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Delineation of a Human Mendelian Disorder of the DNA Demethylation Machinery: TET3 Deficiency
Beck, David B; Petracovici, Ana; He, Chongsheng; Moore, Hannah W; Louie, Raymond J; Ansar, Muhammad; Douzgou, Sofia; Sithambaram, Sivagamy; Cottrell, Trudie; Santos-Cortez, Regie Lyn P; Prijoles, Eloise J; Bend, Renee; Keren, Boris; Mignot, Cyril; Nougues, Marie-Christine; Õunap, Katrin; Reimand, Tiia; Pajusalu, Sander; Zahid, Muhammad; Saqib, Muhammad Arif Nadeem; Buratti, Julien; Seaby, Eleanor G; McWalter, Kirsty; Telegrafi, Aida; Baldridge, Dustin; Shinawi, Marwan; Leal, Suzanne M; Schaefer, G Bradley; Stevenson, Roger E; Banka, Siddharth; Bonasio, Roberto; Fahrner, Jill A
Germline pathogenic variants in chromatin-modifying enzymes are a common cause of pediatric developmental disorders. These enzymes catalyze reactions that regulate epigenetic inheritance via histone post-translational modifications and DNA methylation. Cytosine methylation (5-methylcytosine [5mC]) of DNA is the quintessential epigenetic mark, yet no human Mendelian disorder of DNA demethylation has yet been delineated. Here, we describe in detail a Mendelian disorder caused by the disruption of DNA demethylation. TET3 is a methylcytosine dioxygenase that initiates DNA demethylation during early zygote formation, embryogenesis, and neuronal differentiation and is intolerant to haploinsufficiency in mice and humans. We identify and characterize 11 cases of human TET3 deficiency in eight families with the common phenotypic features of intellectual disability and/or global developmental delay; hypotonia; autistic traits; movement disorders; growth abnormalities; and facial dysmorphism. Mono-allelic frameshift and nonsense variants in TET3 occur throughout the coding region. Mono-allelic and bi-allelic missense variants localize to conserved residues; all but one such variant occur within the catalytic domain, and most display hypomorphic function in an assay of catalytic activity. TET3 deficiency and other Mendelian disorders of the epigenetic machinery show substantial phenotypic overlap, including features of intellectual disability and abnormal growth, underscoring shared disease mechanisms.
PMCID:7010978
PMID: 31928709
ISSN: 1537-6605
CID: 5006852
Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease
Lalaoui, Najoua; Boyden, Steven E; Oda, Hirotsugu; Wood, Geryl M; Stone, Deborah L; Chau, Diep; Liu, Lin; Stoffels, Monique; Kratina, Tobias; Lawlor, Kate E; Zaal, Kristien J M; Hoffmann, Patrycja M; Etemadi, Nima; Shield-Artin, Kristy; Biben, Christine; Tsai, Wanxia Li; Blake, Mary D; Kuehn, Hye Sun; Yang, Dan; Anderton, Holly; Silke, Natasha; Wachsmuth, Laurens; Zheng, Lixin; Moura, Natalia Sampaio; Beck, David B; Gutierrez-Cruz, Gustavo; Ombrello, Amanda K; Pinto-Patarroyo, Gineth P; Kueh, Andrew J; Herold, Marco J; Hall, Cathrine; Wang, Hongying; Chae, Jae Jin; Dmitrieva, Natalia I; McKenzie, Mark; Light, Amanda; Barham, Beverly K; Jones, Anne; Romeo, Tina M; Zhou, Qing; Aksentijevich, Ivona; Mullikin, James C; Gross, Andrew J; Shum, Anthony K; Hawkins, Edwin D; Masters, Seth L; Lenardo, Michael J; Boehm, Manfred; Rosenzweig, Sergio D; Pasparakis, Manolis; Voss, Anne K; Gadina, Massimo; Kastner, Daniel L; Silke, John
RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage1-7. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis8. Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy-a condition we term 'cleavage-resistant RIPK1-induced autoinflammatory syndrome'. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1D325A mutant mouse strain. Whereas Ripk1-/- mice died postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3, but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life.
