【作者】 赵宁；

【导师】 金春莲；

【作者基本信息】 中国医科大学 ， 遗传学， 2007， 硕士

【摘要】 前言单纯性马蹄内翻足(idiopathic congenital talipes equinovar，ICTEV)是一种出生时即可见的复杂的先天性足畸形。在不同的人群中，ICTEV的发病率有很大差异，在我国约为0.6‰～1‰，男女之比约为2～2.5：1。近年来，国内外对于ICTEV的发病机制争论较多，但均缺乏有力的证据。临床资料和流行病学研究表明，遗传因素在ICTEV的发病过程中发挥重要的作用，但其遗传方式、外显率等均不清楚。目前认为ICTEV主要是在肢芽发育的易损期，由于遗传因素和环境因素的共同作用，某些与肢体发育有关的基因及其产物出现异常的结果。近年来，国内外对ICTEV致病基因的分子水平上的研究很少，研究多集中于对一些合并有类似足部畸形的综合征和多发畸形的研究。这些研究提供了ICTEV致病基因可能的侯选区域：5q15-31.1；6q12-13；7号染色体长臂；17号染色体长臂；10p11.2-15；9号染色体中心粒周围区等，通过生物信息学可知这些区域内存在着一些与骨骼、肌肉、肌腱、神经发育相关的基因，这为我们探寻ICTEV的易感基因提供了有利的线索。目前的研究表明，在ICTEV患侧组织中存在纤维组织明显增加，其中Ⅰ型胶原增加最显著。Ⅰ型胶原是由两条α1链及一条α2链组成的三股螺旋结构。Ⅰ型胶原的α1链和α2链分别由COL1A1和COL1A2基因编码。COL1A1基因和COL1A2基因分别位于17q21.3-q22和7q21.3-q22。目前已发现上百种COL1A1基因突变，主要集中在外显子。COL1A1基因突变可导致多种疾病的发生，主要可引起先天性成骨不全、Ehlers Dahlos综合征(EDS)、骨质疏松症(Osteoporosis)。但是对于COL1A1基因与ICTEV相关性的进一步研究至今仍未见报道。本研究以COL1A1基因为候选基因，将从分子水平上进一步探讨Ⅰ型胶原基因与ICTEV发生的相关性。为了从RNA水平上进一步观察ICTEV中的COL1A1基因表达水平的变化，本研究中我们首先构建了马蹄内翻足大鼠模型，并采用半定量RT-PCR的方法，对后肢发育不同时期的模型胎鼠和正常胎鼠后肢组织中Collal基因的表达水平进行了比较。接下来，我们又采用半定量RT-PCR方法，对20例ICTEV患儿和4例正常儿童的肌肉和肌腱组织中COLlAl基因的表达水平进行了比较。此外，为了探讨COLlAl基因表达水平变化的原因，我们选择了COLlAl基因转录起始点上游-1031bp～+30bp和第1内含子，对84例ICTEV患者进行了突变筛查。材料与方法一、研究对象：Wistar大鼠86只，包括雌鼠65只和雄鼠21只，均购自校动物部。肌肉及肌腱标本来自于20例手术矫正的ICTEV患儿，取4例外伤及尸检患儿相应组织作为正常对照，所有标本均经患者知情同意。84例单纯性马蹄内翻足患者的静脉抗凝血，标本由中国医科大学第二临床医院小儿外科提供。其中男性60例，女性24例(男女之比为2.5：1)；年龄最大者28岁，最小者4个月；单侧马蹄内翻足者51例，双侧马蹄内翻足者33例(双侧者占39.28％)；所有患者均具有典型的临床症状，经X线和手术确诊。100例正常人DNA，研究对象均来自中国东北地区正常汉族人群。二、实验方法1、大鼠模型的构建及半定量RT-PCR检测模型鼠后肢Collal基因的表达(1)构建大鼠模型通过在孕十天给孕鼠灌全反式维甲酸(135mg／kg体重)和矿物油分别获得马蹄内翻足模型胎鼠及正常对照胎鼠。(2)观察Collal基因在动物模型中的表达情况分别取孕15天，17天，19天及21天的实验组和对照组胎鼠的后肢，常规提取总RNA并进行反转录得到cDNA，以β-actin为内对照采用半定量RT-PCR方法研究Collal基因在后肢发育不同阶段的表达情况。2．半定量RT-PCR分析COLlAl基因在患儿及正常对照肌肉、肌腱组织中的表达(1)按常规方法提取患儿及正常对照肌肉、肌腱组织中的总RNA，并进行反转录得到cDNA。(2)以β-actin为内对照采用半定量RT-PCR方法研究COLlAl基因在患儿及正常对照肌肉、肌腱组织中的表达情况。3．PCR-DGGE(1) PCR扩增明确COLlAl基因上游转录调控区和第1内含子的基因序列，并应用Primer5软件设计引物(上海生工合成)，在上游引物的5′端加上40bp的[GC]夹。对84名单纯性马蹄内翻足患者DNA目的片段进行PCR扩增，并用1.5％的琼脂糖凝胶电泳检测。(2) DGGE检测以变性剂浓度范围为20％—80％的聚丙烯酰胺凝胶做水平DGGE，并根据带形及其位置进一步调节变性剂浓度范围及电泳时间；电泳后，下胶，溴化乙锭染色，在紫外灯下观察结果。(3)确定突变位点对电泳带形异常的PCR产物进行DNA测序以确定序列改变的位置与性质，并对正常人DNA的相同位点进行筛查，以判断该碱基置换是突变还是多态。实验结果一、模型胎鼠RT-PCR结果RT-PCR结果显示在模型胎鼠后肢发育的各个时期均存在Collal基因表达的上调。二、人COLlAl基因RT-PCR结果ICTEV患儿肌肉及肌腱组织中COLlAl基因表达水平高于正常组织。三、PCR-DGGE检测结果经DGGE筛查并测序发现一名患者-161bp处存在C→T的杂合突变，另一名患者+274bp处存在C→G的纯合突变。分别经过对100名正常人的同一位点进行筛查，没有发现相同的改变，排除其为多态。经查找人类基因突变数据库(The Human Gene Mutation Database，HGMD)，证实是尚未见报道的新突变。结论1、应用半定量RT-PCR的方法发现在马蹄内翻足模型胎鼠后肢发育的不同时期均存在Collal基因表达的上调，同时发现单纯性马蹄内翻足患儿患侧肌肉及肌腱组织中COLlAl基因表达水平高于正常组织，证明了该基因与马蹄内翻足畸形的发生具有相关性。2、在COLlAl基因-161bp处和+274bp处发现了新突变，提示该基因转录调控序列的突变可能是ICTEV的致病原因之一。更多还原

