Abstract
BACKGROUND
Trophectoderm (TE) biopsy and next generation sequencing (NGS) are currently the preferred techniques for preimplantation genetic testing for aneuploidies (PGT-A). Although this ...approach delivered important improvements over previous testing strategies, increased sensitivity has also prompted a rise in diagnoses of uncertain clinical significance. This includes reports of chromosomal mosaicism, suggesting the presence of karyotypically distinct cells within a single TE biopsy. Given that PGT-A relies on the chromosomal constitution of the biopsied cells being representative of the entire embryo, the prevalence and clinical implications of blastocyst mosaicism continue to generate considerable controversy.
OBJECTIVE AND RATIONALE
The objective of this review was to evaluate existing scientific evidence regarding the prevalence and impact of chromosomal mosaicism in human blastocysts. We discuss insights from a biological, technical and clinical perspective to examine the implications of this diagnostic dilemma for PGT-A.
SEARCH METHODS
The PubMed and Google Scholar databases were used to search peer-reviewed publications using the following terms: ‘chromosomal mosaicism’, ‘human’, ‘embryo’, ‘blastocyst’, ‘implantation’, ‘next generation sequencing’ and ‘clinical management’ in combination with other keywords related to the subject area. Relevant articles in the English language, published until October 2019 were critically discussed.
OUTCOMES
Chromosomal mosaicism predominately results from errors in mitosis following fertilization. Although it appears to be less pervasive at later developmental stages, establishing the true prevalence of mosaicism in human blastocysts remains exceedingly challenging. In a clinical context, blastocyst mosaicism can only be reported based on a single TE biopsy and has been ascribed to 2–13% of embryos tested using NGS. Conversely, data from NGS studies disaggregating whole embryos suggests that mosaicism may be present in up to ~50% of blastocysts. However, differences in testing and reporting strategies, analysis platforms and the number of cells sampled inherently overshadow current data, while added uncertainties emanate from technical artefacts. Moreover, laboratory factors and aspects of in vitro culture generate further variability. Outcome data following the transfer of blastocysts diagnosed as mosaic remain limited. Current studies suggest that the transfer of putative mosaic embryos may lead to healthy live births, but also results in significantly reduced ongoing pregnancy rates compared to the transfer of euploid blastocysts. Observations that a subset of mosaic blastocysts has the capacity to develop normally have sparked discussions regarding the ability of embryos to self-correct. However, there is currently no direct evidence to support this assumption. Nevertheless, the exclusion of mosaic blastocysts results in fewer embryos available for transfer, which may inevitably compromise treatment outcomes.
WIDER IMPLICATIONS
Chromosomal mosaicism in human blastocysts remains a perpetual diagnostic and clinical dilemma in the context of PGT-A. This review offers an important scientific resource, informing about the challenges, risks and value of diagnosing mosaicism. Elucidating these uncertainties will ultimately pave the way towards improved clinical and patient management.
Here, we present LNCipedia (http://www.lncipedia.org), a novel database for human long non-coding RNA (lncRNA) transcripts and genes. LncRNAs constitute a large and diverse class of non-coding RNA ...genes. Although several lncRNAs have been functionally annotated, the majority remains to be characterized. Different high-throughput methods to identify new lncRNAs (including RNA sequencing and annotation of chromatin-state maps) have been applied in various studies resulting in multiple unrelated lncRNA data sets. LNCipedia offers 21 488 annotated human lncRNA transcripts obtained from different sources. In addition to basic transcript information and gene structure, several statistics are determined for each entry in the database, such as secondary structure information, protein coding potential and microRNA binding sites. Our analyses suggest that, much like microRNAs, many lncRNAs have a significant secondary structure, in-line with their presumed association with proteins or protein complexes. Available literature on specific lncRNAs is linked, and users or authors can submit articles through a web interface. Protein coding potential is assessed by two different prediction algorithms: Coding Potential Calculator and HMMER. In addition, a novel strategy has been integrated for detecting potentially coding lncRNAs by automatically re-analysing the large body of publicly available mass spectrometry data in the PRIDE database. LNCipedia is publicly available and allows users to query and download lncRNA sequences and structures based on different search criteria. The database may serve as a resource to initiate small- and large-scale lncRNA studies. As an example, the LNCipedia content was used to develop a custom microarray for expression profiling of all available lncRNAs.
Neuroblastoma (NB) is a heterogeneous disease characterized by distinct clinical features and by the presence of typical copy-number alterations (CNAs). Given the strong association of these CNA ...profiles with prognosis, analysis of the CNA profile at diagnosis is mandatory. Therefore, we tested whether the analysis of circulating cell-free DNA (cfDNA) present in plasma samples of patients with NB could offer a valuable alternative to primary tumor DNA for CNA profiling.
In 37 patients with NB, cfDNA analysis using shallow whole genome sequencing (sWGS) was compared with arrayCGH analysis of primary tumor tissue.
