Silvio Notari

Variably protease-sensitive prionopathy: a new sporadic disease of the prion protein

The objective of the study is to report 2 new genotypic forms of protease‐sensitive prionopathy (PSPr), a novel prion disease described in 2008, in 11 subjects all homozygous for valine at codon 129 of the prion protein (PrP) gene (129VV). The 2 new PSPr forms affect individuals who are either homozygous for methionine (129MM) or heterozygous for methionine/valine (129MV).

Incidence and spectrum of sporadic Creutzfeldt-Jakob disease variants with mixed phenotype and co-occurrence of PrPSc types: An updated classification

Six subtypes of sporadic Creutzfeldt–Jakob disease with distinctive clinico-pathological features have been identified largely based on two types of the abnormal prion protein, PrPSc, and the methionine (M)/valine (V) polymorphic codon 129 of the prion protein. The existence of affected subjects showing mixed phenotypic features and concurrent PrPSc types has been reported but with inconsistencies among studies in both results and their interpretation. The issue currently complicates diagnosis and classification of cases and also has implications for disease pathogenesis. To explore the issue in depth, we carried out a systematic regional study in a large series of 225 cases. PrPSc types 1 and 2 concurrence was detected in 35% of cases and was higher in MM than in MV or VV subjects. The deposition of either type 1 or 2, when concurrent, was not random and always characterized by the coexistence of phenotypic features previously described in the pure subtypes. PrPSc type 1 accumulation and related pathology predominated in MM and MV cases, while the type 2 phenotype prevailed in VVs. Neuropathological examination best identified the mixed types 1 and 2 features in MMs and most MVs, and also uniquely revealed the co-occurrence of pathological variants sharing PrPSc type 2. In contrast, molecular typing best detected the concurrent PrPSc types in VV subjects and MV cases with kuru plaques. The present data provide an updated disease classification and are of importance for future epidemiologic and transmission studies aimed to identify etiology and extent of strain variation in sporadic Creutzfeldt–Jakob disease.

Prions in the Urine of Patients with Variant Creutzfeldt–Jakob Disease

BACKGROUND Prions, the infectious agents responsible for transmissible spongiform encephalopathies, consist mainly of the misfolded prion protein (PrP(Sc)). The unique mechanism of transmission and the appearance of a variant form of Creutzfeldt-Jakob disease, which has been linked to consumption of prion-contaminated cattle meat, have raised concerns about public health. Evidence suggests that variant Creutzfeldt-Jakob disease prions circulate in body fluids from people in whom the disease is silently incubating. METHODS To investigate whether PrP(Sc) can be detected in the urine of patients with variant Creutzfeldt-Jakob disease, we used the protein misfolding cyclic amplification (PMCA) technique to amplify minute quantities of PrP(Sc), enabling highly sensitive detection of the protein. We analyzed urine samples from several patients with various transmissible spongiform encephalopathies (variant and sporadic Creutzfeldt-Jakob disease and genetic forms of prion disease), patients with other degenerative or nondegenerative neurologic disorders, and healthy persons. RESULTS PrP(Sc) was detectable only in the urine of patients with variant Creutzfeldt-Jakob disease and had the typical electrophoretic profile associated with this disease. PrP(Sc) was detected in 13 of 14 urine samples obtained from patients with variant Creutzfeldt-Jakob disease and in none of the 224 urine samples obtained from patients with other neurologic diseases and from healthy controls, resulting in an estimated sensitivity of 92.9% (95% confidence interval [CI], 66.1 to 99.8) and a specificity of 100.0% (95% CI, 98.4 to 100.0). The PrP(Sc) concentration in urine calculated by means of quantitative PMCA was estimated at 1×10(-16) g per milliliter, or 3×10(-21) mol per milliliter, which extrapolates to approximately 40 to 100 oligomeric particles of PrP(Sc) per milliliter of urine. CONCLUSIONS Urine samples obtained from patients with variant Creutzfeldt-Jakob disease contained minute quantities of PrP(Sc). (Funded by the National Institutes of Health and others.).

