唐氏综合征高危孕妇血浆中游离胎儿DNA的Y染色体微缺失筛查

目的 利用孕妇血浆中游离胎儿DNA在孕早期进行Y染色体微缺失筛查,诊断男性胎儿无精子症因子(AZF)缺失情况。方法 留取2013年6月～2014年8月参加产前检测的16～34孕周唐氏综合征筛查高危孕妇的外周血标本89例,提取出全血基因组DNA后利用Y染色体微缺失检测试剂盒检测AZF微缺失。结果 86例孕妇妊娠至胎儿出生,其中男胎孕妇45例,女胎孕妇41例。妊娠女性胎儿的孕妇血浆DNA仅扩增出ZFX/ZFY对照基因,而妊娠男性胎儿的孕妇血浆DNA同时扩增出SRY,ZFX/ZFY对照基因,且有3例样本检测出AZF基因微缺失。结论 通过提取孕妇血浆中游离胎儿DNA能够检测出胎儿是否伴有AZF基因微缺失,从而提前预测胎儿今后罹患生精障碍的风险。     

用不提取核酸PCR扩增孕妇血浆中胎儿游离DNA的SRY和FMR1基因

摘要：目的：建立一种不提取核酸的孕妇血浆中胎儿游离DNA的检测方法。 方法：构建不提取核酸PCR法,用巢式PCR扩增Y染色体上的SRY基因序列和X染色体上的FMR1基因序列,检测44例孕妇血浆中游离DNA。根据随访判断结果的准确性。 结果：44例孕妇经随访确认24例有男性胎儿,20例有女性胎儿。24例孕男性胎儿的孕妇血浆标本中有21例扩增出SRY基因和FMR1基因,敏感性为87.5%（21/24);20例孕女性胎儿的孕妇血浆标本中有17例扩增出FMR1基因,敏感性为85.0%（17/20)。 结论：建立的不提取核酸PCR法具有快速、简便等优点,可用于性连锁遗传病的产前诊断。     

无创产前基因检测技术的临床应用价值评价

目的探讨无创产前基因检测技术在筛查胎儿染色体非整倍体中的作用。 方法选择需做产前诊断的74名孕妇,采用高通量基因测序技术检测母体血浆胎儿游离DNA,分析胎儿染色体拷贝数。依据患者产前诊断的临床指征分为4个组(高龄妊娠组、母体血清筛查高危组、B超胎儿异常组及生育过21-三体患儿组),计算每组高危患者的比率。对无创产前基因检测显示胎儿染色体非整倍体高危的11例患者,行羊膜腔穿刺进行胎儿染色体核型分析。 结果在各组中显示胎儿染色体非整倍体高危的比率是：高龄妊娠组10.50%(4/38),母体血清高危组17.40%(4/23),B超异常组30.00%,生育过21三体患儿组0.00%(0/3),总高危率14.90%(11/74)。在11例无创产前基因检测显示胎儿染色体非整倍体高危患者中,9例的无创产前筛查结果与胎儿染色体核型一致,一致率81.80%。2例患者为假阳性。 结论无创产前基因检测技术是一种筛查胎儿染色体非整倍体的有效方法,其在胎儿染色体异常疾病的产前检测中具有广泛的应用前景。     

产前筛查先天性缺陷与胎儿染色体异常的研究

目的 探讨孕中期血清标志物在产前筛查胎儿染色体异常的作用和价值.方法 对2555例孕中期(14～22周)孕妇血清甲胎蛋白、β-HCG和uE3 3项指标进行检测,并结合孕妇年龄、孕周、体质量、是否双胎、有无糖尿病等,采用仪器配套软件计算风险概率,对南风险孕妇进行染色体检查确认.结果 2555例孕妇中筛查出唐氏综合征高风险210例,占筛查总数的8.2％;18-三体综合征高风险26例,占筛查总数的1.0％;神经管缺陷高风险29例,占筛查总数的1.1％.高风险孕妇中有207例自愿进行了羊水细胞染色体检查或胎儿脐血染色体检查,检测出染色体异常核型12例,异常率为5.8％.结论 孕中期孕妇血清唐氏综合征、18-三体综合征、神经管缺陷联合筛查胎儿先天缺陷是行之有效的方法,可以作为产前筛查的常规项目.     

