scholarly journals Correction of NR2E3 Associated Enhanced S-cone Syndrome Patient-specific iPSCs using CRISPR-Cas9

Genes ◽  
2019 ◽  
Vol 10 (4) ◽  
pp. 278 ◽  
Author(s):  
Laura Bohrer ◽  
Luke Wiley ◽  
Erin Burnight ◽  
Jessica Cooke ◽  
Joseph Giacalone ◽  
...  
Keyword(s):  
Photoreceptor Cell ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Model Systems ◽  
Patient Specific ◽  
Rod Photoreceptor ◽  
Cone Photoreceptor ◽  
Retinal Cells ◽  
Enhanced S Cone Syndrome

Enhanced S-cone syndrome (ESCS) is caused by recessive mutations in the photoreceptor cell transcription factor NR2E3. Loss of NR2E3 is characterized by repression of rod photoreceptor cell gene expression, over-expansion of the S-cone photoreceptor cell population, and varying degrees of M- and L-cone photoreceptor cell development. In this study, we developed a CRISPR-based homology-directed repair strategy and corrected two different disease-causing NR2E3 mutations in patient-derived induced pluripotent stem cells (iPSCs) generated from two affected individuals. In addition, one patient’s iPSCs were differentiated into retinal cells and NR2E3 transcription was evaluated in CRISPR corrected and uncorrected clones. The patient’s c.119-2A>C mutation caused the inclusion of a portion of intron 1, the creation of a frame shift, and generation of a premature stop codon. In summary, we used a single set of CRISPR reagents to correct different mutations in iPSCs generated from two individuals with ESCS. In doing so we demonstrate the advantage of using retinal cells derived from affected patients over artificial in vitro model systems when attempting to demonstrate pathophysiologic mechanisms of specific mutations.

scholarly journals Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dwaipayan Adhya ◽  
George Chennell ◽  
James A. Crowe ◽  
Eva P. Valencia-Alarcón ◽  
James Seyforth ◽  
...  
Keyword(s):  
High Resolution ◽  
Human Brain ◽  
Light Sheet ◽  
Autism Spectrum ◽  
Model Systems ◽  
Large Field ◽  
Autism Spectrum Conditions ◽  
Patient Specific ◽  
Airy Beam

Abstract Background The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes—in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points. Results Conventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes. Conclusion We present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro.

scholarly journals Membrane blebbing as an assessment of functional rescue of dysferlin-deficient human myotubes via nonsense suppression

2010 ◽  
Vol 109 (3) ◽  
pp. 901-905 ◽  
Author(s):  
Bingjing Wang ◽  
Zhaohui Yang ◽  
Becky K. Brisson ◽  
Huisheng Feng ◽  
Zhiqian Zhang ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Nonsense Suppression ◽  
Muscle Membrane ◽  
Membrane Repair ◽  
Vitro Assay ◽  
Membrane Blebbing ◽  
Human Myotubes

Mutations that result in the loss of the protein dysferlin result in defective muscle membrane repair and cause either a form of limb girdle muscular dystrophy (type 2B) or Miyoshi myopathy. Most patients are compound heterozygotes, often carrying one allele with a nonsense mutation. Using dysferlin-deficient mouse and human myocytes, we demonstrated that membrane blebbing in skeletal muscle myotubes in response to hypotonic shock requires dysferlin. Based on this, we developed an in vitro assay to assess rescue of dysferlin function in skeletal muscle myotubes. This blebbing assay may be useful for drug discovery/validation for dysferlin deficiency. With this assay, we demonstrate that the nonsense suppression drug, ataluren (PTC124), is able to induce read-through of the premature stop codon in a patient with a R1905X mutation in dysferlin and produce sufficient functional dysferlin (∼15% of normal levels) to rescue myotube membrane blebbing. Thus ataluren is a potential therapeutic for dysferlin-deficient patients harboring nonsense mutations.

scholarly journals Murine cytomegalovirus open reading frame m29.1 augments virus replication both in vitro and in vivo

2007 ◽  
Vol 88 (11) ◽  
pp. 2941-2951 ◽  
Author(s):  
Mohammad M. Ahasan ◽  
Clive Sweet
Keyword(s):  
Virus Replication ◽  
Post Infection ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Open Reading Frame ◽  
Murine Cytomegalovirus ◽  
Reading Frame ◽  
Codon Mutation

