Diagnostic Accuracy of a Combined Analysis of Cerebrospinal Fluid t-PrP, t-tau, p-tau, and Aβ42 in the Differential Diagnosis of Creutzfeldt-Jakob Disease from Alzheimer’s Disease with Emphasis on Atypical Disease Variants

Abstract

According to recent studies, the determination of cerebrospinal fluid (CSF) total tau (t-tau)/phosphorylated tau (p-tau) ratio and total prion protein (t-PrP) levels significantly improves the accuracy of the diagnosis of Alzheimer’s disease (AD) in atypical cases with clinical or laboratory features mimicking Creutzfeldt-Jakob disease (CJD). However, this has neither been validated nor tested in series including atypical CJD variants. Furthermore, the added diagnostic value of amyloid-β (Aβ)42 remains unclear. To address these issues, we measured t-PrP, 14-3-3, t-tau, p-tau, and Aβ42 CSF levels in 45 typical and 44 atypical/rapidly progressive AD patients, 54 typical and 54 atypical CJD patients, and 33 controls. CJD patients showed significantly lower CSF t-PrP levels than controls and AD patients. Furthermore, atypical CJD was associated with lower t-PrP levels in comparison to typical CJD. T-tau, 14-3-3, or t-PrP alone yielded, respectively, 80.6, 63.0, and 73.0% sensitivity and 75.3, 92.1, and 75% specificity in distinguishing AD from CJD. On receiver operating characteristic (ROC) curve analyses of biomarker combinations, the (t-tau×Aβ42)/(p-tau×t-PrP) ratio achieved the best accuracy, with 98.1% sensitivity and 97.7% specificity overall, and 96.2% sensitivity and 95.5% specificity for the “atypical” disease groups. Our results show that the combined analysis of CSF t-PrP, t-tau, p-tau, and Aβ42 is clinically useful in the differential diagnosis between CJD and AD. Furthermore, the finding of reduced CSF t-PrP levels in CJD patients suggest that, likewise Aβ42 in AD, CSF t-PrP levels reflect the extent of PrPc conversion into abnormal PrP (PrPSc) and the burden of PrPSc deposition in CJD.

Domain-specific quantification of prion protein in cerebrospinal fluid by targeted mass spectrometry

“…For the six PrP peptides harboring sequence differences between species ( Figure 1A, Table S2), we observed excellent selectivity, with peptides consistently detected in sequence-matched species above the background level observed in non-sequence-matched species (Figure S5A-B) and with technical replicate mean coefficients of variation (CVs) all <15% (Table S3). In a doseresponse experiment, 15 N-labeled recombinant human PrP added to human CSF in doseresponse was recovered with good dilution linearity over at least the two orders of magnitude chosen for this experiment ( Figure S5C). Dilution linearity for endogenous CSF PrP was confirmed by mixing high-PrP and low-PrP human CSF samples in different proportions ( Figure S5D).…”

“…Untagged recombinant HuPrP23-230 (MW=22,878) and MoPrP23-231 (MW=23,151), corresponding to full-length post-translationally modified human and mouse PrP without the signal peptide or GPI signal but retaining an N-terminal methionine, were purified by denaturation and Ni-NTA affinity from E. coli inclusion bodies as previously described 31,32 , using a vector generously provided by Byron Caughey (NIAID Rocky Mountain Labs, Hamilton, MT). 15 N incorporation was achieved by growing the E. coli in 15 N cell growth medium (Cambridge Isotope Laboratories CGM-1000-N) induced with 15 N auto-induction medium (Millipore 71759-3). Protein concentration was determined by amino acid analysis (AAA, New England Peptide).…”

“…Percent 15 N isotopic incorporation was estimated using LC-MS/MS. 15 N labeled human recombinant prion protein (10 µg) was digested and desalted following the procedure as described in PrP MRM assay and analyzed as described in Pilot LC-MS/MS analysis. Precursor masses for 15 N were extracted from the chromatograms using XCalibur software Qualbrowser software (Thermo) 3.0.63 with a 6 mz window of centered on the precursors and charge states listed in Table S1.…”

“…In addition to properly identifying the endogenous light peptides by MRM, these heavy peptides control for variability in retention on the LC and the ionization on the MS, caused by the presence of a large number of peptides in the mixture, with over 4,000 peptides identified in CSF pilot study. To further control for the analytical variability that can occur during enzymatic proteolysis and solid phase extraction (SPE) using StageTips 38 , we also added uniformly 15…”

“…PrP is sufficiently abundant in CSF, at concentrations of tens or hundreds of nanograms per milliliter, to be readily quantified with enzyme-linked immunosorbent assay (ELISA). Studies using ELISA have reproducibly found that CSF PrP is decreased in the symptomatic phase of prion disease 3,[13][14][15][16] . Therefore, even though CSF PrP is brain-derived and exhibits good withinsubject test-retest reliability in individuals without prion disease 3 , it might be difficult to use this biomarker to read out the effect of a PrP-lowering drug in symptomatic individuals, because it is unclear whether to expect that such a drug should cause a further decrease in CSF PrP as a direct pharmacodynamic effect, or an increase in CSF PrP due to alleviation of the disease process.…”