The Association Study Of PAX1/SIM2/WNT3A/TBX6/DVL2/LMX1A Genes Polymorphisms With Susceptibility To Congenital Scoliosis In A Chinese Han Population

BackgroundCongenital scoliosis(CS) is defined as a lateral curvature of the spine due to a developmental abnormality,which arise from defects in the development of the axial skeleton.During embryogenesis,the axial skeleton is formed by a process called somitogcnesis,which produces transient segments of tissue known as somites. Disruptions in somitogenesis have been shown to result in vertebral malformations, including uneven segments(hemivertebrae and wedge vertebrae),fused segments (block vertebrae),and problems in midline fusion(butterfly vertebrac).Vertebral defects can arise from the disruption of genes involved in development. environmental insults during gestation,or a combination of these two factors.As the development of genetics and molecular biology in recent years,several preliminary studies about candidate gene contributed to vertebral malformation in human were reported and the genetic etiology hypothesis of CS has caused more and more interests.More evidences suggest that CS may be multigenetic disease.The interaction of environmental factors and the genes that play a role in regulation of somite segmentation,is thought to be disrupted in congenital vertebral deformities such as congenital scoliosis.A multigenic mechanism could be responsible for the type and spacial identity associated with a particular type of congenital scoliosis.Within the past two decades a great deal of information has been learned about the molecular embryology of spine development through the study of mouse and chick embryos.Human and mouse genes share high degree of homology.due the relatively recent evolutionary divergence of these mammals.By studying what happens to the axial skeleton when these genetic instructions are disrupted in the mouse,we have learned a great deal about the most important genes in spinal development.To date,some abnormalities of genes involved in mouse somitogenesis have been found to cause human spinal deformities.Synteny conservation was used as the basis to identify potential human candidate genes by map position.Giampietro therefore undertook a systematic review of mouse mutations with skeletal,tail.or neuromuscular phenotypes and identified 27 potential human candidate genes sites for CS in 2003.Seven of these genes including PAXI,DLL3,WNT3A,MYLK,LMXIA,FBN2 and SIM2 are briefly discussed.Through latest mouse genome database,TBX6 and DVL2 genes were also considered as candidate genes contributed to CS.At present,the etiology of congenital scoliosis is still unknown,no molecular analyses and few clinical genetic studies have been reported.ObjectsTo identify the relationship between candidate genes(PAXI,SIM2,WNT3A,TBX6,DVL2,LMXIA,DLL3)associated with somite developmental pathway and CS with SNPs in Chinese Han population.■To identify the relationship between the genotypes of SNPs and the clinical phenotypes of CS.■To explore possible etiologic hypothesis contributed to the development of CS and the different clinical phenotypes of CS from gene level.Methods■A hospital-based case-control design was applied in this study.■A total of 127 patients(55 boys,72 girls,mean age 12.90 y/o)diagnosed with clinically confirmed congenital scoliosis admitted in Peking Union Medical College(PUMC) Hospital were enrolled in this study according the inclusion and exclusion criterias between October 2005 and September 2007.The diagnosis of scoliosis-free control subjects(55 boys,72 girls, mean age 13.27y/o) consisted of infection(43%),inflammatory disease (41%),trauma(11%),hypersensitivity disease and others(5%) at the same hospital during the same study period.All the control subjects were frequency-matched to the cases on age(±3 years),gender,and nation (Chinese Han).■Genomic DNA was extracted from peripheral blood leukocytes of each subject who had signed informed consent,using QIAamp DNA Blood Mini Kit.■Based on genotype data from the International HapMap project (http://www.Hapmap.org),the tagging single nucleotide polymorphisms (tSNPs) initially were selected using Haploview 4.0 software.The MAF(minor allele frequency) of all selected SNPs were above 10%.The missense or nonsense mutation SNPs in exons,or the SNPs located in promoters,5'UTR or 3'UTR were prefered.■Hardy-Weinberg equilibrium both in control and in case groups were analyzed through Goodness-of fit Chi-square test.■Case group were classified into different clinical phenotypes according to vertebral defect type,the location of deformity,the extent of developmental disruption,combined rib malformations and neural canal deformity.■Genotying of all selected SNPs was done by SNPstream technology (Beckman Couher SNPstream) which is the combination of the fidelity polymerase mediate reaction,single base primer extension and microarray methods.As a high throughput genotyping system,SNPstream technology has been widely used in biochemistry and genetic research.■All the data of 20 SNPs with polymorphism are analyzed by the association analysis based on a single SNP,the association analysis between phenotypes and SNPs.■Odds radios(ORs) and 95%confidence intervals(CIs) were computed by the unconditional logistic regression to estimate the relative risk for the single locus genotypes using online software—SNPstats.