Prenatal Detection Of Fetal Aneuploidies Research Articles

Prenatal Diagnosis of Chromosomal Aneuploidies by Quantitative Pyrosequencing®.

One of the major reasons for pregnant women to ask for prenatal diagnosis is to detect fetal chromosomal aneuploidies. Analysis of allele ratios of SNPs has been used for prenatal detection of fetal aneuploidies using MALDI-TOF mass spectrometry (MS). However, quantitative SNP genotyping by MALDI-TOF MS is challenging. To obtain a better quantification of allelic ratios, a Pyrosequencing(®) protocol for SNP genotyping has been developed to perform prenatal diagnosis of aneuploidies.To avoid the laborious process and risk of cross-contamination brought in by DNA extraction procedures, a PCR assay, which can amplify DNA directly from cells in amniotic fluid, has been developed. Pre-amplification steps such as cell enrichment and heating are required to obtain sufficient amounts of amplification products.In this chapter, SNPs on chromosome 21 are used to detect trisomy 21 as an example of aneuploidy by quantifying the allele ratio using Pyrosequencing. Primer selection for PCRs and Pyrosequencing reactions, optimization of nucleotide dispensation orders, establishment of cutoff values for trisomy 21, and interpretation of data are all factors essential for a successful diagnosis and are discussed in detail herein.

Detecting fetal subchromosomal aberrations by MPS: an unexpected discrepancy between amniocyte DNA and ccffDNA

What's already known about this topic? Noninvasive prenatal detection of fetal aneuploidies by massively parallel sequencing of circulating cell‐free fetal DNA has recently become widely available. Few studies report the detection of submicroscopic chromosomal aberrations.What does this study add? A ~400 kb gain and a ~27 Mb loss were detected on chromosome 13q by genomic microarray analysis of amniocyte DNA. Testing circulating cell‐free fetal DNA not only revealed the same loss but also a much larger gain (~12 Mb), indicating discrepancies between DNA of true fetal and placental origin. The use of a windowed approach is recommended in case of unexpected ‘whole chromosome’ noninvasive prenatal testing results and/or when fetal anomalies are suggestive for subchromosomal aberrations.

Feasibility Study of Semiconductor Sequencing for Noninvasive Prenatal Detection of Fetal Aneuploidy

Noninvasive prenatal detection of common fetal aneuploidies with cell-free DNA from maternal plasma has been achieved with high-throughput next-generation sequencing platforms. Turnaround times for previously tested platforms are still unsatisfactory for clinical applications, however, because of the time spent on sequencing. The development of semiconductor sequencing technology has provided a way to shorten overall run times. We studied the feasibility of using semiconductor sequencing technology for the noninvasive detection of fetal aneuploidy. Maternal plasma DNA from 13 pregnant women, corresponding to 4 euploid, 6 trisomy 21 (T21), 2 trisomy 18 (T18), and 1 trisomy 13 (T13) pregnancies, were sequenced on the Ion Torrent Personal Genome Machine sequencer platform with 318 chips. The data were analyzed with the T statistic method after correcting for GC bias, and the T value was calculated as an indicator of fetal aneuploidy. We obtained a mean of 3 524 401 high-quality reads per sample, with an efficiency rate of 77.9%. All of the T21, T13, and T18 fetuses could be clearly distinguished from euploid fetuses, and the time spent on library preparation and sequencing was 24 h. Semiconductor sequencing represents a suitable technology for the noninvasive prenatal detection of fetal aneuploidy. With this platform, sequencing times can be substantially reduced; however, a further larger-scale study is needed to determine the imprecision of noninvasive fetal aneuploidy detection with this system.

Cell-free nucleic acid based non-invasive prenatal diagnosis of fetal aneuploidies

Prenatal detection of fetal aneuploidies is one of the main goals of the prenatal diagnostic approach. As a benefit of the development of advanced ultrasound equipment and advances in molecular biology in the last decade, there is a significant progress in screening methods for fetal aneuploidies, although invasive methods remain the gold standard for aneuploidy detection. Non-invasive prenatal diagnosis has substantial medical impact as it targets the development of safer and more effective methods to avoid the risk of fetal loss associated with currently used invasive methods. Identification of fetal-specific messenger ribonucleic acids, digital polymerase chain reaction and next-generation sequencing give the real chance for non-invasive prenatal diagnosis of fetal aneuploidies. Although all these methods have both advantages and limitations, some of them are moving closer to clinical implementation. In this review the authors highlight the most recent advances in methods for non-invasive prenatal diagnosis of aneuploidies.

