Review: Creutzfeldt-Jakob disease: prion protein type, disease phenotype and agent strain.

The human transmissible spongiform encephalopathies or human prion diseases are one of the most intensively investigated groups of rare human neurodegenerative conditions. They are generally held to be unique in terms of their complex epidemiology and phenotypic variability, but they may also serve as a paradigm with which other more common protein misfolding disorders might be compared and contrasted. The clinico-pathological phenotype of human prion diseases appears to depend on a complex interaction between the prion protein genotype of the affected individual and the physico-chemical properties of the neurotoxic and transmissible agent, thought to comprise of misfolded prion protein. A major focus of research in recent years has been to define the phenotypic heterogeneity of the recognized human prion diseases, correlate this with molecular-genetic features and then determine whether this molecular-genetic classification of human prion disease defines the biological properties of the agent as determined by animal transmission studies. This review seeks to survey the field as it currently stands, summarize what has been learned, and explore what remains to be investigated in order to obtain a more complete scientific understanding of prion diseases and to protect public health.

RNA integrity in post mortem human variant Creutzfeldt-Jakob disease (vCJD) and control brain tissue.

AIMS: To determine premortem and post mortem factors affecting quality and yield of RNA isolated from the unique archived brain material in the UK National Creutzfeldt-Jakob Disease Surveillance Unit Brain and Tissue Bank and to compare this to control brain tissue with no neurological disease. METHODS: In parallel and in replicate, RNA was prepared from the frontal parasagittal or subfrontal cortex of samples dissected from half brains (frozen intact) or from brain samples snap frozen or placed in RNALater. A total of 350 RNA samples from 78 human autopsy cases, 21 variant Creutzfeldt-Jakob disease, 26 other neurological diseases and 31 non-neurological diseases were studied. RESULTS: There was no difference in the quality or yield of RNA isolated from variant Creutzfeldt-Jakob disease, other neurological disease and non-neurological disease brains. RNA preparations from archived frozen half brains or snap frozen autopsy samples were generally of poor quality (RNA integrity number<5). There was a highly significant negative correlation between the number of times frozen half brains had been sampled and the quality of RNA. Samples stored in RNALater provided higher-quality RNA (RNA integrity number>5). Age at death, gender, post mortem interval and freezer storage time had no effect on RNA quality. CONCLUSION: Reasonable-quality RNA can be isolated from samples dissected from archived frozen human half brains but repeated sampling results in RNA degradation. Better-quality RNA is obtained from samples placed in RNALater than from snap frozen samples. The quality and yield of RNA are not affected by age at death, gender, post mortem interval of >6 h or freezer storage time.

The first case of protease-sensitive prionopathy (PSPr) in The Netherlands: a patient with an unusual GSS-like clinical phenotype.

An atypical case of prion disease is described in a 54-year-old Dutch man, homozygous for valine at codon 129 of the prion protein gene (PRNP). The clinical phenotype was characterised by progressive dementia, spastic paraplegia and sensorimotor polyneuropathy. The disease duration was 20 months. Genetic analysis of PRNP did not reveal any abnormalities. Neuropathologically, only mild spongiform change and a coarse granular immunohistochemical staining for the abnormal prion protein, PrP(Sc), was observed, with poorly formed plaques in the molecular layer of the cerebellar cortex. However, Western blotting showed low but detectable levels of proteinase K(PK)-resistant PrP(Sc) occurring in an unusual ladder-like profile. These features define a phenotype that corresponds to the recently described protease-sensitive prionopathy (PSPr). Our report on the first Dutch patient with PSPr further expands the spectrum of prionopathies and exemplifies the need to re-evaluate cases of atypical prion disease.

Variant CJD infection in the spleen of a neurologically asymptomatic UK adult patient with haemophilia.

