Diagnostic value of cerebrospinal fluid alpha-synuclein seed quantification in synucleinopathies

Abstract

Several studies have confirmed the α-synuclein real-time quaking-induced conversion (RT-QuIC) assay to have high sensitivity and specificity for Parkinson's disease. However, whether the assay can be used as a robust, quantitative measure to monitor disease progression, stratify different synucleinopathies and predict disease conversion in patients with idiopathic REM sleep behaviour disorder remains undetermined. The aim of this study was to assess the diagnostic value of CSF α-synuclein RT-QuIC quantitative parameters in regard to disease progression, stratification and conversion in synucleinopathies. We performed α-synuclein RT-QuIC in the CSF samples from 74 Parkinson's disease, 24 multiple system atrophy and 45 idiopathic REM sleep behaviour disorder patients alongside 55 healthy controls, analysing quantitative assay parameters in relation to clinical data. α-Synuclein RT-QuIC showed 89% sensitivity and 96% specificity for Parkinson's disease. There was no correlation between RT-QuIC quantitative parameters and Parkinson's disease clinical scores (e.g. Unified Parkinson's Disease Rating Scale motor), but RT-QuIC positivity and some quantitative parameters (e.g. Vmax) differed across the different phenotype clusters. RT-QuIC parameters also added value alongside standard clinical data in diagnosing Parkinson's disease. The sensitivity in multiple system atrophy was 75%, and CSF samples showed longer T50 and lower Vmax compared to Parkinson's disease. All RT-QuIC parameters correlated with worse clinical progression of multiple system atrophy (e.g. change in Unified Multiple System Atrophy Rating Scale). The overall sensitivity in idiopathic REM sleep behaviour disorder was 64%. In three of the four longitudinally followed idiopathic REM sleep behaviour disorder cohorts, we found around 90% sensitivity, but in one sample (DeNoPa) diagnosing idiopathic REM sleep behaviour disorder earlier from the community cases, this was much lower at 39%. During follow-up, 14 of 45 (31%) idiopathic REM sleep behaviour disorder patients converted to synucleinopathy with 9/14 (64%) of convertors showing baseline RT-QuIC positivity. In summary, our results showed that α-synuclein RT-QuIC adds value in diagnosing Parkinson's disease and may provide a way to distinguish variations within Parkinson's disease phenotype. However, the quantitative parameters did not correlate with disease severity in Parkinson's disease. The assay distinguished multiple system atrophy patients from Parkinson's disease patients and in contrast to Parkinson's disease, the quantitative parameters correlated with disease progression of multiple system atrophy. Our results also provided further evidence for α-synuclein RT-QuIC having potential as an early biomarker detecting synucleinopathy in idiopathic REM sleep behaviour disorder patients prior to conversion. Further analysis of longitudinally followed idiopathic REM sleep behaviour disorder patients is needed to better understand the relationship between α-synuclein RT-QuIC signature and the progression from prodromal to different synucleinopathies.

Keywords: biomarker; prodromal; seeding; stratification; α-synuclein.

Figure 1

RT-QuIC parameters and other CSF biomarkers in different patient cohorts. (A) The lag phase (T_lag) = time each positive reaction exceeds the threshold (RFU > 5 SD); (B) the maximum fluorescence value (F_max) = highest mean fluorescence value achieved; (C) T₅₀ = time latency to reach 50% of the F_max; (D) the maximum slope (V_max) = maximum increase per unit of time; and (E) the area under curve (AUC). Bars show the average ± SD. (F) Levels of total αSyn and (G) phospho-tau in the Discovery cohort. Bars show the average ± SD. (H) No correlation detected between total αSyn levels and T₅₀ values in the Discovery cohort including all Parkinson’s disease, iRBD and heathy controls (same cases showed in G). (I) Kinetic curves of αSyn seeding activity measured by RT-QuIC in Parkinson’s disease (red line, n = 74) and MSA (purple line, n = 24) clinical cases. Each curve depicts the average percentage of ThT fluorescence from duplicate reactions of each group. Each dot depicts average RFU value at 10-h interval. Vertical bars represent the mean ± SD.

Figure 2

RT-QuIC parameters (A–E) and ELISA biomarkers (F–I) in the Discovery cohort against validated Parkinson’s disease clinical clusters. Data are mean ± SD (range) unless otherwise stated. No other changes were detected except (D) V_max was different across the clusters (P = 0.02), with cluster 1 having higher V_max than clusters 2 (P = 0.02) and 3 (P = 0.03) and cluster 4 having higher V_max than clusters 2 and 3 (P = 0.05).

Figure 3

Kaplan–Meier analysis of iRBD patients showing rates of neurological disease-free survival according to time from baseline lumbar puncture. No evidence was seen that RT-QuIC positive iRBD patients had a higher risk of conversion than RT-QuIC negative counterparts.