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MedRxiv : the Preprint Server For... Mar 2024To identify genetic etiologies and genotype/phenotype associations for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs).
PURPOSE
To identify genetic etiologies and genotype/phenotype associations for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs).
METHODS
We coupled phenotyping with exome or genome sequencing of 467 pedigrees with genetically unsolved oCCDDs, integrating analyses of pedigrees, human and animal model phenotypes, and variants to identify rare candidate single nucleotide variants, insertion/deletions, and structural variants disrupting protein-coding regions. Prioritized variants were classified for pathogenicity and evaluated for genotype/phenotype correlations.
RESULTS
Analyses elucidated phenotypic subgroups, identified pathogenic/likely pathogenic variant(s) in 43/467 probands (9.2%), and prioritized variants of uncertain significance in 70/467 additional probands (15.0%). These included known and novel variants in established oCCDD genes, genes associated with syndromes that sometimes include oCCDDs (e.g., genes that fit the syndromic component of the phenotype but had no prior oCCDD association (e.g., ), genes with no reported association with oCCDDs or the syndromic phenotypes (e.g., ), and genes associated with oCCDD phenocopies that had resulted in misdiagnoses.
CONCLUSION
This study suggests that unsolved oCCDDs are clinically and genetically heterogeneous disorders often overlapping other Mendelian conditions and nominates many candidates for future replication and functional studies.
PubMed: 38585811
DOI: 10.1101/2024.03.22.24304594 -
Neurobiology of Disease Jun 2024Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes,...
Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by YARS1 mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRS mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of Yars mice modelling DI-CMT. We determined that Yars homozygotes display a selective, age-dependent impairment in in vivo axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRS, but not TyrRS, into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy.
Topics: Animals; Charcot-Marie-Tooth Disease; Brain-Derived Neurotrophic Factor; Mice; Axonal Transport; Disease Models, Animal; Tyrosine-tRNA Ligase; Humans; Mice, Transgenic; Muscle, Skeletal; Receptor, trkB; Mutation
PubMed: 38583640
DOI: 10.1016/j.nbd.2024.106501 -
The Journal of Investigative Dermatology Apr 2024Chronic non-healing wounds negatively impact quality of life and are a significant financial drain on health systems. The risk of infection that exacerbates...
Chronic non-healing wounds negatively impact quality of life and are a significant financial drain on health systems. The risk of infection that exacerbates comorbidities in patients necessitates regular application of wound care. Understanding the mechanisms underlying impaired wound healing are therefore a key priority to inform effective new-generation treatments. In this study, we demonstrate that 14-3-3-mediated suppression of signaling through ROCK is a critical mechanism that inhibits the healing of diabetic wounds. Accordingly, pharmacological inhibition of 14-3-3 by topical application of the sphingo-mimetic drug RB-11 to diabetic wounds on a mouse model of type II diabetes accelerated wound closure more than 2-fold than vehicle control, phenocopying our previous observations in 14-3-3ζ-knockout mice. We also demonstrate that accelerated closure of the wounded epidermis by 14-3-3 inhibition causes enhanced signaling through the Rho-ROCK pathway and that the underlying cellular mechanism involves the efficient recruitment of dermal fibroblasts into the wound and the rapid production of extracellular matrix proteins to re-establish the injured dermis. Our observations that the 14-3-3/ROCK inhibitory axis characterizes impaired wound healing and that its suppression facilitates fibroblast recruitment and accelerated re-epithelialization suggest new possibilities for treating diabetic wounds by pharmacologically targeting this axis.
PubMed: 38582367
DOI: 10.1016/j.jid.2024.03.032 -
RSC Chemical Biology Apr 2024Phenotypic screening is a valuable tool to both understand and engineer complex biological systems. We demonstrate the functionality of this approach in the development...
