Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization: clinical experience with 4,500 specimens.

Detection of chromosome aneuploidies in uncultured amniocytes is possible using fluorescence in situ hybridization (FISH). We herein describe the results of the first clinical program which utilized FISH for the rapid detection of chromosome aneuploidies in uncultured amniocytes. FISH was performed on physician request, as an adjunct to cytogenetics in 4,500 patients. Region-specific DNA probes to chromosomes 13, 18, 21, X, and Y were used to determine ploidy by analysis of signal number in hybridized nuclei. A sample was considered to be euploid when all autosomal probes generated two hybridization signals and when a normal sex chromosome pattern was observed in greater than or equal to 80% of hybridized nuclei. A sample was considered to be aneuploid when greater than or equal to 70% of hybridized nuclei displayed the same abnormal hybridization pattern for a specific probe. Of the attempted analyses, 90.2% met these criteria and were reported as informative to referring physicians within 2 d of receipt. Based on these reporting parameters, the overall detection rate for aneuploidies was 73.3% (107/146), with an accuracy of informative results for aneuploidies of 93.9% (107/114). Compared to cytogenetics, the accuracy of all informative FISH results, euploid and aneuploid, was 99.8%, and the specificity was 99.9%. In those pregnancies where fetal abnormalities had been observed by ultrasound, referring physicians requested FISH plus cytogenetics at a significantly higher rate than they requested cytogenetics alone. The current prenatal FISH protocol is not designed to detect all chromosome abnormalities and should only be utilized as an adjunctive test to cytogenetics. This experience demonstrates that FISH can provide a rapid and accurate clinical method for prenatal identification of chromosome aneuploidies.     

An automatic system for chromosomal analysis applied to prenatal diagnosis

Summary Computer-assisted metaphase analysis for prenatal diagnosis can be economically performed by a preexisting hardware-software system. The program can be run by technician-level (i.e., nonspecialist) personnel who can opt for the automatic operational mode for good quality metaphases or for the semimanual mode for problem metaphases (numerous superpositions, artifacts, etc.). The karyotype can be obtained in conversational mode. Hardcopy output is available for all modes.     

Proteomic analysis of amniotic fluid for the diagnosis of fetal aneuploidies

Advances in technologies associated with mass spectrometry-based proteomic techniques have added a new dimension to the field of biomedical research. Most of the existing research on human gestation has focused on the application of these high-throughput methodologies in the study of amniotic fluid. In cases of fetal aneuploidies, the use of proteomic platforms has contributed to the identification of relevant protein biomarkers that could potentially change early diagnosis and treatment. The current article focuses on studies of normal amniotic fluid using proteomic technologies and describes alterations noted in the amniotic fluid proteome in the presence of fetal aneuploidies.     

Haplotype-based approach for noninvasive prenatal diagnosis of congenital adrenal hyperplasia by maternal plasma DNA sequencing

Prenatal diagnosis of congenital adrenal hyperplasia (CAH) is of clinical significance because in utero treatment is available to prevent virilization of an affected female fetus. However, traditional prenatal diagnosis of CAH relies on genetic testing of fetal genomic DNA obtained using amniocentesis or chorionic villus sampling, which is associated with an increased risk of miscarriage. The aim of this study was to demonstrate the feasibility of a new haplotype-based approach for the noninvasive prenatal testing of CAH due to 21-hydroxylase deficiency. Parental haplotypes were constructed using target-region sequencing data of the parents and the proband. With the assistance of the parental haplotypes, we recovered fetal haplotypes using a hidden Markov model (HMM) through maternal plasma DNA sequencing. In the genomic region around the CYP21A2 gene, the fetus inherited the paternal haplotype ‘0’ alleles linked to the mutant CYP21A2 gene, but the maternal haplotype ‘1’ alleles linked to the wild-type gene. The fetus was predicted to be an unaffected carrier of CAH, which was confirmed by genetic analysis of fetal genomic DNA from amniotic fluid cells. This method was further validated by comparing the inferred SNP genotypes with the direct sequencing data of fetal genomic DNA. The result showed an accuracy of 96.41% for the inferred maternal alleles and an accuracy of 97.81% for the inferred paternal alleles. The haplotype-based approach is feasible for noninvasive prenatal testing of CAH.     

