胎儿游离RNA在产前诊断中的研究进展

传统产前诊断依靠有创性操作,具有潜在风险。目前无创性产前诊断方法集中于从母体血液中分离胎儿细胞或从血清或血浆中提取胎儿游离DNA,二者在临床的应用尚存不足。近年,妊娠妇女血循环中游离胎儿RNA的发现和应用,是无创性产前诊断领域的突破。已经证实,游离胎儿RNA分子主要来源于胎盘,在母体血循环中比较稳定,易于检测。同时,由于其无需依赖于胎儿性别或是父源性多态性位点,临床应用领域优于胎儿游离DNA;结合对分子标记物的定量检测方法更适用于产前诊断领域,并已取得初步成果。预测其具有很好的应用前景。     

检测孕妇血循环中胎儿DNA进行无创产前诊断的研究进展

孕妇血循环中胎儿DNA以两种形式存在，一是存在于进入母体血循环中的胎儿的完整细胞内，二是游离于母体血浆中。分析孕妇外周血循环中完整胎儿细胞DNA或游离DNA，是无创性产前诊断领域的一个日益引人注目的技术，这一技术可以单独使用或与其他无创性检查方法，如超声诊断、孕妇血清学分析等联合应用，使无创性产前诊断技术提高到一个新的水平。近年采用先进的磁珠细胞分选法(magnetic cell sorting，MACS)、流式细胞分选术和激光捕获显微分离(1aser capture microdissection，LCM)技术富集分离。     

细胞游离胎儿DNA产前诊断胎儿非整倍体异常

细胞游离胎儿DNA(cffDNA)已被成功在妊娠妇女血循环中检测到。与母体血循环中胎儿细胞不同,cffDNA最早可在妊娠4周检测出,并能在产后迅速消失。cffDNA可能源于胎盘部位的滋养细胞等并通过凋亡机制释放进入母体血循环中。通过检测cffDNA可产前诊断胎儿非整倍体异常,但因其在妊娠妇女血中含量较少,检测具有挑战性。目前已有多种新检测方法用于非整倍体异常的产前诊断,尚需不断完善。     

细胞游离胎儿DNA产前诊断胎儿非整倍体异常

细胞游离胎儿DNA（cffDNA）已被成功在妊娠妇女血循环中检测到。与母体血循环中胎儿细胞不同,cffDNA最早可在妊娠4周检测出,并能在产后迅速消失。cffDNA可能源于胎盘部位的滋养细胞等并通过凋亡机制释放进入母体血循环中。通过检测cffDNA可产前诊断胎儿非整倍体异常,但因其在妊娠妇女血中含量较少,检测具有挑战性。目前已有多种新检测方法用于非整倍体异常的产前诊断,尚需不断完善。     

孕妇血浆中胎儿DNA在产前诊断中的应用

目的依据孕妇血浆中存在游离胎儿DNA的理论,寻找一种无创性产前基因诊断的新方法。方法提取42例孕14~40周的产妇血浆中游离胎儿DNA,采用引物延伸预扩增(primerextensionpreamplification,PEP)后行PCR,特异性扩增其Y染色体重复序列(DYZ3)基因。同时采用9个短串联重复序列(shorttandemrepeat,STR)多态性位点的多重扩增方法以检出血浆中胎儿DNA。结果DYZ3-PCR中,32例妊娠男性胎儿孕妇中有28例血浆中出现基因扩增带,检出率为87.5%,10例妊娠女性胎儿孕妇血浆有2例假阳性结果。性别总符合率为85.7%,STR-PCR中,检测出孕妇血浆中父源性胎儿DNA的存在。结论应用孕妇血浆中胎儿DNA作产前诊断敏感性和特异性较高,是一种无创性产前基因诊断方法,具有广泛的临床应用前景。     

孕妇血浆中游离胎儿DNA测定在产前诊断中的应用

传统的羊膜腔穿刺和绒毛吸取是目前许多医院较为常用的产前诊断技术，因其操作易导致流产、胎儿损伤或死亡、宫内感染、羊膜破裂等，使其在临床上的推广和应用受到了限制。利用孕妇外周血中的胎儿细胞进行遗传学诊断一直是无创性产前诊断的重要探索途径。但由于孕妇外周血中的胎儿细胞数量少，而且存在个体差异，目前很难从外周血中分离出完整的胎儿细胞，这无疑限制了这种方法在临床上的应用。1997年，有学者研究发现，孕妇血液循环中存在较丰富的游离胎儿DNA，这一发现为无创性产前诊断和筛查开辟了新领域。     

血浆/血清游离核酸与无创性产前诊断

循环核酸包括循环DNA和RNA,是近年发现的一类新的血清/血浆分子生物标志物。循环核酸的发现、研究开创了无创性产前诊断的新领域。本文主要阐述了母体血浆中胎儿游离核酸的生物学特性及其在产前胎儿性别鉴定和RHD血型分型、孟德尔疾病、异倍体检测、妊娠合并症方面的应用。     

