Publications by authors named "Annekatrien Boel"

23 Publications

  • Page 1 of 1

Sex-Specific Isolation and Propagation of Human Premeiotic Fetal Germ Cells and Germ Cell-Like Cells.

Cells 2021 May 16;10(5). Epub 2021 May 16.

Ghent-Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, 9000 Ghent, Belgium.

The second trimester of human development is marked by asynchronous gonadal development hampering the isolation of homogenous populations of early and late fetal germ cells (FGCs). We evaluated the feasibility of using surface markers TNAP, PDPN, EPCAM and ITGA6 to isolate FGCs as well as human primordial germ cell-like cells (hPGCLCs) derived from embryonic stem cells (hESCs) from both sexes by fluorescence-activated cell sorting (FACS). Our results suggest that a combination of TNAP and PDPN was sufficient to separate populations of premeiotic FGCs and hPGCLCs in both sexes. This combination of antibodies also proved efficient in separating 'mitotic' from 'retinoic-acid responsive' female FGCs. Furthermore, we report that the differentiation efficiency of TNAP+PDPN+ hPGCLCs from hESCs was sex-independent, but the ability to propagate differed considerably between the sexes. In contrast to male, female hPGCLCs retained their characteristics and exhibited robust colony-forming ability when cultured for five days in medium containing LIF, forskolin and FGF2. We conclude that marked sex differences exist in the isolation and propagation of human FGCs and hPGCLCs. Our study provides novel insights relevant for the optimization of in vitro gametogenesis in humans.
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http://dx.doi.org/10.3390/cells10051214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8156680PMC
May 2021

Lrp5 Mutant and Crispant Zebrafish Faithfully Model Human Osteoporosis, Establishing the Zebrafish as a Platform for CRISPR-Based Functional Screening of Osteoporosis Candidate Genes.

J Bone Miner Res 2021 May 6. Epub 2021 May 6.

Center for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.

Genomewide association studies (GWAS) have improved our understanding of the genetic architecture of common complex diseases such as osteoporosis. Nevertheless, to attribute functional skeletal contributions of candidate genes to osteoporosis-related traits, there is a need for efficient and cost-effective in vivo functional testing. This can be achieved through CRISPR-based reverse genetic screens, where phenotyping is traditionally performed in stable germline knockout (KO) mutants. Recently it was shown that first-generation (F0) mosaic mutant zebrafish (so-called crispants) recapitulate the phenotype of germline KOs. To demonstrate feasibility of functional validation of osteoporosis candidate genes through crispant screening, we compared a crispant to a stable KO zebrafish model for the lrp5 gene. In humans, recessive loss-of-function mutations in LRP5, a co-receptor in the Wnt signaling pathway, cause osteoporosis-pseudoglioma syndrome. In addition, several GWAS studies identified LRP5 as a major risk locus for osteoporosis-related phenotypes. In this study, we showed that early stage lrp5 KO larvae display decreased notochord mineralization and malformations of the head cartilage. Quantitative micro-computed tomography (micro-CT) scanning and mass-spectrometry element analysis of the adult skeleton revealed decreased vertebral bone volume and bone mineralization, hallmark features of osteoporosis. Furthermore, regenerating fin tissue displayed reduced Wnt signaling activity in lrp5 KO adults. We next compared lrp5 mutants with crispants. Next-generation sequencing analysis of adult crispant tissue revealed a mean out-of-frame mutation rate of 76%, resulting in strongly reduced levels of Lrp5 protein. These crispants generally showed a milder but nonetheless highly comparable skeletal phenotype and a similarly reduced Wnt pathway response compared with lrp5 KO mutants. In conclusion, we show through faithful modeling of LRP5-related primary osteoporosis that crispant screening in zebrafish is a promising approach for rapid functional screening of osteoporosis candidate genes. © 2021 American Society for Bone and Mineral Research. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
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http://dx.doi.org/10.1002/jbmr.4327DOI Listing
May 2021

Digital Polymerase Chain Reaction for Assessment of Mutant Mitochondrial Carry-over after Nuclear Transfer for In Vitro Fertilization.