A metabolomics-based approach for non-invasive diagnosis of chromosomal anomalies

Introduction

Chromosomal anomalies (CA) are the most frequent fetal anomalies.

Objective

To evaluate the diagnostic performance of a machine learning ensemble model based on the maternal serum metabolomic fingerprint of fetal aneuploidies during the second trimester .

Methods

This is a case-control pilot study. Metabolomic profiles have been obtained on serum of 328 mothers (220 controls and 108 cases), using gas chromatography coupled to mass spectrometry. Eight machines learning and classification models were built and optimized. An ensemble model was built using a voting scheme. All samples were randomly divided into two sets. One was used as training set, the other one for diagnostic performance assessment.

Results

Ensemble machine learning model correctly classified all cases and controls. The accuracy was the same for trisomy 21 and 18; also, the other CA were correctly detected. Elaidic, stearic, linolenic, myristic, benzoic, citric and glyceric acid, mannose, 2-hydroxy butyrate, phenylalanine, proline, alanine and 3-methyl histidine were selected as the most relevant metabolites in class separation.

Conclusion

The proposed model, based on the maternal serum metabolomic fingerprint of fetal aneuploidies during the second trimester, correctly identifies all the cases of chromosomal abnormalities. Overall, this preliminary analysis appeared suggestive of a metabolic environment conductive to increased oxidative stress and a disturbance in the fetal central nervous system development. Maternal serum metabolomics can be a promising tool in the screening of chromosomal defects. Moreover, metabolomics allows to extend our knowledge about biochemical alterations caused by aneuploidies and responsible for the observed phenotypes.

     

Exclusion of chromosomal mosaicism in prenatal diagnosis

Summary For use in prenatal diagnosis, tables were prepared giving the number of metaphases or clones, respectively, which must be analysed in order to detect fetal mosaicism of a given degree (=percentage of the aberrant cell population) or higher with at least 95% or 99% probability. Different tables are provided for the two techniques of chromosomal preparation: the colony method and the flask method.     

Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection

Current methods for prenatal diagnosis of chromosomal aneuploidies involve the invasive sampling of fetal materials using procedures such as amniocentesis or chorionic villus sampling and constitute a finite risk to the fetus. Here, we outline a strategy for fetal chromosome dosage assessment that can be performed noninvasively through analysis of placental expressed mRNA in maternal plasma. We achieved noninvasive prenatal diagnosis of fetal trisomy 21 by determining the ratio between alleles of a single-nucleotide polymorphism (SNP) in PLAC4 mRNA, which is transcribed from chromosome 21 and expressed by the placenta, in maternal plasma. PLAC4 mRNA in maternal plasma was fetal derived and cleared after delivery. The allelic ratios in maternal plasma correlated with those in the placenta. Fetal trisomy 21 was detected noninvasively in 90% of cases and excluded in 96.5% of controls.     

Ultrasound diagnostic schema for the determination of increased risk for chromosomal fetal aneuploidies in the first half of pregnancy

The aim of the study was to develop an early ultrasound diagnostic schema for the determination of increased risk for fetal chromosomal aneuploidies. The study was conducted on a population of 1318 pregnant women divided into 2 groups: 1255 women with the normal course of pregnancy and 63 women with diagnosed fetal abnormalities. There were 34 cases of chromosomal abnormalities (trisomy 21, 18, 13; triploidy; unbalanced inversion 9; deletion 16) and 29 cases of structural malformations. The estimation of the range of normal values was performed for the nuchal translucency (NT) measurement between 11 and 13 weeks and the nasal bone length (NB) measurement between 12 and 20 week. The results obtained in the collective set of normal pregnancies constituted the basis for the calculation of the range of normal values. The measurements of NB and NT showed a linear value increase with the pregnancy course. The following test characteristics (correlation to CRL) were recorded: NB - sensitivity 60%, specificity 98%, positive predictive value (PPV+) 43%, negative predictive value (NPV-) 98.9%. For the assumption that the test outcome means the presence or absence of the nasal bone in the ultrasound scan the sensitivity was 40%, but specificity 100%; NT - sensitivity 63.6%, specificity 98.2%, PPV+ 38.9%, NPV - 98.2%; NT + NB - presents similar characteristic to the NB or NT alone - sensitivity 55.6%, specificity 98.6%, PPV+ 50%, NPV - 98.9%. The following test characteristics for chromosomal aberration markers (correlation to BPD) were observed: NB - sensitivity 68.4%, specificity 97.4%, PPV+ 56.5%, NPV - 98.4%; NT - sensitivity 73.9%, specificity 97.9%, PPV+ 54.8%, NPV- 99.2%; NT + NB - sensitivity 94.7%, specificity 98.9%, PPV+ 90%, NPV - 99.7%, respectively. The "genetic sonogram" protocol for the structural defect detection was analysed: sensitivity was 80%, specificity 100%, PPV+ 100%, NPV - 99.7%. It is concluded that the new biometric parameter--nasal bone length (NB) and the corrected one--nuchal translucency thickness (NT) are useful markers for fetal abnormalities, especially for chromosomal aberrations. High predictive values of the diagnostic schema for the detection of aneuploidies and structural defects indicate that its application in correlation with the biparietal diameter (BPD) is highly recommended. The proposed schema is an effective algorithm for prenatal diagnostics characterised by high prognostic values. The possible introduction of the schema could result in a decrease of the invasive procedure rates, which could minimise the rate of miscarriages as a complication of amniocenteses.     