PMCID:6930849
PMID: 31827281
ISSN: 1476-4687
CID: 5006842
Human iPSC-Derived Neuronal Cells From CTBP1-Mutated Patients Reveal Altered Expression of Neurodevelopmental Gene Networks
Vijayalingam, S; Ezekiel, Uthayashanker R; Xu, Fenglian; Subramanian, T; Geerling, Elizabeth; Hoelscher, Brittany; San, KayKay; Ganapathy, Aravinda; Pemberton, Kyle; Tycksen, Eric; Pinto, Amelia K; Brien, James D; Beck, David B; Chung, Wendy K; Gurnett, Christina A; Chinnadurai, G
A recurrent de novo mutation in the transcriptional corepressor CTBP1 is associated with neurodevelopmental disabilities in children (Beck et al., 2016, 2019; Sommerville et al., 2017). All reported patients harbor a single recurrent de novo heterozygous missense mutation (p.R342W) within the cofactor recruitment domain of CtBP1. To investigate the transcriptional activity of the pathogenic CTBP1 mutant allele in physiologically relevant human cell models, we generated induced pluripotent stem cells (iPSC) from the dermal fibroblasts derived from patients and normal donors. The transcriptional profiles of the iPSC-derived "early" neurons were determined by RNA-sequencing. Comparison of the RNA-seq data of the neurons from patients and normal donors revealed down regulation of gene networks involved in neurodevelopment, synaptic adhesion and anti-viral (interferon) response. Consistent with the altered gene expression patterns, the patient-derived neurons exhibited morphological and electrophysiological abnormalities, and susceptibility to viral infection. Taken together, our studies using iPSC-derived neuron models provide novel insights into the pathological activities of the CTBP1 p.R342W allele.
PMCID:7653094
PMID: 33192249
ISSN: 1662-4548
CID: 5006922
A pathogenic CtBP1 missense mutation causes altered cofactor binding and transcriptional activity
Beck, David B; Subramanian, T; Vijayalingam, S; Ezekiel, Uthayashankar R; Donkervoort, Sandra; Yang, Michele L; Dubbs, Holly A; Ortiz-Gonzalez, Xilma R; Lakhani, Shenela; Segal, Devorah; Au, Margaret; Graham, John M; Verma, Sumit; Waggoner, Darrel; Shinawi, Marwan; Bönnemann, Carsten G; Chung, Wendy K; Chinnadurai, G
We previously reported a pathogenic de novo p.R342W mutation in the transcriptional corepressor CTBP1 in four independent patients with neurodevelopmental disabilities [1]. Here, we report the clinical phenotypes of seven additional individuals with the same recurrent de novo CTBP1 mutation. Within this cohort, we identified consistent CtBP1-related phenotypes of intellectual disability, ataxia, hypotonia, and tooth enamel defects present in most patients. The R342W mutation in CtBP1 is located within a region implicated in a high affinity-binding cleft for CtBP-interacting proteins. Unbiased proteomic analysis demonstrated reduced interaction of several chromatin-modifying factors with the CtBP1 W342 mutant. Genome-wide transcriptome analysis in human glioblastoma cell lines expressing -CtBP1 R342 (wt) or W342 mutation revealed changes in the expression profiles of genes controlling multiple cellular processes. Patient-derived dermal fibroblasts were found to be more sensitive to apoptosis during acute glucose deprivation compared to controls. Glucose deprivation strongly activated the BH3-only pro-apoptotic gene NOXA, suggesting a link between enhanced cell death and NOXA expression in patient fibroblasts. Our results suggest that context-dependent relief of transcriptional repression of the CtBP1 mutant W342 allele may contribute to deregulation of apoptosis in target tissues of patients leading to neurodevelopmental phenotypes.
