Prenatal Diagnosis Of Common Aneuploidies Research Articles

Rapid Detection of Aneuploidies in Spontaneous Aborted Fetal Samples by Quantitative Fluorescence-PCR: A Descriptive Study

Chromosomal aneuploidies are the most chromosomal abnormalities at birth due to maternal meiosis I errors. Pregnancies with autosomal chromosomal aneuploidies that survive are namely trisomies 13 (Patau syndrome), 18 (Edward syndrome), and 21 (Down syndrome), account for 89% of chromosome abnormalities. Quantitative fluorescent polymerase chain reaction (QF-PCR) which amplifies specific DNA sequences called short tandem repeats (STRs), by using fluorescently labeled primers is a rapid technique for prenatal diagnosis of common aneuploidies. In this study, DNA extraction was performed from 100 samples isolated from muscle tissue of aborted fetuses. The analysis was performed by multiplex QF-PCR using a panel of 25 STRs markers for chromosomes X, Y, 13, 18, and 21. Our results showed that 20% of abortions were due to aneuploidy. 53% of mothers who had abortions were aged 26-35 years old and 32% of them were aged 36-45 years old. The analysis of muscle samples of aborted fetuses indicated that 20 samples showed chromosomal aneuploidy. Of the abnormal cases, 10 cases (~50 %) showed trisomy 21 followed by trisomy 18 (7 cases, ~35%), Klinefelter syndrome (2 cases, ~10 %), and showed trisomy X (1 case, ~5 %). Our results indicated that the D21S1414 marker showed the highest rate of heterozygosity in the study population. Besides some limitations of this study such as sample size, these results suggest that one of the causes of these abortions could be maternal age. We concluded that QF-PCR could be a rapid and reliable method to screen prenatal chromosomal aneuploidy and allow appropriate counseling.

Application of the BACs-on-Beads assay for the prenatal diagnosis of chromosomal abnormalities in Quanzhou, China

BackgroundAn increasing number of techniques have been used for prenatal diagnosis of genetic abnormalities. Our initial objective was to explore the value of the BACs-on-Beads (BoBs) assay for the prenatal diagnosis of aneuploidies and microdeletion/microduplication syndromes in Quanzhou, Southeast China.MethodsA total of 1409 pregnant women with high-risk factors for chromosomal abnormalities admitted to Quanzhou Women’s and Children’s Hospital were enrolled in this study. BoBs assays and karyotype analyses were conducted for all subjects. Subsequently, chromosome microarray analysis (CMA) or fluorescence in situ hybridization (FISH) was performed to validate the findings.ResultsIn this study, karyotype analysis and BoBs assay failed in 4 cases, and 2 cases, respectively. A total of 1403 cases were successfully analyzed, with success rates of 99.72% (1405/1409) and 99.85% (1407/1409) for karyotype analysis and Bobs assay, respectively. BoBs assay rapidly detected chromosomal aneuploidies in line with the karyotyping data. Additionally, 23 cases of microdeletions/microduplications were detected by BoBs assay but missed by karyotyping, including 22q11.2 microdeletions/microduplications, 5p15.32p15.33 microdeletion, Xp22.31 microdeletions/microduplications, Xq27.3 microdeletion, and Yp11.2 and Yq11.22q11.222 microduplication. In comparison with karyotyping, fewer mosaicisms were identified by BoBs assay. A high detection rate of chromosomal abnormalities was observed in the high-risk group during noninvasive prenatal testing (NIPT) (41.72%) and the abnormal ultrasound group (13.43%).ConclusionsBoBs assay can be used for the rapid and efficient prenatal diagnosis of common aneuploidies and microdeletion/microduplication syndromes. Moreover, the combined use of BoBs assay and karyotyping in prenatal diagnosis may allow for a more effective detection of chromosomal abnormalities.

Integrated FISH, Karyotyping and aCGH Analyses for Effective Prenatal Diagnosis of Common Aneuploidies and Other Cytogenomic Abnormalities.

