【作者】 龙兴江；

【导师】 龙桂芳；

【作者基本信息】 广西医科大学 ， 儿科学， 2007， 博士

【摘要】 背景与目的:产前诊断(prenatal diagnosis)作为减少出生缺陷(birth defects)发生率的有效手段越来越多地在临床单位得以应用,但是目前使用的技术大多数属于创伤性产前诊断(invasive prenatal diagnosis)方法,存在一定的局限性,建立安全有效准确的无创性产前诊断(non-invasive prenataldiagnosis)方法一直是研究人员追求的目标。而孕妇外周血浆中游离胎儿DNA(cell-free fetal DNA,cffDNA)的发现,为无创性产前基因诊断开辟了新的领域。地中海贫血(thalassemia)作为出生缺陷的一种,是世界范围最常见的常染色体遗传病之一,也是广西地区高发的遗传病。无创性产前诊断方法的建立无疑对地中海贫血的防治,控制出生缺陷,提高人口出生素质,维护妇女身心健康具有非常重要意义。本研究针对孕妇外周血游离胎儿DNA的相关特性,建立起一套富集cffDNA的方法,并进行相关的检测,以探讨对地中海贫血高危胎儿进行无创性产前诊断的可行性途径。第一部分利用孕妇外周血浆中小片段游离胎儿DNA检测胎儿SRY基因目的探索从孕妇外周血浆中提取小片段cffDNA来提高检测胎儿SRY(sexdetermining region of Y chromosome,SRY)基因准确率的方法,评估利用小片段cffDNA进行无创性产前诊断的可行性。方法收集157例孕妇的外周血,利用柱吸收的方法提取血浆中的cffDNA,经琼脂糖凝胶电泳分离富集小片段cffDNA,使用二重PCR反应(dulex-PCR)检测SRY基因和磷酸甘油醛脱氢酶(glycerol-dehyde-phosphatedehydrogenase,GAPDH)基因。结果来源于86例孕男胎的孕妇血浆标本的小片段cffDNA均检出SRY和GAPDH基因,来源于71例孕女胎的孕妇血浆标本的小片段cffDNA只检出GAPDH基因。与绒毛/羊水标本检测结果以及产后随访结果相符。特异性和敏感性分别为100%(157/157)和100%(86/86)。结论利用琼脂糖凝胶电泳,切胶回收,可以选择性富集孕妇外周血中的小片段cffDNA,相对地提高胎儿DNA的含量,结合二重PCR扩增SRY基因技术可用于无创性产前性连锁遗传疾病和单基因突变疾病的产前诊断。第二部分利用孕妇外周血浆中cffDNA进行STR-PCR检测胎儿基因型目的:利用小片段cffDNA进行D5S818、D7S820、D13S317三个短串联重复序列(short tandem repeat,STR)基因型的分析,观察提取出来的小片段cffDNA中母源性DNA背景对实验的影响。方法:收集了62例孕妇外周血标本,提取小片段游离胎儿DNA,利用多重PCR(mutilplex-PCR)的方法分析胎儿的STR基因型,并与父母基因型相比对。结果:62例小片段cffDNA的扩增结果中,49例标本的扩增结果完全与父母基因型相匹配,未发现母源性DNA的污染。其余13例在D13S317基因座的扩增中出现了母源性DNA的污染。结论:经过对小片段cffDNA进行富集后,仍有可能出现母源性DNA对实验的影响,并且可能影响对实验结果的判读。使用多重PCR反应结合多基因座的扩增,可能可以有助于减少母源性DNA的影响,有助于结果的判读。第三部分利用孕妇外周血浆中小片段游离胎儿DNA进行β-地中海贫血无创性产前基因诊断目的:利用cffDNA对胎儿进行广西、广东地区常见的17种β-地中海贫血基因型的检测,与传统创伤性产前诊断相比较,探讨该方法的准确性和可行性。方法:针对广西、广东地区常见的β-地中海贫血突变的基因型设计三对不同引物,并用生物素标记。对cffDNA进行二次PCR反应后,使用反向斑点杂交(revert dot-blot hybridization,RDB)检测胎儿的β-珠蛋白基因型,与创伤性产前诊断结果比较,观察准确率。结果:37例cffDNA标本检测中,检出重型β-地中海贫血19例,轻型β-地中海贫血11例,正常7例,与创伤性产前诊断结果相比较出现3例误诊,准确率为91.9%(34/37)。结论:利用cffDNA进行β-地中海贫血的检测,可能出现母源性DNA背景的污染,当胎儿基因型与母亲基因型相同时,必须提高警惕,进一步分析或者复查。因为该技术取样容易,对孕妇胎儿无风险,不受孕期时间影响,易为与孕妇接受,因此进一步改进该技术后有望可用于β-地中海贫血的诊断。第四部分利用孕妇外周血浆中小片段游离胎儿DNA进行巴氏水肿胎儿检测目的:通过检测cffDNA以及母源性cfDNA在PCR反应中扩增效率的不同,进行检测巴氏水肿胎儿(Hb Bart’s hydrops foetus)的方法学研究。方法:对来源于拟诊孕水肿胎儿孕妇的小片段cffDNA,进行荧光PCR(Fluorescence PCR)扩增,利用毛细管电泳(capillary electrophoresis,CE)技术判断两种产物峰面积比(peak area ratio)来检测巴氏水肿胎儿。结果:30例拟诊巴氏水肿胎儿的小片段cffDNA扩增结果提示,巴氏水肿胎儿两种产物锋面积比远远＜1。而其他原因所致的水肿胎儿的cffDNA模板扩增结果显示两种产物封面值比近似等于1。结论:通过荧光PCR和毛细管电泳的方法,有望利用cffDNA进行的巴氏水肿胎儿的无创性产前诊断。更多还原

