多重定量荧光PCR在常见染色体非整倍体疾病快速产前诊断中的应用研究

目的:建立适合于中国人群特点的常见染色体非整倍体疾病快速产前诊断技术。方法:随机抽取50例正常无关个体的外周静脉血和9例羊水标本以及3例血清学筛查为阳性的胎儿羊水,应用定量荧光PCR技术分析13、18、21、X/Y染色体上的短串联重复序列(STR)的多态信息含量(PIC)。结果:中国人13、18、21、X/Y染色体上STR:D13S631、D13S634、D13S742、D18S386、D18S391、D18S535、D21S11、D21S1270、IFNAR、X22、XHPRT的PIC分别为0.800、0.749、0.821、0.914、0.623、0.760、0.876、0.848、0.848和0.807。应用以上STR对血清学筛查阳性的胎儿羊水DNA标本进行诊断,证实了18-三体综合征、21-三体综合征和Klinefelter综合征胎儿各1例,与染色体核型分析结果相一致。结论:本文研究所选STR位标在中国人群中呈现较高的遗传多态性,符合Hardy-Weinberg平衡定律,建立了快速产前诊断常见染色体非整倍体疾病的多重定量荧光PCR技术。     

Prenatal diagnosis and genetic analysis of X chromosome polysomy 49, XXXXY

We report on the prenatal diagnosis, genetic analysis and clinical manifestations of a 49,XXXXY fetus. A 31-year-old, primigravida woman was referred for genetic counselling at 17 weeks' gestation with the sonographic findings of intrauterine growth retardation, generalized oedema, a large septated cystic hygroma colli measuring 5x4 cm, and abnormal posturing of the lower extremities. Quantitative fluorescent polymerase chain reaction (QF-PCR) with small tandem repeat (STR) markers specific for chromosome X and a pentanucleotide marker X22 for the Xq/Yq pseudoautosomal region PAR2 rapidly detected the X-chromosome polysomy from amniotic fluid cells. This abnormality appeared to arise from successive non-disjunction during maternal meiosis I and meiosis II. Cytogenetic analysis revealed a karyotype of 49,XXXXY. Our case shows that a 49,XXXXY fetus in the second trimester may demonstrate hydrops fetalis and a large septated cystic hygroma colli by prenatal ultrasound. Our case also shows that QF-PCR assays with sex chromosome specific STR markers provide rapid prenatal diagnosis of numerical sex chromosome aneuploidy as well as its genetic cause in fetal cystic hygroma.     

多重荧光定量PCR方法的建立及其在快速产前诊断中的应用

目的 探讨用于快速产前诊断的多重荧光定量PCR(QF-PCR)方法的建立并评价其临床应用价值.方法 2008年5月到2009年7月在北京协和医院产前诊断中心进行产前诊断的孕妇170例,其中收集羊水123例,新鲜绒毛9例,脐血20例,自然流产绒毛18例.孕妇均为汉族,平均年龄(34.1±4.6)岁,平均孕周为(19.6±1.0)周.采用基因组DNA提取试剂盒提取羊水、绒毛及脐血标本中DNA.采用3种荧光素标记的引物,针对人类染色体中的短串联重复序列(STR)位点参照基因库(GenBank)和文献资料设计并合成20对引物,其中21号染色体6对引物,18号染色体4对引物,13号染色体4对引物,X和Y染色体1对通用引物,另有X染色体4对引物,Y染色体1对引物.设计检测方案为每份标本均进行两套8重QF-PCR(8×QF-PCR),共检测21、18、13号染色体及性染色体各4个位点;如果无法达到诊断要求,再追加第3套4个位点.同时与染色体核型分析结果进行对照.结果 (1)核型分析:170例标本均成功进行了核型分析,其中正常核型151例(89%,151/170),异常核型19例(11%,19/170).(2)QF-PCR检测:170例标本中,QF-PCR成功检测167例(98%),失败3例,QF-PCR检测均在2～3 d得出结果.QF-PCR检测结果正常134例,均与核型分析结果一致;核型分析异常的19例中,QF-PCR检测出异常18例(其中8例21三体及3例18三体).167例QF-PCR成功检测标本中,第1套+第2套引物组合共确诊150例(90%,150/167),加用第3套引物组合共检测3例(2%,3/167),另有14例不提供信息(8%,14/167).(3)QF-PCR诊断效率:QF-PCR用于常见非整倍体异常产前诊断的敏感度为95%(18/19),特异度为100%(134/134),假阳性率0(0/134),假阴性率5%(1/19),阳性预测值为100%(18/18),阴性预测值为99%(134/135).(4)QF-PCR检测常染色体及性染色体结果:21号常染色体STR位点中D21S1270和D21S1411杂合度最高,性染色体中DXS8377杂合度最高,扩增较稳定.结论 多重QF-PCR技术能成功用于常见非整倍体异常的快速产前诊断,检测结果准确,适合于规模较大的产前诊断中心进行大样本检测.     