Molecular biology and pathology of prion strains in sporadic human prion diseases

Prion diseases are believed to propagate by the mechanism involving self-perpetuating conformational conversion of the normal form of the prion protein, PrPC, to the misfolded, pathogenic state, PrPSc. One of the most intriguing aspects of these disorders is the phenomenon of prion strains. It is believed that strain properties are fully encoded in distinct conformations of PrPSc. Strains are of practical relevance to human prion diseases as their diversity may explain the unusual heterogeneity of these disorders. The first insight into the molecular mechanisms underlying heterogeneity of human prion diseases was provided by the observation that two distinct disease phenotypes and their associated PrPSc conformers co-distribute with distinct PrP genotypes as determined by the methionine/valine polymorphism at codon 129 of the PrP gene. Subsequent studies identified six possible combinations of the three genotypes (determined by the polymorphic codon 129) and two common PrPSc conformers (named types 1 and 2) as the major determinants of the phenotype in sporadic human prion diseases. This scenario implies that each 129 genotype–PrPSc type combination would be associated with a distinct disease phenotype and prion strain. However, notable exceptions have been found. For example, two genotype–PrPSc type combinations are linked to the same phenotype, and conversely, the same combination was found to be associated with two distinct phenotypes. Furthermore, in some cases, PrPSc conformers naturally associated with distinct phenotypes appear, upon transmission, to lose their phenotype-determining strain characteristics. Currently it seems safe to assume that typical sporadic prion diseases are associated with at least six distinct prion strains. However, the intrinsic characteristics that distinguish at least four of these strains remain to be identified.

Characterization of Truncated Forms of Abnormal Prion Protein in Creutzfeldt-Jakob Disease

In prion disease, the abnormal conformer of the cellular prion protein, PrPSc, deposits in fibrillar protein aggregates in brain and other organs. Limited exposure of PrPSc to proteolytic digestion in vitro generates a core fragment of 19–21 kDa, named PrP27–30, which is also found in vivo. Recent evidence indicates that abnormal truncated fragments other than PrP27–30 may form in prion disease either in vivo or in vitro. We characterized a novel protease-resistant PrP fragment migrating 2–3 kDa faster than PrP27–30 in Creutzfeldt-Jakob disease (CJD) brains. The fragment has a size of about 18.5 kDa when associated with PrP27–30 type 1 (21 kDa) and of 17 kDa when associated with type 2 (19 kDa). Molecular mass and epitope mapping showed that the two fragments share the primary N-terminal sequence with PrP27–30 types 1 and 2, respectively, but lack a few amino acids at the very end of C terminus together with the glycosylphosphatidylinositol anchor. The amounts of the 18.5- or 17-kDa fragments and the previously described 13-kDa PrPSc C-terminal fragment relatively to the PrP27–30 signal significantly differed among CJD subtypes. Furthermore, protease digestion of PrPSc or PrP27–30 in partially denaturing conditions generated an additional truncated fragment of about 16 kDa only in typical sporadic CJD (i.e. MM1). These results show that the physicochemical heterogeneity of PrPSc in CJD extends to abnormal truncated forms of the protein. The findings support the notion of distinct structural “conformers” of PrPSc and indicate that the characterization of truncated PrPSc forms may further improve molecular typing in CJD.

Agent strain variation in human prion disease: insights from a molecular and pathological review of the National Institutes of Health series of experimentally transmitted disease

Six clinico-pathological phenotypes of sporadic Creutzfeldt-Jakob disease have been characterized which correlate at the molecular level with the type (1 or 2) of the abnormal prion protein, PrP(TSE), present in the brain and with the genotype of polymorphic (methionine or valine) codon 129 of the prion protein gene. However, to what extent these phenotypes with their corresponding molecular combinations (i.e. MM1, MM2, VV1 etc.) encipher distinct prion strains upon transmission remains uncertain. We studied the PrP(TSE) type and the prion protein gene in archival brain tissues from the National Institutes of Health series of transmitted Creutzfeldt-Jakob disease and kuru cases, and characterized the molecular and pathological phenotype in the affected non-human primates, including squirrel, spider, capuchin and African green monkeys. We found that the transmission properties of prions from the common sporadic Creutzfeldt-Jakob disease MM1 phenotype are homogeneous and significantly differ from those of sporadic Creutzfeldt-Jakob disease VV2 or MV2 prions. Animals injected with iatrogenic Creutzfeldt-Jakob disease MM1 and genetic Creutzfeldt-Jakob disease MM1 linked to the E200K mutation showed the same phenotypic features as those infected with sporadic Creutzfeldt-Jakob disease MM1 prions, whereas kuru most closely resembled the sporadic Creutzfeldt-Jakob disease VV2 or MV2 prion signature and neuropathology. The findings indicate that two distinct prion strains are linked to the three most common Creutzfeldt-Jakob disease clinico-pathological and molecular subtypes and kuru, and suggest that kuru may have originated from cannibalistic transmission of a sporadic Creutzfeldt-Jakob disease of the VV2 or MV2 subtype.

Inter-laboratory assessment of PrPSc typing in Creutzfeldt-Jakob disease: a western blot study within the NeuroPrion consortium.

