Abstract
BackgroundHypermethylation of the fragile X mental retardation 1 gene FMR1 results in decreased expression of FMR1 protein FMRP, which is the underlying cause of Fragile X syndrome – an incurable neurological disorder characterized by mental retardation, anxiety, epileptic episodes and autism. Disease-modifying therapies for Fragile X syndrome are thus aimed at treatments that increase the FMRP expression levels in the brain. We describe the development and characterization of two assays for simple and quantitative detection of FMRP protein.MethodAntibodies coupled to fluorophores that can be employed for time-resolved Förster’s resonance energy transfer were used for the development of homogeneous, one-step immunodetection. Purified recombinant human FMRP and patient cells were used as control samples for assay development.ResultsThe assays require small sample amounts, display high stability and reproducibility and can be used to quantify endogenous FMRP in human fibroblasts and peripheral blood mononuclear cells. Application of the assays to FXS patient cells showed that the methods can be used both for the characterization of clinical FXS patient samples as well as primary readouts in drug-discovery screens aimed at increasing endogenous FMRP levels in human cells.ConclusionThis study provides novel quantitative detection methods for FMRP in FXS patient cells. Importantly, due to the simplicity of the assay protocol, the method is suited to be used in screening applications to identify compounds or genetic interventions that result in increased FMRP levels in human cells.
Highlights
Hypermethylation of the fragile X mental retardation 1 gene Fragile X mental retardation 1 (FMR1) results in decreased expression of FMR1 protein FMRP, which is the underlying cause of Fragile X syndrome – an incurable neurological disorder characterized by mental retardation, anxiety, epileptic episodes and autism
Application of the assays to Fragile X syndrome (FXS) patient cells showed that the methods can be used both for the characterization of clinical FXS patient samples as well as primary readouts in drug-discovery screens aimed at increasing endogenous FMRP levels in human cells
Due to the simplicity of the assay protocol, the method is suited to be used in screening applications to identify compounds or genetic interventions that result in increased FMRP levels in human cells
Summary
Hypermethylation of the fragile X mental retardation 1 gene FMR1 results in decreased expression of FMR1 protein FMRP, which is the underlying cause of Fragile X syndrome – an incurable neurological disorder characterized by mental retardation, anxiety, epileptic episodes and autism. Animal experiments with FMR1 knockout mice with a 50% reduction in mGluR5 expression showed a rescue of behavioral deficits [9] and early findings in clinical trials suggest that behavioral deficits can be reduced by treatment with selective mGluR5 inhibitors in a subset of patients with full methylation of the FMR1 promoter region [10] Despite those encouraging results, mGluR5-directed approaches remain symptomatic treatments that do not target the underlying disease cause – decreased postsynaptic levels of FMRP in the central nervous system. The further identification of disease-modifying therapies that can increase the postsynaptic FMRP levels will rely on the development of quantitative and screening compatible readouts
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