Prion Protein Gene Sequence Research Articles

The first report of prion protein gene sequences in Dybowski’s frog and the American bullfrog: high amyloid propensity of the frog prion protein

Prion diseases are fatal infectious neurodegenerative diseases caused by the proteinase K-sensitive form of prion protein (PrPSc). The exact origin of prion seeding and the transition factor of PrPSc has not been elucidated. The main hosts of prion diseases are herbivores, so the feces and corpses of Amphibians can seed PrPSc through ecosystems. The frog is an excellent candidate for transmission studies for this reason, but genetic analyses of the prion protein gene (PRNP) in the context of prion-related characteristics of frog species are lacking. We amplified frog PRNP gene sequences in Dybowski’s frog and the American bullfrog by polymerase chain reaction (PCR) and amplicon sequencing. In addition, we carried out multiple sequencing alignments and annotated major PrP components including signal peptide, tandem repeat domain, and PrPC-PrPSc interaction region of frog PrPs by bioinformatics tools. We predicted secondary and tertiary structures and amyloid propensities of frog PrPs using AlphaFold2 and AMYCO, respectively. We obtained DNA sequences of the PRNP gene in Dybowski’s frog and the American bullfrog, as well as a partially conserved palindromic sequence (PrPC-PrPSc interaction region) and absence of tandem repeat region of PrP in seven frog species. We analyzed protein structure of among these frog species and found that the high Himalaya frog has high aggregation propensity and the western clawed frog does not have the N-terminal signal peptide. To the best of our knowledge, this was the first comparative genetic study regarding prion-related features of frog species.

Influence of the geographic distribution of prion protein gene sequence variation on patterns of chronic wasting disease spread in white-tailed deer (Odocoileus virginianus)

ABSTRACTManaging and controlling the spread of diseases in wild animal populations is challenging, especially for social and mobile species. Effective management benefits from information about disease susceptibility, allowing limited resources to be focused on areas or populations with a higher risk of infection. Chronic wasting disease (CWD), a transmissible spongiform encephalopathy that affects cervids, was detected in Colorado in the late 1960s. CWD was detected in Illinois and Wisconsin in 2002 and has since spread through many counties. Specific nucleotide variations in the prion protein gene (PRNP) sequence have been associated with reduced susceptibility to CWD in white-tailed deer. Though genetic resistance is incomplete, the frequency of deer possessing these mutations in a population is an important factor in disease spread (i.e. herd immunity). In this study we sequenced 625 bp of the PRNP gene from a sampling of 2433 deer from Illinois and Wisconsin. In north-central Illinois where CWD was first detected, counties had a low frequency of protective haplotypes (frequency <0.20); whereas in northwestern Illinois counties, where CWD cases have only more recently been detected, the frequency of protective haplotypes (frequency >0.30) was much higher (p < 0.05). Protective haplotype frequencies varied significantly among infected and uninfected geographic areas. The frequency of protective PRNP haplotypes may contribute to population level susceptibility and may shape the way CWD has spread through Illinois. Analysis of PRNP haplotype distribution could be a useful tool to assess CWD risk and allocate resources to contain and reduce the spread of infection.

Neuropathology of Human Prion Diseases.

The human prion diseases comprise sporadic, genetic, and acquired disorders. These are rare conditions with a heterogeneous clinicopathologic phenotype, which can make diagnosis challenging. A combined clinical, genetic, neuropathologic and biochemical approach to diagnosis is therefore essential. Since prion infectivity is the highest in tissues from the central nervous system, special laboratory precautions are required for the safe handling of these tissues. Neuropathologic assessment is generally performed following autopsy, when the fixed brain should be adequately sampled and studied by conventional stains and immunohistochemistry for the abnormal form of the prion protein. Frozen brain tissue is also required for DNA extraction for prion protein gene sequencing and for Western blot analysis of protease-resistant prion protein. The microscopic assessment of the nature and degree of spongiform change, neuronal loss, gliosis, and abnormal prion protein deposition in the brain can be used to determine the major categories of human prion disease. This information can be combined with clinical, genetic data, and biochemical data to allow an accurate diagnosis of a human prion disease and facilitates subclassification into recognized disease subtypes, for example in sporadic Creutzfeldt-Jakob disease. The spectrum of human prion diseases continues to expand and neuropathology will play a key role in the recognition and understanding of any further novel entities or disease variants that may emerge in the future.