Clin Chem 2021 Apr 5. Epub 2021 Apr 5.

Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.

Background: The quantification of mitochondrial DNA heteroplasmy for the diagnosis of mitochondrial disease or after mitochondrial donation, is performed mainly using next-generation sequencing strategies (NGS). Digital PCR (dPCR) has the potential to offer an accurate alternative for mutation load quantification.

Methods: We assessed the mutation load of 23 low-input human samples at the m.11778 locus, which is associated with Leber's hereditary optic neuropathy (LHON) using 2 droplet digital PCR platforms (Stilla Naica and Bio-Rad QX200) and the standard NGS strategy. Assay validation was performed by analyzing a titration series with mutation loads ranging from 50% to 0.01%.

Results: A good concordance in mutation rates was observed between both dPCR techniques and NGS. dPCR established a distinctly lower level of background noise compared to NGS. Minor alleles with mutation loads lower than 1% could still be detected, with standard deviations of the technical replicates varying between 0.07% and 0.44% mutation load. Although no significant systematic bias was observed when comparing dPCR and NGS, a minor proportional bias was detected. A slight overestimation of the minor allele was observed for the NGS data, most probably due to amplification and sequencing errors in the NGS workflow.

Conclusion: dPCR has proven to be an accurate tool for the quantification of mitochondrial heteroplasmy, even for samples harboring a low mutation load (<1%). In addition, this alternative technique holds multiple benefits compared to NGS (e.g., less hands-on time, more straightforward data-analysis, and a lower up-front capital investment).
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http://dx.doi.org/10.1093/clinchem/hvab021DOI Listing
April 2021

Prospects of Germline Nuclear Transfer in Women With Diminished Ovarian Reserve.

Front Endocrinol (Lausanne) 2021 22;12:635370. Epub 2021 Feb 22.

Ghent-Fertility and Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.

Diminished ovarian reserve (DOR) is associated with a reduced quantity and quality of the retrieved oocytes, usually leading to poor reproductive outcomes which remain a great challenge for assisted reproduction technology (ART). Women with DOR often have to seek for oocyte donation, precluding genetically related offspring. Germline nuclear transfer (NT) is a novel technology in ART that involves the transfer of the nuclear genome from an affected oocyte/zygote of the patient to the cytoplast of an enucleated donor oocyte/zygote. Therefore, it offers opportunities for the generation of genetically related embryos. Currently, although NT is clinically applied only in women with serious mitochondrial DNA disorders, this technology has also been proposed to overcome certain forms of female infertility, such as advanced maternal age and embryo developmental arrest. In this review, we are proposing the NT technology as a future treatment option for DOR patients. Strikingly, the application of different NT strategies will result in an increase of the total number of available reconstituted embryos for DOR patients.
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http://dx.doi.org/10.3389/fendo.2021.635370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7937897PMC
February 2021

Activin A-derived human embryonic stem cells show increased competence to differentiate into primordial germ cell-like cells.

Stem Cells 2021 05 2;39(5):551-563. Epub 2021 Feb 2.

Ghent-Fertility and Stem cell Team (G-FAST), Department of Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.