PMID: 31041561
ISSN: 1364-6753
CID: 4778712
The use of leukocytes' secretome to individually target biological therapy in autoimmune arthritis: a case report
Poubelle, Patrice E; Pagé, Nathalie; Longchamps, Marie-Pier; Sampaio Moura, Natalia; Beck, David B; Aksentijevich, Ivona; Tessier, Philippe A; Pelletier, Martin
BACKGROUND:Biological agents have allowed remarkable improvement in controlling autoimmune arthropathies, although none of the numerous biologics readily available represent a universal treatment standard. Moreover, classical and genetic predictors are currently unsatisfactory to predict individual response to a biologic, and the best treatment selection is still based on a trial-and-error approach. Here, we report a clinical case demonstrating the usefulness of examining the leukocytes' secretome of patients. We set up and standardized a protocol that examines a patient's immune responses to establish the secretome of the blood mononuclear leukocytes and personalize the biotherapy. CASE PRESENTATION/METHODS:A 24-year-old woman was diagnosed with active early rheumatoid arthritis. The initial treatment regimen (prednisone, methotrexate, hydroxychloroquine, naproxen) was inefficient, as well as the anti-TNF adalimumab. The diagnosis was revised as possible rheumatoid arthritis-like psoriatic arthritis and adalimumab was replaced by abatacept (IgG1 Fc-CTLA-4) to no avail. Five years later, abatacept was replaced by the anti-IL-12/IL-23 ustekinumab with no objective control over the symptoms. The patient was thus enrolled in a prospective study based on the quantification of cytokines secreted by peripheral blood leukocytes stimulated with well-known immune activators of pattern recognition receptors and cytokine signalling. The results of this study revealed that plasma concentrations of cytokines were similar between the patient and healthy donors. In comparison to leukocytes from healthy donors, the patient's secretome showed a unique overproduction of IL-6. The anti-IL-6 receptor tocilizumab was, therefore, administered with a rapid improvement of her active psoriatic arthritis that remained dependent on low prednisone dosage. Clinical parameters progressively returned to normal levels and her quality of life was greatly improved, despite the major delay to begin the present personalized treatment. CONCLUSIONS:An efficient way to effectively treat patients with complex autoimmune arthropathies, and avoid irreversible disability, is to know their leukocytes' secretome to identify abnormally secreted cytokines and personalize their biotherapy, as exemplified by this case report.
PMCID:6548783
PMID: 31165299
ISSN: 2001-1326
CID: 5006832
Second Case of HOIP Deficiency Expands Clinical Features and Defines Inflammatory Transcriptome Regulated by LUBAC [Case Report]
Oda, Hirotsugu; Beck, David B; Kuehn, Hye Sun; Sampaio Moura, Natalia; Hoffmann, Patrycja; Ibarra, Maria; Stoddard, Jennifer; Tsai, Wanxia Li; Gutierrez-Cruz, Gustavo; Gadina, Massimo; Rosenzweig, Sergio D; Kastner, Daniel L; Notarangelo, Luigi D; Aksentijevich, Ivona
PMCID:6431612
PMID: 30936877
ISSN: 1664-3224
CID: 5006822
Biochemistry of Autoinflammatory Diseases: Catalyzing Monogenic Disease
Beck, David B; Aksentijevich, Ivona
Monogenic autoinflammatory disorders are a group of conditions defined by systemic or localized inflammation without identifiable causes, such as infection. In contrast to classical primary immunodeficiencies that manifest with impaired immune responses, these disorders are due to defects in genes that regulate innate immunity leading to constitutive activation of pro-inflammatory signaling. Through studying patients with rare autoinflammatory conditions, novel mechanisms of inflammation have been identified that bare on our understanding not only of basic signaling in inflammatory cells, but also of the pathogenesis of more common inflammatory diseases and have guided treatment modalities. Autoinflammation has further been implicated as an important component of cardiovascular, neurodegenerative, and metabolic syndromes. In this review, we will focus on a subset of inherited enzymatic deficiencies that lead to constitutive inflammation, and how these rare diseases have provided insights into diverse areas of cell biology not restricted to immune cells. In this way, Mendelian disorders of the innate immune system, and in particular loss of catalytic activity of enzymes in distinct pathways, have expanded our understanding of the interplay between many seemingly disparate cellular processes. We also explore the overlap between autoinflammation, autoimmunity, and immunodeficiency, which has been increasingly recognized in patients with dysregulated immune responses.
