Rapid prenatal diagnosis of Down Syndrome using quantitative fluorescent PCR in uncultured amniocytes

Rapid prenatal diagnosis of common chromosome aneuploidies have been successful through quantitative fluorescent PCR (QF-PCR) assays and small tandem repeat (STR) markers. The purpose of our study was to investigate the clinical feasibility for rapid prenatal detection of Down syndrome using the quantitative fluorescent PCR in uncultured amniocytes. DNA was extracted from uncultured amniotic fluid of normal karyotype (n=200) and of Down syndrome (n=21). It was amplified using QF-PCR with four STR markers located on chromosome 21. Among normal samples, the ranges of diallelic peaks for at least one STR marker were 1.0-1.3 for D21S11, 1.0-1.4 for D21S1411 and 1.0-1.5 for D21S1270. Down syndrome samples showed trisomic triallelic patterns or trisomic diallelic patterns. The sensitivity, specificity, and efficiency of the assay for detecting Down syndrome were 95.4%, 100%, and 99.5%, respectively. Rapid prenatal diagnosis of Down syndrome using QF-PCR is a reliable technique that aids clinical management of pregnancy.     

Human chromosome 21-specific DNA markers are useful in prenatal detection of Down syndrome

Trisomy 21 is the most common chromosomal aberration in live births. In this study we employed human chromosome 21-specific short tandem repeat (STR) DNA markers to determine the numbers of chromosome 21 present in fetal cells. Forty amniotic fluid samples from pregnancies complicated with fetal Down syndrome and 98 samples from euploid pregnancies were analyzed for D21S11 and interferon-alpha receptor (IFNAR) gene intervening sequence. Fluorescent dye-labeled primers were used in PCR amplification of these 2 markers. The PCR amplicon was analyzed with an automatic DNA sequence analyzer. The results showed that 35 of 40 fetal Down syndrome samples analyzed for IFNAR showed 3 distinct peaks, while 24 of 30 cases analyzed for D21S11 showed 3 distinct peaks. Two Down syndrome samples showed two uneven peaks. By analyzing 98 euploid pregnancies as controls, the ratios of area under the peaks were determined to be 1.31 +/- 0.22 and 1.96 +/- 0.18 (mean +/- SD) for the euploid pregnancies and pregnancies complicated by fetal Down syndrome with 2 peaks, respectively. Our data showed that altogether 39 of 40 (97.5%) Down syndrome cases were correctly identified based on either the 3-peak pattern in one or more of the DNA markers or the relative peak area ratio calculation. In conclusion, polymorphic STR DNA markers are useful for determining the numbers of chromosome 21 in fetal cells. The high sensitivity and automation of the procedures suggest a good prospect for use of this method in prenatal detection of fetal Down syndrome. However, this is a preliminary investigation and a large-scale study is necessary to validate the clinical application of this protocol.     

Sex ratios in fetuses and liveborn infants with autosomal aneuploidy

Ten data sources were used substantially to increase the available data for estimating fetal and livebirth sex ratios for Patau (trisomy 13), Edwards (trisomy 18), and Down (trisomy 21) syndromes and controls. The fetal sex ratio estimate was 0.88 (N = 584) for trisomy 13, 0.90 (N = 1702) for trisomy 18, and 1.16 (N = 3154) for trisomy 21. All were significantly different from prenatal controls (1.07). The estimated ratios in prenatal controls were 1.28 (N = 1409) for CVSs and 1.06 (N = 49427) for amniocenteses, indicating a clear differential selection against males, mostly during the first half of fetal development. By contrast, there were no sex ratio differences for any of the trisomies when comparing gestational ages <16 and >16 weeks. The livebirth sex ratio estimate was 0.90 (N = 293) for trisomy 13, 0.63 (N = 497) for trisomy 18, and 1.15 (N = 6424) for trisomy 21, the latter two being statistically different than controls (1.05) (N = 3660707). These ratios for trisomies 13 and 18 were also statistically different than the ratio for trisomy 21. Only in trisomy 18 did the sex ratios in fetuses and livebirths differ, indicating a prenatal selection against males >16 weeks. No effects of maternal age or race were found on these estimates for any of the fetal or livebirth trisomies. Sex ratios for translocations and mosaics were also estimated for these aneuploids. Compared to previous estimates, these results are less extreme, most likely because of larger sample sizes and less sample bias. They support the hypothesis that these trisomy sex ratios are skewed at conception, or become so during embryonic development through differential intrauterine selection. The estimate for Down syndrome livebirths is also consistent with the hypothesis that its higher sex ratio is associated with paternal nondisjunction. © 1996 Wiley-Liss, Inc.     

