【作者】 卢彦平；

【导师】 李亚里； 袁慧军；

【作者基本信息】 中国人民解放军军医进修学院 ， 妇产科， 2010， 博士

【摘要】 产前诊断是预防出生缺陷的重要手段,广义的产前诊断还包括植入前遗传学诊断(preimplantation genetic diagnosis, PGD)。我国产前诊断起步较晚,染色体核型分析需时过长并缺少适合国情的染色体病快速产前诊断方法；目前对胎儿畸形分子致病机制的研究较少。鉴于此,我们进行了以下研究：一、多重荧光定量PCR技术快速诊断21-三体及18-三体方法的建立及临床应用本研究利用收集的20例21-三体、3例18-三体DNA样本及40例正常人DNA样本,选择多对21、18号染色体短串联重复序列分子标记,建立多重荧光定量PCR检测技术用于21-三体和18-三体的快速产前诊断；利用建立的方法对165例产前诊断病例及4例消化道畸形新生儿进行检测,并与核型分析结果相比较。169例病例中共诊断21-三体4例,18-三体1例,所有病例均在1～3 d得到结果,无漏诊和误诊,并利用建立的多重荧光定量PCR技术为5例核型分析失败的病例提供了明确的产前诊断。对于同期B超检查胎儿结构异常的22例胎儿,则采用传统的核型分析,检出1例(45,X),1例(47,XXY)。结果表明建立的多重荧光定量PCR技术可快速、准确地诊断2-1三体和18-三体,减轻核型分析需时过长给孕妇带来的焦虑,可用于血清学筛查21-三体和18-三体高风险者及高龄孕妇,也适用于对其他遗传性疾病如遗传性耳聋进行产前诊断时并行检测以排除21-三体和18-三体。多重荧光定量PCR技术结合传统核型分析可更好地满足产前诊断的临床需求。二、几种单基因病致病基因的鉴定Meckel-Gruber综合征(MKS)为临床罕见的致死性常染色体隐性遗传病,典型临床特征为胎儿枕部颅骨缺损脑组织膨出、多囊性肾发育不良、肝脏纤维化、轴后性多指趾等,并可合并多种其它畸形。目前已明确了5个与之相关的基因,其中MKS3基因突变引起的畸形多数不合并多指。我们收集了一个家系,夫妇连续4次妊娠Meckel-Gruber综合征胎儿,具有典型的临床表型特征,其中前3例胎儿无多指。先证者引产后提取组织RNA进行RT-PCR扩增,c.DNA测序发现MKS3基因c.1645C>T纯合突变,此突变导致第549位氨基酸从精氨酸变为半胱氨酸(R549C)。三个畸形胎儿的DNA检测均有此纯合突变,对家系成员的检测,明确了该突变在家系中的传递方式。对200例正常人酶切分析未发现此基因突变,经检索为一未经报道的新突变位点。遗传性骨软骨发育异常,是胎儿期常见的畸形,对因肢体短小引产的5例畸形胎儿进行了FGFR3基因热点突变区域的检测,发现2例基因突变,1例为较少报道的c.1108G>T(G370C)突变,另1例为c.1138G>A(G380R)突变,2例均为新生突变,此2个家庭再生育时再发风险低。之后对1例软骨生长不全病例进行了FGFR3、SLC26A2及TRIP11全基因检测,发现了3个新的SNP位点,未发现致病突变。三、Meckel-Gruber综合征植入前遗传学诊断(PGD)的研究因连续四次妊娠Meckel-Gruber综合征畸形儿,该夫妇要求进行PGD。取家系成员成纤维细胞30个,多重替代扩增后,取扩增产物进行实时荧光定量PCR (Taqman-MGB探针)检测基因类型,并选取基因附近D8S270,D8S273和D8S1794分子标记进行检测。Taq-man MGB探针检测成功率85.7%,脱扣率30.7%,D8S270成功率80%,脱扣率16.7%。结果显示初步建立的单细胞扩增体系进一步完善后有望临床应用于植入前遗传学诊断。更多还原

【Abstract】 Prenatal diagnosis including preimplantation genetic diagnosis (PGD) can be used to avoid delivering of fetuses with severe genetic defects. In China, the techniques of prenatal diagnosis hasn’t been fully developed so far. Pregnant women must wait a long time for their results of full karyotype analysis and we do not have suitable techniques for rapid prenatal diagnosis for common aneuploidy. The molecular pathogenic mechanism underlying most of fetal malformations remains to be discovered. In this study, we have conducted some exploring as follows:Part 1:Development of multiple quantitative fluorescent PCR for rapid diagnosis of commom aneuploidy and its clinical applicationTo establish a multiple quantitative fluorescent polymerase chain reaction (QF-PCR) method for trisomy 21 and trisomy 18 test and its potential clinical application.20 cases of trisomy 21,3 cases of trisomy 18 and 40 cases normal controls were used to establish multiple QF-PCR. Several short tandem repeat markers (STR-marker) were chosen from chromosome 21 and 18. The established methods were used for 165 cases of prenatal diagnosis (including 30 cases for hearing loss gene test) and for 4 cases newborn infants with digestive tract obstruction. Among 169 cases, we identified 4 cases of trisomy 21,1 case of trisomy 18. The results were concordant with those of karyotyping.22 cases fetuses with structure malformation were only performed karyotyping analysis.1cases of 45, X, and 1case of 47, XXY were identified.In conclusion, QF-PCR testing is an efficient, rapid and accurate technique for the detection of trisomy 21 and trisomy 18. It should be offered to pregnant women who were identified as having a high risk by serum screening and because of advanced maternal age. It can also be used for pregnant women who were undertaken other prenatal diagnosis. QF-PCR testing combines with karyotype analysis can provide better service for clinical demanding of prenatal diagnosisPart 2:Study of molecular mechanism of some kinds of fetal malformationMeckel-Gruber syndrome (MKS) is an autosomal recessive monogenic lethal disease, characterized by occipital encephalocele, renal cystic dysplasia, hepatic ductal proliferation, fibrosis and polydactyly. MKS is genetically heterogeneous with six loci and five identified genes. We ascertained a Chinese family with four affected fetuses. Three of them had typical characteristics of MKS excepted for polydactyly. We screened the candidate gene of MKS3 by cDNA direct sequencing in this family. We found a c.1645C>T mutation, changed the 549 amino acid of arginine to cysteine (R549C). The R549C mutation was confirmed in two siblings of the proband. All of family members were detected for this mutation and found the couple and one of their parents carried heterozygous R549C mutation. This mutation was not present in 200 normal controls,We also screened the hot mutated exons of FGFR3 gene for five fetuses with short limbs malformations. One fetus carried mutation of c.1108G>T (G370C) and another of c.1138G>A(G380R).They were both de novo mutations. These families have low recurrent risk. For one fetus, we also screened FGFR3, SLC26A2 and TRIP11 gene and found three benign single nucleotide polymorphisms (SNP).Part 3:The establishment of preimplantation genetic diagnosis (PGD) for Meckel-Gruber syndromeThe couple had consecutively got four affected MKS fetuses and they want PGD service to give birth to a healthy baby. We did some basic research for the purpose of providing the PGD service clinically. Multiple displacement amplification (MDA) was used to amplify the whole-genome DNA directly from a single cell of acquired from a heterozygote. MDA products were used for genotyping by real time PCR and for STR markers. The success rate were 85.7% with allele drop out (ADO) of 30.7% in real time PCR and were 80% with ADO of 16.7% in D8S270 genotyping. This protocol of PGD could be used for clinical application after modification.更多还原