Comprehensive High-Depth Proteomic Analysis of Plasma Extracellular Vesicle-Containing Preparations in CDKL5 Deficiency Disorder.

Background/

Objectives

CDKL5 deficiency disorder (CDD) is a rare X-linked developmental and epileptic encephalopathy characterized by early onset refractory epilepsy and severe neurodevelopmental impairment with autistic features. Despite advances in genetic diagnosis, objective biomarkers reflecting disease mechanisms remain limited. Extracellular vesicles (EVs) circulating in the blood may contain disease-related proteins derived from the central nervous system. This study aimed to characterize the plasma EV proteome in CDD in a hypothesis-generating exploratory framework and identify the candidate molecular pathways associated with this disorder.

Methods

Plasma samples from seven patients with genetically confirmed CDD and seven neurotypical developmental controls were analyzed. Extracellular vesicle-containing preparations (EVs-cp) were isolated via immunoprecipitation using antibodies against CD9, CD63, and CD81. Proteomic profiling was performed using data-independent mass spectrometry. Differentially expressed proteins were identified using Welch's t-test with a false discovery rate correction. Functional enrichment, protein interaction network, and correlation analyses were performed using CDKL5 Clinical Severity Assessment (CCSA) scores.

Results

In total, 5617 proteins were identified, of which 3510 were used for quantitative analysis. Compared to the controls, 2108 proteins were upregulated and 158 were downregulated in the CDD samples. Enrichment analysis revealed alterations in vesicle-mediated transport, cytoskeletal organization, and immune-related pathways. Several proteins were also correlated with clinical severity scores.

Conclusions

Plasma EV proteomics revealed molecular alterations associated with CDD and provided a potential approach for biomarker discovery and mechanistic investigation.