Current prenatal genetic evaluation showed a significantly increase in non-invasive screening and the reduction of invasive diagnostic procedures. To evaluate the diagnostic efficacy on detecting common aneuploidies, structural chromosomal rearrangements, and pathogenic copy number variants (pCNV), we performed a retrospective analysis on a case series initially analyzed by aneuvysion fluorescence in situ hybridization (FISH) and karyotyping then followed by array comparative genomic hybridization (aCGH). Of the 386 cases retrieved from the past decade, common aneuploidies were detected in 137 cases (35.5%), other chromosomal structural rearrangements were detected in four cases (1%), and pCNV were detected in five cases (1.3%). The relative frequencies for common aneuploidies suggested an under detection of sex chromosome aneuploidies. Approximately 9.5% of cases with common aneuploidies showed a mosaic pattern. Inconsistent results between FISH and karyotyping were noted in cases with pseudo-mosaicism introduced by culture artifact or variable cellular proliferation from cells with mosaic karyotypic complements under in vitro cell culture. Based on findings from this case series, cell-based FISH and karyotyping should be performed to detect common aneuploidies, structural chromosomal abnormalities, and mosaic pattern. DNA-based aCGH and reflex FISH should be performed to detect and confirm genomic imbalances and pCNV. Practice points to ensure the diagnostic accuracy and efficacy were summarized.

Multiplex Ligation-Dependent Probe Amplification (MLPA) for Prenatal Diagnosis of Common Aneuploidies.

Multiplex Ligation-dependent Probe Amplification (MLPA) is a method to determine the copy number of up to 60 genomic DNA sequences in a single multiplex PCR based reaction.MLPA probes consist of two oligonucleotides that can hybridize next to each other on a certain DNA sequence of interest, where they are ligated. All ligated probes are subsequently amplified by PCR using a single set of primers. Each amplified MLPA probe has a unique length and can be visualized and quantified by capillary electrophoresis. As the primers are almost 100% consumed in the PCR reaction, the quantity of each PCR amplicon is proportional to the number of copies of each probe target sequence in the DNA sample. A trisomy 21 can therefore be detected by an approximately 50% increased signal of each chromosome 21 specific probe relative to reference samples.MLPA with the P095 Aneuploidy probemix for chromosomes 13, 18, 21, X and Y has been used as a rapid detection method on large numbers of samples from uncultured amniotic fluid or from chorionic villi. As compared to FISH and karyotyping, MLPA is more rapid, has a higher throughput, and is less expensive. MLPA however cannot detect low grade mosaicism, female triploidies, and copy number neutral chromosome abnormalities such as inversions and translocations.

Rapid and simultaneous detection of common aneuploidies by quadruplex real-time polymerase chain reaction combined with melting curve analysis.

BackgroundDuring the prenatal period, the number variation of chromosomes 13, 18, 21, X and Y accounts for more than 80% of the clinically significant chromosomal abnormalities diagnosed. Rapid tests for prenatal diagnosis of these abnormalities can improve pregnancy management and alleviate parental anxiety. Here, we present a molecular alternative method for detecting common aneuploidies.MethodsThis method is based on co-amplification of segmental duplications located on two different chromosomes using a single pair of primers. Segmental duplications have a high degree of sequence identity, but have single-nucleotide differences in some regions. These sequence differences can be quantified using melting curve analysis of dual-labeled probes to estimate the relative dosages of different chromosomes. We designed two quadruplex real-time PCR assays to detect aneuploidies of chromosomes 13, 18, 21, X and Y.ResultsWe examined 75 aneuploid DNA samples and 56 unaffected DNA control samples using these two assays and correctly identified all samples. Four cases of unbalanced translocation were also accurately detected. The observed averaged ratio for each chromosomal disorder was similar to the theoretically expected value.ConclusionsOur real-time assay is a robust, rapid, and easy to conduct technique for prenatal diagnosis of common aneuploidies, representing a competitive alternative for use in diagnostic laboratories.

Noninvasive prenatal diagnosis of common aneuploidies by semiconductor sequencing.

