【作者】 王谦；

【导师】 金春莲；

【作者基本信息】 中国医科大学 ， 遗传学， 2009， 博士

【摘要】 前言假肥大性肌营养不良症是一种严重的神经肌肉疾病,分为DMD(Duchennemuscular dystrophy)和BMD(Becker muscular dystrophy)。均是由于抗肌营养不良蛋白基因(DYSTROPHIN)突变所致。DMD发病率为1/3500新生男婴,是最常见的X-连锁隐性致死性遗传病。患儿出生后的活动如抬头、坐姿等均正常,自1岁以后开始逐渐出现站立和行走困难,首先影响骨盆带肌肉,以后累及肩胛带肌肉。患儿动作笨拙,易跌倒,走路摇摇晃晃,登楼梯或由坐、卧位起立困难。患儿双侧腓肠肌逐渐呈假性肥大,腱反射减弱或消失。部分患者表现为行为异常。病变呈进行性加重,常到10岁时已不能行走,大多数患儿最终卧床不起,并发痉挛、褥疮、肺炎而在20岁前死亡。BMD是一种较轻的神经肌肉疾病,发病率约为1/12000,典型的BMD患者首发症状后15-20年仍有行走能力。1/3的DMD和BMD患者有智力减退。此外,很多患者会有语言障碍,IO平均值比正常值约低一个百分点。鉴于本病至今没有有效的治疗方法,因此高效、准确的DMD病基因诊断,携带者检测,产前诊断及正确的遗传咨询是预防本病的关键。Schouten等在多重可扩增探针杂交(Multiplex Amplifiable Probe Hybridisation,MAPH)术的基础上改进并设计了多重依赖连接探针扩增(multiplex ligation-dependent probeamplification,MLPA)技术,是利用可与样本DNA正确杂交并被连接酶连接的探针进行扩增和定量分析。MLPA是对于缺失和重复突变最有效的检测技术。因此本研究拟用MLPA方法筛查DMD患者,对缺失和重复突变的患者和携带诊断。自从法国医学家Guillaume-Benjamin-Amand Duchenne于1861年首次详细描述Duchenne型肌营养不良(DMD)以来,不少遗传学家对该病进行了系统而深入的研究,近年来对Duchenne型肌营养不良患者的治疗有了新的进展,病毒载体介导的基因治疗仍处于前沿领域,一些非病毒载体介导的治疗方法已经应用在DMD的动物和细胞模型上。包括质粒载体介导的基因转染,反义链介导的外显子跳跃和寡核苷酸介导的基因编辑。在过去几年中,非病毒载体的治疗方法已经逐渐从实验室过度到临床。有充足的证据表明由于UTROPHIN与DYSTROPHIN的功能相似,在DMD肌纤维中UTROPHIN可以代替DYSTROPHIN的功能,UTROPHIN与DYSTROPHIN有高度的序列一致性,并且和DYSTROPHIN蛋白复合物(Dystrophin-associated protein complex,DPC)结合。而且,在DMD模型鼠中的研究表明,UTROPHIN蛋白水平升高可以减轻DMD的病理症状,而且这种作用在转基因的体细胞和生殖细胞中都存在。这些都说明,外源性的增加DMD肌肉中UTROPHIN的表达水平似乎可以对DMD起到治疗作用。方法59个假肥大性营养不良症家系外周血来自2005年至2007年在中国医科大学盛京医院儿科和北京协和医科大学遗传系就诊的患者及其父母。其中有51例是DMD,8例BMD,他们表型正常的父母,19例羊水穿刺标本。诊断标准依赖体格检查,家族史,肌酸激酶水平,发病年龄,疾病的进展情况等。所有标本使用均经患者知情并同意。一、多重PCR检测DYSTROPHIN基因12个外显子缺失突变应用多重PCR方法检测了59例患者DYSTROPHIN基因12个外显子缺失突变情况二、MLPA检测DYSTROPHIN基因79个外显子拷贝数改变情况应用MLPA(multiplex ligation-dependent probe amplification)技术重复检测59例患者的DYSTROPHIN基因79个外显子拷贝数改变情况并明确突变位点三、统计分析比较多重PCR方法与MLPA技术的优越性四、产前基因诊断应用MLPA和STR连锁分析技术对19例高度怀疑孕有假肥大性肌营养不良症胎儿的孕妇进行产前基因诊断五、利用相关文献及P-MATCH软件预测UTROPHIN基因5’上游序列转录因子的结合位点获取UTROPHIN基因5’上游1500bp序列信息(http://www.ensembl.org),应用P-MATCH软件预测其转录因子结合位点。六、染色质免疫沉淀和凝胶阻滞实验提取Hela细胞的染色质,甲醛交联、酶切后应用EN1抗体进行沉淀,沉淀下来的染色质通过PCR扩增检测结果。Hela细胞核蛋白和3’生物素标记的探针(含有位点2)在凝胶阻滞缓冲液中室温结合1小时,10%的聚丙烯酰胺凝胶电泳,转膜、紫外交联后,应用化学发光检测系统检测结果。七、RNA干扰试验选择合适的EN1-siRNA干扰序列转染Hela细胞,Real time PCR检测Hela细胞中EN1和UTROPHIN基因mRNA的表达情况结果一、多重PCR检测DYSTROPHIN基因12个外显子缺失用多重PCR方法在59例假肥大性肌营养不良症患者中检测到30例患者有外显子缺失。二、MLPA检测DYSTROPHIN基因79个外显子拷贝数改变MLPA检测出33例外显子缺失,6例外显子重复和1例点突变三、MLPA比多重PCR多检测出10例DYSTROPHIN基因突变,两种方法共同检测出的30例突变中MLPA检测出16例基因突变范围比多重PCR大。四、应用MLPA和STR连锁分析技术在19例高度怀疑孕有假肥大性肌营养不良症胎儿的孕妇中检测出5例男性DMD胎儿,4例女性携带者,10例正常胎儿(7例男性,3例女性)。五、在人类UTROPHIN基因启动子区域存在2个EN1的可能结合位点,分别命名为EN1结合位点1(-1074～-1080)和EN1结合位点2(-892～-899)。六、在体外EN1蛋白可以和UTROPHIN基因启动子区EN1结合位点2直接结合。在Hela细胞中EN1可以和UTROPHIN基因启动子区EN1结合位点2直接结合。我们应用染色质免疫沉淀技术验证在体内EN1和UTROPHIN基因上游序列的结合作用。沉淀的Hela细胞染色质中有EN1结合位点2的扩增,无对照位点的扩增。EMSA结果表明当EN1蛋白质存在时出现阻滞的DNA-蛋白质复合体,当加入EN1抗体时出现超阻滞条带。证明在体外EN1和预测的EN1结合位点2可以直接结合。七、EN1-2 siRNA干扰后,EN1表达下降,UTROPHIN基因表达升高。结论1、MLPA能够高通量的检测DYSTROPHIN基因79个外显子拷贝数改变(缺失,重复,携带者),并发现2例新发突变。2、MLPA技术可以适用于对高度怀疑孕有假肥大性肌营养不良症胎儿的孕妇进行羊水DYSTROPHIN基因突变筛查。3、EN1可能对UTROPHIN基因有负调控作用。更多还原