胎儿染色体异常的产前筛查

目的探讨孕早期胎儿染色体异常的产前筛查方案。方法采用时间分辨荧光免疫法对孕11—14周的2739例孕妇血清中的人绒毛膜促性腺激素β亚单位(β-hCG)、妊娠相关血浆蛋白A(PAPP-A)进行检测，同时利用腹式或阴式B超测量胎儿颈部半透明膜厚度；结合孕妇年龄筛查染色体异常高危胎儿，并通过绒毛或羊水细胞染色体核型分析进一步明确诊断。结果通过四联筛查方案筛查出染色体异常高危胎儿325例，经产前诊断确诊22例胎儿为染色体异常；胎儿染色体异常检出率91．67％，假阳性率11．16％。结论孕早期检测胎儿颈部半透明膜厚度、β-hCG、PAPP-A和年龄四联筛查方案对孕早期产妇产前筛查胎儿染色体异常有较好的实用价值。     

孕妇外周血胎儿ABO血型基因分型技术的建立

本研究目的是建立孕妇外周血胎儿ABO血型基因分型技术,用于ABO血型不合引起的新生儿溶血病的产前诊断。根据ABO血型基因DNA全序列和mRNA序列设计4对引物,选择20例健康供者血浆,提取血浆中DNA进行扩增,探索最佳的血浆DNA提取及PCR扩增条件,初步建立单人ABO血型基因分型技术。将O型血浆DNA与A型或B型血浆DNA按1：1、2：1、4：1、8：1、10：1、20：1、40：1、100：1进行混合,模拟孕妇外周血胎儿与孕妇自身ABO基因混合状态,建立混合ABO血型基因分型技术。选取14例孕30周以上的孕妇外周血标本,进行胎儿ABO血型基因型鉴定,并对孕妇进行追踪,尽量获取胎儿出生以后的外周血标本进行ABO血型鉴定,以评价孕妇外周血胎儿ABO血型基因分型技术的灵敏度与准确性。结果表明：单人血浆进行准确血型鉴定的最少模板DNA量约为0．625ng,500μl血浆提取的DNA量即可达到PCR扩增要求;当混合血浆中O型DNA所占比例≤10时,可以准确检测出非0基因的存在;14名O型孕妇外周血标本中9例标本扩增出非O型基因,5例未扩增出非0基因;通过血清学方法对5例胎儿出生后外周血进行ABO血型鉴定,其中A型3例,B型1例,O型1例,与其基因分型结果一致,符合率100％。结论：本研究建立的孕妇外周血胎儿ABO血型基因提取、分型技术,可以对妊娠中晚期胎儿ABO血型基因型进行准确鉴定,从而为新生儿溶血病的产前诊断与预防提供指导意见。     

孕妇外周血胎儿游离DNA在产前筛查胎儿染色体非整倍体疾病中的价值

目的探讨孕妇外周血胎儿游离DNA在产前筛查胎儿染色体非整倍体疾病中的临床应用价值。方法接受产前诊断孕12~26周孕妇4 300例,采集外周血提取胎儿游离DNA,应用高通量测序技术检测风险值,对检测结果提示高风险者进一步行羊水穿刺胎儿染色体核型分析及基因芯片检测,对检测结果提示低风险者电话随访至分娩。结果胎儿游离DNA高通量测序检测提示高风险者45例,其中21-三体综合征29例,18-三体综合征11例,13-三体综合征5例;1例13-三体综合征羊水穿刺胎儿染色体核型分析未见异常,基因芯片检测发现孕妇13号染色体存在微重复,余44例高风险妊娠均经羊水穿刺胎儿染色体核型分析证实;胎儿游离DNA高通量测序提示低风险4 255例,其中3 008例完成随访,新生儿未见明显异常。结论孕妇外周血胎儿游离DNA产前筛查可高效、准确筛查胎儿染色体非整倍体疾病,且较介入性产前诊断易接受,具有较好的临床应用价值。     

高龄孕妇唐氏筛查-产前诊断序贯方案的选择与评估

目的探讨适宜高龄孕妇唐氏综合征(DS)筛查-产前诊断的序贯方案。方法选取2013年1月至2016年9月于佛山市南海区妇幼保健院妇产科接受早期、中孕期DS血清学筛查以及无创DNA产前检测的3012例高龄孕妇为研究对象。在不同的孕周分别进行早期、中孕期DS血清学筛查以及无创DNA产前检测,对血清学筛查风险值≥l/270或者无创DNA结果高风险的孕妇,均建议行羊膜腔穿刺染色体检查,以确诊胎儿是否为DS;对未接受羊膜腔穿刺染色体检查的孕妇,采用电话随访方式随访其妊娠结局。将这3012例孕妇的筛查结果按照两种序贯方案进行模拟筛查(早期血清学筛查-无创DNA产前检测-染色体核型分析;(2)中期血清学筛查-无创DNA产前检测-染色体核型分析)。分别计算两种方案的假阳性率和筛查效率并进行统计学比较。结果当选择方案(1)时,3012例高龄孕妇中,产前筛查高风险孕妇256例,18例最终被确诊为DS,筛查的假阳性率和筛查效率分别为7.90%和7.03%;而当选择方案(2)时,筛查的假阳性率和筛查效率分别为15.10%和3.81%,两种方案相比较,差异有统计学意义(P0.01)。结论相比中期血清学筛查-无创DNA产前检测方法,采用早期血清学筛查-无创DNA产前检测的序贯筛查方案,具有更高的筛查效率,有较高的临床应用价值。     

