Application In Prenatal Diagnosis Research Articles

Rapid Identification of β-Thalassemia, Hb E, and High Hb F Determinants Using a High-Resolution Melting Analysis: Application in Prenatal Diagnosis in Southern Thailand

β-thalassemia (thal), hemoglobin (Hb) E, and high Hb F determinants, which are caused by mutations in the β-globin gene cluster, are common genetic disorders in Thailand and Southeast Asia. Prenatal diagnosis is essential for couples at risk to identify severe forms, including homozygous β-thal and Hb E/β-thal. Conventional methods, including reverse dot-blot hybridization and gap-polymerase chain reaction (PCR) for genotyping of point and large deletion mutations, require post-PCR steps, which are time-consuming and costly. This study aimed to develop a rapid and efficient method using monoplex high-resolution melting (HRM) analysis for genotyping of Hb E and 11 β-thal mutations; multiplex HRM analysis for identifying six deletional mutations, including two β0-thal mutations (3.5 and 45 kb deletion); and a novel method for detecting four high Hb F determinants, namely, δβ0-thal (12.5 kb deletion), HPFH6, Indian inv-del (Aγδβ)0-thal, and Thai del-inv-ins (Aγδβ)0-thal. The developed assays were validated using 182 blinded fetal DNA samples with 41 β-thal genotypes. Different HRM patterns were observed among wild-type, heterozygote, homozygote, and compound heterozygote genotypes. Six deletional mutations showed specific melt curves. This technique demonstrated 100% concordance with conventional methods. The assay showed 100% sensitivity, specificity, and positive and negative predictive values within the limit of detection at DNA concentrations of 8.0 ng/reaction. Finally, this developed assay was efficient in identifying both point mutations and large deletion, convenient, rapid, and cost-effective and did not require post-PCR steps. Thus, this technique has potential for application in prenatal diagnosis of thal and can inform prevention and control programs.

Multisite Evaluation and Validation of Optical Genome Mapping for Prenatal Genetic Testing

Prenatal diagnostic testing of amniotic fluid, chorionic villi, or more rarely, fetal cord blood is recommended following a positive or unreportable noninvasive cell-free fetal DNA test, abnormal maternal biochemical serum screen, abnormal ultrasound, or increased genetic risk for a cytogenomic abnormality based on family history. Although chromosomal microarray is recommended as the first-tier prenatal diagnostic test, in practice, multiple assays are often assessed in concert to achieve a final diagnostic result. The use of multiple methodologies is costly, time consuming, and labor intensive. Optical genome mapping (OGM) is an emerging technique with application for prenatal diagnosis because of its ability to detect and resolve, in a single assay, all classes of pathogenic cytogenomic aberrations. In an effort to characterize the potential of OGM as a novel alternative to traditional standard of care (SOC) testing of prenatal samples, OGM was performed on a total of 200 samples representing 123 unique cases, which were previously tested with SOC methods (92/123=74.7% cases tested with at least two SOCs). OGM demonstrated an overall accuracy of 99.6% when compared with SOC methods, a positive predictive value of 100%, and 100% reproducibility between sites, operators, and instruments. The standardized workflow, cost-effectiveness, and high-resolution cytogenomic analysis demonstrate the potential of OGM to serve as a first-tier test for prenatal diagnosis.

Whole-exome sequencing applications in prenatal diagnosis of fetal bowel dilatation.

This study introduced whole-exome sequencing (WES) in prenatal diagnosis of fetal bowel dilatation to improve the detection outcome when karyotype analysis and copy number variation sequencing (CNV-seq) were uninformative in detecting pathogenic variants. The work reviewed 28 cases diagnosed with fetal bowel dilatation and analyzed the results of karyotype analysis, CNV-seq, and WES. Among the 28 cases, the detection rate in cases with low risk of aneuploidy was 11.54% (3/26), which is lower than 100% (2/2) in cases with high risk of aneuploidy. Ten low-risk aneuploidy cases with isolated fetal bowel dilatation had normal genetic testing results, while the remaining 16 cases with other ultrasound abnormalities were detected for genetic variants at a rate of 18.75% (3/16). The detection rate of gene variation was 3.85% (1/26) by CNV-seq and 7.69% (2/26) by WES. This study suggested that WES could reveal more genetic risk in prenatal diagnosis of fetal bowel dilatation and has value in prenatal diagnosis to reduce birth defects.

