Bovine spongiform encephalopathy (BSE), better known to the public as mad cow disease, is one of the fatal neurodegenerative diseases that are collectively known as transmissible spongiform encephalopathies (TSEs). These also include Kuru and Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, and chronic wasting disease in deer [28]. Most TSEs are characterized by a long incubation period and a neuropathological feature of multifocal spongiform changes such as astrogliosis, neuronal loss, or an absence of inflammatory response. The causative agent of TSEs is an infectious prion protein (PrP^Sc), that is a structurally abnormal and protease-resistant isoform of the prion protein (PrP) [2]. PrP^Sc is derived from the endogenous cellular prion protein (PrP^C) that has undergone post-translational modification resulting in conformational changes [27,29]. PrP^C is known to play a role in copper metabolism [21,37] but its normal function(s) in cells are not well defined.

It is generally believed that BSE epidemics in cattle are caused by the unusual ingestion of meat and bone meal contaminated with PrP^Sc from BSE-affected cattle or scrapie-affected sheep [28]. BSE likely poses a threat not only to cattle but also to humans because variant CJD in humans is also reportedly caused by consuming meat or other products from BSE-affected cattle [3,35]. Therefore, many nations have focused their research efforts on BSE epidemics and implemented protocols to prevent BSE in cattle.

Recent studies on resistance or susceptibility to TSEs have focused on investigating the genetic structure of the prion protein gene (PRNP). In cattle, PRNP is located on chromosome 13 and the prion protein-coding region is positioned in exon 3 [31]. Polymorphisms of the prion protein-coding region can induce structural changes within the protein itself. Eventually, such structural changes in the prion protein reportedly result in the molecular pathology of TSEs. It has been reported that single nucleotide polymorphisms (SNPs) of PRNP have various effects on the susceptibility and incubation time of prion diseases in humans, mice, and sheep [1,22]. In sheep, polymorphisms in codons 136, 154, and 171 are correlated with susceptibility to scrapie and can thus be used to control disease incidence [8], whereas in humans, a polymorphism in codon 129 has a critical influence to variant CJD incidence [38]. To date, there are no known polymorphisms within the prion protein-coding region associated with BSE susceptibility in cattle. Although E211K (which is analogous to the E200K variant in humans that contributes to CJD) has been reported, it is not associated with typical BSE but rather an atypical form of this disease [10,25].

Susceptibility or resistance to TSE is also associated with variations in the non-coding region of PRNP, including the promoter and/or enhancer. Nucleotide changes in the non-coding region may affect mammalian PrP^C expression, which is also known to affect TSE susceptibility or resistance [33]. In cattle, strong associations between susceptibility to classical BSE and two insertion/deletion (indel) polymorphisms located in two regulatory regions of bovine PRNP have been identified [16,33]. These polymorphisms are a 23-bp indel at position -1594 and a 12-bp indel at position +300 (the positions of the polymorphisms are given with respect to the transcription start site in GenBank accession No. AJ298878); both are associated with promoter activity and bovine PrP^C expression levels in vitro [32]. Therefore, these polymorphisms can both seemingly influence BSE incubation times and susceptibility. The two indel polymorphisms and their frequencies have been previously identified and characterized in a number of bovine strains such as Holstein, Brown Swiss, Fleckvieh, and Japanese Black [9,16,24,33].

Hanwoo cattle [Bos (B.) taurus coreanae] were created by crossbreeding between B. indicus and B. taurus, and are now widely raised in Korea as a native strain [12]. To date, no BSE cases have been reported in Hanwoo. Genotypic descriptions of BSE-related genetic factors, such as SNPs or the number of octapeptide repeats in PRNP, have rarely been reported for Hanwoo, and the genetic frequencies and features related to BSE susceptibility have been observed in only a limited number of samples [14,15]. In the present study, we performed a detailed analysis of both sequence variations in the coding region of PRNP and prevalent indel polymorphisms in the non-coding region of PRNP using a large number of samples from Hanwoo cattle. Furthermore, we analyzed the genetic frequency and haplotype of gene variations associated with BSE susceptibility and compared them to those of other cattle breeds.