Prenatal diagnosis of common aneuploidies using quantitative fluorescent PCR

OBJECTIVE: Quantitative fluorescence-polymerase chain reaction (QF-PCR) has recently been used for the detection of common chromosomal aneuploidies in prenatal diagnosis. Here we describe our experience in prenatal diagnosis of 1100 samples. METHODS: Extraction of DNA was performed from amniotic fluid, chorionic villus samples (CVS), fetal blood and fetal tissue samples, using a simple, rapid protocol. Fluorescent multiplex PCR products of single tandem repeats (STRs) located on chromosomes 13, 18, 21, X and Y were then analyzed on an automated laser fluorescent sequencer. All samples were analyzed with at least two polymorphic markers for chromosomes 13, 18 and 21 and one for the X chromosome. The amelogenin locus was used for sexing. Analysis was performed twice on affected samples. When miscellaneous results were obtained extra markers were used. RESULTS: We evaluated the usefulness of different markers in the Greek population. In a total of 1100 samples, 25 chromosome aberrations were identified, including trisomy 13, 18 and 21, XYY, triploidies 69,XXX and 69,XXY and one Turner mosaic. All results but three were consistent with conventional cytogenetic analysis. One mosaic was missed. Most bloodstained samples were successfully analyzed. CONCLUSION: Successful analysis of a large number of prenatal samples proves QF-PCR to be an efficient adjunct in routine prenatal diagnosis.     

QF-PCR技术检测4262例胎儿常见染色体非整倍体结果回顾分析

目的回顾分析荧光定量聚合酶链反应(quantitative fluorescence polymerase chain reaction,QF-PCR)技术对常见染色体(21、18、13、X/Y)非整倍体产前诊断的结果,评价其应用价值。了解不同产前诊断指征诊断为常见染色体非整倍体的阳性率,从而对遗传咨询提供参考依据。方法选取2018年1月至2018年12月在广东省妇幼保健院进行产前诊断的标本4262例,用QF-PCR和染色体核型分析的方法双盲检测,统计QF-PCR的符合率、灵敏度和特异度及不同产前诊断指征诊断为染色体非整倍体的阳性率。结果与金标准染色体核型分析比较,QF-PCR快速诊断常见染色体(21、18、13、X/Y)非整倍体阳性数305例,漏诊4例低比例非整倍体嵌合体,另外有1例21-三体嵌合体QF-PCR阳性,而核型分析阴性,符合率98.8%,灵敏度和特异度分别为85.9%和99.9%,剔除常见5条染色体非整倍体检测范围以外的阳性标本,灵敏度和特异度分别为98.7%和99.9%。不同的产前诊断指征中诊断为常见染色体非整倍体的阳性率:无创产前基因筛查高风险(77.6%);颈项透明层厚度增厚(10.7%);胎儿超声异常(9.0%);唐氏综合征血清学筛查高风险(8.2%);孕妇年龄≥35岁(4.4%);高危妊娠(3.9%);不良孕产史(1.0%)。结论QF-PCR快速诊断常见染色体(21、18、13、X/Y)非整倍体,灵敏度和特异度高,但对低比例嵌合体非常容易漏诊,两种结果不符合时,不能完全依靠染色体核型分析诊断,了解不同产前诊断指征的阳性率,对遗传咨询有一定的指导价值。     

Application of QF-PCR for the prenatal assessment of discordant monozygotic twins for fetal sex

