Associations between genotypes at codon 171 and 136 of the prion protein gene and production traits in market lambs

OBJECTIVE: To determine whether selection for the homozygous A136 R171 genotype that confers resistance to classic scrapie infection negatively affects production traits in sheep. ANIMALS: 996 commercial lambs obtained from 2 flocks at separate locations across 3 consecutive years. Procedures-Genotyping at codon 136 and 171 was performed by use of commercially available testing or a single-nucleotide polymorphism assay. Carcass data were collected without knowledge of genotype approximately 24 hours after slaughter by an experienced grader. The model to analyze associations between prion protein (PRNP) genotype and production traits was based on genotype, breed, or both as fixed effects and days on feed as a covariate. RESULTS: Average daily gain was significantly associated with only combined codons 136 and 171. In flock 1, weaning average daily gain was significantly greater in AA136 sheep than heterozygotes; the difference between QR171 and RR171 sheep, compared with QQ171 sheep, were not significant although QR171 and RR171 sheep had higher values. However, in flock 2, average daily gain was significantly greater in AV136 sheep than AA136 sheep and in QR171 sheep than QQ171 sheep. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggest there is an advantage for average daily gain in lambs with an arginine allele at codon 171, but there were no other genotype effects on production traits. Thus, selection for the resistant arginine allele at codon 171 to comply with USDA scrapie eradication guidelines should not be detrimental to lamb production in commercial flocks. Effects of codon 136 on average daily gain were ambiguous.     

Allelic polymorphism in the ovine DQA1 gene

Variation in the ovine DQA1 gene was investigated by amplification of exon 2 using PCR, followed by single-strand conformational polymorphism (SSCP) analysis, cloning, and DNA sequencing. Fourteen novel SSCP patterns, representing 14 different sequences, were identified. Eight of these 14 sequences were identical to published DQA1 sequences from sheep, whereas the remaining six were novel but similar to the published DQA1 sequences from sheep and cattle. These six new sequences exhibited conserved region and variable region patterns similar to the published sheep DQA1 sequences, but were different than the published DQA2 sequences from sheep. All of these 14 putative sheep DQA1 sequences fulfilled the criteria used by the established bovine leukocyte antigens major histocompatibility complex nomenclature committee for assignment as new alleles. Comparison of the available DQA1 sequences from sheep and cattle revealed several clusters of ovine DQA1 sequences, and some sheep alleles were more similar to cattle alleles than other sheep alleles. The occurrence of trans-species polymorphism suggests the action of balancing selection at the DQA1 locus. Twenty-four percent of the nucleotide positions showed variation within exon 2, and this variation seems to have arisen largely by point mutation and gene conversion. The nonsynonymous and synonymous substitution rates were similar in both the putative antigen-binding site codons and the putative nonantigen-binding site codons. The extensive polymorphism reported in this article is consistent with polymorphism reported at the bovine DQA1 locus.     

Development and usefulness of new polymerase chain reaction-based tests for detection of different alleles at codons 136 and 171 of the ovine prion protein gene.

OBJECTIVE: To develop new and improved tests to detect alleles at codons 136 and 171 of the ovine prion protein locus and to evaluate the frequency of these alleles. ANIMALS: 159 Suffolk sheep belonging to 3 flocks. PROCEDURE: Polymerase chain reaction (PCR) analysis that contained diagnostic restriction site variation for each allele were developed for the relevant gene regions. Alleles were determined by analyzing DNA isolated from buccal swab specimens or blood samples. RESULTS: At codon 136, frequencies of the alanine and valine alleles were found to be 97 and 3%, respectively. At codon 171, frequencies of the glutamine, arginine, and histidine alleles were found to be 57, 41, and 2%, respectively. CONCLUSIONS: Little variation was detected in codon 136, whereas noteworthy variation was found in codon 171; > 40% of the alleles at this locus coded for glutamine. Because the glutamine allele at codon 171 confers susceptibility to scrapie, reduction of its frequency is of importance to management of sheep flocks. CLINICAL RELEVANCE: Genotyping of sheep, using the tests reported here, should facilitate selective breeding programs designed to decrease the risk of scrapie.     

