Preliminary Validation of a Novel WDR45 Mutation in a Male Patient with Lennox-Gastaut Syndrome
Abstract number :
V.095
Submission category :
12. Genetics / 12A. Human Studies
Year :
2021
Submission ID :
1826032
Source :
www.aesnet.org
Presentation date :
12/9/2021 12:00:00 PM
Published date :
Nov 22, 2021, 06:51 AM
Authors :
Jiaqi Han, Master of Medical Science - Xuanwu Hospital, Capital Medical University; Yuchen Wang - Beijing University of Aeronautics and Astronautics; Yicong lin - Xuanwu Hospital, Capital Medical University; Wei Shi - Beijing University of Aeronautics and Astronautics; Yuping Wang - Xuanwu Hospital, Capital Medical University
Rationale: This study intends to validate the pathogenicity of novel WDR45 p.Ser247Ile mutation in a typical Lennox-Gastaut syndrome patient, thus establishing the relationship between autophagy and epileptic encephalopathies such as Lennox-Gastaut syndrome.
Methods: Recombinant expression plasmids of WDR45 gene wild type and mutant type were constructed respectively using human WDR45 cDNA as a template. The expression vectors were then transfected into HEK293T cells and primary hippocampal neurons cultured in vitro separately. Western blotting technique was used to evaluate the expression of LC3-Ⅰ and LC3-Ⅱ, while double immunofluorescence labeling method (GFP-RFP-LC3) was used to observe the formation of autophagy structures in transfected HEK293T cells. The number of red fluorescent spots under microscope representing the formation of autophagosome was counted. Comparations between wild and mutant HEK293T cells were conducted to assess whether autophagy flux was blocked. Morphological changes in transfected neurons were also observed under confocal microscope to evaluate the effects of mutation on growth of neurons.
Results: The LC3 expression in mutant HEK293T cells was significantly higher than control, indicating the mutation caused the accumulation of LC3 positive vesicles. Similarly, the decrease of red fluorescent spots was observed in mutant carriers, representing reduced formation of autophagosome, which confirm that the WDR45 p.Ser247Ile mutation leads to obstruction of autophagy flux in non-neuronal cells. At the same time, reduced branching of neurons and aberrant dendrites were observed in mutant neurons, which may represent the mutation effects.
Conclusions: Patients with WDR45 gene mutations may present with Lennox-Gastaut syndrome. WDR45 p.Ser247Ile mutation can cause autophagy dysfunction in non-neuronal cells and disturb normal development of neurons.
Funding: Please list any funding that was received in support of this abstract.: Beijing Municipal Administration of Hospitals’ Youth Programme; National Natural Science Foundation of China; Beijing Key Clinical Speciality Excellence Project.
Genetics