RESUMO
Defects in mRNA export from the nucleus have been linked to various neurodegenerative disorders. We report mutations in the gene MCM3AP, encoding the germinal center associated nuclear protein (GANP), in nine affected individuals from five unrelated families. The variants were associated with severe childhood onset primarily axonal (four families) or demyelinating (one family) Charcot-Marie-Tooth neuropathy. Mild to moderate intellectual disability was present in seven of nine affected individuals. The affected individuals were either compound heterozygous or homozygous for different MCM3AP variants, which were predicted to cause depletion of GANP or affect conserved amino acids with likely importance for its function. Accordingly, fibroblasts of affected individuals from one family demonstrated severe depletion of GANP. GANP has been described to function as an mRNA export factor, and to suppress TDP-43-mediated motor neuron degeneration in flies. Thus our results suggest defective mRNA export from nucleus as a potential pathogenic mechanism of axonal degeneration in these patients. The identification of MCM3AP variants in affected individuals from multiple centres establishes it as a disease gene for childhood-onset recessively inherited Charcot-Marie-Tooth neuropathy with intellectual disability.
Assuntos
Acetiltransferases/genética , Doença de Charcot-Marie-Tooth/genética , Predisposição Genética para Doença/genética , Deficiência Intelectual/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Acetiltransferases/metabolismo , Adolescente , Adulto , Células Cultivadas , Doença de Charcot-Marie-Tooth/complicações , Criança , Pré-Escolar , Feminino , Fibroblastos/metabolismo , Humanos , Deficiência Intelectual/complicações , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Mutação , Linhagem , Adulto JovemRESUMO
Efficient intracellular delivery is crucial for biotherapeutics, such as proteins, oligonucleotides, and CRISPR/Cas9 gene-editing systems, to achieve their efficacy. Despite the great efforts of developing new intracellular delivery carriers, the lack of straightforward methods for intracellular delivery quantification limits further development in this area. Herein, we designed a simple and versatile bioorthogonal luminescent reaction (BioLure assay) to analyze intracellular delivery. Our results suggest that BioLure can be used to estimate the amount of intracellularly delivered molecules after electroporation, and the estimation by BioLure is in good correlation with the results from complementary methods. Furthermore, we used BioLure assay to correlate the intracellularly-delivered RNase A amount with its tumoricidal activity. Overall, BioLure is a versatile tool for understanding the intracellular delivery process on live cells, and establishing the link between the cytosolic concentration of intracellularly-delivered biotherapeutics and their therapeutic efficacy.