Manipulating the Prion Protein Gene Sequence and Expression Levels with CRISPR/Cas9.

The mammalian prion protein (PrP, encoded by Prnp) is most infamous for its central role in prion diseases, invariably fatal neurodegenerative diseases affecting humans, food animals, and animals in the wild. However, PrP is also hypothesized to be an important receptor for toxic protein conformers in Alzheimer's disease, and is associated with other clinically relevant processes such as cancer and stroke. Thus, key insights into important clinical areas, as well as into understanding PrP functions in normal physiology, can be obtained from studying transgenic mouse models and cell culture systems. However, the Prnp locus is difficult to manipulate by homologous recombination, making modifications of the endogenous locus rarely attempted. Fortunately in recent years genome engineering technologies, like TALENs or CRISPR/Cas9 (CC9), have brought exceptional new possibilities for manipulating Prnp. Herein, we present our observations made during systematic experiments with the CC9 system targeting the endogenous mouse Prnp locus, to either modify sequences or to boost PrP expression using CC9-based synergistic activation mediators (SAMs). It is our hope that this information will aid and encourage researchers to implement gene-targeting techniques into their research program.

Prion protein gene sequence and chronic wasting disease susceptibility in white-tailed deer (Odocoileus virginianus)

ABSTRACTThe sequence of the prion protein gene (PRNP) affects susceptibility to spongiform encephalopathies, or prion diseases in many species. In white-tailed deer, both coding and non-coding single nucleotide polymorphisms have been identified in this gene that correlate to chronic wasting disease (CWD) susceptibility. Previous studies examined individual nucleotide or amino acid mutations; here we examine all nucleotide polymorphisms and their combined effects on CWD. A 626 bp region of PRNP was examined from 703 free-ranging white-tailed deer. Deer were sampled between 2002 and 2010 by hunter harvest or government culling in Illinois and Wisconsin. Fourteen variable nucleotide positions were identified (4 new and 10 previously reported). We identified 68 diplotypes comprised of 24 predicted haplotypes, with the most common diplotype occurring in 123 individuals. Diplotypes that were found exclusively among positive or negative animals were rare, each occurring in less than 1% of the deer studied. Only one haplotype (C, odds ratio 0.240) and 2 diplotypes (AC and BC, odds ratios of 0.161 and 0.108 respectively) has significant associations with CWD resistance. Each contains mutations (one synonymous nucleotide 555C/T and one nonsynonymous nucleotide 286G/A) at positions reported to be significantly associated with reduced CWD susceptibility. Results suggest that deer populations with higher frequencies of haplotype C or diplotypes AC and BC might have a reduced risk for CWD infection – while populations with lower frequencies may have higher risk for infection. Understanding the genetic basis of CWD has improved our ability to assess herd susceptibility and direct management efforts within CWD infected areas.

Genetic and Pathological Follow-Up Study of Goats Experimentally and Naturally Exposed to a Sheep Scrapie Isolate.

