Use of amplicon-based sequencing for testing fetal identity and monogenic traits with Single Circulating Trophoblast (SCT) as one form of cell-based NIPT

A major challenge for cell-based non-invasive prenatal testing (NIPT) is to distinguish individual presumptive fetal cells from maternal cells in female pregnancies. We have sought a rapid, robust, versatile, and low-cost next-generation sequencing method to facilitate this process. Toward this goal, single isolated cells underwent whole genome amplification prior to genotyping. Multiple highly polymorphic genomic regions (including HLA-A and HLA-B) with 10-20 very informative single nucleotide polymorphisms (SNPs) within a 200 bp interval were amplified with a modified method based on other publications. To enhance the power of cell identification, approximately 40 Human Identification SNP (Applied Biosystems) test amplicons were also utilized. Using SNP results to compare to sex chromosome data from NGS as a reliable standard, the true positive rate for genotyping was 83.4%, true negative 6.6%, false positive 3.3%, and false negative 6.6%. These results would not be sufficient for clinical diagnosis, but they demonstrate the general validity of the approach and suggest that deeper genotyping of single cells could be completely reliable. A paternal DNA sample is not required using this method. The assay also successfully detected pathogenic variants causing Tay Sachs disease, cystic fibrosis, and hemoglobinopathies in single lymphoblastoid cells, and disease-causing variants in three cell-based NIPT cases. This method could be applicable for any monogenic diagnosis

Reliable detection of subchromosomal deletions and duplications using cell-based noninvasive prenatal testing

Objective To gather additional data on the ability to detect subchromosomal abnormalities of various sizes in single fetal cells isolated from maternal blood, using low-coverage shotgun next-generation sequencing for cell-based noninvasive prenatal testing (NIPT). Method Fetal trophoblasts were recovered from approximately 30 mL of maternal blood using maternal white blood cell depletion, density-based cell separation, immunofluorescence staining, and high-resolution scanning. These trophoblastic cells were picked as single cells and underwent whole genome amplification for subsequent genome-wide copy number analysis and genotyping to confirm the fetal origin of the cells. Results Applying our fetal cell isolation method to a series of 125 maternal blood samples, we detected on average 4.17 putative fetal cells/sample. The series included 15 cases with clinically diagnosed fetal aneuploidies and five cases with subchromosomal abnormalities. This method was capable of detecting findings that were 1 to 2 Mb in size, and all were concordant with the microarray or karyotype data obtained on a fetal sample. A minority of fetal cells showed evidence of genome degradation likely related to apoptosis. Conclusion We demonstrate that this cell-based NIPT method has the capacity to reliably diagnose fetal chromosomal abnormalities down to 1 to 2 Mb in size

Clinical validity assessment of genes frequently tested on intellectual disability/autism sequencing panels.

[en] PURPOSE: Neurodevelopmental disorders (NDDs), such as intellectual disability (ID) and autism spectrum disorder (ASD), exhibit genetic and phenotypic heterogeneity, making them difficult to differentiate without a molecular diagnosis. The Clinical Genome Resource Intellectual Disability/Autism Gene Curation Expert Panel (GCEP) uses systematic curation to distinguish ID/ASD genes that are appropriate for clinical testing (ie, with substantial evidence supporting their relationship to disease) from those that are not. METHODS: Using the Clinical Genome Resource gene-disease validity curation framework, the ID/Autism GCEP classified genes frequently included on clinical ID/ASD testing panels as Definitive, Strong, Moderate, Limited, Disputed, Refuted, or No Known Disease Relationship. RESULTS: As of September 2021, 156 gene-disease pairs have been evaluated. Although most (75%) were determined to have definitive roles in NDDs, 22 (14%) genes evaluated had either Limited or Disputed evidence. Such genes are currently not recommended for use in clinical testing owing to the limited ability to assess the effect of identified variants. CONCLUSION: Our understanding of gene-disease relationships evolves over time; new relationships are discovered and previously-held conclusions may be questioned. Without periodic re-examination, inaccurate gene-disease claims may be perpetuated. The ID/Autism GCEP will continue to evaluate these claims to improve diagnosis and clinical care for NDDs

