Human genetic prion diseases (gPrDs) are directly associated with mutations and insertions in the PRNP (Prion Protein) gene. We collected and analyzed the data of 218 Chinese gPrD patients identified between Jan 2006 and June 2020. Nineteen different subtypes were identified and gPrDs accounted for 10.9% of all diagnosed PrDs within the same period. Some subtypes of gPrDs showed a degree of geographic association. The age at onset of Chinese gPrDs peaked in the 50-59 year group. Gerstmann-Sträussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI) cases usually displayed clinical symptoms earlier than genetic Creutzfeldt-Jakob disease (gCJD) patients with point mutations. A family history was more frequently recalled in P105L GSS and D178N FFI patients than T188K and E200K patients. None of the E196A gCJD patients reported a family history. The gCJD cases with point mutations always developed clinical manifestations typical of sporadic CJD (sCJD). EEG examination was not sensitive for gPrDs. sCJD-associated abnormalities on MRI were found in high proportions of GSS and gCJD patients. CSF 14-3-3 positivity was frequently detected in gCJD patients. Increased CSF tau was found in more than half of FFI and T188K gCJD cases, and an even higher proportion of E196A and E200K gCJD patients. 63.6% of P105L GSS cases showed a positive reaction in cerebrospinal fluid RT-QuIC. GSS and FFI cases had longer durations than most subtypes of gCJD. This is one of the largest studies of gPrDs in East Asians, and the illness profile of Chinese gPrDs is clearly distinct. Extremely high proportions of T188K and E196A occur among Chinese gPrDs; these mutations are rarely reported in Caucasians and Japanese.
14-3-3 Proteins/cerebrospinal fluid* ; China ; Creutzfeldt-Jakob Syndrome/genetics* ; Humans ; Mutation/genetics* ; Prion Diseases/genetics* ; Prion Proteins/genetics* ; Prions/genetics* ; tau Proteins/cerebrospinal fluid*

OBJECTIVETo investigate epidemiological, clinical and genetic features of the first Chinese case of Creutzfeldt-Jakob disease (CJD ) with mutation of E200K in PRNP.

METHODSThe general epidemiological and clinical data were collected; CSF 14-3-3 protein was analyzed by Western blot; The PRNP was amplified by PCR and analyzed.

RESULTSA missense mutation in codon 200 (E200K) of the PRNP was identified in this patient; CSF 14-3-3 protein was positive; sleep disturbance was the initial sign and the other symptoms gradually appeared, including memory loss, dizziness and ataxia.

CONCLUSIONThe CJD patient who was first reported in China has a missense mutation in codon 200 (E200K) of the PRNP, and the codon 129 is a methionine homozygous genotype.

Asian Continental Ancestry Group ; China ; Creutzfeldt-Jakob Syndrome ; genetics ; Humans ; Male ; Middle Aged ; Mutation, Missense ; Prion Proteins ; Prions ; genetics

OBJECTIVETo create transgenic mice expressing hamster- and human-PRNP as a model for understanding the physiological function and pathology of prion protein (PrP), as well as the mechanism of cross-species transmission of transmissible spongiform encephalopathies (TSEs).

METHODSHamster and human-PRNP transgenic mice were established by conventional methods. The copy number of integrated PRNP in various mouse lines was mapped by real-time PCR. PRNP mRNA and protein levels were determined by semi-quantitative RT-PCR, real-time RT-PCR, and western blot analysis. Histological analyses of transgenic mice were performed by hematoxylin and eosin (H & E) staining and immunohistochemical (IHC) methods.

RESULTSIntegrated PRNP copy number in various mouse lines was 53 (Tg-haPrP1), 18 (Tg-huPrP1), 3 (Tg-huPrP2), and 16 (Tg-huPrP5), respectively. Exogenous PrPs were expressed at both the transcriptional and translational level. Histological assays did not detect any abnormalities in brain or other organs.

CONCLUSIONWe have established one hamster-PRNP transgenic mouse line and three human-PRNP transgenic mouse lines. These four transgenic mouse lines provide ideal models for additional research.

Animals ; Blotting, Western ; Cricetinae ; DNA ; genetics ; Disease Models, Animal ; Humans ; Immunohistochemistry ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Organ Specificity ; Plasmids ; Prion Diseases ; genetics ; Prion Proteins ; Prions ; genetics ; Real-Time Polymerase Chain Reaction ; Transcription, Genetic

OBJECTIVEThe present study was conducted to understand the effects of PrP in different octapeptide repeats on proliferation of HeLa cells.

METHODS AND RESULTSMutant PrPs with octapeptide repeat insertion were transiently expressed in HeLa cells and their results of MTT assay showed stronger cytotoxic effect on the proliferation of cells than wild-type PrP. Annexin V/PI assay also demonstrated that the expression of mutant PrPs was much easier to induce apoptosis than wild-type in HeLa cells. The percentage of both early and late stage apoptosis in mutant groups were significantly higher than that of wild type.

CONCLUSIONThese data suggest that the expression of mutant PrPs associated with familial CJD is much easier to induce apoptosis in cultured cells than expression of wild type PrP.

Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Proliferation ; Cell Survival ; genetics ; physiology ; Colorimetry ; HeLa Cells ; Humans ; Mutation ; Oligopeptides ; genetics ; Plasmids ; genetics ; Prion Proteins ; Prions ; genetics ; metabolism ; physiology ; Transfection

alphaB-crystallin is a member of the sHSP (Small heat shock protein) family, which plays an impor tant role in multiple neurodegeneration diseases. To give insight into the possible alternation and the role of aB-crystallin in prion disease, the alphaB-crystallin levels in the brain tissues of agent 263K-infected hamsters were evaluated. Western blots and IHC revealed that at the terminal stages of the diseases, the levels of alphaB-crystallin were increased up to 3-fold in the brain tissues of scrapie infected 263K hamsters compared with normal controls. Immunofluorescent assays revealed that the up-regulated alphaB-crystallin was mainly observed in astrocytes, but not in neurons. The co-localization between alphaB-crystallin and abnormal deposition of PrPsc in the brain tissues of the scrapie infected hamsters was not observed. The study may provide a foundation for further revealing the potential role of alphaB-crystallin in prion disease.
Animals ; Brain ; metabolism ; pathology ; Cricetinae ; Humans ; PrPSc Proteins ; metabolism ; Prion Diseases ; genetics ; metabolism ; pathology ; Up-Regulation ; alpha-Crystallin B Chain ; genetics ; metabolism

14-3-3 Proteins ; genetics ; metabolism ; Animals ; Brain ; pathology ; Cricetinae ; Internal Ribosome Entry Sites ; Open Reading Frames ; Prion Diseases ; genetics ; metabolism

OBJECTIVETo report a protocol using biotin-labelled PrP protein in cell free conversion assay instead of isotope.

METHODSA hamster PrP protein (HaPrP) was expressed in E. coli and purified with HIS-tag affinity chromatograph. After being labelled with biotin, HaPrP was mixed with PrPSc preparation from scrapie strain 263K.

RESULTSProtease-resistant bands were detected after four-day incubation.

CONCLUSIONThe new conversion model provides a reliable, easily handling, and environment-friendly method for studies of prion and transmissible spongiform encephalopathies.

Animals ; Biotin ; Blotting, Western ; Cell-Free System ; Chromatography, Affinity ; methods ; Cricetinae ; Escherichia coli ; genetics ; Gene Expression Regulation ; Molecular Weight ; Peptide Hydrolases ; analysis ; metabolism ; PrPSc Proteins ; genetics ; metabolism ; Prion Diseases ; genetics ; pathology ; Recombinant Proteins ; genetics ; metabolism ; Staining and Labeling

OBJECTIVETo describe the incidence condition and characteristic of Creutzfeldt-Jakob disease (CJD) in China.

METHODSThe clinical and epidemical information of patients from China CJD surveillance network was analyzed. Blood and cerebrospinal fluid (CSF) specimens from these patients were differently collected to be used to detect the 14-3-3 protein in the CSF and to analyze the PRNP gene.

RESULTSTen possible and 8 probable clinically diagnosed CJD patients were found. These patients had sporadic CJD. There were no geographic clustering and occupational risk with these patients. The mean age at onset of disease was approximately 60 years. The male to female ratio was approximately 1:1; rapidly progressive dementia was the main early symptom, which was present in 44% of patients.

CONCLUSIONThe geographic distribution, occupation, the ratio of male to female and the mean age of onset were consistent with the characteristics of sporadic CJD.

14-3-3 Proteins ; cerebrospinal fluid ; Adult ; Aged ; Blotting, Western ; China ; epidemiology ; Creutzfeldt-Jakob Syndrome ; cerebrospinal fluid ; epidemiology ; genetics ; Female ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; Population Surveillance ; Prion Proteins ; Prions ; genetics

OBJECTIVETo clone and express human neuron-specific enolase (HuNSE) protein and prepare NSE-specific antibody for prion disease diagnosis.

METHODSHuNSE gene was amplified by RT-PCR and subcloned into a HIS-tagged expression vector pQE30 after sequence verification. HIS-NSE fusion protein expression was obtained in E. coli M15 after IPTG induction followed by purification of the fusion protein by Ni-NTA affinity chromatography. Two male rabbits were immunized for 4 times with the purified protein, and the antiserum against NSE protein was collected and evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemistry.

RESULTSSDS-PAGE assay yielded an approximately 22 kD HIS-NSE fusion protein. The prepared antiserum could recognize both recombinant NSE protein and native NSE protein extracted from the brain tissues of different mammalian species as shown by Western blotting and immunohistochemistry.

CONCLUSIONHigh expression of HuNSE is obtained in E. coli and the prepared antiserum against HuNSE can be used potentially for diagnosis of prion-associated diseases and other nervous degeneration diseases.

Animals ; Blotting, Western ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Humans ; Immune Sera ; blood ; immunology ; Immunohistochemistry ; Male ; Phosphopyruvate Hydratase ; biosynthesis ; genetics ; immunology ; Prion Diseases ; diagnosis ; immunology ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

Binding sites of five monoclonal antibodies were obtained by reinforceable method of overlapping recombinant prion protein and synthetic peptide. Overlapping peptides of PrP core were expressed in Escherichia coli by insertion of serial PCR amplicons of ovine PrP gene fragments into pET32a. The expressed fusion peptides were then tested for the binding activity to PrP monoclonal antibodies in Western blotting. The binding sites of 5 monoclonal antibodies of ovine PrP were located respectively as follows: 2H3 in 199 aa-213 aa, 4C6, 5F11 and 7F11 in 139 aa-168 aa and 7F1 in 214 aa-227 aa. There oligo peptides were synthesized and used in ELISA test for more accurate localization of the binding sites. The binding sites of 4C6, 5F11 and 7F11 were further confirmed to be in 149 aa-158 aa. This conclusion may contribute to the research for pathogenesis and diagnostic method of scrapie and bovine transmissible spongiform encephalopathy.
Animals ; Antibodies, Monoclonal ; immunology ; metabolism ; Binding Sites, Antibody ; immunology ; Epitopes ; immunology ; Escherichia coli ; genetics ; metabolism ; Prion Diseases ; diagnosis ; Prions ; genetics ; immunology ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Scrapie ; diagnosis ; Sheep