Abstract

Detection techniques for pathological α-synuclein (αSynP), such as Real-time quaking-induced conversion（RT-QuIC）, have been applied to diagnose neurodegenerative diseases. However, limitations remain in genotyping, sensitivity for peripheral biomarkers, and standardized detection systems. This study systematically evaluates the diagnostic efficacy of the novel αSyn-Quiescent seed amplification assay (QSAA) in skin, serum, and lymphocytes and explores its association with genotypes and disease progression.

We analyzed total αSyn and αSyn oligomer levels in skin samples (n=400) and serum samples (n=200) from a cohort including GBA/LRRK2-mutant Parkinson’s disease (PD), sporadic PD, MSA-P/MSA-C, dementia with Lewy bodies (DLB), and healthy controls. Compared to traditional αSyn-RT-QuIC, QSAA demonstrated higher sensitivity (92.1% vs. 78.6%, p<0.001) and genotype specificity (seeding dose SD₅₀ = 10⁻⁴.² in GBA-mutant groups vs. 10⁻³.⁷ in LRRK2 groups, p=0.008). Cryo-electron microscopy revealed that αSyn fibrils from GBA-mutant patients exhibited a left-handed superhelical conformation (diameter: 8.2±0.3 nm), whereas LRRK2 groups showed a straight filamentous morphology (diameter: 6.5±0.2 nm). αSyn-QSAA seed concentration significantly correlated with motor symptoms (UPDRS-III: r=0.68, p<0.001).

For lymphocytes, magnetic bead-sorted CD4⁺T/CD8⁺T/CD68⁺ monocytes/CD56⁺NK cells pretreated at pH 6.8 and 60°C for 30 minutes with one mM S₂O₄²⁻ achieved a QSAA positivity rate of 91.3% (vs. 47.2% in untreated controls, p<0.001), with 82.4% detection consistency in samples stored >10 months.

These findings suggest that:

1. αSyn-QSAA enables precise genotype-conformation-clinical phenotype correlations in cutaneous pathology, offering a new dimension for individualized diagnosis;
2. The serum and lymphocyte QSAA system overcomes limitations in peripheral biomarker detection, improving sensitivity 3-5-fold;
3. Conformational differences revealed by cryo-EM may provide a structural biological basis for disease subtyping.

Keywords: α-Synuclein; QSAA; genotype-conformation correlation; peripheral biomarkers

Significance: This study advances αSyn detection from cerebrospinal fluid to minimally invasive peripheral samples, facilitating early diagnosis and therapeutic monitoring