Method For Noninvasive Prenatal Diagnosis Research Articles

Targeted Linked-Read Sequencing for Direct Haplotype Phasing of Parental GJB2/SLC26A4 Alleles: A Universal and Dependable Noninvasive Prenatal Diagnosis Method Applied to Autosomal Recessive Nonsyndromic Hearing Loss in At-Risk Families

Noninvasive prenatal diagnosis (NIPD) for autosomal recessive nonsyndromic hearing loss (ARNSHL) has been rarely reported until recent years. However, the previous method could not be performed on challenging genome loci (e.g. CNVs, deletions, inversions, or gene recombinants) or on families without proband genotype. Here, this study assesses the performance of relative haplotype dosage analysis (RHDO)-based NIPD for identifying fetal genotyping in pregnancies at risk of ARNSHL. Fifty couples carrying pathogenic variants associated with ARNSHL in either GJB2 or SLC26A4 were recruited. The RHDO-based targeted linked-read sequencing combined with whole gene coverage probes was used to genotype the fetal cell-free DNA (cfDNA) of 49 families that met the quality control standard.. Fetal amniocyte samples were genotyped using invasive prenatal diagnosis (IPD) to assess the performance of NIPD. The NIPD results showed 100%(49/49) concordance with those obtained through IPD. Two families with copy number variation and recombination were also successfully identified. Sufficient specific informative SNPs for haplotyping, as well as the fetal cfDNA concentration and sequencing depth, are prerequisites for RHDO-based NIPD. This method has the merits of covering the entire genes of GJB2 and SLC26A4, qualifying for copy number variation and recombination analysis with remarkable sensitivity and specificity. Therefore, it has clinical potential as an alternative to traditional IPD for ARNSHL.

The current applications of cell-free fetal DNA in prenatal diagnosis of single-gene diseases: A review.

Prenatal diagnosis of hereditary diseases has substantially altered the way medical geneticists are helping families affected by genetic disorders. However, the risk of miscarriage and fear of invasive diagnostic procedures may discourage many couples from seeking prenatal diagnosis. With the discovery of maternal plasma cell-free fetal DNA, prenatal diagnosis has entered a new era of progress. Cell-free DNA is released during normal physiological functions as well as through the cell death programs of apoptosis and necrosis. It can be found in the plasma and other body fluids. Although this method has the advantage of being noninvasive, it is still rather expensive and requires advanced hardware and comprehensive data analysis. Promising implications of noninvasive prenatal diagnosis methods for the diagnosis of common trisomy disorders have paved the way for the development of more complicated assays of single-gene disorders. Relative mutation dosage and relative haplotype dosage are the most widely implemented assays for noninvasive prenatal diagnosis of single-gene disorders. However, each assay has its own advantages and disadvantages. Relative mutation dosage is based on the droplet digital polymerase chain reaction (PCR) technique which includes quantification features of real-time PCR assays. Relative haplotype dosage is based on next-generation sequencing that includes analysis of the maternal and paternal genome followed by sequencing of maternal plasma cell-free DNA. Co-amplification at a lower denaturation temperature PCR is another approach that is based on forming heteroduplexes between alleles to selectively amplify paternal mutations. In this review, we have described the most common noninvasive prenatal diagnosis approaches and compared their applications in genetic disorder diagnosis with different inheritance patterns.

Noninvasive fetal genotyping of single nucleotide variants and linkage analysis for prenatal diagnosis of monogenic disorders

BackgroundHigh-cost, time-consuming and complex processes of several current approaches limit the use of noninvasive prenatal diagnosis (NIPD) for monogenic disorders in clinical application. Thus, a more cost-effective and easily implementable approach is required.MethodsWe established a low-cost and convenient test to noninvasively deduce fetal genotypes of the mutation and single nucleotide polymorphisms (SNPs) loci by means of targeted amplification combined with deep sequencing of maternal genomic and plasma DNA. The sequential probability ratio test was performed to detect the allelic imbalance in maternal plasma. This method can be employed to directly examine familial pathogenic mutations in the fetal genome, as well as infer the inheritance of parental haplotypes through a group of selected SNPs linked to the pathogenic mutation.ResultsThe fetal mutations in 17 families with different types of monogenic disorders including hemophilia A, von Willebrand disease type 3, Duchenne muscular dystrophy, hyper-IgM type 1, glutaric acidemia type I, Nagashima-type palmoplantar keratosis, and familial exudative vitreoretinopathy were identified in the study. The mutations included various forms: point mutations, gene inversion, deletions/insertions and duplication. The results of 12 families were verified by sequencing of amniotic fluid samples, the accuracy of the approach in fetal genotyping at the mutation and SNPs loci was 98.85% (172/174 loci), and the no-call rate was 28.98% (71/245 loci). The overall accuracy was 12/12 (100%). Moreover, the approach was successfully applied in plasma samples with a fetal fraction as low as 2.3%.ConclusionsWe have shown in this study that the approach is a cost-effective, less time consuming and accurate method for NIPD of monogenic disorders.

