Noninvasive prenatal sequencing for multiple Mendelian monogenic disorders among fetuses with skeletal dysplasia or increased nuchal translucency.

OBJECTIVES: To evaluate the performance of noninvasive prenatal sequencing for multiple Mendelian monogenic disorders (NIPS-M) among fetuses with skeletal abnormalities or increased nuchal translucency (NT). METHODS: Pregnancies with fetal skeletal abnormalities or increased NT (≥3.0 mm) observed by ultrasonography were recruited between October 2017 and March 2019. Parental blood from 13 couples were collected for NIPS-M testing reported. All the NIPS-M results were followed up by invasive diagnostic testing or neonatal examination. RESULTS: Among the 13 cases, 8 (61.5%) yielded positive results for pathogenic variants in the FGFR3, COL1A1, RAF1, PTPN11 and SOS1 genes by NIPS-M. One case was excluded for further analysis due to insufficient fetal DNA (<4.5%). De novo mutations were reported in six of the eight positive cases (75%). The other two were inconclusive as the pathogenic variants were detected in both plasma and genomic DNA of the mothers. The sensitivity of NIPS-M was 100%. CONCLUSIONS: Our pilot study demonstrates that NIPS-M is an accurate approach for detection of multiple monogenic disorders among fetuses with skeletal abnormalities or increased NT. It serves as an alternative and highly sensitive method to provide valuable molecular information for these groups of women who are reluctant to undergo invasive procedure.

The utility of genome-wide cell-free DNA screening in the prenatal diagnosis of Pallister-Killian syndrome.

OBJECTIVE: To report genome-wide cell-free DNA (cfDNA) screening facilitating the diagnosis of Pallister-Killian syndrome (PKS). METHODS: This is a retrospective cohort analysis of positive genome-wide cfDNA screening results showing increased signal from chromosome 12 and the detection of PKS. The genome-wide cfDNA screening results and the subsequent investigations were reviewed. RESULTS: Three singleton pregnancies (3/29007) from 2016 to 2017 yielded positive results indicating large gains on the entire p-arm of chromosome 12. In two cases, multiple structural abnormalities were detected by prenatal ultrasound and the couples opted for termination of pregnancy. Chromosomal microarray performed on fetal skin tissues of the two abortuses detected mosaic tetrasomy 12p, consistent with PKS. In the third case, karyotype and chromosomal microarray performed on an amniotic fluid sample also showed mosaic tetrasomy 12p. In each of the three cases, genome-wide cfDNA screening revealed a large gain on chromosome 12p; subsequent prenatal or postnatal diagnostic testing confirmed the diagnosis of PKS. CONCLUSION: We report the ability of genome-wide cfDNA screening to provide early suspicion and facilitate the subsequent genetic diagnosis of PKS. As genome-wide cfDNA screening becomes increasingly available, incidental diagnosis of partial aneuploidies is expected to increase.

Characteristics and mode of inheritance of pathogenic copy number variants in prenatal diagnosis.

BACKGROUND: Microdeletions and microduplications can occur in any pregnancy independent of maternal age. The spectrum and features of pathogenic copy number variants including the size, genomic distribution, and mode of inheritance are not well studied. These characteristics have important clinical implications regarding expanding noninvasive prenatal screening for microdeletions and microduplications. OBJECTIVES: The aim was to investigate the spectrum and characteristics of pathogenic copy number variants in prenatal genetic diagnosis and to provide recommendations for expanding the scope of noninvasive prenatal screening for microdeletions and microduplications. STUDY DESIGN: This was a retrospective study of 1510 pregnant women who underwent invasive prenatal diagnostic testing by chromosomal microarray analysis. Prenatal samples were retrieved by amniocentesis or chorionic villus sampling and sent to our prenatal genetic diagnosis laboratory for chromosomal microarray analysis. The risk of carrying a fetus with pathogenic copy number variants is stratified by the patients' primary indication for invasive testing. We searched the literature for published prenatal chromosomal microarray data to generate a large cohort of 23,865 fetuses. The characteristics and spectrum of pathogenic copy number variants including the type of aberrations (gains or losses), genomic loci, sizes, and the mode of inheritance were studied. RESULTS: Overall, 375 of 23,865 fetuses (1.6%) carried pathogenic copy number variants for any indication for invasive testing, and 44 of them (11.7%) involve 2 or more pathogenic copy number variants. A total of 428 pathogenic copy number variants were detected in these fetuses, of which 280 were deletions and 148 were duplications. Three hundred sixty (84.1%) were less than 5 Mb in size and 68 (15.9%) were between 5 and 10 Mb. The incidence of carrying a pathogenic copy number variant in the high-risk group is 1 in 36 and the low-risk group is 1 in 125. Parental inheritance study results were available for 311 pathogenic copy number variants, 71 (22.8%) were maternally inherited, 36 (11.6%) were paternally inherited, and 204 (65.6%) occurred de novo. CONCLUSION: Collectively, pathogenic copy number variants are common in pregnancies. High-risk pregnancies should be offered invasive testing with chromosomal microarray analysis for the most comprehensive investigation. Detection limits on size, parental inheritance, and genomic distribution should be carefully considered before implementing copy number variant screening in expanded noninvasive prenatal screening.

