应用PCR对脆性X综合征进行产前筛查与诊断

目的对脆性X综合征进行产前基因筛查与诊断。方法采用聚合酶链式反应（polymerase chain reaction,PCR）和聚丙烯酰胺凝胶电泳技术,对46例孕妇及其胎儿的脆性X基因（CGG）n重复序列进行检测,同时采用PCR扩增牙幼基因对胎儿性别进行鉴定。结果在46例孕妇及其胎儿中,检出2例前突变携带者孕妇,2例男性患者胎儿。结论采用PCR扩增脆性X基因（CGG）n重复序列,结合扩增牙幼基因进行性别鉴定,可对脆性X综合征进行产前筛查与诊断。     

利用孕妇血浆中游离胎儿DNA进行胎儿性别诊断

目的评价从母血浆中分离游离胎儿DNA的可行性和它在临床的应用性。方法用巢式PCR复合扩增技术检测31例孕妇血浆中Y染色体上的SRY特异序列和X染色体上的ATL1特异序列。结果在15例怀有男性胎儿的孕妇血浆中检测到Y染色体的特异序列，在怀有女性胎儿的孕妇血浆中仅检测到X染色体的特异序列。胎儿性别检测的敏感性和特异性为100％。结论从母血浆中分离游离胎儿DNA是可行的，该技术可在临床上早期探测胎儿性别，预防X-连锁的遗传病患儿的出生。     

孕妇血浆中游离胎儿DNA的检测与分析

目的 建立不依赖于胎儿性别和父本DNA、可适用于染色体数目异常和单基因遗传病的无创性产前诊断方法.方法 应用降落PCR和二次PCR扩增技术,联合检测41例正常孕妇(其中孕龄7 w 1例,14～20 w 39例,32 w 1例)血浆中游离胎儿DNA的SRY基因和D17S1293、D21S11、DXS8377等3个短串联重复序列(short tandem repeat,STR)位点.SRY基因扩增产物进行琼脂糖凝胶电泳,STR位点扩增产物进行变性聚丙烯酰胺凝胶电泳并银染显色.结果 41例孕妇血浆DNA中均检出非母源性等位基因条带;联合SRY基因和X-STR位点鉴定胎儿性别,38例判断明确,3例不能明确判断性别.结论 检测孕妇血浆中的游离胎儿DNA,可快捷地获得男性胎儿和女性胎儿的父源性DNA信息,不仅适用于性连锁遗传疾病,而且适用于常染色体遗传疾病等的产前基因诊断.     

产前胎儿性别PCR鉴定方法及其在X连锁遗传病诊断中的应用

作者采用ＰＣＲ技术扩增Ｙ染色体长臂上的ＤＹＺ－１基因，对２２８例羊水进行了产前胎儿性别鉴定，其中４例为ＤＭＤ／ＢＭＤ的高危患儿，１例为甲型血友病的高危患儿。同时用常规细胞遗传学检查作比较，结果表明ＰＣＲ技术用于产前胎儿性别鉴定具有快速、简便、灵敏、特异性强、方法稳定可靠等优点，对Ｘ连锁遗传病的产前诊断具有重要作用。     

孕早期宫颈分泌物中获取胎儿细胞DNA进行产前诊断的可行性

目的：探索获取胎儿细胞DNA用于产前基因诊断的可行性，及能否用于预期胎儿性别。方法：收集92例孕早期人工流产妇女宫颈分泌物和绒毛组织，提取宫颈分泌物中DNA，扩增Y染色体特异重复序列DNA。短期培养制备绒毛染色体，分析核型确定流产胎儿性别。结果：92例经绒毛染色体核型分析的人工流产胎儿中，正确预期胎儿性别72例，准确率78％。结论：采集孕早期宫颈分泌物，可获得胎儿滋养层细胞DNA用于产前诊断，其准确性和可靠性有待进一步提高。     