Comparison of CNA profiles on cfDNA showed highly concordant patterns, particularly in high-stage patients. Numerical chromosome imbalances as well as large and focal structural aberrations including
and
amplification and
deletion could be readily detected with sWGS using a low input of cfDNA.
In conclusion, sWGS analysis on cfDNA offers a cost-effective, noninvasive, rapid, robust and sensitive alternative for tumor DNA copy-number profiling in most patients with NB.
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Plants produce a wide range of allelochemicals to defend against herbivore attack, and generalist herbivores have evolved mechanisms to avoid, sequester, or detoxify a broad spectrum of natural ...defense compounds. Successful arthropod pests have also developed resistance to diverse classes of pesticides and this adaptation is of critical importance to agriculture. To test whether mechanisms to overcome plant defenses predispose the development of pesticide resistance, we examined adaptation of the generalist two-spotted spider mite, Tetranychus urticae , to host plant transfer and pesticides. T. urticae is an extreme polyphagous pest with more than 1,100 documented hosts and has an extraordinary ability to develop pesticide resistance. When mites from a pesticide-susceptible strain propagated on bean were adapted to a challenging host (tomato), transcriptional responses increased over time with ∼7.5% of genes differentially expressed after five generations. Whereas many genes with altered expression belonged to known detoxification families (like P450 monooxygenases), new gene families not previously associated with detoxification in other herbivores showed a striking response, including ring-splitting dioxygenase genes acquired by horizontal gene transfer. Strikingly, transcriptional profiles of tomato-adapted mites resembled those of multipesticide-resistant strains, and adaptation to tomato decreased the susceptibility to unrelated pesticide classes. Our findings suggest key roles for both an expanded environmental response gene repertoire and transcriptional regulation in the life history of generalist herbivores. They also support a model whereby selection for the ability to mount a broad response to the diverse defense chemistry of plants predisposes the evolution of pesticide resistance in generalists.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Abstract
Shallow whole-genome sequencing to infer copy number alterations (CNAs) in the human genome is rapidly becoming the method par excellence for routine diagnostic use. Numerous tools exist to ...deduce aberrations from massive parallel sequencing data, yet most are optimized for research and often fail to redeem paramount needs in a clinical setting. Optimally, a read depth-based analytical software should be able to deal with single-end and low-coverage data-this to make sequencing costs feasible. Other important factors include runtime, applicability to a variety of analyses and overall performance. We compared the most important aspect, being normalization, across six different CNA tools, selected for their assumed ability to satisfy the latter needs. In conclusion, WISECONDOR, which uses a within-sample normalization technique, undoubtedly produced the best results concerning variance, distributional assumptions and basic ability to detect true variations. Nonetheless, as is the case with every tool, WISECONDOR has limitations, which arise through its exclusiveness for non-invasive prenatal testing. Therefore, this work presents WisecondorX in addition, an improved WISECONDOR that enables its use for varying types of applications. WisecondorX is freely available at https://github.com/CenterForMedicalGeneticsGhent/WisecondorX.
Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a naïve pluripotent state. Efforts have been made ...to trigger naïve pluripotency in hESCs for subsequent unbiased lineage‐specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines. This required either ectopic expression of naïve genes such as NANOG and KLF2 or inclusion of multiple pluripotency‐associated factors. We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor + Ascorbic Acid + Forskolin) facilitating rapid induction of transgene‐free naïve pluripotency in hESCs, as well as in mESCs, which has not been shown earlier. The converted naïve hESCs survived long‐term single‐cell passaging, maintained a normal karyotype, upregulated naïve pluripotency genes, and exhibited dependence on signaling pathways similar to naïve mESCs. Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile. We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward naïve pluripotency. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs that is fast, reproducible, supports naïve mESC derivation, and allows efficient differentiation. Stem Cells 2015;33:2686–2698
Different Shades of Naïve Pluripotency in Human Embryonic Stem Cells.