Retinal tissue preparation for high-resolution live imaging of photoreceptors expressing multiple transgenes

Live imaging has become the favorite method in recent years to study the protein transport, localization and dynamics in live cells. Protein transport is extremely essential for proper function of photoreceptors. Aberration in the proper transport of proteins gives rise to the loss of photoreceptor and blindness. On the other hand, the ease of generation of transgenic Xenopus laevis tadpoles and the advantage of high resolution live confocal imaging provide new insight into understanding protein dynamics in photoreceptors. There are several steps for quantifying and visualizing fluorescently tagged proteins in photoreceptors starting with assembly of plasmids, generation of transgenic tadpoles, preparation of retinal tissues, imaging the transgenic photoreceptors and finally analyzing the recorded data. The focus of this manuscript is to describe how to prepare retinal tissues suited for live cell imaging and provide our readers with a tutorial video. We also give a summary of steps leading to a successful experiment that might be designed for imaging the ultrastructures of photoreceptors, the expression of two or more different fluorescently tagged proteins, their localization, distribution, or protein dynamics within photoreceptors. • Retinal tissue live imaging demonstrates the ultrastructures of photoreceptors. • High resolution live confocal imaging provides new insight into understanding the pathophysiology of photoreceptors. Method name: Retinal tissue preparation for high-resolution live imaging, Keywords: Live imaging, Retinal, Photoreceptor, Xenopu

With 3 Types of Respiratory Acquisition: 3.0 T Respiratory Triggered Acquisition Can Obtain Higher Quality DWI Images of the Upper Abdomen

Objective. To compare the effects of 1.5 T and 3.0 T upper abdominal magnetic resonance diffusion-weighted imaging (DWI) under three acquisition techniques of breath holding, breath triggering, and free breathing, so as to provide a reference for the usage of upper abdominal DWI scanning. Methods. Twenty-one healthy subjects were selected from social volunteers and underwent routine magnetic resonance imaging (MRI) and DWI on 1.5 T and 3.0 T, respectively. DWI included three acquisition methods: breath triggering, breath holding, and free breathing, and b values were 100 and 800. The DWI image artifacts, image quality, apparent diffusion coefficient (ADC), and the signal-to-noise ratio (SNR) obtained through the three acquisition methods were compared. Results. The 1.5 T free-breathing DWI image quality was the best, while the 3.0 T had the best breath-triggered DWI image quality. The 3.0 T breath-triggered DWI image quality was better than the 1.5 T free-breathing DWI image (P=0.012), and the SNR of free-breathing DWI was the highest. Between the two field intensities, the SNR of the liver in the 3.0 T group was much lower than that in the 1.5 T group, and obvious differences were not observed in ADC values of normal liver, gallbladder, kidney, spleen, and pancreas. Conclusion. 3.0 T respiratory-triggered acquisition can obtain higher quality DWI images. But in the case of only 1.5 T field strength, free-breathing acquisition of DWI images should be selected

An Inducible Expression System to Measure Rhodopsin Transport in Transgenic <i>Xenopus</i> Rod Outer Segments

<div><p>We developed an inducible transgene expression system in <i>Xenopus</i> rod photoreceptors. Using a transgene containing mCherry fused to the carboxyl terminus of rhodopsin (Rho-mCherry), we characterized the displacement of rhodopsin (Rho) from the base to the tip of rod outer segment (OS) membranes. Quantitative confocal imaging of live rods showed very tight regulation of Rho-mCherry expression, with undetectable expression in the absence of dexamethasone (Dex) and an average of 16.5 µM of Rho-mCherry peak concentration after induction for several days (equivalent to >150-fold increase). Using repetitive inductions, we found the axial rate of disk displacement to be 1.0 µm/day for tadpoles at 20 °C in a 12 h dark /12 h light lighting cycle. The average distance to peak following Dex addition was 3.2 µm, which is equivalent to ~3 days. Rods treated for longer times showed more variable expression patterns, with most showing a reduction in Rho-mCherry concentration after 3 days. Using a simple model, we find that stochastic variation in transgene expression can account for the shape of the induction response. </p> </div

An inducible expression system for <i>Xenopus</i> rods.