A proteomic approach to studying the differentiation of neural stem cells

The mechanisms that regulate the maintenance of stem cell self-renewal versus differentiation are complex and remain mostly unknown. Understanding neurogenesis and neural cell differentiation presents a unique challenge for the treatment of nervous system disorders. To gain more insight into molecular mechanisms of the differentiation of neural cells, we combined the advantage of porcine fetal neural stem cells (NSCs) in vitro differentiation model and proteomic analysis. Using 2-DE followed by MS, we profiled constituent proteins of NSCs and their differentiated progenies at first and then indicated protein species that were significantly up- or down-regulated during the differentiation. The largest identified group of constituent proteins was related to RNA and protein metabolism and processing, including chaperones, and the second largest consisted of proteins involved in cell organization (cytoskeleton and annexins). Differentiation of neural cells was found to be accompanied by changes in the expression of proteins involved in DNA and RNA binding, mRNA processing and transport, stress responses, iron storage, and redox regulation. Additional immunoblot analysis verified the induction of alpha-B crystallin and heterogeneous nuclear ribonucleoproteins (hnRNPs) A1 and A2/B1. Furthermore, immunocytochemistry demonstrated specific localization of alpha-B crystallin in the cytoplasm or nucleus of glial cells and confirmed cellular expression patterns of hnRNPs A1 and A2/B1. These findings represent a significant step towards understanding neural cell differentiation and identification of the regulatory proteins associated with this process.     

Correlation between preimplantation genetic diagnosis for chromosomal aneuploidies and the efficiency of establishing human ES cell lines

There are several sources from which human embryonic stem cell (hESC) lines can be generated: surplus embryos after in vitro fertilization procedures, one- and three-pronuclear zygotes, early arrested or highly fragmented embryos that have reached the blastocyst stage, or otherwise chromosomally or genetically abnormal embryos after preimplantation genetic diagnosis (PGD). We report on the efficiency of establishing hESC lines from blastocysts with proven meiotic or mitotic errors after sequential testing of both polar bodies and blastomere analysis on day 3. The success rate of establishing hESC lines originating from blastocysts carrying a meiotic error was as low as 2.4% and differed significantly from the success rate of establishing hESC lines originating from blastocysts with balanced meiotic errors (21.6%) or mitotic errors (after sequential testing (9.1%) and after blastomere testing alone (12.2%)). This suggests that it may be reasonable to apply sequential PGD prior to the initiation of hESC culture. Information about the karyotype may in the future help refine the methods and possibly improve the efficiency by which hESC lines are derived from embryos with prezygotic abnormalities. Additionally, it may in general prove very difficult to obtain abnormal hESC lines for scientific study from aneuploid PGD embryos, which will limit our ability to study the biological consequences of chromosomal abnormalities. Furthermore, the success rates for generating aneuploid cell lines originating from fertilized oocytes carrying a prezygotic nondisjunction error seem to mirror the miscarriage rates during pregnancy of embryos carrying such errors.     

