Development of an Automated Capillary Immunoassay to Detect Prion Glycotypes in Creutzfeldt-Jakob Disease.

Creutzfeldt-Jakob disease (CJD) comprises a group of transmissible neurodegenerative diseases with vast phenotypic diversity. Sporadic CJD heterogeneity is predominantly influenced by the genotype at codon 129 of the prion-encoding gene and the molecular weight of PrPSc fragments after protease digestion, resulting in a classification of 6 subtypes of CJD (MM1, MM2, MV1, MV2, VV1, and VV2). The majority of cases with CJD can be distinguished using this classification system. However, a number of reported CJD cases are phenotypically unique from others within their same subtype, such as variably protease-sensitive prionopathies, or exist as a mixture of subtypes within the same patient. Western blotting of brain tissue, along with the genotyping of codon 129 of the prion-encoding gene, is considered the "gold standard" for the biochemical characterization of CJD. Western blotting requires a significant amount of prion protein for detection, is labor-intensive, and is also associated with high interassay variability. In addition to these limitations, a growing body of research suggests that unique subtypes of CJD are often undetected or misdiagnosed using standard diagnostic western blotting protocols. Consequently, we successfully optimized and developed a capillary-based western assay using the JESS Simple Western (ProteinSimple) to detect and characterize prion proteins from patients with CJD. We found that this novel assay consistently differentiated CJD type 1 and type 2 cases with a limit of detection 10 to 100× higher than traditional western blotting. Cases with CJD in which type 1 and type 2 coexist within the same brain region can be detected using type 1-specific and type 2-specific antibodies, and we found that there was remarkable specificity for the detection of cases with variably protease-sensitive prionopathy. The assay presented displays outstanding sensitivity, allowing for the preservation of valuable samples and enhancing current detection methods.

A novel Gerstmann-Sträussler-Scheinker disease mutation defines a precursor for amyloidogenic 8 kDa PrP fragments and reveals N-terminal structural changes shared by other GSS alleles.

To explore pathogenesis in a young Gerstmann-Sträussler-Scheinker Disease (GSS) patient, the corresponding mutation, an eight-residue duplication in the hydrophobic region (HR), was inserted into the wild type mouse PrP gene. Transgenic (Tg) mouse lines expressing this mutation (Tg.HRdup) developed spontaneous neurologic syndromes and brain extracts hastened disease in low-expressor Tg.HRdup mice, suggesting de novo formation of prions. While Tg.HRdup mice exhibited spongiform change, PrP aggregates and the anticipated GSS hallmark of a proteinase K (PK)-resistant 8 kDa fragment deriving from the center of PrP, the LGGLGGYV insertion also imparted alterations in PrP's unstructured N-terminus, resulting in a 16 kDa species following thermolysin exposure. This species comprises a plausible precursor to the 8 kDa PK-resistant fragment and its detection in adolescent Tg.HRdup mice suggests that an early start to accumulation could account for early disease of the index case. A 16 kDa thermolysin-resistant signature was also found in GSS patients with P102L, A117V, H187R and F198S alleles and has coordinates similar to GSS stop codon mutations. Our data suggest a novel shared pathway of GSS pathogenesis that is fundamentally distinct from that producing structural alterations in the C-terminus of PrP, as observed in other prion diseases such as Creutzfeldt-Jakob Disease and scrapie.

A case cluster of variant Creutzfeldt-Jakob disease linked to the Kingdom of Saudi Arabia.

