母体循环中游离胎儿DNA的起源及诊断潜力研究进展

有关母体循环中游离胎儿DNA的研究一直是热点.胎儿核酸以惊人的稳定性存在于母体循环中,可能由于凋亡体中存在某些保护机制,游离胎儿DNA在母体循环中大量产生和快速变化更为妊娠过程提供了动力学依据.综合各学科理论和现有数据,认为母体循环中游离胎儿核酸主要来源于胎盘,其次也有部分来自胎儿造血细胞系统.由于母体循环中胎儿DNA的相对容易检测,胎儿非整倍体病、子痫前期、胎儿RhD基因型和一些单基因遗传病的诊断与鉴别手段应运而生.有关非性别依赖基因多态性、外遗传标志物以及循环mRNA序列研究的进展最终将使此类技术不受胎儿性别限制而推广应用.     

C21orf25片段在母胎间的甲基化差异及其临床价值

目的:阐明C21orf25片段在母胎间甲基化状态的差异,探讨其用于无创性产前诊断的价值。方法:收集388例孕妇血浆,其中126例同时提取外周血细胞及胎盘(或绒毛)组织DNA,采用甲基化敏感性限制性酶切联合荧光定量PCR(MSRE+PCR)技术,检测母胎间C21orf25基因、RASSF1A基因甲基化状态的差异,分析其影响因素,并分别以胎源性RASSF1A或SRY基因为参照位点,计算孕妇血浆中胎源性C21orf25与参照位点的浓度比,判断胎儿21号染色体数量。结果:C21orf25及RASSF1A在胎盘或绒毛组织中均呈高甲基化状态,而在母体外周血细胞呈现低甲基化状态。C21orf25在母胎间的甲基化差异程度受孕周的影响,早孕期小于中、晚孕期(P=0.002,P<0.01)。采用MSRE+PCR技术对孕妇血浆中C21orf25、RASSF1A片段的检出率分别为100%和96.9%。计算184例正常妊娠孕妇血浆中胎源性C21orf25/RASSF1A比值,确定其95%的参考值范围为0.20～3.46,以此为标准判断71例胎儿21号染色体数量,其中67例二倍体妊娠的准确率为94.0%(63/67),4例21三体综合征妊娠,2例C21orf25/RASSF1A比值高于参考区间上限而获得诊断。以SRY为参照位点,94例正常二倍体男胎妊娠中甲基化C21orf25/SRY比值为3.16±1.20,95%参考值范围为0.81～5.51,以此为标准判断35例男胎21号染色体数量,准确率为97.1%(34/35),2例21三体妊娠均获准确诊断。结论:高甲基化C21orf25基因可作为孕妇血浆中胎儿DNA的标志物,通过计算该位点与参照基因的浓度比值,有望进行唐氏综合征的无创性产前诊断。     

母血中胎儿游离核酸检测在诊断胎儿非整倍体中的应用

胎儿染色体非整倍体是常见且严重的染色体异常类出生缺陷。目前常用的侵入性检查方式因存在诸多弊端而被部分孕妇拒绝。在过去的15年中,母体血浆中胎儿游离核酸的研究进展使得无创性产前诊断胎儿非整倍体成为可能。而最近胎儿与母体表观遗传学差异及胎儿特异性RNA的发现使得这项技术摆脱了胎儿性别的限制。随着数字PCR及大规模平行测序等新技术的运用,胎儿非整倍体检测的敏感性和特异性得到了提高。本文综述了无创性产前诊断胎儿非整倍体的研究进展,并讨论了其临床应用的可行性。     

母血中胎儿mRNA用于无创伤性产前诊断的研究进展

约4%的新生儿受一种或多种先天性疾病的影响,而其中35%是遗传性疾病.因此,及早进行产前诊断,对有效预防和控制遗传病患儿的出生具有十分重要的意义.传统的产前诊断方法,如羊膜腔穿刺、绒毛膜取样、脐带取血等均具有创伤性,对胎儿和孕妇都有一定的危险性,有的甚至造成流产、胎儿肢体损伤、短肢畸形、宫内感染、死胎等,所以人们一直在探求无创伤性的产前诊断方法.     