孕妇血浆中胎儿游离DNA的提取

目的 探索从孕妇外周血浆中检测胎儿游离DNA（cfDNA）,评估利用cfDNA进行无创性产前基因诊断的可行性.方法 收集30例孕妇的外周血,利用柱吸收的方法提取血浆中的cfDNA,经琼脂糖凝胶电泳分离富集小片段cfDNA,用巢式PCR技术扩增胎儿SRY基因.结果 18例孕男胎的孕妇血浆中有16例经巢式PCR扩增出Y染色体SRY基因,12例孕女胎的孕妇血浆中均未扩增出SRY基因.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胎儿cfDNA,可用于无创性产前诊断.     

探讨胎儿超声异常孕妇无创DNA产前检测结果

目的:研究分析胎儿超声异常对于胎儿染色体异常的筛查应用价值,并分析无创DNA产前检测技术在产前胎儿缺陷筛查中的临床应用策略。方法:选取2018年1月至2018年12月在本院进行产前检查的孕妇2182例。先行对胎儿超声检查,对于超声检查中提示胎儿异常的孕妇进行无创DNA检查,根据两种检查结果分析产前胎儿超声检查对于胎儿染色体异常的筛查效果以及无创DNA产前检查的应用策略。结果:超声检查胎儿异常24例,无创DNA检查结果显示高风险3例,胎儿羊水细胞染色体核型分析核型异常2例。结论:胎儿产前超声检查可作为产前胎儿异常的重要筛查手段,无创DNA产前检测可作为准确性较高的补救性筛查方式,从而可提高筛查结果的准确率。     

Non-invasive fetal sex determination using a conventional nested PCR analysis of fetal DNA in maternal plasma

BACKGROUND: In order to provide a reliable non-invasive method for fetal sex determination in a routine setting, we evaluated the possibility of identifying the fetal Y chromosome-specific sequence in maternal plasma using conventional PCR analysis. METHODS: Fetal gender was determined in 31 pregnant women including one with a dizygotic twin pregnancy between 7 and 32 weeks of gestation using DNA extracted from 200 microl of each maternal plasma. The 198 bp SRY gene-specific sequence on Y chromosome and the 261 bp ATL1 gene-specific sequence on X chromosome were co-amplified in a multiplex nested PCR manner. The result was confirmed by routine analysis of fetal tissue obtained by invasive procedure or examination of newborns after delivery. RESULTS: The 198 bp SRY-specific sequence was detected in 15 plasma samples obtained from pregnant women carrying male fetuses. In the remaining cases bearing female fetuses, only the 261 bp ATL1 gene sequence was detected, producing 100% sensitivity and specificity of fetal sex prediction. The result was completely concordant with the conventional fetal tissue analysis and examination of the newborns after delivery. CONCLUSIONS: A conventional nested PCR analysis of maternal plasma could be used for accurate fetal gender detection and enable a reliable prospective non-invasive fetal sex determination which should enhance prenatal diagnostic options especially for sex-linked disorders.     

Specific magnetic bead-based capture of free fetal DNA from maternal plasma

An automated magnetic capture hybridization (MCH) method for the extraction and enrichment of fetal RHD specific DNA fragments from maternal plasma was developed using plasma from 1000 D-negative pregnant women. A real time PCR protocol for RHD exon 7 was applied. MCH was compared with the QIAamp DSP Virus Kit (QIAamp) as a reference. Compared with the QIAamp method, the percentage of fetal DNA increased from 2.86% to 4.83% (p < 0.05, n = 8). The 95% detection limit of MCH was determined at 286 pg/ml (43 geg/ml) compared with 138 pg/ml (21 geq/ml) for the QIAamp DSP Virus Kit.     