Protocols for specifying human primordial germ cell-like cells (hPGCLCs) from human embryonic stem cells (hESCs) remain hindered by differences between hESC lines, their derivation methods, and maintenance culture conditions. This poses significant challenges for establishing reproducible in vitro models of human gametogenesis. Here, we investigated the influence of activin A (ActA) during derivation and maintenance on the propensity of hESCs to differentiate into PGCLCs. We show that continuous ActA supplementation during hESC derivation (from blastocyst until the formation of the post-inner cell mass intermediate [PICMI]) and supplementation (from the first passage of the PICMI onwards) is beneficial to differentiate hESCs to PGCLCs subsequently. Moreover, comparing isogenic primed and naïve states prior to differentiation, we showed that conversion of hESCs to the 4i-state improves differentiation to (TNAP [tissue nonspecific alkaline phosphatase]+/PDPN [podoplanin]+) PGCLCs. Those PGCLCs expressed several germ cell markers, including TFAP2C (transcription factor AP-2 gamma), SOX17 (SRY-box transcription factor 17), and NANOS3 (nanos C2HC-type zinc finger 3), and markers associated with germ cell migration, CXCR4 (C-X-C motif chemokine receptor 4), LAMA4 (laminin subunit alpha 4), ITGA6 (integrin subunit alpha 6), and CDH4 (cadherin 4), suggesting that the large numbers of PGCLCs obtained may be suitable to differentiate further into more mature germ cells. Finally, hESCs derived in the presence of ActA showed higher competence to differentiate to hPGCLC, in particular if transiently converted to the 4i-state. Our work provides insights into the differences in differentiation propensity of hESCs and delivers an optimized protocol to support efficient human germ cell derivation.
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http://dx.doi.org/10.1002/stem.3335DOI Listing
May 2021

Diagnosis and Treatment of Male Infertility-Related Fertilization Failure.

J Clin Med 2020 Dec 1;9(12). Epub 2020 Dec 1.

Ghent-Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium.

Infertility affects approximately 15% of reproductive-aged couples worldwide, of which up to 30% of the cases are caused by male factors alone. The origin of male infertility is mostly attributed to sperm abnormalities, of which many are caused by genetic defects. The development of intracytoplasmic sperm injection (ICSI) has helped to circumvent most male infertility conditions. However, there is still a challenging group of infertile males whose sperm, although having normal sperm parameters, are unable to activate the oocyte, even after ICSI treatment. While ICSI generally allows fertilization rates of 70 to 80%, total fertilization failure (FF) still occurs in 1 to 3% of ICSI cycles. Phospholipase C zeta (PLCζ) has been demonstrated to be a critical sperm oocyte activating factor (SOAF) and the absence, reduced, or altered forms of PLCζ have been shown to cause male infertility-related FF. The purpose of this review is to (i) summarize the current knowledge on PLCζ as the critical sperm factor for successful fertilization, as well as to discuss the existence of alternative sperm-induced oocyte activation mechanisms, (ii) describe the diagnostic tests available to determine the cause of FF, and (iii) summarize the beneficial effect of assisted oocyte activation (AOA) to overcome FF.
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http://dx.doi.org/10.3390/jcm9123899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761017PMC
December 2020

Cutis laxa: A comprehensive overview of clinical characteristics and pathophysiology.

Clin Genet 2021 Jan 27;99(1):53-66. Epub 2020 Oct 27.

Center for Medical Genetics Ghent, Ghent University Hospital, Ghent, Belgium.

Cutis laxa (CL) syndromes comprise a rare group of multisystem disorders that share loose redundant skin folds as hallmark clinical feature. CL results from impaired elastic fiber assembly and homeostasis, and the known underlying gene defects affect different extracellular matrix proteins, intracellular trafficking, or cellular metabolism. Due to the underlying clinical and molecular heterogeneity, the diagnostic work-up of CL patients is often challenging. In this review, we provide a practical approach to the broad differential diagnosis of CL syndromes, provide an overview of the molecular pathogenesis of the different subtypes, and suggest general management guidelines.
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http://dx.doi.org/10.1111/cge.13865DOI Listing
January 2021

Maximizing CRISPR/Cas9 phenotype penetrance applying predictive modeling of editing outcomes in Xenopus and zebrafish embryos.

Sci Rep 2020 09 4;10(1):14662. Epub 2020 Sep 4.

Department of Biomedical Molecular Biology, Ghent University, Technologiepark 71, 9052, Ghent (Zwijnaarde), Belgium.