基因DNA甲基化在子痫前期中的研究进展

子痫前期是一种妊娠期特有的疾病,以高血压和蛋白尿为主要临床特征,同时可伴有全身多脏器损伤。由于其病因不明,除终止妊娠之外缺乏有效的临床治疗方法,因此,子痫前期是导致孕产妇死亡的主要原因之一。子痫前期的发病机制还未得到具体的阐述,近年来随着表观遗传学领域的发展,尤其是基因甲基化的研究,为子痫前期的诊断和治疗提供了一个新的视角。研究发现,异常的全基因组甲基化以及Cp G岛甲基化与子痫前期中许多重要功能基因表达异常有关,推测其间接参与了子痫前期的发生。综述基因DNA甲基化在该疾病发生中的研究现状和研究进展综述。     

子痫前期孕妇外周血浆中高甲基化SIM2基因的含量变化

目的:通过检测高甲基化SIM2基因来分析游离胎儿DNA在正常妊娠孕妇及子痫前期孕妇外周血浆中含量的变化。方法:共收集114例外周血浆样本(包括健康未妊娠女性志愿者10例,产后48小时健康女性10例,正常妊娠孕妇57例,子痫前期孕妇37例),利用HpaⅡ、MspⅠ分别对提取的血浆DNA进行甲基化酶切反应后再进行实时荧光定量PCR反应,相对定量法检测高甲基化SIM2基因的含量。结果:①10例健康未妊娠女性志愿者及产后48小时健康女性外周血浆中均未检测出高甲基化SIM2基因;②57例正常妊娠孕妇中有43例检测出高甲基化SIM2基因,在不同妊娠阶段高甲基化SIM2基因含量的差异有统计学意义,高甲基化SIM2基因的含量在中期妊娠是早期妊娠的2.33倍,晚期妊娠是早期妊娠的5.28倍;③37例子痫前期孕妇中有36例检出高甲基化SIM2基因,高甲基化SIM2基因的含量在轻度子痫前期孕妇血浆中是正常晚期妊娠的3.05倍,在重度子痫前期孕妇中是正常晚期妊娠的6.32倍。结论:高甲基化SIM2基因是可靠的胎儿特异性标记,在孕妇外周血浆中高甲基化SIM2基因的含量随妊娠进展而增加,与孕周有关。高甲基化SIM2基因在子痫前期孕妇血浆中含量明显增加,能够反应子痫前期疾病的严重程度。     

CDH1基因异常甲基化在上皮性卵巢癌中的检测及临床意义

目的 探讨CDH1基因异常甲基化在上皮性卵巢癌发生发展中的作用及临床意义。方法 对中国医科大学附属第一医院妇科、辽宁省肿瘤医院妇科1999年至2006年63例上皮性卵巢癌组织原发灶、41例相应的盆腹腔转移灶、10例癌旁卵巢组织及20例正常卵巢组织,采用甲基化特异性聚合酶链式反应（MSP）法检测CDH1基因启动子区甲基化状态。结果 上皮性卵巢癌组织原发灶及相应的盆腹腔转移灶中存在CDH1基因启动子区异常甲基化,发生率分别为28.6%、39.0%,显著高于正常卵巢组织（P〈0.01）。10例癌旁卵巢组织中1例检测到CDH1基因启动子区甲基化,20例正常卵巢组织未检测到CDH1基因启动子区甲基化。CDH1基因启动子区甲基化的发生率在临床Ⅰ期和Ⅱ期显著低于Ⅲ期和Ⅳ期（P〈0.05）,在高分化癌中低于低分化癌（P〈0.05）。结论 CDH1基因启动子区异常甲基化与上皮性卵巢癌发生密切相关,并可能与上皮性卵巢癌转移、临床分期及分化程度有关。     