胎儿游离核酸与子痫前期的研究进展

子痫前期(preeclampsia,PE)是全球范围内致母婴或新生儿死亡与致残的主要原因之一。目前尚无早期的诊断标准。一经检出,母婴已受到不同程度的损伤。因此,在PE临床症状出现前做出早期诊断对降低孕产妇与围生儿病死率、提高母婴健康具有重要意义。现有的常规产前诊断技术虽然可靠但可能危及母婴安全,因此寻找一种特异、无创、快速的诊断方法具有重要的临床意义。循环的胎儿细胞游离核酸,包括胎儿游离DNA、胎儿游离RNA以及微RNA(miRNA),作为一种特异性的无创性生物学标记物,其不仅可以有效地对PE进行预测,而且为无创性产前诊断开辟了新的方法,具有良好的发展前景。近年来,学者们在游离核酸与PE早期预测的相关性方面做了大量的研究。综述胎儿游离DNA、mRNA以及miRNA的发现、组织来源及其在PE中的早期预测作用。     

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。     

应用Rh阴性孕妇外周血胎儿游离DNA进行RhD血型产前基因诊断

目的探讨利用Rh阴性孕妇血浆中游离胎儿DNA进行无创性产前诊断胎儿RhD血型的方法。方法通过SRY基因确定胎儿DNA的存在，采用PCR-SSP技术对32例妊娠11～40周的单胎RhD阴性孕妇血浆中胎儿游离DNA进行RHD基因外显子5、7、10和内含子4的特异性扩增，其基因型结果与产后新生儿脐血血清学检测结果进行对比性分析，回顾性评价胎儿基因分型结果的准确性。结果32例样本中，28例基因定型结果与血清学表型相符，准确率达到87．5％，通过检测RHD1227A等位基因鉴定了RhD放散型，使结果达到93．75％（30／32）的准确率。结论利用Rh阴性孕妇血浆中游离胎儿DNA进行无创性诊断胎儿RhD血型，是一种简便、快速且无损伤性的产前基因诊断的可行性方法，可用于新生儿溶血病的预防和诊断，值得临床推广应用。     

Non-invasive prenatal diagnosis from the perspective of a low-resource country

Current clinical practice in obstetrics has shifted the paradigm from a conventional prenatal approach based on invasive procedures, risking both fetus and mother, to non-invasive prenatal testing for some fetal conditions via the analysis of cell-free fetal DNA in maternal blood. In the past 15 years, much research has been devoted to refining the methodology for measuring cell-free fetal DNA in maternal circulation and to exploring clinical applications of this technology as a potential tool for prenatal diagnosis. Since the rapid spread around the world of prenatal diagnosis based on cell-free fetal DNA, it is time to start thinking how this cutting-edge technology might influence current practice of obstetrics in low-resource countries.     

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.     

Noninvasive prenatal diagnosis of aneuploidy using cell-free nucleic acids in maternal blood: promises and unanswered questions

The discovery of cell-free fetal (cff) DNA and RNA in the maternal circulation has driven developments in noninvasive prenatal diagnosis (NIPD) for the past decade. Detection of paternally derived alleles in cff DNA is becoming well established. Now much interest is focussing on NIPD of fetal chromosomal abnormalities, such as trisomy 21, which is a considerable challenge because this demands accurate quantitative measurements of the amounts of specific cff DNA or cff RNA sequences in maternal blood samples. Emerging strategies for distinguishing and quantifying the fetal nucleic acids in the maternal circulation promise continued development of the field, and pose a number of unanswered questions.     

Tests non invasifs et maladies géniques : que peut-on faire ?

Analysis of cell-free fetal DNA (cffDNA) in maternal plasma is very promising for early diagnosis ofmonogenic diseases. However, noninvasive prenatal diagnosis (NIPD) of these disorders has been limited by the lack of sensitivity of the basic biomolecular methods, by the availability of suitable technical platforms, and the need to set up patient- or disease-specific custom-made approaches. Initially, it was only possible to identify DNA sequences that are either paternal in origin or have arisen de novo in some autosomal dominant conditions. NIPD can also be applied to autosomal recessive conditions if the parents carry different mutant alleles by excluding the presence of paternal mutant alleles in maternal plasma. More recently, advances in DNA technology, such as next-generation sequencing (NGS), have allowed accurate quantification of specific sequences in maternal plasma, and subsequently enabled noninvasive testing for conditions such as thalassaemia. Today, the hospital of Montpellier provides the first NIPD test for cystic fibrosis from maternal blood by analysis of the cffDNA by searching the paternal mutation in families with CFTR compound heterozygosity.     