应用STR-荧光定量PCR行产前唐氏综合征诊断的初步研究

目的建立快速高效的产前诊断唐氏综合征的方法。方法选取21号染色体上唐氏综合征关键区域内的4个串联重复序列（STR）（D21S1413,D21S1414,D21S1446,D21S1437）作为遗传标记,采用荧光定量PCR扩增技术（QF—PCR）及片段分析技术来检测178例羊水标本,并与羊水标本的染色体核型分析结果相比对,评价QF—PCR方法的特异性和灵敏度。结果所有178例羊水标本中经核型分析证实有19例为21三体,另有2例为性染色体数目异常,19例唐氏综合征样本均可由QF—PCR法扩增检出。19例唐氏综合症样本,采用4对STR引物同时检测,阳性诊断率达100％。结论基于STR的QF—PCR技术具有简单、快速及准确等优点,对唐氏综合征大规模的产前基因诊断具有很好的临床价值。     

STR新位点产前诊断21、18、13三体综合征

目的短串联重复新位点D13S252、D13S305、D13S628、D13S325,D18S390、D18S819、D18S978、D21S1409、D21S1435在产筛高危孕妇中产前诊断21、18、13三体。方法选择产前血清学筛查高危和或胎儿B超异常100例孕妇羊水和脐血样本为研究对象,包含上述位点的单管多重体系检测21、18、13三体,PCR产物测序检测,Gene MapperID3.2软件分析处理。计算体系中各位点杂合度和多态信息含量。结果 97例扩增,检测出25例染色体非整倍体,包括18例21-三体,6例18-三体,1例13-三体,和72例正常二体,3例扩增失败。所有结果与染色体核型分析相符。体系中各STR位点的等位基因数4～14个,H为0.61～0.88,PIC为0.556～0.870。新位点的PIC为0.575～0.793,均0.5。21号染色体联合使用4个以上STR位点,18号染色体5个以上位点检出率才能达100%。结论上述STR新位点杂合度和多态信息含量高,可用于21、18、13三体产前诊断。     

Rapid prenatal diagnosis of trisomy 21 by real-time quantitative polymerase chain reaction with amplification of small tandem repeats and S100B in chromosome 21

Trisomy 21 (Down syndrome) is the most common congenital anomaly, and it occurs in one out of 700-1000 births. Current techniques such as amniocentesis and chorionic villi sampling (CVS) require lengthy laboratory culture procedures and high costs. This study was undertaken to establish a rapid prenatal diagnosis of trisomy 21 using real-time quantitative polymerase chain reaction (PCR) of fetal DNA from amniotic fluid. Real-time quantitative PCR was performed with DNA templates obtained from 14 normal blood samples, 10 normal amniotic fluid samples, 14 Down syndrome blood samples, and 7 Down syndrome amniotic fluid samples. Primers for D21S167 and S100B of chromosome 21 were used. Primers that direct the amplification of the 165-bp fragment of the insulin-like growth factor (IGF)-1 gene on chromosome 12 using a PCR primer were included to generate an internal standard for quantitation. The relative levels of D21S167 and S100B were 2.6 and 2.4 times higher in the blood of Down syndrome patients than those in the control group. The differences between these two groups were statistically significant (p-values were 0.0012 and 0.0016, respectively). The relative levels of D21S167 and S100B were 2.1 and 2.7 times higher in the amniotic fluid of Down syndrome fetuses than those in the control group. The difference between these two groups was statistically significant (p-values were 0.0379 and 0.0379, respectively). Prenatal diagnosis of trisomy 21 by real-time quantitative PCR using STR (small tandem repeats) amplification of D21S167 and S100B is a useful, accurate and rapid diagnostic method. Furthermore, it may also be useful for prenatal diagnosis with fetal DNA from maternal blood, and for preimplantation genetic diagnosis and prenatal counseling.     