Clinical Significance of Non-Invasive Prenatal Screening for Trisomy 7: Cohort Study and Literature Review.

Trisomy 7 is the most frequently observed type of rare autosomal trisomies in genome-wide non-invasive prenatal screening (NIPS). Currently, the clinical significance of trisomy 7 NIPS-positive results is still unknown. We reviewed two independent cohorts from two laboratories where similar NIPS metrics were applied. A total of 70,441 singleton cases who underwent genome-wide NIPS were analyzed, among which 39 pregnancies were positive for trisomy 7, yielding a screen-positive rate of 0.055% (39/70,441). There were 28 cases with invasive testing results available; the positive predictive value (PPV) was 3.6% (1/28). We then searched the published NIPS studies to generate a large cohort of 437,873 pregnancies and identified 247 cases (0.056%) that were screened positive for trisomy 7. The overall PPV was 3.4% (4/118) in the combined data. The presence of uniparental disomy 7 was not detected in the NIPS trisomy 7-positive pregnancies with normal fetal karyotype. Among the 85 cases with pregnancy outcome available in combined data, 88.2% were normal live births, 14.1% had intrauterine growth restriction, preterm birth or low birth weight, 3.5% presented with ultrasound abnormality, and no fetal loss was observed. Our data provide valuable information for counseling and management of trisomy 7-positive NIPS pregnancies.

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy.

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60-80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2-4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Validation of a high-throughput and robust technique: BACs-on-beads assay (KaryoLite BoBs) for pre-implantation aneuploidy screening.

OBJECTIVE: This study aims to validate the BACs-on-Beads (BoB) technology as a robust and high throughput method for pre-implantation genetic screening (PGS) for aneuploidy. MATERIAL AND METHODS: The performances with respect to the sensitivity, specificity, success rate and detection rate of this technique from new BoBs technology and traditional array chromosomal genomic hybridization (aCGH) were compared. And the use of BoBs as a screening tool for euploid embryos in PGS was evaluated. RESULT: In the first part of validation study, there were total 75 embryos completed PGS by both BoBs and aCGH. The success rate of PGS was 97.4%, and the results showed 100% concordance between BoBs and aCGH for aneuploidy. In the second part, a total 219 embryos were involved. The success rate of PGS by BoBs was 100%. BoBs identified 28% (62/219) euploidy which were further confirmed to be euploidy by aCGH. CONCLUSION: This new strategic approach using BoBs as a first tier PGS screening tool and aCGH as a confirmatory tool can increase the throughput of PGS with a reduced cost and time to meet the demand in high volume units.

LncRNA Dum interacts with Dnmts to regulate Dppa2 expression during myogenic differentiation and muscle regeneration.

Emerging studies document the roles of long non-coding RNAs (LncRNAs) in regulating gene expression at chromatin level but relatively less is known how they regulate DNA methylation. Here we identify an lncRNA, Dum (developmental pluripotency-associated 2 (Dppa2) Upstream binding Muscle lncRNA) in skeletal myoblast cells. The expression of Dum is dynamically regulated during myogenesis in vitro and in vivo. It is also transcriptionally induced by MyoD binding upon myoblast differentiation. Functional analyses show that it promotes myoblast differentiation and damage-induced muscle regeneration. Mechanistically, Dum was found to silence its neighboring gene, Dppa2, in cis through recruiting Dnmt1, Dnmt3a and Dnmt3b. Furthermore, intrachromosomal looping between Dum locus and Dppa2 promoter is necessary for Dum/Dppa2 interaction. Collectively, we have identified a novel lncRNA that interacts with Dnmts to regulate myogenesis.

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.