Murine cytomegalovirus mutant Rc29, with a premature stop codon mutation in the m29 open reading frame (ORF), produced no apparent phenotype in cell culture or following infection of BALB/c mice. In contrast, a similar mutant virus, Rc29.1, with a premature stop codon mutation in its m29.1 ORF, showed reduced virus yields (2–3 log10 p.f.u. ml−1) in tissue culture. Mutant virus yields in BALB/c mice were delayed, reduced (∼1 log10 p.f.u. per tissue) and persisted less well in salivary glands compared with wild-type (wt) and revertant (Rv29.1) virus. In severe combined immunodeficiency mice, Rc29.1 virus showed delayed and reduced replication initially in all tissues (liver, spleen, kidneys, heart, lung and salivary glands). This delayed death until 31 days post-infection (p.i.) compared with wt (23 days p.i.) but at death virus yields were similar to wt. m29 gene transcription was initiated at early times post-infection, while production of a transcript from ORF m29.1 in the presence of cycloheximide indicated that it was an immediate-early gene. ORFs m29.1 and M28 are expressed from a bicistronic message, which is spliced infrequently. However, it is likely that each ORF expresses its own protein, as antiserum derived in rabbits to the m29.1 protein expressed in bacteria from the m29.1 ORF detected only one protein in Western blot analysis of the size predicted for the m29.1 protein. Our results suggest that neither ORF is essential for virus replication but m29.1 is important for optimal viral growth in vitro and in vivo.

Abstract P482: Elucidating And Characterizing The Molecular Mechanistic Role Of Phospholamban L39 Stop In The Pathophysiology Of Cardiomyopathy Using Patient-derived Human Induced Pluripotent Stem Cells And Humanized Knock-in Mouse Model Systems

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Anichavezhi Devendran ◽  
Rasheed Bailey ◽  
Sumanta Kar ◽  
Francesca Stillitano ◽  
Irene Turnbull ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Mononuclear Cells ◽  
Embryonic Stem ◽  
Model Systems ◽  
Patient Specific ◽  
Induced Pluripotent

Background: Heart failure (HF) is a complex clinical condition associated with substantial morbidity and mortality worldwide. The contractile dysfunction and arrhythmogenesis related to HF has been linked to the remodelling of calcium (Ca ++ ) handling. Phospholamban (PLN) has emerged as a key regulator of intracellular Ca ++ concentration. Of the PLN mutations, L39X is intriguing as it has not been fully characterized. This mutation is believed to be functionally equivalent to PLN null (KO) but contrary to PLN KO mice, L39X carriers develop a lethal cardiomyopathy (CMP). Our study aims at using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from homozygous L39X carriers to elucidate the role of L39X in human pathophysiology. Our plan also involves the characterization of humanized L39X knock-in mice (KM), which we hypothesize will develop a CMP from mis-localization of PLN and disruption of Ca ++ signalling. Methodology and Results: Mononuclear cells from Hom L39X carriers were obtained to generate 11 integration-free patient-specific iPSC clones. The iPSC-CMs were derived using established protocols. Compared to the WT iPSC-CMs, the Hom L39X derived-CMs PLN had an abnormal cytoplasmic distribution and formed intracellular aggregates, with the loss of perinuclear localization. There was also a 70% and 50% reduction of mRNA and protein expression of PLN respectively in L39X compared to WT iPSC-CMs. These findings indicated that L39X PLN is both under-expressed and mis-localized within the cell. To validate this observation in-vivo, we genetically modified FVB mice to harbour the human L39X. Following electroporation, positively transfected mouse embryonic stem cells were injected into host blastocysts to make humanized KM that were subsequently used to generate either a protamine-Cre (endogenous PLN driven expression) or a cardiac TNT mouse (i.e., CMP specific). Conclusion: Our data confirm an abnormal intracellular distribution of PLN, with the loss of perinuclear accumulation and mis-localization, suggestive of ineffective targeting to or retention of L39X. The mouse model will be critically important to validate the in-vitro observations and provides an ideal platform for future studies centred on the development of novel therapeutic strategies including virally delivered CRISPR/Cas9 for in-vivo gene editing and testing of biochemical signalling pathways.

scholarly journals Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Budd A Tucker ◽  
Robert F Mullins ◽  
Luan M Streb ◽  
Kristin Anfinson ◽  
Mari E Eyestone ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Protein Misfolding ◽  
Autosomal Recessive ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Photoreceptor Cells ◽  
Precursor Cells ◽  
Patient Specific ◽  
Basal Bodies ◽  
Induced Pluripotent

Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient’s keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient’s mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient’s other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1−/− mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration.