Molecular typing is of considerable importance for the surveillance and epidemiology of human transmissible spongiform encephalopathies (TSEs). It relies on the detection of distinct protease‐resistant prion protein (PrPSc) core fragments that differ in molecular mass and/or glycoform ratio. In this collaborative study, we tested the inter‐laboratory agreement in TSE molecular typing. Sixteen characterized brain specimens from sporadic TSEs and variant Creutzfeldt‐Jakob disease (vCJD) cases were distributed blindly to seven laboratories for molecular characterization by a defined protocol and classification. Agreement between laboratories in the classification of samples was excellent. In particular, there were no differences in the distinction between PrPSc type 1, type 2A, and type 2B with one exception, which eventually was identified as a case with types 1 and 2 co‐occurrence. This shows that the general technique and particular classification system used here are robust and represent a reliable basis for diagnostic and epidemiologic purposes. The subtle further distinction of subtypes among type 1 and type 2 groups requires high‐sensitivity gel electrophoresis protocols that are unsuitable for routine diagnostic needs and must be reserved for research investigations. Further research is necessary on the identification and significance of co‐occurrence of PrPSc types 1 and 2 within one brain.

A refined method for molecular typing reveals that co-occurrence of PrPSc types in Creutzfeldt-Jakob disease is not the rule

Molecular typing in Creutzfeldt–Jakob disease (CJD) relies on the detection of distinct protease-resistant prion protein (PrPSc) core fragments, which differ in molecular mass or glycoform ratio. However, the definition and correct identification of CJD cases with a co-occurrence of PrPSc types remains a challenge. With antibodies recognizing a linear epitope in the octapeptide repeat PrP region, supposed to distinguish between the two major PrPSc isoforms (ie, types 1 and 2), it was recently shown that all type 2 cases display an associated band with a type 1 migration pattern, which led to the conclusion that multiple PrPSc types regularly coexist in CJD. We studied brain samples from 53 sporadic CJD and 4 variant CJD subjects using a high-resolution electrophoresis, a wide range of proteinase K (PK) activities, the ‘type 1-selective’ antibody 12B2, and several unselective antibodies. We found that the type 1-like band detected by 12B2 in all CJD subtypes associated with PrPSc type 2 is not a PK-resistant PrPSc core but rather matches the physicochemical properties of partially cleaved fragments, which result from the several PK cleavage sites included in the N-terminal portion of PrPSc. Furthermore, using gels with high resolution and a relatively high PK activity, we were able to increase the detection sensitivity of either type 1 or 2, when coexisting, to amount corresponding to 3–5% of the total PrPSc signal (ie, weak band of one type/total PrPSc). Our results show that there are many pitfalls associated with the use of ‘type 1 selective’ antibodies for CJD typing studies and that co-occurrence of PrPSc types in CJD is not the rule. The present results further validate the rationale basis of current CJD classification and the qualitative nature of molecular typing in CJD.

Multiorgan detection and characterization of protease-resistant prion protein in a case of variant CJD examined in the United States.

Background Variant Creutzfeldt–Jakob disease (vCJD) is a prion disease thought to be acquired by the consumption of prion-contaminated beef products. To date, over 200 cases have been identified around the world, but mainly in the United Kingdom. Three cases have been identified in the United States; however, these subjects were likely exposed to prion infection elsewhere. Here we report on the first of these subjects. Methodology/Principal Findings Neuropathological and genetic examinations were carried out using standard procedures. We assessed the presence and characteristics of protease-resistant prion protein (PrPres) in brain and 23 other organs and tissues using immunoblots performed directly on total homogenate or following sodium phosphotungstate precipitation to increase PrPres detectability. The brain showed a lack of typical spongiform degeneration and had large plaques, likely stemming from the extensive neuronal loss caused by the long duration (32 months) of the disease. The PrPres found in the brain had the typical characteristics of the PrPres present in vCJD. In addition to the brain and other organs known to be prion positive in vCJD, such as the lymphoreticular system, pituitary and adrenal glands, and gastrointestinal tract, PrPres was also detected for the first time in the dura mater, liver, pancreas, kidney, ovary, uterus, and skin. Conclusions/Significance Our results indicate that the number of organs affected in vCJD is greater than previously realized and further underscore the risk of iatrogenic transmission in vCJD.

Creutzfeldt-Jakob disease associated with the R208H mutation in the prion protein gene

The authors investigated a patient who died of apparent sporadic Creutzfeldt-Jakob disease (CJD) but carried a R208H substitution in the prion protein (PrP). The patient phenotype was indistinguishable from typical sporadic CJD (i.e., MM1 subtype). In addition, pathologic PrP, PrPSc, originated from both the normal and the mutated PRNP allele and had the same characteristics as PrPSc type 1. The authors propose that the R208H mutation influences disease susceptibility without significantly affecting PrPSc properties or disease phenotype.