Prion diseases, update]

In prion diseases, genotypic classification has been useful to understand the clinical course and pathological changes. However, among patients with the same prion protein (PrP) genotype, there are variations in the pathological and clinical phenotype. Recently, PrP typing was proposed by the molecular weight of protease-resistant PrP (PrPres). Combined with genotype and PrPres typing, sporadic Creutzfeldt-Jakob disease (CJD) could be classified precisely. In addition, we found the fragmented PrP molecules to differentiate between dura-classic CJD and dura-plaque type CJD. We report herein that the fragmented PrP is a useful marker to classify human prion diseases, and also a clue to analyze abnormal PrP structures. The fragmented PrP was detected in patients with classic-type CJD, sporadic thalamic-type CJD, familial CJD with codon 200 or 232 mutation, or familial Gerstmann-Straussler syndrome (GSS) with codon 102 mutation. Among patients with type-1 abnormal PrP, the transmission study was successful in sporadic CJD with type 1 PrPres and the fragment PrP, but not in CJD without the fragmented PrP. Thus, in the prion field, type-specific PrP structure contributes to the clinicopathology and transmissibility.     

Rapid detection of Creutzfeldt-Jakob disease and scrapie prion proteins

Creutzfeldt-Jakob disease (CJD) and Gerstmann-Str?ussler syndrome (GSS) of humans as well as scrapie of animals are caused by prions. The scrapie prion protein isoform (PrPSc) is the only macromolecule identified to date which is a component of the infectious prion particle. PrPSc is converted to PrP 27-30 by limited proteolysis while the cellular isoform, designated PrPC, is completely digested under the same conditions. ELISA studies demonstrated that native PrP 27-30 bound to plastic surfaces resisted proteolysis and exhibited little or no immunoreactivity but after denaturation with guanidinium thiocyanate (GdnSCN), immunoreactivity was greatly enhanced. PrPSc bound to nitrocellulose also exhibited enhanced immunoreactivity after denaturation. PrPSc was readily detected in brain extracts from scrapie-infected hamsters, mice, and sheep by dot-blot immunoassays using limited proteolysis followed by GdnSCN denaturation. The high sensitivity and specificity of the immunoassay allowed detection of regional differences in PrPSc in sheep brain. CJD prion protein isoform (PrPCJD) was also detected in the brains of all 10 patients tested with neuropathologically confirmed CJD and in 1 patient with GSS. Enhanced immunoreactivity of PrPSc or PrPCJD after denaturation cannot only be used for immunodiagnosis of prion diseases but may also form the basis of new assays in experimental studies directed at the chemical structure of the prion particle.     

Rationale for diagnosing human prion disease.

Human prion diseases (PrD) like Creutzfeldt-Jakob disease (CJD) include sporadic, acquired and familial neurodegenerative disorders. The central events in the neuropathological process of PrDs are severe neuronal loss, spongiform change and accumulation of abnormal prion protein (PrPSc). The latter is a conformational variant of the host-encoded cellular PrP (PrPC), a copper-binding protein. The physiological role of PrPC is debated. Definitive diagnosis of PrD is based on post mortem demonstration of PrPSc by immunohistochemistry or Western blot. Mutations in the PrP gene (PRNP), the polymorphic site at codon 129, and the molecular characteristic of protease resistant PrP influence the phenotype. Clinical symptoms, cranial MRI scan, EEG and investigation of 14-3-3 protein in cerebrospinal fluid (CSF) suggest a diagnosis of probable CJD. Variant CJD, related to bovine spongiform encephalopathy, shows a different clinical course, symmetrical high intensity MRI signal in the pulvinar, presence of PrPSc in tonsil biopsy tissue, and a lower sensitivity of CSF 14-3-3 protein compared to sporadic CJD. Future possibilities in diagnosis of PrDs include either the demonstration of PrPSc in body fluids or disease associated changes in laboratory variables or gene expression.     

