Genomic Sequence Variability of the Prion Gene (PRNP) in Korean Cattle

In this study, we have investigated sequence variants in the PRNP gene of 20 individuals belonging to the Korean cattle, and have analyzed and compared genetic features between varieties of other cattle breeds. Of the 73 sequence variants identified in Korean cattle, 27 were identified for the first time in this study, whereas 46 of these polymorphisms had previously been isolated. We discovered a 2.6 kb SNP hot spot region localized on the putative promoter region of the PRNP gene. Furthermore, the copy numbers of the octapeptide repeat (24 bp indel) which is detected on the coding sequence (CDS) of the PRNP exhibited a completely homozygous 6/6 genotype which is dominant in other cattle breeds. We also characterized a new 19 bp/10 bp allele located on the putative promoter region of the PRNP gene, which represented 0.71 in allele frequency. To the best of our knowledge, this report is the first to address polymorphisms of the PRNP gene structure in Korean cattle in which BSE has yet to be discovered. Therefore, our findings may prove useful with regard to our current understanding of allelic diversity in bovine species, and may also provide new insights into the genetic factors associated with susceptibility or resistance to BSE.


INTRODUCTION
Transmissible spongiform encephalopathies (TSEs), also commonly referred to as prion diseases, are fatal neurodegenerative diseases. Examples of these diseases include scrapie in sheep, Kuru and Creuzfeldt-Jakob disease (CJD) in humans, and bovine spongiform encephalopathy (BSE) in cattle. The pathogenesis of these diseases involves the conversion of the normal-cellular prion protein (PrP C ) into a pathological protein (PrP sc ), which is infectious (Prusiner 1998). The accumulation of these PrP sc in the brain has been implicated in the development of the diseases themselves. Bovine spongiform encephalopathy (BSE) is generally believed to be caused by the ingestion of scrapie and/or BSE-infected meat or bone meal (Wilesmith 1991). The development of a variant of CJD (vCJD) in humans has also been determined to occur as the result of the ingestion of meat products originating from BSEinfected cattle (Bruce et al., 1997;Scott et al., 1999;Collinge et al., 2001;Asante et al., 2002). Thus, in order to adequately eliminate BSE from the food chain, the determination and identification of genetic factors that confer resistance to BSE would constitute the solution to a fundamental problem.
The PRNP gene, which is known to code for the prion protein, has become the focus of increasing attention in the quest to determine the molecular mechanisms underlying the infectious behavior of these prion diseases, as well as the mechanisms relevant to the transmission of prion species between species, most notably transmission between bovines and humans (Bruce et al., 1997;Scott et al., 1999;Collinge et al., 2001;Asante et al., 2002). In studies of the bovine PRNP gene, great efforts have been made to identify genetic mutations occurring within the global population, and this gene has been previously examined in such species as Japanese cattle (Takasuga et al., 2003), American beef cattle (Heaton et al., 2003), Holstein-Friesian cattle (Hills et al., 2003), Chinese cattle (Zhang et al., 2004) and German cattle (Sander et al., 2004), as well as 36 other cattle breeds (Seabury et al., 2004b). At present, variations associated with resistance and/or susceptibility to the BSE diseases have been fairly regularly reported. Some of these variations include the following; the octapeptide repeat polymorphism in CDS (Goldmann et al., 1991;Hunter et al., 1994;Walawski et al., 2003a;Walawski et al., 2003b;Sander et al, 2004), the large indel polymorphism of 12 bp in the first intron and 23 bp in the putative promoter (Sander et al., 2004;Seabury et al., 2004a), and SNPs (Heaton et al., 2003;Takasuga et al., 2003;Seabury et al., 2004b).
The Hanwoo strain of Korean cattle is one of the few known breeds of cattle in the world in which no individuals have been determined to suffer from or carry BSE. This could be attributable either to adequate control of the food chain in this case, or to a certain specific genetic factor inherent to the Hanwoo lineage. Therefore, the Hanwoo strain may carry important information with which we can more thoroughly understand the genetic features involved with susceptibility to BSE. Nevertheless, the genetic features of this strain of cattle, with regard to BSE, have yet to be determined. It is most that polymorphism research associated with Korean cattle is involved with meat quality and weight (Cheong et al., 2006;Kong et al., 2006;Zhou et al., 2006), and structure of Korean cattle PRNP gene was demonstrated by recent study (Choi et al., 2006). In this study, we evaluated PRNP polymorphisms in 20 individuals of the Hanwoo breed, which is a well-conserved genetic lineage, maintained carefully by the national breeding system of the Republic of Korea. In this study, we investigated polymorphisms in 31,236 bp region covering putative promoter, three exons and two introns of PRNP. Moreover, we discovered several genetically reliable differences between the Hanwoo breed and foreign cattle breeds, which were expected to generate useful information for our understanding of BSE disease in general.

