To define serum proteomic features and identify candidate biomarkers that distinguish double seronegative neuromyelitis optica spectrum disorder (DN-NMOSD) from AQP4-IgG positive neuromyelitis optica spectrum disorder (AQP4-IgG+ NMOSD) and relapsing–remitting multiple sclerosis (RRMS). 

Diagnostic challenges persist particularly in DN-NMOSD. This clinically heterogeneous subgroup lacks validated biomarkers and its pathogenesis is unclear, highlighting the need for serum-based biomarkers to support diagnosis and guide treatment.

Clinico-radiological data and serum samples were collected from age-matched participants with double seronegative (AQP4-IgG and MOG-IgG negative) NMOSD (n=17), AQP4-IgG+ NMOSD (n=18), RRMS (n=28), and healthy controls (n=29). Mass spectrometry-based proteomic analysis was performed. Differentially expressed proteins from group comparisons were analysed for pathway enrichment using the Reactome database.

Both DN-NMOSD (16/17, 94%) and AQP4-IgG+ NMOSD (17/18, 94%) showed marked female predominance. Age at onset differed across groups (p=0.005). Nano-LC-MS/MS identified a total of 461 serum proteins. Pathway enrichment analysis indicated that innate immune system and neutrophil degranulation were the main dysregulated pathways seperating both DN-NMOSD and AQP4-IgG+ NMOSD from MS and healthy controls. AQP4-IgG+ NMOSD showed additional enrichment of platelet degranulation and hemostasis, while the complement cascade was prominently enriched in DN-NMOSD vs. RRMS.

Our findings indicate that both DN-NMOSD and AQP4-IgG+ NMOSD share dysregulated pathways, especially innate immunity, neutrophil-related responses, and the complement system, while the dysregulation patterns suggest different mechanisms. These results may improve understanding of DN-NMOSD and indicate potential serum biomarkers. Further validation of our findings in larger cohorts is warranted.