CRISPR/Cas9 genome editing has revolutionized functional genomics in vertebrates. However, CRISPR/Cas9 edited F animals too often demonstrate variable phenotypic penetrance due to the mosaic nature of editing outcomes after double strand break (DSB) repair. Even with high efficiency levels of genome editing, phenotypes may be obscured by proportional presence of in-frame mutations that still produce functional protein. Recently, studies in cell culture systems have shown that the nature of CRISPR/Cas9-mediated mutations can be dependent on local sequence context and can be predicted by computational methods. Here, we demonstrate that similar approaches can be used to forecast CRISPR/Cas9 gene editing outcomes in Xenopus tropicalis, Xenopus laevis, and zebrafish. We show that a publicly available neural network previously trained in mouse embryonic stem cell cultures (InDelphi-mESC) is able to accurately predict CRISPR/Cas9 gene editing outcomes in early vertebrate embryos. Our observations can have direct implications for experiment design, allowing the selection of guide RNAs with predicted repair outcome signatures enriched towards frameshift mutations, allowing maximization of CRISPR/Cas9 phenotype penetrance in the F generation.
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http://dx.doi.org/10.1038/s41598-020-71412-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473854PMC
September 2020

Slc2a10 knock-out mice deficient in ascorbic acid synthesis recapitulate aspects of arterial tortuosity syndrome and display mitochondrial respiration defects.

Hum Mol Genet 2020 06;29(9):1476-1488

Center for Medical Genetics Ghent, Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium.

Arterial tortuosity syndrome (ATS) is a recessively inherited connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutations in SLC2A10, encoding the facilitative glucose transporter GLUT10. Former studies implicated GLUT10 in the transport of dehydroascorbic acid, the oxidized form of ascorbic acid (AA). Mouse models carrying homozygous Slc2a10 missense mutations did not recapitulate the human phenotype. Since mice, in contrast to humans, are able to intracellularly synthesize AA, we generated a novel ATS mouse model, deficient for Slc2a10 as well as Gulo, which encodes for L-gulonolactone oxidase, an enzyme catalyzing the final step in AA biosynthesis in mouse. Gulo;Slc2a10 double knock-out mice showed mild phenotypic anomalies, which were absent in single knock-out controls. While Gulo;Slc2a10 double knock-out mice did not fully phenocopy human ATS, histological and immunocytochemical analysis revealed compromised extracellular matrix formation. Transforming growth factor beta signaling remained unaltered, while mitochondrial function was compromised in smooth muscle cells derived from Gulo;Slc2a10 double knock-out mice. Altogether, our data add evidence that ATS is an ascorbate compartmentalization disorder, but additional factors underlying the observed phenotype in humans remain to be determined.
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http://dx.doi.org/10.1093/hmg/ddaa071DOI Listing
June 2020

Erratum. Chromosomal mosaicism in human blastocysts: the ultimate diagnostic dilemma.

Hum Reprod Update 2020 04;26(3):450-451

Ghent-Fertility and Stem Cell Team (G-FAST), Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium.

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http://dx.doi.org/10.1093/humupd/dmaa015DOI Listing
April 2020

Chromosomal mosaicism in human blastocysts: the ultimate diagnostic dilemma.

Hum Reprod Update 2020 04;26(3):313-334

Ghent-Fertility and Stem Cell Team (G-FAST), Department for Reproductive Medicine, Ghent University Hospital, Corneel Heymanslaan 10, 9000 Ghent, Belgium.

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.
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http://dx.doi.org/10.1093/humupd/dmz050DOI Listing
April 2020

Vitrification negatively affects the Ca-releasing and activation potential of mouse oocytes, but vitrified oocytes are potentially useful for diagnostic purposes.

Reprod Biomed Online 2020 Jan 28;40(1):13-25. Epub 2019 Sep 28.

Ghent-Fertility and Stem cell Team (G-FaST), Department of Human Structure and Repair, Ghent University Hospital, C. Heymanslaan 10, Ghent 9000, Belgium.

Research Question: To what extent does vitrification affect the Ca-releasing and activation potential of mouse oocytes, which are commonly used to determine the oocyte activation potential of human spermatozoa?

