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Shigeo Nishimata - One of the best experts on this subject based on the ideXlab platform.
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identification of a high frequency somatic nlrc4 mutation as a cause of autoinflammation by Pluripotent Cell based phenotype dissection
Arthritis & Rheumatism, 2017Co-Authors: Yuri Kawasaki, Ryosuke Seki, Atsushi Hijikata, Isao Asaka, Ayako Nagahashi, Mitsujiro Osawa, Akira Watanabe, Akira Niwa, Takayuki Tanaka, Shigeo NishimataAbstract:OBJECTIVE:To elucidate the genetic background of a patient with neonatal-onset multisystem inflammatory disease (NOMID) with no NLRP3 mutation. METHODS:A Japanese male child diagnosed as having NOMID was studied. The patient did not have any NLRP3 mutation, even as low-frequency mosaicism. We performed whole-exome sequencing on the patient and his parents. Induced Pluripotent stem Cells (iPSCs) were established from the patient's fibroblasts. The iPSCs were then differentiated into monocyte lineage to evaluate the cytokine profile. RESULTS:We established multiple iPSC clones from a patient with NOMID and incidentally found that the phenotypes of monocytes from iPSC clones were heterogeneous and could be grouped into disease and normal phenotypes. Because each iPSC clone was derived from a single somatic Cell, we hypothesized that the patient had somatic mosaicism of an interleukin-1β-related gene. Whole-exome sequencing of both representative iPSC clones and the patient's blood revealed a novel heterozygous NLRC4 mutation, p.T177A (c.529A>G), as a specific mutation in diseased iPSC clones. Knockout of the NLRC4 gene using the clustered regularly interspaced short palindromic repeat/Cas9 system in a mutant iPSC clone abrogated the pathogenic phenotype. CONCLUSION:Our findings indicate that the patient has somatic mosaicism of a novel NLRC4 mutation. To our knowledge, this is the first case showing that somatic mutation of NLRC4 causes autoinflammatory symptoms compatible with NOMID. The present study demonstrates the significance of prospective genetic screening combined with iPSC-based phenotype dissection for individualized diagnoses.
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Pluripotent Cell based phenotypic dissection identifies a high frequency somatic nlrc4 mutation as a cause of autoinflammation
Arthritis & Rheumatism, 2016Co-Authors: Yuri Kawasaki, Ryosuke Seki, Atsushi Hijikata, Isao Asaka, Ayako Nagahashi, Mitsujiro Osawa, Akira Watanabe, Akira Niwa, Takayuki Tanaka, Shigeo NishimataAbstract:Objective To elucidate the genetic background of a patient with neonatal-onset multisystem inflammatory disease (NOMID) who does not carry any NLRP3 mutation. Methods A Japanese male diagnosed as NOMID was recruited. The patient had no NLRP3 mutation even as low frequency mosaicism. We performed whole exome sequencing (WES) of the patient and his parents. Induced Pluripotent stem Cells (iPSCs) were established from the fibroblasts of the patient. iPSCs were then differentiated into monocytic lineage to evaluate the cytokine profile. Results We established multiple iPSC clones from an NOMID patient and incidentally found that the phenotype of monocytes from iPSC clones were heterogeneous, and could be grouped into “diseased” and “normal” phenotype. Because each iPSC clone was derived from a single somatic Cell, we hypothesized the patient had somatic mosaicism of an IL-1β-related gene. WES of both representative iPSC clones and patient's blood identified a novel heterozygous NLRC4 mutation, p.T177A (c.529A>G), as a specific mutation in “diseased” iPSC clones. Knockout of the NLRC4 gene using CRISPR/Cas9 system in a mutant iPSC clone abrogated the pathogenic phenotype. Conclusion We concluded the patient as having somatic mosaicism of a novel NLRC4 mutation. To our knowledge, this is the first case showing somatic NLRC4 mutation causes autoinflammatory symptoms compatible to NOMID. The present study demonstrates the significance of prospective genetic screening combined with iPSC-based phenotypic dissection for individualized diagnoses. This article is protected by copyright. All rights reserved.
Yuri Kawasaki - One of the best experts on this subject based on the ideXlab platform.