OBJECTIVE: To establish the utility of quantitative fluorescent polymerase chain reaction (QF-PCR) in order to determine the zygosity of multiple pregnancies, as well as to define the origin of the most frequent aneuploidies in amniotic fluid samples. METHODS: We describe the case of a monochorionic (MC) diamniotic (DA) pregnancy with phenotypically discordant twins (nuchal cystic hygroma and non-immune hydrops in twin A and no anomalies in twin B). QF-PCR was performed for rapid prenatal diagnosis in uncultured amniocytes and subsequently in cultured cells. Polymorphic markers for chromosomes X, Y, 13, 18 and 21 were used for determination of zygosity as well as sex chromosome aneuploidy. RESULTS: Twin A showed a Turner Syndrome (TS) mosaicism pattern by QF-PCR in uncultured amniocytes. The monozygotic origin of the pregnancy was determined. Interphase fluorescence in situ hybridization (I-FISH) in this sample showed a mosaicism X0/XY (83/17%). Cytogenetic analysis revealed a 45,X0 karyotype in twin A and a 46,XY karyotype in twin B. CONCLUSIONS: QF-PCR is a reliable tool for the determination of the zygosity independently of the chorionicity and the fetal sex in case of twin pregnancy. Testing both direct and cultured cells can provide useful results for genetic counselling in chromosomal mosaicisms.     

Very early prenatal diagnosis on coelomic cells using quantitative fluorescent polymerase chain reaction

Quantitative fluorescent PCR (QF-PCR) has been shown to be an accurate assay for the rapid prenatal diagnosis of chromosome disorders. The extra-embryonic coelom develops during week 4 of gestation and it can be aspirated from the following week, making coelocentesis the earliest possible method of prenatal diagnosis after implantation. The possibility of using the QF-PCR assay performed on DNA extracted from cells present in the extra-embryonic coelom has been evaluated for the detection of aneuploidies. QF-PCR amplification using several markers for chromosomes X, Y, 21, 18 and 13 was successfully achieved on all 17 serial samples of exo-coelomic fluid (ECF), placental tissue and maternal blood. Multiplex analyses of maternal blood samples and chorionic tissues allowed the distinction of fetal from maternal patterns and, eventually, the identification of maternal contamination of the ECF samples. Prenatal detection of fetal gender was successful in all cases. When tested with autosomal primers, seven samples were found to contain exclusively fetal DNA. Eight samples contained small amounts of maternal DNA that did not interfere with the QF-PCR analysis. One fetus showed trisomy 13. QF-PCR requires very small volumes of sample compared with cell culture, suggesting that coelocentesis may prove useful for very early prenatal diagnosis.     

Rapid prenatal diagnosis of aneuploidy for chromosomes 21, 18, 13, and X by quantitative fluorescence polymerase chain reaction

OBJECTIVE: Quantitative fluorescence polymerase chain reaction (QF-PCR) is a rapid method for detection of chromosome copy number by amplification of repeat sequences at polymorphic loci. Our objective was to assess the performance of QF-PCR in detecting common aneuploidies in prenatal diagnosis. STUDY DESIGN: The study group consisted of pregnant women referred for amniocentesis or chorionic villus sampling (CVS) due to increased risk of fetal aneuploidy. Samples were collected from known affected and normal pregnancies. These were blindly screened for trisomy of chromosomes 21, 18, 13, and sex chromosome abnormalities, using QF-PCR. DNA from uncultured amniocytes was directly extracted using a modified alkaline lysis method. DNA from CVS was extracted by the phenol-chloroform procedure. Ten short tandem repeat (STR) markers were used for detection of fetal aneuploidy and gender. The STRs were selected for high heterozygosity rates and efficiency of the PCR amplification. The forward primer of each pair was labeled with a unique fluorescent dye. Amplified products were detected by an ABI Prism 310 Genetic Analyzer and results were analyzed using GeneScan Analysis Software. RESULTS: A total of 65 amniotic fluid and CVS samples were collected from affected and normal pregnancies. Two samples were contaminated with blood and were therefore excluded from the analysis. All 29 cases of aneuploidy were correctly diagnosed by QF-PCR, including 17 cases of trisomy 21, 7 cases of trisomy 18, and 5 cases with trisomy 13. The 34 normal samples were also correctly diagnosed as such. Thus, all results were in agreement with the standard cytogenetic results. There were no false-positive or false-negative results. CONCLUSION: We conclude that QF-PCR is a rapid, reliable, and reproducible method that may be used to provide rapid results in prenatal diagnosis of aneuploidy.     