As of mid-2016, 231 cases of variant Creutzfeldt-Jakob disease-the human form of a prion disease of cattle, bovine spongiform encephalopathy-have been reported from 12 countries. With few exceptions, the affected individuals had histories of extended residence in the UK or other Western European countries during the period (1980-96) of maximum global risk for human exposure to bovine spongiform encephalopathy. However, the possibility remains that other geographic foci of human infection exist, identification of which may help to foreshadow the future of the epidemic. We report results of a quantitative analysis of country-specific relative risks of infection for three individuals diagnosed with variant Creutzfeldt-Jakob disease in the USA and Canada. All were born and raised in Saudi Arabia, but had histories of residence and travel in other countries. To calculate country-specific relative probabilities of infection, we aligned each patient's life history with published estimates of probability distributions of incubation period and age at infection parameters from a UK cohort of 171 variant Creutzfeldt-Jakob disease cases. The distributions were then partitioned into probability density fractions according to time intervals of the patient's residence and travel history, and the density fractions were combined by country. This calculation was performed for incubation period alone, age at infection alone, and jointly for incubation and age at infection. Country-specific fractions were normalized either to the total density between the individual's dates of birth and symptom onset ('lifetime'), or to that between 1980 and 1996, for a total of six combinations of parameter and interval. The country-specific relative probability of infection for Saudi Arabia clearly ranked highest under each of the six combinations of parameter × interval for Patients 1 and 2, with values ranging from 0.572 to 0.998, respectively, for Patient 2 (age at infection × lifetime) and Patient 1 (joint incubation and age at infection × 1980-96). For Patient 3, relative probabilities for Saudi Arabia were not as distinct from those for other countries using the lifetime interval: 0.394, 0.360 and 0.378, respectively, for incubation period, age at infection and jointly for incubation and age at infection. However, for this patient Saudi Arabia clearly ranked highest within the 1980-96 period: 0.859, 0.871 and 0.865, respectively, for incubation period, age at infection and jointly for incubation and age at infection. These findings support the hypothesis that human infection with bovine spongiform encephalopathy occurred in Saudi Arabia.

The prion protein modulates A-type K+ currents mediated by Kv4.2 complexes through dipeptidyl aminopeptidase-like protein 6.

Widely expressed in the adult central nervous system, the cellular prion protein (PrP(C)) is implicated in a variety of processes, including neuronal excitability. Dipeptidyl aminopeptidase-like protein 6 (DPP6) was first identified as a PrP(C) interactor using in vivo formaldehyde cross-linking of wild type (WT) mouse brain. This finding was confirmed in three cell lines and, because DPP6 directs the functional assembly of K(+) channels, we assessed the impact of WT and mutant PrP(C) upon Kv4.2-based cell surface macromolecular complexes. Whereas a Gerstmann-Sträussler-Scheinker disease version of PrP with eight extra octarepeats was a loss of function both for complex formation and for modulation of Kv4.2 channels, WT PrP(C), in a DPP6-dependent manner, modulated Kv4.2 channel properties, causing an increase in peak amplitude, a rightward shift of the voltage-dependent steady-state inactivation curve, a slower inactivation, and a faster recovery from steady-state inactivation. Thus, the net impact of wt PrP(C) was one of enhancement, which plays a critical role in the down-regulation of neuronal membrane excitability and is associated with a decreased susceptibility to seizures. Insofar as previous work has established a requirement for WT PrP(C) in the Aß-dependent modulation of excitability in cholinergic basal forebrain neurons, our findings implicate PrP(C) regulation of Kv4.2 channels as a mechanism contributing to the effects of oligomeric Aß upon neuronal excitability and viability.

Evidence for synergistic effects of PRNP and ATP7B mutations in severe neuropsychiatric deterioration.

BACKGROUND: Wilson's disease (WD), a rare cause of neuropsychiatric deterioration, is associated with mutations in the ATP7B gene. Prion diseases are also rare causes of neuropsychiatric deterioration that can occur sporadically without an identifiable cause, or can be attributed to mutations in the PRNP gene. CASE PRESENTATION: Here we describe a biological "experiment of nature" in which a patient presented with severe neuropsychiatric decline and strong biochemical evidence of WD. Genetic analysis revealed that he was a compound heterozygote for two ATP7B sequence variants (c.2165dupT, p.Arg723Glufs*32; and c.4039G > A, p.Gly1347Ser), the first having been reported once previously, and the second being novel. In addition, the patient was heterozygous for a PRNP variant, c.160G > A, p.Gly54Ser, that has been reported in a neuropsychiatric patient only once previously in association with a similarly severe clinical course of neuropsychiatric disease and early age of onset, but no accompanying information on ATP7B genotype. Of particular interest was the observation that the patient's older sister, who carried the same ATP7B genotype and laboratory evidence for biochemical WD but was clinically asymptomatic, lacked the PRNP variant allele. CONCLUSIONS: We propose that synergism may occur between at least some allelic variants of ATP7B and PRNP, possibly exerted through effects on cellular copper metabolism.

