Somatic cell mapping of the bovine prion protein gene and restriction fragment length polymorphism studies in cattle and sheep

Brains affected by the progressive neurological disease bovine spongiform encephalopathy (BSE) contain scrapie-associated fibrils and the protease-resistant isoform of prion protein. The gene encoding the normal host prion protein (PRNP) has been mapped to human chromosome 20 and mouse chromosome 2 with the hamster cDNA probe pEA974. Using this probe and a panel of bovine-rodent hybrid somatic cells, we have mapped PRNP to bovine syntenic group U11 (100% concordancy). PRNP restriction fragment length polymorphisms (RFLPs) were detected with five of six enzymes (BglII, EcoRI, HindIII, MspI and TaqI) in sheep, in contrast to one of 16 enzymes (HincII) in cattle. Codominant segregation of the bovine HincII RFLP was demonstrated in six backcross pedigrees. While PRNP RFLPs are tightly linked to scrapie incubation period, and consequently susceptibility or resistance to disease in rodents and sheep, the relationship between the PRNP RFLPs and BSE incubation period has not been determined.     

Analysis of polymorphic microsatellites within the bovine and ovine prion protein (PRNP) genes

Twenty-four microsatellite sites with at least three repeats were found in the bovine prion protein gene (PRNP) and 23 in the ovine PRNP gene. Eight microsatellite sites were polymorphic in cattle and six in sheep with up to 10 alleles per site. In many cases allelic DNA fragments had variants in microsatellite sites and in flanking regions. Distances between microsatellite sites in eight genes from cattle and sheep occurred on average every 0.9 kb. The numerous polymorphic microsatellite sites will improve analysis of phylogenetic origin of different PRNP alleles and trait association studies for bovine spongiform encephalopathy (BSE) and scrapie.     

Iatrogenic and sporadic Creutzfeldt-Jakob disease in 2 sisters without mutation in the prion protein gene

ABSTRACT

Human genetic prion diseases have invariably been linked to alterations of the prion protein (PrP) gene PRNP. Two sisters died from probable Creutzfeldt-Jakob disease (CJD) in Switzerland within 14 y. At autopsy, both patients had typical spongiform change in their brains accompanied by punctuate deposits of PrP. Biochemical analyses demonstrated proteinase K-resistant PrP. Sequencing of PRNP showed 2 wild-type alleles in both siblings. Retrospectively, clinical data revealed a history of dural transplantation in the initially deceased sister, compatible with a diagnosis of iatrogenic CJD. Clinical and familial histories provided no evidence for potential horizontal transmission. This observation of 2 siblings suffering from CJD without mutations in the PRNP gene suggests potential involvement of non-PRNP genes in prion disease etiology.     

p.Asn176Lys and p.Met137Thr dimorphisms of the PRNP gene significantly decrease the susceptibility to classical scrapie in ARQ/ARQ sheep

In this study, we investigated the susceptibility to scrapie of Sarda breed sheep carrying the genotype ARQ/ARQ with additional polymorphisms at the PRNP gene. To do this, we examined 256 scrapie-affected sheep and 320 flock-mate negative controls from 24 flocks. Logistic regression analysis demonstrated that sheep carrying the ARQ/ARQ genotype with additional dimorphisms had lower risk of becoming scrapie affected when compared with those with ARQ/ARQ_wildtype genotype. ARQ/ARQ genotypes that were detected with heterozygous or homozygous p.Asn176Lys and p.Met137Thr dimorphisms were associated with the lowest susceptibility to the disease. A significant lower risk was also associated with the p.Arg154His dimorphism, while p.Leu141Phe had a protective effect that was not statistically significant.     

Novel polymorphisms in ovine immune response genes and their association with abortion

The sheep has worldwide agricultural importance, yet the genetic control of the immune responses underlying susceptibility or resistance to ovine disease is little understood. Here, we identify six novel polymorphisms in the ovine immune response genes interferon-γ (IFNG), tumour necrosis factor-α (TNF), interleukin-1β (IL1B) and interleukin-4 (IL4) in pedigree Charollais flocks. We confirm the presence of previously reported polymorphisms in IFNG and IL1B in Charollais. Restriction fragment length polymorphism (RFLP) genotyping assays have been developed for four polymorphisms, IFNGg.168C>T, IFNGg.285A>G, IL1Bg.689C>T and TNFg.3UTRA>G, and a Taqman genotyping assay has been developed for IL4g.485C>T. The previously described IL2g.647C>T polymorphism is adapted for RFLP analysis. Allele frequencies are described in Charollais, Lleyn and Suffolk cross sheep. Polymorphisms are typed in both Charollais ewes and lambs and analysed against abortion phenotypes. A subset of animals have also been analysed for the presence of Toxoplasma gondii, an abortion-causing protozoan. The IFNGg.168T allele is shown to be associated with increased risk of a ewe having an abortion, while the IFNGg.285G allele is associated with increased risk of a lamb being aborted. These assays provide tools for the investigation of the genetic basis of other phenotypes in sheep, including infectious disease susceptibility.     