Design: The effect of mouse oocyte vitrification on Ca dynamics and developmental competence after oocyte activation was assessed and compared with fresh mouse oocytes. Moreover, the Ca store content of the endoplasmic reticulum was determined at different time points during the vitrification-warming procedure. Finally, the Ca pattern induced by cryoprotectant exposure was determined.

Results: After human sperm injection into mouse oocytes, Ca dynamics but not fertilization rates were significantly altered by vitrification warming (P < 0.05). Ca dynamics in response to SrCl or ionomycin were also altered by oocyte vitrification. In contrast, activation and blastocyst rates after SrCl exposure were not affected (P > 0.05), whereas activation rates after ionomycin exposure were significantly lower in vitrified-warmed oocytes (P < 0.05); blastocyst rates were not affected (P > 0.05). Cryoprotectant exposure was associated with a strong drop in endoplasmic reticulum Ca store content. Oocytes rapidly recovered during warming and recovery in Ca-containing media; a threshold area under the curve of Ca dynamics to obtain activation rates above 90% was determined.

Conclusions: Vitrified-warmed mouse oocytes display reduced Ca-releasing potential upon oocyte activation, caused by cryoprotectant exposure. With adapted classification criteria, these oocytes could be used for diagnosing oocyte activation deficiencies in patients. Evaluating the Ca-signalling machinery in vitrified-warmed human oocytes is required.
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http://dx.doi.org/10.1016/j.rbmo.2019.09.012DOI Listing
January 2020

Arterial Tortuosity Syndrome: An Ascorbate Compartmentalization Disorder?

Antioxid Redox Signal 2021 04 14;34(11):875-889. Epub 2019 Nov 14.

Institute of Clinical Experimental Research, Molecular Biology, and Pathobiochemistry, Semmelweis University, Budapest, Hungary.

Cardiovascular disorders are the most important cause of morbidity and mortality in the Western world. Monogenic developmental disorders of the heart and vessels are highly valuable to study the physiological and pathological processes in cardiovascular system homeostasis. The arterial tortuosity syndrome (ATS) is a rare, autosomal recessive connective tissue disorder showing lengthening, tortuosity, and stenosis of the large arteries, with a propensity for aneurysm formation. In histopathology, it associates with fragmentation and disorganization of elastic fibers in several tissues, including the arterial wall. ATS is caused by pathogenic variants in encoding the facilitative glucose transporter (GLUT)10. Although several hypotheses have been forwarded, the molecular mechanisms linking disrupted GLUT10 activity with arterial malformations are largely unknown. The vascular and systemic manifestations and natural history of ATS patients have been largely delineated. GLUT10 was identified as an intracellular transporter of dehydroascorbic acid, which contributes to collagen and elastin cross-linking in the endoplasmic reticulum, redox homeostasis in the mitochondria, and global and gene-specific methylation/hydroxymethylation affecting epigenetic regulation in the nucleus. We revise here the current knowledge on ATS and the role of GLUT10 within the compartmentalization of ascorbate in physiological and diseased states. Centralization of clinical, treatment, and outcome data will enable better management for ATS patients. Establishment of representative animal disease models could facilitate the study of pathomechanisms underlying ATS. This might be relevant for other forms of vascular dysplasia, such as isolated aneurysm formation, hypertensive vasculopathy, and neovascularization. . 34, 875-889.
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http://dx.doi.org/10.1089/ars.2019.7843DOI Listing
April 2021

Assisted oocyte activation significantly increases fertilization and pregnancy outcome in patients with low and total failed fertilization after intracytoplasmic sperm injection: a 17-year retrospective study.

Fertil Steril 2019 08 24;112(2):266-274. Epub 2019 May 24.

Ghent Fertility and Stem Cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium.

Objective: To investigate the extent to which assisted oocyte activation (AOA) improves clinical outcomes in patients diagnosed with oocyte activation deficiencies (OADs).

Design: Retrospective cohort study comparing AOA cycles and previous intracytoplasmic sperm injection (ICSI) cycles in couples experiencing low or total failed fertilization after ICSI. Importantly, the sperm-related oocyte-activating capacity was examined in all patients before AOA with the use of the mouse oocyte activation test (MOAT).