Prion genotypes of scrapie-infected Canadian sheep 1998-2008

This report describes the genetics of the prion protein gene (PRNP) at codons 136, 154, and 171 for sheep diagnosed with naturally acquired classical scrapie in Canada between 1998 and 2008. Genotyping analysis was performed on 249 sheep with confirmed classical scrapie infection representing 98 flocks from 6 provinces. A further case-control analysis of 3 of these flocks compared the genotypes between infected sheep (n = 72) and those of their healthy flockmates (n = 1990). The incidence of classical scrapie in the Canadian sheep population was highly associated with the ARQ haplotype (91.8%) and the ARQ/ARQ genotype (91.6%). In addition, the ARQ haplotype was found at significantly higher frequency in scrapie-infected sheep when compared with their healthy flockmates. Comparison with other published data suggests that the scrapie risk of PRNP genotypes differs between Canada and countries where the VRQ allele is associated with the highest susceptibility to infection.     

绵羊朊蛋白等位基因多态性的研究

绵羊痒病的发生和易感性可受绵羊朊蛋白等位基因密码子136,154和171多态性的影响。利用SSCP电泳和非变性聚丙烯酰胺凝胶结合的方法对10只健康绵羊的朊蛋白等位基因154,171两个多态性位点进行检测,发现8只纯合子羊基因型为154RR171QQ,2只杂合子羊基因型为154RR171HR。表明该方法可以应用于此等位基因多态性的研究中。     

PRNP haplotype and genotype frequencies in Brazilian sheep: issues for conservation and breeding programs

Polymorphisms of PRNP gene have been strongly correlated to the susceptibility/resistance to scrapie in sheep. Variants at the coding positions 136, 154 and 171 have been the most frequently associated to susceptibility to classical scrapie. The aim of this study was to estimate PRNP haplotype and genotype frequencies in a sample of 1400 sheep from 13 different breeds that are representative of the main production regions in Brazil. A total of four different alleles (ARR, ARQ, AHQ and VRQ) and nine genotypes were observed at different frequencies among the investigated breeds. There were distinct patterns of allelic distribution between naturalized and commercial/specialized breeds and different geographic regions. These results will influence the development and management of breeding and conservation programs and will help to develop Brazilian efforts to avoid scrapie epidemics.     

Detection of polymorphisms in the prion protein gene in the Belgian sheep population: some preliminary data

The development of clinical signs of TSE/scrapie in sheep has been linked to polymorphisms in the prion protein (PRNP) gene. The most important polymorphisms appear to be at codons 136, 154, and 171. The objective of this study was to investigate the polymorphisms at these codons in the Belgian sheep population, including clinical healthy animals, healthy animals at the slaughterhouse and animals in TSE/scrapie positive farms (including a Nor98 farm).     

Characterization of the prion protein gene (PRNP) region in Swiss sheep breeds

Polymorphisms in the PRNP gene have both an influence on the progress of scrapie and also of bovine spongiform encephalopathy (BSE) in sheep. In the light that sheep are potentially susceptible to BSE the goal of this project was to characterize 50 animals of each of the four major Swiss sheep breeds, namely Swiss Oxford Down, Swiss Black‐Brown Mountain, Valais Blacknose and Swiss White Alpine with respect to polymorphisms in the PRNP region. A 628‐bp fragment of exon 3 of PRNP comprising codons 112–211 was amplified by PCR and analysed by direct sequencing. Heterozygous genotypes were further verified by restriction fragment length polymorphism analyses. Polymorphisms could be observed at codons 112, 136, 137, 141, 143, 154 and 171 but not at codons 138, 151 and 211. One of the scrapie cases in Switzerland, a sheep of Swiss White Alpine, was also genotyped. Based on the polymorphisms observed at codons 136, 154 and 171 all four sheep breeds should be considered potentially susceptible to scrapie and BSE.     

我国部分地区传染性羊痒病基因型的分布情况调查

引起羊痒病的病原朊蛋白是一种正常的唾液酸糖蛋白(PrPc)在三级结构发生改变后形成的异常蛋白(PrPsc)。该病的发生与绵羊朊蛋白编码基因PRNP遗传多样性密切相关,主要表现在PRNP第136、154和171位密码子组成的PRNP基因型与绵羊对痒病的抗病性的联系。根据已建立的一种利用荧光定量PCR扩增反应对羊痒病抗性基因进行筛选的方法,对我国部分地区的无角多赛特绵羊羊痒病基因分布情况进行调查,从而从品种选育水平上杜绝羊痒病的发生。     

新疆地方绵羊品种PRNP基因多态性研究

从新疆采取了8个地方绵羊品种的血液样品171份,提取绵羊基因组DNA,用PCR方法扩增绵羊PRNP基因,通过序列测定,对它们的PRNP基因型进行研究,确定了PRNP基因136、154、171位密码子的多态性为136(A/A),154(H/R)和171(Q/R/H/K),结果发现所检测的新疆地方绵羊品种PRNP基因136位密码子均为A,其基因型均为A型痒病抵抗性基因型。     