Introduction of the QF-PCR analysis for the purposes of prenatal diagnosis in Bulgaria--estimation of applicability of 6 STR markers on chromosomes 21 and 18

OBJECTIVE: The aim of our study was to estimate the observed heterozygosity and informativeness of 6 STR markers on chromosomes 18 and 21 in the Bulgarian population. We have evaluated the applicability of these markers used from other investigators for QF-PCR prenatal diagnosis of the most common autosomal aneuploidies in Bulgaria. METHODS: DNA samples (n = 486) were extracted from different fetal tissues (amniotic fluid cells, chorionic villus samples, and fetal tissue after abortions). PCR amplifications of 4 STR markers located on chromosome 21 (D21S11, D21S1411, D21S1270, and D21S1440) and 2 on chromosome 18 (D18S535 and D18S51) were performed. They were analysed on an automated sequencer, and the allele dosage ratios were calculated. RESULTS: The results indicate the selected markers as highly informative for our population and suitable for QF-PCR prenatal diagnosis in Bulgaria. All samples with trisomy 21 (n = 8), trisomy 18 (n = 4) and triploidy (n = 1) were correctly detected by our analysis. Thus, no false-negative results were observed. CONCLUSION: QF-PCR analysis could be an applicable alternative in prenatal and postnatal diagnosis in cases with a strong suspicion for particular autosomal aneuploidies (including chromosomes 21, 18, and 13) in small countries with limited resources like Bulgaria.     

Rapid prenatal diagnosis of aneuploidies and zygosity in multiple pregnancies by amniocentesis with single insertion of the needle and quantitative fluorescent PCR

OBJECTIVE: To investigate amniotic fluid (AF) samples retrieved in multiple pregnancies by single insertion of the needle, for rapid assessment of chromosome copy number, zygosity, and cross-contamination between fetuses, using Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) amplification of highly polymorphic microsatellite markers. METHODS: Fifty-two multiple pregnancies were selected (47 twins, 5 triplets) and 108 samples of amniotic fluid were sampled between 12 to 20 weeks of gestation (mean 15.5) using the single-needle technique. Aneuploidy screening by QF-PCR amplification of short tandem repeats (STRs) on chromosomes X, Y, 21, 13, and 18 was carried out within 24 h of collection. Owing to the sampling procedure, the eventual presence of contamination between fetuses was also evaluated in every case. RESULTS: Normal and aneuploid fetuses were readily identified by QF-PCR. Fetal reduction was made available, for trisomic fetuses, without further waiting for completion of fetal karyotyping. In twin gestations, the ultrasound examination of chorionicity was always in agreement with the molecular assessment of zygosity. Contamination between fetuses due to the sampling procedure with a single puncture was never observed. CONCLUSION: Rapid prenatal diagnosis of aneuploidies by QF-PCR is a sensitive, efficient, and reliable assay. When applied in multiple pregnancies, it has the added value of allowing the assessment of zygosity in all cases, independently of chorionicity and fetal sex.     

Development and targeted application of a rapid QF-PCR test for sex chromosome imbalance

OBJECTIVES: A QF-PCR test has been developed to diagnose sex chromosome imbalances in prenatal samples and has been applied to a diagnostic service. METHODS: The test uses a PCR multiplex with eight primer pairs: six X-chromosome polymorphic markers, including two markers from Xp (a region not included in previously published sex chromosome aneuploidy tests), one polymorphic marker for a locus common to the long arms of the X and Y chromosomes, and the non-polymorphic amelogenin marker. Homozygosity for all X-chromosome markers and the absence of the Y-chromosome amelogenin marker is highly likely (907 : 1) to represent monosomy X (Turner syndrome), but interphase FISH is always used to confirm such a result. RESULTS: Blind studies were carried out to validate the test and the first year of clinical use has been reported. Results are usually issued within one working day, and the test is more efficient than interphase FISH. CONCLUSIONS: The sex chromosome imbalance test has been targeted to prenatal samples displaying a clear ultrasound indication consistent with Turner syndrome, and has also been used to identify fetal sex in pregnancies at risk of inheriting a sex-linked molecular disorder. No misdiagnoses were made. It is concluded that QF-PCR can rapidly and accurately diagnose sex chromosome status and imbalances, reducing maternal anxiety and aiding in efficient pregnancy management.     