PMCID:6365650
PMID: 30766537
ISSN: 1664-3224
CID: 5006812
Bi-allelic Mutations in Phe-tRNA Synthetase Associated with a Multi-system Pulmonary Disease Support Non-translational Function
Xu, Zhiwen; Lo, Wing-Sze; Beck, David B; Schuch, Luise A; Oláhová, Monika; Kopajtich, Robert; Chong, Yeeting E; Alston, Charlotte L; Seidl, Elias; Zhai, Liting; Lau, Ching-Fun; Timchak, Donna; LeDuc, Charles A; Borczuk, Alain C; Teich, Andrew F; Juusola, Jane; Sofeso, Christina; Müller, Christoph; Pierre, Germaine; Hilliard, Tom; Turnpenny, Peter D; Wagner, Matias; Kappler, Matthias; Brasch, Frank; Bouffard, John Paul; Nangle, Leslie A; Yang, Xiang-Lei; Zhang, Mingjie; Taylor, Robert W; Prokisch, Holger; Griese, Matthias; Chung, Wendy K; Schimmel, Paul
The tRNA synthetases catalyze the first step of protein synthesis and have increasingly been studied for their nuclear and extra-cellular ex-translational activities. Human genetic conditions such as Charcot-Marie-Tooth have been attributed to dominant gain-of-function mutations in some tRNA synthetases. Unlike dominantly inherited gain-of-function mutations, recessive loss-of-function mutations can potentially elucidate ex-translational activities. We present here five individuals from four families with a multi-system disease associated with bi-allelic mutations in FARSB that encodes the beta chain of the alpha2beta2 phenylalanine-tRNA synthetase (FARS). Collectively, the mutant alleles encompass a 5'-splice junction non-coding variant (SJV) and six missense variants, one of which is shared by unrelated individuals. The clinical condition is characterized by interstitial lung disease, cerebral aneurysms and brain calcifications, and cirrhosis. For the SJV, we confirmed exon skipping leading to a frameshift associated with noncatalytic activity. While the bi-allelic combination of the SJV with a p.Arg305Gln missense mutation in two individuals led to severe disease, cells from neither the asymptomatic heterozygous carriers nor the compound heterozygous affected individual had any defect in protein synthesis. These results support a disease mechanism independent of tRNA synthetase activities in protein translation and suggest that this FARS activity is essential for normal function in multiple organs.
PMCID:6035289
PMID: 29979980
ISSN: 1537-6605
CID: 5006802
Extending the phenotypic spectrum of Sengers syndrome: Congenital lactic acidosis with synthetic liver dysfunction
Beck, David B; Cusmano-Ozog, Kristina; Andescavage, Nickie; Leon, Eyby
Sengers syndrome is a rare autosomal recessive mitochondrial disease characterized by lactic acidosis, hypertrophic cardiomyopathy and bilateral cataracts. We present here a case of neonatal demise, within the first day of life, who initially presented with severe lactic acidosis, with evidence of both chorioamnionitis and cardiogenic shock. Initial metabolic labs demonstrated a severe lactic acidosis prompting genetic testing which revealed a homozygous pathogenic variant for Sengers syndrome in AGK, c.979A >  T; p.K327*. In addition to the canonical features of Sengers syndrome, our patient is the first reported case with liver dysfunction extending the phenotypic spectrum both in terms of severity and complications. This case also highlights the importance of maintaining a broad differential for congenital lactic acidosis.
PMCID:5904566
PMID: 29682452
ISSN: 2214-6490
CID: 5006792
Photoactivated In Vivo Proximity Labeling
Beck, David B; Bonasio, Roberto
Identification of molecular interactions is paramount to understanding how cells function. Most available technologies rely on co-purification of a protein of interest and its binding partners. Therefore, they are limited in their ability to detect low-affinity interactions and cannot be applied to proteins that localize to difficult-to-solubilize cellular compartments. In vivo proximity labeling (IPL) overcomes these obstacles by covalently tagging proteins and RNAs based on their proximity in vivo to a protein of interest. In IPL, a heterobifunctional probe comprising a photoactivatable moiety and biotin is recruited by a monomeric streptavidin tag fused to a protein of interest. Following UV irradiation, candidate interacting proteins and RNAs are covalently biotinylated with tight spatial and temporal control and subsequently recovered using biotin as an affinity handle. Here, we describe experimental protocols to discover novel protein-protein and protein-RNA interactions using IPL. © 2017 by John Wiley & Sons, Inc.
PMID: 28628203
ISSN: 2160-4762
CID: 5006782