Detection of Novel Pathogenic Variants in Two Families with Recurrent Fetal Congenital Heart Defects.

Congenital heart disease (CHD) is the most common birth defect with strong genetic heterogeneity. To date, about 400 genes have been linked to CHD, including cell signaling molecules, transcription factors, and structural proteins that are important for heart development. Genetic analysis of CHD cases is crucial for clinical management and etiological analysis. Whole-exome sequencing (WES) was performed to identify the genetic variants in two independent CHD cases with DNA samples from fetuses and their parents, followed by the exclusion of aneuploidy and large copy number variations (CNVs). The WES results were verified by Sanger sequencing. In family A, a compound heterozygous variation in PLD1 gene consisting of c.1132dupA (p.I378fs) and c.1171C>T (p.R391C) was identified in the fetus. The two variants were inherited from the father (c.1132dupA) and the mother (c.1171C>T), respectively. In family B, a hemizygous variant ZIC3: c.861delG (p.G289Afs*119) was identified in the fetus, which was inherited from the heterozygous mother. We further confirmed that these variants PLD1: c.1132dupA and ZIC3: c.861delG were novel. The findings in our study identified novel variants to the mutation spectrum of CHD and provided reliable evidence for the recurrent risk and reproductive care options to the affected families. Our study also demonstrates that WES has considerable prospects of clinical application in prenatal diagnosis.

Application of copy number variation squencing for prenatal diagnosis

To assess the value of next-generation sequencing-based copy number variation sequencing (CNV-seq) for the detection of copy number variations (CNVs) in prenatal diagnosis. The results of single nucleotide polymorphism array (SNP-array) for prenatal diagnosis from May 2018 to December 2020 were reviewed. Selected cases of CNVs of clinical significance or low-percentage mosaic aneuploidies were included. Preserved DNA samples of amniotic fluid DNA were detected by CNV-seq. The results of CNV-seq and CMA were analyzed. A total of 16 488 data of SNP-array were re-analyzed, and 343 DNA samples were selected for the CNV-seq assay. All samples were successfully analyzed. Compared with the SNP-array, the proportion of full concordance, partial concordance and missed detection was 91.5% (314/343), 1.2% (4/343) and 7.3% (25/343), respectively. The non-detection zones of CNV-seq were confirmed, which have encompassed the SHOX gene and AZFc region. With a high accuracy and wide genome-wide coverage, CNV-seq is worthy for a wide application in prenatal diagnosis, though the limitation of testing should be taken into consideration, and the appropriate prenatal diagnosis method should be selected for different populations to reduce the occurrence of birth defects.

Direct Chromosomal Phasing: An Easy and Fast Approach for Broadening Prenatal Diagnostic Applicability

The assignment of alleles to haplotypes in prenatal diagnostic assays has traditionally depended on family study analyses. However, this prevents the wide application of prenatal diagnosis based on haplotype analysis, especially in countries with dispersed populations. Here, we present an easy and fast approach using Droplet Digital PCR for the direct determination of haplotype blocks, overcoming the necessity for acquiring other family members’ genetic samples. We demonstrate this approach on nine families that were referred to our center for a prenatal diagnosis of β-thalassaemia using four highly polymorphic single nucleotide variations and the most common pathogenic β-thalassaemia variation in our population. Our approach resulted in the successful direct chromosomal phasing and haplotyping for all nine of the families analyzed, demonstrating a complete agreement with the haplotypes that are ascertained based on family trios. The clinical utility of this approach is envisaged to open the application of prenatal diagnosis for β-thalassaemia to all cases, while simultaneously providing a model for extending the prenatal diagnostic application of other monogenic diseases as well.