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identification of a high frequency somatic nlrc4 mutation as a cause of autoinflammation by Pluripotent Cell based phenotype dissection
Arthritis & Rheumatism, 2017Co-Authors: Yuri Kawasaki, Ryosuke Seki, Atsushi Hijikata, Isao Asaka, Ayako Nagahashi, Mitsujiro Osawa, Akira Watanabe, Akira Niwa, Takayuki Tanaka, Shigeo NishimataAbstract:OBJECTIVE:To elucidate the genetic background of a patient with neonatal-onset multisystem inflammatory disease (NOMID) with no NLRP3 mutation. METHODS:A Japanese male child diagnosed as having NOMID was studied. The patient did not have any NLRP3 mutation, even as low-frequency mosaicism. We performed whole-exome sequencing on the patient and his parents. Induced Pluripotent stem Cells (iPSCs) were established from the patient's fibroblasts. The iPSCs were then differentiated into monocyte lineage to evaluate the cytokine profile. RESULTS:We established multiple iPSC clones from a patient with NOMID and incidentally found that the phenotypes of monocytes from iPSC clones were heterogeneous and could be grouped into disease and normal phenotypes. Because each iPSC clone was derived from a single somatic Cell, we hypothesized that the patient had somatic mosaicism of an interleukin-1β-related gene. Whole-exome sequencing of both representative iPSC clones and the patient's blood revealed a novel heterozygous NLRC4 mutation, p.T177A (c.529A>G), as a specific mutation in diseased iPSC clones. Knockout of the NLRC4 gene using the clustered regularly interspaced short palindromic repeat/Cas9 system in a mutant iPSC clone abrogated the pathogenic phenotype. CONCLUSION:Our findings indicate that the patient has somatic mosaicism of a novel NLRC4 mutation. To our knowledge, this is the first case showing that somatic mutation of NLRC4 causes autoinflammatory symptoms compatible with NOMID. The present study demonstrates the significance of prospective genetic screening combined with iPSC-based phenotype dissection for individualized diagnoses.
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Pluripotent Cell based phenotypic dissection identifies a high frequency somatic nlrc4 mutation as a cause of autoinflammation
Arthritis & Rheumatism, 2016Co-Authors: Yuri Kawasaki, Ryosuke Seki, Atsushi Hijikata, Isao Asaka, Ayako Nagahashi, Mitsujiro Osawa, Akira Watanabe, Akira Niwa, Takayuki Tanaka, Shigeo NishimataAbstract:Objective To elucidate the genetic background of a patient with neonatal-onset multisystem inflammatory disease (NOMID) who does not carry any NLRP3 mutation. Methods A Japanese male diagnosed as NOMID was recruited. The patient had no NLRP3 mutation even as low frequency mosaicism. We performed whole exome sequencing (WES) of the patient and his parents. Induced Pluripotent stem Cells (iPSCs) were established from the fibroblasts of the patient. iPSCs were then differentiated into monocytic lineage to evaluate the cytokine profile. Results We established multiple iPSC clones from an NOMID patient and incidentally found that the phenotype of monocytes from iPSC clones were heterogeneous, and could be grouped into “diseased” and “normal” phenotype. Because each iPSC clone was derived from a single somatic Cell, we hypothesized the patient had somatic mosaicism of an IL-1β-related gene. WES of both representative iPSC clones and patient's blood identified a novel heterozygous NLRC4 mutation, p.T177A (c.529A>G), as a specific mutation in “diseased” iPSC clones. Knockout of the NLRC4 gene using CRISPR/Cas9 system in a mutant iPSC clone abrogated the pathogenic phenotype. Conclusion We concluded the patient as having somatic mosaicism of a novel NLRC4 mutation. To our knowledge, this is the first case showing somatic NLRC4 mutation causes autoinflammatory symptoms compatible to NOMID. The present study demonstrates the significance of prospective genetic screening combined with iPSC-based phenotypic dissection for individualized diagnoses. This article is protected by copyright. All rights reserved.
Michael J Ziller - One of the best experts on this subject based on the ideXlab platform.
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targeted disruption of dnmt1 dnmt3a and dnmt3b in human embryonic stem Cells
Nature Genetics, 2015Co-Authors: Jing Liao, Michael J Ziller, Rahul Karnik, Kendell Clement, Alexander M Tsankov, Veronika Akopian, Casey A Gifford, Julie Donaghey, Christina Galonska, Ramona PopAbstract:DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases (DNMTs) in human embryonic stem Cells (ESCs) using CRISPR/Cas9 genome editing to further investigate the roles and genomic targets of these enzymes. Disruption of DNMT3A or DNMT3B individually as well as of both enzymes in tandem results in viable, Pluripotent Cell lines with distinct effects on the DNA methylation landscape, as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to findings in mouse, deletion of DNMT1 resulted in rapid Cell death in human ESCs. To overcome this immediate lethality, we generated a doxycycline-responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1-mutant lines. However, doxycycline-mediated repression of exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive Cell death. Our data provide a comprehensive characterization of DNMT-mutant ESCs, including single-base genome-wide maps of the targets of these enzymes.