Recent advances in the research of Creutzfeldt-Jakob disease (CJD) and Gerstmann-Strüssler syndrome (GSS)]

The abnormal isoform of prion protein (PrP) was detected by Western blotting and immunohistochemistry in all brains of 53 CJD and 20 GSS patients. Formic acid pretreatment on formalin fixed, paraffin-embedded thin sections enhanced immunostaining of PrP in both congophilic and non-congophilic kuru plaques which were absent in sporadic CJD patients with short clinical courses. Newly developed pretreatment on tissue sections, called hydrolytic autoclaving, could detect fine granular deposits of PrP in the synaptic structures. The fine PrP grains were detected in almost all CJD patients, regardless of the length of clinical courses, but never in control brains. This method can be applied to long preserved paraffin blocks. We analysed the PrP gene and found following variations. Proline-to-leucine change at codon 102 was found in 10 Japanese families with GSS and 7 sporadic CJD patients with kuru plaques. Alanine-to-valine change at codon 117 was found in a big Alsatian family with cerebral neurologic signs and dementia. In one Japanese family, 4 members died from typical CJD and showed glutamate-to-lysine change at codon 200. A 168 bp insertion which codes for 56 amino acids corresponding to 7 extra uninterrupted repeats of proline-glycine rich octapeptide (PHGGGWGQ) was detected in the N terminal region of PrP gene. This new insertion was found in a Japanese woman who showed slowly progressive dementia for 7 years but lacked particular pathological changes, except for a few kuru-like plaques in the cerebellum.     

Distinctive cerebellar immunoreactivity for the prion protein in familial (E200K) Creutzfeldt-Jakob disease

We have compared the immunomorphological spectrum of the deposition of the disease-associated prion protein (PrP(Sc)) in the cerebral and cerebellar cortex of 32 Creutzfeldt-Jakob disease (CJD) patients with the PrP gene (PRNP) E200K mutation to 45 sporadic CJD and 14 other genetic prion disease cases. PrP deposits correlate with the genotype at the methionine/valine (MV) polymorphic codon 129. While the diffuse/synaptic and patchy/perivacuolar PrP deposits and PrP plaques have a similar distribution and correlation with the genotype at codon 129 as in sporadic CJD, an additional peculiar PrP immunostaining pattern occurs in the cerebellum in 81% E200K mutation brains including 93% of M129M, 71% of M129V, but not in the single V129V case. It is localized to the molecular layer and consists of coarse granular PrP deposits arranged in a stripe-like manner predominantly perpendicular to the surface, closely resembling the parasagittal arborization of climbing fibers. Our results suggest that (1) the type of PrP deposits in the cerebellum may suggest genetic disease and the need for genetic testing; and (2) the peculiar stripes of PrP deposits might reflect selective vulnerability of cerebellar structures.     

Distribution of prion protein in German patients with Creutzfeldt-Jakob disease is different from that in Japanese patients

We investigated the distribution of prion protein (PrP) in 14 German patients with sporadic Creutzfeldt-Jakob disease (CJD) and compared it with that observed in Japanese patients. Immunohistochemical study revealed diffuse gray matter stainings including synaptic structures in all cases. In addition, 4 patients showed plaque-type deposition which was very rarely observed among sporadic Japanese patients without known mutation of the PrP gene but with valine at codon 129. A higher incidence of PrP plaques in German sporadic CJD may be related to the racial difference in the PrP gene.     

Cortical propagation of Creutzfeldt–Jakob disease with codon 180 mutation

A patient with Creutzfeldt–Jakob disease (CJD) with prion protein (PrP) gene codon 180 mutation (CJD 180) experienced cognitive decline over the 1.5-year period before her death. Serial magnetic resonance imaging (MRI) studies tracked stepwise propagation of cortical abnormal swelling and T2 elongations.     

A novel phenotype in familial Creutzfeldt-Jakob disease: prion protein gene E200K mutation coupled with valine at codon 129 and type 2 protease-resistant prion protein.