Thirty-seven goats carrying different prion protein genotypes (PRNP) were orally infected with a classical scrapie brain homogenate from wild-type (ARQ/ARQ) sheep and then mated to obtain 2 additional generations of offspring, which were kept in the same environment and allowed to be naturally exposed to scrapie. Occurrence of clinical or subclinical scrapie was observed in the experimentally infected goats (F0) and in only one (F1b) of the naturally exposed offspring groups. In both groups (F0 and F1b), goats carrying the R154H, H154H, R211Q, and P168Q-P240P dimorphisms died of scrapie after a longer incubation period than wild-type, G37V, Q168Q-P240P, and S240P goats. In contrast, D145D and Q222K goats were resistant to infection. The immunobiochemical signature of the scrapie isolate and its pathological aspects observed in the sheep donors were substantially maintained over 2 goat generations, i.e., after experimental and natural transmission. This demonstrates that the prion protein gene sequence, which is shared by sheep and goats, is more powerful than any possible but unknown species-related factors in determining scrapie phenotypes. With regard to genetics, our study confirms that the K222 mutation protects goats even against ovine scrapie isolates, and for the first time, a possible association of D145 mutation with scrapie resistance is shown. In addition, it is possible that the sole diverse frequencies of these genetic variants might, at least in part, shape the prevalence of scrapie among naturally exposed progenies in affected herds. This study was aimed at investigating the genetic and pathological features characterizing sheep-to-goat transmission of scrapie. We show that in goats with different prion protein gene mutations, the K222 genetic variant is associated with scrapie resistance after natural and experimental exposure to ovine prion infectivity. In addition, we observed for the first time a protective effect of the D145 goat variant against scrapie. Importantly, our results demonstrate that the phenotypic characteristic of the wild-type sheep scrapie isolate is substantially preserved in goats carrying different susceptible PRNP gene variants, thus indicating that the prion protein gene sequence, which is shared by sheep and goats, plays a fundamental role in determining scrapie phenotypes.

Correlation between the Insertion/Deletion Mutations of Prion Protein Gene and BSE Susceptibility and Milk Performance in Dairy Cows

Abstract Objective To investigate the 23 bp and 12 bp insertion/deletion (indel) mutations within the bovine prion protein (PRNP) gene in Chinese dairy cows, and to detect the associations of two indel mutations with BSE susceptibility and milk performance. Methods Based on bovine PRNP gene sequence, two pairs of primers for testing the 23 bp and 12 bp indel mutations were designed. The PCR amplification and agarose electrophoresis were carried out to distinguish the different genotypes within the mutations. Moreover, based on previous data from other cattle breeds and present genotypic and allelic frequencies of two indels mutations in this study, the corrections between the two indel mutations and BSE susceptibility were tested, as well as the relationships between the mutations and milk performance traits were analyzed in this study based on the statistical analyses. Results In the analyzed Chinese Holstein population, the frequencies of two “del” alleles in 23 bp and 12 bp indel muations were more frequent. The frequency of haplotype of 23del-12del was higher than those of 23del- 12ins and 23ins-12del. From the estimated r2 and D’ values, two indel polymorphisms were linked strongly in the Holstein population (D’ = 57.5%, r2 = 0.257). Compared with the BSE-affected cattle populations from the reported data, the significant differences of genotypic and allelic frequencies were found among present Holstein and some BSE-affected populations (P &lt; 0.05 or P &lt; 0.01). Similarly, there were significant frequency distribution differences of genotypes and alleles among Chinese Holstein and several previous reported healthy dairy cattle (P &lt; 0.05 or P &lt; 0.01). Moreover, association of genotype and combined genotypes of two indel polymorphisms with milk performance and resistant mastitis traits were analyzed in Holstein population, but no significant differences were found (P &gt; 0.05). Conclusions These observations revealed that the influence of two indel mutations within the bovine PRNP gene on BSE depended on the breed and they did not affect the milk production traits, which layed the foundation for future selection of resistant animals, and for improving health conditions for dairy breeding against BSE in China.

Prion protein gene polymorphism in four West African sheep populations

A total of 162 individuals, belonging to three Burkinabé and one Niger sheep populations, were analysed for prion protein (PrP) gene polymorphism at codons 136, 154 and 171. The ARQ allele was the most frequent in both the Burkinabé (86.7%) and the Niger (67.5%) sheep populations. The highly sensitive allele VRQ was not found in the sampled individuals. The highly resistant ARR allele was in very low frequency in the Burkina-Sahel (4.4%) and Mossi (3.2%) populations and was not present in the Djallonké and Touareg populations. Only 4 out of 15 possible PrP genotypes were identified in the sampled individuals. No favourable ARR/ARR genotypes were found in either of the breeds. Sequencing a subgroup of the samples allowed the identification of other five polymorphisms on the PrP gene sequence at codons 116, 138, 151, 237 and 240. The very low frequency of the ARR allele in the West African sheep should dissuade the implementation of a preventive selection programme aimed to increase resistance to scrapie, to avoid an extreme erosion of the genetic stock.