SUMMARY: All UK patients with bleeding disorders treated with any UK-sourced pooled factor concentrates between 1980 and 2001 have been informed that they may be at an increased risk of infection with variant Creutzfeldt-Jakob disease (vCJD). We describe a study to detect disease-associated, protease-resistant prion protein (PrP(res)) in 17 neurologically aymptomatic patients with haemophilia considered to be at increased risk of vCJD. Materials from 11 autopsy and seven biopsy cases were analysed for PrP(res). The tissues available from each case were variable, ranging from a single biopsy sample to a wide range of autopsy tissues. A single specimen from the spleen of one autopsy case gave a strong positive result on repeated testing for PrP(res) by Western blot analysis. This tissue came from a 73-year-old male patient with no history of neurological disease, who was heterozygous (methionine/valine) at codon 129 in the prion protein gene. He had received over 9000 units of factor VIII concentrate prepared from plasma pools known to include donations from a vCJD-infected donor, and some 400,000 units not known to include donations from vCJD-infected donors. He had also received 14 units of red blood cells and had undergone several surgical and invasive endoscopic procedures. Estimates of the relative risks of exposure through diet, surgery, endoscopy, blood transfusion and receipt of UK-sourced plasma products suggest that by far the most likely route of infection in this patient was receipt of UK plasma products.

Panencephalopathic Creutzfeldt-Jakob disease in the Netherlands and the UK: clinical and pathological characteristics of nine patients.

AIMS: The panencephalopathic type of Creutzfeldt-Jakob disease (PECJD) has extensive abnormalities in cerebral white matter as well as the cortex. PECJD has rarely been described in Caucasians and debate continues on its classification and pathogenesis. We describe our experience of PECJD over a 14-year period of surveillance for CJD in the Netherlands and the UK. METHODS: Between 1993 and 2006, nine cases of PECJD were identified. Clinical, histological and biochemical characteristics of all patients were analysed and compared; all cases were classified clinically as sporadic CJD. RESULTS: The median age at onset was 57.8 years and median disease duration was 22 months. The average brain weight was 887 g. Most patients showed a two-stage clinical course with initial rapid deterioration to a state of akinetic mutism, which then persisted over a longer time scale. Neuropathological findings were characterized by severe global atrophy with status spongiosus. Cerebral white matter involvement tended to be associated with either disease duration or severity of cerebral cortical lesions. Five patients could be classified into the MM1 subtype of sporadic CJD, one patient into the MM2 subgroup and another into the MV2 subgroup. Two patients were heterozygous at codon 129 in the prion protein gene and contained both type 1 and type 2 PrP(res) isoforms in the brain. CONCLUSIONS: We believe that white matter pathology in PECJD represents an end-stage pattern that reflects secondary degeneration due to widespread cortical neuronal loss that occurs in the early part of the disease, rather than representing a primary lesion.

Disease-associated prion protein is not detectable in human systemic amyloid deposits.

Cerebral and cardiac amyloid deposits have been reported after scrapie infection in transgenic mice expressing variant prion protein (PrP(C)) lacking the glycophosphatidylinositol anchor. The amyloid fibril protein in the systemic amyloid deposits was not characterized, and there is no clinical or pathological association between prion diseases and systemic amyloidosis in humans. Nevertheless, in view of the potential clinical significance of these murine observations, we tested both human amyloidotic tissues and isolated amyloid fibrils for the presence of PrP(Sc), the prion protein conformation associated with transmissible spongiform encephalopathy (TSE). We also sequenced the complete prion protein gene, PRNP, in amyloidosis patients. No specific immunohistochemical staining for PrP(Sc) was obtained in the amyloidotic cardiac and other visceral tissues of patients with different types of systemic amyloidosis. No protease-resistant prion protein, PrP(res), was detectable by Western blotting of amyloid fibrils isolated from cardiac and other systemic amyloid deposits. Only the complete normal wild-type PRNP gene sequence was identified, including the usual distribution of codon 129 polymorphisms. These reassuringly negative results do not support the idea that there is any relationship of prions or TSE with human systemic amyloidosis, including cardiac amyloid deposition.

In vitro amplification and detection of variant Creutzfeldt-Jakob disease PrPSc.