Setting: Infertility center at a university hospital.

Patient(s): A total of 122 couples with a history of low or total failed fertilization after ICSI.

Intervention(s): ICSI, MOAT, AOA, and embryo transfer.

Main Outcome Measure(s): Fertilization, pregnancy, and live birth rates.

Result(s): MOAT revealed 19 patients with a sperm-related OAD (MOAT group 1), 56 patients with a diminished sperm-related oocyte-activating capacity (MOAT group 2), and 47 patients with a suspected oocyte-related OAD (MOAT group 3). AOA (191 cycles) significantly improved fertilization, pregnancy, and live birth rates in all MOAT groups compared with previous ICSI attempts (243 cycles). Fertilization rates after AOA were significantly different among MOAT groups 1 (70.1%), 2 (63.0%), and 3 (57.3%). Between MOAT group 1 and 3, significant differences in pregnancy (49.0% vs. 29.4%) and live birth (41.2% vs. 22.1%) rates were observed. In total, 225 embryo transfers resulted in 60 healthy live births following AOA.

Conclusion(s): Patients undergoing diagnostic testing before AOA show a significant improvement in clinical outcomes compared with previous cycles. Our findings highlight that AOA should be reserved for patients with clear OADs.
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http://dx.doi.org/10.1016/j.fertnstert.2019.04.006DOI Listing
August 2019

De Novo and Inherited Pathogenic Variants in KDM3B Cause Intellectual Disability, Short Stature, and Facial Dysmorphism.

Am J Hum Genet 2019 04 28;104(4):758-766. Epub 2019 Mar 28.

Department of Human Genetics, Radboud University Medical Center, 6525GA Nijmegen, the Netherlands; Donders Institute for Brain, Cognition and Behavior, Radboud University Nijmegen, 6525GA Nijmegen, the Netherlands; Department of Clinical Genetics and School for Oncology & Developmental Biology (GROW), Maastricht University Medical Center, 6202AZ Maastricht, the Netherlands.

By using exome sequencing and a gene matching approach, we identified de novo and inherited pathogenic variants in KDM3B in 14 unrelated individuals and three affected parents with varying degrees of intellectual disability (ID) or developmental delay (DD) and short stature. The individuals share additional phenotypic features that include feeding difficulties in infancy, joint hypermobility, and characteristic facial features such as a wide mouth, a pointed chin, long ears, and a low columella. Notably, two individuals developed cancer, acute myeloid leukemia and Hodgkin lymphoma, in childhood. KDM3B encodes for a histone demethylase and is involved in H3K9 demethylation, a crucial part of chromatin modification required for transcriptional regulation. We identified missense and truncating variants, suggesting that KDM3B haploinsufficiency is the underlying mechanism for this syndrome. By using a hybrid facial-recognition model, we show that individuals with a pathogenic variant in KDM3B have a facial gestalt, and that they show significant facial similarity compared to control individuals with ID. In conclusion, pathogenic variants in KDM3B cause a syndrome characterized by ID, short stature, and facial dysmorphism.
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http://dx.doi.org/10.1016/j.ajhg.2019.02.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451728PMC
April 2019

Decreased Nuclear Ascorbate Accumulation Accompanied with Altered Genomic Methylation Pattern in Fibroblasts from Arterial Tortuosity Syndrome Patients.

Oxid Med Cell Longev 2019 13;2019:8156592. Epub 2019 Jan 13.

Institute of Clinical Experimental Research, Semmelweis University, Budapest 1094, Hungary.

