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Discrimination of MSA-P and MSA-C by RT-QuIC analysis of olfactory mucosa: the first assessment of assay reproducibility between two specialized laboratories.

ABSTRACT: Detection of the pathological and disease-associated alpha-synuclein (αSynD) in the brain is required to formulate the definitive diagnosis of multiple system atrophy (MSA) and Parkinson's disease (PD). We recently showed that αSynD can be detected in the olfactory mucosa (OM) of MSA and PD patients. For this reason, we have performed the first interlaboratory study based on α-synuclein Real-Time Quaking-Induced Conversion (αSyn_RT-QuIC) analysis of OM samples collected from PD and MSA patients with the parkinsonian (MSA-P) and cerebellar (MSA-C) phenotypes. OM samples were prospectively collected from patients with a probable diagnosis of MSA-P (n = 20, mean disease duration 4.4 years), MSA-C (n = 10, mean disease duration 4 years), PD (n = 13, mean disease duration 8 years), and healthy control subjects (HS) (n = 11). Each sample was analyzed by αSyn_RT-QuIC in two independent specialized laboratories, one located in Italy (ITA-lab) and one located in the USA (USA-lab). Both laboratories have developed and used harmonized αSyn_RT-QuIC analytical procedures. Results were correlated with demographic and clinical data. The αSyn_RT-QuIC analysis reached a 96% interrater agreement of results (IAR) between laboratories (Kappa = 0.93, 95% CI 0.83-1.00). In particular, αSyn_RT-QuIC seeding activity was found in the OM of 9/13 patients with PD (sensitivity 69%, IAR 100%) and 18/20 patients with MSA-P (sensitivity 90%, IAR 100%). Interestingly, samples collected from patients with MSA-C did not induce αSyn_RT-QuIC seeding activity, except for one subject in USA-lab. Therefore, we found that MSA-P and MSA-C induced opposite effects. Regardless of disease diagnosis, the αSyn_RT-QuIC seeding activity correlated with some clinical parameters, including the rigidity and postural instability. Our study provides evidence that OM-αSynD may serve as a novel biomarker for accurate clinical diagnoses of PD, MSA-P, and MSA-C. Moreover, αSyn_RT-QuIC represents a reliable assay that can distinguish patients with MSA-P from those with MSA-C, and may lead to significant advancements in patients stratification and selection for emerging pharmacological treatments and clinical trials.

SUBMITTER: Bargar C

PROVIDER: S-EPMC8665327 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundParkinson's disease (PD) is a neurodegenerative disorder whose diagnosis is often challenging because symptoms may overlap with neurodegenerative parkinsonisms. PD is characterized by intraneuronal accumulation of abnormal ?-synuclein in brainstem while neurodegenerative parkinsonisms might be associated with accumulation of either ?-synuclein, as in the case of Multiple System Atrophy (MSA) or tau, as in the case of Corticobasal Degeneration (CBD) and Progressive Supranuclear Palsy (PSP), in other disease-specific brain regions. Definite diagnosis of all these diseases can be formulated only neuropathologically by detection and localization of ?-synuclein or tau aggregates in the brain. Compelling evidence suggests that trace-amount of these proteins can appear in peripheral tissues, including receptor neurons of the olfactory mucosa (OM).MethodsWe have set and standardized the experimental conditions to extend the ultrasensitive Real Time Quaking Induced Conversion (RT-QuIC) assay for OM analysis. In particular, by using human recombinant ?-synuclein as substrate of reaction, we have assessed the ability of OM collected from patients with clinical diagnoses of PD and MSA to induce ?-synuclein aggregation, and compared their seeding ability to that of OM samples collected from patients with clinical diagnoses of CBD and PSP.ResultsOur results showed that a significant percentage of MSA and PD samples induced ?-synuclein aggregation with high efficiency, but also few samples of patients with the clinical diagnosis of CBD and PSP caused the same effect. Notably, the final RT-QuIC aggregates obtained from MSA and PD samples owned peculiar biochemical and morphological features potentially enabling their discrimination.ConclusionsOur study provide the proof-of-concept that olfactory mucosa samples collected from patients with PD and MSA possess important seeding activities for ?-synuclein. Additional studies are required for (i) estimating sensitivity and specificity of the technique and for (ii) evaluating its application for the diagnosis of PD and neurodegenerative parkinsonisms. RT-QuIC analyses of OM and cerebrospinal fluid (CSF) can be combined with the aim of increasing the overall diagnostic accuracy of these diseases, especially in the early stages.

Project description:The clinical diagnosis of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), is challenging, especially at an early disease stage, due to the heterogeneous and often non-specific clinical manifestations. The discovery of reliable specific markers for synucleinopathies would consequently be of great aid to the diagnosis and management of these disorders. Real-Time Quaking-Induced Conversion (RT-QuIC) is an ultrasensitive technique that has been previously used to detect self-templating amyloidogenic proteins in the cerebrospinal fluid (CSF) and other biospecimens in prion disease and synucleinopathies. Using a wild-type recombinant ?-synuclein as a substrate, we applied RT-QuIC to a large cohort of 439 CSF samples from clinically well-characterized, or post-mortem verified patients with parkinsonism or dementia. Of significance, we also studied patients with isolated REM sleep behavior disorder (iRBD) (n?=?18) and pure autonomic failure (PAF) (n?=?28), representing clinical syndromes that are often caused by a synucleinopathy, and may precede the appearance of parkinsonism or cognitive decline. The results show that our RT-QuIC assay can accurately detect ?-synuclein seeding activity across the spectrum of Lewy Body (LB)-related disorders (LBD), including DLB, PD, iRBD, and PAF, with an overall sensitivity of 95.3%. In contrast, all but two patients with MSA showed no ?-synuclein seeding activity in the applied experimental setting. The analysis of the fluorescence response reflecting the amount of ?-synuclein seeds revealed no significant differences between the clinical syndromes associated with LB pathology. Finally, the assay demonstrated 98% specificity in a neuropathological cohort of 101 cases lacking LB pathology. In conclusion, ?-synuclein RT-QuIC provides an accurate marker of synucleinopathies linked to LB pathology and may have a pivotal role in the early discrimination and management of affected patients. The finding of no ?-synuclein seeding activity in MSA seems to support the current view that MSA and LBD are associated with different conformational strains of ?-synuclein.

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Discrimination of MSA-P and MSA-C by RT-QuIC analysis of olfactory mucosa: the first assessment of assay reproducibility between two specialized laboratories.

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