Diagnostic accuracy of cerebrospinal fluid protein markers for sporadic Creutzfeldt-Jakob disease in Canada: a 6-year prospective study.

BACKGROUND: To better characterize the value of cerebrospinal fluid (CSF) proteins as diagnostic markers in a clinical population of subacute encephalopathy patients with relatively low prevalence of sporadic Creutzfeldt-Jakob disease (sCJD), we studied the diagnostic accuracies of several such markers (14-3-3, tau and S100B) in 1000 prospectively and sequentially recruited Canadian patients with clinically suspected sCJD. METHODS: The study included 127 patients with autopsy-confirmed sCJD (prevalence = 12.7%) and 873 with probable non-CJD diagnoses. Standard statistical measures of diagnostic accuracy were employed, including sensitivity (Se), specificity (Sp), predictive values (PVs), likelihood ratios (LRs), and Receiver Operating Characteristic (ROC) analysis. RESULTS: At optimal cutoff thresholds (empirically selected for 14-3-3, assayed by immunoblot; 976 pg/mL for tau and 2.5 ng/mL for S100B, both assayed by ELISA), Se and Sp respectively were 0.88 (95% CI, 0.81-0.93) and 0.72 (0.69-0.75) for 14-3-3; 0.91 (0.84-0.95) and 0.88 (0.85-0.90) for tau; and 0.87 (0.80-0.92) and 0.87 (0.84-0.89) for S100B. The observed differences in Sp between 14-3-3 and either of the other 2 markers were statistically significant. Positive LRs were 3.1 (2.8-3.6) for 14-3-3; 7.4 (6.9-7.8) for tau; and 6.6 (6.1-7.1) for S100B. Negative LRs were 0.16 (0.10-0.26) for 14-3-3; 0.10 (0.06-0.20) for tau; and 0.15 (0.09-0.20) for S100B. Estimates of areas under ROC curves were 0.947 (0.931-0.961) for tau and 0.908 (0.888-0.926) for S100B. Use of interval LRs (iLRs) significantly enhanced accuracy for patient subsets [e.g., 41/120 (34.2%) of tested sCJD patients displayed tau levels > 10,000 pg/mL, with an iLR of 56.4 (22.8-140.0)], as did combining tau and S100B [e.g., for tau > 976 pg/mL and S100B > 2.5 ng/mL, positive LR = 18.0 (12.9-25.0) and negative LR = 0.02 (0.01-0.09)]. CONCLUSIONS: CSF 14-3-3, tau and S100B proteins are useful diagnostic markers of sCJD even in a low-prevalence clinical population. CSF tau showed better overall diagnostic accuracy than 14-3-3 or S100B. Reporting of quantitative assay results and combining tau with S100B could enhance case definitions used in diagnosis and surveillance of sCJD.

Identification of circulating microRNA signatures as potential biomarkers in the serum of elk infected with chronic wasting disease.

Chronic wasting disease (CWD) is an emerging infectious prion disorder that is spreading rapidly in wild populations of cervids in North America. The risk of zoonotic transmission of CWD is as yet unclear but a high priority must be to minimize further spread of the disease. No simple diagnostic tests are available to detect CWD quickly or in live animals; therefore, easily accessible biomarkers may be useful in identifying infected animals. MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules that circulate in blood and are promising biomarkers for several infectious diseases. In this study we used next-generation sequencing to characterize the serum miRNA profiles of 35 naturally infected elk that tested positive for CWD in addition to 35 elk that tested negative for CWD. A total of 21 miRNAs that are highly conserved amongst mammals were altered in abundance in sera, irrespective of hemolysis in the samples. A number of these miRNAs have previously been associated with prion diseases. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the discriminative potential of these miRNAs as biomarkers for the diagnosis of CWD. We also determined that a subgroup of 6 of these miRNAs were consistently altered in abundance in serum from hamsters experimentally infected with scrapie. This suggests that common miRNA candidate biomarkers could be selected for prion diseases in multiple species. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses pointed to a strong correlation for 3 of these miRNAs, miR-148a-3p, miR-186-5p, miR-30e-3p, with prion disease.