MLPA联合遗传连锁分析在假肥大型肌营养不良症产前诊断中的价值

目的 探讨多重连接依赖探针扩增技术(multiplex ligation-dependent probe amplification,MLPA)联合短串联重复序列(short tandem repeat,STR)基因连锁分析用于Duchenne型假肥大型肌营养不良症(Duchenne muscular dystrophy,DMD)产前诊断的价值.方法 通过检测Y染色体性别决定基因(Y chromosome sex-determining gene,SR Y)判断胎儿性别;MLPA检测45个DMD家系中先证者、孕妇以及胎儿Dystrophin基因突变情况,并对家系成员和胎儿进行第45、49、50内含子以及5′和3 ′端STR的连锁分析.结果 45个进行产前诊断的家系中,SRY阳性31例,其中6例为DMD患病胎儿;阴性14例,其中4例为携带者,余未见异常.结论 MLPA能检测胎儿Dystrophin基因外显子突变情况,STR连锁能分析胎儿是否继承母源性风险X染色体,因此,STR连锁分析能发现MLPA技术检测不到的外显子突变胎儿.将两种方法结合起来用于DMD的产前诊断准确性更高.     

两种遗传标记系统鉴定母血中有核红细胞来源

目的探讨母血中胎儿有核红细胞的鉴定方法及其对无创性产前诊断的意义。方法86例孕妇外周血,以血红蛋白γ链染色结合显微操作分离获取胎儿有核红细胞。经PEP扩增胎儿细胞基因组后,共70例进行SRY基因检测;32例进行X-STR位点DXS101、DXS6797分析并与双亲基因型对比,判断模板细胞来源及胎儿性别。结果所有孕妇血样均检出胎儿有核红细胞。SRY基因判断胎儿性别的准确率为97.14%(68/70)。联用DXS101、DXS6797对胎儿细胞鉴定及性别判断的准确率达100%(32/32)。16例同时接受2种方法,检测结果均一致。结论采用SRY基因或多X-STR位点作为母血中胎源细胞鉴定系统,两种方法均能有效判断细胞来源,且各有优缺点,两者结合将有更广阔的应用前景。     

超声测量胎儿侧脑室轻度增宽与临床预后的相关性分析

目的探讨胎儿轻度侧脑室增宽与临床预后之间的关系。方法选择产前检查后发现的胎儿侧脑室后角轻度增宽34例作为观察组,胎儿正常的30例作为对照组,进行新生儿神经行为测定(NBNA)评分,两组比较分析。结果34例胎儿侧脑室轻度增宽中33例做了染色体检查,仅1例染色体为21-三体,1例未查染色体;有3例在妊娠中期被首次发现后因加重而引产;与胎儿性别无统计学意义;观察组的出生后新生儿NBNA评分较对照组高,有统计学意义。结论侧脑室轻度增宽的胎儿,尤其是>12mm的胎儿,出生后发生神经系统发育不良的风险较正常者增加;侧脑室轻度增宽与胎儿性别无关;因胎儿脑室扩张是一动态的过程,如不合并染色体和其他的结构异常,大部分预后良好。     

两例Y染色体部分缺失胎儿的产前诊断

目的对两例Y染色体部分缺失胎儿进行产前诊断。方法采用常规G显带及C显带技术分析胎儿及父亲的核型,采用荧光原位杂交(fluorescence in situ hybridization,FISH)、染色体拷贝数变异检测技术(copy number varaition sequencing,CNV-seq)性别决定基因(sex region of Y chromosome,SRY)检测技术及无精子因子(azoospermia factor,AZF)检测技术检测胎儿DNAO结果2例胎儿羊水染色体在320〜400条带水平均提示46,XN,del(Y)(qll.2),Y染色体着丝粒探针FISH检测结果均提示Y染色体数目未见异常。2例胎儿父亲外周血染色体核型均未见明显异常。胎儿羊水DNA拷贝数检测提示一例胎儿Y染色体q 11.221-ql2处缺失12.88 Mb,涉及全部AZFb+AZFc区域;另一例胎儿Y染色体qll.21-ql2处缺失14.84 Mb,涉及全部AZF区域。2例胎儿羊水SRY基因检测提示SRY基因阳性,SKY基因编码区未检测到已报道的致病点突变。2例胎儿基因检测提示存在AZF部分或全部缺失。结论联合多种技术有助于明确诊断Y染色体结构异常。CNV-seq检测有利于快速筛查胎儿Y染色体微缺失,可做为对染色体核型分析的补充和验证的方法。     