【Abstract】 IntroductionDuchenne and Becker muscular dystrophies（DMD and BMD） are X-link drecessive lethal disease with an incidence of～1 in 3500 newborns,and it has been estimated that approximately one third of the cases result from new mutations.As the disease progresses,the contractures increasingly develop,leading to the asymmetrical spinal deformities.Most patients die at the age of 20 of pneumonia related to chronic respiratory insufficiency.The allelic disorder BMD has a milder clinical course and a slower disease progression.With an incidence of～1 per 120,000 newborns.Patients with BMD have a reduced life expectancy,but the majority of patients survived as normal and the affected patients remain ambulant until 16 years of age,although onset in the 3^rd or 4^th decade,even later.Cardiac involvement is invariably associated with DMD and BMD,one third of DMD is associated with mild but potentially significant difficulties in a range of neurobehavioral areas.Besides,many patients have language handicap.IQ is below normal one percentage point.There is currently no effective treatment.so exploring the molecular mechanism of DMD/BMD will provide theories for improving genetic cohort,theatment of DMD/BMD.Multiplex Ligation-dependent Probe Amplification（MLPA） has become widely used for detecting deletions or duplications of the DYSTROPHIN gene among patients and carriers.It was established by Schouten based on MLPA.After DMD was characterized by the France sicientist Guillaume-Benjamin-Amand Duchenne in 1861,many genetist working for optimal management of DMD.Several curative therapeutic strategies including cell and gene therapy are being pursued but are still at an experimental stage.Although viral-mediated gene therapy has been at the forefront of the field,several non-viral gene therapy approaches have been applied to animal and cellular models of DMD. These include plasmid-mediated gene delivery,antisense-mediated exon skipping,and oligonucleotide-mediated gene editing.In the past several years,non-viral gene therapy has moved from the laboratory to the clinic.UTROPHIN might be able to serve as a surrogate for DYSTROPHIN in DMD muscle fibers because there is convincing evidence for functional redundancy between these two proteins.Indeed,UTROPHIN shares a high degree of sequence identity with DYSTROPHIN and also associates with members of the DAPC.Moreover,studies in the mdx mouse,a DYSTROPHIN negative model of DMD,have established that the elevation of UTROPHIN levels in dystrophic muscle fibers can restore sarcolemmal expression of DAPC members and alleviate the dystrophic pathology.This elevation can be accomplished by both germline gene transfer and somatic gene transfer of UTROPHIN.Together,these findings indicate that a gene-therapy approach focusing on induction of exogenously supplied UTROPHIN to DMD muscle fibers is a plausible treatment for this disorder.MethodsSamples from 59 families were collected with informed consent.All subjects were referred from the Department of Pediatrics,Shengjing Hospital of China Medical University and the Department of Medical Genetics,Peking Union Medical University between January 2005 and December 2007.The research plan was approved by the ethics committees of both universities.The subjects included 51 boys diagnosed with DMD and 8 with BMD,their unaffected parents and 19 amniotic samples.Clinical diagnosis was made based on physical examination,family history,serum creatine phosphokinase（CPK） levels,age of onset,calf pseudohypertrophy,wheelchair confinement,presence of cardiomyopathy and electromyographic patterns.Multiplex