Massively parallel sequencing (MPS) of cell-free fetal DNA from maternal plasma has revolutionized our ability to perform noninvasive prenatal diagnosis. This approach avoids the risk of fetal loss associated with more invasive diagnostic procedures. The present study developed an effective method for noninvasive prenatal diagnosis of common chromosomal aneuploidies using a benchtop semiconductor sequencing platform (SSP), which relies on the MPS platform but offers advantages over existing noninvasive screening techniques. A total of 2,275 pregnant subjects was included in the study; of these, 515 subjects who had full karyotyping results were used in a retrospective analysis, and 1,760 subjects without karyotyping were analyzed in a prospective study. In the retrospective study, all 55 fetal trisomy 21 cases were identified using the SSP with a sensitivity and specificity of 99.94% and 99.46%, respectively. The SSP also detected 16 trisomy 18 cases with 100% sensitivity and 99.24% specificity and 3 trisomy 13 cases with 100% sensitivity and 100% specificity. Furthermore, 15 fetuses with sex chromosome aneuploidies (10 45,X, 2 47,XYY, 2 47,XXX, and 1 47,XXY) were detected. In the prospective study, nine fetuses with trisomy 21, three with trisomy 18, three with trisomy 13, and one with 45,X were detected. To our knowledge, this is the first large-scale clinical study to systematically identify chromosomal aneuploidies based on cell-free fetal DNA using the SSP and provides an effective strategy for large-scale noninvasive screening for chromosomal aneuploidies in a clinical setting.

Non-invasive prenatal diagnosis of the most common aneuploidies with cell-free fetal DNA in maternal serum – preliminary results

The aim of the study was to present initial results of non-invasive prenatal diagnosis of common aneuploidies of chromosomes 21, 18 and 13 based on cell-free fetal DNA in maternal serum in high-risk patients, and to compare the results with routine karyotyping. Before the invasive procedure, 10 ml of peripheral blood from 10 patients was collected to isolate cell-free fetal DNA and to perform a non-invasive fetal trisomy test (NIFTY provided by Beijing Genomics Institute, BGI, Shenzen, China). Three out of 10 samples showed an abnormal karyotype in traditional karyotyping. There were 9 conclusive NIFTY results. NIFTY detected 1 out of 2 trisomies 18. The quantity of cell-free fetal DNA in maternal plasma in the second probe with trisomy 18 was unsatisfactory fora conclusive NIFTY result. In 1 case traditional karyotyping revealed mosaicism impossible to detect with NIFTY

Effectiveness of multiplex ligation dependent probe amplification (MLPA) in prenatal diagnosis of common aneuploidies

To assess the effectiveness of the MLPA method (multiplex ligation dependent probe amplification) in prenatal diagnosis of common aneuploidies and compare its concordance with traditional karyotyping. From October 2008 until July 2012 we performed 195 MLPA (MRC-Holland) tests with the P095 probe mix on DNA extracted from chorionic villi or amniotic fluid from pregnant women with elevated risk for abnormal fetal karyotype and 5 tests on miscarriage DNA samples. Cell culture and traditional karyotyping were performed in parallel. Traditional karyotyping was successfully performed in 192 cases (98.5%; 192/195). In 52 cases the fetal karyotype was abnormal (26.8%). The most common findings included aneuploidies of the following chromosomes: 13, 18, 21, X, Y (86.5%, 45/52). There were 179 conclusive and 1 inconclusive MLPA result (92.3%; 180/195). The absolute specificity and sensitivity of the MLPA test were 100%. In 9 cases traditional karyotyping revealed aberrations impossible to detect with the MLPA P095 kit. The MLPA reaction was successfully performed on all miscarriage DNA samples. MLPA is an effective method for detecting common aneuploidies. Its effectiveness for miscarriage DNA samples remains to be elucidated in further studies.