It has been established that polymorphisms in the PRNP gene are associated with susceptibility to prion diseases in humans, sheep, and mice [1,22,38]. Many studies have also explored the relationships between PRNP polymorphisms and BSE susceptibility in cattle [16,26,33]. The majority of these studies have been focused on investigating both the protein-coding and non-coding regions of the PRNP gene to search for polymorphisms that affect BSE susceptibility. It has been demonstrated that non-synonymous alterations in the protein-coding region of PRNP can cause structural changes whereas nucleotide changes in the regulatory region including the promoter can modulate PRNP expression [33]. Of note, these variations may influence BSE susceptibility or resistance. To date, there are no known polymorphisms associated with susceptibility to BSE in cattle with the exception of the E211K variant, an analogue of the E200K variant in humans that affects CJD [10]. However, E211K has been reported in only two animals, one of which had BSE; this case of BSE was atypical rather than typical [10,25].

In the present study, we identified four variations (G234A, C555T, C576T, and C630T) in the PRNP coding region that were synonymous polymorphisms. In previous studies of the PRNP gene in Hanwoo cattle, only a few genetic variations in the PRNP coding region were reported. Jeong et al. [15] analyzed the open reading frame (ORF) region of the PRNP gene in 120 Hanwoo cattle and found two polymorphisms: the third position of codon 78 (G234A) and the third position of codon 192 (C576T). However, these authors did not find any variation in the octapeptide repeat. These polymorphisms (G234A and C576T), which were also found in our present study, are commonly found in both B. taurus and B. indicus [4]. However, C555T and C630T detected in our study have been previously found only in B. indicus [4]. Two SNPs (A467G and A669C) identified in our study are point mutations because their minor allele frequencies were especially low (less than 0.01) in the experimental population. Thus, our findings indicate that the polymorphisms found in this study cannot induce structural changes in the prion protein.

The octapeptide repeat region near the N-terminal portion of the prion protein has a binding affinity to divalent cations, and may affect protein structure and function [7]. It has been established that this protein commonly have five or six repeats [24]. The presence of extra repeats is correlated with increased TSE susceptibility as has been previously observed in transgenic mice expressing bovine PrP^C with four extra octapeptide repeats as well as humans with nine octapeptide repeat insertion in the PRNP gene [6,19]. It has also been shown that Brown Swiss cattle expressing seven octapeptide repeats are more susceptible to BSE than animals of other breeds [4,34,36]. Collectively, these findings indicate that bovine PrP^C containing extra octapeptide repeats may enhance susceptibility to BSE. All Hanwoo animals sampled in the present study had six octapeptide repeats, which was different from animals of other breeds with either five or six octapeptide repeats. Thus, Hanwoo may have a relatively lower risk of being infected with BSE based on their conserved number of octapeptide repeats encoded in the PRNP gene.

A tentative association between PRNP promoter polymorphisms, including the 23-bp and 12-bp indels, and BSE susceptibility has been previously reported in German cattle [33]. This study revealed that the frequencies of these two polymorphisms are significantly different between BSE-affected and healthy cattle populations in terms of both allele and genotype frequencies. These polymorphisms are thought to be responsible for differences in BSE incubation time and/or susceptibility, but are not involved in resistance to atypical BSE [5,33]. Results of previous reporter gene assays have suggested that both the 23-bp and 12-bp indel polymorphisms are involved in modulating the expression of bovine PRNP [32]. It has been established that the 23-bp indel polymorphism within the 5'-flanking region is responsible for a binding site for the RP58 repressor protein, which is expressed mainly in the brain, and the 12-bp indel polymorphism within intron 1 is responsible for a binding site for the SP1 transcription factor involved in the regulation of numerous promoters [13,32].