Quantitative analysis of the bidirectional fetomaternal transfer of nucleated cells and plasma DNA

BACKGROUND: Recently, much interest has been generated on the fetomaternal transfer of nucleated cells and plasma DNA. However, there has been no systematic quantitative comparison of these two directions and two modalities of trafficking within the same study population. METHODS: The fetus-to-mother transfer of nucleated cells and plasma DNA in pregnant women carrying male babies was studied using a real-time quantitative PCR assay for the S:RY gene. For mother-to-fetus transfer, real-time quantitative PCR assays for the insertion/deletion polymorphisms involving the glutathione S:-transferase M1 and angiotensin-converting enzyme genes were used. RESULTS: Of the 50 informative mother-baby pairs, maternal DNA was detected in the cellular fraction of umbilical cord blood in 24% of cases (12 of 50), at a median fractional concentration of 2.6 x 10(-4) (interquartile range, 1.7 x 10(-4) to 3.6 x 10(-4)). In the plasma fraction of cord blood, maternal DNA was detected in 30% (15 of 50) of cases at a median fractional concentration of 3 x 10(-3) (interquartile range, 1 x 10(-3) to 1.6 x 10(-2)). For the other direction of trafficking, fetus-to-mother transfer of nucleated cells was detected in 26% of cases (13 of 50) at a median fractional concentration of 3.2 x 10(-4) (interquartile range, 0.6 x 10(-4) to 7.6 x 10(-4)). In the plasma fraction, fetal DNA was detected in 100% of maternal plasma (50 of 50) at a median fractional concentration of 3 x 10(-2) (interquartile range, 1.4 x 10(-2) to 5. 3 x 10(-2)). CONCLUSIONS: This study indicated that significantly more fetal DNA is present in the plasma of pregnant women compared with DNA from the cellular fraction of maternal blood. In addition, maternal DNA was demonstrated in both the cellular and plasma fractions of cord blood after delivery. This study has therefore determined the fundamental quantitative values for the bidirectional fetomaternal cellular and plasma DNA traffic.     

Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma

BACKGROUND: Circulating fetal DNA (cfDNA) in maternal plasma has been measured to investigate its possible relationship with pregnancy-related disorders, including fetal trisomy 21 and preeclampsia. The circulating concentrations of single-copy fetal genes, however, are close to the detection limits of PCR methods. METHODS: We optimized a protocol for the real-time quantitative PCR amplification of the multicopy sequence DYS14 on the Y-chromosome. This was compared with an established real-time PCR assay for the single-copy SRY gene. RESULTS: By probit regression analysis, the measurements of male DNA by the DYS14 assay had a 10-fold lower detection limit (0.4 genome equivalents) than did measurements of SRY. For plasma samples from women in the first trimester of pregnancy, imprecision (CV) was 2%-22% when amplifying DYS14 compared with 26%-140% for SRY. CONCLUSIONS: The low copy numbers of fetal DNA in plasma of women in the first trimester of pregnancy cannot be measured precisely when targeting single-copy sequences. Better results are obtained by amplifying a sequence that is present in multiple copies per male genome.     

Noninvasive determination of fetal RHD status by examination of cell-free DNA in maternal plasma

BACKGROUND: Cell-free fetal DNA in maternal plasma opens the way for routine risk-free diagnosis of fetal D status of D- mothers. The focus was on accuracy of RHD typing and confirmation of fetal DNA in maternal plasma while RHD was not detected. STUDY DESIGN AND METHODS: Plasma DNA was extracted (by manual and/or automatic method) from 255 D- pregnant women and amplified in exons 7 and 10 and intron 4 of RHD gene with real-time polymerase chain reaction. The presence of fetal DNA was confirmed by testing SRY and, when negative, by one of 11 different polymorphisms found in the father but not in the mother. The results were compared with the D status of the newborns. RESULTS: After exclusion of 25 cases (10%) because of material shortage, in 230 cases (90%) available for complete study, the predictive value of the procedure of fetal RHD testing (RHD genotyping plus confirmation of fetal DNA) was 99.6 percent. SRY detection confirmed fetal DNA presence in maternal plasma in all boys, whereas the detection of various polymorphisms in all girls but one. CONCLUSIONS: Fetal RHD genotyping from maternal plasma may be used with confidence, although additional polymorphisms for confirmation of fetal DNA should be included for 100 percent predictive value (instead of 99.6%).     

