De novo pathogenic variants in neuronal differentiation factor 2 (NEUROD2) cause a form of early infantile epileptic encephalopathy

2018 ◽  
Vol 56 (2) ◽  
pp. 113-122 ◽  
Author(s):  
Annalisa G Sega ◽  
Emily K Mis ◽  
Kristin Lindstrom ◽  
Saadet Mercimek-Andrews ◽  
Weizhen Ji ◽  
...  
Keyword(s):  
Genome Editing ◽  
Candidate Genes ◽  
Neuronal Differentiation ◽  
Protein Function ◽  
De Novo ◽  
Single Gene ◽  
Epileptic Encephalopathy ◽  
Epileptic Encephalopathies ◽  
Differentiation Factor

BackgroundEarly infantile epileptic encephalopathies are severe disorders consisting of early-onset refractory seizures accompanied often by significant developmental delay. The increasing availability of next-generation sequencing has facilitated the recognition of single gene mutations as an underlying aetiology of some forms of early infantile epileptic encephalopathies.ObjectivesThis study was designed to identify candidate genes as a potential cause of early infantile epileptic encephalopathy, and then to provide genetic and functional evidence supporting patient variants as causative.MethodsWe used whole exome sequencing to identify candidate genes. To model the disease and assess the functional effects of patient variants on candidate protein function, we used in vivo CRISPR/Cas9-mediated genome editing and protein overexpression in frog tadpoles.ResultsWe identified novel de novo variants in neuronal differentiation factor 2 (NEUROD2) in two unrelated children with early infantile epileptic encephalopathy. Depleting neurod2 with CRISPR/Cas9-mediated genome editing induced spontaneous seizures in tadpoles, mimicking the patients’ condition. Overexpression of wild-type NEUROD2 induced ectopic neurons in tadpoles; however, patient variants were markedly less effective, suggesting that both variants are dysfunctional and likely pathogenic.ConclusionThis study provides clinical and functional support for NEUROD2 variants as a cause of early infantile epileptic encephalopathy, the first evidence of human disease caused by NEUROD2 variants.

scholarly journals A Novel Heterozygous Missense Variant in YWHAG Gene Cause Early-Onset Epilepsy in a Chinese Family

2020 ◽  
Author(s):  
Zhi Yi ◽  
Zhenfeng Song ◽  
Jiao Xue ◽  
Chengqing Yang ◽  
Fei Li ◽  
...  
Keyword(s):  
Early Onset ◽  
Seizure Control ◽  
De Novo ◽  
Missense Variant ◽  
Epileptic Encephalopathy ◽  
Chinese Family ◽  
Gene Variants ◽  
Case Presentation ◽  
Epileptic Encephalopathies ◽  
Refractory Seizures

Abstract Background: Developmental and epileptic encephalopathies (DEE) are a heterogeneous group of severe disorders which are characterized by early-onset, refractory seizures and developmental slowing or regression. Genetic variations are significant causes for them. De novo variants in an increasing number of candidate genes have been found to be causal. YWHAG gene variants have been reported to cause developmental and epileptic encephalopathy 56 (DEE56). Case presentation: Here, we report a novel heterozygous missense variant c.170G>A (p.R57H) in YWHAG gene cause early-onset epilepsy in a Chinese family. Both the proband and his mother exhibit early onset seizures, intellectual disability, developmental delay. While the proband achieve seizure control with sodium valproate, his mother's seizures were not well controlled. Conclusions: Our report further confirming the haploinsufficiency of YWHAG results in developmental and epileptic encephalopathies.

scholarly journals Dynamic Genome Editing Using In Vivo Synthesized Donor ssDNA in Escherichia coli

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 467
Author(s):  
Min Hao ◽  
Zhaoguan Wang ◽  
Hongyan Qiao ◽  
Peng Yin ◽  
Jianjun Qiao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Editing ◽  
Genome Engineering ◽  
Single Gene ◽  
Rolling Circle Replication ◽  
Rolling Circle ◽  
Cas9 Nuclease ◽  
Dynamic Genome ◽  
Circular Ssdna

As a key element of genome editing, donor DNA introduces the desired exogenous sequence while working with other crucial machinery such as CRISPR-Cas or recombinases. However, current methods for the delivery of donor DNA into cells are both inefficient and complicated. Here, we developed a new methodology that utilizes rolling circle replication and Cas9 mediated (RC-Cas-mediated) in vivo single strand DNA (ssDNA) synthesis. A single-gene rolling circle DNA replication system from Gram-negative bacteria was engineered to produce circular ssDNA from a Gram-positive parent plasmid at a designed sequence in Escherichia coli. Furthermore, it was demonstrated that the desired linear ssDNA fragment could be cut out using CRISPR-associated protein 9 (CRISPR-Cas9) nuclease and combined with lambda Red recombinase as donor for precise genome engineering. Various donor ssDNA fragments from hundreds to thousands of nucleotides in length were synthesized in E. coli cells, allowing successive genome editing in growing cells. We hope that this RC-Cas-mediated in vivo ssDNA on-site synthesis system will be widely adopted as a useful new tool for dynamic genome editing.