Variant Creutzfeldt-Jakob disease (vCJD) poses a serious risk of secondary transmission and the need to detect infectivity in asymptomatic individuals is therefore of major importance. Following infection, it is assumed that minute amounts of disease-associated prion protein (PrP(Sc)) replicate by conversion of the host cellular prion protein (PrP(C)). Therefore, methods of rapidly reproducing this conversion process in vitro would be valuable tools in the development of such tests. We show that one such technique, protein misfolding cyclic amplification (PMCA), can amplify vCJD PrP(Sc) from human brain tissue, and that the degree of amplification is dependent upon the substrate PRNP codon 129 polymorphism. Both human platelets and transgenic mouse brain are shown to be suitable alternative substrate sources, and amplified PrP(Sc) can be detected using a conformation-dependent immunoassay (CDI), allowing the detection of putative proteinase K sensitive forms of PrP(Sc).

Predicting susceptibility and incubation time of human-to-human transmission of vCJD.

BACKGROUND: Identification of possible transmission of variant Creutzfeldt-Jakob disease (vCJD) via blood transfusion has caused concern over spread of the disease within the human population. We aimed to model iatrogenic spread to enable a comparison of transmission efficiencies of vCJD and bovine spongiform encephalopathy (BSE) and an assessment of the effect of the codon-129 polymorphism on human susceptibility. METHODS: Mice were produced to express human or bovine prion protein (PrP) by direct replacement of the mouse PrP gene. Since the human PrP gene has variation at codon 129, with MM, VV, and MV genotypes, three inbred lines with an identical genetic background were produced to express human PrP with the codon-129 MM, MV, and VV genotypes. Mice were inoculated with BSE or vCJD and assessed for clinical and pathological signs of disease. FINDINGS: BSE was transmitted to the bovine line but did not transmit to the human lines. By contrast, vCJD was transmitted to all three human lines with different pathological characteristics for each genotype and a gradation of transmission efficiency from MM to MV to VV. INTERPRETATION: Transmission of BSE to human beings is probably restricted by the presence of a significant species barrier. However, there seems to be a substantially reduced barrier for human-to-human transmission of vCJD. Moreover, all individuals, irrespective of codon-129 genotype, could be susceptible to secondary transmission of vCJD through routes such as blood transfusion. A lengthy preclinical disease is predicted by these models, which may represent a risk for further disease transmission and thus a significant public-health issue.

Dura mater-associated Creutzfeldt-Jakob disease: experience from surveillance in the UK.

Between 1970 and 2003, seven cases of human dura mater-associated Creutzfeldt-Jakob disease (CJD) were identified in the UK. Furthermore, we identified a case of CJD in a porcine dura graft recipient. The mean incubation period of the human dura mater cases was 93 (range 45-177) months. The clinico-pathological features of the cases are described and compared with cases previously reported in the world literature.

Raised CSF phospho-tau concentrations in variant Creutzfeldt-Jakob disease: diagnostic and pathological implications.

OBJECTIVE: To investigate whether phosphorylated tau protein (tau-pT181) is increased in variant Creutzfeldt-Jakob disease (vCJD) and if the tau-pT181/tau protein ratio is useful for distinguishing between patients with and without CJD. METHODS: CSF tau protein and tau-pT181 were measured in 50 patients with sporadic CJD (sCJD), 51 patients with vCJD, 46 sCJD controls, and 37 vCJD controls, using Innotest hTau and Innotest P-Thr181, Innogenetics. RESULTS: Concentrations of CSF tau protein were increased in sCJD (5120 v 367 pg/ml in controls, p < 0.001) and vCJD (952 v 106 pg/ml, p < 0.001); tau-pT181 was also raised in sCJD (61 v 35 pg/ml in controls, p = 0.002) and vCJD (114 v 33 pg/ml, p < 0.001). Median concentrations of tau-pT181 were higher in vCJD than in sCJD (p < 0.001). The tau-pT181/tau protein ratio was lower than in controls in both sCJD (12 v 128 (p < 0.001)) and vCJD (119 v 279 (p < 0.001)). Mean tau-pT181/tau protein ratio was 10-fold higher in vCJD than in sCJD. Raised CSF tau protein had the highest efficiency for distinguishing sCJD and vCJD from controls. CONCLUSIONS: CSF tau-pT181 concentrations are raised in vCJD and are higher than in sCJD. Measurement of CSF tau-pT181/tau protein ratio does not improve the diagnostic efficiency of CSF tau protein alone for either vCJD or sCJD. The higher concentration of CSF tau-pT181 found in vCJD suggests that unexplained pathogenic factors influence the phosphorylation of tau protein in vCJD patients.