Recently, it has been reported that polymorphic differences could be the reason for changes in PRNP expression levels based on the significant correlation between 23-bp indel polymorphisms and PRNP mRNA expression in two Japanese cattle breeds [23]. Thus, the 23-bp deletion can restrict the access of the RP58 repressor and induce overexpression of the prion protein, thereby increasing BSE susceptibility, whereas the 12-bp deletion can decrease PRNP promoter activity and reduce expression of the prion protein [32]. However, the allele frequency of the 12-bp deletion has been shown to be higher in BSE-affected animals than in healthy ones [33]. These apparently contradictory results can be explained by the following logic. First, there is strong linkage between the 23-bp and 12-bp indels. In other words, the rate of crossover between the 23-bp and 12-bp indels is quite low, which leads to linkage disequilibrium. In our haplotype analysis of combinations including both 23-bp and 12-bp indels, we found that the 23 del/12 del and 23 ins/12 ins haplotypes were common (more than 80%) while the 23 del/12 ins frequency was relatively low. Very few cases of the 23 ins/12 del haplotype, which were considered statistically negligible (less than 1%), were observed in the UK Holstein, German Holstein, German Brown, German Fleckvieh, or Hanwoo cattle. Second, the 23-bp deletion seems to have a more crucial role in BSE susceptibility than the 12-bp insertion. According to the reporter gene assays previously performed by Sander et al. [32], the 23 ins/12 ins haplotype showed weaker promoter activity than the 23 del/12 del haplotype although the promoter activity of the 12-bp insertion was stronger than that of the 23-bp deletion. These results can be explained by the previous finding that, with the 23 ins/12 ins haplotype, SP1 binding on the 12-bp insertion can be repressed by RP58 binding on the 23-bp insertion [20]. Promoter activity associated with the 12-bp indel is thus dependent upon the 23-bp indel. Therefore, although the 12-bp deletion has a negative effect on BSE susceptibility according to previous in vitro experiments, a higher frequency of the 12-bp deletion with the 23-bp deletion can be found in BSE-affected animals compared to healthy controls.

In China, the frequency distribution of genotypes, alleles, and haplotypes for these 12-bp and 23-bp indel polymorphisms of 349 native Chinese cattle were recently investigated [39]. Relatively low frequencies of the 12-bp deletion allele and 23 del/12 del haplotype, which have been suggested to affect BSE susceptibility, were observed. Another Chinese group identified SNPs in exon 3, a 23-bp indel in the promoter region, and a 12-bp indel in intron 1 in the PRNP gene from two Chinese indigenous cattle breeds of northeast China [30]. They found that there three different genotypes exist among both Yanbian and Chinese Red Steppes breeds in the 23-bp indel and 12-bp indel loci, and that 23del/12del is the main haplotype accounting for more than 50% of all Yanbian and Chinese Red Steppes cattle.

Since there were no BSE cases in the Hanwoo population, we could not compare differences in the 23-bp and 12-bp indel polymorphisms between healthy and BSE-affected Hanwoo cattle. Therefore, we compared our data for the Hanwoo cattle with data for other breeds from previous studies by Juling et al. [16] and Nakamitsu et al. [24]. We found that the deletion allele frequencies of 23-bp and 12-bp indel polymorphisms were lower for Hanwoo than those for the other breeds except for German Brown. In addition, the difference in allele frequencies between Hanwoo cattle and BSE-affected animals was significant (p < 0.05). According to the haplotype analysis, Hanwoo had a lower 23 del/12 del frequency than the other breeds except for German Brown (both BSE-affected and healthy) and German Holstein (healthy). These data indicate that the Hanwoo population is likely to have a lesser risk for BSE compared to other cattle breeds. Kim et al. [18] also surveyed the allelic, genotypic, and haplotypic distribution of the 23-bp and 12-bp indel PRNP polymorphisms from Korean cattle. This study revealed that the allelic frequencies for the 23-bp indel were 44.3% (insertion) and 57.7% (deletion), whereas they were 44.4% (insertion) and 55.6% (deletion) for the 12-bp indel [18]. These particular results are similar to ones from our own investigation. However, the genotypes examined in the Kim et al. [18] study had more homologous patterns with a lower del/ins frequency than those in our investigation. Allelic and genotypic frequencies for the 23-bp and 12-bp indels similar to those found by Kim et al. [18] have also been observed in another previous study of Hanwoo cattle.

In conclusion, our results demonstrated that Hanwoo cattle do not have any unique alterations that cause structural changes in the prion protein associated with BSE susceptibility. In addition, we found that all of the Hanwoo cattle included in this study had six octapeptide repeats, which are typically found in cattle and do not influence BSE susceptibility. For both the 23-bp and 12-bp indel polymorphisms, Hanwoo exhibited low levels of deletion frequencies. Since these 23-bp and 12-bp deletion alleles are associated with a higher susceptibility to BSE as reported in other breeds [33], it is likely that Hanwoo cattle are less susceptible to BSE than those of other breeds based on their lower deletion frequency in the 23-bp and 12-bp indel polymorphisms. To further clarify the relationship between genetic variations and functional alterations of the PRNP gene in Hanwoo cattle, further functional analyses should be performed.