Detection of Microdeletion 22q11.2 in a Fetus by Next-Generation Sequencing of Maternal Plasma

Efforts have been undertaken recently to assess the fetal genome through analysis of circulating cell-free (ccf) fetal DNA obtained from maternal plasma. Sequencing analysis of such ccf DNA has been shown to enable accurate prenatal detection of fetal aneuploidies, including trisomies of chromosomes 21, 18, and 13. We sought to extend these analyses to examine subchromosomal copy number variants through the sequencing of ccf DNA. We examined a clinically relevant genomic region, chromosome 22q11.2, the location of a series of well-characterized deletion anomalies that cause 22q11.2 deletion syndrome. We sequenced ccf DNA isolated from maternal plasma samples obtained from 2 patients with confirmed 22q11.2 deletion syndrome and from 14 women at low risk for fetal chromosomal abnormalities. The latter samples were used as controls, and the mean genomic coverage was 3.83-fold. Data were aligned to the human genome, repetitive regions were removed, the remaining data were normalized for GC content, and z scores were calculated for the affected region. The median fetal DNA contribution for all samples was 18%, with the affected samples containing 17%-18% fetal DNA. Using a technique similar to that used for sequencing-based fetal aneuploidy detection from maternal plasma, we detected a statistically significant loss of representation of a portion of chromosome 22q11.2 in both of the affected fetal samples. No such loss was detected in any of the control samples. Noninvasive prenatal diagnosis of subchromosomal fetal genomic anomalies is feasible with next-generation sequencing.

Sensitivity of Noninvasive Prenatal Detection of Fetal Aneuploidy from Maternal Plasma Using Shotgun Sequencing Is Limited Only by Counting Statistics

We recently demonstrated noninvasive detection of fetal aneuploidy by shotgun sequencing cell-free DNA in maternal plasma using next-generation high throughput sequencer. However, GC bias introduced by the sequencer placed a practical limit on the sensitivity of aneuploidy detection. In this study, we describe a method to computationally remove GC bias in short read sequencing data by applying weight to each sequenced read based on local genomic GC content. We show that sensitivity is limited only by counting statistics and that sensitivity can be increased to arbitrary precision in sample containing arbitrarily small fraction of fetal DNA simply by sequencing more DNA molecules. High throughput shotgun sequencing of maternal plasma DNA should therefore enable noninvasive diagnosis of any type of fetal aneuploidy.

Application of proteomics for diagnosis of fetal aneuploidies and pregnancy complications

Proteomic technologies represent new strategies towards high-throughput, simultaneous analysis of thousands of biological molecules leading to the discovery of biomarkers for early diagnosis, prognosis and prediction of pregnancy outcome. Proteomics have additional relevance in understanding pathophysiology and the development of molecularly targeted therapeutics. Comparison of normal human amniotic fluid proteome with that coming from pregnancies carrying fetuses with chromosomal abnormalities facilitated the detection of panels of potential biomarkers for prenatal detection of fetal aneuploidies. Candidate biomarkers for the early prediction of preeclampsis are also available, while four biomarkers (defensins-2 and -1, calgranulin-C, and calgranulin-A), which were called the "MR score", can quickly and accurately detect potentially dangerous infections and predict premature birth. Researchers remain hopeful that proteomic studies will allow for the identification of either one protein marker or of a panel of markers for prenatal detection of fetal aneuploidies and pregnancy complications that could be usefully employed for diagnostic purposes or improvement of the current screening methods. For maximum predictive power however, biomarkers should be selected for further comparative analysis of expression and structural modifications in large numbers of samples from chromosomally normal and abnormal pregnancies obtained from different populations.

Promises and pitfalls of first trimester sonographic markers in the detection of fetal aneuploidy

First trimester sonographic markers are the only markers achieving detection rates above 50% in the prenatal detection of fetal aneuploidy. Although potentially they are the best markers, some concerns have arisen about its clinical application. Pitfalls may be due to inability to examine the markers, incorrect assessment, or incorrect interpretation of the findings. Markers may be unable to be examined due to maternal (maternal body habitus, previous surgery) or fetal reasons (incompatible fetal position or fetal movements). Causes of incorrect interpretation may be insufficient image magnification, incorrect caliper placement (nuchal translucency), incorrect insonation angle (nasal bone), venous contamination (ductus venosus), or arterial contamination (tricuspid regurgitation), among others. Venous contamination in ductus venosus waveforms may mimic an abnormal blood flow when it is normal, and the opposite can also occur. Finally, incorrect interpretation of a substantially increased nuchal translucency may lead to a false impression of an ominous fetal prognosis or may be confounded with a cystic hygroma.