Validation of quantitative fluorescent-PCR for rapid prenatal diagnosis of common aneuploidies in the Chinese population

Quantitative fluorescent polymerase chain reaction (QF-PCR) is an accurate and reliable method for rapid detection of aneuploidy; however, it is not routinely used in China. We aimed to validate QF-PCR as a means for prenatal common aneuploidy screening and to analyze the heterozygosities of short tandem repeat (STR) markers in the Chinese population. The sequences of 19 STR markers in chromosomes 21, 18, 13, X, and Y were designed; three kinds of fluoresceins were used to label the primers, and the QF-PCR detecting conditions were explored and optimized. The results of analysis of 210 prenatal samples by multiplex QF-PCR were compared with karyotyping analysis. All cases were successfully tested by QF-PCR and conventional cytogenetic analysis. QF-PCR results were consistent with the results of cytogenetic analyses, with the exception of two cases. The sensitivity and specificity of QF-PCR to diagnose common aneuploidies were 94.74 and 100%, respectively. The heterozygosities of most of the markers were lower than reported for Western populations, but relatively similar to those of other Asian populations. We conclude that QF-PCR is able to detect the common aneuploidies for prenatal diagnosis with high detection efficacy; therefore it is suitable for rapid prenatal diagnosis and for large-scale testing in laboratories. However, we need to add new STR markers or to find alternative STR markers with high heterozygosity in order to make this technique useful for routine diagnosis.

Quantitative fluorescent-polymerase chain reaction for rapid prenatal diagnosis of common aneuploidies

Objective To evaluate the clinical value of quantitative fluorescent polymerase chain reaction (QF-PCR) in rapid prenatal diagnosis of aneuploidies. Methods Twenty-two short tandem repeats (STR) and AMXY located on chromosome 13,18,21,X and Y were used as markers to examine 1740 samples from high risk pregnant women in Down syndrome screening and advanced maternal age(≥35 yrs) by QF-PCR.Samples were also tested by karyotype analysis and the results of the two methods were compared. Results Karyotype analysis and QF-PCR results were successfully obtained from 1690 samples. All QF-PCR reports were obtained within 48 hours after sample collection.For 1639 samples,normal results were obtained by both karyotype analysis and QF-PCR.Among 51 samples that were found abnormal by karyotype analysis,41 were abnormal in QF-PCR.The rapid tests found all numerical abnormalities involving chromosome 21,18,13,X and Y in prenatal diagnosis,including trisomy 21 (n =30),trisomy 18 (n =6),45,XO (n =1 ),47,XYY (n=1),47,XXX (n=1),69,XXX (n=1) and mosaic 47,XXY[94]/46,XX[6] (n=1)(47,XXY in QF-PCR).No false positive results were found.The results obtained by QF-PCR were consistent with those of cytogenetic studies in 99.4％ of the samples (1680/1690).Only ten cases of mosain and structural abnormality could not be found (0.6％,10/1690) by QF-PCR. Conclusions Rapid QF-PCR test might diagnose all aneuploidies involving chromosome 21,18,13,X and Y.It could provide rapid and accurate diagnosis for 99.4％ pregnant women with positive Down syndrome screening and advanced maternal age. Key words: Aneuploidy; Prenatal diagnosis; Polymerase chain reaction

Application of multiplex ligation-dependent probe amplification to diagnosis and prenatal diagnosis of common aneuploidies

To investigate the application value of the multiplex ligation-dependent probe amplification (MLPA) technique in diagnosis and prenatal diagnosis of chromosomes 13, 18, 21, X and Y aneuploidy. Forty-four cases including 30 peripheral blood samples, 10 fetal cord blood samples, and 4 amniotic fluid samples were collected in this study. DNA was isolated from the samples and detected by MLPA, followed by analyzing in ABI310 Genetic Analyzer. Analysis of copy number changes for chromosomes 13, 18, 21, X and Y was carried out with RH-MLPA-analysis software. The routine karyotype analyses were also done for all the samples. Of 44 samples, the results of 42 by MLPA method was consistent with that by chromosome karyotyping. Only one case with trisomy 21 chimerism was failed to reach conclusion. In addition, one case of mark chromosome segment was identified as Y-chromosome segment by MLPA, while karyotyping failed to make judgment. The accurate rate of MLPA was 97.7% (43/44). The MLPA technique can simultaneously detect dozens of different target sequences and their copy number changes in a single reaction. It showed high specificity, good reproducibility, was fast and high-throughput. The MLPA technique can be applied to diagnosis and prenatal diagnosis of the common chromosomal aneuploidy.