A novel phenotype of familial Creutzfeldt-Jakob disease (CJD) with mutated codon 200 of the prion protein gene (PRNP) coupled with the valine codon 129 (E200K-129V haplotype) has two features never observed in subjects carrying the pathogenic mutation coupled with the methionine codon 129 (E200K-129M haplotype): (1) plaque-like prion protein (PrP) deposits in the cerebellum and (2) type 2 protease-resistant prion protein (PrP(res)). This observation further underlines the role of codon 129 on the mutated PRNP allele in modulating the phenotype of familial prion diseases.     

国人Creutzfeldt—Jakob病PrP基因表达变化的研究

目的探讨国人朊病毒病PrP基因变异与临床病理变化的关系及同欧亚病例的对比。方法取5例散发型Creutzfeldt－Jakob病（CJD）患者静脉血提取DNA，以多聚酶联反应（PCR）扩增PrP基因，用7种限制性内切酶酶切密码子102、105、129、145、178、180、200、219和232位点及DNA序列测定，并设14例非CJD痴呆和16例健康人为对照。结果发现3组中各1例具密码子129多态性，为缬氨酸／甲硫氨酸杂合子，其余均为甲硫氨酸纯合子，无缬氨酸129纯合子。结论国人CJD患者PrP基因变异及其相关的临床特征与欧洲患者不同。     

Analysis of the PrP gene in a Tunisian family with Creutzfeldt-Jakob disease]

Results of PrP gene analysis in 5 of 9 members from a Jewish Tunisian family with Creutzfeldt-Jakob disease (CJD) showed a mutation at codon 200 involving substitution of lysine (Lys200) for glutamic acid (Glu200). This observation suggests that Lys200 allele probably tracks with CJD in this family and supports the possible genetic basis of the disease in the Mediterranean cluster. A second PrP variant not associated with Lys200 allele involving a short deletion in the coding sequence has also been found in only one subject.     

Prion protein glycotype analysis in familial and sporadic Creutzfeldt-Jakob disease patients

Creutzfeldt-Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) are characterised by the accumulation of a pathological conformer of PrP, named PrPsc. Molecular weight and glycosylation of the protease-resistant core of PrPsc (PrP27-30) are heterogeneous in different forms of TSEs. We analysed PrP27-30 glycotypes in a large number of TSE-affected patients: 50 sporadic CJD (sCJD), 1 iatrogenic CJD, 1 Gerstmann-Str?ussler-Scheinker syndrome (GSS) with the Pro102Leu mutation of PrP, 3 familial CJD (fCJD) with the Glu200Lys mutation and, for the first time, 7 fCJD with the Val210ll3e mutation. All patients were screened for the polymorphic codon 129 of the PrP gene. PrP27-30 deglycosylation and PrPsc immunohistochemistry were performed in selected cases. We found that two PrP27-30 glycotypes (type 1A and type 2A) are produced in sCJD. Type 1A is more frequently associated with methionine than valine in position 129. Type 1A is also formed in Val210lle fCJD. In Glu200Lys fCJD and GSS patients, we found that PrP27-30 has the same mobility of type 1 but different glycosylation ratios (type 1B). Our findings indicate that the polymorphic residue 129 of PrP has a leading role in determining the proteinase degradation site of PrPsc while mutant residues 102 or 200 influence only the glycosylation pattern.     

Mutation in the prion protein gene at codon 232 in Japanese patients with Creutzfeldt-Jakob disease: a clinicopathological,immunohistochemical and transmission study

We describe the clinical, neuropathological, immunohistochemical and transmission findings in three patients with Creutzfeldt-Jakob disease (CJD) with a substitution from methionine to arginine at codon 232 (M232R) in the prion protein (PrP) gene. The patients with M232R presented clinically with rapidly progressive dementia, myoclonus, and periodic synchronous discharges in the electroencephalogram. These findings were mostly consistent with those for sporadic CJD. All patients reached the stage of akinetic mutism between 2 and 6 months, and died between 4 and 24 months after the onset of the disease. Histopathological examination revealed spongiform changes, neuronal loss and severe astrocytosis. Immunohistochemical staining for PrP showed diffuse gray matter staining, including synaptic structures. However, no plaque-type PrP deposition was observed in the affected brain tissue sections. The brain homogenates from two patients were successfully transmitted to experimental animals. Since the same mutation was not found in 100 healthy control individuals, the mutation might be associated with the disease. The clinicopathological and experimental transmission studies of CJD patients with this PrP gene mutation may thus help us to determine both phenotypic variations and the potential infectivities in different forms of prion diseases. Received: 27 December 1995 / Revised, accepted: 25 March 1995     