Loss of cysteine 584 impairs the storage and release, but not the synthesis of von Willebrand factor

2014 ◽  
Vol 112 (12) ◽  
pp. 1159-1166 ◽  
Author(s):  
Viviana Daidone ◽  
Giovanni Barbon ◽  
Elena Pontara ◽  
Grazia Cattini ◽  
Lisa Gallinaro ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Von Willebrand Disease ◽  
Hek293 Cells ◽  
Loss Of Function ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryCysteines play a key part in von Willebrand factor (VWF) dimerisation and polymerisation, and their loss may severely affect VWF structure and function. We report on three patients with type 3 von Willebrand disease carrying the new c.1751G>T missense mutation that induces the substitution of cysteine 584 by phenylalanine (C584F), and the deletion of seven nucleotides in exon 7 (c.729_735del), producing a premature stop codon at position 454 (E244Lfs*211). VWF was almost undetectable in the patients’ plasma and platelets, while a single, poorly represented, oligomer emerged on plasma VWF multimer analysis. No post-DDAVP increase in VWF and factor VIII was observed. Expressing human recombinant C584F-VWF in HEK293T cells showed that C584F-VWF was synthesised and multimerised but not secreted – apart from the first oligomer, which was slightly represented in the conditioned medium, with a pattern similar to the patients’ plasma VWF. The in vitro expression of the E244Lfs*211–VWF revealed a defective synthesis of the mutated VWF, with a behavior typical of loss of function mutations. Cellular trafficking, investigated in HEK293 cells, indicated a normal C584F-VWF content in the endoplasmic reticulum and Golgi apparatus, confirming the synthesis and multimerisation of C584F-VWF. No pseudo-Weibel Palade bodies were demonstrable, however, suggesting that C584F mutation impairs the storage of C584F-VWF. These findings point to cysteine 584 having a role in the release of VWF and its targeting to pseudo-Weibel Palade bodies in vitro, as well as in its storage and release by endothelial cells in vivo.

scholarly journals Identification and characteristics of the structural gene for the Drosophila eye colour mutant sepia, encoding PDA synthase, a member of the Omega class glutathione S-transferases

2006 ◽  
Vol 398 (3) ◽  
pp. 451-460 ◽  
Author(s):  
Jaekwang Kim ◽  
Hyunsuk Suh ◽  
Songhee Kim ◽  
Kiyoung Kim ◽  
Chiyoung Ahn ◽  
...  
Keyword(s):  
Structural Gene ◽  
Stop Codon ◽  
Gel Filtration ◽  
Premature Stop Codon ◽  
Genomic Region ◽  
Gene Encoding ◽  
Colour Mutant ◽  
Eye Colour ◽  
Glutathione S Transferases

The eye colour mutant sepia (se1) is defective in PDA {6-acetyl-2-amino-3,7,8,9-tetrahydro-4H-pyrimido[4,5-b]-[1,4]diazepin-4-one or pyrimidodiazepine} synthase involved in the conversion of 6-PTP (2-amino-4-oxo-6-pyruvoyl-5,6,7,8-tetrahydropteridine; also known as 6-pyruvoyltetrahydropterin) into PDA, a key intermediate in drosopterin biosynthesis. However, the identity of the gene encoding this enzyme, as well as its molecular properties, have not yet been established. Here, we identify and characterize the gene encoding PDA synthase and show that it is the structural gene for sepia. Based on previously reported information [Wiederrecht, Paton and Brown (1984) J. Biol. Chem. 259, 2195–2200; Wiederrecht and Brown (1984) J. Biol. Chem. 259, 14121–14127; Andres (1945) Drosoph. Inf. Serv. 19, 45; Ingham, Pinchin, Howard and Ish-Horowicz (1985) Genetics 111, 463–486; Howard, Ingham and Rushlow (1988) Genes Dev. 2, 1037–1046], we isolated five candidate genes predicted to encode GSTs (glutathione S-transferases) from the presumed sepia locus (region 66D5 on chromosome 3L). All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro. The molecular mass of recombinant CG6781 was estimated to be 28 kDa by SDS/PAGE and 56 kDa by gel filtration, indicating that it is a homodimer under native conditions. Sequencing of the genomic region spanning CG6781 revealed that the se1 allele has a frameshift mutation from ‘AAGAA’ to ‘GTG’ at nt 190–194, and that this generates a premature stop codon. Expression of the CG6781 open reading frame in an se1 background rescued the eye colour defect as well as PDA synthase activity and drosopterins content. The extent of rescue was dependent on the dosage of transgenic CG6781. In conclusion, we have discovered a new catalytic activity for an Omega class GST and that CG6781 is the structural gene for sepia which encodes PDA synthase.

