The first clinical validation of whole-genome screening on standard trophectoderm biopsies of preimplantation embryos.

Objective: To validate the performance of our laboratory-developed whole-genome screening assay within clinical preimplantation genetic testing environments. Design: Perform a laboratory-developed whole-genome assay on both cell lines and trophectoderm biopsies, subsequently employing the next-generation sequencing procedure to reach a sequencing depth of 30X. Adhere to the Genome Analysis Toolkit best practices for accuracy, sensitivity, specificity, and precision calculations by comparing samples with references. Our assay was then applied to cell lines and biopsies harboring known pathogenic variants, aiming to ascertain these changes solely from the next-generation sequencing data, independent of parental genome information. Settings: Clinical laboratory. Patients: Coriell cell lines and research embryos with known chromosomal or genetic variants. Research trophectoderm biopsies from a couple that are heterozygous carriers for distinct variants in the same autosomal recessive gene (HOGA1). Intervention: Not applicable. Main Outcome Measures: Accuracy, sensitivity, specificity, and precision were assessed by comparing the samples to their references. For samples with known variants, we calculated our sensitivity to detecting established variants. For the research embryos, noncarrier, carrier, and compound heterozygous states of inherited HOGA1 variants were distinguished independently of parental samples. Results: Amplification of DNA from cell lines and embryos yielded success rates exceeding 99.9% and 98.2%, respectively, although maintaining an accuracy of >99.9% for aneuploidy assessment. The accuracy (99.99%), specificity (99.99%), sensitivity (98.0%), and precision (98.1%) of amplified genome in the bottle (reference NA12878) and embryo biopsies were comparable to results on genomic DNA, including mitochondrial heteroplasmy. Using our assay, we achieved >99.99% sensitivity when examining samples with known chromosomal and genetic variants. This encompassed pathogenic CFTR, BRCA1, and other variants, along with uniparental isodisomies and microdeletions such as DiGeorge syndrome. Our research study identified noncarrier, carrier, and compound heterozygous states within trophectoderm biopsies while simultaneously screening for 1,300 other severe monogenic diseases. Conclusion: To our knowledge, this is the first clinical validation of whole-genome embryo screening. In this study, we demonstrated high accuracy for aneuploidy calls (>99.9%) and genetic variants (99.99%), even in the absence of parental genomes. This assay demonstrates advancements in genomic screening and an extended scope for testing capabilities in the realm of preimplantation genetic testing.

Eye, Ocular Adnexa, and Facial Manifestations of Tetrasomy 18p.

Tetrasomy 18p is often the result of an additional isochromosome for the short arm of chromosome 18. Although many organ systems are affected phenotypically, the ocular manifestations associated with tetrasomy 18p have not been well characterized in the literature. This case report presents the ocular and facial features associated with tetrasomy 18p in a 4-year-old Black girl, along with a review of clinical presentations previously reported in the literature. A systematic review of the literature in PubMed was conducted to summarize the reported eye, ocular adnexa, and distinctive facial features in individuals with confirmed tetrasomy 18p. Searching "Tetrasomy 18p" generated 65 article results, of which 28 articles had sufficient eye and facial descriptions. Including the patient in this report, 90 patients had confirmed tetrasomy 18p. The most common features noted in these 90 patients, with a roughly equal male-to-female ratio of impact (7:8), were as follows: microcephaly (57%), triangular facies (18%), anomalous palpebral fissures (31%), strabismus (48%), low-set ears (52%), hearing loss to some extent (16%), depressed or flat nasal bridge (18%), smooth philtrum (41%), thin upper lip (27%), and highly arched palate (21%). Additionally, many were noted to have feeding difficulties (28%), developmental delay (58%), and abnormal brain findings on imaging (20%). Muscle tone was abnormal in 23% of the patients. This report elucidates the reoccurring eye, ocular adnexa, and distinctive facial features associated with tetrasomy 18p. This knowledge may assist in timely diagnosis and encourage providers to use a multidisciplinary approach for the treatment of affected individuals. [J Pediatr Ophthalmol Strabismus. 2021;58(6):e44-e48.].

Monosomy X rescue explains discordant NIPT results and leads to uniparental isodisomy.

Noninvasive prenatal testing accurately detects trisomy for chromosomes 13, 21, and 18, but has a significantly lower positive predictive value for monosomy X. Discordant monosomy X results are often assumed to be due to maternal mosaicism, usually without maternal follow-up. We describe a case of monosomy X-positive noninvasive prenatal testing that was discordant with the 46,XX results from amniocentesis and postnatal testing. This monosomy X pregnancy doubled the single X chromosome, leading to 45,X/46,XX mosaicism in the placenta and uniparental isodisomy X in the amniotic fluid. Thus, at least some discordant monosomy X results are due to true mosaicism in the pregnancy, which has important implications for clinical outcome and patient counseling.

The Gyc76C Receptor Guanylyl Cyclase and the Foraging cGMP-Dependent Kinase Regulate Extracellular Matrix Organization and BMP Signaling in the Developing Wing of Drosophila melanogaster.