Sporadic Creutzfeldt-Jakob Disease in a Young Girl With Unusually Long Survival.

Creutzfeldt-Jakob disease (CJD) is a rapidly progressive, fatal degenerative encephalopathy caused by a pathologically altered form of the prion protein (PrP). CJD is rare, with 1 and 2 cases per million per year reported in the general population, mostly in individuals over 50 years of age. It is almost unknown in the pediatric population. Sporadic CJD with unusually long survival (sCJD-LS), an unusual clinicopathological variant of CJD, has been described mostly in Japanese patients. We present here the first case report of pediatric CJD-LS occurring sporadically in a teenage girl of European descent, with initially rapid neurocognitive decline followed by a prolonged (∼10 years) clinical course. Neuropathological findings at autopsy included generalized cerebral and cerebellar atrophy with relative sparing of the hippocampi, cerebral and cerebellar white and gray matter involvement, minimal spongiform change, PrP deposits in the neocortex, striatum and cerebellum by immunohistochemistry, and protease-resistant PrP by Western immunoblot. With its longer disease duration and atypical manifestations of white matter loss, CJD-LS can be clinically mistaken for other neurodegenerative diseases, or in the pediatric setting for metabolic/genetic conditions. This case clearly demonstrates that with rapid-onset encephalopathy, prion disease should be carefully considered, even in younger patients with slower disease progression.

Species barriers for chronic wasting disease by in vitro conversion of prion protein.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that can affect North American cervids (deer, elk, and moose). Using a novel in vitro conversion system based on incubation of prions with normal brain homogenates, we now report that PrP(CWD) of elk can readily induce the conversion of normal cervid PrP (PrP(C)) molecules to a protease-resistant form, but is less efficient in converting the PrP(C) of other species, such as human, bovine, hamster, and mouse. However, when substrate brain homogenates are partially denatured by acidic conditions (pH 3.5), PrP(CWD)-induced conversion can be greatly enhanced in all species. Our results demonstrate that PrP(C) from cervids (including moose) can be efficiently converted to a protease-resistant form by incubation with elk CWD prions, presumably due to sequence and structural similarities between these species. Moreover, partial denaturation of substrate PrP(C) can apparently overcome the structural barriers between more distant species.

On the phylogenetic placement of human T cell leukemia virus type 1 sequences associated with an Andean mummy.

Recently, the putative finding of ancient human T cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR) DNA sequences in association with a 1500-year-old Chilean mummy has stirred vigorous debate. The debate is based partly on the inherent uncertainties associated with phylogenetic reconstruction when only short sequences of closely related genotypes are available. However, a full analysis of what phylogenetic information is present in the mummy data has not previously been published, leaving open the question of what precisely is the range of admissible interpretation. To fulfill this need, we re-analyzed the mummy data in a new way. We first performed phylogenetic analysis of 188 published LTR DNA sequences from extant strains belonging to the HTLV-1 Cosmopolitan clade, using the method of statistical parsimony which is designed both to optimize phylogenetic resolution among sequences with little evolutionary divergence, and to permit precise mapping of individual sequence mutations onto branches of a divergence network. We then deduced possible phylogenetic positions for the two main categories of published Chilean mummy sequences, based on their published 157-nucleotide LTR sequences. The possible phylogenetic placements for one of the mummy sequence categories are consistent with a modern origin. However, one of these placements for the other mummy sequence category falls very close to the root of the Cosmopolitan clade, consistent with an ancient origin for both this mummy sequence and the Cosmopolitan clade.

A case of familial Creutzfeldt-Jakob disease presenting with dry cough.

BACKGROUND: Clinical diagnosis of Creutzfeldt-Jakob disease (CJD) is based on the classical triad of rapidly progressive dementia, myoclonus and abnormal EEG. The 200k mutation within the gene encoding PrP, located on the short arm of chromosome 20, accounts for more than 70% of families with CJD worldwide. CASE REPORT: Herein, we report a patient who developed persistent dry cough and classical signs of CJD, including severe cognitive decline, cerebellar signs, and myoclonic jerks, leading to death a few weeks after disease onset. Mutation screening showed that he had the 200k point mutation in the PRNP gene. His mother had died twenty years earlier with neuropathologically confirmed CJD. She had presented a rapidly progressive ataxia with myoclonus, dementia, visual hallucinations, and the same persistent dry cough. CONCLUSIONS: The clinical presentation of this familial CJD case with persistent dry cough is quite unusual. Therefore, a neurological etiology should be sought when confronted with an unexplained persistent cough.