Validation of QF-PCR for Rapid Prenatal Diagnosis of Common Chromosomal Aneuploidies in Korea

Sung-Hee Han, M.D., Jae-Song Ryu, M.S., Jeong-Wook An, M.T., Ok-Kyoung Park, M.T., Hye-Ryoung Yoon, M.D., Young-Ho Yang, M.D. and Kyoung-Ryul Lee, M.D.. J Genet Med 2010;7:59-66. https://doi.org/10.5734/JGM.2010.7.1.59

OmniPlex—a new QF‐PCR assay for prenatal diagnosis of common aneuploidies based on evaluation of the heterozygosity of short tandem repeat loci in the Czech population

The aim of our study was to assess the utility of commonly used multiplex assays of short tandem repeat markers used for quantitative fluorescent polymerase chain reaction (QF-PCR) for prenatal rapid aneuploidy detection (RAD) in routine prenatal diagnosis in the Czech population. Two previously published RAD multiplexes (2M test) were tested on 2906 local prenatal samples and used to calculate the rates of heterozygosity within this population. Most of the markers used in both multiplexes were highly informative. However, some had little utility, either due to a low heterozygosity rate (D21S499, D18S978 and P39) or because they were difficult to evaluate (DXS1283E). After evaluation of the 2M test results, a new multiplex assay (OmniPlex) was designed, developed and tested on 960 samples. This new assay was evaluated for heterozygosity rates and for the probability of having two or more informative markers on each chromosome. OmniPlex assay significantly improved the QF-PCR methodology for rapid prenatal aneuploidy detection in the Czech population. Based on detected heterozygosity of markers used for QF-PCR in this population, OmniPlex is a robust assay for the detection of chromosomes 13, 18, 21, X and Y in a single reaction.

Multiplex ligation‐dependent probe amplification (MLPA) in prenatal diagnosis—experience of a large series of rapid testing for aneuploidy of chromosomes 13, 18, 21, X, and Y

Multiplex ligation-dependent probe amplification (MLPA) is a relatively new method for rapid prenatal diagnosis of common aneuploidies, and larger series to evaluate its performance remain to be reported. A total of 2400 prenatal chorionic villus samples (CVS) and 1525 prenatal samples of amniotic fluids (AF) were analyzed using a commercial MLPA kit (SALSA P095) for aneuploidy of chromosomes 13, 18, 21, X, and Y, and subsequent G-banding. MLPA gave conclusive results in 2330 (97.1%) CVS and 1417 (92.9%) AF samples. MLPA and G-banding showed concordant results except for five CVS and two AF. These were acceptable differences, as MLPA is not expected to detect all cases of mosaicism or partial deletions. MLPA gave inconclusive results for 19 (0.79%) CVS and 20 (1.31%) AF samples in which mosaicism, triploidy, contamination by maternal cells, or structural abnormalities were suspected by MLPA. Finally, 30 (1.97%) AF were discarded because of maternal blood staining, and 51 (2.1%) CVS and 58 (3.8%) AF were discarded because of technical problems. The data presented confirm that MLPA is a rapid, simple and reliable method for large scale testing for nonmosaic aneuploidy of chromosomes 13, 18, 21, X, or Y in trypsin-digested CVS and in AF.

Rapid prenatal diagnosis of common chromosome aneuploidies by QF-PCR: Evaluation of two in vitro diagnostic test kits (Chromoquant and Aneufast)

Rapid prenatal diagnosis of common aneuploidies (21, 18, 13 trisomy and sex chromosome) can be performed by the Quantitative Fluorescence Polymerase Chain Reaction (QF-PCR) of Short Tandem Repeat (STR). The main advantages of the QF-PCR are its rapidity (24–48 h), accuracy and automation. Furthermore, its provides an additional and useful contribution for the karyotype analysis. Recently, three kits are commercially available for the in vitro diagnosis of common chromosome aneuploidies by QF-PCR. In this study we have evaluated two of these kits: Chromoquant Version 2 and Aneufast.