DNA sample
DNA was isolated from the blood of 20 individual healthy Korean Hanwoo cattle (HW) using the Core-One TM Blood DNA Purification Kit (CoreBioSystem, Seoul, Korea). As was mentioned in the introduction section, these cattle represent a well-conserved genetic lineage, maintained by systematic breeding conducted by the National Livestock Research Institute of Korea.

DNA amplification
In order to generate genomic fragments spanning the PRNP gene, 61 different PCR primer sets were designed using the GenBank reference sequence AJ298878, which includes a promoter, three exons, two introns, and a 3'untranslated region (UTR). These primers were designed to overlap to a length of 100-150 bp between each of the fragments, and to collectively encompass a span of 31,236 bp. Our PCR reactions were conducted using 1 µl (10-20 ng/µl) of genomic DNA, 0.5 µl of 5 pmol primer, 1 µl of 10×buffer, 0.25 µl of dNTP mixture, 6.65 µl of distilled water, and 0.1 µl of Taq DNA polymerase (SolGent, Daejeon, Korea), in a total volume of 10 µl. Amplification was conducted using the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA), followed by 35 cycles of denaturation at 96°C for 10 s, annealing for 5 s at 50°C, and 4 minutes of extension at 60°C. All of the PCR products were assessed for both yield and purity on agarose gel. The PCR products were purified via ethanol precipitation.

DNA sequencing
The purified PCR products were sequenced directly with a BigDye Terminator (Ver 3.1) Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). The sequencing PCR reactions were conducted using 10 ng of amplified DNA, 0.5 µl of 3 pmol primer, 0.87 µl of 5× buffer, 1.38 µl of distilled water, and 0.25 µl of BigDye, in a total volume of 3 µl. Amplification was conducted with a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA), followed by 35 cycles of denaturation at 96°C for 10 s, annealing for 5 s at 50°C, and 4 minutes of extension at 60 °C. The PCR products were purified via ethanol precipitation, and resolved with an ABI 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA). The individual sequences were initially edited and assembled using Sequencher 4.1.4 (Gene Codes Corp., Ann Arbor, USA) software. The complete sequences were generated via a final editing process, which included manual visual confirmation performed using the original chromatograms, with the Sequencher program.

Statistical analysis
Allelic and genotypic distributions were evaluated for significant differences using Fisher's exact test, which was applied using the STAT-SAK program (Dallal GE, freeware).

Sequence variants
We re-sequenced a 31,236 bp genomic region in 20 individual animals. This area encompassed the promoter, three exons, two introns, and the 3'-UTR of the PRNP gene. In this region, we were able to identify 73 total polymorphisms, including 65 single nucleotide polymorphisms (SNPs), one polymorphism involving two adjacent nucleotides, four indels (12 bp, 14 bp, 23 bp, and 24 bp), two allelic variants (19/10 bp and 10/3 bp), and one different region containing a 97 bp sequence (Table 1). Comparing with the variants referenced in previous reports (Heaton et al., 2003;Hills et al., 2003;Sander et al., 2004), 27 of the 73 polymorphisms isolated in Korean cattle were confidently identified to be newly-discovered variants that had never been previously reported, which included 26 SNPs, and one 19 bp/10 bp allelic variant. And also, the remaining 46 polymorphisms matched with that had been previously described. In addition, we also discovered a 97 bp-difference in the 3'-UTR from the Korean cattle, as compared to the previously-used genomic sequence, AJ298878, from Bos taurus (Hills et al., 2001).

SNPs of PRNP gene in Korean cattle
The SNPs were the most frequently observed form of variation, at 89% (65/73). They were located as follows: on the putative promoter (18), on intron 1 (16), on intron 2 (20), within the 3'-UTR (6), and on exon 3 (5). The positions of SNPs newly identified in this study (27)  summarized and schematically diagrammed in Figure 1. As a result of this, we discovered two remarkable things; the first of these is the existence of a SNP hot spot region, which is located on the putative promoter region, and spans 2.6 kb in length (47,470). In all of the breeds thus far examined, 1.9 kb (48,584-50,470) of this 2.6 kb region is highly conserved. The second remarkable finding was that the Korean cattle also harbored no SNPs in the CDS region of the PRNP gene, although this region contains abundant SNPs in most other breeds of cattle (Heaton et al., 2003;Hills et al., 2003;Takasuga et al., 2003;Sander et al., 2004;Seabury et al., 2004b;Zhang et al., 2004).

