PMCA optimization for the detection of prions in blood and urine of patients with fatal familial insomnia

Aims: Fatal Familial Insomnia (FFI) is a rare genetic prion disease caused by a point mutation at codon 178 of the PRNP gene encoding the cellular prion protein (PrPC), resulting in the substitution of asparagine with aspartic acid (D178N). Genetic testing can identify FFI individuals years before the clinical phase, however, it is necessary to identify biomarkers useful to treat FFI before its onset. Two sensitive techniques named protein misfolding cyclic amplification (PMCA) and Real-Time Quaking-Induced Conversion (RT-QuIC) allows the detection of minute amount of prions (PrPSc) in peripheral tissues of patients with prion diseases, including the olfactory mucosa of FFI individuals. In this study, we developed a new PMCA protocol with the aim of detecting prions in both urine and blood of FFI mutation carriers. Materials: The brain homogenates (BH) of two FFI cases, with the MM and MV polymorphisms at PRNP codon 129, were serially diluted (from 10-4 to 10-12) in PBS 1X, urine or plasma of a healthy control subject and subjected to PMCA. The BH of a patient with Alzheimer’s disease (AD) was used as negative control. Longitudinally collected urine samples from D178N mutation carriers (n=18) and negative controls (n=19) were tested by PMCA. Moreover, we analyzed one plasma sample collected from a patient at an advanced stage of FFI along with plasma samples (n=2) from negative controls. Methods: Before PMCA analyses, samples of urine and plasma were subjected to an high-speed centrifugation treatment. PMCA was performed using the BH of transgenic mice expressing bank vole PrPC (TgVole 1X) as a reaction substrate. The amplified products were analyzed by Western blot following proteinase K digestion. Results: All the dilutions of FFI prions prepared in PBS, urine or plasma were successfully amplified within two rounds of PMCA, achieving detection up to the 10-12 dilution. The AD-BH diluted in all matrices remained always negative. Notably, 5 out of the 18 urine samples from FFI mutation carriers tested positive, one from a patient in the clinical phase and four from asymptomatic carriers. All the urine samples collected from the negative controls resulted negative. Additionally, a plasma sample from a FFI mutation carrier in an advanced disease stage yielded a positive result too. Discussion: We successfully amplified brain-derived FFI prions spiked in both urine and plasma samples. The possibility to detect prions in the urine of an asymptomatic carrier of FFI mutation suggests that the PMCA can be used to recognize early biomarkers of disease. Further analyses on these urine samples will be performed to assess other clinical parameters, including kidney function, to better understand their positivity. Conclusions: More samples of urine and plasma will be analyzed to evaluate the reliability and robustness of our PMCA protocol results. Identifying a reliable biomarker useful to predict the onset in mutation carriers and monitor disease progression will allow patients to be enrolled at the correct stage of disease with the most appropriate treatment.