Phenotypic screening is a valuable tool to both understand and engineer complex biological systems. We demonstrate the functionality of this approach in the development of cell-free protein synthesis (CFPS) technology. Phenotypic screening identified numerous compounds that enhanced protein production in yeast lysate CFPS reactions. Notably, many of these were competitive ATP kinase inhibitors, with the exploitation of their inherent substrate promiscuity redirecting ATP flux towards heterologous protein expression. Chemoproteomic-guided strain engineering partially phenocopied drug effects, with a 30% increase in protein yield observed upon deletion of the ATP-consuming SSA1 component of the HSP70 chaperone. Moreover, drug-mediated metabolic rewiring coupled with template optimization generated the highest protein yields in yeast CFPS to date using a hitherto less efficient, but more cost-effective glucose energy regeneration system. Our approach highlights the utility of target-agnostic phenotypic screening and target identification to deconvolute cell-lysate complexity, adding to the expanding repertoire of strategies for improving CFPS.
PubMed: 38576719
DOI: 10.1039/d4cb00004h -
BioRxiv : the Preprint Server For... Mar 2024Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes,...
Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRS mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of mice modelling DI-CMT. We determined that homozygotes display a selective, age-dependent impairment in axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRS, but not TyrRS, into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy.
PubMed: 38559020
DOI: 10.1101/2023.04.09.536152 -
Infection and Immunity May 2024The mouse pathogen is utilized as a model organism for studying infections caused by the human pathogens enteropathogenic (EPEC) and enterohemorrhagic (EHEC) and to...
The mouse pathogen is utilized as a model organism for studying infections caused by the human pathogens enteropathogenic (EPEC) and enterohemorrhagic (EHEC) and to elucidate mechanisms of mucosal immunity. In response to infection, innate lymphoid cells and T cells secrete interleukin (IL)-22, a cytokine that promotes mucosal barrier function. IL-22 plays a pivotal role in enabling mice to survive and recover from infection, although the exact mechanisms involved remain incompletely understood. Here, we investigated whether particular components of the host response downstream of IL-22 contribute to the cytokine's protective effects during infection. In line with previous research, mice lacking the IL-22 gene ( mice) were highly susceptible to infection. To elucidate the role of specific antimicrobial proteins modulated by IL-22, we infected the following knockout mice: (calprotectin), (lipocalin-2), (Reg3β), (Reg3γ), and (C3). All knockout mice tested displayed a considerable level of resistance to infection, and none phenocopied the lethality observed in mice. By investigating another arm of the IL-22 response, we observed that -infected mice exhibited an overall decrease in gene expression related to intestinal barrier integrity as well as significantly elevated colonic inflammation, gut permeability, and pathogen levels in the spleen. Taken together, these results indicate that host resistance to lethal infection may depend on multiple antimicrobial responses acting in concert, or that other IL-22-regulated processes, such as tissue repair and maintenance of epithelial integrity, play crucial roles in host defense to attaching and effacing pathogens.
Topics: Animals; Mice; Citrobacter rodentium; Disease Models, Animal; Enterobacteriaceae Infections; Interleukin-22; Mice, Inbred C57BL; Mice, Knockout; Pancreatitis-Associated Proteins
PubMed: 38557196
DOI: 10.1128/iai.00099-24 -
Nature Communications Mar 2024The Y-linked SRY gene initiates mammalian testis-determination. However, how the expression of SRY is regulated remains elusive. Here, we demonstrate that a conserved...
The Y-linked SRY gene initiates mammalian testis-determination. However, how the expression of SRY is regulated remains elusive. Here, we demonstrate that a conserved steroidogenic factor-1 (SF-1)/NR5A1 binding enhancer is required for appropriate SRY expression to initiate testis-determination in humans. Comparative sequence analysis of SRY 5' regions in mammals identified an evolutionary conserved SF-1/NR5A1-binding motif within a 250 bp region of open chromatin located 5 kilobases upstream of the SRY transcription start site. Genomic analysis of 46,XY individuals with disrupted testis-determination, including a large multigenerational family, identified unique single-base substitutions of highly conserved residues within the SF-1/NR5A1-binding element. In silico modelling and in vitro assays demonstrate the enhancer properties of the NR5A1 motif. Deletion of this hemizygous element by genome-editing, in a novel in vitro cellular model recapitulating human Sertoli cell formation, resulted in a significant reduction in expression of SRY. Therefore, human NR5A1 acts as a regulatory switch between testis and ovary development by upregulating SRY expression, a role that may predate the eutherian radiation. We show that disruption of an enhancer can phenocopy variants in the coding regions of SRY that cause human testis dysgenesis. Since disease causing variants in enhancers are currently rare, the regulation of gene expression in testis-determination offers a paradigm to define enhancer activity in a key developmental process.
