The Association Analysis Of Genes Ploymorphisms With Susceptibility To Congenital Scoliosis In A Chinese Han Population

BackegroundCongenital scoliosis(CS) is defined as a lateral curvature of the spine due to developmental abnormality,which arises from defects in the development of the axial skeleton.The spinal curvature can be observed in coronal,sagittal,or horizontal plane. During embryogenesis,the axial skeleton is formed by a process called somitogenesis, which produces transient segmental tissue known as somites.Disruptions in somitogenesis have been shown to result in vertebral malformations,including segmentation failure and formation failure.The exact etiology of vertebral malformation is still unknown by now.The animal experiments showed that the vertebral malformation can be induced by abnormal expression of development-related genes,radioactive ray,medicine and intoxication.Recently, various mouse mutants and knock-out mice have been used in the study of gene function in development,and the results demonstrate the developmental abnormality caused by the dysfunction of single gene.The animal experiments have shown that a variety of genes play a role during development,and the abnormal expression of these genes can result in obvious vertebral malformation.The genetic etiology of CS has drawn more and more attention of biologists.The recent studies support that CS is a disease of multifactorial inheritance.The dysfunction of different genes can result in different phenotypes.At present,the study of candidate genes of CS is preliminary internationally and blank nationally.Within the past two decades,a great deal of information has been learned about the molecular embryology of spine development through the studies of mouse and chick embryos.Different species show the relatively high conservation in nucleotide sequences and amino acid sequences during species evolvement.By reviewing the mouse gene database,we identified some genes related to vertebral malformation.The human candidate genes of CS were further determined by synteny analysis.Objective●To identify the relationship between ten candidate genes and CS through SNPs genotype.●To identify the relationship between the gene polymorphisms and the clinical phenotypes of CS.●To explore possible etiologic hypothesis contributed to the onset of CS.Methods●A hospital-based case-control design was applied in this study.●A total of 154 patients with CS and 144 controls were enrolled in this study according to inclusion and exclusion criteria from Oct,2005 to May,2008.●Genomic DNA was extracted from peripheral blood leukocytes of each subject.●75 SNP loci,distributed in 10 genes,were selected based on the genotype data from the NCBI.●Case group were classified into different clinical phenotypes according to vertebral malformation type,malformed levels and rib malformation type.●All the SNPs were genotyped by VeraCode GoldenGate Genotyping Assay system.●Hardy-Weinberg equilibrium both in case and control groups were analyzed through Goodness-of-fit Chi-square test.●The association analysis and logistic regression analysis of single locus were performed to evaluate the association between the single locus and onset risk of CS.●The software of Haploview and SNPstats were used to perform linkage disequilibrium analysis,haplotype association analysis and haplotype logistic regression analysis of the multiple loci in each gene to evaluate the association between the haplotype and onset risk of CS.Results●Total 75 loci including 10 genes were examined and 11 loci were abandoned.Of the 11 abandoned loci,8 were given up because of the low recall ratio and other 3 were for non-polymorphisms.The recall ratios of the rest 65 loci were higher than 99.3%.●Four loci in 2 genes in control group were not in Hardy-Weinberg equilibrium and the P values were less than 0.05.These loci were DLL3-rs2304222, MYLK-rs1254392,MYLK-rs820463,and MYLK-rs9422.Seven loci in 3 genes in case group were not in Hardy-Weinberg equilibrium and the P values were less than 0.05.The seven loci were LMX1A-rs12029324,LMX1A-rs12023709, LMX1A-rs1819768,LMX1A-rs16841013,MYLK-rs9422,and TBX6-rs3809624.●The linkage disequilibrium blocks were found in 7 genes,including four loci (rs2304223,rs1110627,rs2304214 and rs3212276) in DLL3,four loci(rs2074222, rs222837,rs222836,and rs2074216) in DVL2,two loci(rs1442849 and rs1348325) in HES7,ten loci(rs1819768,rs12023709,rs16840972 and rs16841013;rs6671290,rs16841029 and rs1354510;rs4657412,rs6667188,and rs1532815) in LMX1A,three loci(rs12172926,rs3732487 and rs1350152) in MYLK,two loci(rs8060511 and rs3809624) in TBX6,fifteen loci(rs708114, rs6426490,rs6672559,rs3094913,rs881398,rs708121,rs3094911,rs3094912, rs708122 and rs10916258;rs7539664,rs10916262,rs11589513,rs6675092 and rs4653533) in WNT3A.●Five loci in DLL3 were examined.No positive result was found in association analysis of single locus based on allele and genotype between case group and control group.Logistic regression analysis of single locus also showed negative result between these two groups.No positive result was found in haplotype association analysis and logistic regression analysis of multiple loci.The further stratification analysis based on the clinical phenotypes also showed no positive result.These results suggest the genetic polymorphisms of five loci are not associated with the susceptibility of CS.For Chinese Han population,the genetic variants of DLL3 gene might not play a role in the onset of CS.