Creutzfeldt-Jakob disease associated with a V203I homozygous mutation in the prion protein gene

We report a Japanese patient with Creutzfeldt-Jakob disease (CJD) with a V203I homozygous mutation of the prion protein gene (PRNP). A 73-year-old woman developed rapidly progressive gait disturbance and cognitive dysfunction. Four months after the onset, she entered a state of an akinetic mutism. Gene analysis revealed a homozygous V203I mutation in the PRNP. Familial CJD with a V203I mutation is rare, and all previously reported cases had a heterozygous mutation showing manifestations similar to those of typical sporadic CJD. Although genetic prion diseases with homozygous PRNP mutations often present with an earlier onset and more rapid clinical course than those with heterozygous mutations, no difference was found in clinical phenotype between our homozygous case and reported heterozygous cases.     

The first report of polymorphisms and genetic characteristics of the prion protein gene (PRNP) in horses

ABSTRACT

Prion diseases have a wide host range, but prion-infected cases have never been reported in horses. Genetic polymorphisms that can directly impact the structural stability of horse prion protein have not been investigated thus far. In addition, we noticed that previous studies focusing on horse-specific amino acids and secondary structure predictions of prion protein were performed for limited parts of the protein. In this study, we found genetic polymorphisms in the horse prion protein gene (PRNP) in 201 Thoroughbred horses. The identified polymorphism was assessed to determine whether this polymorphism impedes stability of protein using PolyPhen-2, PROVEAN and PANTHER. In addition, we evaluated horse-specific amino acids in horse and mouse prion proteins using same methods. We found only one single nucleotide polymorphism (SNP) in the horse prion protein, and three annotation tools predicted that the SNP is benign. In addition, horse-specific amino acids showed different effects on horse and mouse prion proteins, respectively.

Abbreviations: PRNP: prion protein gene; SNP: single nucleotide polymorphism; CJD: Creutzfeldt-Jakob disease; CWD: chronic wasting disease; TME: transmissible mink encephalopathy; FSE: feline spongiform encephalopathy; MD: molecular dynamics; ER: endoplasmic reticulum; GPI: glycosylphosphatidylinositol; NMR: nuclear magnetic resonance; ORF: open reading frame; GWAS: genome-wide association study; NAPA: non-adaptive prion amplification; HMM: hidden Markov model; NCBI: National Center for Biotechnology Information     

PRNP polymorphisms in Chinese ovine, caprine and bovine breeds

Transmissible spongiform encephalopathy (TSE) is a neurodegenerative disorder in humans and animals. Polymorphisms and mutations in the prion protein (PRNP) gene have been associated with the incidence of natural and experimental TSE and the incubation period length. In this study, we determined PRNP polymorphisms in Chinese ovine, caprine and bovine breeds using 400 samples from 13 ovine and caprine breeds and 250 samples from nine bovine breeds. In the ovine and caprine PRNP gene, we found five previously unreported amino acid polymorphisms and two silent nucleotide alterations. In bovine PRNP, we found eight previously unreported amino acid polymorphisms and six silent nucleotide alterations.     

Cellular prion protein in ovine milk

Cellular prion protein, PrP(C), is essential for the development of prion diseases where it is considered to be a substrate for the formation of the disease-associated conformer, PrP(Sc). In sheep, PrP(C) is abundant in neuronal tissue and is also found at lower concentrations in a range of non-neuronal tissues, including mammary gland. Here, we demonstrate the presence of soluble PrP(C) in the non-cellular, non-lipid fraction of clarified ovine milk. Compared with brain-derived PrP(C), ovine milk PrP(C) displays an increased electrophoretic mobility. Ovine milk PrP(C) is mainly present as three species that differ in the extent of their N-linked glycosylation, with glycoform profiles varying among animals. Similar PrP(C) species are also present in fresh and commercial homogenised/pasteurised bovine milk, with additional N-terminal PrP(C) fragments detectable in ruminant milk and commercial milk products.     