Prenatal Diagnosis of Fetal Cleft Lip and Palate with Three-Dimensional Ultrasound Information Technology

Objective. To evaluate the three-dimensional ultrasound paper cleft lip and palate deformities in applications in prenatal diagnosis. Methods. 25 cases of cleft lip and palate fetus, 20–32 weeks of gestational age, with the maternal age of 22–44 years, were examined by prenatal ultrasound in our hospital; conventional two-dimensional ultrasound examination was performed after a cleft lip, and the application of three-dimensional ultrasound imaging surface and a transparent imaging showed the alveolar process and the palate of the fetus. Also, the results of two-dimensional ultrasound and postnatal (or after induction) results were compared. Results. Of the 25 cases, there were 6 cases of postpartum induction or simply unilateral cleft lip, 17 cases of unilateral cleft palate, and two cases of bilateral cleft lip palate. There was no significant ( P > 0.05 ) difference of two- and three-dimensional ultrasound detection rate of pure cleft lip; two-dimensional ultrasound cleft palate detection rate was 36.8% (7/19), and three-dimensional ultrasound cleft palate detection rate was 89.5% (17/19). The two methods showed a statistically significant ( P < 0.05 ) difference in the detection rate of cleft palate. Conclusion. Three-dimensional ultrasound can significantly improve the diagnostic accuracy of prenatal cleft palate.

A Rapid PCR-Free Next-Generation Sequencing Method for the Detection of Copy Number Variations in Prenatal Samples.

Next-generation sequencing (NGS) is emerging as a new method for the detection of clinically significant copy number variants (CNVs). In this study, we developed and validated rapid CNV-sequencing (rCNV-seq) for clinical application in prenatal diagnosis. Low-pass whole-genome sequencing was performed on PCR libraries prepared from amniocyte genomic DNA. From 10–40 ng of input DNA, PCR-free libraries consistently produced sequencing data with high unique read mapping ratios, low read redundancy, low coefficient of variation for all chromosomes and high genomic coverage. In validation studies, reliable and accurate CNV detection using PCR-free-based rCNV-seq was demonstrated for a range of common trisomies and sex chromosome aneuploidies as well as microdeletion and duplication syndromes. In reproducibility studies, CNV copy number and genomic intervals closely matched those defined by chromosome microarray analysis. Clinical testing of genomic DNA samples from 217 women referred for prenatal diagnosis identified eight samples (3.7%) with known chromosome disorders. We conclude that PCR-free-based rCNV-seq is a sensitive, specific, reproducible and efficient method that can be used in any NGS-based diagnostic laboratory for detection of clinically significant CNVs.

Application of KaryoLite™ BACs on Beads™ assay for prenatal diagnosis to detect chromosome aneuploidy in amniotic fluid cells

Prenatal diagnosis (PND) is a technique to detect chromosome abnormalities in fetus before birth. The techniques currently used for PND are conventional karyotyping and fluorescent in situ hybridization (FISH). Both techniques still have limitations, e.g., a high error rate of detection. The purpose of this study was to evaluate the efficiency of KaryoLite BACs-on-Beads (KL-BoBs™) as a routine diagnostic assay in a clinical application for PND by comparison with conventional karyotyping. A total of 37 amniotic fluid cell samples from 18 patients were used to validate KL-BoBs™ technique. The results of the KL-BoBs™ were 100% concordant with karyotyping for trisomy 18, trisomy 21, triple X, normal XX and normal XY. In conclusion, our results indicated that KL-BoBs™ had high concordance with conventional karyotying for prenatal diagnosis to detect chromosome status in amniotic fluid cells. Moreover, using an appropriate reference could reduce the coefficient of variation (CV) and increase accuracy of interpretation.

BACs-on-beads: a new robust and rapid detection method for prenatal diagnosis

Karyotyping, the gold standard used for diagnosis of chromosomal abnormalities, is being progressively replaced by rapid aneuploidy testing (RAT) techniques such as quantitative fluorescence-PCR, FISH and multiplex ligation-dependent probe amplification for diagnosing the common aneuploidies or chromosomal microarray analysis for comprehensive genome-wide testing. However, due to technical limitations, current RATs are confined to the detection of common aneuploidies 13, 18, 21 and sex chromosomes. To overcome the limitations of RATs, a bacterial artificial chromosomes-on-beads (BoBs™) assay technology has been introduced for the detection of the common aneuploidies as well as specific microdeletion syndromes. The BoBs assay is a bead-based multiplex assay using polystyrene beads impregnated with two spectrally distinct infrared fluorochromes to create a liquid array of up to 100 unique spectral signatures that supports the analysis of that scale of simultaneous hybridization assays on a minute DNA sample. This review gives an overview on the collective experiences of BoBs applications in prenatal diagnosis.

