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

Background 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. Methods 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. Results 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. Conclusions 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.

Initiation and progression of α-synuclein pathology in Parkinson’s disease

“…Currently there is no PET ligand available for α-synuclein aggregates but the resolution of their structure by cryo-electron microscopy may facilitate this urgently needed biomarker. The concept of self-templating assembly of α-synuclein has led to the adaptation of assays previously used for prions (RT-QuIC or PMCA) to measure α-synuclein seeding when incubated with CSF [ 118 , 119 ] and more recently peripheral tissue homogenates [ 120 ]. α-Synuclein seeding assays in the CSF showed sensitivities and specificities for PD diagnosis at 80–90% with a high degree of overlap between the two assay types [ 121 ].…”

The Future of Seed Amplification Assays and Clinical Trials

“…De Luca and colleagues reported the first application of α-synuclein RT-QuIC to a more accessible sample than CSF: nasal brushings, which seeded positive responses in 10/18 PD samples, 9/11 MSA samples, and only 1/6 CBD samples and 2/12 PSP samples (De Luca et al, 2019 ). Subsequent work established reproducibility of a modified version of the same assay between laboratories with 96% inter-rater agreement of results with good sensitivity for MSA with predominant parkinsonism (18/20 results positive, giving a sensitivity of 90%) but a somewhat lower sensitivity for PD (9/13 results positive, giving a sensitivity of 69%) and interestingly only 1/20 positive results in MSA with predominant cerebellar ataxia, which could potentially be attributable to differences in α-synuclein strain, such as tissue tropism (Bargar et al, 2021a ). Only 1/22 healthy controls seeded a positive reaction indicating a specificity of 95% (Bargar et al, 2021a ).…”

“…Subsequent work established reproducibility of a modified version of the same assay between laboratories with 96% inter-rater agreement of results with good sensitivity for MSA with predominant parkinsonism (18/20 results positive, giving a sensitivity of 90%) but a somewhat lower sensitivity for PD (9/13 results positive, giving a sensitivity of 69%) and interestingly only 1/20 positive results in MSA with predominant cerebellar ataxia, which could potentially be attributable to differences in α-synuclein strain, such as tissue tropism (Bargar et al, 2021a ). Only 1/22 healthy controls seeded a positive reaction indicating a specificity of 95% (Bargar et al, 2021a ). Stefani et al ( 2021 ) assessed α-synuclein RT-QuIC of nasal brushings as a test for prodromal α-synucleinopathy and reported a sensitivity of 44.4% for iRBD, unfavourable when compared to CSF studies (Iranzo et al, 2021 ) but also reported a sensitivity of 46.3% for Parkinson’s disease (Stefani et al, 2021 ), which is lower than reported in previous studies on nasal brushings (De Luca et al, 2019 ; Bargar et al, 2021a ), although specificity was high (89.8%).…”

The Alpha-Synuclein RT-QuIC Products Generated by the Olfactory Mucosa of Patients with Parkinson’s Disease and Multiple System Atrophy Induce Inflammatory Responses in SH-SY5Y Cells

“…Thanks to the development of the Seed Amplification Assays (SAAs), traces of αSyn D were found in peripheral tissues of patients with α-synucleinopathies, including cerebrospinal fluid (CSF) [ 20 , 21 , 22 , 23 , 24 , 25 ], skin [ 26 , 27 , 28 , 29 ], submandibular gland [ 26 , 30 , 31 ] and olfactory mucosa [ 25 , 32 , 33 , 34 ]. Recently, we have optimized the Real-Time Quaking-Induced Conversion (RT-QuIC) assay for detecting αSyn D in OM samples of patients with PD and MSA [ 33 , 34 ].…”

“…Thanks to the development of the Seed Amplification Assays (SAAs), traces of αSyn D were found in peripheral tissues of patients with α-synucleinopathies, including cerebrospinal fluid (CSF) [ 20 , 21 , 22 , 23 , 24 , 25 ], skin [ 26 , 27 , 28 , 29 ], submandibular gland [ 26 , 30 , 31 ] and olfactory mucosa [ 25 , 32 , 33 , 34 ]. Recently, we have optimized the Real-Time Quaking-Induced Conversion (RT-QuIC) assay for detecting αSyn D in OM samples of patients with PD and MSA [ 33 , 34 ]. The αSyn_RT-QuIC exploited the ability of αSyn D (also referred to as seed) present in OM tissues to template the conformational conversion of recombinant αSyn (rec-αSyn, used as RT-QuIC reaction substrate) which then aggregated to form αSyn amyloid fibrils [ 20 , 24 , 26 ].…”