scholarly journals C. elegans PVD Neurons: A Platform for Functionally Validating and Characterizing Neuropsychiatric Risk Genes

2016 ◽  
Author(s):  
Cristina Aguirre-Chen ◽  
Nuri Kim ◽  
Olivia Mendivil Ramos ◽  
Melissa Kramer ◽  
W. Richard McCombie ◽  
...  
Keyword(s):  
Protein Function ◽  
Molecular Mechanisms ◽  
Neuronal Development ◽  
De Novo ◽  
Genetic Interaction ◽  
Cost Effective ◽  
Chromatin Remodelers ◽  
C Elegans ◽  
Effective Manner

AbstractOne of the primary challenges in the field of psychiatric genetics is the lack of an in vivo model system in which to functionally validate candidate neuropsychiatric risk genes (NRGs) in a rapid and cost-effective manner1−3. To overcome this obstacle, we performed a candidate-based RNAi screen in which C. elegans orthologs of human NRGs were assayed for dendritic arborization and cell specification defects using C. elegans PVD neurons. Of 66 NRGs, identified via exome sequencing of autism (ASD)4 or schizophrenia (SCZ)5−9 probands and whose mutations are de novo and predicted to result in a complete or partial loss of protein function, the C. elegans orthologs of 7 NRGs were found to be required for proper neuronal development and represent a variety of functional classes, including transcriptional regulators and chromatin remodelers, molecular chaperones, and cytoskeleton-related proteins. Notably, the positive hit rate, when selectively assaying C. elegans orthologs of ASD and SCZ NRGs, is enriched >14-fold as compared to unbiased RNAi screening10. Furthermore, we find that RNAi phenotypes associated with the depletion of NRG orthologs is recapitulated in genetic mutant animals, and, via genetic interaction studies, we show that the NRG ortholog of ANK2, unc-44, is required for SAX-7/MNR-1/DMA-1 signaling. Collectively, our studies demonstrate that C. elegans PVD neurons are a tractable model in which to discover and dissect the fundamental molecular mechanisms underlying neuropsychiatric disease pathogenesis.

Trio deep-sequencing does not reveal unexpected mutations in Cas9-edited monkeys

2018 ◽  
Author(s):  
Xin Luo ◽  
Yaoxi He ◽  
Chao Zhang ◽  
Xiechao He ◽  
Lanzhen Yan ◽  
...  
Keyword(s):  
Genome Editing ◽  
Deep Sequencing ◽  
De Novo ◽  
Clinical Applications ◽  
Preclinical Model ◽  
Whole Genome ◽  
Spontaneous Mutations ◽  
Target Effect

CRISPR-Cas9 is a widely-used genome editing tool, but its off-target effect remains a concern, especially in view of future clinical applications. Non-human primates (NHPs) share close genetic and physiological similarities with humans, making them an ideal preclinical model for developing Cas9-based therapies. However, no comprehensive in vivo off-target assessment has been conducted in NHPs. Here we performed whole genome trio sequencing of Cas9-treated monkeys. We found they only carried a small number of de novo mutations that can be explained by expected spontaneous mutations, and no unexpected mutations were detected.

scholarly journals De novo SCN8A and inherited rare CACNA1H variants associated with severe developmental and epileptic encephalopathy

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Robin N. Stringer ◽  
Bohumila Jurkovicova-Tarabova ◽  
Ivana A. Souza ◽  
Judy Ibrahim ◽  
Tomas Vacik ◽  
...  
Keyword(s):  
Ion Channel ◽  
De Novo ◽  
Epileptic Encephalopathy ◽  
Gene Encoding ◽  
Epileptic Encephalopathies ◽  
Voltage Gated ◽  
Voltage Gated Calcium Channels ◽  
Whole Exome ◽  
Heterozygous Variant

AbstractDevelopmental and epileptic encephalopathies (DEEs) are a group of severe epilepsies that are characterized by seizures and developmental delay. DEEs are primarily attributed to genetic causes and an increasing number of cases have been correlated with variants in ion channel genes. In this study, we report a child with an early severe DEE. Whole exome sequencing showed a de novo heterozygous variant (c.4873–4881 duplication) in the SCN8A gene and an inherited heterozygous variant (c.952G > A) in the CACNA1H gene encoding for Nav1.6 voltage-gated sodium and Cav3.2 voltage-gated calcium channels, respectively. In vitro functional analysis of human Nav1.6 and Cav3.2 channel variants revealed mild but significant alterations of their gating properties that were in general consistent with a gain- and loss-of-channel function, respectively. Although additional studies will be required to confirm the actual pathogenic involvement of SCN8A and CACNA1H, these findings add to the notion that rare ion channel variants may contribute to the etiology of DEEs.