An efficient method for noninvasive prenatal diagnosis of fetal trisomy 13, trisomy 18, and trisomy 21.

BackgroundMolecular size determination of circulating free fetal DNA in maternal plasma is an important detection method for noninvasive prenatal testing (NIPT). The fetal DNA molecule is the primary factor determining the overall performance of NIPT and its clinical interpretation. The proportion of cell-free fetal DNA molecules is expressed as the fetal DNA fraction in the plasma of pregnant women.MethodsWe proposed an effective method to deduce fetal chromosomal aneuploidy based on the proportion of a certain range of DNA fragment lengths from maternal plasma. We gradually narrowed the range of the upper and lower boundary via a traversing algorithm.ResultsWe explored the optimal range of the upper and lower boundary by using size-based DNA fragment length. Using this range, the accuracy of the sensitivity and specificity could be improved by up to 100% for detecting the three most common autosomal aneuploidies, namely trisomy 13, trisomy 18, trisomy 21 in the sample set.ConclusionsNumerical experiments demonstrate that our method is effective and efficient. The program is available upon request.

Targeted linked-read sequencing for direct haplotype phasing of maternal DMD alleles: a practical and reliable method for noninvasive prenatal diagnosis

For the noninvasive prenatal diagnosis (NIPD) of X-linked recessive diseases such as Duchenne muscular dystrophy (DMD), maternal haplotype phasing is a critical step for dosage analysis of the inherited allele. Until recently, the proband-based indirect haplotyping method has been preferred despite its limitations for use in clinical practice. Here, we describe a method for directly determining the maternal haplotype without requiring the proband’s DNA in DMD families. We used targeted linked-read deep sequencing (mean coverage of 692×) of gDNA from 5 mothers to resolve their haplotypes and predict the mutation status of the fetus. The haplotype of DMD alleles in the carrier mother was successfully phased through a targeted linked-read sequencing platform. Compared with the proband-based phasing method, linked-read sequencing was more accurate in differentiating whether the recombination events occurred in the proband or in the fetus. The predicted inheritance of the DMD mutation was diagnosed correctly in all 5 families in which the mutation had been confirmed using amniocentesis or chorionic villus sampling. Direct haplotyping by this targeted linked-read sequencing method could be used as a phasing method for the NIPD of DMD, especially when the genomic DNA of the proband is unavailable.

Improving Equivalency in Metagenomics: A Harmonized Process to Extract Fecal DNA.

In the field of clinical chemistry, there is a major push to harmonize and standardize laboratory processes and materials to improve the equivalency of reported test results and reduce the risk of postanalytical errors. The impetus of harmonized results is expanding to the field of molecular pathology and is seen in the development of standardized reference materials for the measurement of pathogens in clinical samples, and the harmonization of PCR and fluorescence in situ hybridization methods for noninvasive prenatal diagnosis and cancer screening, respectively. Molecular methods are used to study microbial communities in the human body to assess contributions of microbiota to human health. Microbiota in the gastrointestinal tract comprise one of the best studied ecosystems owing to their large volume, high diversity, and …

Haplotype-based Noninvasive Prenatal Diagnosis of Hyperphenylalaninemia through Targeted Sequencing of Maternal Plasma