Evaluation of associations between prion haplotypes and growth, carcass, and meat quality traits in a Dorset x Romanov sheep population

There is concern about potential antagonistic correlated responses due to intensive selection for scrapie-resistant haplotypes of the prion (PRNP) gene in sheep. The objective of the present research was to test for associations of PRNP haplotypes for codons 136, 154, and 171 with growth, carcass, and meat quality traits in an F2 Dorset x Romanov population (n = 415) segregating the 2 callipyge alleles. Haplotypes of the 3 PRNP codons were determined for each sheep, and breed of origin of each gamete was predicted by genotyping 6 microsatellite markers flanking the PRNP locus. Twenty-five growth, carcass, and meat quality traits were evaluated. Data were analyzed using a basic model consisting of fixed effects of year, sex, and callipyge genotype, the random effect of sire, and 7 covariates corresponding to the probability that a lamb inherited a specific PRNP haplotype of either Dorset or Romanov origin. A fixed effect of litter size was added to the model for growth traits. The model for carcass traits contained the linear and quadratic effects of chilled carcass weight and the interactions among callipyge genotype and linear and quadratic terms. For meat quality traits, the model contained chilled carcass weight as a covariate and the interaction between callipyge genotype and chilled carcass weight. A contrast between the resistant ARR haplotype and the average effect of other PRNP haplotypes was tested to investigate the effects of potential selection for ARR within each breed of origin (Dorset, ARR vs. ARQ, VRQ, and AHQ; Romanov, ARR vs. ARQ and VRQ). There was limited evidence that selecting for scrapie resistance would cause correlated responses due to linkage disequilibrium. Associations of only 3 traits with PRNP haplotypes were detected in either breed of origin. In Romanov, the ARR haplotype was associated with longer carcasses (P < 0.013), narrower rumps (P = 0.038), and less marbling (P = 0.022) than the average of ARQ and VRQ haplotypes. No significant contrasts were detected for Dorset. This study is the first to account for breed of origin while investigating haplotype associations in an F2 population. This study provided limited evidence of associations between PRNP haplotypes and growth, carcass, and meat quality traits.     

PrP polymorphisms in Basque sheep breeds determined by PCR-restriction fragment length polymorphism and real-time PCR

Two new PCR-based methods were developed to decode prion protein (PrP) gene polymorphisms at codons 136, 154 and 171: a PCR-restriction fragment length polymorphism (RFLP) analysis consisting of two PCR reactions followed by three enzymatic digestions, and a real-time PCR consisting of four reactions with seven fluorogenic probes. Both methods were used to study the distribution of PrP gene polymorphisms in a representative sample (1297 animals) of the populations of the two native breeds of sheep of the Spanish Basque Country, Latxa and Carranzana. Fourteen genotypes were found in the Latxa breed, in which ARQ/ARQ was the genotype most frequently observed (49.3 per cent), followed by ARR/ARQ (32.6 per cent) and ARQ/ARH (5.8 per cent). The genotype associated with the highest resistance to scrapie (ARR/ARR) was present in 5 per cent of the animals analysed. Similar results were observed in the Carranzana sheep.     

Identification of a new leucine haplotype (ALQ) at codon 154 in the ovine prion protein gene in Spanish sheep

Genetic susceptibility to scrapie is closely linked to variations at codons 136, 154, and 171 of the prion protein (PRNP) gene. This association between the PRNP genotype and susceptibility to scrapie is the basis of breeding programs for scrapie resistance in different countries. In this paper, we describe the method used with 2 Spanish dairy sheep breeds (Churra and Castellana) to ascertain the initial status of protection against scrapie as a first step toward adapting their breeding schemes to include resistance as a complementary selection criterion. The procedure for genotype identification is based on multiplex minisequencing methodology and has been shown to be accurate, easy to interpret, and to have a medium throughput. The frequency of the ARQ allele was similar in the 2 populations at nearly 70%. The ARR allele, associated with resistance in the homozygous state, reaches around 23% in Churras and nearly 20% in Castellanas. The high-risk VRQ allele appeared at a relatively low frequency in both breeds. No other haplotypes were found in these 2 breeds. Furthermore, in this screening we found a new allele carrying leucine at codon 154. This new genetic variant might play a role in susceptibility to scrapie because codon 154 belongs to a region considered to have an important role in conformational conversion of the cellular to the pathogenic protein.     