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genomic distribution and inter sample variation of non cpg methylation across human Cell types
PLOS Genetics, 2011Co-Authors: Michael J Ziller, Christoph Bock, Fabian Muller, Jing Liao, Yingying Zhang, Patrick Boyle, Charles B Epstein, Bradley E BernsteinAbstract:DNA methylation plays an important role in development and disease. The primary sites of DNA methylation in vertebrates are cytosines in the CpG dinucleotide context, which account for roughly three quarters of the total DNA methylation content in human and mouse Cells. While the genomic distribution, inter-individual stability, and functional role of CpG methylation are reasonably well understood, little is known about DNA methylation targeting CpA, CpT, and CpC (non-CpG) dinucleotides. Here we report a comprehensive analysis of non-CpG methylation in 76 genome-scale DNA methylation maps across Pluripotent and differentiated human Cell types. We confirm non-CpG methylation to be predominantly present in Pluripotent Cell types and observe a decrease upon differentiation and near complete absence in various somatic Cell types. Although no function has been assigned to it in pluripotency, our data highlight that non-CpG methylation patterns reappear upon iPS Cell reprogramming. Intriguingly, the patterns are highly variable and show little conservation between different Pluripotent Cell lines. We find a strong correlation of non-CpG methylation and DNMT3 expression levels while showing statistical independence of non-CpG methylation from pluripotency associated gene expression. In line with these findings, we show that knockdown of DNMTA and DNMT3B in hESCs results in a global reduction of non-CpG methylation. Finally, non-CpG methylation appears to be spatially correlated with CpG methylation. In summary these results contribute further to our understanding of cytosine methylation patterns in human Cells using a large representative sample set.
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reference maps of human es and ips Cell variation enable high throughput characterization of Pluripotent Cell lines
Cell, 2011Co-Authors: Christoph Bock, Evangelos Kiskinis, Griet Verstappen, Gabriella L Boulting, Zachary D Smith, Michael J ZillerAbstract:The developmental potential of human Pluripotent stem Cells suggests that they can produce disease-relevant Cell types for biomedical research. However, substantial variation has been reported among Pluripotent Cell lines, which could affect their utility and clinical safety. Such Cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced Pluripotent stem (iPS) Cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS Cell lines, and we have measured the in vitro differentiation propensity of these Cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS Cells and to predict the differentiation efficiency of individual Cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of Pluripotent Cell lines.
Gary S Stein - One of the best experts on this subject based on the ideXlab platform.
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epigenetic control of Cell cycle dependent histone gene expression is a principal component of the abbreviated Pluripotent Cell cycle
Molecular and Cellular Biology, 2012Co-Authors: Ricardo F Medina, Janet L Stein, Gary S Stein, Andre J. Van Wijnen, Prachi N Ghule, Fernando Cruzat, Rasim A Barutcu, Martin MontecinoAbstract:Self-renewal of human Pluripotent embryonic stem Cells proceeds via an abbreviated Cell cycle with a shortened G(1) phase. We examined which genes are modulated in this abbreviated period and the epigenetic mechanisms that control their expression. Accelerated upregulation of genes encoding histone proteins that support DNA replication is the most prominent gene regulatory program at the G(1)/S-phase transition in Pluripotent Cells. Expedited expression of histone genes is mediated by a unique chromatin architecture reflected by major nuclease hypersensitive sites, atypical distribution of epigenetic histone marks, and a region devoid of histone octamers. We observed remarkable differences in chromatin structure--hypersensitivity and histone protein modifications--between human embryonic stem (hES) and normal diploid Cells. Cell cycle-dependent transcription factor binding permits dynamic three-dimensional interactions between transcript initiating and processing factors at 5' and 3' regions of the gene. Thus, progression through the abbreviated G(1) phase involves Cell cycle stage-specific chromatin-remodeling events and rapid assembly of subnuclear microenvironments that activate histone gene transcription to promote nucleosomal packaging of newly replicated DNA during stem Cell renewal.
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reprogramming the Pluripotent Cell cycle restoration of an abbreviated g1 phase in human induced Pluripotent stem ips Cells
Journal of Cellular Physiology, 2011Co-Authors: Prachi N Ghule, Janet L Stein, Andre J. Van Wijnen, Ricardo F Medina, Christopher J Lengner, Matthew Mandeville, Meng Qiao, Zbigniew Dominski, Jane B Lian, Gary S SteinAbstract:Induced Pluripotent stem (iPS) Cells derived from terminally differentiated human fibroblasts are re-programmed to possess stem Cell like properties. However, the extent to which iPS Cells exhibit unique properties of the human embryonic stem (hES) Cell cycle remains to be established. Human ES Cells are characterized by an abbreviated G1 phase (~2.5 h) and accelerated organization of subnuclear domains that mediate the assembly of regulatory machinery for histone gene expression [i.e., histone locus bodies (HLBs)]. We therefore examined Cell cycle parameters of iPS Cells in comparison to hES Cells. Analysis of DNA synthesis (BrdU incorporation), Cell cycle distribution (FACS analysis and Ki67 staining) and subnuclear organization of HLBs [immuno-fluorescence microscopy and fluorescence in situ hybridization (FISH)] revealed that human iPS Cells have a short G1 phase (~2.5 h) and an abbreviated Cell cycle (16–18 h). Furthermore, HLBs are formed and reorganized rapidly after mitosis (within0.5 to 1.5 h). Thus, reprogrammed iPS Cells have Cell cycle kinetics and dynamic subnuclear organization of regulatory machinery that are principal properties of Pluripotent hES Cells. Our findings support the concept that the abbreviated Cell cycle of hES and iPS Cells is functionally linked to pluripotency.