scholarly journals SAC1p is an integral membrane protein that influences the cellular requirement for phospholipid transfer protein function and inositol in yeast

1993 ◽  
Vol 122 (1) ◽  
pp. 79-94 ◽  
Author(s):  
EA Whitters ◽  
AE Cleves ◽  
TP McGee ◽  
HB Skinner ◽  
VA Bankaitis
Keyword(s):  
Membrane Protein ◽  
Actin Cytoskeleton ◽  
Protein Function ◽  
De Novo ◽  
Integral Membrane Protein ◽  
Secretory Function ◽  
Transfer Protein ◽  
Phospholipid Transfer ◽  
Golgi Secretory Function

Mutations in the SAC1 gene exhibit allele-specific genetic interactions with yeast actin structural gene defects and effect a bypass of the cellular requirement for the yeast phosphatidylinositol/phosphatidylcholine transfer protein (SEC14p), a protein whose function is essential for sustained Golgi secretory function. We report that SAC1p is an integral membrane protein that localizes to the yeast Golgi complex and to the yeast ER, but does not exhibit a detectable association with the bulk of the yeast F-actin cytoskeleton. The data also indicate that the profound in vivo effects on Golgi secretory function and the organization of the actin cytoskeleton observed in sac1 mutants result from loss of SAC1p function. This cosuppression of actin and SEC14p defects is a unique feature of sac1 alleles as mutations in other SAC genes that result in a suppression of actin defects do not result in phenotypic suppression of SEC14p defects. Finally, we report that sac1 mutants also exhibit a specific inositol auxotrophy that is not exhibited by the other sac mutant strains. This sac1-associated inositol auxotrophy is not manifested by measurable defects in de novo inositol biosynthesis, nor is it the result of some obvious defect in the ability of sac1 mutants to utilize inositol for phosphatidylinositol biosynthesis. Thus, sac1 mutants represent a novel class of inositol auxotroph in that these mutants appear to require elevated levels of inositol for growth. On the basis of the collective data, we suggest that SAC1p dysfunction exerts its pleiotropic effects on yeast Golgi function, the organization of the actin cytoskeleton, and the cellular requirement for inositol, through altered metabolism of inositol glycerophospholipids.

SCN8A and Its Related Epileptic Phenotypes

2021 ◽  
Author(s):  
Andrea Praticò ◽  
Carmela Gulizia ◽  
Gloria Gangi ◽  
Claudia Oliva ◽  
Catia Romano ◽  
...  
Keyword(s):  
De Novo ◽  
Wide Spectrum ◽  
Single Gene ◽  
Great Majority ◽  
Epileptic Encephalopathy ◽  
Specific Gene ◽  
Channel Blockers ◽  
Germline Mosaicism ◽  
Pathogenic Variants ◽  
Genetic Epilepsies

AbstractSodium channelopathies are among the most common single-gene causes of epilepsy and have been considered model disorders for the study of genetic epilepsies. Epilepsies due to SCN8A pathogenic variants can present with a broad range of phenotypes varying from a severe epileptic encephalopathy with multiple types of drug-resistant seizure to neurodevelopmental delay, mental retardation, and electroencephalogram (EEG) findings of multifocal spike and waves (mostly in the temporal/parietal/occipital areas). In rare cases, benign familial infantile seizures and developmental delay with/without ataxia have been reported. A first-level, specific SCN8A Sanger's sequencing, although available, is rarely performed because the clinical phenotype is not strictly characteristic and several overlaps with other genetic epilepsies may occur. Given its indistinctive phenotype, diagnosis is usually performed through a specific gene panel for epileptic encephalopathies, early epilepsies, or genetic epilepsy in general, or through whole exome sequencing (WES) and more rarely through whole genome sequencing (WGS). Mutations in SCN8A occur as an autosomal dominant trait. The great majority of individuals diagnosed with SCN8A epilepsy do not have an affected parent, because usually SCN8A patients do not reproduce, and mutations are inherited as a “de novo” trait. In rare cases, SCN8A mutations may be inherited in the setting of parental germline mosaicism. SCN8A-related epilepsies have not shown a clear genotype–phenotype correlation, the same variants have been described with different clinical expressivity and this could be due to other genetic factors or to interacting environmental factors. There is no standardized treatment for SCN8A-related epilepsy because of the rarity of the disease and the unavailability of specific, targeted drugs. Treatment is based mainly on antiepileptic drugs which include classic wide-spectrum drugs such as valproic acid, levetiracetam, and lamotrigine. Sodium-channel blockers (phenytoin, carbamazepine, oxcarbazepine, and lamotrigine) have shown appreciable results in terms of seizure reduction, in particular, in patients presenting gain-of-function mutations. Nowadays, new potentially transformative gene therapy treatment approaches are currently being explored, allowing in the next future, a precision-based treatment directed against the gene defect and protein alterations.

