Zebrafish models of Okur-Chung Neurodevelopmental Syndrome: Exploring Genotype-Phenotype Relationships

Description

Okur-Chung Neurodevelopmental syndrome (OCNDS) is a rare disorder characterized by hypotonia, developmental delay, dysmorphic features, and more. It is caused by pathogenic variants on CSNK2A1, the α subunit of protein kinase CK2. CK2 is considered a master regulator involved in…

Okur-Chung Neurodevelopmental syndrome (OCNDS) is a rare disorder characterized by hypotonia, developmental delay, dysmorphic features, and more. It is caused by pathogenic variants on CSNK2A1, the α subunit of protein kinase CK2. CK2 is considered a master regulator involved in many cell functions from cell differentiation and proliferation to apoptosis. Here, we create a potential zebrafish model of OCNDS with CK2 inhibition and characterize fibroblast cells with, K198R, D156E, and R47G variants of CSNK2A1. RNAseq results display a wide range of effects notably in the Myosin Protein superfamily, Insulin-like Growth Factor family, and in proteins related to mitochondrial function and cell metabolism. Factors in cell growth and metabolism across the nervous system and neuromuscular interactions appear to be most affected with similarities in markers to oncogenic states in some cases.

Date Created
2023-05
Agent

mTOR Signaling Dynamics in Two Families with Tuberous Sclerosis Complex Exhibiting Intrafamilial Phenotypic Variability

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Description

Tuberous sclerosis complex (TSC) is a rare genetic disease caused by heterozygous dominant mutations in the TSC1 or TSC2 genes that affects 1/6000 newborns (Curatalo et al., 2002; de Vries & Howe, 2007). TSC has a variety of clinical manifestations…

Tuberous sclerosis complex (TSC) is a rare genetic disease caused by heterozygous dominant mutations in the TSC1 or TSC2 genes that affects 1/6000 newborns (Curatalo et al., 2002; de Vries & Howe, 2007). TSC has a variety of clinical manifestations ranging from hypomelanotic macules to neurological conditions such as epilepsy (Neuman & Henske, 2011; de Vries & Howe, 2007). In cases where the TSC mutations are inherited from parent to offspring (familial TSC)- the child can still exhibit more severe symptoms despite having the same TSC mutation as the parent, a phenomenon known as intrafamilial phenotypic variability (IPV) (Curatalo et al, 2002). We hypothesize that the variants in genes of the mTOR signaling pathway (genetic modifiers) may enhance or suppress mTOR pathway activity, resulting in IPV. Patient derived primary fibroblasts cell lines from two families exhibiting IPV were studied as well as an unrelated control cell line. We identified variants in IRS1, FZD5, and PIK32CG genes from children with severe phenotype in one family and variants in PIK3R3, TNFRSF19, and EIF4G1 in a severe child in another pathway. We explored the functional impact of these genes on mTOR pathway activity.

Date Created
2022-05
Agent

Development of a Novel Zebrafish Model for Dynamin-1 Epileptic Encephalopathy

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Description
Epileptic encephalopathies (EE) are genetic or environmentally-caused conditions that cause “catastrophic” damage or degradation to the sensory, cognitive, and behavioral centers of the brain. Whole-exome sequencing identified de novo heterozygous missense mutations within the DNM1 gene of five pediatric patients…

Epileptic encephalopathies (EE) are genetic or environmentally-caused conditions that cause “catastrophic” damage or degradation to the sensory, cognitive, and behavioral centers of the brain. Whole-exome sequencing identified de novo heterozygous missense mutations within the DNM1 gene of five pediatric patients with epileptic encephalopathies. DNM1 encodes for the dynamin-1 protein which is involved in endocytosis and synaptic recycling, and it is a member of dynamin GTPase. The zebrafish, an alternative model system for drug discovery, was utilized to develop a novel model for dynamin-1 epileptic encephalopathy through a small molecule inhibitor. The model system mimicked human epilepsy caused by DNM1 mutations and identified potential biochemical pathways involved in the production of this phenotype. The use of microinjections of mutated DNM1 verified phenotypes and was utilized to determine safe and effective antiepileptic drugs (AEDs) for treatment of this specific EE. This zebrafish dynamin-1 epileptic encephalopathy model has potential uses for drug discovery and investigation of this rare childhood disorder.
Date Created
2019-05
Agent

