【作者】 原所茂；

【导师】 邱贵兴； 翁习生； 王以朋；

【作者基本信息】 中国协和医科大学 ， 外科学， 2009， 博士

【摘要】 研究背景先天性脊柱侧凸(Congenital scoliosis,CS)是胚胎期脊椎发育异常而引起的脊柱侧凸,可在脊柱冠状面、矢状面和水平面引起侧凸、后凸和旋转畸形。在脊柱发育的胚胎期,形成一种重要的过渡性组织-体节(somite),体节决定中轴骨的形成,已经证明体节发育受影响可导致脊椎畸形,包括脊椎形成障碍和脊椎分节不良。脊椎畸形目前确切的病因尚不清楚,动物实验表明发育相关基因的表达异常可导致脊椎畸形,而射线、药物或中毒也可诱导脊椎畸形。近年来,基因突变鼠和基因敲除鼠被广泛应用于基因在发育过程中功能作用的研究,人们逐渐了解了单一基因功能障碍可导致的发育异常。大量的动物实验均显示了多种基因在体节发育中起到重要作用,若其表达异常,则可导致明显的脊椎畸形,因此目前CS的遗传病学研究引起了越来越多学者的重视。目前的研究支持CS是一种多基因遗传病,不同的基因异常可导致不同的表型。目前对于人类CS候选基因的研究尚处于初始的阶段,在国内尚是空白。近20年来对小鼠和鸡胚胎发育的研究使我们对脊椎发育的分子胚胎学信息有了越来越多的了解。对于物种进化而言,不同物种间在核苷酸序列和氨基酸序列上有较高的保守性。我们可以通过系统回顾小鼠基因组数据库就可以找到那些与脊椎畸形相关的基因,再通过基因图的位置以同线性保守基因序列为基础进一步确立人类CS可能的候选基因。研究目的●通过对10个候选基因SNPs的筛查,探索候选基因与人类CS之间的关联,依据实验结果提出可能的病因学假说●通过对候选基因SNPs基因型与CS临床表型的关联分析,探索可能的致病基因,从遗传学角度解释CS临床表型●从基因水平探索候选基因与CS及CS临床表型之间的关系研究方法本研究采用病例-对照研究设计。●研究对象:根据入选和排除标准入选2005年10月-2008年5月期间在北京协和医院脊柱中心收治且确诊的154例中国汉族先天性脊柱侧凸(Cs)患者和144例非先天性脊柱侧凸的对照组。●研究方法:(?) QIAamp DNA Blood Mini Kit试剂盒提取全血DNA(?)在NCBI-SNPs数据库(http://www.ncbi.nlm.nih.gov/SNP)中查找上述10个基因中已知的SNPs。优先选取杂合度高于10%,位于外显子或有错义突变的SNPs和位于3’-和5’-调控区的SNPs作为遗传标志,共选取了75个SNPs。(?)将病例组根据脊椎畸形临床特点、脊椎畸形部位、是否合并肋骨畸形将病例组进行分层。(?)所有研究对象应用VeraCode GoldenGate Genotyping Assay对所选的SNPs进行基因型鉴定。(?)拟和优度x~2检验(Goodness-of-fit Chi-square test)分析病例、对照组基因型频率的分布是否符合H-W平衡。(?)对单个位点行基于基因型/等位基因频率的关联分析和基于基因型频率的Logistic回归分析,评估单个位点与CS发病风险的相关性。(?)应用Haploview和在线软件SNPstats对单个基因内的多个位点进行连锁不平衡分析、单倍型关联分析以及非条件Logistic回归分析,评估单倍型与CS发生风险的相关性。研究结果●我们共检测了10个基因的75个位点,最终有11个位点被放弃,其中8个位点是因为检出率较低,无法进行进一步的分析,另外3个位点是由于检测结果显示该位点无多态性而放弃,其余65个位点检出率均＞99.3%。●对照组中有2个基因共4个SNP位点的H-W平衡检验时P值＜0.05,不符合H-W平衡,它们是DLL3-rs2304222,MYLK-rs1254392,MYLK-rs820463,MYLK-rs9422。病例组中有3个基因共7个SNP位点的P值＜0.05,不符合H-W平衡,它们是LMX1A-rs12029324,LMX1A-rs12023709,LMX1A-rs1819768,LMX1A-rs16841013,MYLK-rs9422,TBX6-rs3809624。