Michael B. Coulthart

MRI lesion profiles in sporadic Creutzfeldt–Jakob disease

Background: With respect to sporadic Creutzfeldt–Jakob disease (sCJD), six molecular subtypes (MM1, MM2, MV1, MV2, VV1, and VV2) have been described, which vary with respect to age at disease onset, disease duration, early symptoms, and neuropathology. MRI signal alterations were reported to correlate with distinct Creutzfeldt–Jakob disease (CJD) subtypes. This multicenter, international study aimed to describe the brain MRI findings associated with each of the sCJD molecular subtypes. Methods: Pathologically confirmed sCJD cases with codon 129 genotype (MM, MV, and VV), PrPSc type, and fluid-attenuated inversion recovery (FLAIR) or diffusion-weighted imaging (DWI) were collected in seven countries. All MRI scans were assessed for signal changes according to a standard protocol encompassing seven cortical regions, basal ganglia, thalamus, and cerebellum. Results: MRI scans were evaluated in 211 CJD patients (98 MM1, 23 MM2, 19 MV1, 30 MV2, 9 VV1, and 32 VV2). Basal ganglia hyperintensities occurred most frequently in MV2, VV2, and MM1 subtypes (79, 77, and 70%). Wide cerebral cortical signal increase was most common in VV1, MM2, and MV1 subtypes (86, 77, and 77%). Thalamic hyperintensities occurred most often in VV2 (45%) and MV2 (43%). The most consistent finding across most subtypes was high signal in basal ganglia, with these abnormalities found in 63% (FLAIR) and 71% (DWI). Conclusion: Cortical signal increase and hyperintensities in the basal ganglia and thalamus are detected by MRI across all molecular sporadic Creutzfeldt–Jakob disease subtypes. Our findings argue that characteristic MRI lesion patterns may occur for each molecular subtype.

Rapid and Sensitive RT-QuIC Detection of Human Creutzfeldt-Jakob Disease Using Cerebrospinal Fluid

ABSTRACT  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. 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.

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

BackgroundTo 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.MethodsThe 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.ResultsAt 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)].ConclusionsCSF 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.

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 (PrPres). 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 PrPres, 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 PrPres 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 PrPres 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.

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

BackgroundThe objective of this study was to describe the diagnostic panorama of human transmissible spongiform encephalopathies across 11 countries.MethodsFrom 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.ResultsIn 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).ConclusionConsiderable 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.

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

Background: Prion protein (PrP) interacts with dipeptidyl aminopeptidase-like protein 6 (DPP6), but the functional significance was unknown. Results: PrP formed complexes with and impacted the function of potassium channels containing DPP6 and Kv4.2. Conclusion: PrP modulates voltage-dependent and kinetic properties of Kv4.2 channels. Significance: This could explain a phenotype of PrP knock-out mice and the effects of amyloid β oligomers. Widely expressed in the adult central nervous system, the cellular prion protein (PrPC) is implicated in a variety of processes, including neuronal excitability. Dipeptidyl aminopeptidase-like protein 6 (DPP6) was first identified as a PrPC 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 PrPC 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 PrPC, 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 PrPC 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 PrPC in the Aβ-dependent modulation of excitability in cholinergic basal forebrain neurons, our findings implicate PrPC regulation of Kv4.2 channels as a mechanism contributing to the effects of oligomeric Aβ upon neuronal excitability and viability.

Molecular evolution of influenza A/H3N2 viruses in the province of Québec (Canada) during the 1997–2000 period ☆