Objective To add evidence that massive parallel sequencing (MPS) is a valuable substitute for array comparative genomic hybridization (arrayCGH) with a resolution that is more appropriate for ...preimplantation genetic diagnosis (PGD) in translocation carriers. Design Study of diagnostic accuracy. Setting University hospital. Patient(s) Fifteen patients with a balanced structural rearrangement were included in the study: eight reciprocal translocations, four Robertsonian translocations, two inversions, and one insertional translocation. Intervention(s) Trophectoderm biopsy was performed on 47 blastocysts. Main Outcome Measure(s) In the current study, shallow whole genome MPS on a NextSeq500 (Illumina) and Ion Proton (Life Technologies) instrument was performed in parallel on 47 whole genome amplified trophectoderm samples. Data analyses were performed using the QDNAseq algorithm implemented in Vivar. Result(s) In total, 5 normal and 42 abnormal embryos were analyzed. All aberrations previously detected with arrayCGH could be readily detected in the MPS data using both technologies and were correctly identified. The smallest detected abnormality was a ∼4.5 Mb deletion/duplication. Conclusion(s) This study demonstrates that shallow whole genome sequencing can be applied efficiently for the detection of numerical and structural chromosomal aberrations in embryos, equaling or even exceeding the resolution of the routinely used microarrays.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Targeted mutagenesis by the CRISPR/Cas9 system is currently revolutionizing genetics. The ease of this technique has enabled genome engineering in-vitro and in a range of model organisms and has ...pushed experimental dimensions to unprecedented proportions. Due to its tremendous progress in terms of speed, read length, throughput and cost, Next-Generation Sequencing (NGS) has been increasingly used for the analysis of CRISPR/Cas9 genome editing experiments. However, the current tools for genome editing assessment lack flexibility and fall short in the analysis of large amounts of NGS data. Therefore, we designed BATCH-GE, an easy-to-use bioinformatics tool for batch analysis of NGS-generated genome editing data, available from https://github.com/WouterSteyaert/BATCH-GE.git. BATCH-GE detects and reports indel mutations and other precise genome editing events and calculates the corresponding mutagenesis efficiencies for a large number of samples in parallel. Furthermore, this new tool provides flexibility by allowing the user to adapt a number of input variables. The performance of BATCH-GE was evaluated in two genome editing experiments, aiming to generate knock-out and knock-in zebrafish mutants. This tool will not only contribute to the evaluation of CRISPR/Cas9-based experiments, but will be of use in any genome editing experiment and has the ability to analyze data from every organism with a sequenced genome.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Myxoid pleomorphic liposarcoma is a recently defined subtype of liposarcoma, which preferentially involves the mediastinum of young patients and shows mixed histological features of conventional ...myxoid liposarcoma and pleomorphic liposarcoma. While myxoid pleomorphic liposarcoma is known to lack the EWSR1/FUS-DDIT3 fusions characteristic of the former, additional genetic data are limited. To further understand this tumor type, we extensively examined a series of myxoid pleomorphic liposarcomas by fluorescence in situ hybridization (FISH), shallow whole genome sequencing (sWGS) and genome-wide DNA methylation profiling. The 12 tumors occurred in 6 females and 6 males, ranging from 17 to 58 years of age (mean 33 years, median 35 years), and were located in the mediastinum (n = 5), back, neck, cheek and leg, including thigh. Histologically, all cases consisted of relatively, bland, abundantly myxoid areas with a prominent capillary vasculature, admixed with much more cellular and less myxoid foci containing markedly pleomorphic spindled cells, numerous pleomorphic lipoblasts and elevated mitotic activity. Using sWGS, myxoid pleomorphic liposarcomas were found to have complex chromosomal alterations, including recurrent large chromosomal gains involving chromosomes 1, 6–8, 18–21 and losses involving chromosomes 13, 16 and 17. Losses in chromosome 13, in particular loss in 13q14 (including RB1, RCTB2, DLEU1, and ITM2B genes), were observed in 4 out of 8 cases analyzed. Additional FISH analyses confirmed the presence of a monoallelic RB1 deletion in 8/12 cases. Moreover, nuclear Rb expression was deficient in all studied cases. None showed DDIT3 gene rearrangement or MDM2 gene amplification. Using genome-wide DNA methylation profiling, myxoid pleomorphic liposarcomas and conventional pleomorphic liposarcomas formed a common methylation cluster, which segregated from conventional myxoid liposarcomas. While the morphologic, genetic and epigenetic characteristics of myxoid pleomorphic liposarcoma suggest a link with conventional pleomorphic liposarcoma, its distinctive clinical features support continued separate classification for the time being.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Objective
Facial dysostosis is a group of rare craniofacial congenital disabilities requiring multidisciplinary long-term care. This report presents the phenotypic and genotypic information from ...South India.
Design
The study is a case series.
Setting
This was an international collaborative study involving a tertiary craniofacial clinic and medical genetics unit.
Patients, Participants
The participants were 9 families with 17 affected individuals of facial dysostosis.
Intervention
Exome analysis focused on known genes associated with acrofacial and mandibulofacial syndromes.
Main Outcome Measure
The outcome measure was to report phenotyptic and genetic heterogeneity in affected individuals.
Results
A Tessier cleft was seen in 7 (41%), lower eyelid coloboma in 12 (65%), ear anomalies in 10 (59%), uniolateral or bilateral aural atresia in 4 (24%), and deafness in 6 (35%). The facial gestalt of Treacher Collins syndrome (TCS) showed extensive phenotypic variations. Pathogenic variants in TCOF1 (Treacher Collins syndrome) were seen in six families, POLR1A (acrofacial dysostosis, Cincinnati type) and EFTUD2 (mandibulofacial dysostosis with microcephaly) in one each. One family (11.1%) had no detectable variation. Five out of six probands with Treacher Collins syndrome had other affected family members (83.3%), including a non-penetrant mother, identified after sequencing.
Conclusion
Our report illustrates the molecular heterogeneity of mandibulofacial dysostosis in India.
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CMK, NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
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