荧光定量PCR产前快速诊断唐氏综合征

目的探索荧光定量PCR方法在产前快速诊断唐氏综合征中的作用。方法收集65例妊娠20~24w,进行羊水穿刺胎儿细胞培养染色体核型分析的胎儿羊水,采用荧光定量PCR技术对标本D21S11位点进行PCR扩增、检测,并与染色体核型分析的结果进行对照分析。结果65例未培养羊水标本经过荧光定量PCR检测,发现2例唐氏综合征,显示1:1:1三条或2:1两条D21S11位点等位基因带,63例显示1:1两条带;与细胞培养染色体核型分析的结果基本一致。结论荧光定量PCR技术具有检测速度快、需要模板量少,检测结果准确等特点,可用于染色体异常的产前快速诊断。     

Examination of trisomy 13, 18 and 21 foetal tissues at different gestational ages using FISH

In man high levels of aneuploidy are seen in spontaneous abortions. Very few autosomal trisomies survive to birth, the three most common being those for chromosome 13, 18 and 21 giving rise to the syndromes named Patau, Edwards and Down respectively. Since the majority of these spontaneously abort, what makes the survivors different from the aborters? Could it be that they have tissue specific mosaicism with the additional normal cell line supporting survival? In this study fluorescence in situ hybridisation was used as a convenient way to detect trisomy in interphase cells. To study the level of mosaicism across gestation, different tissues from 21 trisomic foetuses were analysed using probes for chromosome 13, 18, 21, X and Y. Two trisomy 18 foetuses exhibited mosaicism. Two others, one trisomy 13 and one trisomy 18 had mosaic placentas. There was no clear association between the limited mosaicism seen and severity of the phenotype. We conclude that at least for this sample set, tissue-specific mosaicism was not likely to be responsible for potential survival to birth.     

21三体综合征的快速基因诊断

目的探讨一种快速、简便、准确检测21三体综合征的基因诊断方法。方法选用位于21号染色体Down′s综合征核心区（21q21-21q22）内及其附近的短串联重复序列（STR）（D21S1432,D21S11,D21S1436,D21S1442,D21S1270,D21S1411,D21S1446）,建立STR-PCR快速诊断平台。通过对经染色体核型分析的10例正常胎儿的羊水脱落细胞DNA,及4例判定为21三体综合征胎儿的绒毛组织DNA进行21号染色体STR分析,从而进行方法评价。结果在上述7个STR位点中,10名正常胎儿除在D21S1436位点存在2∶1条带的占5/10,其余位点杂合度（64.29%-92.86%）及相互间的符合率为100%。4例21三体胎儿经6个STR位点联合分析,均被确诊为21三体综合征。结论6个STR位点（D21S1432,D21S11,D21S1442,D21S1270,D21S1411,D21S1446）可作为21三体综合征有价值的遗传标记,可用于对其做出快速、准确的基因诊断。     

Systematic evidence-based review: The application of noninvasive prenatal screening using cell-free DNA in general-risk pregnancies

PurposeNoninvasive prenatal screening (NIPS) using cell-free DNA has been assimilated into prenatal care. Prior studies examined clinical validity and technical performance in high-risk populations. This systematic evidence review evaluates NIPS performance in a general-risk population.MethodsMedline (PubMed) and Embase were used to identify studies examining detection of Down syndrome (T21), trisomy 18 (T18), trisomy 13 (T13), sex chromosome aneuploidies, rare autosomal trisomies, copy number variants, and maternal conditions, as well as studies assessing the psychological impact of NIPS and the rate of subsequent diagnostic testing. Random-effects meta-analyses were used to calculate pooled estimates of NIPS performance (P < .05). Heterogeneity was investigated through subgroup analyses. Risk of bias was assessed.ResultsA total of 87 studies met inclusion criteria. Diagnostic odds ratios were significant (P < .0001) for T21, T18, and T13 for singleton and twin pregnancies. NIPS was accurate (≥99.78%) in detecting sex chromosome aneuploidies. Performance for rare autosomal trisomies and copy number variants was variable. Use of NIPS reduced diagnostic tests by 31% to 79%. Conclusions regarding psychosocial outcomes could not be drawn owing to lack of data. Identification of maternal conditions was rare.ConclusionNIPS is a highly accurate screening method for T21, T18, and T13 in both singleton and twin pregnancies.     