【Abstract】 ObjectiveIdiopathic congenital talipes equinovarus (ICTEV) is a complex footdeformity presented at birth with 0.6‰～1‰birth rate in Chinese.The cause ofICTEV remains controversial. Many factors as skeleton development anomaly,neuromuscular disease,developmental arrest in uterus and so on seem to beresponsible. It is considered that the genetic factors play an important role in thepathogenesis of ICTEV, and the heritability of ICTEV is about 65%, it is yetunknown about the hereditary patterns and penetrance of ICTEV.Recently, it isbelieved that abnormal expression of some limb development-related genes leadsto ICTEV. At present,researches on human ICTEV mainly focus on manysyndromes with foot malformations,and little advances have been made in theresearches on the susceptibility genes of ICTEV. The researches provid somecandidate regions and genes for ICTEV such as 5q15-31.1,10p11.2-15 and soon,which provide an useful clue for us to look for the candidate genes of ICTEV.Researches indicat that the content of typeⅠcollagen is remarkable increasedin ICTEV.TypeⅠcollagen is the most abundant and widly expressed collagen inhuman. It provides the structural support for the body in skeleton, skin, bloodvessele,nerves,intestines,and in the fibous capsules of organs. TypeⅠcollagen is aheterotrimer of two identicalαl(Ⅰ) chains and one chα2(Ⅰ) chain. Theαl(Ⅰ) andchα2(Ⅰ) chains of typeⅠcollagen are encoded by the unlinked loci COL1A1 andCOL1A2 respectively. COL1A1 and COL1A2 locate in 17q21.3-q22 and7q21.3-q22 respectively.Now, it has discovered hundred of mutations in COL1A1gene.Mutations at these loci result primarily in the connective tissue disorders: osteogenesis imperfecta,Ehler-Danlos syndrome typesⅦA andⅦB,Osteoporosis.But there is no report about association between COL1A1 andICTEV.In our study, we will probe into the association between COL1A1 andICTEV furtherly on molecular level.In our studies,we constructed animal model of talipes equinovarus withWistar rats, and discovered that there was up-regulation of Collal gene expressionin different stage of limb development in the animal model by RT-PCR. Then,wealso discovered that there was up-regulation of COL1A1 gene expression in themusclar and tenden tissues of ICTEV patients by RT-PCR. At the sametime,mutation sites in transcription regular sequences(-1031bp～+30bp) and intron1 of COL1A1 gene were detected in 84 patients with ICTEV by denaturinggradient gel electrophoresis technique.The result was that we found a -161C→Theterozygous mutation and a +274C→G homozygous mutation in COL1A1gene.So,we thought COL1A1 gene was confirmed to be associated with ICTEV.Material and Methods1. Samples86 Wistar rats were bought from the animal department of China MedicalUniversity.Musclar and tenden tissues of 20 patients with ICTEV and correspondingnormal tissues came from the Second Affiliated Hospital, China MedicalUniversity.84 patients with ICTEV came from the Second Affiliated Hospital, ChinaMedical University. All patients had typical manifestation and were confirmed byX-rays and surgical operation. 100 normal DNA come from normal crowd ofnortheastern china.2.Construction of rat model and Study Collal gene in thehindlimbs of rat models with RT-PCR135mg/kg body weight of ATRA was used to construct rat model on day 10 ofgestation, which has shown a good consistency with talipes equinovarusmalformation as that in humans. On day 15、17、19 and 21 of gestation,thehindlimbs were cut from model fetus and normal controls,and total RNA was extracted,then reverse transcribed to cDNA. RT-PCR was used to detecte theexpression of COL1A1.3.Study COL1A1 gene in musclar and tenden tissues of ICTEVwith RT-PCRThe total RNA was extracted from musclar and tenden tissues of patients withICTEV and normal controls,then reverse transcribed to cDNA. RT-PCR was usedto detecte the expression of COL1A1.4.PCR-DGGEMutation sites in transcription regular sequences(-1031bp～+30bp) andintron 1 of COL1A1 gene were detected in 84 patients with ICTEV by denaturinggradient gel electrophoresis technique.Results1.The expression of Collal gene in rat modelsRT-PCR results found the expression of Collal gene up-regulated in differentstage of limb development in rat models with the clubfoot like deformity.2. The expression of COL1A1 gene in muscles and tenden ofpatients with ICTEVRT-PCR results found the expression of COL1A1 gene in musclar and tendentissues of ICTEV is higher than normal tissues.3. Result of PCR-DGGEThe result was that we found a-161C→T heterozygous mutation and a+274C→G homozygous mutation in COL1A1 gene.Conclusions1.RT-PCR results found the expression of Collal gene up-regulated indifferent stage of limb development, and the expression of COL1A1 geneup-regulated in musclar and tenden tissues of patients with ICTEV,whichconfirmed the association between COL1A1 gene and talipes equinovarusdeformation.2. We discovered a-161T→C heterozygous mutation and a +274C→Ghomozygous mutation in COL1A1 gene of patients with ICTEV. It illustrated that the mutation in transcription regular sequences could caused ICTEV.更多还原