Detection of Four Novel Polymorphisms in PrP gene of Pakistani sheep (Damani and Hashtnagri) and goats (Kamori and Local Hairy) breeds

Scrapie is a fatal neurodegenerative disorder of sheep and goats caused by post-translational conformational change in the host-encoded prion protein (PrPC). Susceptibility or resistance to scrapie has been associated with the presence of polymorphisms in the prion protein (PrP) gene. In the present study, we analyzed the PrP gene sequence to determine the frequency of polymorphisms in 56 sheep (28 each from Damani and Hashtnagri breeds) and 56 goats (28 each from Kamori and Local Hairy breeds). A total of 7 amino acid polymorphisms were detected in the PrP gene for sheep and 4 for goats. These amino acid polymorphisms were combined in 13 alleles and 15 genotypes in sheep and 5 alleles and 6 genotypes in goats. The overall frequency of the most sheep scrapie-resistant polymorphism (Q171R) was calculated to be 0.107. The most scrapie-susceptible polymorphism (A136V) was not detected in any of the studied sheep. The overall frequency of scrapie-associated polymorphism (H143R) in goats was found to be 0.152. Along with already known amino acid polymorphisms, two novel polymorphisms were also detected for each of sheep (Q171N and T191I) and goats (G22C and P63L). However, the overall frequency of these polymorphisms was extremely low.

PRNP genetic variability and molecular typing of natural goat scrapie isolates in a high number of infected flocks

One hundred and four scrapie positive and 77 negative goats from 34 Greek mixed flocks were analysed by prion protein gene sequencing and 17 caprine scrapie isolates from 11 flocks were submitted to molecular isolate typing. For the first time, the protective S146 variant was reported in Greece, while the protective K222 variant was detected in negative but also in five scrapie positive goats from heavily infected flocks. By immunoblotting six isolates, including two goat flockmates carrying the K222 variant, showed molecular features slightly different from all other Greek and Italian isolates co-analysed, possibly suggesting the presence of different scrapie strains in Greece.

PRNP allelic series from 19 years of prion protein gene sequencing at the MRC Prion Unit

Mutation of the human prion protein gene (PRNP) open reading frame (ORF) accounts for almost all reported familial concurrence of prion disease. The more common mutations globally: octapeptide repeat insertions, P102L, D178N, E200K, and V210I have occurred in large multigenerational pedigrees and display autosomal dominant inheritance, however, many rare genetic changes have been reported that are of uncertain pathogenicity. Based on 19 years of PRNP sequencing at the MRC Prion Unit, London, and analysis of 3664 samples from patients referred with suspected prion disease and healthy populations, we present novel allele combinations, healthy control population data, results of screening the PRNP ORF in DNA from the entire referral series and the CEPH human genome diversity cell line panel. Of the 10 alleles detected in patients for which detailed cases histories are presented, 4 are unreported (G54S, D167N, V209M, Q212PP), two changes are thought to be pathogenic but have not been described in our regions (P105L from the UK, G114V from India and Turkey), and the remainder reported in healthy control populations or in trans to known pathogenic mutations suggesting non- or low pathogenicity (G54S, 1-OPRI, G142S, N171S, V209M, E219K). New genotype-phenotype correlations and population frequencies presented will help the diagnosis and genetic counselling of those with suspected inherited prion disease.

Structure of the Prion Protein and Its Gene: An Analysis Using Bioinformatics and Computer Simulation