<p>(A) Schematic diagram of a <i>Xenopus</i> rod. In <i>Xenopus</i>, there is a daily synthesis of approximately 80 discs, and the previous disks are displaced apically. Thus, the distance of disks from the base of the OS is linearly related to the time after induction. (B) Schematic diagram of a XOP:G3U-Rho-mCherry system (left) and a XOP:Rho-mCherry constitutive expression system (right). Rod-specific expression is accomplished using the <i>Xenopus</i> rhodopsin promoter (XOP) driving transcription of G3. Dex treatment of animals transgenic for both XOP:G3 and pUAS:Rho-mCherry induces synthesis of Rho-mCherry that is transported and integrated into the rod outer segment (OS) disk membranes. Rods with XOP:Rho-mCherry express the Rho-mCherry constitutively. (C) Constitutive expression of XOP:Rho-mCherry transgene (top). There are two kinds axial variation of Rho-mCherrry expression in the OS: diurnal variation (middle) and long-term variation (lower). (D) Dex induction treatment paradigm 1. Tadpoles (St. 54) were treated with 10 µM Dex for 7 days and then sacrificed immediately before imaging. (E) Dex induction treatment paradigm II. Tadpoles (St. 54) received repetitive 3-day 10 µM Dex inductions (black boxes), each followed by a 5 day interval without Dex. Seven days after the last induction, retinas were explanted immediately before imaging.</p

Disk displacement measured from the spatial distribution of induction response peak.

<p>(A) Diagram of the Dex treatment paradigm. (B) Correlation of distance of the peak response to the IS/OS junction and time of Dex treatment (Error bar is standard deviation, dash line is the linear regression line. (C) Histogram of peak-to-peak distances in following repetitive inductions with the eight day paradigm. The distance distribution was fit to a Gaussian curve with an R² = 0.96. The mean of peak-peak distance was 8.0 μm (SD = 2.4, n = 72).</p

Distribution of Rho-mCherry in live rods after repetitive 3-day induction.

<p>(A) Live rods with one to three responses in a retina chip are shown with the fluorescence merge with DIC. Scale bar is 10 μm. (B) Five individual rods with two (2,3) or one (4,5) responses are shown with fluorescence and merged with DIC . Scale bar is 5 μm. (C) Relative mCherry fluorescence intensity profiles of several different live rods, which received same treatment but exhibited different responses. Top scan is from the cell in A with three responses and the others from cells indicated in B. Scale bar on the x-axis represents 10 μm.</p

Model to simulate induction response.

<p>(A) Schematic of the G3 inducible system. This model includes transcription, translation and transport of Rho-mCherry reporter with both nuclear and cytoplasmic mRNA and protein degradation. (B) Simplified model of inducible system. Transcription and translation steps were combined to yield a model with five parameters: variable sinusoidal synthesis of G3, Dex, k_act, γ_cyto and γ_nuc for degradation (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082629#pone.0082629.s001" target="_blank">Appendix S1</a> for details). (C) Simulation of induction responses to seven Dex treatments. Upper panel shows constant G3 expression and lower panel shows variable G3 expression with 10-day period. Color arrows in left-most panel indicate induction at different phases of a 10-day period. The corresponding average induction responses (Rho expression) are plotted in the corresponding color. These responses were obtained by averaging hundreds of simulations responses with randomized phases. The rightmost plots show how the phase of G3 expression changes the characteristic positions in induction response: rising midpoint (red), peak (purple) and falling midpoint (green). </p