718 were statistically different between caseg roup and control group(P=0.0196, 0.0196,0.0378 respectively).In the unconditional logistic regression analysis,after adjustment for age and gender,rs2300496 and rs2300500 both showed significant difference in Codominant model(OR=0.12,95%, CI=0.01-0.99,p=0.015,AIC=402.4),Dominant model(OR=0.56,95Z%,CI= 0.34-0.92,p=0.022,AIC=403.6) and Log-additive model(OR=0.54, 95%CI=0.35-0.86,p=0.0073,AIC=401.6).The Log-additive model was accepted as the best inheritance model because of the smallest AIC (Akaikeinformation) value.rs3213718 showed significant difference in Log-additive model(OR=0.60,95%CI=0.39-0.93,p=0.022,AIC=403.5).(2)Haplotype-AIS:we got 1 positive haplotype:SNP8C-SNP1A-SNP13C-SNP14C-SNP2C-SNP3T-SNP4G-SNP5T-SNP11C.(3)The genotypes distributions of rs2300496 and rs2300500 were statistically different between PUMCⅡAIS group and control group(P=0.0470,0.0470 respectively).(4)The genotypes distributions of rs2300496,rs2300500 and rs3213718 were statistically different between thoracic curve group and control group(P=0.0180,0.0180,0.0402 respectively).(5)There was no statistical difference between main curve severity and genotypes distributions of all of 12 SNPs.Conclusion1.Genetic variants of CALM1 gene are associated with AIS and may play an important role in the development of AIS.2.The development of PUMCⅡAIS might be related to genotype polymorphisms of rs2300496 and rs2300500.The development of thoracic curve AIS might be related to genotype polymorphisms of rs2300496,rs2300500 and rs3213718.3.The different clinical phenotype of AIS might be related to the different SNP site.It was preliminarily suggested that PUMC classification of AIS is not only the criterion of treatment,but also the logical combination of genetic mechanism and phenotype.4.There was no statistically different between main curve severity and genotypes distributions of all of 12 SNPs in CALM1 gene.5.Although it's too early to draw a conclusion,it was suggested that different clinical phenotype of AIS might be related to different SNP loci.It's the first time to research the association of genetic mechanism of AIS with to clinical classificiation.