●DLL3、DVL2、HES7、LMX1A、MYLK、TBX6、WNT3A等7个基因内的位点之间有连锁不平衡关系。DLL3有四个位点(rs2304223、rs1110627、rs2304214和rs3212276)存在连锁不平衡关系,组成连锁不平衡block。DVL2的rs2074222、rs222837、rs222836、rs2074216四个位点组成连锁不平衡block。HES7的rs1442849和rs1348325两个位点组成连锁不平衡block。LMX1A内发现了有三个连锁不平衡block,分别是rs1819768、rs12023709、rs16840972、rs16841013四个位点组成的block 1,rs6671290、rs16841029和rs1354510三个位点组成的block 2,rs4657412、rs6667188、rs1532815三个位点组成的block 3。MYLK的rs12172926、rs3732487、rs1350152三个位点组成连锁不平衡block。TBX6的rs8060511和rs3809624两个位点组成连锁不平衡block。WNT3A内发现有两个连锁不平衡block,分别是rs708114、rs6426490、rs6672559、rs3094913、rs881398、rs708121、rs3094911、rs3094912、rs708122、rs10916258十个位点组成的block 1,rs7539664、rs10916262、rs11589513、rs6675092、rs4653533五个位点组成的block 2。●共筛选了DLL3的5个SNP位点:rs2304222,rs2304214,rs2304223,rs3212276,rs1110627。在对病例组和对照组行基于等位基因和基因型的单个位点关联分析中我们并没有得到与这5个位点相关的阳性结果;在对单个位点的Logistic回归分析中也未发现阳性位点;对DLL3中多个位点进行单倍型关联分析和Logistic回归分析,均未发现阳性结果。在对病例组的临床表型的进一步分层分析中也没有发现阳性结果。这说明这5个位点的遗传多态性和CS易感性无关,对中国汉族人群来说,DLL3基因的遗传变异可能不是引起CS发病的主要因素。●共筛选了DVL2的5个SNP位点:rs222850,rs222837,rs222836,rs2074216,rs2074222。在对病例组和对照组行基于等位基因和基因型的单个位点关联分析中我们并没有得到与这5个位点相关的阳性结果;在对单个位点的Logistic回归分析中也未发现阳性位点;对DVL2中多个位点进行单倍型关联分析和Logistic回归分析,均未发现阳性结果。在对病例组的临床表型的进一步分层分析中也没有发现阳性结果,这说明这5个位点的遗传多态性和CS易感性无关,对中国汉族人群来说,DVL2基因的遗传变异可能不是引起CS发病的主要因素。●共筛选了HES7的3个SNP位点:rs1442849,rs3027279,rs1348325。在对病例组和对照组行基于等位基因和基因型的单个位点关联分析中我们并没有得到与这3个位点相关的阳性结果;在对单个位点的Logistic回归分析中也未发现阳性位点;对HES7中多个位点进行单倍型关联分析发现,HEST-rs1442849和HES7-rs1348325所组成的连锁不平衡块中,ht2-GA在两组中存在显著性差异(x~2=3.876,P=0.049);但是在进一步的单倍型Logistic回归分析中未发现阳性结果。在对病例组的临床表型的进一步分层分析中发现,HES7-rs1442849和HES7-rs3027279两个位点在有分节障碍的CS组和对照组间比较时,等位基因在两组间存在显著性差异(x~2=4.472,P=0.035;x~2=4.786,P=0.029);但在基因型关联分析中这两组间无阳性结果发现;对这两组行单个位点Logistic回归分析未见阳性结果;在进一步的单倍型关联分析和Logistic回归分析中也无阳性结果发现。