Allelic variants and indel polymorphisms
Because indel polymorphisms are more likely to exert regulatory effects than are single nucleotide substitutions or SNPs, indel polymorphisms have frequently been the focus of searches for associations with susceptibility to BSE (Heaton et al., 2003;Hills et al., 2003;Takasuga et al., 2003;Sander et al., 2004;Seabury et al., 2004a;Zhang et al., 2004). Four indels (12 bp, 14 bp, 23 bp and 24 bp) have been reported previously in a variety of other cattle breeds (Goldmann et al., 1991;Hunter et al., 1994;Walawski et al., 2003a;Walawski et al., 2003b;Sander et al., 2004;Seabury et al., 2004a), all of them were confirmed in Korean cattle. Furthermore, we investigated the allelic frequencies of these polymorphic indels using 96 individuals by using PCR amplification method (data not shown), and then compared these frequencies with those found for other breeds of cattle, including both healthy and BSE-affected German cattle breeds, as well as U.S. sires ( Table 2). The allele frequencies of the 23 bp indel in Korean cattle were significantly higher than those determined for healthy German cattle breeds, U.S. sires, and BSE-affected German cattle. However, the frequencies of the 12 bp indel were similar to those of BSE-affected German cattle breeds, but differed from above two healthy cattle breeds. Most significant result of the Korean cattle were obtained from the 24 bp indel (octapeptide repeat) and 14 bp indel, which are located on the coding sequence and 3' UTR region respectively. The copy numbers of the octapeptide repeat exhibited a completely homozygous 6/6 genotype, and the allelic frequencies of 14 bp indel were completely fixed into two repeats. In addition, we detected a novel allelic variant, 19 bp/10 bp (CAGAATCTCTAAGGTGGGC→TTAGGAT GGA) (Figure 2), which is located on the putative promoter region, upstream from the 23 bp indel and represented 0.71 in allele frequency.  46327  46795  47004  64177  64590  64611  47221  47238  47450  47836~47837  48584  48689  48695  48732  48773  48815  48921  48890  49246  49345  49416  49729~49730  49542  49834  50000  57359  57904  50743  50121  50138  50297  50308  50319  50352  50376  51199  51208  51586  51862  51879  52183  52445  53793  54420  54428  55903~55905  55360  58212  58455  58999  59254  60291  60511  60835  61326  61426  65754~65825  73544~73640  67490~67491  66877  66948  70637  67864  69085  69359  69774  73493  74256 Figure 1. Diagram illustrating the genomic architecture of the bovine PRNP gene. All positions of SNPs, allele variants, and indels, whether they are previously described polymorphisms (Heaton et al., 2003;Hills et al., 2003;Sander et al., 2004) or were first detected in this study, are indicated with regard to the PRNP reference sequence (Hills et al., 2001; AJ298878). a) Three PRNP exons are represented by black boxes, and the protein coding region is depicted as a hatched box within exon 3. The black vertical lines indicate the positions of the previously reported polymorphisms. b) The positions of the polymorphic sites detected in Korean cattle are also represented with regard to the PRNP reference sequence (AJ298878). Long vertical lines represent common polymorphic sites between them, and the short lines represent specific polymorphisms on the respective cattle breeds. The seven large indel polymorphisms are indicated by arrows. The particularly highly polymorphic region surrounding the putative promoter is represented by a dotted circle. Furthermore, we also found 97 bp sequences difference between the Korean cattle and the breeds of Bos taurus (AJ298878: Hills et al., 2001, BN000291: Sander et al., 2004 previously reported, the sequences is located 3,884 bp downstream from exon 3 within the 3'-UTR. In order to further evaluate this discrepancy, we amplified the genomic region via PCR, using 10 individual Korean cattle and five individual animals from each of four strains (Simmental, Limousin, Holstein, and Brown Swiss) and determined their sequences. The alignment of these sequences with AJ298878 and BN000291 revealed that all of these sequences, except for two reference sequences, agreed perfectly (data not shown). Our result strongly suggest a possibility that the 97 bp sequence difference between them appears to have been caused by technical errors in AJ298878 and BN000291, although this confirmation is not sufficiently confident so as to completely exclude the possibility of strain specificity, due to the insufficient sample number used in this study.