【Abstract】 Background:Since the introduction of prenatal diagnosis to prevent birth defects,the risk of invasive prenatal diagnosis for both mother and foetus has been considered.Noninvasive prenatal diagnosis is one of the many long-sought goals in human genetics.The discovery of cell-free fetal DNA(cffDNA)in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis and monitoring.As a result of its development,fetal DNA in maternal plasma is considered as a hope for the noninvaSive prenatal diagnosis of single gene disorders such as thalassemia,which is one of the most common autosomal recessive single gene disorders and has markedly high incidence in Guangxi province.The development of noninvasive prenatal diagnosis has a significant contribution to women physical and mental health.In order to investigate the feasible noninvasive prenatal diagnosis of thalassemia,we established a whole set methodology for cffDNA enrichment and detection based upon its correlation characteristics.PartⅠDetection of Fetal SRY Gene Using Size-Fractionated Cell-Free Fetal DNA in Maternal PlasmaObject:To evaluate the feasibility of cffDNA-based noninvasive prenatal diagnosis,we developed a precise technique for fetal SRY gene detection using size-fractionated cell-free DNA in maternal plasma.Methods:Peripheral blood samples were collected form 157 pregnant women. CffDNA was extracted based on a column absorbent method and isolated by agarose gel electrophoresis.A dulex-polymerase chain reaction(PCR)was used to detected SRY gene and glycerol-dehyde-phosphate dehydrogenase(GAPDH) gene.Results:Both SRY and GAPDH gene were detected in 86 cffDNA samples form women bearing male fetuses.And only GAPDH gene was detected in 71 cffDNA samples from women bearing female fetuses.These results have a coincidence whit those of villus or amniotic fluid samples and postpartum follow-up.The specificity and sensitivity reached to 100%(157/157)and 100% (86/86),respectively.Conclusion:By agarose gel electrophoresis,re-extratedand and dulex PCR, size-fractionated cell-free fetal DNA in maternal plasma can be selective enriched and used to noninvasive prenatal diagnosis of sex-linked disorders and single gene disorders.PartⅡ.Detection of fetal STR genotypes Using Size-Fractionated Cell-Free Fetal DNA in Maternal PlasmaObject:To evaluate the reliable discrimination between the fetal and maternal DNA in maternal plasma,we analyzed 3 short tandem repeat(STR)genotypes (D5S818,D7S820 and D13S317)on basis of size-fractionated cell-free DNA in maternal plasma.Methods:CffDNA samples from 62 pregnant women were collected according to the method established in PartⅠ.Then foetus and its corresponding parents STR genotypes were analyzed by a mutilplex-PCR.Results:According to STR-PCR amplification,the fetal genotypes of 49/62 samples completely matched to those of corresponding parents.However,we observed maternal DNA contamination to D 13S317 amplification in 13 cases.Conclusion:The reliable discrimination between fetal and maternal DNA in maternal plasma has hitherto been a technical challenge.By selective enriching of cffDNA,we still can’t completely exclude maternal DNA contamination,and such DNA background unavoidably interfered the conclusively identification. However,in our study,a specific amplification of maternal DNA was less likely based on the mutilplex-PCR technology.PartⅢ.Noninvasive prenatal diagnosis ofβ-thalassaemia using Size-Fractionated Cell-Free Fetal DNA in Maternal PlasmaObject:To investigated the clinical feasibility of cffDNA-based noninvasive prenatal diagnosis forβ-thalassaemia.Methods:According to commom mutational genotypes of 17 kinds ofβ-thalassaemia in Guangxi and Guangdong province,we designed 3 pairs of primers,biotin labeled.Then cffDNA was amplificated by a duplex PCR,and fetalβ-globin genotype was analyzed by revert dot-blot hybridization.The results were conclusively identified by traditional invasive methodology.Conclusion:Among 37 cffDNA samples,19 and 11 were identified asβ-thalassaemia major andβ-thalassaemia minor,respectively,and 7 were identified as normal.By comparing with that of invasive methods,3 cases were stated as misdiagnosis.The accurate rate reached to 91.9%(34/37).Conclusion:When fetal genotype is coincident with maternal,further analysis or re-examination is necessary for the unavoidable contamination of background DNA.Due to its facilely sampling,no risk for both of gravida and foetus,and no limited by duration of pregnancy,the cffDNA-based noninvasive prenatal screening forβ-thalassaemia has been expected clinical application.PartⅣ.Noninvasive prenatal diagnosis of Hb Bart’s hydrops foetus using Size-Fractionated Cell-Free Fetal DNA in Maternal Plasma.Object:To explore a new noninvasive methodology for Hb Bart’s hydrops foetus,we investigated PCR amplification efficiency discrimination between fetal cffDNA and maternal cfDNA.Methods:CffDNA samples form women bearing possible Hb Bart’s hydrops foetus were collected.Fluorescence PCR and capillary electrophoresis(CE) were performed.And Hb Bart’s hydrops foetus was conclusively identified because of different peak area ratio of products.Results:The peak area ratio of cffDNA sample from Hb Bart’s hydrops foetus was significantly less than 1.However,the peak area ration of cffDNA sample from hydrops foetus due to another reason was approximately equal to 1.Conclusion:By fluorescence PCR and capillary electrophoresis,we developed a cffDNA-based prenatal screening for Hb Bart’s hydrops foetus among large population.Additionally,traditional invasive methodology can provid sufficient foetus DNA samples to the PCR technology established in this study.As the result of these,a rapid screening for Hb Bart’s hydrops foetus is under considered.更多还原