Abstract 19944: A PDE5A Gene Mutation Affecting Risk of Myocardial Infarction

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Tan An Dang ◽  
Ingrid Braenne ◽  
Redouane Aherrahrou ◽  
Stephanie Tennstedt ◽  
Thorsten Kessler ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Promoter Activity ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Alternative Promoter ◽  
Gain Function ◽  
Lod Score ◽  
X Ray ◽  
Hek Cells

INTRODUCTION: Multiple frequent genetic variants were shown to affect myocardial infarction (MI) risk. Genetic causes for familial clustering of MI are less clear. We aimed to identify and characterize the molecular underpinnings of premature MI in a family with 9 affected individuals. METHODS AND RESULTS: Employing cosegregation analysis and exome sequencing we identified a mutation in the phosphodiesterase 5A ( PDE5A ) gene in all affected individuals (LOD score 3.16). It is located in an alternative promoter site of PDE5A and leads to a premature stop codon in one of the PDE5A isoforms (p.Lys7Ter). PDE5A encodes for three isoforms catalyzing cGMP, a second messenger mediating vasodilation and platelet passivation. Effects of the stop codon were investigated by western blot analysis after in vitro mutagenesis. Overexpression in HEK cells did not reveal a loss of transcript but the expression of a N-terminally truncated protein. Deeper analyses of translation initiation by deletion of possible transcription starts via in vitro mutagenesis uncovered a protein lacking 91 amino acids compared to the full-length isoform. Activity of the truncated PDE5A was measured using PDEGlo (Promega). Moreover, the effect of the variant on the alternative promoter site was analyzed by luciferase assays. Therefore, a 600 bp fragment containing either the mutated or WT allele was cloned into a pGL4.10 vector (Promega). Overexpression in HEK cells showed 40% increase of promoter activity with the mutated allele (p<0.05). Similar results could be shown in other cell lines. CONCLUSION: We identified a mutation in the PDE5A gene associated with premature MI. While a gain of function of PDE5A makes sense from a pathophysiological stance, the particular variant seemed to result in a premature stop codon. However, we could demonstrate that the variant might lead to increased promoter activity and that the presumable stop codon does not result into a loss of transcript but rather a truncated, potentially more active PDE5A isoform. Along with our homology modeling results and x-ray crystallographic and biochemical studies of Wang et al. (2010), these data support the idea of overexpression of a truncated though functional PDE5A in mutation carriers, possibly resulting in a gain function.

Diffusible rod-promoting signals in the developing rat retina

Development ◽  
1992 ◽  
Vol 114 (4) ◽  
pp. 899-906 ◽  
Author(s):  
T. Watanabe ◽  
M.C. Raff
Keyword(s):  
Amacrine Cell ◽  
Photoreceptor Cells ◽  
Neural Retina ◽  
Rod Photoreceptor ◽  
Cell Culture System ◽  
Retinal Cells ◽  
Neuroepithelial Cells ◽  
Developing Rat

We previously developed a reaggregate cell culture system in which embryonic rat retinal neuroepithelial cells proliferate and give rise to opsin-expressing rod photoreceptor cells (rods) on the same schedule in vitro as they do in vivo. We showed that the proportion of neuroepithelial cells in the embryonic day 15 (E15) retina that differentiated into opsin+ rods after 5–6 days in such cultures increased by approximately 40-fold when the E15 cells were cultured in the presence of an excess of postnatal day 1 (P1) neural retinal cells. In the present study, we have further analyzed this rod-promoting activity of neonatal neural retinal cells. We show that the activity is mediated by a diffusible signal(s) that seems to act over a relatively short distance. Whereas neonatal (P1-P3) neural retina has rod-promoting activity, E15 and adult neural retina, neonatal thymus, cerebrum and cerebellum do not. Finally, we show that neonatal neural retina promotes rod but not amacrine cell development.

scholarly journals New insights into the interplay between the translation machinery and nonsense-mediated mRNA decay factors

2018 ◽  
Vol 46 (3) ◽  
pp. 503-512 ◽  
Author(s):  
Etienne Raimondeau ◽  
Joshua C. Bufton ◽  
Christiane Schaffitzel
Keyword(s):  
Mrna Decay ◽  
Stop Codon ◽  
Translation Termination ◽  
Premature Stop Codon ◽  
Molecular Function ◽  
Translation Machinery ◽  
New Findings ◽  
Nascent Chain ◽  
Nonsense Mediated Mrna Decay

Faulty mRNAs with a premature stop codon (PTC) are recognized and degraded by nonsense-mediated mRNA decay (NMD). Recognition of a nonsense mRNA depends on translation and on the presence of NMD-enhancing or the absence of NMD-inhibiting factors in the 3′-untranslated region. Our review summarizes our current understanding of the molecular function of the conserved NMD factors UPF3B and UPF1, and of the anti-NMD factor Poly(A)-binding protein, and their interactions with ribosomes translating PTC-containing mRNAs. Our recent discovery that UPF3B interferes with human translation termination and enhances ribosome dissociation in vitro, whereas UPF1 is inactive in these assays, suggests a re-interpretation of previous experiments and modification of prevalent NMD models. Moreover, we discuss recent work suggesting new functions of the key NMD factor UPF1 in ribosome recycling, inhibition of translation re-initiation and nascent chain ubiquitylation. These new findings suggest that the interplay of UPF proteins with the translation machinery is more intricate than previously appreciated, and that this interplay quality-controls the efficiency of termination, ribosome recycling and translation re-initiation.

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