Prion protein (PrP) gene encodes cellular PrP (PrPC), a glycosylphosphatidylinositol (GPI)-anchored cell membrane protein indispensable for infections of prion, which causes Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep. Although PrPC is known to be converted into an abnormal isoform (PrPSc) upon prion infection and play an important role in prion diseases, the mechanisms involved remain unclear, partly due to the insolubility of PrPSc, which prevents experimental biochemical and biophysical analyses. Recently, with improvements in computer power and methods, computer analyses have been contributing more to prion studies. A comparison of PrP gene sequences revealed mutations and polymorphisms in the open reading frame (ORF) of the human PrP gene related to prion diseases. In contrast, little mutations or polymorphisms related to susceptibility to BSE were found in the ORF of the bovine PrP gene, though relationships between insertion/deletion (Ins/Del) polymorphisms of the PrP gene promoter and susceptibility to BSE have been found. Our results have shown that the specific protein 1 (Sp1) plays important role in the activity of PrP gene promoter, which is influenced by polymorphisms in the Sp1 binding sites. The potential structural dynamics of PrP have been simulated by computational methods such as molecular dynamics (MD) and quantum mechanics (QM). The proposed mechanisms of conversion have revealed new insights in prion diseases. In this review, we will introduce the gene structure, polymorphisms, and potential structural dynamics of PrP revealed by basic and advanced computational analyses. The possible contribution of these methods to elucidation of the pathogenicity of prion diseases and functions of PrPC is discussed.

Creutzfeldt–Jakob disease with E200K PRNP mutation: a case report and revision of the literature

Creutzfeldt-Jakob disease (CJD) is typically characterized by rapidly progressive dementia and myoclonus, and it is caused by a conformational change of the prion protein. The heritable forms are associated with mutation in the gene encoding the prion protein (PRNP). We report a 63-year-old Italian woman harboring the E200K PRNP mutation. Electroencephalogram, cerebrospinal fluid analysis, PRNP gene sequencing, histopathologic examination, immunohistochemical studies, and Western blotting analysis confirmed the diagnosis of CJD. Pyramidal involvement was the first sign and the prominent clinical feature. Later on, she developed also myoclonus, ataxia, spastic tetraplegia, and at last dementia with akinetic mutism. Usually, signs of degeneration of the pyramidal tracts occur in a small number of patients as the disease advances. Our report supports the variability of the clinical expression of the E200K genetic CJD. Further studies are needed to understand the molecular basis underlying the phenotypic variability among patients carrying this mutation.

Identification and Characterization of two Bovine Spongiform Encephalopathy cases Diagnosed in the United States

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy of cattle, first detected in 1986 in the United Kingdom and subsequently in other countries. It is the most likely cause of variant Creutzfeldt-Jakob disease (vCJD) in humans, but the origin of BSE has not been elucidated so far. This report describes the identification and characterization of two cases of BSE diagnosed in the United States. Case 1 (December 2003) exhibited spongiform changes in the obex area of the brainstem and the presence of the abnormal form of the prion protein, PrP(Sc), in the same brain area, by immunohistochemistry (IHC) and Western blot analysis. Initial suspect diagnosis of BSE for case 2 (November 2004) was made by a rapid ELISA-based BSE test. Case 2 did not exhibit unambiguous spongiform changes in the obex area, but PrP(Sc) was detected by IHC and enrichment Western blot analysis in the obex. Using Western blot analysis, PrP(Sc) from case 1 showed molecular features similar to typical BSE isolates, whereas PrP(Sc) from case 2 revealed an unusual molecular PrP(Sc) pattern: molecular mass of the unglycosylated and monoglycosylated isoform was higher than that of typical BSE isolates and case 2 was strongly labeled with antibody P4, which is consistent with a higher molecular mass. Sequencing of the prion protein gene of both BSE-positive animals revealed that the sequences of both animals were within [corrected] the range of the prion protein gene sequence diversity previously reported for cattle.

Comparative analysis of the prion protein ( PrP) gene in cetacean species

The partial PrP gene sequence and the deduced protein of eight cetacean species, seven of which have never been reported so far, have been determined in order to extend knowledge of sequence variability of the PrP genes in different species and to aid in speculation on cetacean susceptibility to prions. Both the nucleotide and the deduced amino acid sequences have been analysed in comparison with some of the known mammalian PrPs. Cetacean PrPs present typical features of eutherian PrPs. The PrP gene from the species of the family Delphinidae gave identical nucleic acid sequences, while differences in the PrP gene were found in Balaenopteridae and Ziphidae. The phylogenetic tree resulting from analysis of the cetacean PrP gene sequences, together with reported sequences of some ungulates, carnivores and primates, showed that the PrP gene phylogenesis mirrors the species phylogenesis. The PrP gene of cetaceans is very close to species where natural forms of TSEs are known. From an analysis of the sequences and the phylogenesis of the PrP gene, susceptibility to or occurrence of prion diseases in cetaceans can not be excluded.