天津地区D21S1411和D21S1413 2个STR基因座遗传多态性的研究

目的研究天津地区D21S1411和D21S14132个STR基因座的遗传多态性,探讨其在唐氏综合征基因诊断及产前基因诊断应用的可行性。方法随机选择301例天津地区无亲缘关系的汉族个体及313例染色体核型分析正常的胎儿样本,盐析法提取DNA,聚合酶链式反应扩增,非变形聚丙烯酰胺凝胶电泳,硝酸银染色,凝胶图像分析管理系统分析扩增产物。结果D21S1411基因座共发现9种等位基因、45种基因型;D21S1413基因座共发现7种等位基因、28种基因型。外周血样本D21S1411基因座的PIC、DP、PE和Ho分别为0．85、0．967、0．483和73．4％,D21S1413基因座的PIC、DP、PE和Ho分别为n83、0．949、0.719和86．20k;胎儿样本D21S1411基因座的PIC、DP、PE和H0分别为0．80、n927、0．494和78．5％,D21S1413基因座的PIC、DP、PE和Ho分别为0．76、0．915、0．808和87．2％。结论D21S1411和D21S14132个STR基因座基因杂合度高、具有高鉴别力,是21号染色体上理想的遗传标记,可用于DS的基因诊断及产前基因诊断。     

Prenatal detection of trisomy 21 and 18 from amniotic fluid by quantitative fluorescent polymerase chain reaction.

Prenatal diagnosis of fetal trisomies is usually performed by cytogenetic analysis on amniotic fluid. This requires lengthy laboratory procedures and high costs, and is unsuitable for large scale screening of pregnant women. An alternative method, which is both rapid and inexpensive and suitable for diagnosing trisomies even from single fetal cells, is the fluorescent polymerase chain reaction using polymorphic small tandem repeats (STRs). In this paper we present the preliminary results of a larger study comparing parallel prenatal diagnoses of trisomies 21 and 18 using cytogenetics with quantitative fluorescent polymerase chain reaction using STR markers. The results obtained by the two techniques were concordant in all cases. This is the first study reporting significant numbers of prenatal diagnoses using the quantitative fluorescent polymerase chain reaction. We believe that further studies on greater numbers of samples will determine the absolute reliability of this technique. These results also provide a model for diagnosis of trisomy from single fetal cells isolated from maternal blood.     

A cytogenetic register of trisomies in Scotland: results of the first 2 years (1989, 1990)

A population-based register of all autosomal trisomies diagnosed in Scotland was established in 1989. Data were provided by all cytogenetic service laboratories, and included karyotype, date and place of outcome, indication for analysis, maternal age and place of residence. The Register includes all foetuses diagnosed prenatally and all cytogenetically-confirmed live- and still-births with autosomal trisomy, including partial, mosaic and familial cases. In the 2 years 1989-90, 76 prenatal and 147 postnatal diagnoses were notified. For Down syndrome karyotypes the estimated rate, assuming no terminations and after adjusting for spontaneous foetal losses following diagnosis, was 1.23 per 1000 livebirths. This was almost identical to that expected by applying published maternal age-specific rates to the maternal age distribution in Scotland, indicating a very high level of ascertainment. The adjusted rates for trisomies 13 and 18 were also close to expected values derived from published data. Prenatal screening was estimated to reduce the newborn incidence of trisomy 21 by about one quarter overall, and about one half in mothers over 35 years. For trisomy 18, the estimated overall reduction was also about one quarter. It is concluded that the Register provides a practical and cost-effective means of monitoring the effects of prenatal screening, with near-complete ascertainment. In the longer term it will provide a database for studies of the actiology of these conditions.     