荧光定量PCR检测孕妇外周血中游离胎儿DNA在子痫前期中的价值

目的探讨孕妇外周血中的游离胎儿DNA水平作为子痫前期预测指标的可能。方法普通PCR检测子痫前期孕妇22例和正常孕妇25例的血浆中Y染色体性别决定基因（SRY）,然后应用荧光定量PCR技术对有SRY基因表达者的胎儿DNA水平进行定量分析并比较子痫前期孕妇组和正常孕妇组胎儿DNA水平的差异。结果22例子痫前期患者及25例对照组中各有12例出现SRY阳性信号。子痫前期组的胎儿DNA水平明显高于正常对照组（P〈0.01）。结论孕妇外周血中的游离胎儿DNA可望作为预测子痫前期的指标之一。     

Identification of fetal mesenchymal stem cells in maternal blood: implications for non-invasive prenatal diagnosis

Strategies for genetic prenatal diagnosis on fetal cells in the maternal circulation have been limited by lack of a cell type present only in fetal blood. However, the recent identification of mesenchymal stem cells (MSC) in first trimester fetal blood offers the prospect of targeting MSC for non-invasive prenatal diagnosis. We developed protocols for fetal MSC enrichment from maternal blood and determined sensitivity and specificity in mixing experiments of male fetal MSC added to female blood, in dilutions from 1 in 10(5) to 10(8). We then used the optimal protocol to isolate fetal MSC from maternal blood in the first trimester, using blood taken after surgical termination of pregnancy as a model of increased feto-maternal haemorrhage. In model mixtures, we could amplify one male fetal MSC in 2.5 x 10(7) adult female nucleated cells, yielding a 100% pure population of fetal cells, but not one fetal MSC in 10(8) nucleated cells. Fetal MSC were identified in one of 20 post-termination maternal blood samples and confirmed as fetal MSC by XY fluorescence in-situ hybridization (FISH), immunophenotyping and osteogenic and adipogenic differentiation. We report the isolation of fetal MSC from maternal blood; however, their rarity in post-termination blood suggests they are unlikely to have a role in non-invasive prenatal diagnosis. Failure to locate these cells routinely may be attributed to their low frequency in maternal blood, to sensitivity limitations of enrichment technology, and/or to their engraftment in maternal tissues soon after transplacental passage. We speculate that gender microchimerism in post-reproductive maternal tissues might result from feto-maternal trafficking of MSC in early pregnancy.     

Quantification of all fetal nucleated cells in maternal blood between the 18th and 22nd weeks of pregnancy using molecular cytogenetic techniques

Different types of nucleated fetal cells (trophoblasts, erythroblasts, lymphocytes, and granulocytes) have been recovered in maternal peripheral blood. In spite of many attempts to estimate the number of fetal cells in maternal circulation, there is still much controversy concerning this aspect. The numbers obtained vary widely, ranging from 1 nucleated cell per 104 to 1 per 109 nucleated maternal cells. The purpose of our project was to determine the absolute number of all different types of male fetal nucleated cells per unit volume of peripheral maternal blood. Peripheral blood samples were obtained from 12 normal pregnant women known to carry a male fetus between 18 and 22 weeks of pregnancy. Three milliliters (3 ml) of maternal blood has been processed without any enrichment procedures. Fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS) were performed, and fetal XY cells were identified (among maternal XX cells) and scored by fluorescent microscopy screening. The total number of male fetal nucleated cells per milliliter of maternal blood was consistent in each woman studied and varied from 2 to 6 cells per milliliter within the group of normal pregnancies. The number of fetal cells in maternal blood, at a given period, is reproducible and can therefore be assessed by cytogenetic methods. This confirms the possibility of developing a non-invasive prenatal diagnosis test for aneuploidies. Furthermore, we demonstrate that it is possible to repeatedly identify an extremely small number of fetal cells among millions of maternal cells.     

Fetal cells in the maternal circulation: isolation by multiparameter flow cytometry and confirmation by polymerase chain reaction.

During pregnancy, nucleated fetal erythrocytes enter the maternal circulation and can be isolated efficiently from the maternal cells by multiparameter flow cytometry. Male DNA, implying presence of a male fetus, can be identified in flow-sorted maternal blood by polymerase chain reaction with oligonucleotide primers flanking single-copy Y-specific DNA sequences. Among flow-sorted samples, we correctly identified fetal sex in 17/18 (94%) pregnancies of 10-21 weeks gestation. Maternal blood thus provides a potential opportunity for prenatal diagnosis that could preclude the need for invasive procedures in current use.     