Chromosomal microarray as first‐tier approach in low‐risk pregnancies: detection rate should not be the only criterion for its application

We read with interest the paper by Hillman et al.1 concerning the use of chromosomal microarray (CMA) in prenatal diagnosis. Presented datasets included: a prospective cohort of pregnant women with ultrasound abnormalities and a systematic review and meta-analysis of studies on prenatal cases referred for any indication, fetal ultrasound abnormalities included. The authors corroborated the usefulness of CMA in pregnancies with ultrasound abnormalities, and reported an additional detection rate of abnormalities by CMA compared with karyotyping of 4.1% in their cohort and of 10% in the whole meta-analysis. These different figures were likely due to the different analytical sensitivity of the CMA platforms used (i.e. bacterial artificial chromosome (BAC) vs oligoarray). A BAC platform comparable to that utilized in the Hillman study has been tested by other groups2, 3, who claimed higher detection rates, by also incorporating into their results large imbalances detectable by means of good-quality chromosomal preparations. Accordingly, Hillman et al. emphasize the relevance of high-quality laboratory practices to compare CMA and karyotyping results in order to avoid a significant bias in any conclusion. Furthermore, copy number variations (CNVs) such as the PMP22 deletion, classified as a ‘positive’ result, should be regarded as incidental findings1, 2 and should not be considered in the detection rate estimation of CMA when comparing with karyotyping; moreover, such results are unlikely to address the particular concerns the couple may have when seeking prenatal diagnosis for abnormal ultrasound findings. However, the results reported by Hillman et al. agree with published guidelines and statements indicating that pregnancies with ultrasound abnormalities would benefit from CMA. Subanalysis of the pregnancies referred for ‘any indication’ disclosed highly heterogeneous results, with the additional detection rate of abnormalities by CMA compared with karyotyping ranging from 0.4 to 50%, while the detection rate of VOUS (variants of unknown significance) was 1.4%. Therefore, given the heterogeneity and increasing resolution over the years of CMA platforms and the merging of low- and high-risk populations, it is difficult to draw any final conclusion, particularly regarding CMA use in low-risk pregnancies. A recent report has indeed shown that when advanced maternal age or anxiety were the only or main indications for referral, the detection gain by CMA ranged from 1.0% and 0.6%, respectively4. These figures are evidently low compared with the VOUS detection rate. In our opinion, the higher diagnostic capacity of CMA should not be the only criterion for updating health policy committee statements; such decisions should be planned only after careful assessment of the large-scale application of CMA to unselected populations. In this perspective, several crucial issues await resolution, including the appropriate array design, assessment of the clinical relevance of a CNV with variable penetrance and expressivity, consensus on reporting VOUS and the related emotional burden of such findings. In order to assess comprehensively the large-scale application of CMA in low-risk pregnancies, we reiterate the advocacy for a model such as ACCE5, which is formulated based on the main criteria for evaluation of a genetic test (analytical validity, clinical validity and clinical utility) but which also consciously considers ethical, legal and social implications. The latter considerations are of particular importance when proposing genetic testing in a prenatal setting. We think that to better balance the costs and benefits of CMA application in prenatal diagnosis without specific indications, teamwork between geneticists, obstetricians and public health methodologists should be established, with partnership of the non-scientific stakeholders (i.e. pregnant couples/family and patient support organizations). A. Baroncini†, L. Sinibaldi*‡, L. Bernardini‡, P. Cavalli§, F. Faravelli¶, M. Gentile**, M. Lituania††, P. Volpe‡‡, L. Camurri§§, A. Novelli‡, and B. Dallapiccola¶¶ †ASL di Imola, Italy, UOC di Genetica Medica, Dip. Materno-Infantile, Imola, Italy; ‡IRCCS Fondazione Casa Sollievo della Sofferenza, Laboratorio Mendel, San Giovanni Rotondo, Italy; §Azienda Istituti Ospitalieri di Cremona, Servizio di Genetica, Cremona, Italy; ¶IRCCS Ospedali Galliera, SSD Genetica Medica Genova, Genova, Italy; **Ospedale di Venere, ASL di Bari, UOC Genetica Medica, Bari, Italy; ††IRCCS Ospedali Galliera, SSD Fisiopatologia Preconcezionale e Prenatale, Genova, Italy; ‡‡Ospedale di Venere, ASL di Bari, UOC Medicina Fetale, Bari, Italy; §§RDI Rete Diagnostica Italiana, Genetica Medica, Padova, Italy; ¶¶IRCCS Ospedale Pediatrico Bambino Gesù, Roma, Italy *Correspondence. (e-mail: [email protected])

Chromosomal Microarrays in Prenatal Diagnosis: Time for a Change of Policy?