Noninvasive prenatal diagnosis of Down syndrome: current knowledge and novel insights

The noninvasive prenatal diagnosis of trisomy 21 (Down syndrome) is an actively researched area of prenatal medicine, as this is the most common aneuploidy compatible with life and a major cause of mental retardation. The isolation of intact fetal cells, and most importantly, the successful detection of fetal-origin nucleic acids (cell-free fetal DNA and RNA), in maternal plasma even from the early stages of pregnancy has inspired scientists to develop discriminative genetic markers for the prenatal detection of aneuploidy. In the near future, the development of epigenetic fetal-specific markers will possibly allow the universal application of a cell-free fetal DNA-based diagnostic test regardless of the gender of the fetus or its polymorphic status. Other promising approaches rely upon the detection of free placentally derived RNA transcribed from genes located on chromosome 21 and the application of highly sensitive techniques, such as digital polymerase chain reaction and high-throughput shotgun sequencing. However, irrespective of which strategy is selected for isolating or distinguishing fetal genetic material in maternal plasma, the small quantity of fetal origin nucleic acids poses severe technical challenges. In this review article, we present an overview of the current knowledge in the field of noninvasive prenatal assessment of fetuses with Down syndrome and the future perspectives regarding new fetal markers and novel molecular techniques that may eventually be applied in the clinical setting as a valid and safe option for women who opt for noninvasive accurate prenatal diagnosis.     

Uses of cell free fetal DNA in maternal circulation

For over a decade, researchers have focused their attention on the development of non-invasive prenatal diagnosis tests based on cell-free fetal DNA circulating in maternal blood. With the possibility of earlier and safer testing, non-invasive prenatal diagnosis has the potential to bring many positive benefits to prenatal diagnosis. Non-invasive prenatal diagnosis for fetal sex determination for women who are carriers of sex-linked conditions is now firmly established in clinical practice. Other non-invasive prenatal diagnosis-based tests are set to follow, as future applications, such?as the detection of single-gene disorders and chromosomal abnormalities, are now well within reach. Here, we review recent developments in non-invasive prenatal diagnosis for genetic conditions and chromosomal abnormalities, and provide an overview of research into ethical concerns, social issues and stakeholder view points.     

Cell-free fetal nucleic acid testing: a review of the technology and its applications

Cell-free fetal nucleic acids circulating in the blood of pregnant women afford the opportunity for early, noninvasive prenatal genetic testing. The predominance of admixed maternal genetic material in circulation demands innovative means for identification and analysis of cell-free fetal DNA and RNA. Techniques using polymerase chain reaction, mass spectrometry, and sequencing have been developed for the purposes of detecting fetal-specific sequences, such as paternally inherited or de novo mutations, or determining allelic balance or chromosome dosage. Clinical applications of these methods include fetal sex determination and blood group typing, which are currently available commercially although not offered routinely in the United States. Other uses of cell-free fetal DNA and RNA being explored are the detection of single-gene disorders, chromosomal abnormalities, and inheritance of parental polymorphisms across the whole fetal genome. The concentration of cell-free fetal DNA may also provide predictive capabilities for pregnancy-associated complications. The roles that cell-free fetal nucleic acid testing assume in the existing framework of prenatal screening and invasive diagnostic testing will depend on factors such as costs, clinical validity and utility, and perceived benefit-risk ratios for different applications. As cell-free fetal DNA and RNA testing continues to be developed and translated, significant ethical, legal, and social questions will arise that will need to be addressed by those with a stake in the use of this technology. Target Audience: Obstetricians & Gynecologists and Family Physicians Learning Objectives: After participating in this activity, physicians should be better able to evaluate techniques and tools for analyzing cell-free fetal nucleic acids, assess clinical applications of prenatal testing, using cell-free fetal nucleic acids and barriers to implementation, and distinguish between relevant clinical features of cell-free fetal nucleic acid testing and existing prenatal genetic screening and diagnostic procedures.     

Cell-free Nucleic Acids as Potential Markers for Preeclampsia

Preeclampsia is one of the leading causes of maternal and fetal/neonatal mortality and morbidity worldwide. Therefore, widely applicable and affordable tests are needed to make an early diagnosis before the occurrence of the clinical symptoms. Circulating cell-free nucleic acids in plasma and serum are novel biomarkers with promising clinical applications in different medical fields, including prenatal diagnosis.Quantitative changes of cell-free fetal (cff)DNA in maternal plasma as an indicator for impending preeclampsia have been reported in different studies, using real-time quantitative PCR for the male-specific SRY or DYS 14 loci. In case of early onset preeclampsia, elevated levels may be already seen in the first trimester. The increased levels of cffDNA before the onset of symptoms may be due to hypoxia/reoxygenation within the intervillous space leading to tissue oxidative stress and increased placental apoptosis and necrosis. In addition to the evidence for increased shedding of cffDNA into the maternal circulation, there is also evidence for reduced renal clearance of cffDNA in preeclampsia. As the amount of fetal DNA is currently determined by quantifying Y-chromosome specific sequences, alternative approaches such as the measurement of total cell-free DNA or the use of gender-independent fetal epigenetic markers, such as DNA methylation, offer a promising alternative. Cell-free RNA of placental origin might be another potentially useful biomarker for screening and diagnosis of preeclampsia in clinical practice. Fetal RNA is associated with subcellular placental particles that protect it from degradation. Its levels are ten-fold higher in pregnant women with preeclampsia compared to controls. In conclusion, through the use of gender-independent sequences, the universal incorporation of fetal nucleic acids into routine obstetric care and into screening or diagnostic settings using combined markers may soon become a reality. Effort has now to be put into the establishment of standardized and simplified protocols for the analysis of these biomarkers in a clinical setting.