Nucleated erythrocytes in maternal blood: quantity and quality of fetal cells in enriched populations

The discovery of nucleated erythrocytes in maternal circulationprovides a potential source for non-invasive prenatal diagnosis.We have evaluated the use of a three-stage procedure to determinethe number of cells that are of fetal rather than maternal origin.First, monoclonal antibodies specific for CD45 and CD14 wereused in conjunction with a magnetic (MACS) column to depleteunwanted leukocytes from maternal blood. This was followed bya positive MACS enrichment for nucleated erythrocytes, usingan anti-CD71 (transferrin receptor) monoclonal antibody. Todiscriminate between fetal nucleated erythrocytes and thoseof maternal origin, enriched fractions were simultaneously stainedwith an anti-fetal haemoglobin (HbF) antibody and hybridizedwith probes specific for X and Y chromosomes. Samples were thensubjected to blind analysis along with negative control samplesfrom non-pregnant volunteers. Using this dual analysis, we wereable to determine that less than one nucleated erythrocyte perml of maternal blood was of fetal origin. Small numbers of thesefetal cells were found in 87.5% of pregnancies, ranging from6 to 35 weeks gestational age. Comparison of HbF and X/Y probedata also suggests that the fetal cells are less suitable forfluorescence in-situ hybridization (FISH) analysis than similarpreparations from other sources. cell separation methods/fluorescence in-situ hybridization/hereditary diseases/polymerase chain reaction/pregnancy     

Fetomaternal cell trafficking: a new cause of disease?

Isolation of fetal cells from maternal blood is under active investigation as a noninvasive method of prenatal diagnosis. In the context of studying cell surface antigens expressed on fetal cells we discovered that fetal cells from a prior pregnancy also could be detected. This led to the appreciation of the persistence of fetal cells in maternal blood for as long as 27 years postpartum, and the realization that following pregnancy, a woman becomes a chimera. Quantitative polymerase chain reaction analyses have shown that a term pregnancy is not required for the subsequent development of fetal cell microchimerism. As many as 500,000 fetal nucleated cells are transfused following an elective first trimester termination of pregnancy. The relationship between fetal cell microchimerism and maternal disease is currently being explored. During pregnancy, fetal cells in the maternal skin are related to polymorphic eruptions of pregnancy and increased fetomaternal trafficking is detectable in cases of preeclampsia. After delivery, more male DNA of presumed fetal origin is present in the blood and skin of women with scleroderma as compared with healthy controls. Scleroderma is of particular interest because it shows a strong female predilection and it is an autoimmune disease with clinical similarities to graft-versus-host disease. Fetomaternal cell trafficking provides a potential explanation for the increased prevalence of autoimmune disorders in adult women following their childbearing years.     

用荧光原位杂交从母血中检测胎儿细胞

目的 从母血中分离胎儿细胞并确定其来自胎儿。方法 从孕早、中期各２０名、分娩后１５名母血中富集并分离有核细胞。用Ｙ特异性探针（ＰＹ３．４）行荧光原位杂交,从中识别胎儿细胞。结果 孕早、中期孕妇各怀１５名男胎。阳性细胞比例是１：６５２８．０及１：２７３．８。与同期１０名女胎阳性细胞相比,差别有高度显著性。分娩１周内的３名,阳必民孕中期的差别没有显著性。分娩３个月后的阳性率与比,差别有高度显著性。分娩     

Prenatal diagnosis from maternal blood: simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative magnetic cell sorting.

Fetal nucleated cells in the maternal circulation constitute a potential source of cells for the non-invasive prenatal diagnosis of fetal genetic abnormalities. We have investigated the use of the Magnetic Activated Cell Sorter (MACS) for enriching fetal nucleated erythrocytes. Mouse monoclonal antibodies specific for CD45 and CD32 were used to deplete leucocytes from maternal blood using MACS sorting, thus enriching for fetal nucleated erythrocytes which do not express either of these antigens. However, significant maternal contamination was present even after MACS enrichment preventing the accurate analysis of fetal cells by interphase fluorescence in situ hybridisation (FISH). To overcome this problem, we used simultaneous immunophenotyping of cells with the mouse antifetal haemoglobin antibody, UCH gamma, combined with FISH analysis using chromosome X and Y specific DNA probes. This approach enables selective FISH analysis of fetal cells within an excess of maternal cells. Furthermore, we have confirmed the potential of the method for clinical practice by a pilot prospective study of fetal sex in women referred for amniocentesis between 13 and 17 weeks of gestation.     