利用母体血浆游离胎儿DNA进行胎儿SRY基因定量检测

目的探讨胎儿游离DNA在无创性产前诊断中的应用价值。方法行产前诊断的孕妇87例,分为中期妊娠(16～24周)61例(中孕组)和早期妊娠(11～14周)26例(早孕组),采用实时荧光定量PCR方法,利用Taqman探针对妊娠早、中期孕妇血浆游离的胎儿Y染色体性别决定基因(sex-determining region of Y-chromosome,SRY)进行扩增和定量分析。结果孕13周后的样本均得到特异性扩增,特异度和灵敏度均为100%;中孕组男性胎儿SRY检测的平均DNA浓度为155.46copies/mL血浆,实测范围为43.6～326.8copies/mL血浆;早孕组平均浓度为26.42copies/mL血浆,实测范围为14.3～73.3copies/mL血浆。结论采用荧光实时定量PCR在妊娠早、中期可稳定检测到胎儿游离DNA,可作为孕期性连锁遗传性疾病研究或产前诊断的有效补充手段。     

Size distributions of maternal and fetal DNA in maternal plasma

BACKGROUND: The discovery of fetal DNA in maternal plasma has opened up an approach for noninvasive prenatal diagnosis. Despite the rapid expansion in clinical applications, the molecular characteristics of plasma DNA in pregnant women remain unclear. METHODS: We investigated the size distribution of plasma DNA in 34 nonpregnant women and 31 pregnant women, using a panel of quantitative PCR assays with different amplicon sizes targeting the leptin gene. We also determined the size distribution of fetal DNA in maternal plasma by targeting the SRY gene. RESULTS: The median percentages of plasma DNA with size >201 bp were 57% and 14% for pregnant and nonpregnant women, respectively (P <0.001, Mann-Whitney test). The median percentages of fetal-derived DNA with sizes >193 bp and >313 bp were 20% and 0%, respectively, in maternal plasma. CONCLUSION: Plasma DNA molecules are mainly short DNA fragments. The DNA fragments in the plasma of pregnant women are significantly longer than those in the plasma of nonpregnant women, and the maternal-derived DNA molecules are longer than the fetal-derived ones.     

Hypermethylated RASSF1A in maternal plasma: A universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis

BACKGROUND: We recently demonstrated that the promoter of the RASSF1A gene is hypermethylated in the placenta and hypomethylated in maternal blood cells. This methylation pattern allows the use of methylation-sensitive restriction enzyme digestion for detecting the placental-derived hypermethylated RASSF1A sequences in maternal plasma. METHODS: We performed real-time PCR after methylation-sensitive restriction enzyme digestion to detect placental-derived RASSF1A sequences in the plasma of 28 1st-trimester and 43 3rd-trimester pregnant women. We used maternal plasma to perform prenatal fetal rhesus D (RhD) blood group typing for 54 early-gestation RhD-negative women, with hypermethylated RASSF1A as the positive control for fetal DNA detection. RESULTS: Hypermethylated RASSF1A sequences were detectable in the plasma of all 71 pregnant women. The genotype of plasma RASSF1A after enzyme digestion was identical to the fetal genotype in each case, thus confirming its fetal origin. Nineteen of the 54 pregnant women undergoing prenatal fetal RhD genotyping showed undetectable RHD sequences in their plasma DNA samples. The fetal DNA control, RASSF1A, was not detectable in 4 of the 19 women. Subsequent chorionic villus sample analysis revealed that 2 of these 4 women with negative RHD and RASSF1A signals were in fact carrying RhD-positive fetuses. CONCLUSIONS: Hypermethylated RASSF1A is a universal marker for fetal DNA and is readily detectable in maternal plasma. When applied to prenatal RhD genotyping, this marker allows the detection of false-negative results caused by low fetal DNA concentrations in maternal plasma. This new marker can also be applied to many other prenatal diagnostic and monitoring scenarios.     

利用孕妇血浆中胎儿游离DNA进行无创伤性产前基因诊断的研究进展

孕妇血浆中胎儿游离DNA,源于胎儿细胞和/或胎盘的凋亡,经核酸内切酶选择性地剪切为313 bp以下的短片段分子,因相对含量丰富,成为了当前无创伤性产前分子遗传诊断中胎儿DNA的重要来源,业已开展了胎儿性别鉴定、RhD阴性孕妇的Rh（D）基因检测、胎儿非整倍体病的诊断、STR遗传标记检测等应用研究。根据胎儿游离DNA的浓度、纯度、分子片段大小分布的特征和检测的靶基因,采用相应的分子基因诊断策略和实验设计,限制扩增产物的片段长度,有助于提高无创伤性产前胎儿分子遗传诊断的成功率。当前,大规模并行基因组测序技术为直接利用胎儿游离DNA进行无创伤性产前基因诊断开辟了新途径。