Topics: Animals; Female; Humans; Male; Cell Line; Gonadal Dysgenesis; Mammals; Regulatory Sequences, Nucleic Acid; Sertoli Cells; Sex-Determining Region Y Protein; Steroidogenic Factor 1; Testis
PubMed: 38555298
DOI: 10.1038/s41467-024-47162-2 -
Cell Reports Apr 2024Motor neuron (MN) demise is a hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Post-transcriptional gene regulation can...
Motor neuron (MN) demise is a hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Post-transcriptional gene regulation can control RNA's fate, and defects in RNA processing are critical determinants of MN degeneration. N-methyladenosine (mA) is a post-transcriptional RNA modification that controls diverse aspects of RNA metabolism. To assess the mA requirement in MNs, we depleted the mA methyltransferase-like 3 (METTL3) in cells and mice. METTL3 depletion in embryonic stem cell-derived MNs has profound and selective effects on survival and neurite outgrowth. Mice with cholinergic neuron-specific METTL3 depletion display a progressive decline in motor behavior, accompanied by MN loss and muscle denervation, culminating in paralysis and death. Reader proteins convey mA effects, and their silencing phenocopies METTL3 depletion. Among the mA targets, we identified transactive response DNA-binding protein 43 (TDP-43) and discovered that its expression is under epitranscriptomic control. Thus, impaired mA signaling disrupts MN homeostasis and triggers neurodegeneration conceivably through TDP-43 deregulation.
Topics: Animals; Humans; Mice; Adenosine; Amyotrophic Lateral Sclerosis; Cholinergic Neurons; DNA-Binding Proteins; Methyltransferases; Motor Neurons; Neuromuscular Diseases
PubMed: 38554281
DOI: 10.1016/j.celrep.2024.113999 -
Cell Reports Apr 2024The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and...
The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (Pten) retinas. Wnt signaling molecules are also enriched in Pten retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.
Topics: Animals; PTEN Phosphohydrolase; Retina; Wnt Signaling Pathway; Cell Adhesion; Mice; Endocytosis; Amacrine Cells; Mice, Knockout; Protein Transport; Wnt Proteins; Cell Adhesion Molecules
PubMed: 38551961
DOI: 10.1016/j.celrep.2024.114005 -
Biomedicines Mar 2024Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy. It follows an autosomal dominant inheritance pattern in most cases, with incomplete... (Review)
Review
Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy. It follows an autosomal dominant inheritance pattern in most cases, with incomplete penetrance and heterogeneity. It is familial in 60% of cases and most of these are caused by pathogenic variants in the core sarcomeric genes (, , , , , , , ). Genetic testing using targeted disease-specific panels that utilize next-generation sequencing (NGS) and include sarcomeric genes with the strongest evidence of association and syndrome-associated genes is highly recommended for every HCM patient to confirm the diagnosis, identify the molecular etiology, and guide screening and management. The yield of genetic testing for a disease-causing variant is 30% in sporadic cases and up to 60% in familial cases and in younger patients with typical asymmetrical septal hypertrophy. Genetic testing remains challenging in the interpretation of results and classification of variants. Therefore, in 2015 the American College of Medical Genetics and Genomics (ACMG) established guidelines to classify and interpret the variants with an emphasis on the necessity of periodic reassessment of variant classification as genetic knowledge rapidly expands. The current guidelines recommend focused cascade genetic testing regardless of age in phenotype-negative first-degree relatives if a variant with decisive evidence of pathogenicity has been identified in the proband. Genetic test results in family members guide longitudinal clinical surveillance. At present, there is emerging evidence for genetic test application in risk stratification and management but its implementation into clinical practice needs further study. Promising fields such as gene therapy and implementation of artificial intelligence in the diagnosis of HCM are emerging and paving the way for more effective screening and management, but many challenges and obstacles need to be overcome before establishing the practical implications of these new methods.
PubMed: 38540296
DOI: 10.3390/biomedicines12030682