散发性Creutzfeldt-Jakob病患者10例prion基因研究

目的 检测10例Creutzfeldt-Jakob病（CJD）患者prion基因（PRNP）外显子突变情况.方法 抽取患者外周静脉血,提取DNA,PCR法扩增PRNP外显子后直接测序,并用限制性内切酶Nsp Ⅰ检测PRNP 129位点密码子基因型.结果 2例肯定CJD患者中,1例PRNP检测未见异常,另1例PRNP第729碱基G被C取代（729G→C）,使编码prion第211个氨基酸的密码子GAG变成了GAC,翻译后第211个氨基酸由谷氨酸变为天冬氨酸（E211D）.8例很可能CJD患者中,2例PRNP第751碱基G被A取代（751G→A）,使编码prion第219个氨基酸的密码子GAG变成了AAG,翻译后第219个氨基酸由谷氨酸变为赖氨酸（E219K）.10例CJD患者PRNP 129位点密码子基因型都是甲硫氨酸纯合型.结论 1例肯定CJD患者的prion基因外显子存在一种新的点突变E211D,这很可能是导致遗传prion病发生的原因.2例很可能CJD患者的prion基因突变E219K,与M129V同属于基因多态性,而不是致病原因.prion基因检测有助于prion病的诊断.     

Genetic and environmental factors determining the development of Creutzfeldt-Jakob disease in Libyan Jews.

The cluster of Creutzfeldt-Jakob disease (CJD) among Jews of Libyan origin is one of the largest in the world. A number of hypotheses have been proposed to account for this cluster, the most prevalent but unsubstantiated hypothesis being that a transmissible agent was ingested in the form of scrapie-infected sheep brains. It has, however, been shown that a modified host protein encoded by the gene specifying the scrapie amyloid precursor is critically involved in the pathogenesis of transmissible spongiform encephalopathies such as CJD, Gerstmann-Strüssler-Scheinker syndrome and Kuru. A mutation at codon 200 in the open reading frame of this gene has recently been linked to a cluster of CJD patients in Slovakia. We examined the prevalence of this mutation among CJD patients of Libyan descent in Israel. All patients were found to have the same codon 200 mutation. These findings implicate this mutation in the high prevalence of CJD among Libyan Jews and Sephardic Jews from other Mediterranean countries.     

Diffusion-weighted MRI in familial Creutzfeldt-Jakob disease with the codon 200 mutation in the prion protein gene

Magnetic resonance imaging (MRI) with diffusion-weighted imaging (DWI) has been reported to be a useful tool for early diagnosis of sporadic Creutzfeldt-Jakob disease (CJD). We report MRI findings with DWI, as well as with fluid-attenuated inversion recovery (FLAIR) and T1-weighted imaging (T1WI), in a case of familial CJD with a mutation at codon 200 of the prion protein gene. DWI in this patient showed high signal intensity in the basal ganglia and the cerebral cortex, similar to findings in sporadic CJD. In addition, T1WI showed areas of high signal intensity bilaterally in the globus pallidus. Despite the clinical diversity and atypical laboratory findings seen in familial CJD with the codon 200 mutation, these neuroimaging studies suggest that common regional distributions and a common pathogenesis might underlie the clinical progression both in sporadic CJD and in familial CJD with the codon 200 mutation in the prion protein gene. DWI abnormalities may be characteristic features that should be considered in the diagnosis of familial as well as of sporadic CJD.     