In this study, we compared antigenic (hemagglutination inhibition (HI) assay) and molecular (sequencing of the hemagglutinin (HA1) gene) characterization of influenza isolates collected in the Province of Québec (Canada) during the last three flu seasons (1997-2000). Twenty-three isolates were tested by a standard HI assay and 37 by sequencing of the HA1 gene for their homology to the A/H3N2 vaccine strains A/Wuhan/359/95 (1997-1998) and A/Sydney/5/97 (1998-1999 and 1999-2000). By HI, two isolates were antigenically similar to A/Wuhan/359/95 (both from 1997 to 1998), 16 were similar to A/Sydney/5/97 (1997-2000) and no conclusions could be inferred for the other five isolates due to identical HI titers for the two vaccine strains (n=4) or insufficient viral titer (n=1). Sequence analysis revealed that four isolates from 1997 to 1998 were related to A/Wuhan/359/95 whereas the others (n=4) from 1997 to 1998, as well as all isolates from 1998 to 1999 (n=18) and 1999 to 2000 (n=11) were closer to A/Sydney/5/97. The mean number of amino acid differences for the 33 A/Sydney/5/97-like isolates compared with the homologous vaccine strain was 6.3 (1.9%), 9.2 (2.8%) and 13.6 (4.1%) for those collected in 1997-1998, 1998-1999, and 1999-2000, respectively. Phylogenetic analysis confirmed that a progressive drift occurred among our A/H3N2 influenza isolates over the last three flu seasons. Furthermore, it revealed that isolates collected during the last two flu seasons were in fact more related to A/Panama/2007/99 (2000-2001 vaccine strain) than to A/Sydney/5/97. Our studies suggest that molecular analysis of the HA1 gene should complement the HI assay for a more accurate analysis of influenza A virus drift.

Evolution of Surface and Nonstructural-1 Genes of Influenza B Viruses Isolated in the Province of Québec, Canada, during the 1998–2001 Period

After 2 minor winter seasons, influenza B viruses were predominantly isolated in the Province of Quebec, Canada, during the 2000–2001 season representing 74% of laboratory-confirmed influenza viruses. We performed an antigenic study of the hemagglutinin (HA) protein and a molecular characterization of the HA1 region, nonstructural-1 (NS1) and neuraminidase (NA)/NB genes of 20 influenza B strains isolated in the Province of Quebec during the 1998–2001 period. Our isolates were compared to recent vaccine strains (B/Harbin/7/94 in 1998–1999, B/Yamanashi/166/98 in 1999–2000 and 2000–2001, and B/Sichuan/379/99 in 2001–2002). The hemagglutination inhibition (HI) test revealed that all isolates were different from B/Harbin/7/94 and were more related to the 2 other vaccine strains although precise identification was often impossible. Molecular analysis of the HA1 gene revealed that both B/Yamanashi/166/98-like and B/Sichuan/379/99-like isolates co-circulated during the 1998–1999 season whereas isolates from the 2 subsequent years were more related to B/Sichuan/379/99. Most isolates (8/9) of the 2000–2001 season contained a N126D substitution recently associated with altered antigenicity in recent influenza B/Yamagata/16/88-related viruses. Although the HA1 and NS1 protein sequences of viruses isolated during the 1998–1999 season were clearly different from those of the respective vaccine strain (B/Harbin/7/94), the NA protein sequence of those isolates was slightly more related to B/Harbin/7/94 than B/Yamanashi/166/98 suggesting distinct patterns of evolution for these genes. This study confirms the importance of a detailed molecular analysis for understanding the evolution of influenza B viruses.

Exploring Physical and Chemical Factors Influencing the Properties of Recombinant Prion Protein and the Real-Time Quaking-Induced Conversion (RT-QuIC) Assay

Real-time quaking-induced conversion (RT-QuIC), a highly specific and sensitive assay able to detect low levels of the disease-inducing isoform of the prion protein (PrPd) in brain tissue biopsies and cerebral spinal fluid, has great potential to become a method for diagnosing prion disease ante mortem. In order to standardize the assay method for routine analysis, an understanding of how physical and chemical factors affect the stability of the recombinant prion protein (rPrP) substrate and the RT-QuIC assay’s sensitivity, specificity, and reproducibility is required. In this study, using sporadic Creutzfeldt-Jakob Disease brain homogenate to seed the reactions and an in vitro-expressed recombinant prion protein, hamster rPrP, as the substrate, the following factors affecting the RT-QuIC assay were examined: salt and substrate concentrations, substrate storage, and pH. Results demonstrated that both the generation of the quality and quantities of rPrP substrate critical to the reaction, as well as the RT-QuIC reaction itself required strict adherence to specific physical and chemical conditions. Once optimized, the RT-QuIC assay was confirmed to be a very specific and sensitive assay method for sCJD detection. Findings in this study indicate that further optimization and standardization of RT-QuIC assay is required before it can be adopted as a routine diagnostic test.

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

BackgroundWilson’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 presentationHere 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.ConclusionsWe propose that synergism may occur between at least some allelic variants of ATP7B and PRNP, possibly exerted through effects on cellular copper metabolism.