母血清筛查21三体、18三体阳性病例的产前诊断

目的探讨孕中期母血清筛查21-三体，18-三体阳性病例的染色体异常情况。方法AFP和Free-heG13用美国PE公司的时间分辨免疫荧光分析系统测定，风险值计算21-三体以1／270，18-三体以1／350为切割值。阳性病例进行羊膜腔穿刺羊水细胞培养。结果在54580例产前筛中有2615例为21-三体或18-三体阳性病例，占总筛查人数的4．79％。其中对1161例阳性病例进行了染色体分析（占总阳性数的44．4％）。结果发现29例异常核型，其中21-三体14例，18-三体5例，性染色体异常3例，其他异常核型7例，异常发生率2．49％。结论母血清产前筛查结合羊水诊断能够有效的避免胎儿染色体异常的发生。     

Cytological and epidemiological findings in trisomies 13, 18, and 21: England and Wales 2004-2009

This study describes the cytological and epidemiological findings in 985 trisomy 13 and 2512 trisomy 18 compared with 10,255 trisomy 21 diagnoses between 2004 and 2009 included in the National Down Syndrome Cytogenetic Register of England and Wales. The frequency of occurrence, proportions diagnosed prenatally, sex ratios, mean maternal age, and proportions of mothers with recurrences were analyzed. Ninety-seven, 98%, and 92% were free karyotypes for trisomy 21, 18, and 13, respectively; 3% of 21, 1% of 18, and 8% of trisomy 13 were translocations; and under 1% of trisomies 21 and 18 were double or triple aneuploids. Overall 1% of each trisomy had mosaicism, but 48% of the trisomy 21 double aneuploids, and 10% of trisomy 18 multiple aneuploids had mosaicism. The proportion of livebirths was 40% of trisomy 21, 11% of 18, and 13% of 13, respectively. Free trisomies 21 and 13 had an excess of males, and 18 had an excess of females, as did mosaic free trisomies 21 and 18. Mean maternal ages were 35.9 years in trisomy 21, 36.4 years in 18, and 34.6 years in 13. During the 6 years of data collection 1% of the mothers had recurrences, most recurrent trisomy 21 or 18 were identical translocations, but hetero-trisomic recurrences included 21 and 18, and 21 and 13. There are significant differences between the trisomic karyotypes and attributes, possibly related to their variable origins. Notable are the relative excess of trisomy 13 translocations, mosaicism in cases with multiple aneuploidy, and the types of homo- and hetero-recurrences.     

Assessment of new markers for the rapid detection of aneuploidies by quantitative fluorescent PCR (QF–PCR)

Rapid prenatal diagnoses of major chromosome aneuploidies have been achieved successfully using quantitative fluoresent PCR (QF–PCR) assays and small tandem repeat (STR) markers. Here we report the results of evaluating the use of previously untested X-linked STRs, (DXS6803) and (DXS6809), together with modified amelogenin (AMXY) sequences and the X22 marker that maps in the pseudoautosomal region PAR2 on the long arm of the X and Y chromosomes. These markers will allow prenatal diagnoses of sex chromosome aneuploidies such as 45,X (pure Turner Syndrome), 47,XXY and 47,XYY, while assessing the sex of the fetuses. Data are also presented concerning the difficulties associated with the evaluation of the frequencies of the various types of sub-populations of cells in amniotic fluid samples collected from fetuses with sex chromosome mosaicism. The results of evaluating the use of new markers for the rapid diagnosis of aneuploidies affecting chromosomes 21,18 and 13 are also presented. Three chromosome 21 specific STRs have been found to produce trisomic triallelic or diallelic patterns from all amniotic samples retrieved from fetuses with Down Syndrome. Since all samples tested were amplified and no false positive or negative results were observed, the present results confirm the diagnostic value of QF–PCR for the prenatal detection of major numerical chromosome disorders.     