Here we developed a haplotype-based noninvasive prenatal diagnosis method for hyperphenylalaninemia (HPA) and demonstrated its accuracy and feasibility during early pregnancy. Capture sequencing was performed on genomic DNA from parents and probands using customized hybridization probes targeting highly heterozygous single-nucleotide polymorphisms located within the 1 M region flanking phenylalanine hydroxylase (PAH) and 6-pyruvoyltetrahydropterin (PTS) and its coding region to determine the parental haplotypes and linkage to pathogenic mutations. Maternal plasma DNA obtained at 12–20 weeks of gestation was also subjected to targeted sequencing to deduce the fetal haplotypes based on the parental haplotypes. The fetal genotypes were further validated by invasive prenatal diagnosis. Haplotype-based noninvasive prenatal testing was successfully performed in 13 families. Five fetuses were identified to harbor bi-allelic pathogenic variants of PAH, four fetuses were carriers of one heterozygous PAH variant, three fetuses were normal, and the fetus of the 6-pyruvoyl tetrahydrobiopterin synthase family was identified as normal. The fetal genotypes at two gestational weeks from the same PAH family were identical. All results were consistent with the prenatal diagnosis based on amniotic fluid. Haplotype-based noninvasive prenatal testing for HPA through targeted sequencing is accurate and feasible during early gestation.

ҰРЫҚ ДАМУЫНЫҢ АҚАУЛАРЫНА БИOХИМИЯЛЫҚ СКРИНИНГТІҢ НӘТИЖЕЛЕРІ

The leading cause of malformations of the human fetus is chromosomal abnormalities. In connection with this, one of the main tasks of medical genetics is to reduce the incidence of chromosomal pathology in the human population. To solve this problem, invasive and non-invasive methods of prenatal diagnosis are used. The article presents the results of biochemical screening of blood serum of pregnant women and, in particular, the results of a cytogenetic study of the fetus of pregnant women at risk. Biochemical screening was conducted for 30335 pregnant women. The screening was conducted in the I and II trimester at 11-13 and 14-20 weeks of pregnancy. In the first trimester, a double test was used to determine the level of the β-subunit of the hCG hormone and the pregnancy-related plasma protein RAPP-A. In the second trimester, a triple test was used - determination of the level of AFP, hCG and free estriol E3. Based on the use of the enzyme immunoassay, the results of the indices of these biomarkers determined the possibility of the presence of chromosomal abnormalities in the fetus. According to biochemical screening, a risk group was identified from 662 (2.2%) pregnant women who underwent invasive diagnosis. Cytogenetic studies of metaphase cells of villi chorion were carried out. Fetal development disorders were detected in 41 cases, which was 6.2%. In two cases, cystic gigrum and spina bifida were identified. Karyotype disorders were found in 39 (5.9%) of the fetuses. Among them, the karyotype of Down syndrome was revealed in 29 cases, which was 74.4%. Comparative analysis by age factor showed an increase in the frequency of fetuses with Down's syndrome by 2.2 times in pregnant women 30 and older. Key words: biochemical screening, biomarkers, enzyme immunoassay, chromosomal abnormalities, fetal karyotype.

Proof-of-principle rapid noninvasive prenatal diagnosis of autosomal recessive founder mutations.

Noninvasive prenatal testing can be used to accurately detect chromosomal aneuploidies in circulating fetal DNA; however, the necessity of parental haplotype construction is a primary drawback to noninvasive prenatal diagnosis (NIPD) of monogenic disease. Family-specific haplotype assembly is essential for accurate diagnosis of minuscule amounts of circulating cell-free fetal DNA; however, current haplotyping techniques are too time-consuming and laborious to be carried out within the limited time constraints of prenatal testing, hampering practical application of NIPD in the clinic. Here, we have addressed this pitfall and devised a universal strategy for rapid NIPD of a prevalent mutation in the Ashkenazi Jewish (AJ) population. Pregnant AJ couples, carrying mutation(s) in GBA, which encodes acid β-glucosidase, were recruited at the SZMC Gaucher Clinic. Targeted next-generation sequencing of GBA-flanking SNPs was performed on peripheral blood samples from each couple, relevant mutation carrier family members, and unrelated individuals who are homozygotes for an AJ founder mutation. Allele-specific haplotypes were constructed based on linkage, and a consensus Gaucher disease-associated founder mutation-flanking haplotype was fine mapped. Together, these haplotypes were used for NIPD. All test results were validated by conventional prenatal or postnatal diagnostic methods. Ten parental alleles in eight unrelated fetuses were diagnosed successfully based on the noninvasive method developed in this study. The consensus mutation-flanking haplotype aided diagnosis for 6 of 9 founder mutation alleles. The founder NIPD method developed and described here is rapid, economical, and readily adaptable for prenatal testing of prevalent autosomal recessive disease-causing mutations in an assortment of worldwide populations. SZMC, Protalix Biotherapeutics Inc., and Centogene AG.