Human transmissible spongiform encephalopathies in eleven countries: diagnostic pattern across time, 1993-2002.

BACKGROUND: The objective of this study was to describe the diagnostic panorama of human transmissible spongiform encephalopathies across 11 countries. METHODS: From data collected for surveillance purposes, we describe annual proportions of deaths due to different human transmissible spongiform encephalopathies in eleven EUROCJD-consortium countries over the period 1993-2002, as well as variations in the use of diagnostic tests. Using logistic models we quantified international differences and changes across time. RESULTS: In general, pre-mortem use of diagnostic investigations increased with time. International differences in pathological confirmation of sporadic Creutzfeldt-Jakob disease, stable over time, were evident. Compared to their counterparts, some countries displayed remarkable patterns, such as: 1) the high proportion, increasing with time, of variant Creutzfeldt-Jakob disease in the United Kingdom, (OR 607.99 95% CI 84.72-4363.40), and France (OR 18.35, 95% CI 2.20-152.83); 2) high, decreasing proportions of iatrogenic Creutzfeldt-Jakob disease in France, (OR 5.81 95% CI 4.09-8.24), and the United Kingdom, (OR 1.54 95% CI 1.03-2.30); and, 3) high and stable ratios of genetic forms in Slovakia (OR 21.82 95% CI 12.42-38.33) and Italy (OR 2.12 95% CI 1.69-2.68). CONCLUSION: Considerable international variation in aetiological subtypes of human transmissible spongiform encephalopathies was evident over the observation period. With the exception of variant Creutzfeldt-Jakob disease and iatrogenic Creutzfeldt-Jakob disease in France and the United Kingdom, these differences persisted across time.

Rapid and sensitive RT-QuIC detection of human Creutzfeldt-Jakob disease using cerebrospinal fluid.

UNLABELLED: Fast, definitive diagnosis of Creutzfeldt-Jakob disease (CJD) is important in assessing patient care options and transmission risks. Real-time quaking-induced conversion (RT-QuIC) assays of cerebrospinal fluid (CSF) and nasal-brushing specimens are valuable in distinguishing CJD from non-CJD conditions but have required 2.5 to 5 days. Here, an improved RT-QuIC assay is described which identified positive CSF samples within 4 to 14 h with better analytical sensitivity. Moreover, analysis of 11 CJD patients demonstrated that while 7 were RT-QuIC positive using the previous conditions, 10 were positive using the new assay. In these and further analyses, a total of 46 of 48 CSF samples from sporadic CJD patients were positive, while all 39 non-CJD patients were negative, giving 95.8% diagnostic sensitivity and 100% specificity. This second-generation RT-QuIC assay markedly improved the speed and sensitivity of detecting prion seeds in CSF specimens from CJD patients. This should enhance prospects for rapid and accurate ante mortem CJD diagnosis. IMPORTANCE: A long-standing problem in dealing with various neurodegenerative protein misfolding diseases is early and accurate diagnosis. This issue is particularly important with human prion diseases, such as CJD, because prions are deadly, transmissible, and unusually resistant to decontamination. The recently developed RT-QuIC test allows for highly sensitive and specific detection of CJD in human cerebrospinal fluid and is being broadly implemented as a key diagnostic tool. However, as currently applied, RT-QuIC takes 2.5 to 5 days and misses 11 to 23% of CJD cases. Now, we have markedly improved RT-QuIC analysis of human CSF such that CJD and non-CJD patients can be discriminated in a matter of hours rather than days with enhanced sensitivity. These improvements should allow for much faster, more accurate, and practical testing for CJD. In broader terms, our study provides a prototype for tests for misfolded protein aggregates that cause many important amyloid diseases, such as Alzheimer's, Parkinson's, and tauopathies.

Insights into origins of Human T-cell Lymphotropic Virus Type 1 based on new strains from aboriginal people of Canada.