Comparison of Recombinant Growth/Differentiation Factor-5 (rGDF-5) and Bone Morphogenetic Protein-2 (rBMP-2) in the In Vivo Bone Formation in Porous Hydroxyapatite Ceramic

2005 ◽  
Vol 284-286 ◽  
pp. 945-948
Author(s):  
H. Shimaoka ◽  
Y. Dohi ◽  
K. Narikawa ◽  
Hajime Ohgushi ◽  
M. Ikeuchi ◽  
...  
Keyword(s):  
Growth Factors ◽  
Bone Formation ◽  
Bone Morphogenetic Protein ◽  
De Novo ◽  
Bone Morphogenetic Protein 2 ◽  
Osteogenic Potential ◽  
Differentiation Factor ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 5

Various recombinant growth factors have been used for promoting osteoblastic differentiation cascade. To compare the growth/differentiation factor-5 (GDF-5) and bone morphogenetic protein-2 (BMP-2) in the in vivo osteogenic potential of bone marrow mesenchymal stem cells (MSCs), the bone formation was assessed by rat subcutaneous implantation of 5 kinds of hydroxyapatite (HA) implants; namely GDF/HA composites, BMP/HA composites, MSCs/HA composites and the MSCs/HA composites supplemented with recombinant mouse GDF-5 (GDF/MSCs/HA) or recombinant human BMP-2 (BMP/MSCs/HA). Neither the GDF/HA nor the BMP/HA composites exhibited any bone formation at any time after implantation. At both 2 and 4 weeks after implantation, obvious de novo bone formation together with active osteoblasts was seen histologically in many pores of the GDF/MSCs/HA and BMP/MSCs/HA composites. The GDF/MSCs/HA and BMP/MSCs/HA composites also showed high alkaline phosphatase (ALP) and osteocalcin expression determined at both the protein and gene levels. Compared with GDF/MSCs/HA, the BMP/MSCs/HA composites exhibited excellent osteogenesis with relatively early osteoblastic phenotype expression. These findings indicate that the two growth factors synergistically enhance de novo bone formation capability of MSCs/HA composites and the importance of ceramic surface to retain and to deliver the molecules of growth factors for the cell differentiation and maturation.

scholarly journals CRISPR Genome Editing Applied to the Pathogenic Retrovirus HTLV-1

2020 ◽  
Vol 10 ◽  
Author(s):  
Amanda R. Panfil ◽  
Patrick L. Green ◽  
Kristine E. Yoder
Keyword(s):  
Genome Editing ◽  
De Novo ◽  
Leukemia Virus ◽  
Strand Break ◽  
Cell Leukemia Virus Type ◽  
Guide Rna ◽  
Human T Cell ◽  
T Cell Leukemia Virus

CRISPR editing of retroviral proviruses has been limited to HIV-1. We propose human T-cell leukemia virus type 1 (HTLV-1) as an excellent model to advance CRISPR/Cas9 genome editing technologies against actively expressing and latent retroviral proviruses. HTLV-1 is a tumorigenic human retrovirus responsible for the development of both leukemia/lymphoma (ATL) and a neurological disease (HAM/TSP). The virus immortalizes and persists in CD4+ T lymphocytes that survive for the lifetime of the host. The most important drivers of HTLV-1-mediated transformation and proliferation are the tax and hbz viral genes. Tax, transcribed from the plus-sense or genome strand, is essential for de novo infection and cellular immortalization. Hbz, transcribed from the minus-strand, supports proliferation and survival of infected cells in both its protein and mRNA forms. Abrogating the function or expression of tax and/or hbz by genome editing and mutagenic double-strand break repair may disable HTLV-1-infected cell growth/survival and prevent immune modulatory effects and ultimately HTLV-1-associated disease. In addition, the HTLV-1 viral genome is highly conserved with remarkable sequence homogeneity, both within the same host and even among different HTLV isolates. This offers more focused guide RNA targeting. In addition, there are several well-established animal models for studying HTLV-1 infection in vivo as well as cell immortalization in vitro. Therefore, studies with HTLV-1 may provide a better basis to assess and advance in vivo genome editing against retroviral infections.

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