重度子痫前期患者胎盘组织中缺氧适应相关基因的表达及其临床意义

目的 探讨胎盘组织中缺氧诱导因子脯氨酸羟化酶1(HPH1)和因子抑制缺氧诱导因子1(FIH-1)在重度子痫前期发病和疾病进展中的作用.方法 选择2006年7月至2007年7月在中国医科大学附属盛京医院妇产科住院选择剖宫产终止妊娠的重度子痫前期孕妇34例(重度子痫前期组)及正常晚期妊娠孕妇24例(对照组),采用RT-PCR和蛋白印迹法检测两组孕妇胎盘组织中的HPH1和FIH-1 mRNA及蛋白的表达,并与临床指标进行相关性分析.结果 (1)重度子痫前期组孕妇胎盘组织巾HPH1 mRNA及蛋白表达水平分别为0.40±0.04和59.5 ±3.4,显著低于对照组的0.84±0.12和71.6±1.7(P均<0.01);重度子痫前期组孕妇FIH-1 mRNA及蛋白表达水平分别为0.31±0.05和45.6±2.4,也显著低于对照组的0.43±0.04和54.9±2.1(P均<0.01).(2)重度子痫前期组孕妇胎盘组织中HPH1和FIH-1 mRNA及其蛋白表达水平与平均动脉压(MAP)均呈显著负相关关系,r均为-0.854(P均<0.01);与24 h尿蛋白定量均呈显著负相关关系,r均为-0.936(P均<0.01);与有无眼底动脉痉挛均呈显著负相关关系,r均为-0.854(P均<0.01).(3)两组58例孕妇胎盘组织中的HPH1 mRNA表达水平高于FIH-1 mRNA的表达,分别为0.58±0.27和0.39 ±0.10,两者呈正相关关系,,为0.686(P<0.01);HPH1蛋白表达水平高于FIH-1蛋白的表达水平,分别为64.5±6.7和49.4±5.2,两者也呈正相关关系,r为0.947(P<0.01).结论 HPHl和FIH-1的表达变化可能与蕈度子痫前期的发病及疾病进展相关.     

新生儿血培养中凝固酶阴性葡萄球菌细胞间粘附基因A和D的检测及临床意义

目的 探讨细胞间粘附基因(ica)A和icaD在新生儿凝固酶阴性葡萄球菌(CNS)败血症中的诊断意义。方法 收集北京儿童医院新生儿病房2001年11月至2003年3月间血培养为CNS的患儿为研究入选病例。通过应用聚合酶链反应(PCR)方法检测icaA及icaD的存在。结果 在80例入选患儿中，血培养均分离出CNS。根据新生儿败血症的诊断标准，临床诊断败血症27例(33.8%)，其中早产儿7例，足月小样儿2例。在菌种的分布上，表皮葡萄球菌18例(66.7%)，溶血葡萄球菌7例(25.9%)，人葡萄球菌2例(7.4%)。对90株CNS菌株应用PCR方法检测icaA及icaD，共有8株阳性，均为表皮葡萄球菌，并且icaA和icaD同时阳性。在经PCR扩增后所产生的图谱中，icaA在814bp，icaD在282bp。在ica阳性的8例病例中，临床诊断为败血症7例，一致率为87.5%，在临床排除败血症的53例患儿中，仅有1例ica阳性(1.8%)。ica的阳性预测值为259%，阴性预测值为981%。结论 ica基因PCR检测是临床诊断CNS败血症或导管相关感染的一种潜在的实验室方法。 Abstract Objective The purpose of this study was to assess whether the person making the clinical decision may benefit from the detection of icaA and icaD genes encoding putative virulent factors.Methods From Nov.2001 to Mar.2003,we detected the icaA and icaD genes in 80 neonates with a collection of clinical isolates from blood cultures by using a simple,rapid,and reliable PCR method.Results An overall total of 80 neonates with CNS strains from blood cultures were identified.There were 27 cases(33.8%)diagnosed neonatal sepsis clinically according to the standard,of which 7 were premature,2 were small for gestational age of full term infants.The distribution of species among the clinical CNS strains,18(66.7%)cases were Staphylococcus epidermidis,which was the leading cause,and then S.hemolyticus (n=7),S.hominis (n=2).There was a statistical difference in them.Of the 90 strains,eight were positive to icaA and icaD genes,with PCR products being obtained for the icaA and icaD genes in all of these strains,a 814 bp band for the icaA gene and a 282 bp band for the icaD gene.All of them were Staphylococcus epidermidis.Among 8 cases with positive ica genes,7 were diagnosed as sepsis clinically.The coincidence rate was 87.5%.Positive predictive value of the ica genes were 25.9%，and negative predictive value were 98.1%.Conclusion Ica genes may potentiate the clinical criteria used for the diagnosis of neonatal sepsis,and may discriminate between contamination and infections. Key words Coagulase negative staphylococcus;PCR;Newborn     