Optimized criteria for using fluorescence in situ hybridization in the prenatal diagnosis of common aneuploidies

To evaluate the medical and economic performance of three strategies for selecting patients eligible for interphase FISH in the prenatal diagnosis of common aneuploidies. We evaluated three protocols on the same population that was referred for prenatal diagnosis between June 2001 and December 2006. The number of aneuploidies detected by FISH and the relative cost (reagent and technical staff cost) are reported for each strategy. 2707 women were referred for prenatal diagnosis either because of advanced maternal age over 38 (48%), abnormal maternal serum screening (35%) or prenatal ultrasound anomalies (17%). A total of 4.8% chromosomal anomalies (balanced and unbalanced) were diagnosed after karyotyping. Theoretically, interphase FISH should have detected 79.4% of the unbalanced anomalies. We observed a significant improvement in the trisomy 21 detection by selecting the probes according to the reason for referral. The last protocol adopted, which offers a rapid test to 57% of women undergoing amniocentesis, presents the best aneuploidy detection rate (68% of total aneuploidies, 87% of trisomy 21). Selecting probes according to medical criteria patients combined with a technical procedure modification allows medico-economic improvement of interphase FISH in routine diagnosis.

Prenatal Diagnosis of Common Aneuploidies in Transcervical Samples Using Quantitative Fluorescent-PCR Analysis

The aim of this study was to test the feasibility of diagnosing common fetal chromosomal aneuploidies using quantitative fluorescent (QF)-PCR on transcervical cell (TCC) samples collected in the first trimester of pregnancy by means of intrauterine lavage (IUL). A total of 181 TCC samples were retrieved from pregnant women between 5 and 12 weeks of gestation, immediately before elective termination of pregnancy, at which time corresponding placental tissue and maternal blood specimens were also obtained. Isolation of trophoblastic cells by micromanipulation was attempted in all TCC samples. Micromanipulated specimens were analyzed by multiplex QF-PCR, including short tandem repeats for the chromosomes X, Y, 21, 18, and 13. The micromanipulation was successful in 152 of 181 cases (84.8%) where chorionic villous filaments and/or cell clumps of seeming trophoblastic origin could be isolated. All 152 samples were tested by QF-PCR analysis and peaks of paternal origin could be documented in all cases. Two cases of trisomy 21 and two cases of monosomy X0 were detected by means of QF-PCR assay, in accordance with the results obtained in corresponding placental samples. This study provides evidence that the use of multiplex QF-PCR amplification of selected microsatellites could be applied to micromanipulated TCC samples and in particular to IUL samples, which often contain trophoblastic cells, for the detection of chromosomal aneuploidies. The approach described in this study appears, therefore, a very promising tool toward non-invasive prenatal genetic diagnosis in the early stage of gestation.

Prenatal diagnosis of common aneuploidies using quantitative fluorescent PCR.

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. 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. 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. Successful analysis of a large number of prenatal samples proves QF-PCR to be an efficient adjunct in routine prenatal diagnosis.

Interphase FISH for prenatal diagnosis of common aneuploidies.

Cytogenetic analysis is currently a standard prenatal diagnostic test. It is routinely offered to pregnant patients who have an increased risk of carrying chromosomally abnormal fetuses. The traditional “gold standard” for prenatal diagnosis of chromosome abnormalities is metaphase analysis by G-banding. The primary advantages of standard cytogenetic analysis are the ability to detect aneuploidies as well as structural chromosomal aberrations with great accuracy (<99.5%). Traditional karyotyping, however, requires isolation of metaphase chromosomes from cultured fetal cells and therefore is time consuming. Although reporting time has decreased dramatically during the last 3 decades, conventional karyotyping still requires 7–12 d of which culture time is the most time consuming ().