Microarrays have replaced conventional karyotyping as a first-tier test for unbalanced chromosome anomalies in postnatal cytogenetics mainly due to their unprecedented resolution facilitating the detection of submicroscopic copy number changes at a rate of 10–20% depending on indication for testing. A number of studies have addressed the performance of microarrays for chromosome analyses in high risk pregnancies due to abnormal ultrasound findings and reported an excess detection rate between 5% and 10%. In low risk pregnancies, clear pathogenic copy number changes at the submicroscopic level were encountered in 1% or less. Variants of unclear clinical significance, unsolicited findings, and copy number changes with variable phenotypic consequences are the main issues of concern in the prenatal setting posing difficult management questions. The benefit of microarray testing may be limited in pregnancies with only moderately increased risks (advanced maternal age, positive first trimester test). It is suggested to not change the current policy of microarray application in prenatal diagnosis until more data on the clinical significance of copy number changes are available.

Prenatal screening and diagnosis of genetic deafness by microarray

To evaluate a microarray-based mutation screening method for genetic deafness and its application in prenatal diagnosis. Mutation screening of common deafness genes was performed in pregnant women and volunteers spouses. Nine common mutations in four major deafness genes, GJB2, GJB3, SLC26A4 and mitochondrial 12S rRNA, were detected simultaneously by a microarray-based method. Genetic counseling was given based on their testing results. 5.11% of pregnant women carried at least one mutation. Among them, seven carried mutation in the mitochondria 12S rRNA gene and were offered aminoglycoside-induced ototoxicity warning. For other mutation carriers of GJB2 or SLC26A4 genes, additional mutation screening was performed in their husbands by direct sequencing. A total of 20 couples were at risk of giving birth to children with genetic deafness. Of five couples who selected to undergo prenatal diagnostic testing of the fetus, four were diagnosed as wild type or heterozygous for the tested genes and one as p.V37I/c.235delC compound heterozygous for GJB2. DNA microarray is a quick, easy and reliable method to screen mutations in genetic deafness genes. Application of this method in prenatal screening and diagnosis might effectively reduce the occurrence of genetic deafness.

Fetal magnetic resonance imaging and three-dimensional ultrasound in clinical practice: Applications in prenatal diagnosis

Three-dimensional ultrasound and magnetic resonance imaging are powerful imaging techniques that are used increasingly in evaluating fetal anatomy. In this chapter, we review the main applications of these imaging modalities in current practice and present an overview of the malformations that may benefit from assessment with three-dimensional ultrasound and magnetic resonance imaging.

Re: Microarray application in prenatal diagnosis: a position statement from the cytogenetics working group of the Italian Society of Human Genetics (SIGU), November 2011

Re: Microarray application in prenatal diagnosis: a position statement from the cytogenetics working group of the Italian Society of Human Genetics (SIGU), November 2011

Re: Microarray application in prenatal diagnosis: a position statement from the cytogenetics working group of the Italian Society of Human Genetics (SIGU), November 2011

Re: Microarray application in prenatal diagnosis: a position statement from the cytogenetics working group of the Italian Society of Human Genetics (SIGU), November 2011

Microarray application in prenatal diagnosis: a position statement from the cytogenetics working group of the Italian Society of Human Genetics (SIGU), November 2011

