Goats with aspartic acid or serine at codon 146 of the PRNP gene remain scrapie-negative after lifetime exposure in affected herds in Cyprus.

The results of the study reported here are part of an ongoing integrated research programme aimed at producing additional, robust, evidence on the genetic resistance to classical scrapie in goats, with particular reference to codon 146. The study targeted animals aged ⩾6 years, which were born and raised in infected herds and were being culled for management reasons. A total of 556 animals were tested, and all positive animals (n = 117) were of the susceptible NN genotype. A total of 246 goats heterozygous or homozygous for putatively resistant alleles (S146 and D146) were screened with no positive results. The outcome of this study supports the hypothesis that the D146 and S146 alleles could be used as the basis for a nationwide strategy for breeding for resistance in the Cypriot goat population.

Prion protein genotype survey confirms low frequency of scrapie-resistant K222 allele in British goat herds.

Scrapie in goats is a transmissible, fatal prion disease, which is endemic in the British goat population. The recent success in defining caprine PRNP gene variants that provide resistance to experimental and natural classical scrapie has prompted the authors to conduct a survey of PRNP genotypes in 10 goat breeds and 52 herds to find goats with the resistant K222 allele. They report here the frequencies in 1236 tested animals of the resistance-associated K222 and several other alleles by breed and herd. Eight animals were found to be heterozygous QK222 goats (0.64 per cent genotype frequency, 95 per cent CI 0.28 to 1.27 per cent) but no homozygous KK222 goats were detected. The K222 allele was found in Saanen, Toggenburg and Anglo-Nubian goats. The fact that only a few goats with the K222 allele have been identified does not preclude the possibility to design and implement successful breeding programmes at national level.

Allelic variants at codon 146 in the PRNP gene show significant differences in the risk for natural scrapie in Cypriot goats.

Previous studies have shown the association between the polymorphisms serine (S) or aspartic acid (D) at codon 146 of the PRNP gene and resistance to scrapie. All goats aged >12 months (a total of 1075 animals) from four herds with the highest prevalence of scrapie in the country were culled and tested, of which 234 (21·7%) were positive by either the rapid test or immunohistochemistry (IHC) for any of the tissues tested. The odds of scrapie infection occurring in NN146 goats was 101 [95% credible interval (CrI) 19-2938] times higher than for non-NN146 or unknown genotypes. IHC applied to lymphoreticular tissue produced the highest sensitivity (94%, 95% CrI 90-97). The presence of putatively resistant non-NN146 alleles in the Cypriot goat population, severely affected by scrapie, provides a potential tool to reduce/eradicate scrapie provided that coordinated nationwide breeding programmes are implemented and maintained over time.

Four BSE cases with an L-BSE molecular profile in cattle from Great Britain.

Bovine spongiform encephalopathy (BSE) is a prion disease of cattle which was first observed in Great Britain (GB) in 1986. Throughout the subsequent BSE epidemic, cases identified by passive surveillance have shown consistent histopathological, immunohistochemical, biochemical and biological properties. However, since the start of active surveillance in 2001, across Europe and elsewhere, approximately 67 cases with different biochemical characteristics have been identified by Western blotting (WB). These cases fall into two categories; 'H-type' (H-BSE) or 'L-type' (L-BSE), based on the relatively heavy (H-BSE) or light (L-BSE) mass of the unglycosylated band of the prion protein, as compared with WB against that obtained from classical BSE (C-BSE) cases. Here we report the detection and confirmation of the first four L-BSE cases by active surveillance in GB, two of which were born after the reinforced feed ban of 1996 (BARB cases). These four L-BSE cases were found in relatively old cattle (age range; 11-21 years old) and the carcases did not enter the human food chain or animal feed chains.

Protective effect of the T112 PrP variant in sheep challenged with bovine spongiform encephalopathy.

Sheep with an ARQ/ARQ PRNP genotype at codon positions 136/154/171 are highly susceptible to experimental infection with bovine spongiform encephalopathy (BSE). However, a number of sheep challenged orally or intracerebrally with BSE were clinically asymptomatic and found to survive or were diagnosed as BSE-negative when culled. Sequencing of the full PRNP gene open reading frame of BSE-susceptible and -resistant sheep indicated that, in the majority of Suffolk sheep, resistance was associated with an M112T PRNP variant (TARQ allele). A high proportion (47 of 49; 96%) of BSE-challenged wild-type (MARQ/MARQ) Suffolk sheep were BSE-infected, whereas none of the 20 sheep with at least one TARQ allele succumbed to BSE. Thirteen TARQ-carrying sheep challenged with BSE are still alive and some have survival periods equivalent to, or greater than, reported incubation periods of BSE in ARR/ARR and VRQ/VRQ sheep.