Evaluation of sex chromosome aneuploidies in women with Turner's syndrome by G-banding and FISH. A serial case study

OBJECTIVE: To evaluate sex chromosome aneuploidies in patients with Turner's syndrome using two cytogenetic techniques. STUDY DESIGN: A sample of 35 women with a clinical suspicion of Turner syndrome was examined in the Hospital of Obstetrics and Gynecology, Instituto Mexicano del Seguro Social, Monterrey, Mexico. They were subjected to a conventional cytogenetic technique with G-banding and to fluorescence in situ hybridization (FISH) using a specific alpha satellite X chromosome (DXZ1) and specific alpha satellite Y chromosome (DYZ1). RESULTS: Using both techniques, 17 cases (48.57%) showed the same karyotype. Using FISH: (1) in 8 cases the presence of the Y chromosome was confirmed, (2) in 18 cases (51.43%) a new cell line was identified, (3) in 2 cases (5.71%) the derivative X was clarified, and (4) in 3 cases (8.57%) the origin of the chromosome markers (1 of X chromosome and 2 of Y chromosome) was delineated. FISH highlighted the differences between the initial diagnosis, based on G-banding, and the final diagnosis, determined by specific probes for the X and Y chromosomes. CONCLUSION: FISH is a useful tool in the detection of low-frequency cell lines and identification of the nature and origin of derivative chromosomes and unknown chromosome markers that have important implications for the treatment of patients with Turner's syndrome.     

Detection of chromosome abnormalities by quantitative fluorescent PCR in ectopic pregnancies

OBJECTIVE: To evaluate the potential value of quantitative fluorescent polymerase chain reaction (QF-PCR) in the detection of chromosome abnormalities in ectopic pregnancies. METHODS: Seventy chorionic villi samples of ectopic pregnancies were studied by QF-PCR. Primers for chromosomes 16, 21, X and Y in chorionic villi were evaluated. Fluorescence in situ hybridization (FISH) was performed when results of QF-PCR showed aneuploidy, in case of unexplicable QF-PCR peaks, and in 10 cases with normal QF-PCR results. RESULTS: QF-PCR produced a result for chromosomes X and Y in 66 cases (94%), for chromosome 16 in 62 cases (89%) and for chromosome 21 in 55 cases (79%). Overall, QF-PCR produced a result for the chromosomes tested in 54 ectopic pregnancy cases (77%). Fifty-two of these results were normal disomic (96%) and two were abnormal, one trisomy 16 (2%) and one triploidy (2%). In 16 cases (23%) no definite QF-PCR results could be obtained for all chromosomes, 11 due to amplification failure, and 5 due to unexplicable QF-PCR peaks. In 10 cases with normal QF-PCR results, disomy was confirmed by FISH. The trisomy 16 was also confirmed by FISH. Furthermore, a result was obtained with FISH in 5 of the cases without definite QF-PCR results. CONCLUSION: Although QF-PCR can establish the chromosomal status in ectopic pregnancies for chromosomes 16, 21, X and Y in the majority of cases, the technical failure rate is still considerable and does not improve results when compared to cytogenetic techniques.     

Complete discrepancy between abnormal fetal karyotypes predicted by QF-PCR rapid testing and karyotyped cultured cells in a first-trimester CVS

A chorion villus sample (CVS) biopsied at 11 weeks' gestation for raised nuchal translucency, revealed monosomy X (presumptive 45,X karyotype) by QF-PCR for rapid aneuploidy testing for chromosomes 13, 18, 21, X and Y. Long-term culture gave the karyotype: 47,XY,+ 21[66]/49,XYY,+ 21,+ 21 [22]. This discrepancy prompted redigestion of the combined residual villus fragments from the original QF-PCR assay. The repeat QF-PCR assay identified the presence of trisomy 21 and a Y chromosome consistent with a 47,XY,+ 21 karyotype. A double non-disjunction event early in embryogenesis in a 47,XY,+ 21 conceptus with subsequent cell lineage compartmentalisation of the three observed cell lines (45,X; 47,XY,+ 21 and 49,XYY,+ 21,+ 21) would account for these results. This is the first reported case to describe complete discrepancy at diagnosis between abnormal karyotypes detected by QF-PCR rapid aneuploidy testing and a cultured karyotype in the same CVS.     