Prenatal detection of fetal aneuploidy by sonographic ear length.

To determine the usefulness of a fetal ear length nomogram in the prenatal detection of fetal aneuploidy and to determine whether ear smallness in cases of aneuploidy is a primary or secondary event. Ear lengths of 447 singleton fetuses (October 1996 to October 1997)were prospectively evaluated between 14 and 41 weeks to establish a nomogram created by modeling the mean and SD separately. Records of aneuploid fetuses were retrospectively reviewed, and their ear lengths were plotted against the nomogram to determine detection rates, with ear length in or below the 10th and 50th percentiles for a given gestational age and biparietal diameter used as abnormal cutoffs. The nomogram for fetal ear length measurements provided sufficient data to derive the 10th, 50th, and 90th percentiles on the basis of gestational age and biparietal diameter. The ear length of euploid fetuses was significantly correlated with gestational age (R2 = 0.96; P < .001) and biparietal diameter (R2 = 0.95; P < .001). From 96 aneuploid fetuses identified, 63 had ear lengths in or below the 10th percentile for gestational age (sensitivity, 66%). When using ear length against biparietal diameter, the sensitivities for all aneuploid fetuses for cutoffs at or below the 10th and 50th percentiles were 43% (40 of 93) and 83% (77 of 93), respectively. Most aneuploid fetuses have sonographically small ears (< or = 10th percentile for gestational age). This smallness is not entirely related to overall small fetal size, but in almost half the cases, the fetal ear length is disproportionately smaller than the biparietal diameter.

Fetal DNA Analyzed in Plasma from a Mother’s Three Consecutive Pregnancies to Detect Paternally Inherited Aneuploidy

The recent demonstration of fetal DNA in maternal plasma and serum at concentrations much higher than those present in the cellular fraction has introduced new possibilities for noninvasive prenatal diagnosis of paternally inherited dominant disorders (1)(2)(3). To date, prenatal detection of fetal aneuploidy in maternal blood has focused on searching intact cells using fluorescence in situ hybridization. The use of fetal DNA in maternal plasma to determine fetal aneuploidy has rarely been described. We previously reported prenatal detection of a paternally inherited fetal aneuploidy from fetal DNA in maternal plasma (4). Here we report the application of such a technique in an additional case involving a mother’s three consecutive pregnancies. We studied fetal DNA in maternal plasma from a pregnant woman whose fetuses possibly had paternally inherited aneuploidy. Her husband had a balanced reciprocal translocation between the long arm of chromosome 10 and the short arm of chromosome 22, 46,XY,t(10;22)(q24.1;p11.2). The woman’s karyotype was normal. During her first pregnancy, genetic amniocentesis was performed at 19 weeks of gestation, and the maternal blood sample was collected at 22 gestational weeks before termination of the pregnancy. In contrast, during her second and third pregnancies, the …

Efficacy of Maternal Serum Screening in the Prenatal Detection of Fetal Chromosome Abnormalities in Japanese Women

Objective: This prospective study assesses the efficacy of maternal serum screening for use in prenatal diagnosis of fetal anomaly and chromosome imbalance in Japanese women. Methods: Maternal serum α-fetoprotein, human chorionic gonadotropin, and unconjugated estriol were measured in 1,055 singleton pregnant women between 14 and 20 weeks of gestation. A calculated risk for trisomy 21 of ≥1/299 or α-fetoprotein ≥2.5 multiples of the median was adopted as positive. Results: Three hundred and seventy-eight of the 1, 055 women screened (35.8%) were identified as positive. Sensitivity, false-positive rate, and positive predictive value in women aged <35 years were 60.0, 10.6, and 6.8%, respectively, and these values were 87.5, 49.3, and 4.2%, respectively, in women aged ≥35 years. The false-positive rate in women aged <35 years was significantly lower than that for women aged ≥35 years (p < 0.001). Chromosomal abnormalities were identified in 21 cases, including 10 with trisomy 21, 5 with trisomy 18, 2 with trisomy 13, and 4 with other chromosomal disorders. Seventeen of the 21 cases (81.0%) showed screen-positive results, and among these all 10 cases with trisomy 21 were detectable. Two cases with trisomy 18, 1 with trisomy 13 and 1 with isochromosome X showed extremely low human chorionic gonadotropin levels (0.4 ± 0.1 multiples of the median, mean ± SE), although they were screen negative. Of the 264 women who did not undergo amniocentesis, none had any clinical findings consistent with aneuploidy after birth. Conclusions: Our results suggest that the evaluation of each serum marker, as well as of the calculated risk, was significantly important in the prenatal detection of fetal aneuploidy.