Identification in Israel of 2 Jewish Creutzfeldt — Jakob disease patients with a 178 mutation at their PrP gene

Among the dozen known mutations in the PrP gene which segregate with the inherited prion diseases, only 2 mutations have been described in Israel so far: the codon 200 mutation in Creutzfeldt-Jakob disease (CJD) affected Libyan Jews, and the codon 102 mutation in 1 Jewish Gerstmann–Straussler–Scheinker (GSS) affected pedigree of German origin. We report here 2 unrelated CJD ¹⁷⁸ cases affected by a unique phenotype: aphemia¹ apraxia² uncontrolled laugh and no ataxia. As opposed to other CJD ¹⁷⁸ patients, in these patients, the signal transduction protein 14-3-3, recently suggested as a CJD marker, was detected in the cerebrospinal fluid samples by immunostaining. The D178N mutation, known to be linked to 2 different phenotypes: Fatal Familial Insomnia (FFI) and CJD, was not described so far among Jews. The phenotype reported here³ although it shares a common Val¹²⁹/Asn¹⁷⁸ haplotype with the previously described CJD¹⁷⁸, may point to a different clinical subtype of CJD¹⁷⁸.     

Basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease

OBJECTIVE: To determine the correlation of clinical and pathologic features with prion protein (PrP) gene polymorphism at codon 129 and with biochemical characteristics of the protease-resistant PrP (PrPres) in sporadic Creutzfeldt-Jakob disease (CJD). METHODS: Clinical data acquisition, determination of the codon 129 genotype of the PrP gene, brain pathologic study, and immunoblot analysis of crude brain extracts were carried out in 14 patients. RESULTS: The first group of 10 subjects showed the classic clinical triad, with dementia, myoclonus, and periodic sharp waves on EEG. None of the subjects had amyloid plaques, but PrP immunoreactivity was of diffuse synaptic type in the cerebellar cortex. All subjects were methionine-methionine at codon 129 and the PrPres had a biochemical profile of type 1 (unglycosylated band of 21.5 kD). A second group of three patients showed cerebellar ataxia and later dementia. Periodic sharp waves on EEG were absent. PrP amyloid plaques predominated in the cerebellar cortex, along with diffuse PrP immunoreactivity. These subjects were valine-valine at codon 129 and had a type 2 PrPres (unglycosylated band of 19.4 kD). In the last patient cerebellar ataxia and dementia appeared simultaneously. Many Kuru-type plaques were present in the cerebellar cortex; many PrP amyloid plaques were present in the basal ganglia. This patient was methionine-valine at codon 129 and the PrPres was of type 1. CONCLUSIONS: The codon 129 genotype is only one of the factors determining CJD phenotype, and the biochemical pattern of PrP has no direct correlation with this phenotype.     

New haplotype of familial Creutzfeldt-Jakob disease with a codon 200 mutation and a codon 219 polymorphism of the prion protein gene in a Japanese family

We report a new haplotype of familial Creutzfeldt-Jakob disease (CJD) with a codon 200 mutation and a codon 219 polymorphism of the prion protein gene in a Japanese family. There were four cases diagnosed with CJD neuropathologically, one of which was identified with a codon 200 mutation (glutamic acid to lysine) and a codon 219^Lys polymorphism on the same allele. Clinicopathologically, two cases had a long clinical course, whereas the others were similar to the cases with a codon 200 mutation. Three cases was diagnosed with the panencephalopathic-type CJD neuropathologically and the other was diagnosed with the subacute spongiform encephalopathy, a subtype of CJD. We consider that the clinicopathological features in familial CJD are not steadily uniform and that it is impossible to state definitely from this study whether the codon 219 polymorphism influences the clinicopathological aspects in familial CJD with a codon 200 mutation (glutamic acid to lysine). Received: 10 August 1998 / Revised: 2 December 1998, 30 May 1999 / Accepted: 31 May 1999     

Neuronal and astrocytic responses involving the serotonergic system in human spongiform encephalopathies