Noninvasive prenatal diagnosis experience in the Çukurova Region of Southern Turkey: detecting paternal mutations of sickle cell anemia and β‐thalassemia in cell‐free fetal DNA using high‐resolution melting analysis

This study used a high-resolution melting (HRM) technique to detect paternal mutations for the noninvasive prenatal diagnosis (NIPD) of β-thalassemia and sickle cell anemia (HbS). We also determined the levels of cell-free fetal DNA and total cell-free DNA. We used the HRM technique for fetal genotyping of paternal mutations in maternal plasma from 32 pregnancies at risk of β-thalassemia and 57 pregnancies at risk of HbS. The DNA levels in maternal plasma were measured using real-time quantitative PCR. Multiples of the median (MoM) values were calculated in women at risk for β-thalassemia or HbS. Twenty-two paternal mutations were detected in 89 pregnant women. Although we were successfully able to detect the paternal β-thalassemia mutations, the mutant HbS fetuses could not be distinguished from maternal background in the early weeks of pregnancy. The detection of DYS14 in male fetuses was 100%. The MoM values of women at high risk of having HbS-affected fetuses were higher than those for the other groups. High-resolution melting is a useful method for NIPD of β-thalassemias by detecting paternal mutations in the maternal plasma. Cell-free fetal DNA quantification and MoM values were not informative for HbS or β-thalassemias in early pregnancy.

The End of Amniocentesis? From TriTest to PrenaTestTM

ABSTRACT Purpose The rapid development in molecular biological technologies makes it possible to screen and to diagnosis thousands of genetic conditions, mutations and also predispositions to chronic diseases or traits, either prenatally or after birth. Clinical application of noninvasive prenatal diagnosis (NIPD) using fetal deoxyribonucleic acid (DNA) in maternal plasma has become a reality. We review the latest developments in screening and diagnosis of chromosomal diseases and a new noninvasive method of prenatal diagnosis. Materials and methods PrenaTest™ (LifeCodexx AG, Konstanz, Germany, patented and licensed by Sequenom Inc., San Diego, California, USA) is a molecular genetic test that can detect fetal trisomy 21 with a high precision level from maternal blood using new generation multiplex genome sequencing techniques. The test is based on the analysis of extracellular fetal DNA measured in the blood of pregnant women: Cell-free fetal DNA (cffDNA). Results In the case of trisomy 21, there were found 105 specific fragments of chromosome 21, 15 of fetal origin and 90 of maternal origin. The validity of PrenaTest™ has been demonstrated by many tests (427 cases), and the sensitivity was 95%, with a false negative rate of 5% and a specificity of 99.5%. Anyway, an additional ultrasound can always be performed to assess fetal morphology. Conclusion The arrival of the molecular genetic era also leads to many new ethical, social and medicolegal problems and dilemmas that obstetricians will have to face in the near future. There is an urgent need for the development of a new model for provision of genetic screening and diagnosis. How to cite this article Borruto F, Treisser A, Abdelkrim SB, Comparetto C. The End of Amniocentesis? From TriTest to PrenaTestTM. Donald School J Ultrasound Obstet Gynecol 2013;7(2):213-218.

Advances in pregnancy management in carriers of hemophilia

Recent advances in prenatal diagnosis and multidisciplinary management has improved the reproductive outlook for carriers of hemophilia and their offspring. Pre-pregnancy planning allows the woman and her partner to explore reproductive options and prepares the family for the potential delivery of an affected male infant. Non-invasive methods of prenatal diagnosis are advancing, with the aim of providing a definitive test for hemophilia. Carriers of hemophilia have an increased risk of primary and secondary postpartum hemorrhage. The affected male offspring is potentially at risk of cranial bleeding during delivery. A multidisciplinary approach provides advanced planning for the optimum mode of delivery and provision of appropriate haemostatic cover to reduce the risk of bleeding complications. This review summarises current recommendations for pregnancy management in carriers of hemophilia, from the initial pre-pregnancy counseling through to delivery and care of the neonate.