QF-PCR-based prenatal detection of aneuploidy in a southeast Asian population

OBJECTIVES: We have investigated the efficacy of using quantitative fluorescent polymerase chain reaction (QF-PCR) for the prenatal recognition of aneuploidy in chromosomes 13, 18, 21, X and Y. A total of 1115 samples, from mainly southeast Asian patients, were analysed and compared in a blind trial to the results previously obtained cytogenetically. METHODS: A multiplex PCR involving 15 short tandem repeat (STR) sequences was used. The probability of two or more of these markers being informative was calculated, and this required the multiplex PCR to be modified. RESULTS: The QF-PCR and previous cytogenetic results concurred, except for two products of conception (POC). One of these may be a case of complete uniparental disomy that was not recognized cytogenetically. The other was tetraploid, and as such appeared normal using QF-PCR. A mosaic trisomy 18 was correctly identified. The population sample was of a mainly Chinese, ethnic origin, and the allele frequency, size and heterozygosity appeared more restricted than the population groups analysed hitherto. CONCLUSION: The QF-PCR methodology is an efficient cost-effective method of screening for major chromosome aneuploidy, and, for certain referral categories, could be used alone. It also appears to be applicable to patients of different ethnic origins.     

A case of 48,XXYY syndrome detected prenatally by QF-PCR

We report on the prenatal diagnosis and genetic analysis of a 48,XXYY fetus. A 28-year-old woman was referred for amniocentesis at 18 weeks' gestation because of advanced paternal age. Quantitative-fluorescence polymerase chain reaction (QF-PCR) with small tandem repeat (STR) markers rapidly detected the sex chromosome polysomy from amniotic fluid cells. This abnormality appeared to arise from successive non-disjunction during paternal meiosis I and meiosis II. Cytogenetic analysis revealed a karyotype of 48,XXYY. This case adds to the evidence that an age-related increase in sex chromosomal aneuploidies occurs in sperm. This case also shows that QF-PCR assays can provide rapid prenatal diagnosis of numerical sex chromosome aneuploidy.     

Prenatal diagnosis of mos46,X,del(Y)(q11.2)/45,X by cytogenetic and molecular studies with multiplex STR analysis

OBJECTIVES: To present the prenatal diagnosis of a fetus of mos46,X,del(Y)(q11.2)/45,X by cytogenetic and molecular analysis. CASE AND METHODS: A 35-year-old pregnant woman came to our hospital for amniocentesis, and fetal chromosomal aberrations with mos46,X, + mar/45,X were found. Fluorescence in situ hybridization revealed the existence of a Y centromere on the marker chromosome. Analysis with six pairs of short tandem repeat markers showed that the genomic DNA extracted from the uncultured amniotic fluid cells contained a deletion of Yq11.1-Yq11.2. Spermatogenesis loci of the Y chromosome were studied using four sets of multiplex PCR. The proximal two markers DYS271 and KALY were present and the other 16 distal markers were deleted. No deletion was noted in the Y chromosome of the father. RESULTS: Cytogenetic and molecular analyses revealed deletions of AZFb, d, and c regions on Yq11.2-Yqter in the fetal Y chromosome. Postmortem examination of the fetus showed a grossly normal male fetus with normal external genitalia and testes. CONCLUSION: The present report demonstrates that molecular analysis using polymorphic microsatellite markers and multiplex PCR is a useful complement to cytogenetic methods for the identification and the characterization of Y-chromosomal deletions.     

Validation of QF-PCR for prenatal aneuploidy screening in the United States

OBJECTIVE: QF-PCR is an inexpensive and reliable method for aneuploidy screening; however, despite its obvious advantages, it is not in routine use in the United States. Our objective in the present study was to validate QF-PCR as a means for prenatal aneuploidy screening in our institution. METHODS: A QF-PCR assay using 15 primer pairs located on chromosomes 13, 18, 21 X and Y was established for aneuploidy screening. Amniotic fluid (AF) and chorionic villus sampling (CVS) samples consisting only of the cells recovered from the plasticware discarded by our institutional cytogenetics laboratory were collected and DNA was prepared by a simple and inexpensive microwave procedure. QF-PCR was then performed and interpreted using established criteria. RESULTS: 687 consecutive prenatal samples were screened in a blinded prospective manner, and results were compared to those obtained by conventional cytogenetics. 100% of autosomal trisomies were detected, and there were zero false positives. A single case each of XXY and 45X were missed. CONCLUSIONS: QF-PCR for prenatal aneuploidy screening was validated in our laboratory and has now been approved by the New York State Clinical Laboratory Evaluation Program. We propose a simple protocol for integrating QF-PCR into the normal cytogenetics laboratory workflow.     