MLPA技术在检测胎儿非整倍体异常中的应用

目的探讨多重探针连接依赖式扩增（multiplex ligation—dependent probe amplification,MLPA）在快速检测胎儿染色体非整倍体异常中的应用价值。方法344例产前诊断标本同时进行MLPA检测及核型分析,所有样本进行MLPA获得的扩增产物信息经Coffalyserv9．4软件（Holland—MRC公司）进行定量分析,观察样本DNA拷贝数的变化,并将所得结果与染色体核型分析结果进行比较,计算其检测的敏感度、特异度及阳性预测值。结果MLPA分析在标本接收后24h内即可得出结果,共检出染色体倍体异常产前诊断标本5例,包括唐氏综合征（Downsyndrome,47,＋21）6例、爱德华氏综合征（Edwardssyndrome,47,＋18）l例。MLPA检测24h报告结果临床符合率为97．7％。结论MLPA检测21、18、13、X、Y等染色体非整倍体疾病时,与核型分析相比较,MLPA是一种快速、高效的分析非整倍体的产前诊断的方法,具有临床应用价值。     

Diagnosis of Down syndrome and other aneuploidies using quantitative polymerase chain reaction and small tandem repeat polymorphisms

Pregnant women over 35 years of age are routinely offered screeningtests and karyotyping to detect Down syndrome and certain otheraneuploidies because the risk of these disorders increases exponentiallywith maternal age. It is, however, only cost-effective to karyotypehigh-risk pregnancies and a substantial number of remaininganeuploidy cases go undetected. We describe a rapid, inexpensivemethod to detect trisomy using polymorphic small tandem repeat(STR) markers and the polymerase chain reaction (PCR) to amplifyamniocyte DNA. STR patterns obtained on patients with trisomy21, trisomy 18 and triplo-X syndromes are distinct from controls.Polymorphic trisomy genotypes either show three fragment peaksof equal intensity or two fragments at a 2:1 dosage ratio. Inaddition, Turner syndrome (45, XO) DNA can be distinguishedfrom normal male DNA because it fails to amplify a Y-chromosomespecific PCR marker yet contains only a single dose of X-specificSTR markers. Quantitative analysis of peak heights and areasfrom STR markers show that the two peak patterns separate intocompletely non-overlapping groups. The high level of heterozygosityof most STR markers result in a predominance of heterozygouscontrols and trisomy patterns with multiple alleles, the easiestpatterns to differentiate. Homozygosity, and hence and uninformativeSTR pattern, is more common in controls than in trisomy samples.We anticipate as few as three STR markers per chromosome shouldbe over 99% informative.     

SNP等位位点比例法产前诊断唐氏综合征方法的建立

目的建立一种简便直观的产前诊断唐氏综合征的方法。方法提取63例羊水细胞中胎儿DNA,利用分子开关技术对PLAC4基因的rs8130833位点进行特异性扩增,最后采用荧光分光光度计对扩增产物进行光谱分析,根据产物的荧光强度鉴定SNP等位位点比例,从而区分出正常胎儿和唐氏患儿。结果63例羊水细胞中有33例杂合样本,对33例杂合样本进行检测,诊断特异性达94．1％。结论通过荧光光谱进行的SNP等位位点比例分析法简便直观,可以进一步应用在唐氏综合征的非创伤性产前诊断。     

Outcome of publicly funded nationwide first-tier noninvasive prenatal screening

PurposeNoninvasive prenatal screening (NIPS) using cell-free DNA has transformed prenatal care. Belgium was the first country to implement and fully reimburse NIPS as a first-tier screening test offered to all pregnant women. A consortium consisting of all Belgian genetic centers report the outcome of two years genome-wide NIPS implementation.MethodsThe performance for the common trisomies and for secondary findings was evaluated based on 153,575 genome-wide NIP tests. Furthermore, the evolution of the number of invasive tests and the incidence of Down syndrome live births was registered.ResultsTrisomies 21, 18, and 13 were detected in respectively 0.32%, 0.07%, and 0.06% of cases, with overall positive predictive values (PPVs) of 92.4%, 84.6%, and 43.9%. Rare autosomal trisomies and fetal segmental imbalances were detected in respectively 0.23% and 0.07% of cases with PPVs of 4.1% and 47%. The number of invasive obstetric procedures decreased by 52%. The number of trisomy 21 live births dropped to 0.04%.ConclusionExpanding the scope of NIPS beyond trisomy 21 fetal screening allows the implementation of personalized genomic medicine for the obstetric population. This genome-wide NIPS approach has been embedded successfully in prenatal genetic care in Belgium and might serve as a framework for other countries offering NIPS.