Effects of polymorphisms in ovine and caprine prion protein alleles on cell-free conversion

ABSTRACT: In sheep polymorphisms of the prion gene (PRNP) at the codons 136, 154 and 171 strongly influence the susceptibility to scrapie and bovine spongiform encephalopathy (BSE) infections. In goats a number of other gene polymorphisms were found which are suspected to trigger similar effects. However, no strong correlation between polymorphisms and TSE susceptibility in goats has yet been obtained from epidemiological studies and only a low number of experimental challenge data are available at present. We have therefore studied the potential impact of these polymorphisms in vitro by cell-free conversion assays using mouse scrapie strain Me7. Mouse scrapie brain derived PrPSc served as seeds and eleven recombinant single mutation variants of sheep and goat PrPC as conversion targets. With this approach it was possible to assign reduced conversion efficiencies to specific polymorphisms, which are associated to low frequency in scrapie-affected goats or found only in healthy animals. Moreover, we could demonstrate a dominant-negative inhibition of prion polymorphisms associated with high susceptibility by alleles linked to low susceptibility in vitro.     

Protective effect of the AT137RQ and ARQK176 PrP alleles against classical scrapie in Sarda breed sheep

The susceptibility of sheep to scrapie is under the control of the host’s prion protein (PrP) gene and is also influenced by the strain of the agent. PrP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (Q/R/H) are the main determinants of susceptibility/resistance of sheep to classical scrapie. They are combined in four main variants of the wild-type ARQ allele: VRQ, AHQ, ARH and ARR. Breeding programmes have been undertaken on this basis in the European Union and the USA to increase the frequency of the resistant ARR allele in sheep populations. Herein, we report the results of a multi-flock study showing the protective effect of polymorphisms other than those at codons 136, 154 and 171 in Sarda breed sheep. All ARQ/ARQ affected sheep (n = 154) and 378 negative ARQ/ARQ controls from four scrapie outbreaks were submitted to sequencing of the PrP gene. The distribution of variations other than those at the standard three codons, between scrapie cases and negative controls, was statistically different in all flocks. In particular, the AT₁₃₇RQ and ARQK₁₇₆ alleles showed a clear protective effect. This is the first study demonstrating a protective influence of alleles other than ARR under field conditions. If further investigations in other sheep breeds and with other scrapie sources confirm these findings, the availability of various protective alleles in breeding programmes of sheep for scrapie resistance could be useful in breeds with a low frequency of the ARR allele and would allow maintaining a wider variability of the PrP gene.     

PrP genotyping of Austrian sheep breeds

Scrapie, an ovine and caprine transmissible spongiforme encephalopathy, is widely spread among sheep populations in many European countries. As it is known that susceptibility to scrapie is determined genetically, breeding programmes aiming at providing scrapie-resistant flocks have been established. Selection is based on the prion protein (PrP) genotype, which is used to classify animals into risk groups of susceptibility (R1-R5) according to the amino acids encoded by codons at positions 136, 154 and 171, respectively. At position 136 (136V-->136A) alanine and at position 154 (154H-->154R) as well as 171 (171Q-->171R) arginine are the favoured amino acids. Whereas PrP genotyping data are available for many of the European sheep breeds, comparable data for local Austrian sheep breeds are missing. The most known among these are Tyrolean mountain sheep, forest sheep. Tyrolean stone sheep and Carynthian sheep. The genotypes of 112 sheep from these four local breeds were determined. In terms of PrP genetics, Austrian breeds belong to the group of non-valine-breeds, with the exception of the Carynthian sheep, that exhibited a frequency of 136V of 4.2%. The most frequent allele was ARQ with 64.6-71.2% (depending on the breed), followed by ARR (14.8-25.8%). In contrast to the above-mentioned findings, scrapie has never been diagnosed in any of the Austrian sheep breeds. Native Austrian sheep breeds exhibit a very robust constitution, a pronounced adaptation to harsh climates and good reproduction parameters as well as a marked mother instinct. Therefore, these breeds are often used in crossbreeding programmes. Beside the above-mentioned characteristics, our results indicate that the investigated breeds may be effectively used in crossing-out breeding programmes for eliminating valine at position 136 of PrP.     

Application of temperature-gradient gel electrophoresis for detection of prion protein gene polymorphisms in Polish Swiniarka sheep.