●Five loci in DVL2 were examined.No positive result was found in association analysis of single locus based on allele and genotype between case group and control group.Logistic regression analysis of single locus also showed negative result between these two groups.No positive result was found in haplotype association analysis and logistic regression analysis of multiple loci.The further stratification analysis based on the clinical phenotypes also showed no positive result.These results suggest the genetic polymorphisms of five loci are not associated with the susceptibility of CS.For Chinese Han population,the genetic variants of DVL2 gene might not play a role in the onset of CS.●Three loci in HES7 were examined.No positive result was found in association analysis of single locus based on allele and genotype between case group and control group.Logistic regression analysis of single locus also showed negative result between these two groups.In the haplotype association analysis,significant difference(x~2=3.876,P=0.049) was found in ht2-GA between two groups. However,no positive result was found in the further haplotype logistic regression analysis.In the further stratification analysis based on clinical phenotypes, significant differences were found in HES7-rs1442849 and HES7-rs3027279 between CS with segmentation failure group and control group in allele association analysis.However,no positive result was found in genotype association analysis and single locus logistic regression analysis between these two groups.When the analysis was performed between CS with thoracic malformation group and control group,significant differences were found in HES7-rs1442849 and HES7-rs3027279 in allele association analysis,and HES7-rs3027279 in genotype association analysis.In the single locus logistic regression analysis,A/G-A/A was found to be a risk factor compared to G/G (OR=1.74,95%CI=1.05-2.86,P=0.029).In the haplotype analysis of the LD block comprised by HES7-rs1442849 and HES7-rs3027279,significant differences were found in ht1-AA and ht2-GA between CS with thoracic malformation group and control group.These results suggest that HES7 gene might be predisposing gene for CS with segmentation failure and CS with thoracic malformation.●Thirteen loci in LMX1A were examined.No positive result was found in association analysis of single locus based on allele and genotype between case group and control group.Logistic regression analysis of single locus showed some positive results.For LMX1A-rs1354510 and LMX1A-rs16841013,A/G was a protective factor compared with G/G-A/A(OR=0.58,95%CI=0.37-0.92,P=0.02; OR=0.57,95%CI=0.36-0.91,P=0.018).The further stratification analysis was performed based on the clinical phenotype.The analysis between CS with formation failure group and control group showed significant differences in three loci,including LMX1A-rs6671290(x~2=7.839,P=0.019),LMX1A-rs1354510 (x~2=10.015,P=0.007),LMX1A-rs16841013(x~2=10.350,P=0.006).Single locus logistic regression analysis demonstrated that A/G was a protective factor compared with G/G-A/A in LMX1A-rs4657412(OR=0.57,95%CI=0.33-0.96, P=0.035),LMX1A-rs6671290(OR=0.66,95%CI=0.37-1.18,P=0.025), LMX1A-rs1354510(OR=0.42,95%CI=0.25-0.73,P=0.0017),LMX1A-rs16841013 (R=0.42,95%CI=0.24-0.73,P=0.0016),and A/A was a risky factor in LMX1A-rs6671290(OR=2.10,95%CI=0.81-5.41,P=0.025).No positive result was found in haplotype association analysis and logistic regression analysis of multiple loci.The analysis between CS with thoracic malformation group and control group showed that A/G was a risky factor compared with G/G-A/A in LMX1A-rs1354510(OR=0.53,95%CI=0.30-0.93,P=0.025) and LMX1A-rs16841013(OR=0.52,95%CI=0.30-0.93,P=0.022).The haplotype association analysis and logistic regression analysis between these two groups showed no positive result.These results suggest that LMX1A might be the predisposing gene for CS with formation failure and CS with rib malformation.●Eight loci in MYLK were examined.No positive result was found in association analysis of single locus based on allele and genotype between case group and control group.Logistic regression analysis of single locus also showed negative result between these two groups.No positive result was found in haplotype association analysis and logistic regression analysis of multiple loci.The further stratification analysis based on the clinical phenotypes showed the genotype difference between CS with lumbar malformation group and control group was significant in MYLK-rs13080634(x~2=80.073,P=0.000) and MYLK-rs12172926 (x~2=12.178,P=0.000).Single locus logistic regression analysis showed A/C was a protective factor compared with A/A-C/C in MYLK-rs3732487(OR=0.47, 95%CI=0.23-0.96,P=0.035) between these two groups.No positive result was found in the association analysis and logistic regression analysis of multiple loci. These results suggest that MYLK gene might play a role in the onset of CS with lumbar malformation.●Five loci in NOTCH1 were examined.No positive result was found in association analysis of single locus based on allele and genotype between case group and control group.Logistic regression analysis of single locus also showed negative result between these two groups.No positive result was found in haplotype association analysis and logistic regression analysis of multiple loci.The further stratification analysis based on the clinical phenotypes of also showed no positive result.These results suggest the genetic polymorphisms of five loci are not associated with the susceptibility of CS.For Chinese Han population,the genetic variants of NOTCH1 gene might not play a role in the onset of CS.