A novel approach to studying the migratory morphology of embryonic mesenchymal cells

A polarized morphology, defined by extension of an anterior pseudopod, is essential for the amoeboid migration of embryonic mesenchymal cells. Leukocytes adopt a similar morphology immediately following suspension in simple buffers containing chemotactic factors. Polarization in suspension therefore provides a rapid and sensitive screening assay for putative regulators of leukocyte migration. The aim of the present study was to investigate whether this assay might also be used to study the motile behaviour of embryonic mesenchymal cells. Primary cultures of mesenchymal cells were established from explants of stage 28 chick embryo corneal-limbal stroma. Serum-starved, subconfluent cultures were harvested using ethylene-diamine tetra-acetic acid and resuspended in Hanks' solution for up to 15 min at 37 degrees C. A variety of cell shapes, including spherical cells, blebbed cells, and cells with either non-polarized or polarized pseudopodia were observed. The proportions of cells with pseudopodia increased significantly over time. Treatment of cells with the chemotactic mitogen platelet derived growth factor-BB (PDGF-BB, homodimer isoform) suppressed blebbing and increased both pseudopod formation and polarization. Optimal polarization occurred in concentrations of PDGF-BB that are similar to those required for optimal chemotaxis (10 ng x mL(-1)). The polarization observed in the absence of PDGF-BB suggests that the migration of cells examined in this study might be controlled at least in part by some intrinsic mechanism. In addition, the strong polarization response to PDGF-BB confirms the role for this growth factor during corneal development. Observations of mesenchymal cell morphology in suspension, therefore provide novel data regarding the motile behaviour of embryonic cells.     

Clinical utility of chromosomal microarray analysis in invasive prenatal diagnosis

Novel methodologies for detection of chromosomal abnormalities have been made available in the recent years but their clinical utility in prenatal settings is still unknown. We have conducted a comparative study of currently available methodologies for detection of chromosomal abnormalities after invasive prenatal sampling.A multicentric collection of a 1-year series of fetal samples with indication for prenatal invasive sampling was simultaneously evaluated using three screening methodologies: (1) karyotype and quantitative fluorescent polymerase chain reaction (QF-PCR), (2) two panels of multiplex ligation-dependent probe amplification (MLPA), and (3) chromosomal microarray-based analysis (CMA) with a targeted BAC microarray. A total of 900 pregnant women provided informed consent to participate (94% acceptance rate). Technical performance was excellent for karyotype, QF-PCR, and CMA (~1% failure rate), but relatively poor for MLPA (10% failure). Mean turn-around time (TAT) was 7 days for CMA or MLPA, 25 for karyotype, and two for QF-PCR, with similar combined costs for the different approaches. A total of 57 clinically significant chromosomal aberrations were found (6.3%), with CMA yielding the highest detection rate (32% above other methods). The identification of variants of uncertain clinical significance by CMA (17, 1.9%) tripled that of karyotype and MLPA, but most alterations could be classified as likely benign after proving they all were inherited. High acceptability, significantly higher detection rate and lower TAT, could justify the higher cost of CMA and favor targeted CMA as the best method for detection of chromosomal abnormalities in at-risk pregnancies after invasive prenatal sampling.     

Fetal-specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21

The trials performed worldwide toward noninvasive prenatal diagnosis (NIPD) of Down's syndrome (or trisomy 21) have shown the commercial and medical potential of NIPD compared to the currently used invasive prenatal diagnostic procedures. Extensive investigation of methylation differences between the mother and the fetus has led to the identification of differentially methylated regions (DMRs). In this study, we present a strategy using the methylated DNA immunoprecipitation (MeDiP) methodology in combination with real-time quantitative PCR (qPCR) to achieve fetal chromosome dosage assessment, which can be performed noninvasively through the analysis of fetal-specific DMRs. We achieved noninvasive prenatal detection of trisomy 21 by determining the methylation ratio of normal and trisomy 21 cases for each tested fetal-specific DMR present in maternal peripheral blood, followed by further statistical analysis. The application of this fetal-specific methylation ratio approach provided correct diagnosis of 14 trisomy 21 and 26 normal cases.     

Maternal and fetal chromosomal aberrations in mice following prenatal exposure to subembryotoxic doses of lead nitrate

Maternal exposure to low levels of lead nitrate (12.5, 25, 50 mg/kg body weight), administered on the 9th day of gestation, did not cause embryonic resorption and fetal lethality, but induced chromosomal deletions and other forms of aberrations in fetal liver and maternal bone marrow cells.