在对胸椎畸形的CS组和对照组进行比较时发现,HES7-rs1442849和HES7-rs3027279两个位点的等位基因在两组间存在显著性差异(x~2=4.211,P=0.040;x~2=4.781,P=0.029);HES7-rs3027279位点的基因型在两组间也存在显著性差异(x~2=5.979,P=0.049);在对单个位点Logistic回归分析时发现HES7-rs1442849位点的A/G-A/A相对于G/G来说是风险因素(OR=1.74,95%CI=1.05-2.86,P=0.029);在对HES7-rs1442849和HES7-rs3027279两个位点组成的连锁不平衡块行关联分析时发现,htl-AA和ht2-GA在两组中均存在显著性差异(x~2=4.211,p=0.0402;x~2=5.242,P=0.022)。这些结果提示HES7基因对于有分节障碍和有胸椎畸形的先天性脊柱侧凸发病来说是易感基因。●共筛选了LMX1A的13个SNP位点:rs4657412,rs12029324,rs12023709,rs12040140,rs6667188,rs1532815,rs1819768,rs6671290,rs16841029,rs4656435,rs1354510,rs16841013,rs16840972。在对病例组和对照组行基于等位基因和基因型的单个位点关联分析中我们并没有得到与这15个位点相关的阳性结果;在对单个位点行Logistic回归分析发现,LMX1A-rs1354510的A/G相对于G/G-A/A为保护性因素(OR=0.58,95%CI=0.37-0.92,P=0.02),LMX1A-rs16841013的A/G相对于G/G-A/A为保护性因素(OR=0.57,95%CI=0.36-0.91,P=0.018);在对多位点组成的单倍型行关联分析和Logistic回归分析时未发现阳性结果。在对病例组的临床表型的进一步分层分析中发现,在对有形成障碍的CS组和对照组两组进行比较时,有三个位点的基因型在两组间存在显著性差异,这三个位点分别是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);进一步对这两组行单个位点Logistic回归分析发现,LMX1A-rs4657412的A/G相对于G/G-A/A为保护性因素(OR=0.57,95%CI=0.33-0.96,P=0.035),LMX1A-rs6671290的A/G相对于G/G为保护性因素(OR=0.66,95%CI=0.37-1.18,P=0.025),而A/A为危险性因素(OR=2.10,95%CI=0.81-5.41,P=0.025),LMX1A-rs1354510的A/G相对于G/G-A/A为保护性因素(OR=0.42,95%CI=0.25-0.73,P=0.0017),LMX1A-rs16841013的A/G相对于G/G-A/A,为保护性因素(R=0.42,95%CI=0.24-0.73,P=0.0016);在对多位点组成的单倍型行关联分析和Logistic回归分析时无阳性发现。在对有肋骨畸形的CS组和对照组进行分析时发现,单个位点Logistic回归分析示LMX1A-rs1354510的A/G相对于A/A-G/G为保护性因素(OR=0.53,95%CI=0.30-0.93,P=0.025),LMX1A-rs16841013的A/G相对于G/G-A/A为保护性因素(OR=0.52,95%CI=0.30-0.93,P=0.022);而对多位点组成的单倍型行关联分析和Logistic回归分析时无阳性发现。以上结果说明,LMX1A可能是有形成障碍的先天性脊柱侧凸和有肋骨畸形的先天性脊柱侧凸的易感基因。●共筛选了MYLK的8个SNP位点:rs1254392,rs3732487,rs40305,rs1350152,rs820463,rs9422,rs13080634,rs12172926。在对病例组和对照组行基于等位基因和基因型的单个位点关联分析中我们并没有得到与这8个位点相关的阳性结果;在对单个位点的Logistic回归分析中也未发现阳性位点;对MYLK中多个位点进行单倍型关联分析和Logistic回归分析,均未发现阳性结果。