Identification of a proteinase K resistant protein for use as an internal positive control marker in PrP Western blotting.

The routine use of an internal positive control (IPC) marker could prove useful in the diagnosis of transmissible spongiform encephalopathy (TSE) diseases, particularly in surveillance programmes where large numbers of negative results are reported. Detection of an endogenous IPC protein in a negative sample adds confidence to the correct sample processing throughout the analytical procedure and could avoid the reporting of false negative diagnoses. Proteinase K (PK) resistance is one of the key diagnostic determinants of the disease-associated form of PrP (PrP(Sc)), the only disease-specific macromolecule currently associated with TSE disease. Additional PK resistant proteins, endogenous to TSE-suspect diagnostic tissue samples, were therefore assessed for use as IPC markers in the Western blot diagnosis of BSE and scrapie. Results indicated that, whilst essentially maintaining a standard PrP extraction and detection protocol, a ferritin heavy chain sub-unit of approximately 22kDa, was consistently detected in all PK treated TSE positive and negative tissue samples tested. Its presence in a range of sample types, any of which could be submitted under BSE and scrapie surveillance programmes, confirmed it as a suitable protein for an IPC marker in PrP(Sc) Western blotting.

Polymorphisms of the prion protein gene coding region in born-after-the-reinforced-ban (BARB) bovine spongiform encephalopathy cattle in Great Britain.

Polymorphisms of the prion protein gene are associated with differing susceptibilities to transmissible spongiform encephalopathy diseases, as shown for variant Creutzfeldt-Jakob disease in humans and scrapie in sheep, but not yet in cattle. Imposition of control measures in the UK, including a reinforced ruminant feed ban in 1996, has led to a reduction in the incidence of bovine spongiform encephalopathy (BSE). BSE-affected cattle born after 1996 in Great Britain have been termed born-after-the-reinforced-ban (BARB) cases. In this study, the PrP gene coding region from 100 BARB BSE cases and 66 matched healthy-control cattle was sequenced to investigate whether this would reveal a genetic basis to their origin. Polymorphisms identified were not found to be associated with increased susceptibility to BSE in the BARB cases. Analysis of BARB cases grouped either by clinical status or by whether they formed an isolated or clustered case was also undertaken, but differences were not found to be significant.

PrP genotypes of atypical scrapie cases in Great Britain.

Great Britain and elsewhere have detected atypical scrapie infection in sheep with PrP genotypes thought to be genetically resistant to the classical form of scrapie. DNA sequencing of the PrP gene of British atypical scrapie cases (n=69), classical scrapie cases (n=59) and scrapie-free controls (n=138) was undertaken to identify whether PrP variants, other than the three well-characterized polymorphic codons, influenced susceptibility to atypical scrapie infection. Four non-synonymous changes, M112T, M137T, L141F and P241S, were detected that are most probably associated with the A(136)R(154)Q(171) haplotype. Only the PrP variant containing a phenylalanine residue at amino acid position 141 was found to be associated more commonly with the atypical scrapie cases. In addition to the single nucleotide polymorphisms associated with the ARQ allele, two out of nine atypical scrapie cases with the ARR/ARR genotype were found to contain a 24 bp insertion, leading to an additional octapeptide repeat. In terms of PrP genetics, one classification of the GB scrapie cases examined in this study would place animals carrying any homozygous or heterozygous combination of ARR, AHQ or AF(141)RQ alleles, or any one of these alleles when paired with ARQ, as being susceptible to atypical scrapie infection, and animals heterozygous or homozygous for VRQ or homozygous for ARQ as being susceptible to classical scrapie disease. The AHQ PrP allele was associated with the highest incidence of atypical scrapie (263 per 100 000 alleles), whilst VRQ was associated with the lowest incidence (10 per 100 000 alleles).

Significant association of a M129V independent polymorphism in the 5' UTR of the PRNP gene with sporadic Creutzfeldt-Jakob disease in a large German case-control study.