Phenotypic variability in human prion diseases.

Human prion diseases are rare neurodegenerative disorders that can occur as sporadic, familial or acquired disorders. Within each of these categories there is a wide range of phenotypic variation that is not encountered in other neurodegenerative disorders. The identification of the prion protein and its key role in the pathogenesis of this diverse group of diseases has allowed a fuller understanding of factors that influence disease phenotype. In particular, the naturally occurring polymorphism at codon 129 in the prion protein gene has a major influence on the disease phenotype in sporadic, familial and acquired prion diseases, although the underlying mechanisms remain unclear. Recent technical advances have improved our ability to study the isoforms of the abnormal prion protein in the brain and in other tissues. This has lead to the concept of molecular strain typing, in which different isoforms of the prion protein are proposed to correspond to individual strains of the transmissible agent, each with specific biological properties. In sporadic Creutzfeldt-Jakob disease there are at least six major combinations of codon 129 genotype and prion protein isotype, which appear to relate to distinctive clinical subgroups of this disease. However, these relationships are proving to be more complex than first considered, particularly in cases with more than a single prion protein isotype in the brain. Further work is required to clarify these relationships and to explain the mechanism of neuropathological targeting of specific brain regions, which accounts for the diversity of clinical features within human prion diseases.

Abnormal prion protein in the retina of the most commonly occurring subtype of sporadic Creutzfeldt-Jakob disease.

BACKGROUND: Involvement of the eye has been reported in patients with variant Creutzfeldt-Jakob disease (vCJD), but there is disagreement on whether retinal involvement occurs in sporadic Creutzfeldt-Jakob disease (sCJD). METHODS: Western blotting, paraffin embedded tissue blotting, and immunohistochemistry were used to test whether the abnormal form of the prion protein (PrPSc) accumulates to detectable levels in the eye in a case of the most common subtype of sCJD (MM1). RESULTS: Low levels of PrPSc were detectable in the retina, localised to the plexiform layers of the central retina. PrPSc was not detectable in other ocular tissues. CONCLUSIONS: The abnormal form of the prion protein is present in the retina in the most common sCJD subtype (MM1), albeit at levels lower than those found previously in vCJD and in sCJD of the VV2 subtype.

Evaluation of urinary PrPSc as a diagnostic test for sporadic, variant, and familial CJD.

Previously collected urine specimens from 100 patients referred to the UK National CJD Surveillance Unit as suspected cases of Creutzfeldt-Jakob disease (CJD) were analyzed, testing for abnormal prion protein (PrP(Sc)). In this context, the test had a low sensitivity and was not completely specific for CJD. Additionally, the proteins detected by this assay were not PrP(Sc) but appeared to be immunoglobulins.

Variant Creutzfeldt-Jakob disease: risk of transmission by blood and blood products.

Variant Creutzfeldt-Jakob disease (CJD) is a novel acquired human prion disease apparently resulting from exposure to the bovine spongiform encephalopathy (BSE) agent. Variant CJD differs from other human prion diseases in that the disease-associated form of the prion protein and infectivity are readily detectable in lymphoid tissues throughout the body. Lymphoid tissues and lymphocytes are implicated in the peripheral pathogenesis of prion diseases (where infectivity may be detected during the preclinical phase of the illness), giving rise to concerns that blood and blood products may also contain infectious particles, representing a possible source of iatrogenic spread of variant CJD. This concern has been reinforced following the experimental transmission of BSE in a sheep model by transfusion of blood and buffy coat from animals in the preclinical phase of the illness, and the recent identification of a UK case of variant CJD in a patient who had received packed red blood cells that had been donated by an individual who subsequently died from variant CJD. Studies in animal models suggest that most prion infectivity in blood may be cell-associated, with lower levels in the plasma, and there is evidence to suggest that any infectivity present may be reduced during the process of plasma fractionation. However, the possibility that plasma or blood products could transmit the disease cannot be excluded. Further studies are required to develop more sensitive means to detect disease-associated prion protein in blood; such techniques could be employed for screening purposes to reduce exposure to contaminated products and to assist with risk management in potentially exposed individuals.