This study presents preliminary data on the polymorphism in the prion protein gene of Swiniarka sheep using temperature gradient gel electrophoresis (TGGE). Available data indicate that sensitivity to scrapie is associated with polymorphisms in three codons of prion protein gene: 136,154, and 171. The TGGE method was used to detect point mutations in these codons responsible for sensitivity or resistance to scrapie. This study revealed presence of an allele encoding valine (V) in codon 136, which is associated with high sensitivity to scrapie and occurred in the form of heterozygous allele together with alanine (AV). The highest variability was observed in codon 171, with presence of arginine (R) and glutamine (Q) in the homozygous (RR or QQ) as well as the heterozygous form (RQ). The results of examination of fifty sheep DNA samples with mutations in codons 136, 154, and 171 demonstrated that TGGE can be used as a simple and rapid method to detect mutations in the PrP gene of sheep. Several samples can be run at the same time, making TGGE ideal for the screening of large numbers of samples.     

Assessment of the genetic risk and impact of lateral transmission in a valine-associated scrapie outbreak in sheep

OBJECTIVE: To characterize an outbreak of valine-associated scrapie, assess the relative risk of scrapie infection in relation to allele frequency at codon 136, and investigate lateral transmission of infection in a sheep flock within the United States. ANIMALS: 1,006 sheep. PROCEDURE: To determine genotypes, blood or semen samples were assessed via commercial testing; in 190 slaughtered sheep, scrapie status was determined via immunohistochemical evaluation of tissues. Scrapie-positive sheep born to scrapie-negative dams and sheep infected after 1 year of age were identified to assess lateral transmission. RESULTS: Genotypes were determined for codon 171 (164 sheep) or codons 136 and 171 (842 sheep). Forty-four of 160 slaughtered sheep of known genotype were scrapie positive. In these sheep, the presence of at least 1 valine allele at codon 136 (V136) was highly correlated with scrapie-positive status. Lateral transmission was the probable source of infection for 4 scrapie-positive sheep born to scrapie-negative dams and for 11 sheep in which scrapie was diagnosed at > 50 months of age. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the outbreak of scrapie was associated with a relatively high frequency of V136 in the flock, introduction of a valine-dependent scrapie strain, and the occurrence of lateral transmission. Genotyping of sheep may assist management decisions following diagnosis of scrapie in a sheep with at least 1 V136. It may be prudent to remove sheep of the diploid genotype AVQR (at codons 136 and 171) from infected flocks.     

Polymorphism of the DQA2 gene in goats

Variation in the caprine DQA2 gene was investigated using PCR-single-strand conformational polymorphism (SSCP) and DNA sequencing. Eleven DQA2 alleles were defined by SSCP patterns from 23 goats. All the caprine alleles shared high sequence homology to ovine DQA2 sequences, and exhibited a pattern of polymorphism similar to DQA2 alleles from sheep and cattle but different from caprine DQA1 sequences. Thirty-eight AA positions in the alpha1 domain of caprine DQA2 molecules were polymorphic, and a high degree of polymorphism was observed in the putative antigen-binding region, with 74% of the positions being polymorphic. Phylogenetic analysis of caprine, ovine, and bovine DQA sequences revealed that the caprine DQA2 sequences identified here grouped with ovine DQA2, bovine DQA2, DQA3, and DQA4 sequences but are separate from the group of caprine DQA1 alleles. Nine of the caprine DQA2 sequences were more similar to ovine DQA2 alleles, whereas the remaining two were more closely related to ovine DQA2-like and bovine DQA3 alleles. This finding suggests that the caprine DQA2 sequences may represent two loci, which probably arose by either gene duplication or gene conversion events. Allelic lineages were evident for both DQA2 and DQA2-like loci, supporting the trans-species mode of evolution of major histocompatibilitly complex genes. The high level of polymorphism and similarity between caprine and ovine DQA2 alleles suggests that the DQA2 gene may play an important role in immune responses to shared pathogens.     

小尾寒羊PRNP等位基因密码子136、154和171的多态性研究

为研究小尾寒羊PRNP等位基因密码子多态性,本研究以绵羊全血基因组DNA为模版,通过PCR,扩增绵羊PRNP等位基因目的片段,并将其克隆于pMD-18T载体。经SSCP法筛选出阳性克隆后测序,确定各绵羊PRNP136位、154位和171位密码子等位基因型及频率分布。在104只绵羊中共检出15个PRNP136位、154位和171位密码子等位基因型,痒病易感基因型占优势(53.85%,56/104)。本研究证明,该绵羊群属于痒病易感/易发群体,一旦痒病传入具有罹患痒病的极大可能性。