在对病例组的临床表型的进一步分层分析中发现,在对腰椎畸形的CS组和对照组进行比较时,两个位点的基因型在两组间存在显著性差异,这两个位点是MYLK-rs13080634(x~2=80.073,P=0.000),MYLK-rs12172926(x~2=12.178,P=0.000);对这两组行单个位点Logistic回归分析示MYLK-rs3732487的A/C相对于A/A-C/C,为保护性因素(OR=0.47,95%CI=0.23-0.96,P=0.035);在对多位点单倍型的关联分析和Logistic回归分析时无阳性结果。这说明MYLK基因可能是腰椎畸形的先天性侧凸发病的易感基因。●共筛选了NOTCH1的5个SNP位点:rs2229971,rs10521,rs2229974,rs6563,rs4489420。在对病例组和对照组行基于等位基因和基因型的单个位点关联分析中我们并没有得到与这5个位点相关的阳性结果,对单个位点Logistic回归分析、多位点单倍型关联分析和Logistic回归分析时也无阳性结果。在对病例组的临床表型的进一步分层分析中也没有发现阳性结果,这说明这5个位点的遗传多态性和CS易感性无关,对中国汉族人群来说,NOTCH1基因的遗传变异可能不是引起CS发病的主要因素。●共筛选了PAX1的3个SNP位点:rs17861031,rs6047590,rs6035934。在对病例组和对照组行基于等位基因和基因型的单个位点关联分析中我们并没有得到与这3个位点相关的阳性结果,对单个位点Logistic回归分析、多位点单倍型关联分析和Logistic回归分析时也无阳性结果。在对病例组的临床表型的进一步分层分析中也没有发现阳性结果,这说明这3个位点的遗传多态性和CS易感性无关,对中国汉族人群来说,PAX1基因的遗传变异可能不是引起CS发病的主要因素●共筛选了SIM2的3个SNP位点:rs2073416,rs2051397,rs2073601。在对病例组和对照组行基于等位基因和基因型的单个位点关联分析中我们并没有得到与这3个位点相关的阳性结果,对单个位点Logistic回归分析、多位点单倍型关联分析和Logistie回归分析时也无阳性结果。在对病例组的临床表型的进一步分层分析中也没有发现阳性结果,这说明这3个位点的遗传多态性和CS易感性无关,对中国汉族人群来说,SIM2基因的遗传变异可能不是引起CS发病的主要因素。●筛选了TBX6基因的2个SNP位点:rs8060511和rs3809624。在对病例组和对照组行基于等位基因和基因型的单个位点关联分析中发现,TBX6-rs3809624位点的三种基因型构成在病例组和对照组间存在显著性差异(x~2=6.836,P=0.033);进一步的单个位点Logistic回归分析可以看出相对于TBX6-rs3809624的G/G基因型,A/G-A/A为危险性因素(OR=1.89,95%CI=1.14-3.11,P=0.012);在对多位点单倍型行关联分析和Logistic回归分析没有发现阳性结果。在对病例组的临床表型的进一步分层分析中,我们发现在对有分节障碍的CS组和对照组进行比较分析时,TBX6-rs3809624的基因型在两组间存在显著性差异(x~2=7.780,P=0.020);对这两组的单个位点行Logistic回归分析时发现,TBX6-rs8060511的A/C相对于C/C-A/A为危险性因素(OR=1.74,95%CI=1.06-2.87,P=0.029),TBX6-rs3809624的A/G-A/A相对于G/G为危险性因素(OR=2.03,95%CI=1.20-3.43,P=0.008);而在对多位点单倍型关联分析和Logistic回归分析时未发现阳性结果。在对无肋骨畸形的CS组和对照组进行分析时发现,TBX6-rs3809624的基因型在两组间存在显著性差异(x~2=8.263,P=0.016),行单个位点Logistic回归分析示TBX6-rs3809624的A/G-A/A相对于G/G为危险性因素(OR=2.25,95%CI=1.25-4.08,P=0.0072);进一步行多位点单倍型关联分析发现TBX6内的Block中的htl-CG在两组间有显著性差异(x~2=7.379,P=0.007)。