The consanguinity effect on QF-PCR diagnosis of autosomal anomalies

OBJECTIVES: Quantitative Fluorescent PCR (QF-PCR) is a simpler and faster method of detecting common chromosomal abnormalities when compared to cytogenetic analysis. The aim of our study is to investigate the applicability of this methodology in a population where consanguineous marriages are common and to estimate the heterozygous frequency of the PCR markers used. METHODS: Four hundred and twenty-three DNA samples were extracted from uncultured amniocytes and amplified with 18 short tandem repeats (STR) markers specific to chromosomes 13, 18 and 21. Amplification products were analyzed using the GeneScan software. RESULTS: QF-PCR correctly identified all the numerical abnormalities related to chromosomes 13, 18 and 21. A total of 24 autosomal trisomies (5.7%) were detected. The markers D21S1432 and D21S11 were the most consistent in providing unequivocal positive results for chromosome 21 and the heterozygosity percentages of the markers used were lower than the values reported in Western populations. CONCLUSION: QF-PCR is reliable for the prenatal diagnosis of numerical anomalies of the chromosomes 13, 18 and 21 in our study population. The absence of STR heterozygosity data from Lebanon and surrounding countries makes our study very useful for the development of a reliable QF-PCR trisomy detection test.     

Increased nuchal translucency, hydrops fetalis or hygroma colli. A new test strategy for early fetal aneuploidy detection

OBJECTIVES: Nuchal translucency measurement of 3 mm or more (> or = 95th centile for gestation age), hydrops fetalis or hygroma colli between the 11th and 14th weeks of gestation is associated with a higher risk of fetal Down syndrome and other aneuploidies. So far, chromosome preparation of chorionic villi samplings (CVS) after short-term (or direct) culture is the only valid, reliable and rapid method of choice for the early detection of chromosomal aberrations. However, because of the placental mosaicisms detected after short-term culture, CVS has to be confirmed by a second method. Moreover, short-term villi preparation does not always provide a sufficient quantity and quality of metaphases to enable cytogenetic analysis. Unfortunately, a predicative cytogenetic result will be available only after long-term cultivation (usually after 1-2 weeks). An alternative rapid method, inexpensive and suitable for diagnosing autosomal trisomies, is the quantitative fluorescence polymerase reaction (QF-PCR) using different polymorphic small tandem repeats (STRs) on CVS-DNA. Therefore, it was the aim of the study to evaluate whether a new CVS test strategy could be employed in early pregnancies at high risk after the rapid detection of fetal chromosomal abnormalities by QF-PCR for chromosomes 13, 18 or 21 and sexing in conjunction with short-term chromosome analysis. MATERIALS: Nineteen CVS were chosen for QF-PCR detection of trisomy 21, 18 or 13 after an increased nuchal translucency measurement (> or = 95th centile for gestation age), a hydrops fetalis or a hygroma colli. The amelogenin locus of chromosomes X and Y (AMXY) were used for sexing. The QF-PCR results were compared with routine karyotyping after short- and/or long-term cultivation of CVS cells. RESULTS: An informative result was demonstrated in all analysed specimens. Nine CVS were diagnosed as a QF-PCR trisomy either for chromosome 21, 18 and 13. The pathological samples also included 4 cases of mosaicism where the normal cell line was not identified by QF-PCR. In 1 additional case with a normal QF-PCR result, short-term CVS chromosome analysis showed a mosaic trisomy 13, whereas longterm CVS culture revealed a normal karyotype. The malformed aborted fetus showed no clinical signs of trisomy 13, confirming the normal results obtained by QF-PCR and long-term CVS chromosome analysis. One pregnancy with a Turner syndrome was not identified by molecular analysis. CONCLUSIONS: This study showed that all early pregnancies with a clinically relevant autosomal trisomy could be detected prenatally in routine practice by QF-PCR. The combined use of both rapid methods - QF-PCR and short-term chromosome analysis - optimise the results by minimising the possibility of false-positive or false-negative findings. We believe that after verification of a pathological result obtained by two independent methods (QF-PCR and short-term CVS chromosome analysis), long-term villi cultivation is no longer necessary. However, in all cases with discrepancies, especially in samples with mosaic findings at short-term CVS cultivation, further studies are still necessary.