The causes of the worldwide distribution of Human T-cell Lymphotropic Virus Type 1 (HTLV-1) remain incompletely understood, with competing hypotheses regarding the number and timing of events leading to intercontinental spread on historical and prehistoric timescales. Ongoing discovery of this virus in aboriginal populations of Asia and the Americas has been the main source of evidence for the latter. We conducted molecular phylogenetic and dating analyses for 13 newly reported HTLV-1 strains from Canada. We analyzed two full-length proviral genomes from aboriginal residents of Nunavut (an autonomous territory in Northern Canada including most of the Canadian Arctic), 11 long-terminal-repeat (LTR) sequences from aboriginal residents of British Columbia's Pacific coast, and 2 LTR sequences from non-aboriginal Canadians. Phylogenetic analysis demonstrated a well-supported affinity between the two Nunavut strains and two East Asian strains, suggesting the presence of an Asian-American sublineage within the widespread "transcontinental" subgroup A clade of HTLV-1 Cosmopolitan subtype a. This putative sublineage was estimated to be 5400-11,900 years in age, consistent with a long-term presence of HTLV-1 in aboriginal populations of the Canadian Arctic. Phylogenetic affinities of the other 11 Canadian HTLV-1 aboriginal strains were diverse, strengthening earlier evidence for multiple incursions of this virus into coastal aboriginal populations of British Columbia. Our results are consistent with the hypothesis of ancient presence of HTLV-1 in aboriginal populations of North America.

Molecular, biochemical and genetic characteristics of BSE in Canada.

The epidemiology and possibly the etiology of bovine spongiform encephalopathy (BSE) have recently been recognized to be heterogeneous. In particular, three types [classical (C) and two atypical (H, L)] have been identified, largely on the basis of characteristics of the proteinase K (PK)-resistant core of the misfolded prion protein associated with the disease (PrP(res)). The present study was conducted to characterize the 17 Canadian BSE cases which occurred prior to November 2009 based on the molecular and biochemical properties of their PrP(res), including immunoreactivity, molecular weight, glycoform profile and relative PK sensitivity. Two cases exhibited molecular weight and glycoform profiles similar to those of previously reported atypical cases, one corresponding to H-type BSE (case 6) and the other to L-type BSE (case 11). All other cases were classified as C-type. PK digestion under mild and stringent conditions revealed a reduced protease resistance in both of these cases compared to the C-type cases. With Western immunoblotting, N-terminal-specific antibodies bound to PrP(res) from case 6 but not to that from case 11 or C-type cases. C-terminal-specific antibodies revealed a shift in the glycoform profile and detected a fourth protein fragment in case 6, indicative of two PrP(res) subpopulations in H-type BSE. No mutations suggesting a genetic etiology were found in any of the 17 animals by sequencing the full PrP-coding sequence in exon 3 of the PRNP gene. Thus, each of the three known BSE types have been confirmed in Canadian cattle and show molecular characteristics highly similar to those of classical and atypical BSE cases described from Europe, Japan and the USA. The occurrence of atypical cases of BSE in countries such as Canada with low BSE prevalence and transmission risk argues for the occurrence of sporadic forms of BSE worldwide.

Cellular prion protein is released on exosomes from activated platelets.

Cellular prion protein (PrP(C)) is a glycophosphatidylinositol (GPI)-anchored protein, of unknown function, found in a number of tissues throughout the body, including several blood components of which platelets constitute the largest reservoir in humans. It is widely believed that a misfolded, protease-resistant form of PrP(C), PrP(Sc), is responsible for the transmissible spongiform encephalopathy (TSE) group of fatal neurodegenerative diseases. Although the pathogenesis of TSEs is poorly understood, it is known that PrP(C) must be present in order for the disease to progress; thus, it is important to determine the physiologic function of PrP(C). Resolving the location of PrP(C) in blood will provide valuable clues as to its function. PrP(C) was previously shown to be on the alpha granule membrane of resting platelets. In the current study platelet activation led to the transient expression of PrP(C) on the platelet surface and its subsequent release on both microvesicles and exosomes. The presence of PrP(C) on platelet-derived exosomes suggests a possible mechanism for PrP(C) transport in blood and for cell-to-cell transmission.