Isolation of single circulating trophoblasts from maternal circulation for noninvasive fetal copy number variant profiling

ObjectiveTo develop a multi-step workflow for the isolation of circulating extravillous trophoblasts (cEVTs) by describing the key steps enabling a semi-automated process, including a proprietary algorithm for fetal cell origin genetic confirmation and copy number variant (CNV) detection. MethodsDetermination of the limit of detection (LoD) for submicroscopic CNV was performed by serial experiments with genomic DNA and single cells from Coriell cell line biobank with known imbalances of different sizes. A pregnancy population of 372 women was prospectively enrolled and blindly analyzed to evaluate the current workflow. ResultsAn LoD of 800 Kb was demonstrated with Coriell cell lines. This level of resolution was confirmed in the clinical cohort with the identification of a pathogenic CNV of 800 Kb, also detected by chromosomal microarray. The mean number of recovered cEVTs was 3.5 cells per sample with a significant reverse linear trend between gestational age and cEVT recovery rate and number of recovered cEVTs. In twin pregnanices, evaluation of zygosity, fetal sex and copy number profiling was performed in each individual cell. ConclusionOur semi-automated methodology for the isolation and single-cell analysis of cEVTS supports the feasibility of a cell-based noninvasive prenatal test for fetal genomic profiling. © 2022 A. Menarini Biomarkers Singapore Pte Ltd. Prenatal Diagnosis published by John Wiley & Sons Ltd

Prenatal maternal plasma DNA screening for cystic fibrosis: A computer modelling study of screening performance.

Background: Prenatal cystic fibrosis (CF) screening is currently based on determining the carrier status of both parents. We propose a new method based only on the analysis of DNA in maternal plasma. Methods: The method relies on the quantitative amplification of the CF gene to determine the percentage of DNA fragments in maternal plasma at targeted CF mutation sites that carry a CF mutation. Computer modelling was carried out to estimate the distributions of these percentages in pregnancies with and without a fetus affected with CF. This was done according to the number of DNA fragments counted and fetal fraction, using the 23 CF mutations recommended by the American College of Medical Genetics for parental carrier testing. Results: The estimated detection rate (sensitivity) is 70% (100% of those detected using the 23 mutations), the false-positive rate 0.002%, and the odds of being affected given a positive screening result 14:1, compared with 70%, 0.12%, and 1:3, respectively, with current prenatal screening based on parental carrier testing. Conclusions: Compared with current screening practice based on parental carrier testing, the proposed method would substantially reduce the number of invasive diagnostic procedures (amniocentesis or chorionic villus sampling) without reducing the CF detection rate. The expected advantages of the proposed method justify carrying out the necessary test development for use in a clinical validation study.The author(s) declared that no grants were involved in supporting this work

Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like

Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell

Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like

Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell

Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like

Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell

Obstetric professionals’ perceptions of non-invasive prenatal testing for Down syndrome: clinical usefulness compared with existing tests and ethical implications

Background: While non-invasive prenatal testing (NIPT) for fetal aneuploidy is commercially available in many countries, little is known about how obstetric professionals in non-Western populations perceive the clinical usefulness of NIPT in comparison with existing first-trimester combined screening (FTS) for Down syndrome (DS) or invasive prenatal diagnosis (IPD), or perceptions of their ethical concerns arising from the use of NIPT. Methods: A cross-sectional survey among 327 obstetric professionals (237 midwives, 90 obstetricians) in Hong Kong. Results: Compared to FTS, NIPT was believed to: provide more psychological benefits and enable earlier consideration of termination of pregnancy. Compared to IPD, NIPT was believed to: provide less psychological stress for high-risk women and more psychological assurance for low-risk women, and offer an advantage to detect chromosomal abnormalities earlier. Significant differences in perceived clinical usefulness were found by profession and healthcare sector: (1) obstetricians reported more certain views towards the usefulness of NIPT than midwives and (2) professionals in the public sector perceived less usefulness of NIPT than the private sector. Beliefs about earlier detection of DS using NIPT were associated with ethical concerns about increasing abortion. Participants believing that NIPT provided psychological assurance among low-risk women were less likely to be concerned about ethical issues relating to informed decision-making and pre-test consultation for NIPT. Conclusions: Our findings suggest the need for political debate initially on how to ensure pregnant women accessing public services are informed about commercially available more advanced technology, but also on the potential implementation of NIPT within public services to improve access and equity to DS screening services