Non-invasive prenatal diagnosis of fetal trisomy 21 using cell-free fetal DNA in maternal blood

Since the existence of cell-free fetal DNA (cff-DNA) in maternal circulation was discovered, it has been identified as a promising source of fetal genetic material in the development of reliable methods for non-invasive prenatal diagnosis (NIPD) of fetal trisomy 21 (T21). Currently, a prenatal diagnosis of fetal T21 is achieved through invasive techniques, such as chorionic villus sampling or amniocentesis. However, such invasive diagnostic tests are expensive, require expert technicians, and have a miscarriage risk approximately 1%. Therefore, NIPD using cff-DNA in the detection of fetal T21 is significant in prenatal care. Recently, the application of new techniques using single-molecular counting methods and the development of fetal-specific epigenetic markers has opened up new possibilities in the NIPD of fetal T21 using cff-DNA. These new technologies will facilitate safer, more sensitive and accurate prenatal tests in the near future. In this review, we investigate the recent methods for the NIPD of fetal T21 and discuss their implications in future clinical practice.

Noninvasive Fetal Trisomy (NIFTY) test: an advanced noninvasive prenatal diagnosis methodology for fetal autosomal and sex chromosomal aneuploidies

BackgroundConventional prenatal screening tests, such as maternal serum tests and ultrasound scan, have limited resolution and accuracy.MethodsWe developed an advanced noninvasive prenatal diagnosis method based on massively parallel sequencing. The Noninvasive Fetal Trisomy (NIFTY) test, combines an optimized Student’s t-test with a locally weighted polynomial regression and binary hypotheses. We applied the NIFTY test to 903 pregnancies and compared the diagnostic results with those of full karyotyping.Results16 of 16 trisomy 21, 12 of 12 trisomy 18, two of two trisomy 13, three of four 45, X, one of one XYY and two of two XXY abnormalities were correctly identified. But one false positive case of trisomy 18 and one false negative case of 45, X were observed. The test performed with 100% sensitivity and 99.9% specificity for autosomal aneuploidies and 85.7% sensitivity and 99.9% specificity for sex chromosomal aneuploidies. Compared with three previously reported z-score approaches with/without GC-bias removal and with internal control, the NIFTY test was more accurate and robust for the detection of both autosomal and sex chromosomal aneuploidies in fetuses.ConclusionOur study demonstrates a powerful and reliable methodology for noninvasive prenatal diagnosis.

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.

Non-invasive prenatal diagnostics of aneuploidy using next-generation DNA sequencing technologies, and clinical considerations

Rapidly developing next-generation sequencing (NGS) technologies produce a large amount of data across the whole human genome and allow a large number of DNA samples to be analyzed simultaneously. Screening cell-free fetal DNA (cffDNA) obtained from maternal blood using NGS technologies has provided new opportunities for non-invasive prenatal diagnosis (NIPD) of fetal aneuploidies. One of the major challenges to the analysis of fetal abnormalities is the development of accurate and reliable algorithms capable of analyzing large numbers of short sequence reads. Several such algorithms have recently been developed. Here, we provide a review of recent NGS-based NIPD methods as well as the available algorithms for short-read sequence analysis. We furthermore introduce the practical application of these algorithms for the detection of different types of fetal aneuploidies, and compare the performance, cost and complexity of each approach for clinical deployment. Our review identifies several main technologies and trends in NGS-based NIPD. The main considerations for clinical development for NIPD and screening tests using DNA sequencing are: accuracy, intellectual property, cost and the ability to screen for a wide range of chromosomal abnormalities and genetic defects. The cost of the diagnostic test depends on the sequencing method, diagnostic algorithm and volume of the tests. If the cost of sequencing equipment and reagents remains at or around current levels, targeted approaches for sequencing-based aneuploidy testing and SNP-based methods are preferred.

MeDIP real‐time qPCR of maternal peripheral blood reliably identifies trisomy 21

To reevaluate the efficiency of the 12 differentially methylated regions (DMRs) used in the methylated DNA immunoprecipitation (MeDIP) real-time quantitative polymerase chain reaction (real-time qPCR) based approach, develop an improved version of the diagnostic formula and perform a larger validation study. Twelve selected DMRs were checked for copy number variants in the Database of Genomic Variants. The DMRs located within copy number variants were excluded from the analysis. One hundred and seventy-five maternal peripheral blood samples were used to reconstruct and evaluate the new diagnostic formula and for a larger-scale blinded validation study using MeDIP real-time qPCR. Seven DMRs entered the final model of the prediction equation and a larger blinded validation study demonstrated 100% sensitivity and 99.2% specificity. No significant evidence for association was observed between cell free fetal DNA concentration and D value. The MeDIP real-time qPCR method for noninvasive prenatal diagnosis of trisomy 21 was confirmed and revalidated in 175 samples with satisfactory results demonstrating that it is accurate and reproducible. We are currently working towards simplification of the method to make it more robust and therefore easily, accurately, and rapidly reproduced and adopted by other laboratories. Nevertheless, larger scale validation studies are necessary before the MeDIP real-time qPCR-based method could be applied in clinical practice.