●Three loci in PAX1 were examined.No positive result was found in association analysis of single locus based on allele and genotype between case group and control group.Logistic regression analysis of single locus also showed negative result between these two groups.No positive result was found in haplotype association analysis and logistic regression analysis of multiple loci.The further stratification analysis based on the clinical phenotypes also showed no positive result.These results suggest the genetic polymorphisms of six loci are not associated with the susceptibility of CS.For Chinese Han population,the genetic variants of PAX1 gene might not play a role in the onset of CS.●Three loci in SIM2 were examined.No positive result was found in association analysis of single locus based on allele and genotype between case group and control group.Logistic regression analysis of single locus also showed negative result between these two groups.No positive result was found in haplotype association analysis and logistic regression analysis of multiple loci.The further stratification analysis based on the clinical phenotypes also showed no positive result.These results suggest the genetic polymorphisms of three loci are not associated with the susceptibility of CS.For Chinese Han population,the genetic variants of SIM2 gene might not play a role in the onset of CS.●Two loci in TBX6 were examined.Association analysis of single locus based on allele and genotype between case group and control group showed the genotype difference between these two groups was significant in TBX6-rs3809624 (x~2=6.836,P=0.033).Logistic regression analysis of single locus showed A/G-A/A was a risky factor compared with G/G in TBX6-rs3809624(OR=1.89, 95%CI=1.14-3.11,P=0.012).The further stratification analysis based on the clinical phenotype showed the genotype difference was significant between CS with segmentation failure group and control group in TBX6-rs3809624(x~2=7.780, P=0.020).The logistic regression analysis between these two groups demonstrated that A/C was a risky factor compared with C/C-A/A in TBX6-rs8060511 (OR=1.74,95%CI=1.06-2.87,P=0.029),A/G-A/A was a risky factor compared with G/G in TBX6-rs3809624(OR=2.03,95%CI=1.20-3.43,P=0.008).However, the haplotype association analysis and logistic regression analysis showed no positive result.The analysis between CS without rib malformation group and control group showed that the genotype difference was significant in TBX6-rs3809624(x~2=8.263,P=0.016),and A/G-A/A was a risky factor compared with G/G in TBX6-rs3809624(OR=2.25,95%CI=1.25-4.08,P=0.0072).The haplotype analysis showed the difference of ht1-CG in the TBX6 block between these two groups was significant(x~2=7.379,P=0.007).The logistic regression analysis between CS with rib malformation group and control group showed A/G was a risky factor compared with G/G-A/A in TBX6-rs3809624(OR=1.80, 95%CI=1.02-3.18,P=0.041).The comparison between CS with thoracic malformation group and control group demonstrated that the genotype difference was significant(x~2=6.642,P=0.036) in TBX6-rs3809624.A/G-A/A was a risky factor compared with G/G in TBX6-rs3809624(OR=1.88,95%CI=1.12-3.14, P=0.015).The haplotype association analysis and logistic regression analysis showed no positive result.All these results suggest TBX6 gene might be the predisposing gene in Chinese Han population.The further analysis indicates that TBX6 gene might be associated with CS with segmentation failure,CS without rib malformation,CS with rib malformation,and CS with thoracic malformation.●Seventeen loci in WNT3A were examined.No positive result was found in association analysis of single locus based on allele and genotype between case group and control group.Single locus logistic regression analysis showed A/A was a protective factor compared with G/G-A/G in WNT3A-rs6672559(OR=0.43, 95%CI=0.20-0.90,P=0.021).The further stratification analysis based on the clinical phenotype showed the genotype difference between CS with segmentation group and control group was significant in WNT3A-rs6672559(x~2=6.104, P=0.047).The logistic regression analysis showed A/A was a protective factor compared with G/G-A/G in WNT3A-rs6672559(OR=0.40,95%CI=0.18-0.86, P=0.016).The analysis between CS without rib malformation group and control group showed that there was significant difference in genotype of WNT3A-rs6672559,and A/A was a protective factor compared with G/G-A/G in WNT3A-rs6672559.The analysis between CS with rib malformation group and control group showed that A/G was a protective factor compared with G/G-A/A in WNT3A-rs766972(OR=0.53,95%CI=0.30-0.94,P=0.031).The analysis between CS with thoracic malformation group and control group showed that A/A was a protective factor compared with G/G-A/G in WNT3A-rs6672559(OR=0.41, 95%CI=0.19-0.88,P=0.018).These analysis results suggest that the genetic variants of WNT3A might be associated with the onset of CS.The further analysis showed that WNT3A gene might be associated with CS with segmentation failure, CS without rib malformation,CS with rib malformation and CS with thoracic malformation. Conclusion●Genetic polymorphisms of HES7,LMX1A,TBX6 and WNT3A genes are associated with CS and may play an important role in mediating susceptibility to developing CS in a Chinese Han population.●HES7 gene might be the predisposing gene of CS with segmentation failure and CS with thoracic malformation.LMX1A gene might be the predisposing gene of CS with formation failure and CS with rib malformation.TBX6 gene and WNT3A gene might be the predisposing genes of CS without rib malformation,CS with rib malformation and CS with thoracic malformation.●It is suggested genetic polymorphisms of DLL3,DVL2,MYLK,NOTCH1,PAX1, SIM2 may not be associated with the susceptibility to CS in a Chinese Han population.