PCRThe DMD gene of 59 patients were detected by multiplex PCR.MLPA analysis The mutations of DMD gene were detected by MLPAStatistical analysisEfficacy of deletion/duplication detection by MLPA as compared with that of mPCRPrenatal diagnosisFor the 19 couples who were at risk of carrying another DMD/BMD fetus, prenatal diagnoses were achieved for all subjects using combined MLPA and linkage analysis.P-Match software was used to analyze the sequence upstream of the transcription start site of the UTROPHIN gene.The 1500bp sequence uptream of UTROPHIN gene was obtained from http://www.ensembl.org,we used P-Match software to predict the binding sites of transcriptional factors on the sequence.Chromatin immunoprecipitation assay and Electrophoretic mobility shift assayThe chromatin was extracted from Hela cells,sheared with an Enzymatic Shearing Kit to obtain 500-1000bp fragments.EN1 antibody was used at the immunoprecipitation step.Eluted DNA from the sample and control was assessed for the presence of UTROPHIN DNA region by PCR.Nucleoprotein was extracted from Hela cells and incubated with the UTROPHIN upstream region-containing binding site 2 labeled using a Biotin 3′End DNA ing Kit for 60 min at room temperature in a gel shift buffer.Reactions were examined for nucleoprotein binding by electrophoretic mobility shift assays（EMSA） on a 10% nondenaturing polyacrylamide,transfered to the memberane,cross-linked.DNA binding bands were detected using a chemiluminescence system.siRNA EN1 was knockout by siRNA,the expression of UTROPHIN were detected by Real time PCR.ResultsMultiplex PCR31 exon deletions were detected by multiplex PCRMLPA analysis33 exon deletions,6 exon duplications and one point mutation were detected by MLPA.Statistical analysis10 mutations including 3 exonic deletions,1 single base deletion and 6 exonic duplications were only detected by MLPA.16 deletions in fact had involved more exons.Prenatal diagnosis5 were at risk of DMD/BMD,4 carriers,10 normal fetus were detected by MLPA and STR analysis in 19 couples who were at risk of carrying another DMD/BMD fetus.P-Match analyze resultsThe 5’ region of the human UTROPHIN gene contained two potential binding sites for the EN1 protein designated EN1 binding site 1（-1074～-1080） and EN1 binding site 2（-892～-899）.EN1 binds with site 2 in UTROPHIN promoter region in the developing limb in Hela cells.EN1 directly binds to site 2 in UTROPHIN promoter region.To verify the binding in vivo of EN1 to binding site 2 within the UTROPHIN promoter,we used the chromatin formaldehyde cross-linking and immunoprecipitation （CHIP） technique.The immunoprecipitated Hela cell chromatin showed a substantial enrichment only of the sequence containing site 2,indicating that EN1 efficiently bound only to site 2 in vivo.No enrichment was detected for the control site.SiRNAAfter EN1 was knockout by siRNA,the expression of UTROPHIN was increased.Conclusion1.For the comprehensive coverage of all exons of the DYSTROPHIN gene,compared with that of mPCR MLPA should be the method of choice for initial screening of DMD/BMD patients.2.For its sensitivity and robust performance,MLPA can provide a powerful assay for prenatal diagnosis for such diseases.3.EN1 may decreased the expression of UTROPHIN更多还原