The first report of genetic variations in the chicken prion protein gene

ABSTRACT

Abnormal structural changes of the prion protein (PrP) are the cause of prion disease in a wide range of mammals. However, spontaneous infected cases have not been reported in chicken. Genetic variations of the prion protein gene (PRNP) may impact susceptibility to prion disease but have not been investigated thus far. Because an investigation of the chicken PRNP can improve the understanding of characteristics related to resistance to prion disease, research on the chicken PRNP is highly desirable. In this study, we investigated the genetic characteristics of the chicken PRNP gene. For this, we performed direct sequencing in 106 Dekalb White chickens and analyzed the genotype and allele frequencies of chicken PRNP gene. We found two insertion and deletion polymorphisms in the chicken PRNP: c.163_180delAACCCAGGGTACCCCCAT and c.268_269insC. The former is a U2 hexapeptide deletion polymorphism. Of the 106 samples, 13 (12.26%) were insertion homozygotes, 89 (83.96%) were heterozygotes, and 4 (3.77%) were deletion homozygotes in c.163_180delAACCCAGGGTACCCCCAT. In the c.268_269insC polymorphism, 102 (96.23%) were deletion homozygotes, and 4 (3.77%) were heterozygotes. Insertion homozygotes of c.268_269insC were not detected. Two polymorphisms were in perfect linkage disequilibrium (LD) with a D’ value of 1.0, and three haplotypes were identified. Furthermore, PROVEAN evaluates 163_180delAACCCAGGGTACCCCCAT as ‘deleterious’ with a score of – 13.173. Furthermore, single nucleotide polymorphisms (SNPs) in the open reading frame (ORF) of the PRNP gene were not found in the chicken. To the best of our knowledge, this was the first report on the genetic variations of the chicken PRNP gene.     

Genetic effects of PRNP gene insertion/deletion (indel) on phenotypic traits in sheep

Prion protein (PRNP) gene is well known for affecting mammal transmissible spongiform encephalopathies (TSE), and is also reported to regulate phenotypic traits (e.g. growth traits) in healthy ruminants. To identify the insertion/deletion (indel) variations of the PRNP gene and evaluate their effects on growth traits, 768 healthy individuals from five sheep breeds located in China and Mongolia were identified and analyzed. Herein, four novel indel polymorphisms, namely, Intron-1-insertion-7bp (I1-7bp), Intron-2-insertion-15bp (I2-15bp), Intron-2-insertion-19bp (I2-19bp), and 3′ UTR-insertion-7bp (3′ UTR-7bp), were found in the sheep PRNP gene. In five analyzed breeds, the minor allelic frequencies (MAF) of the above indels were in the range of 0.008 to 0.986 (I1-7bp), 0.113 to 0.336 (I2-15bp), 0.281 to 0.510 (I2-19bp), and 0.040 to 0.238 (3′ UTR-7bp). Additionally, there were 15 haplotypes and the haplotype ‘I_I2-15bp-D_3’UTR-7bp-D_I2-19bp-D_I1-7bp’ had the highest frequency, which varied from 0.464 to 0.629 in five breeds. Moreover, association analysis revealed that all novel indel polymorphisms were significantly associated with 13 different growth traits (P < 0.05). Particularly, the influences of I2-15bp on chest width (P = 0.001) in Small Tail Han sheep (ewe), 3′ UTR-7bp on chest circumference (P = 0.003) in Hu sheep, and I2-19bp on tail length (P = 0.001) in Tong sheep, were highly significant (P < 0.01). These findings may be a further step toward the detection of indel-based typing within and across sheep breeds, and of promising target loci for accelerating the progress of marker-assisted selection in sheep breeding.     

Allelic variants of ovine prion protein gene (PRNP) in Oklahoma sheep

1,144 sheep belonging to 21 breeds and known crosses were sequence analyzed for polymorphisms in the ovine PRNP gene. Genotype and allele frequencies of polymorphisms in PRNP known to confer resistance to scrapie, a fatal neurodegenerative disease of sheep, are reported. Known polymorphisms at codons 136 (A/V), 154 (H/R) and 171 (Q/R/H/K) were identified. The frequency of the 171R allele known to confer resistance to type C scrapie was 53.8% and the frequency of the 136A allele known to influence the resistance to type A scrapie was 96.01%. In addition, we report the identification of five new polymorphisms at codons 143 (H/R), 167 (R/S), 180 (H/Y), 195 (T/S) and 196 (T/S). We also report the identification of a novel allele (S/R) at codon 138.     