BACKGROUND: A single nucleotide polymorphism (SNP) in the coding region of the prion protein gene (PRNP) at codon 129 has been repeatedly shown to be an associated factor to sporadic Creutzfeldt-Jakob disease (sCJD), but additional major predisposing DNA variants for sCJD are still unknown. Several previous studies focused on the characterisation of polymorphisms in PRNP and the prion-like doppel gene (PRND), generating contradictory results on relatively small sample sets. Thus, extensive studies are required for validation of the polymorphisms in PRNP and PRND. METHODS: We evaluated a set of nine SNPs of PRNP and one SNP of PRND in 593 German sCJD patients and 748 German healthy controls. Genotyping was performed using MALDI-TOF mass spectrometry. RESULTS: In addition to PRNP 129, we detected a significant association between sCJD and allele frequencies of six further PRNP SNPs. No significant association of PRND T174M with sCJD was shown. We observed strong linkage disequilibrium within eight adjacent PRNP SNPs, including PRNP 129. However, the association of sCJD with PRNP 1368 and PRNP 34296 appeared to be independent on the genotype of PRNP 129. We additionally identified the most common haplotypes of PRNP to be over-represented or under-represented in our cohort of patients with sCJD. CONCLUSION: Our study evaluated previous findings of the association of SNPs in the PRNP and PRND genes in the largest cohorts for association study in sCJD to date, and extends previous findings by defining for the first time the haplotypes associated with sCJD in a large population of the German CJD surveillance study.

Breaking an absolute species barrier: transgenic mice expressing the mink PrP gene are susceptible to transmissible mink encephalopathy.

Transmissible mink encephalopathy (TME) is a rare disease of the North American mink, which has never been successfully transmitted to laboratory mice. We generated transgenic mice expressing the mink prion protein (PrP) and inoculated them with TME or the mouse-adapted scrapie strain 79A. TME infected mink PrP-transgenic mice on a murine PrP knockout background. The absolute species barrier between the infectious agent of TME and mice was therefore broken. Following TME and 79A infection of mice carrying both mink and murine PrP(C), only proteinase-resistant PrP homologous to the incoming agent was detectable. The presence of the murine PrP(C) prolonged the incubation time of TME substantially.

Identification of the pathological prion protein allotypes in scrapie-infected heterozygous bank voles (Clethrionomys glareolus) by high-performance liquid chromatography-mass spectrometry.

Cerebral formation of the pathological isoform of the prion protein (PrP) is a crucial molecular event in prion diseases. The bank vole (Clethrionomys glareolus) is a rodent species highly susceptible to natural scrapie. The PrP gene of bank vole is polymorphic (Met/Ile) at codon 109. Here we show that homozygous 109Met/Met voles have incubation times shorter than heterozygous 109Met/Ile voles after experimental challenge with three different scrapie isolates. An HPLC-MS/MS method was optimized and applied to investigate whether in heterozygous animals both PrP allotypes are able to undergo pathological conversion. The results demonstrate that both allotypes of the prion protein participate to pathological deposition.

Early age of onset in fatal familial insomnia. Two novel cases and review of the literature.

Fatal familial insomnia (FFI) is a prion disease exhibiting the PRNP D178N/129M genotype. Features of this autosomal dominant illness are progressive insomnia, dysautonomia, myoclonus, cognitive decline and motor signs associated with thalamic nerve cell loss and gliosis. In contrast to the new variant of Creutzfeldt-Jakob disease (vCJD) the onset of FFI is in middle to late adulthood. We report two male patients who belong to a large German FFI kindred. They were examined clinically, and postmortem neuropathological examination was carried out in collaboration with the German reference centre for prion disease. Additionally, the prion protein gene (PRNP) was analysed. To identify further patients with disease onset under 30 years of age a comprehensive literature review was carried out. Two male patients presented with typical symptoms of FFI at the age of 23 and 24 years. In their kindred, the age of onset has never before been under 44 years of age. Our literature review identified five additional early onset cases who died at age 21 to 25 years. In all 22 reviewed FFI families the median manifestation age was 49.5 years. Although phenotypic variability of FFI is common, age of onset under 30 years has been considered to be a hallmark of vCJD with a mean manifestation at 27 years of age. Our findings underline that in addition to vCJD, FFI must be considered in cases of young-onset prion disease. This has considerable impact on clinical management and genetic counselling.

Sporadic Creutzfeldt-Jakob disease: magnetic resonance imaging and clinical findings.