DISCUSSION
The association of polymorphisms of the PRNP and BSE susceptibility has been investigated fairly extensively, in a variety of cattle breeds (Heaton et al., 2003;Hills et al., 2003;Sander et al., 2004;Seabury et al., 2004a). However, the Korean cattle there is not one of the breeds with which such polymorphism studies have been conducted, although the breed is one of the only domestic breeds in which BSE has never been detected. Therefore, in this study, we conducted an analysis of the sequence variability of the Korean cattle in the region including the putative promoter, three exons, two introns, and the 3'-UTR region of the PRNP gene locus. Consequently, we identified 73 total polymorphisms, including 65 single nucleotide polymorphisms (SNP), four indel polymorphisms (12 bp, 14 bp, 23 bp, and 24 bp), and four allelic variants exhibiting differences greater than two bp in adjacent nucleotides. A comparison of our findings with database entries and the results of studies performed with other cattle breeds revealed the presence of 27 additional polymorphic sites in Korean cattle, including 25 SNPs, one 19 bp/10 bp allelic variant, and one different region containing a 97 bp sequence. Polymorphisms in exon 3 within the 3'-UTR region have frequently been the subject of scrutiny in other cattle breeds, as sequence variation in the region has often been believed to exert critical influences with regard to mRNA processing, and this is especially true of the CDS region (Heaton et al., 2003;Hills et al., 2003;Takasuga et al., 2003;Zhang et al., 2004;Sander et al., 2004;Seabury et al., 2004b). We detected eight genetic polymorphic sites in exon 3 of the Korean cattle including six SNPs and two indels (24 bp and 14 bp). However, we were unable to detect any SNPs within the CDS region, which is interesting, as this has not been the case in any other breed of cattle.
The octapeptide repeat is localized on the coding sequence (CDS) of the PRNP (Figure 1) and reflected in the occurrence of three alleles: with five, six and seven repeats of the Pro-His/Gln-Gly(Gly)-Gly-Try-Gly-Gln amino-acid sequence. In the previously examined cattle breeds, the 6/6 homozygote genotype showed an extremely higher prevalence than the other genotype, especially in German (Sander et al., 2004), Scottish (Hunter et al., 1994), and Polish Black-and-White cattle breeds ( Walawski et al., 2003a). In this study we didn't observe any animals carrying the allele with five or seven octapeptide repeat, all the animals studied here was the 6/6 homozygote genotype.
In the future, other studies will investigate polymorphisms on the putative promoter region spanning exon 1, because mutations in this region may affect the levels of PRNP expression, and may also exert some influence on BSE incubation time and susceptibility (Bossers et al., 1996). Thus far, two compelling results have been reported; one involves the noticeably low frequency of repetitive elements in the region ~2.5 kb upstream of exon 1, in which the presence of additional functional elements might be speculated to exist; and the other involves the 23 bp indel located upstream of exon 1, which is generally considered to be an interesting candidate for a hypothetical regulatory mutation, assumed to be associated with mRNA processing.
Analyses of all the genetic variants identified here, as well as those identified in previous studies, indicate the presence of a SNP hot spot region which spans the putative promoter. However, the distribution of the SNPs in Korean cattle differs substantially from that observed in other cattle breeds. Furthermore, the allelic frequencies of the 23 bp indel in Korean cattle (0.56/0.44) exhibited a higher degree of similarity with those observed in healthy German cattle (0.43/0.57) and U.S. sires (0.30/0.70) than with BSEaffected German cattle (0.27/0.73). This indicates that the genetic resources of the Korean cattle differ significantly from those of other cattle breeds.
An ingestion of meat and bone meal originating from BSE-infected cattle has been implicated as a factor in the spread of BSE between cattle (Wilesmith, 1991). Therefore, the presence of exhaustive controls to avoid and prevent BSE infections might be the actual reason for the total absence of BSE in Korean cattle. And also, genetic factors, especially genetic variants to have found in CDS region of PRNP gene, can not rule out as the reason (Sander et al., 2004;Seabury et al., 2004b). Therefore, it is worth speculating that the Korean cattle does not show any SNPs within the CDS region of exon3. These factors may, to some degree, contribute to a genetic resistance to BSE in Korean cattle. Our findings of some reliable genetic differences between foreign and Korean cattle may prove vital to our current understanding of genetic factors associated with susceptibility to BSE.