A precise guideline establishing chromosomal microarray analysis (CMA) applications and platforms in the prenatal setting does not exist. The controversial question is whether CMA technologies can or should soon replace standard karyotyping in prenatal diagnostic practice. A review of the recent literature and survey of the knowledge and experience of all members of the Italian Society of Human Genetics (SIGU) Committee were carried out in order to propose recommendations for the use of CMA in prenatal testing. The analysis of datasets reported in the medical literature showed a considerable 6.4% incidence of pathogenic copy number variations (CNVs) in the group of pregnancies with sonographically detected fetal abnormalities and normal karyotype. The reported CNVs are likely to have a relevant role in terms of nosology for the fetus and in the assessment of reproductive risk for the couple. Estimation of the frequency of copy number variations of uncertain significance (VOUS) varied depending on the different CMA platforms used, ranging from 0-4%, obtained using targeted arrays, to 9-12%, obtained using high-resolution whole genome single nucleotide polymorphism (SNP) arrays. CMA analysis can be considered a second-tier diagnostic test to be used after standard karyotyping in selected groups of pregnancies, namely those with single (apparently isolated) or multiple ultrasound fetal abnormalities, those with chromosomal rearrangements, even if apparently balanced, and those with supernumerary marker chromosomes.

Non-medical applications of non-invasive prenatal diagnosis: Ethical issues

<h2>Abstract</h2> Non-invasive prenatal diagnosis (NIPD) is becoming increasingly important and its application in prenatal diagnosis is reaching consensus in the scientific research community. We discuss the opportunities and ethics of non-invasive prenatal testing for non-medical purposes, including forensic genetics. A number of ethical issues arise from non-medical applications of NIPD, such as sex determination and paternity testing in earlier gestational age and subsequent offspring selection. NIPD provides a source of information about the genetic make-up of the foetus, avoiding the small but significant risk of pregnancy loss related to invasive testing such as amniocentesis or chorionic villi sampling. NIPD is characterized by: safety, early detection and easy sampling. These features of NIPD increase the opportunity of prenatal testing also for non-medical reasons. Even if NIPD can be qualified as a good practice prenatal diagnosis tout court remains a topic of ethical judgements. The non-medical use of NIPD will benefit from an informed and open debate involving both pregnant women and physicians.

Identification by array-Comparative Genomic Hybridization (array-CGH) of a large deletion of luteinizing hormone receptor gene combined with a missense mutation in a patient diagnosed with a 46,XY disorder of sex development and application to prenatal diagnosis

This paper reports the case of an infant presenting with sexual ambiguity at birth. The child presented with labia majora synechia, thready genital tubercle and perineal hypospadias. The karyotype was 46,XY. Low testosterone levels with no response to hCG administration, associated with high LH level for her age, high FSH level, high inhibin B levels and normal AMH indicated a lack of LH receptivity and prompted us to screen the LHCGR gene for mutations. A previously described missense mutation (p.Cys131Arg) was identified at homozygous state in the propositus and at heterozygous state in the mother. This variation, however, was not found in the father. Our attention was drawn by the presence of several single nucleotide polymorphisms (SNPs), identified at homozygous state without any paternal contribution from exon 1 to exon 10 of LHCGR, suggesting a paternal deletion. Array DNA analysis was performed revealing a large deletion extending from 61,493 to 135,344 bp and including the LHCGR gene. Adequate genetic counselling was provided. This paper describes the first application of prenatal diagnosis in LHCGR deficiency for 46,XY disorders of sex development with the subsequent delivery of a normal baby.

Flow cytometric methods for prenatal and neonatal diagnosis

Flow cytometry offers a promising alternative to the current methods of amniocentesis or chorionic villus sampling (CVS) for fetal cell sorting for prenatal diagnosis. While flow cytometric methods have been greatly improved to be more sensitive at detecting fetal cells within the maternal circulation, there are still several challenges that need to be overcome before application in prenatal diagnosis. However, flow cytometry is a powerful tool that can be used to enhance molecular testing and other diagnostic testing modalities in prenatal and neonatal diagnosis. It remains the gold standard to identify cellular immunodeficiencies and, for some immunological disorders with established biomarkers, flow cytometric assays can be used to make a definitive diagnosis. In this review, the advantages and disadvantages of using MACS and FACS analysis for fetal cell sorting are discussed. This review also includes an overview of the current flow cytometric assays and biomarkers that may be used for prenatal and neonatal diagnosis of common immunological and hematological abnormalities and the role of flow cytometry in treatment monitoring after bone marrow and stem cell transplantation.