Noninvasive prenatal diagnosis of chromosomal aneuploidies by isolation and analysis of fetal cells from maternal blood.

The isolation and analysis of nucleated fetal cells (NFCs) from maternal blood may represent a new approach to noninvasive prenatal diagnosis. Although promising, these techniques require highly accurate separation of NFCs from nucleated cells of maternal origin; the two major problems limiting these techniques are the relative rarity of fetal cells in maternal blood and the need to establish their fetal origin. We now report a novel procedure that has allowed accurate separation of NFCs from maternal cells. The technique reported involves direct micromanipulator isolation of histochemically identified hemoglobin F-positive nucleated cells to obtain fetal nucleated red blood cells (FNRBCs) of high yield and purity. Using this technique, followed by cell-by-cell multicolor fluorescence in situ hybridization (FISH) analysis of purified FNRBCs, we were able to detect some of the most common human aneuploidies (including Down syndrome, Klinefelter syndrome, and trisomy 13) in 33 pregnant women referred for amniocentesis. The procedure used, which can be completed in <72 hrs, produced complete concordance with the results of amniocentesis. We also confirm findings of prior studies suggesting that the number of FNRBCs in maternal circulation is remarkably higher in abnormal pregnancies than in normal pregnancies, especially in women carrying a fetus with trisomy 21.     

Fetomaternal cell trafficking: A new cause of disease?

Isolation of fetal cells from maternal blood is under active investigation as a noninvasive method of prenatal diagnosis. In the context of studying cell surface antigens expressed on fetal cells we discovered that fetal cells from a prior pregnancy also could be detected. This led to the appreciation of the persistence of fetal cells in maternal blood for as long as 27 years postpartum, and the realization that following pregnancy, a woman becomes a chimera. Quantitative polymerase chain reaction analyses have shown that a term pregnancy is not required for the subsequent development of fetal cell microchimerism. As many as 500,000 fetal nucleated cells are transfused following an elective first trimester termination of pregnancy. The relationship between fetal cell microchimerism and maternal disease is currently being explored. During pregnancy, fetal cells in the maternal skin are related to polymorphic eruptions of pregnancy and increased fetomaternal trafficking is detectable in cases of preeclampsia. After delivery, more male DNA of presumed fetal origin is present in the blood and skin of women with scleroderma as compared with healthy controls. Scleroderma is of particular interest because it shows a strong female predilection and it is an autoimmune disease with clinical similarities to graft‐versus‐host disease. Fetomaternal cell trafficking provides a potential explanation for the increased prevalence of autoimmune disorders in adult women following their childbearing years. Am. J. Med. Genet. 91:22–28, 2000. © 2000 Wiley‐Liss, Inc.     

Noninvasive prenatal diagnosis of chromosomal aneuploidies by isolation and analysis of fetal cells from maternal blood

The isolation and analysis of nucleated fetal cells (NFCs) from maternal blood may represent a new approach to noninvasive prenatal diagnosis. Although promising, these techniques require highly accurate separation of NFCs from nucleated cells of maternal origin; the two major problems limiting these techniques are the relative rarity of fetal cells in maternal blood and the need to establish their fetal origin. We now report a novel procedure that has allowed accurate separation of NFCs from maternal cells. The technique reported involves direct micromanipulator isolation of histochemically identified hemoglobin F‐positive nucleated cells to obtain fetal nucleated red blood cells (FNRBCs) of high yield and purity. Using this technique, followed by cell‐by‐cell multicolor fluorescence in situ hybridization (FISH) analysis of purified FNRBCs, we were able to detect some of the most common human aneuploidies (including Down syndrome, Klinefelter syndrome, and trisomy 13) in 33 pregnant women referred for amniocentesis. The procedure used, which can be completed in <72 hrs, produced complete concordance with the results of amniocentesis. We also confirm findings of prior studies suggesting that the number of FNRBCs in maternal circulation is remarkably higher in abnormal pregnancies than in normal pregnancies, especially in women carrying a fetus with trisomy 21. © 2001 Wiley‐Liss, Inc.