The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, we identify Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. We show that Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. We identify a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases, and show that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. We discuss parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues.

Partial monosomy of 11q22.2q22.3 including the SDHD gene in individuals with developmental delay.

Deletions in the middle portion of 11q are not as well described in the literature as terminal 11q deletions that result in Jacobsen syndrome. One confounding factor in the older literature is that the G-banding pattern of 11q13q21 is very similar to 11q21q23. The advent of fluorescence in situ hybridization and later microarray technologies have allowed for a better resolution of many of these deletions, but genotype-phenotype correlations are still difficult since these deletions are rare events. We present five individuals who presented with developmental delays with de novo 11q22.2q23.3 deletions. Deletions were observed by standard G-banded chromosome analysis with clarification of breakpoints and gene content by SNP microarray analysis. Of note, all individuals had identical distal breakpoints. All deletions include SDHD, which is implicated in hereditary paraganglioma/pheochromocytoma, for which the patients will need to be monitored in adulthood. In spite of the large deletions of 8.6 Mb (Patients 1 and 3), 13.98 Mb (Patient 2), and 12.6 Mb (Patients 4 and 5) all patients show somewhat mild intellectual disability and dysmorphism.

Crossveinless d is a vitellogenin-like lipoprotein that binds BMPs and HSPGs, and is required for normal BMP signaling in the Drosophila wing.

The sensitivity of the posterior crossvein in the pupal wing of Drosophila to reductions in the levels and range of BMP signaling has been used to isolate and characterize novel regulators of this pathway. We show here that crossveinless d (cv-d) mutations, which disrupt BMP signaling during the development of the posterior crossvein, mutate a lipoprotein that is similar to the vitellogenins that comprise the major constituents of yolk in animal embryos. Cv-d is made in the liver-like fat body and other tissues, and can diffuse into the pupal wing via the hemolymph. Cv-d binds to the BMPs Dpp and Gbb through its Vg domain, and to heparan sulfate proteoglycans, which are well-known for their role in BMP movement and accumulation in the wing. Cv-d acts over a long range in vivo, and does not have BMP co-receptor-like activity in vitro. We suggest that, instead, it affects the range of BMP movement in the pupal wing, probably as part of a lipid-BMP-lipoprotein complex, similar to the role proposed for the apolipophorin lipid transport proteins in Hedgehog and Wnt movement.

The microRNA pathway regulates the temporal pattern of Notch signaling in Drosophila follicle cells.

Multicellular development requires the correct spatial and temporal regulation of cell division and differentiation. These processes are frequently coordinated by the activities of various signaling pathways such as Notch signaling. From a screen for modifiers of Notch signaling in Drosophila we have identified the RNA helicase Belle, a recently described component of the RNA interference pathway, as an important regulator of the timing of Notch activity in follicle cells. We found that loss of Belle delays activation of Notch signaling, which results in delayed follicle cell differentiation and defects in the cell cycle. Because mutations in well-characterized microRNA components phenocopied the Notch defects observed in belle mutants, Belle might be functioning in the microRNA pathway in follicle cells. The effect of loss of microRNAs on Notch signaling occurs upstream of Notch cleavage, as expression of the constitutively active intracellular domain of Notch in microRNA-defective cells restored proper activation of Notch. Furthermore, we present evidence that the Notch ligand Delta is an important target of microRNA regulation in follicle cells and regulates the timing of Notch activation through cis inhibition of Notch. Here we have uncovered a complex regulatory process in which the microRNA pathway promotes Notch activation by repressing Delta-mediated inhibition of Notch in follicle cells.

Dynamin participates in the maintenance of anterior polarity in the Caenorhabditis elegans embryo.

Cell polarity is crucial for the generation of cell diversity. Recent evidence suggests that the actin cytoskeleton plays a key role in establishment of embryonic polarity, yet the mechanisms that maintain polarity cues in particular membrane domains during development remain unclear. Dynamin, a large GTPase, functions in both endocytosis and actin dynamics. Here, the Caenorhabditis elegans dynamin ortholog, DYN-1, maintains anterior polarity cues. DYN-1-GFP foci are enriched in the anterior cortex in a manner dependent on the anterior polarity proteins, PAR-6 and PKC-3. Membrane internalization and actin comet formation are enriched in the anterior, and are dependent on DYN-1. PAR-6-labeled puncta are also internalized from cortical accumulations of DYN-1-GFP. Our results demonstrate a mechanism for the spatial and temporal regulation of endocytosis in the anterior of the embryo, contributing to the precise localization and maintenance of polarity factors within a dynamic plasma membrane.

Dynamin and cytokinesis.

Animal and plant cytokineses appear morphologically distinct. Recent studies, however, have revealed that these cellular processes have many things in common, including the requirement of co-ordinated membrane trafficking and cytoskeletal dynamics. At the intersection of these two processes are the members of the dynamin family of ubiquitous eukaryotic GTPases. In this review, we highlight the conserved contribution of classical dynamin and dynamin-related proteins during cytokinesis in both animal and plant systems.