The relationships between the degree of cortical prion protein (PrP) deposition, tissue vacuolation and astrocytosis were studied in the frontal cortex of 27 cases of human spongiform encephalopathy, encompassing 13 cases of sporadic Creutzfeldt-Jakob disease (sCJD), four cases of familial CJD (fCJD) (one owing to E200K mutation, one owing to 144 bp insertion, one owing to P102L mutation and one owing to A117V mutation), five cases of iatrogenic CJD (iCJD) owing to growth hormone therapy and five cases of variant CJD (vCJD). The size and number of tryptophan hydroxylase (TPH) positive cells in the dorsal raphe were determined as an index of the function of the brain's serotonergic system. The amount of PrP deposited in frontal cortex in vCJD was significantly greater than that in both sCJD and iCJD, which did not differ significantly from each other. The extent of grey matter deposition of PrP correlated with that of white matter deposition. Deposition of PrP as plaques was greater in cases of sCJD bearing valine at codon 129 of PrP gene, especially when homozygous. However, all cases of vCJD displayed florid plaque formation yet these were homozygous for methionine at codon 129. Prion protein deposition as plaques was greater in cases of sCJD with 2A PrP isotype than those with 1 PrP isotype, similar to that seen in cases of vCJD all of which are 2B PrP isotype. There were no significant differences in the extent of astrocytosis between the different aetiological groups, in either grey or white matter, as visualized with glial fibrillary acidic protein (GFAP) or 5HT-2A receptor (5HT-2AR) immunostaining, although there was a strong correlation between the severity of 5HT-2AR and GFAP reactions within both grey and white matter. The extent of PrP deposition within the grey, but not white, matter correlated with the degree of astrocytosis for both GFAP and 5HT-2AR and the extent of tissue vacuolation in grey and white matter, although the latter did not correlate with degree of astrocytosis for either GFAP or 5HT-2AR. Astrocytes may be responding directly to the presence of PrP within the tissue, rather than the vacuolar damage to neurones. Although S100beta immunoreactivity was present in astrocytes in control cases, no S100beta staining was seen in astrocytes in either grey or white matter in most CJD cases. There were no differences in the number of TPH-positive cells between CJD and control cases, although the mean TPH-positive cell size was significantly greater, and cells were more intensely stained, in CJD compared to controls, suggesting a pathological overactivity of the brain's serotonergic system in CJD. This may result in excessive release of 5HT within the brain triggering increased 5HT-2AR expression within activated astrocytes leading to release and depletion of S100beta protein from such cells. The clinical symptoms of fluctuating attention and arousal could be mediated, at least in part, by such alterations in function of the serotonergic system.     

How is Creutzfeldt-Jakob disease acquired?

Creutzfeldt-Jakob disease (CJD) is one of several related disorders collectively called prion diseases. These disorders affect man and animals and are now known to be caused by the abnormal configuration of a naturally occurring protein, PrP(c). By mechanisms still not well understood, this natural protein is converted into a pathologic variant, PrP(sc). The disease is 'acquired' spontaneously perhaps by posttranslational conversion of a PrP(c) into a PrP(sc) population. This sporadic form of CJD has been reported worldwide with a frequency of 1/million. Other modes of acquisition include the following: ingestion of brain tissue from deceased victims through ritual cannibalism at burial ceremonies formerly (and no longer) practiced by New Guinea Highlanders; iatrogenically, through corneal transplants from infected donors, inoculation of human growth hormone and gonadotropin prepared from infected human pituitary glands; from inadequately sterilized depth electrodes introduced neurosurgically into the brain during workups of patients with epilepsy, and applications of infected dura mater in neurosurgical procedures. Most recently, an infected bovine source (bovine spongiform encephalopathy) has been implicated and produces a new variant of CJD. Clusters of CJD in families in some populations have been recognized which are inherited in Mendelian fashion. These clusters are related to mutations of the PRNP gene in specific codons (e.g. codon 200). Homozygosity for these mutations increases the chances of manifesting the disease. Other potential methods of acquisition, such as by blood transfusion, surgical sutures, tonometers, consumption of hog brain or other organs and tissue, remain unproven.