Characterization of mTOR Pathway and Reduced Neuronal Size Phenotype in Rett Syndrome Model

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Description
Rett syndrome is a genetically based, X-linked neurodevelopmental disorder that affects 1 in 10,000 live female births. Approximately 95-97% of Rett syndrome cases are attributed to a mutation in the MECP2 gene. In the laboratory setting, key neuropathological phenotypes of…

Rett syndrome is a genetically based, X-linked neurodevelopmental disorder that affects 1 in 10,000 live female births. Approximately 95-97% of Rett syndrome cases are attributed to a mutation in the MECP2 gene. In the laboratory setting, key neuropathological phenotypes of Rett syndrome include small neuronal soma and nuclear size, increased cell packing density, and abnormal dendritic branching. Our lab previously created and characterized the A140V mouse model of atypical Rett syndrome in which the males are viable. Hippocampal and cerebellar granule neurons in A140V male mice have reduced soma and nuclear size compared to wild type. We also found that components of the mTOR pathway including rictor, 4E-BP-1, and mTOR, were reduced in A140V mutant mice. Quantitative PCR analysis also showed reduced IGFPB2 expression in A140V mice along with an upward trend in AKT levels that did not meet statistical significance. The objective of this study is i) to characterize the down regulation of AKT-mTOR pathway, and ii) to examine the effect of a genetic strategy to rescue mTOR pathway deficiencies in Mecp2 mutant mouse model. Genetic rescue of the mTOR pathway downregulation was done by crossing heterozygous female A140V mice with heterozygous male Tsc2 mice. Quantitative PCR analysis of A140V_Tsc2 RNA expression supported genetic rescue of mTOR pathway components, however, more testing is needed to fully characterize the rescue effect. Western blot analysis also showed reduction in phosphorylated AKT in Mecp2 A140V and T158A mutant mice, however, more testing is still needed to characterize the mTOR pathway in A140V_Tsc2 mice. Finally, other methods, such as a pharmacological approach, or transfection to increase mTOR pathway activity in cell lines, will be tested to determine if rescue of mTOR pathway activity ameliorate the Rett syndrome phenotype.
Date Created
2016-12
Agent

Going Beyond the Diagnosis in Rare Childhood Disorders: Development of Personalized Treatment Methods for Mitochondrial Disease in Patients with a Nonsynonymous MTFMT Mutation

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Description
A single splice site mutation in the mitochondrial methionyl-tRNA formyltransferase (MTFMT) gene is described in three patients with mitochondrial disease from two unrelated families. Nuclear-encoded MTFMT localized to the mitochondria is responsible for the formylation of Met-tRNAMet necessary for the…

A single splice site mutation in the mitochondrial methionyl-tRNA formyltransferase (MTFMT) gene is described in three patients with mitochondrial disease from two unrelated families. Nuclear-encoded MTFMT localized to the mitochondria is responsible for the formylation of Met-tRNAMet necessary for the initiation of translation in the mitochondria. This mutation has been associated with mitochondrial disease (oxidative phosphorylation deficiencies due to a decreased expression of MTFMT), Leigh syndrome, and developmental delay. However, there is significant phenotypic variation between patients, which is not uncommon in mitochondrial disease. Though the variation was not clearly elucidated through analysis of gene expression, this data supported two potential gene modifiers as well as proposed an alternative energy producing pathway in the cell—glutamine metabolism. This nonsynonymous mutation at site c.626C>T generates a splicing suppressor in the coding region on exon 4 resulting exon skipping in almost all transcripts in homozygotes during splicing. It is hypothesized that antisense oligotherapy will be effective in rescuing this mutation by inhibiting the splice silencer and promoting exon inclusion as well as an increased expression of MTFMT protein in affected patients. Patient fibroblast cells were treated with MTFMT Oligo 3, which was shown to be promising in previous experiments. Real-Time qPCR was used to measure mRNA expression showing a significant up-regulation of wild-type MTFMT with treatment. In order to test whether this therapy increases mitochondrial function as well, three mitochondrial functional assays measuring superoxide species in the mitochondria, the mitochondrial membrane potential, and calcium uptake in the mitochondria were tested for optimization of results. Success has been shown in the measurement of superoxide species and mitochondrial membrane potential in patient cells without treatment. Oligotherapy will hopefully be considered as a viable therapeutic option in the future as further testing is conducted and perfected.
Date Created
2016-05
Agent
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