BSE-associated polymorphisms in the prion protein gene: an investigation
K. Vernerova1, L. Tothova1, A. Mikova1, P. Vodrazka2, B. Simek2, L. Hanusova1 & J. Citek1 1 Faculty of Agriculture, University of South Bohemia, Ceske Budejovice, Czech Republic 2 State Veterinary Institute Jihlava, Jihlava, Czech Republic
Cattle; BSE; prion gene; 23 indel; 12 indel.
J. Citek, Faculty of Agriculture, University of
South Bohemia, CZ37005 Ceske Budejovice,
Received: 21 August 2013; accepted: 14 March 2014
The aim of this study was to determine the frequency of the 12-bp and 23-bp indel polymorphisms in the prion protein gene (PRNP) in cattle and to investigate the association between these frequencies and the occurrence of bovine spongiform encephalopathy (BSE). There was no significant difference in the 12-bp indel frequency between the BSE animals and control group. For the 23-bp indel, the BSE animals had a significantly lower + + (insins) genotype frequency and + allele frequency compared with the control animals. The / genotype frequency in the
BSE animals was not significantly higher when compared with the control animals. One allele increased the risk of BSE by a factor of 1.55 (i.e. by 55%) for the 12-bp indel and by a factor of 2.10 for the 23-bp indel. When both indels are considered, one allele increased the risk of BSE by a factor of 1.54.
Over the past two or three decades, bovine spongiform encephalopathy (BSE) has been a cause of disquiet to the public and to the veterinary and biological research communities.
At first, slow viruses were thought to be the cause of spongiform encephalopathies (Hadlow 1959; Gajdusek et al. 1966), but more recent findings have shown that prions are the cause. Prions are characterized as unprecedented infectious pathogens that cause several fatal neurodegenerative diseases through an entirely novel mechanism. BSE, scrapie in sheep, and
Creutzfeldt-Jakob disease (CJD) in humans are some of the most notable prion diseases (Prusiner 1998), and these diseases are able to circumvent the species barrier (e.g. Casalone et al. 2004). This hypothesis was widely accepted even though it has been convincingly disputed by some scientists (Scholz 2002).
More recently, the genetic aspects of these diseases have come into focus. The genomes of the affected species have been analysed, and particular attention has focused on the prion protein gene (PRNP). Many polymorphic sites have been identified, and their association with BSE symptoms has been investigated.
Hills et al. (2001) published the full genomic sequence of the bovine PRNP and reported that there is an insertion/deletion polymorphism (indel) within the promoter region. Clawson et al. (2006) reported 388 polymorphisms within a 25.2 kb genomic region that contains PRNP and identified 19 haplotypes. Murdoch et al. (2010a) investigated whether there is an association between the PRNP haplotypes and BSE using a set of haplotype-tagging single-nucleotide polymorphisms (htSNPs) and two indels. Using this technique, they discovered that there is an association between the PRNP haplotypes and the occurrence of classic
BSE. In another study, Murdoch et al. (2010b) identified 27 SNPs in 18 different chromosomes that are associated with the occurrence of BSE.
Particular attention has focused on the 23-bp indel polymorphism in the PRNP promoter region, the 12bp polymorphism in intron 1 and the removal of the putative SP1 binding site (Hills et al. 2001; Sander © 2014 Blackwell Verlag GmbH • J. Anim. Breed. Genet. 131 (2014) 403–408 doi:10.1111/jbg.12090
J. Anim. Breed. Genet. ISSN 0931-2668 et al. 2004). It has been shown that there is a strong correlation between both of these indel polymorphisms and the occurrence of BSE (Sander et al. 2005;
Juling et al. 2006). Msalya et al. (2010) suggested that changes in PRNP expression are linked to these indel polymorphisms and may result in different BSE incubation periods and BSE susceptibility. Another mutation, E211K, has also been reported (Clawson et al. 2008; Heaton et al. 2008; Nicholson et al. 2008).
In this study, the allele and genotype frequencies of the 12- and 23-bp insertion/deletion polymorphisms in cattle, including BSE-positive animals, in the Czech
Republic were determined.
Materials and methods
The 23-bp indel polymorphism in the bovine PRNP promoter region and the 12-bp indel polymorphism in intron 1 were analysed in Czech Simmental (n = 25) and Holstein sires (n = 30). Both of these breeds are part of the artificial insemination (AI) programme in the Czech Republic. Czech Red cows and sires (n = 26), an original Czech breed, were also used in this study. BSE-positive cattle (n = 26) were also analysed.
The BSE status was determined by the State Veterinary Institute Jihlava using immunohistochemical, histopathological, Western blot and ELISA methods.
Genomic DNA was isolated from the sperm, whole blood and muscle tissue using either a commercial kit or a standard chloroform extraction laboratory method. DNA was also isolated from the brains of
BSE-positive animals. PCR was performed using the method described in Jeong et al. (2006). The 23-bp indel forward primer sequence was 50-GTGCCAGCCATGTAAGTG-30, and the 23-bp indel reverse primer sequence was 50-TGGACAGGCACAATGGG-30. The sequences for the 12-bp indel primers were forward 50-TTACCCTCCTGGTTAGGAG-30 and reverse 50CTAGATTCCTACACACCAC-30. The annealing temperature, cycle specifications and reaction mixture were optimized for our laboratory environment. The
PCR products were separated on a 3% agarose gel and visualized using ethidium bromide staining and UV light.
One of the BSE-positive animals developed clinical symptoms of the disease. The other BSE-positive animals were diagnosed after slaughter and had not shown symptoms of encephalopathy. The analyses in this study were conducted in accordance with the contemporary animal welfare law of the Czech