Early non-invasive detection of fetal Y chromosome sequences in maternal plasma using multiplex PCR

Clinical indications for fetal sex determination include risk of X-linked disorders, a family history of conditions associated with ambiguous development of the external genitalia, and some fetal ultrasound findings. It is usually performed in the first trimester from fetal material obtained through CVS and is associated with an approximately 1% risk of miscarriage. Ultrasound fetal sex determination is often performed after 11 weeks of gestation. This study aims to validate a reliable method for non-invasive prenatal diagnosis of fetal gender using maternal plasma cell-free fetal DNA (cffDNA) for fetal sex assessment in the first trimester of pregnancy and test its clinical utility in the diagnosis of potentially affected pregnancies in carriers of X-linked disorders. In the validation study, blood samples from 100 pregnant women at 6-11 weeks of gestation were analysed. In the clinical study, 17 pregnancies at risk of having an affected fetus were tested. 7 ml of maternal blood in EDTA were obtained and cffDNA was extracted using a commercially available kit. DNA was enzymatically digested using a methylation sensitive endonuclease (AciI) to remove maternal unmethylated sequences of the RASSF1A gene. A multiplex PCR was performed for the simultaneous amplification of target sequences of SRY and DYS14 from chromosome Y, along with RASSF1A and ACTB sequences. Amplification of these loci indicates fetal gender, confirms the presence of cffDNA and allows assessment of digestion efficiency. After establishing the appropriate experimental conditions, validation studies were successful in all 100 cases tested with no false negative or false positive results. Y chromosome-specific sequences were detected in 68 samples, and 32 cases were diagnosed as female based on the amplification of RASFF1A sequences only, in the absence of ACTB. In the clinical studies, fetal sex was correctly diagnosed in 16 pregnancies, and one case was reported as inconclusive. Fetal sex assessment by detecting Y chromosome sequences in maternal blood can be routinely used from the 6th week of gestation. Reliable fetal sex determination from maternal blood in the 1st trimester of gestation can avoid conventional invasive methods of prenatal diagnosis.

Non-invasive prenatal diagnosis of aneuploidies: new technologies and clinical applications

Non-invasive prenatal diagnosis (NIPD) has substantial medical importance as it targets the development of safer and more effective methods to avoid the risk of fetal loss associated with currently used invasive methods. Several approaches have been demonstrated as being proof-of concept for NIPD of chromosomal aneuploidies. These approaches include cell-based and cell-free detection methods, involving the investigation of fetal cells in the maternal circulation, formaldehyde treatment of maternal plasma, DNA methylation studies using sodium bisulfite or restriction enzymes, protein-based studies, identification of fetal-specific mRNAs and digital polymerase chain reaction (PCR) approaches, and recently next-generation sequencing and methylated DNA immunoprecipitation real-time quantitative PCR-based approaches. Although all these NIPD methods have both advantages and limitations, some are moving closer to clinical implementation. Biotechnology companies dedicated to the development of NIPD tests such as the sequencing- or methylation-based approaches are finalizing large clinical trials. It is expected that these new technologies will facilitate safer, more sensitive and accurate prenatal diagnostic tests in the near future. In this review, we highlight the most recent advances in methods for NIPD of aneuploidies, and we discuss their future implications in clinical practice.

Preliminary Studies on Cell-free Fetal DNA Separation and Extraction in Glass Lab-on-a-chip for Capillary Gel Electrophoresis

Abstract Preliminary studies on separation and extraction of cell-free fetal DNA in glass lab-on-a-chip (LOC) prior to non-invasive prenatal diagnosis method, are presented. The chip, containing crossed injector, 25 mm long separator, Y-shaped extractor and glass reservoirs for fluid handling, was fabricated of borosilicate glass. DNA fragments in a size range 20-500 base pairs (bp), corresponding to fetal and maternal cell-free DNA, were separated and extracted in less than 2 minutes.