Misbehaviour of XIST RNA in Breast Cancer Cells

A role of X chromosome inactivation process in the development of breast cancer have been suggested. In particular, the relationship between the breast cancer predisposing gene BRCA1 and XIST, the main mediator of X chromosome inactivation, has been intensely investigated, but still remains controversial. We investigated this topic by assessing XIST behaviour in different groups of breast carcinomas and in a panel of breast cancer cell lines both BRCA1 mutant and wild type. In addition, we evaluated the occurrence of broader defects of heterochromatin in relation to BRCA1 status in breast cancer cells. We provide evidence that in breast cancer cells BRCA1 is involved in XIST regulation on the active X chromosome, but not in its localization as previously suggested, and that XIST can be unusually expressed by an active X and can decorate it. This indicates that the detection of XIST cloud in cancer cell is not synonymous of the presence of an inactive X chromosome. Moreover, we show that global heterochromatin defects observed in breast tumor cells are independent of BRCA1 status. Our observations sheds light on a possible previously uncharacterized mechanism of breast carcinogenesis mediated by XIST misbehaviour, particularly in BRCA1-related cancers. Moreover, the significant higher levels of XIST-RNA detected in BRCA1-associated respect to sporadic basal-like cancers, opens the possibility to use XIST expression as a marker to discriminate between the two groups of tumors

Comparison of two high-throughput semiconductor chip sequencing platforms in noninvasive prenatal testing for Down syndrome in early pregnancy

Background: Noninvasive prenatal testing (NIPT) to detect fetal aneuploidy using next-generation sequencing on ion semiconductor platforms has become common. There are several sequencers that can generate sufficient DNA reads for NIPT. However, the approval criteria vary among platforms and countries. This can delay the introduction of such devices and systems to clinics. A comparison of the sensitivity and specificity of two different platforms using the same sequencing chemistry could be useful in NIPT for fetal chromosomal aneuploidies. This would improve healthcare authorities' confidence in decision-making on sequencing-based tests. Methods: One hundred and one pregnant women who were predicted at high risk of fetal defects using conventional prenatal screening tests, and who underwent definitive diagnosis by full karyotyping, were enrolled from three hospitals in Korea. Most of the pregnant women (69.79 %) received NIPT during weeks 11-13 of gestation and 30.21 % during weeks 14-18. We used Ion Torrent PGM and Proton semi-conductor-based sequencers with 0.3x sequencing coverage depth. The average total reads of 101 samples were approximately 4.5 and 7.6 M for PGM and Proton, respectively. A Burrows-Wheeler Aligner (BWA) algorithm was used for the alignment, and a z-score was used to decide fetal trisomy 21. Interactive dot diagrams from the sequencing data showed minimal z-score values of 2.07 and 2.10 to discriminate negative versus positive cases of fetal trisomy 21 for the two different sequencing systems. Results: Our z-score-based discrimination method resulted in 100 % positive and negative prediction values for both ion semiconductor PGM and Proton sequencers, regardless of their sequencing chip and chemistry differences. Both platforms performed well at an early stage (11-13 weeks of gestation) compared with previous studies. Conclusions: These results suggested that, using two different sequencers, NIPT to detect fetal trisomy 21 in early pregnancy is accurate and platform-independent. The data suggested that the amount of sequencing and the application of common, simple, and robust statistical analyses are more important than sequencing chemistry and platform types. This result has practical implications in countries where PGM is approved for NIPT but the Proton system is not.ope