OBJECTIVE: To assess if clinical features, prion protein codon 129, and molecular subtype correlate with MRI basal ganglia hyperintensity in sporadic Creutzfeldt-Jakob disease (CJD). METHODS: The authors studied 219 patients including 153 confirmed CJD cases for their neurologic symptoms and MRI findings. The MRI was assessed by a blinded investigator for the presence of high signal intensity on T2-weighted images in the basal ganglia. RESULTS: Patients with basal ganglia high signal on T2-weighted images were more likely to present with rapid progressive dementia in an early stage and shorter disease duration (median 6.7 months and 8.6 months). Surprisingly, among the CJD cases, patients without signal increase of the basal ganglia were shown to have a higher frequency of extrapyramidal disturbances (82% vs 70%). More striking differences were found for symptoms such as depression and sensory disturbances, which were more frequent among cases without signal increase. MRI was more likely to be diagnostic in patients with MV2 molecular subtype. CONCLUSIONS: Selected clinical and pathologic features correlate with the presence of basal ganglia high signal on T2-weighted MRI in patients with definite or probable CJD.

Efficacy of flupirtine on cognitive function in patients with CJD: A double-blind study.

BACKGROUND: In cell culture experiments, flupirtine maleate (FLU), a triaminopyridine compound, was able to protect neuronal cells from apoptotic cell death induced by prion protein fragments and beta-amyloid peptides. As FLU is a clinically safe drug, the authors started a double-blind placebo-controlled study in patients with Creutzfeldt-Jakob disease (CJD). METHODS: Twenty-eight patients with CJD were randomized to an oral treatment with either FLU (n = 13) or matching placebo (PLA; n = 15). For inclusion and continuing the study, the patients had to achieve at least 50% in two of the subscales of the dementia tests employed. A battery of standardized questionnaires was employed to monitor the progression of the disease. The main outcome variable was the cognitive part of the Alzheimer's Disease Assessment Scale (ADAS-Cog); the difference between baseline and the best score under treatment was defined as the primary efficacy variable for hypothesis testing. RESULTS: CJD types were homogeneously distributed among the treatment groups. Patients treated with FLU showed significantly less deterioration in the dementia tests than patients treated with PLA. The mean change in ADAS-Cog (baseline to best) was +8.4 (+/-15.3) in the FLU group and +20.6 (+/-15.1) in the PLA group (p = 0.02, one-sided t-test). CONCLUSIONS: FLU has beneficial effects on cognitive function in patients with CJD. These positive results also may suggest a treatment potential of FLU in other neurodegenerative disorders. However, further studies are necessary.

Mutant prion protein acquires resistance to protease in mouse neuroblastoma cells.

Conversion of the cellular isoform of the prion protein (PrP(C)) into the pathogenic isoform (PrP(Sc)) is thought to be the causative event in prion diseases. Biochemically, PrP(Sc) differs from PrP(C) in its partial resistance to proteinase K (PK). The amino acid sequence AGAAAAGA, comprising residues 112-119 of the murine PrP(C), has been shown to be amyloidogenic and evolutionarily conserved. To assess the effect of mutations at and around this hydrophobic sequence on protease resistance, the sequence was replaced either by alanines or by glycines and, in a third mutant, a large part surrounding this region was removed. The PrP mutant carrying substitutions of glycines for alanines showed PK resistance and aberrant proteolytic processing. Tetracycline-induced expression of this mutant indicated that resistance to protease is acquired concurrent with the synthesis of the protein. These findings indicate that mutations in the central hydrophobic region lead to immediate alterations in PrP structure and processing.

[Clinically atypical CJD: diagnostic relevance of cerebrospinal fluid markers and molecular genetic analysis?]. / Die klinisch atypische CJK - Diagnostische Bedeutung von Liquormarkern und molekulargenetischen Untersuchungen? -