A pregnancy with discordant fetal and placental chromosome 18 aneuploidies revealed by invasive and noninvasive prenatal diagnosis

This study investigated a pregnancy where the fetus was diagnosed with monosomy 18p by invasive amniocentesis and karyotyping. Additional noninvasive prenatal diagnosis, which detects fetal chromosome abnormalities in the circulating cell-free plasma DNA originating from the placenta revealed a related 18p monosomy/18q trisomy, suggesting confined placental mosaicism. Based on recent observations of chromosomal instability in the early preimplantation embryo, this study speculates on the possible embryonic origin(s) of these related but discordant chromosome 18 aneuploidies in the placental and fetal tissues. The findings highlight the potential for both false-positive and -negative noninvasive prenatal diagnosis results in pregnancies where there is either confined placental mosaicism or placental mosaicism.The study investigated a pregnancy involving a fetus with a chromosome disease syndrome called monosomy 18p where part of the short arm of chromosome 18 was missing in the fetal tissues. Using non-invasive prenatal diagnosis which detects fetal chromosome abnormalities in the circulating cell free plasma DNA originating from the placenta, we also detected monosomy 18p as well a related chromosome 18 abnormality involving duplication of the long arm of chromosome 18. This suggested confined placental mosaicism where the constitution of the chromosomes are different between fetal and placental tissues. We speculated that these related chromosome 18 abnormalities arose during preimplantation embryo development, leading to the formation of different chromosome abnormalities observed in the placental and fetal tissues of this pregnancy. Our findings highlight the potential for both false positive and negative non-invasive prenatal diagnosis test results in pregnancies where there is confined placental mosaicism.     

Quantitative analysis of intact fetal cells in maternal plasma by real-time PCR

OBJECTIVE: Fetal genetic materials in maternal circulation might be potentially used for non-invasive prenatal diagnosis (NIPD). In this study, we quantitatively analysed the intact fetal cells existing in maternal plasma, which would be a special method of NIPD. STUDY DESIGN: Eleven samples from preeclamptic patients, two samples from patients with RhD incompatibilities, and 30 samples from normal pregnancies as controls were collected. The intact cells in the plasma fraction were separated by centrifugation at high speed. The intact cell pellets were washed with PBS to remove any cell-free DNA. RESULTS: We were able to detect intact fetal cells in 3 out of 11 preeclamptic plasma samples from women carrying male fetuses, and from both plasma samples of RhD incompatibility affected pregnancies. However, intact fetal cells were not detected in all the 16 normal pregnancies with a male fetus, although cell-free fetal DNA was detected in all these cases. CONCLUSIONS: Intact fetal cells might be present in maternal plasma. However, the rarity of these cells limits their use for reliable, non-invasive prenatal diagnosis. The detection of such cells was successful in some preeclamptic and RhD incompatibility samples, not in the normal controls. Therefore, as opposed to free fetal DNA in maternal blood the use of intact fetal cells does not provide a reliable mode for NIPD.     