Akira Niwa - One of the best experts on this subject based on the ideXlab platform.
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identification of a high frequency somatic nlrc4 mutation as a cause of autoinflammation by Pluripotent Cell based phenotype dissection
Arthritis & Rheumatism, 2017Co-Authors: Yuri Kawasaki, Ryosuke Seki, Atsushi Hijikata, Isao Asaka, Ayako Nagahashi, Mitsujiro Osawa, Akira Watanabe, Akira Niwa, Takayuki Tanaka, Shigeo NishimataAbstract:OBJECTIVE:To elucidate the genetic background of a patient with neonatal-onset multisystem inflammatory disease (NOMID) with no NLRP3 mutation. METHODS:A Japanese male child diagnosed as having NOMID was studied. The patient did not have any NLRP3 mutation, even as low-frequency mosaicism. We performed whole-exome sequencing on the patient and his parents. Induced Pluripotent stem Cells (iPSCs) were established from the patient's fibroblasts. The iPSCs were then differentiated into monocyte lineage to evaluate the cytokine profile. RESULTS:We established multiple iPSC clones from a patient with NOMID and incidentally found that the phenotypes of monocytes from iPSC clones were heterogeneous and could be grouped into disease and normal phenotypes. Because each iPSC clone was derived from a single somatic Cell, we hypothesized that the patient had somatic mosaicism of an interleukin-1β-related gene. Whole-exome sequencing of both representative iPSC clones and the patient's blood revealed a novel heterozygous NLRC4 mutation, p.T177A (c.529A>G), as a specific mutation in diseased iPSC clones. Knockout of the NLRC4 gene using the clustered regularly interspaced short palindromic repeat/Cas9 system in a mutant iPSC clone abrogated the pathogenic phenotype. CONCLUSION:Our findings indicate that the patient has somatic mosaicism of a novel NLRC4 mutation. To our knowledge, this is the first case showing that somatic mutation of NLRC4 causes autoinflammatory symptoms compatible with NOMID. The present study demonstrates the significance of prospective genetic screening combined with iPSC-based phenotype dissection for individualized diagnoses.
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Pluripotent Cell based phenotypic dissection identifies a high frequency somatic nlrc4 mutation as a cause of autoinflammation
Arthritis & Rheumatism, 2016Co-Authors: Yuri Kawasaki, Ryosuke Seki, Atsushi Hijikata, Isao Asaka, Ayako Nagahashi, Mitsujiro Osawa, Akira Watanabe, Akira Niwa, Takayuki Tanaka, Shigeo NishimataAbstract:Objective To elucidate the genetic background of a patient with neonatal-onset multisystem inflammatory disease (NOMID) who does not carry any NLRP3 mutation. Methods A Japanese male diagnosed as NOMID was recruited. The patient had no NLRP3 mutation even as low frequency mosaicism. We performed whole exome sequencing (WES) of the patient and his parents. Induced Pluripotent stem Cells (iPSCs) were established from the fibroblasts of the patient. iPSCs were then differentiated into monocytic lineage to evaluate the cytokine profile. Results We established multiple iPSC clones from an NOMID patient and incidentally found that the phenotype of monocytes from iPSC clones were heterogeneous, and could be grouped into “diseased” and “normal” phenotype. Because each iPSC clone was derived from a single somatic Cell, we hypothesized the patient had somatic mosaicism of an IL-1β-related gene. WES of both representative iPSC clones and patient's blood identified a novel heterozygous NLRC4 mutation, p.T177A (c.529A>G), as a specific mutation in “diseased” iPSC clones. Knockout of the NLRC4 gene using CRISPR/Cas9 system in a mutant iPSC clone abrogated the pathogenic phenotype. Conclusion We concluded the patient as having somatic mosaicism of a novel NLRC4 mutation. To our knowledge, this is the first case showing somatic NLRC4 mutation causes autoinflammatory symptoms compatible to NOMID. The present study demonstrates the significance of prospective genetic screening combined with iPSC-based phenotypic dissection for individualized diagnoses. This article is protected by copyright. All rights reserved.