Ascorbate requiring Fe/2-oxoglutarate-dependent dioxygenases located in the nucleoplasm have been shown to participate in epigenetic regulation of gene expression via histone and DNA demethylation. Transport of dehydroascorbic acid is impaired in the endomembranes of fibroblasts from arterial tortuosity syndrome (ATS) patients, due to the mutation in the gene coding for glucose transporter GLUT10. We hypothesized that altered nuclear ascorbate concentration might be present in ATS fibroblasts, affecting dioxygenase activity and DNA demethylation. Therefore, our aim was to characterize the subcellular distribution of vitamin C, the global and site-specific changes in 5-methylcytosine and 5-hydroxymethylcytosine levels, and the effect of ascorbate supplementation in control and ATS fibroblast cultures. Diminished nuclear accumulation of ascorbate was found in ATS fibroblasts upon ascorbate or dehydroascorbic acid addition. Analyzing DNA samples of cultured fibroblasts from controls and ATS patients, a lower global 5-hydroxymethylcytosine level was found in ATS fibroblasts, which could not be significantly modified by ascorbate addition. Investigation of the (hydroxy)methylation status of specific regions in six candidate genes related to ascorbate metabolism and function showed that ascorbate addition could stimulate hydroxymethylation and active DNA demethylation at the PPAR- gene region in control fibroblasts only. The altered DNA hydroxymethylation patterns in patient cells both at the global level and at specific gene regions accompanied with decreased nuclear accumulation of ascorbate suggests the epigenetic role of vitamin C in the pathomechanism of ATS. The present findings represent the first example for the role of vitamin C transport in epigenetic regulation suggesting that ATS is a compartmentalization disease.
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http://dx.doi.org/10.1155/2019/8156592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6360052PMC
March 2019

Publisher Correction: BATCH-GE: Batch analysis of Next-Generation Sequencing data for genome editing assessment.

Sci Rep 2018 Oct 29;8(1):15845. Epub 2018 Oct 29.

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-33869-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6206066PMC
October 2018

CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments.

Dis Model Mech 2018 10 18;11(10). Epub 2018 Oct 18.

Center for Medical Genetics, Department of Biomolecular Medicine, Ghent University, Corneel Heymanslaan 10, 9000 Ghent, Belgium

Targeted genome editing by CRISPR/Cas9 is extremely well fitted to generate gene disruptions, although precise sequence replacement by CRISPR/Cas9-mediated homology-directed repair (HDR) suffers from low efficiency, impeding its use for high-throughput knock-in disease modeling. In this study, we used next-generation sequencing (NGS) analysis to determine the efficiency and reliability of CRISPR/Cas9-mediated HDR using several types of single-stranded oligodeoxynucleotide (ssODN) repair templates for the introduction of disease-relevant point mutations in the zebrafish genome. Our results suggest that HDR rates are strongly determined by repair-template composition, with the most influential factor being homology-arm length. However, we found that repair using ssODNs does not only lead to precise sequence replacement but also induces integration of repair-template fragments at the Cas9 cut site. We observed that error-free repair occurs at a relatively constant rate of 1-4% when using different repair templates, which was sufficient for transmission of point mutations to the F1 generation. On the other hand, erroneous repair mainly accounts for the variability in repair rate between the different repair templates. To further improve error-free HDR rates, elucidating the mechanism behind this erroneous repair is essential. We show that the error-prone nature of ssODN-mediated repair, believed to act via synthesis-dependent strand annealing (SDSA), is most likely due to DNA synthesis errors. In conclusion, caution is warranted when using ssODNs for the generation of knock-in models or for therapeutic applications. We recommend the application of in-depth NGS analysis to examine both the efficiency and error-free nature of HDR events.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.035352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6215429PMC
October 2018

BATCH-GE: Analysis of NGS Data for Genome Editing Assessment.

Methods Mol Biol 2018 ;1865:83-90

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Due to its simple nature, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technique is massively used nowadays to modify genomic loci in a wide range of model systems. The possibility to interrogate gene function on a genome-wide scale is revolutionizing fundamental life sciences and will lead to new clinical breakthroughs. Its strength is even more pronounced when it is used in tandem with next-generation sequencing (NGS). The high throughput and low cost cause NGS to be the method of choice for exploring CRISPR-Cas9 experimental results. To analyze the NGS reads from genome editing experiments only few bioinformatics tools are available. BATCH-GE is a flexible and easy-to-use tool, which is especially useful for dealing with large amounts of data. It 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.
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http://dx.doi.org/10.1007/978-1-4939-8784-9_6DOI Listing
May 2019

Arterial tortuosity syndrome: 40 new families and literature review.