Comparative analysis of the prion protein gene sequences in African lion

The prion protein gene of African lion (Panthera Leo) was first cloned and polymorphisms screened. The results suggest that the prion protein gene of eight African lions is highly homogenous. The amino acid sequences of the prion protein (PrP) of all samples tested were identical. Four single nucleotide polymorphisms (C42T, C81A, C420T, T600C) in the prion protein gene (Prnp) of African lion were found, but no amino acid substitutions. Sequence analysis showed that the higher homology is observed to felis catus AF003087 (96.7%) and to sheep number M31313.1 (96.2%) Genbank accessed. With respect to all the mammalian prion protein sequences compared, the African lion prion protein sequence has three amino acid substitutions. The homology might in turn affect the potential intermolecular interactions critical for cross species transmission of prion disease.

Amino acid sequence of the Felis catus prion protein

A new determination of the house cat ( Felis catus) prion protein gene sequence (f Prnp), which has so far been subject of controversy, is reported. The newly determined f Prnp sequence is similar to dog ( Canis familiaris) and mink ( Mustela putorius) Prnp, but differs significantly from both f Prnp sequences that were previously deposited in the GenBank. Comparison of the canine and feline prion protein sequences suggests a set of amino acid replacements relative to bovine PrP that might relate to the observed different susceptibilities of the two species to TSE infection by ingestion of BSE-infected beef.

Additions and corrections: Prion protein gene sequence of Canada's first non-imported case of bovine spongiform encephalopathy (BSE)

On page 1006 of the above-cited article, reference is made to a 2001 report by Horn et al. (Horn, G., Bobrow, M., Bruce, M., Goedert, M., McLean, A., and Webster, J. 2001. Review of the origin of BSE: report to United Kingdom Government. http://147.46.94.112/BSE/bse0927.pdf), with the intention of directing the reader to the most recently published broad discussion of various questions surrounding the origins of BSE, including but not limited to the occurrence of a spontaneous mutation in cattle. Unfortunately, the single reference might suggest incorrectly that it was in this report that the hypothesis of the origin of BSE via spontaneous mutation in cattle was first advanced. In fact, it was in the Report of the BSE Inquiry (http://www.bseinquiry.gov.uk; see Vol. 2, paragraph 7.3) that the idea was first fully articulated. The reader is directed to the Report to obtain a more complete background discussion on the novel-mutation hypothesis, in addition to detailed consideration of alternative hypotheses.

Prion protein gene sequence of Canada's first non-imported case of bovine spongiform encephalopathy (BSE).

In May 2003, Canada became the 22nd country outside of the United Kingdom to report a case of bovine spongiform encephalopathy (BSE) in an animal not known to be imported from a country with cattle previously affected by this fatal, transmissible prion disease. Despite extensive testing of thousands of other animals that may have been exposed to contaminated feed at the same time as the affected animal, no evidence has been found for other infections. This finding leaves room for conjectures that the single confirmed case arose spontaneously, perhaps (by analogy with human Creutzfeldt-Jakob disease) as a result of a somatic protein misfolding event or a novel germline mutation. Here we present DNA sequence data from the affected animal's prion protein coding sequence that argue definitively against the latter hypothesis.

Comparative Analysis of the Prion Protein Open Reading Frame Nucleotide Sequences of Two Wild Ruminants, the Moufflon and Golden Takin

The prion protein (PrP) nucleotide sequences of two ruminants were determined in order to elucidate the differences in susceptibility to spongiform encephalopathy agents in each species. The nucleotide sequences of PrP coding regions of the moufflon and the golden takin encompassed 771 bp in length. The PrP gene sequences of the golden takin were closely related to those of sheep with one amino acid difference. The PrP gene sequence of the moufflon was identical to that of sheep. The similarities between the PrP genes of these two animals and sheep imply that the species barriers between these animals are small or non-existent. These PrP genes could be used to establish transgenic mice with higher susceptibility to prion-related diseases.