HISTORY AND CLINICAL FINDINGS: A 66-year-old woman with known remitting depressive episodes was admitted to a psychiatric hospital where a severe major depression was diagnosed. The patient failed to respond to SSRI medication. In addition, she developed a pronounced speech disturbance and gait ataxia. Therefore she was referred to the department of neurology 5 months after disease onset. She presented with a predominant cerebellar syndrome. Because of a severe dysarthria testing of cognitive functions were impossible. INVESTIGATIONS: EEG revealed a general slowing of the basic activity. Periodic sharp wave complexes were not seen during the entire disease course. Cerebrospinal fluid analysis (CSF) was normal with regard to cell count and blood-csf-barrier function. 14- 3-3 proteins were detected in CSF, and CSF levels of tau-protein and neuron-specific enolase were strongly elevated. A point mutation was detected at codon 196 (E196K) of the prion proteine gene. In addition, codon 129 was found homozygous for valine. TREATMENT AND COURSE: During the following 10 weeks the patient developed pyramidal and extrapyramidal signs, myoclonus and akinetic mutism. Complications were decubital ulcers and infections of the urinary tract. The myoclonus slightly regressed following treatment with benzodiazepines. The patient died 9 months after disease onset. CONCLUSION: A rapid disease course and typical changes of surrogate markers in CSF may support the in-vivo diagnosis of CJD despite incomplete clinical criteria (e. g. lack of dementia). In such cases, detection of mutations by molecular genetic analysis is of importance to further support the diagnosis of CJD.

Creutzfeldt-Jakob disease (CJD) in a case of suspected chronic heavy metal poisoning.

We describe a patient who died of suspected heavy metal poisoning after a nine-month history of rapidly worsening dementia. Autopsy at a forensic-pathological institute established the postmortem diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) based on demonstration of the proteinase-resistant prion protein (PrPsSc) in Western-Blot on native brain tissue. Microscopic examination of the macroscopically largely inconspicuous brain revealed marked spongiform changes in the gray matter--mainly affecting the cerebral cortex, nucleus caudatus, and putamen--with confluent vacuoles. Patchy or perivacuolar deposits of PrPSc were found as well as granular PrPsc deposits. The cerebellum contained focal PrPsc deposits. There was an astrogliosis in the white matter and a proliferation of microglia in the gray matter with a simultaneous clear reduction in neuronal elements. The differential diagnosis is discussed, as is the potential risk to those performing autopsy on forensic cases with a clinical picture of rapidly progressing dementia, especially in cases where a prion disease is not initially suspected.

Prion protein binds copper within the physiological concentration range.

The prion protein is known to be a copper-binding protein, but affinity and stoichiometry data for the full-length protein at a physiological pH of 7 were lacking. Furthermore, it was unknown whether only the highly flexible N-terminal segment with its octarepeat region is involved in copper binding or whether the structured C-terminal domain is also involved. Therefore we systematically investigated the stoichiometry and affinity of copper binding to full-length prion protein PrP(23-231) and to different N- and C-terminal fragments using electrospray ionization mass spectrometry and fluorescence spectroscopy. Our data indicate that the unstructured N-terminal segment is the cooperative copper-binding domain of the prion protein. The prion protein binds up to five copper(II) ions with half-maximal binding at approximately 2 microm. This argues strongly for a direct role of the prion protein in copper metabolism, since it is almost saturated at about 5 microm, and the exchangeable copper pool concentration in blood is about 8 microm.

Current clinical diagnosis in Creutzfeldt-Jakob disease: identification of uncommon variants.

According to the recently established molecular basis for phenotypic heterogeneity of sporadic Creutzfeldt-Jakob disease (CJD), six different phenotypes are characterized by the size of the protease-resistant fragment of the pathological prion protein (types 1 and 2) and homozygosity or heterozygosity for methionine or valine at codon 129 of the prion protein gene (designated by MM1, MM2, MV1, MV2, W1, and W2). In the present investigation, we analyzed the value of commonly used clinical tests (electroencephalogram [EEG], detection of 14-3-3 protein in cerebrospinal fluid [CSF], and hyperintensity of the basal ganglia in magnetic resonance imaging) for the clinical diagnosis in each CJD phenotype. The detection of periodic sharp and slow wave complexes in the EEG is reliable in the clinical diagnosis of MM1 and MV1 patients only. The CSF analysis for 14-3-3 protein showed high sensitivity in all analyzed subgroups with the exception of MV2 patients. Valine-homozygous patients had a negative EEG, but most had detectable levels of neuronal proteins in the CSF. The sensitivity of the magnetic resonance imaging was 70%, irrespective of the subgroup, but was particularly reliable in the clinical diagnosis of MV2 patients. The widening spectrum of diagnostic techniques in CJD is not only useful in the increased accuracy of the clinical diagnosis but should also lead to the identification of more atypical cases of sporadic CJD.