Genetic linkage analysis between protein polymorphisms and the FecB major gene in sheep

A genetically linked marker locus is sought for the Booroola gene (FecB), a major gene which confers increased prolificacy in sheep. We examined 18 polymorphic proteins in sheep and found 10 to be informative in half-sib families where the Booroola gene was segregating. Recombination was observed between each of the protein loci and the Booroola gene. The loci and exclusion distance for each (calculated as the recombination fraction where the lod score was equal to -2.0) are as follows: NADH diaphorase, DIA1 (9.2 cM); arylesterase, EsA (11.9 cM); haemoglobin beta chain, HBB (17.5 cM); leucine amino peptidase, LAP (19.7 cM); malic enzyme, ME1 (14.8 cM); ovine plasminogen antigen, OPA (12.6 cM); alpha-1-protease inhibitor, PI2 (5.7 cM), erythrocyte 'X' protein, Prot-X (25.3 cM); post transferrin, PTF (2.2 cM); transferrin, TF (33.8 cM).     

Prion protein gene (PrP) polymorphisms in healthy sheep in Turkey

Scrapie, a transmissible spongiform encephalopathy (TSE) or prion disease, is a fatal, neurodegenerative disease in sheep and goats. This disease has been known in Europe for more than 250 years. Susceptibility to scrapie is associated with polymorphisms in the sheep prion protein gene (PrP) gene. In sheep, polymorphism in the PrP gene has been identified at a number of codons, and polymorphisms at codons 136, 154 and 171 have reported linkage with susceptibility to scrapie. Polymorphisms at the PrP locus were studied in 413 animals representing three native sheep breeds (Imroz, Chios and Kıvırcık) in Turkey. Genomic DNA was obtained from blood, and genotypes were screened using PCR and direct DNA sequencing. We report 17 genotypes derived from seven different alleles. The most frequent genotype in the Kıvırcık sheep is ARQ/ARQ, whereas the ARR/ARQ genotype is predominant in the Chios and Imroz breeds. In general, the ARQ haplotype was the predominant haplotype. ARQ haplotype was also predominant in the Kıvırcık and Chios sheep breeds, whereas the Imroz sheep predominantly had the ARR haplotype. The susceptibility-associated VRQ haplotype was found in 2.38%, 0.35% and 0.81% of the Imroz, Kıvırcık and Chios sheep, respectively. Moreover, seven additional polymorphisms have been detected at codons G127S, G127V, H143R, G145S, Y172D, N174Y and Q189L. Among these polymorphisms, the N174Y allele is a novel polymorphism, and the G145S allele is a novel allele for a known polymorphic locus.     

Prion protein scrapie and the normal cellular prion protein

Prions are infectious proteins and over the past few decades, some prions have become renowned for their causative role in several neurodegenerative diseases in animals and humans. Since their discovery, the mechanisms and mode of transmission and molecular structure of prions have begun to be established. There is, however, still much to be elucidated about prion diseases, including the development of potential therapeutic strategies for treatment. The significance of prion disease is discussed here, including the categories of human and animal prion diseases, disease transmission, disease progression and the development of symptoms and potential future strategies for treatment. Furthermore, the structure and function of the normal cellular prion protein (PrP^C) and its importance in not only in prion disease development, but also in diseases such as cancer and Alzheimer's disease will also be discussed.     

PCR-based detection of the polymorphism at codon 136 in the ovine prion protein gene

In many breeds of sheep, a polymorphism at codon 136 of the prion protein gene has been shown to be strongly associated with the risk of developing scrapie. A single-step procedure for detecting this allelic variation is described here. When performed on a series of animals, the test was in complete agreement with their genotypes as had been previously determined by sequencing. The test is potentially easier and quicker to perform than any of the variety of methods that are currently used for this purpose.     

The prion protein M129V polymorphism

The PRNP gene encodes the cellular isoform of prion protein (PrP^c). The M129V polymorphism influences the risk of prion diseases and may modulate the rate of neurodegeneration with age. We present the first study of the polymorphism among Polish centenarians. In the control group (n = 165, ages 18 to 56 years) the observed M129V genotype frequencies agreed with those expected according to the Hardy-Weinberg equilibrium (MM, MV, VV): 43%, 44%, 13% (HWE p > 0.05). Among centenarians (n = 150, ages 100 to 107) both homozygotes were more common than expected and HWE was rejected: 46%, 37%, 17% (expected 42%, 46%, 13%; HWE p = 0.025). This finding is consistent with a higher mortality rate among heterozygotes. However, the observed allele and genotype frequencies did not differ significantly between the oldest-old and the young controls. The genotypic frequencies were not related to severe cognitive impairment among the centenarians.