Genet Med 2018 10 11;20(10):1236-1245. Epub 2018 Jan 11.

Pediatrics Department, Kuwait University, Kuwait City, Kuwait.

Purpose: We delineate the clinical spectrum and describe the histology in arterial tortuosity syndrome (ATS), a rare connective tissue disorder characterized by tortuosity of the large and medium-sized arteries, caused by mutations in SLC2A10.

Methods: We retrospectively characterized 40 novel ATS families (50 patients) and reviewed the 52 previously reported patients. We performed histology and electron microscopy (EM) on skin and vascular biopsies and evaluated TGF-β signaling with immunohistochemistry for pSMAD2 and CTGF.

Results: Stenoses, tortuosity, and aneurysm formation are widespread occurrences. Severe but rare vascular complications include early and aggressive aortic root aneurysms, neonatal intracranial bleeding, ischemic stroke, and gastric perforation. Thus far, no reports unequivocally document vascular dissections or ruptures. Of note, diaphragmatic hernia and infant respiratory distress syndrome (IRDS) are frequently observed. Skin and vascular biopsies show fragmented elastic fibers (EF) and increased collagen deposition. EM of skin EF shows a fragmented elastin core and a peripheral mantle of microfibrils of random directionality. Skin and end-stage diseased vascular tissue do not indicate increased TGF-β signaling.

Conclusion: Our findings warrant attention for IRDS and diaphragmatic hernia, close monitoring of the aortic root early in life, and extensive vascular imaging afterwards. EM on skin biopsies shows disease-specific abnormalities.
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http://dx.doi.org/10.1038/gim.2017.253DOI Listing
October 2018

CRISPR/Cas9 mediated knockout of rb1 and rbl1 leads to rapid and penetrant retinoblastoma development in Xenopus tropicalis.

Sci Rep 2016 10 14;6:35264. Epub 2016 Oct 14.

Developmental Biology Unit, Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.

Retinoblastoma is a pediatric eye tumor in which bi-allelic inactivation of the Retinoblastoma 1 (RB1) gene is the initiating genetic lesion. Although recently curative rates of retinoblastoma have increased, there are at this time no molecular targeted therapies available. This is, in part, due to the lack of highly penetrant and rapid retinoblastoma animal models that facilitate rapid identification of targets that allow therapeutic intervention. Different mouse models are available, all based on genetic deactivation of both Rb1 and Retinoblastoma-like 1 (Rbl1), and each showing different kinetics of retinoblastoma development. Here, we show by CRISPR/Cas9 techniques that similar to the mouse, neither rb1 nor rbl1 single mosaic mutant Xenopus tropicalis develop tumors, whereas rb1/rbl1 double mosaic mutant tadpoles rapidly develop retinoblastoma. Moreover, occasionally presence of pinealoblastoma (trilateral retinoblastoma) was detected. We thus present the first CRISPR/Cas9 mediated cancer model in Xenopus tropicalis and the first genuine genetic non-mammalian retinoblastoma model. The rapid kinetics of our model paves the way for use as a pre-clinical model. Additionally, this retinoblastoma model provides unique possibilities for fast elucidation of novel drug targets by triple multiplex CRISPR/Cas9 gRNA injections (rb1 + rbl1 + modifier gene) in order to address the clinically unmet need of targeted retinoblastoma therapy.
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http://dx.doi.org/10.1038/srep35264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064383PMC
October 2016

BATCH-GE: Batch analysis of Next-Generation Sequencing data for genome editing assessment.

Sci Rep 2016 07 27;6:30330. Epub 2016 Jul 27.

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

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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http://dx.doi.org/10.1038/srep30330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4962088PMC
July 2016