在对有肋骨畸形的CS组和对照组进行单个位点Logistic回归分析时发现,TBX6-rs3809624的A/G相对于G/G-A/A,为危险性因素(OR=1.80,95%CI=1.02-3.18,P=0.041)。在对有胸椎畸形的CS组和对照组进行比较时发现,TBX6-rs3809624位点的基因型在两组间存在显著性差异(x~2=6.642,P=0.036);对这两组行单个位点Logistic回归分析示TBX6-rs3809624的A/G-A/A相对于G/G,为危险性因素(OR=1.88,95%CI=1.12-3.14,P=0.015);在对多位点单倍型行关联分析和Logistic回归分析时未发现阳性结果。以上实验结果提示TBX6基因是中国汉族人群先天性脊柱侧凸的易感基因,进一步的分析说明TBX6基因对有分节障碍的先天性脊柱侧凸、无肋骨畸形的先天性脊柱侧凸、有肋骨畸形的先天性脊柱侧凸以及有胸椎畸形的先天性脊柱侧凸均有关联。●共筛选了WNT3A基因中的17个SNP位点。在对病例组和对照组行基于等位基因和基因型的单个位点关联分析中我们并没有得到与这17个位点相关的阳性结果;对单个位点Logistic回归分析时发现,WNT3A-rs6672559的A/A相对于G/G-A/G为保护性因素(OR=0.43,95%CI=0.20-0.90,P=0.021);对多位点单倍型关联分析和Logistic回归分析时未发现阳性结果。在对病例组的临床表型的进一步分层分析中发现,在对有分节障碍的CS组和对照组进行比较分析时发现,WNT3A-rs6672559位点的基因型在两组间存在显著性差异(x~2=6.104,P=0.047);单个位点Logistic回归分析示WNT3A-rs6672559的A/A相对于G/G-A/G为保护性因素(OR=0.40,95%CI=0.18-0.86,P=0.016)。在对无肋骨畸形的CS组和对照组的分析中发现,WNT3A-rs6675092和WNT3A-rs6672559位点的基因型在两组间存在显著性差异(x~2=5.374,P=0.048;x~2=6.900,P=0.032);单个位点Logistic回归分析示WNT3A-rs6672559的A/A相对于G/G-A/G为保护性因素(OR=0.35,95%CI=0.15-0.83,P=0.016)。在对有肋骨畸形的CS组和对照组进行分析时发现,单个位点Logistic回归分析示WNT3A-rs766972的A/G相对于G/G-A/A为保护性因素(OR=0.53,95%CI=0.30-0.94,P=0.031)。在对胸椎畸形的CS组和对照组进行分析时发现,WNT3A-rs6672559的A/A相对于G/G-A/G为保护性因素(OR=0.41,95%CI=0.19-0.88,P=0.018)。这些分析结果说明WNT3A遗传多态性和CS易感性无关,对中国汉族人群来说,WNT3A基因的遗传变异可能与CS发病相关联,进一步的分析说明WNT3A基因对无肋骨畸形的先天性脊柱侧凸、有肋骨畸形的先天性脊柱侧凸以及有胸椎畸形的先天性脊柱侧凸均有关联。结论●在中国汉族人群中HES7、LMX1A、TBX6和WNT3A基因多态性与先天性脊柱侧凸发病相关,有可能是决定CS个体遗传易感性的重要因素。●HES7基因对于有分节障碍和有胸椎畸形的先天性脊柱侧凸发病来说是易感基因;LMX1A可能是有形成障碍的先天性脊柱侧凸和有肋骨畸形的先天性脊柱侧凸的易感基因;TBX6基因可能是无肋骨畸形的先天性脊柱侧凸、有肋骨畸形的先天性脊柱侧凸以及有胸椎畸形的先天性脊柱侧凸的易感基因;WNT3A基因可能是有分节障碍的先天性脊柱侧凸、无肋骨畸形的先天性脊柱侧凸、有肋骨畸形的先天性脊柱侧凸以及有胸椎畸形的先天性脊柱侧凸的易感基因。●在中国汉族人群中DLL3、DVL2、MYLK、NOTCH1、PAX1、SIM2基因可能与先天性脊柱侧凸的发病不相关。更多还原

【Abstract】 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.更多还原