■And pairwise linkage disequilibrium values D' and r~2 were calculated in the control population using Haploview 4.0 software.Haplotype frequencies were estimated and difference in haplotype distributions between cases and controls were assessed using UNPAHSED software.Results■There were no significant difference of average age and sex proportion between case group and control group(p＞0.05).■We failured in designing premier of three tSNPs of DLL3 and discasded the gene in this study.Eventually 20 common SNPs of PAXI.SIM2,WNT3A, TBX6,DVL2,and LMXIA genes were chosen to be tagging SNPs and all SNPs have polymorphism.■SNP1(rs17861031) and SNP2(rs6047590) of PAXI gene were genotyped and both polymorphisms were in Hardy-Weinberg equilibrium both in control and in ease groups.Both polymorphisms were not in linkage disequilibrium.No association is obseiwed between SNP1 and SNP2 genotypes/allele polymorphisms and risk of CS.■Three SNPs including SNP3(rs2073601),SNP4(rs 2073417) and SNP5 (rs 2051307)of SIM2 gene were genotyped and both polymorphisms of SNP3 and SNP5 were in Hardy-Weinberg equilibrium both in control and in case groups.All polymorphisms were not in linkage disequilibrium.No association is observed between SNP3.SNP4 and SNP5 genotypes/allele polymorphisms and risk of CS.■Two SNPs including SNP6(rs964941) and SNP7(rs752107) of WNT3A gent were genotyped and both polymorphisms were in Hardy-Weinberg equilibrium both in control and in case groups.Both polymorphisms were not in linkage disequilibrium.No association is observed between SNP6 and SNP7 genotypes/allele polymorphisms and risk of CS.■Two SNPs including SNP8(rs2289292) and SNP9(rs3809624) of TBX6 gene were genotyped.Both polymorphisms were in Hardy-Weinberg equilibrium both in control group,but not in case group.The minor allele frequencies in cases/controls were,respectively,as follows:SNP8C=108 (0.43)/135(0.53).SNP8T=146(0.57) /119(0.47),SNP9C=144 0.57)/120(0.47).SNP9T=110(0.43) /134(0.53).The single locus analysis revealed the allele frequency distributions of SNP8 and SNP9 were statistically significantly different between case patients and control subjects(P=0.017 and P=0.033).In the unconditional logistic regression analysis,after adjustment for age and gender,SNP8 showed significant difference in both dominant(OR=0.56:95%CI=0.33-0.96) and log-additive (OR=0.69;95%CI=0.50-0.97) models,and the P-values were 0.03 and 0.029,respectively.The log-additive model was accepted as the best inheritance model because of the smaller AIC(Akaike information) value (354.9).Both polymorphisms were in full linkage disequilibrium.In the haplotype analysis,we got 2 positive haplotypes:SNP08T-SNP09C and SNP08C-SNP09T■Five SNPs including SNP10(rs2074222),SNP11(rs222837),SNP12 (rs222835),SNP13(rs10671352) and SNP14(rs222836) of DVL2 gene were selected to be tagging SNPs.All polymorphisms were in Hardy-Weinberg equilibrium both in control and in case groups.All polymorphisms were in strong linkage disequilibrium(D'=1;r~2＞0.5).No association is observed between SNP10.SNP11,SNP12.SNP13 and SNP14 genotypes/haplotype polymorphisms and risk of CS.■Six SNPs including SNP15(rs1819768),SNP16(rs12023709),SNP17 rs16841013),SNP18(rs4656435),SNP19(rs4657412) and SNP20 rs4657411) of LMXIA gene were selected to be tagging SNPs.All polymorphisms were in Hardy-Weinberg equilibrium both in control and in case groups.In the association analysis between the genotypes of SNPs and CS,we got two positive SNPs:SNP15 and SNP16.The minor genotype frequencies in cases/controls were,respectively,as follows:SNP15AA=7 (0.16)/17(0.13).SNP15AC=62(0.49) /45(0.35),SNP15CC= 58(0.46)/65(0.51),SNP16AA=58(0.46)/65(0.51).SNP16AG =62(0.49)/45(0.35),SNP16GG=7(0.16)/17(0.13).The single locus analysis revealed the genotype distributions of SNP15 and SNP16 were statistically significantly different between case patients and control subjects(P=0.026 and P=0.026).In the unconditional logistic regression analysis,after adjustment for age and gender,SNP15 and SNP16 both showed significant difference in both Ressessive model(OR=0.38; 95%CI=0.15-0.94) and Overdominant model(OR=1.73;95%Cl=1.05-2.8), and the P-values were 0.029 and 0.032.respectively.The Resscssive model was accepted as the best inheritance model because of the smaller AIC (Akaike information) value(354.9).SNP15,SNP16;SNP17 and SNP20 were in strong linkage disequilibrium.Both SNP18 and SNP19 were also in strong linkage disequilibrium.But no association is observed between all haplotypes polymorphisms and risk of CS.■In the association analysis between the genotyhpes and the phenotype l(according to vertebral defect type) of CS,we got 5 positive SNPs.SNP15, SNP16 and SNP17 polymorphisms of LMXIA gene may related to CS with failure of formation.SNP8 and SNP9 polymorphisms of TBX6 gene may related to CS with a fallure of segmentation.■In the association analysis between the genotyhpes and other phenotypes of CS,we didn't have got any positive SNPs.Conclusion■Genetie variants of TBX6 and LMXIA genes are associated with CS and may play an important role in mediating susceptibility to developing CS in a Chinese Han population.■Genetic variants of different genes may play an important role in mediating susceptibility to different clinical phenotypes of CS in a Chinese Han population.It was suggested that genetic variants of LMXIA gene may related to CS with failure of formation,and genetic variants of TBX6 gone may related to CS with a failure of segmentation.■It was suggested genetic variants of PAX1,SIM2,WNT3A and DVL2 genes may not be associated with the susceptibility to CS and different clinical phenotypes of CS in a Chinese Han population.Further studies are warranted before definitive conclusion can be drawn.