Advances in the detection of prion protein in peripheral tissues of variant Creutzfeldt-Jakob disease patients using paraffin-embedded tissue blotting.

The accumulation of PrP(Sc), an abnormal and disease-associated form of the normal prion protein (PrP(c)), within the central nervous system (CNS) is a key pathological feature of Creutzfeldt-Jakob disease (CJD). Following limited proteolytic digestion of PrP(Sc), the detection of PrP(res) within lymphoid tissues is a unique characteristic of variant CJD in comparison with other human prion diseases, raising fears of an increased risk of iatrogenic spread. Because levels of PrP(res) in lymphoid tissues are lower than those found in CNS tissue, there is concern that other peripheral tissues may harbour infectivity at levels that current detection systems cannot demonstrate PrP(res). We have modified the paraffin-embedded tissue blot (PET blot), a technique combining immunohistochemistry (IHC), histoblot and Western blotting, for the detection of PrP(res) in paraffin sections in peripheral tissues in variant CJD. Five cases of variant CJD were examined, using a panel of anti-PrP antibodies. In each of these five cases, spleen, tonsil, lymph nodes and dorsal root ganglia showed an increase in the sensitivity and specificity of labelling using the PET blot when compared with optimized PrP(res) IHC methods. Control cases showed no evidence of PrP accumulation in either peripheral or CNS tissues. Autopsy and biopsy brain material from sporadic CJD cases also showed an increased sensitivity of PrP(res) detection with the PET blot, confirming its value as an important diagnostic and research tool in human prion diseases.

Neuropathology and molecular biology of variant Creutzfeldt-Jakob disease.

The neuropathological features of human prion diseases are spongiform change, neuronal loss, astrocytic proliferation and the accumulation of PrP(Sc), the abnormal isoform of prion protein (PrP). The pattern of brain involvement is remarkably variable and is substantially influenced by the host PrP genotype and PrP(Sc) isotype. Variant Creutzfeldt-Jakob disease (vCJD) is a novel human prion disease which results from exposure to the bovine spongiform encephalopathy (BSE) agent. The neuropathology of vCJD shows consistent characteristics, with abundant florid and cluster plaques in the cerebrum and cerebellum, and widespread accumulation of PrP(res) on immunocytochemistry. These features are distinct from all other types of human prion disease. Spongiform change is most marked in the basal ganglia, while the thalamus exhibits severe neuronal loss and gliosis in the posterior nuclei. These areas of thalamic pathology correlate with the areas of high signal seen in the thalamus on magnetic resonance imaging (MRI) examination of the brain. Western blot analysis of PrP(Sc) in the brain in vCJD tissue shows a uniform isotype, with a glycoform ratio characterized by predominance of the diglycosylated band, distinct from sporadic CJD. PrP(Sc) accumulation in vCJD is readily detectable outside the brain, in contrast with other forms of human prion disease, particularly in the lymphoid system and in parts of the peripheral nervous system. This has raised concern about the possible iatrogenic transmission of vCJD by contaminated surgical instruments, or blood. All cases of vCJD are methionine homozygotes at codon 129 of the prion protein gene (PRNP). Continued surveillance is required to investigate cases of vCJD in the UK and other countries where BSE has been reported, particularly as cases of 'human BSE' in individuals who are MV or VV at codon 129 of the PrP gene have not yet been identified. Histological, genetic and biochemical techniques are essential tools for the adequate diagnosis and investigation of human prion diseases.