Recent advances in non-invasive prenatal DNA diagnosis through analysis of maternal blood

Prenatal diagnosis of aneuploidy and single-gene disorders is usually performed by collecting fetal samples through amniocentesis or chorionic villus sampling. However, these invasive procedures are associated with some degree of risk to the fetus and/or mother. Therefore, in recent years, considerable effort has been made to develop non-invasive prenatal diagnostic procedures. One potential non-invasive approach involves analysis of cell-free fetal DNA in maternal plasma or serum. Another approach utilizes fetal cells within the maternal circulation as a source of fetal DNA. At the present time, fetal gender and fetal RhD blood type within RhD-negative pregnant women can be reliably determined through analysis of maternal plasma. Furthermore, genetic alterations can be diagnosed in the maternal plasma when the mother does not have the alterations. However, the diagnosis of maternally inherited genetic disease and aneuploidy is limited using this approach. Non-invasive prenatal diagnosis through examination of intact fetal cells circulating within maternal blood can be used to diagnose a full range of genetic disorders. Since only a limited number of fetal cells circulate within maternal blood, procedures to enrich the cells and enable single cell analysis with high sensitivity are required. Recently, separation methods, including a lectin-based method and autoimage analyzing, have been developed, which have improved the sensitivity of genetic analysis. This progress has supported the possibility of non-invasive prenatal diagnosis of genetic disorders. In the present article, we discuss recent advances in the field of non-invasive prenatal diagnosis.     

The fetal fraction of cell-free DNA in maternal plasma is not affected by a priori risk of fetal trisomy

Objective: To determine the relationship between a priori risk for fetal trisomy and the fraction of fetal cell-free DNA (cfDNA) in maternal blood. Methods: A comparative analysis on fetal cfDNA amounts was performed in subjects stratified into a priori risk groups based on maternal age, prenatal screening results, or nuchal translucency measurement. Results: Across the highest and lowest deciles within each group, there were no significant differences in the fetal cfDNA fraction. Conclusions: These data support the concept that non-invasive prenatal test performance as determined by fetal cfDNA fraction is not predicted to be different based on patient risk classification.     

Non-invasive prenatal screening of fetal Down syndrome by maternal plasma DNA sequencing in twin pregnancies

Non-invasive prenatal screening for fetal Down syndrome (NIFTY) by maternal plasma sequencing was performed in 12 subjects with twin pregnancies, including 11 with normal fetuses and 1 with discordant fetal Trisomy 21. For every sample, it was processed, sequenced and reported as soon as it was collected as other clinical samples for singleton pregnancies. The NIFTY test was negative in the 11 pregnancies carried normal fetuses, and was positive (high risk) in the case with discordant fetal Trisomy 21. The sensitivity and specificity were both 100%. This small case series suggested the NIFTY as a screening test for fetal Trisomy 21 is feasible in twin pregnancies.     

Sharpening the Tools: A summary of a National Institutes of Health workshop on new technologies for detection of fetal cells in maternal blood for early prenatal diagnosis

In 2003 the National Institute of Child Health and Human Development (NICHD) sponsored a workshop entitled “Sharpening the Tools”, which was designed to explore the then current state of prenatal diagnosis and screening using fetal cells in maternal blood. The goals of the workshop were to: review the then current state of the field and assess present capabilities, identify future research needs and challenges in this area, identify promising new and innovative approaches for future exploration, and provide a written summary of the conference for public distribution. The workshop featured brief presentations by experts from a wide range of scientific fields and by innovative bioengineering and technology leaders from academic centers and private industry. The workshop was divided into presentations on target cells, target approaches for separation, genetic and protein analysis, and ‘out of the box’ (bioengineering) approaches. The passage of time since the workshop has allowed an objective assessment of where the research has progressed. A 2006 update on the field is included at the end of the summary.