Gene and haplotype polymorphisms of the Prion gene (PRNP) in Japanese Brown, Japanese native and Holstein cattle

Polymorphisms in the prion protein gene ( PRNP ) are known to be associated with transmissible spongiform encephalopathies in human, sheep and goats. There is tentative association between PRNP promoter polymorphism and bovine spongiform encephalopathy (BSE) susceptibility in cattle. In this study, we genotyped for six bovine PRNP polymorphic sites including a 23-bp indel in the promoter, a 12-bp indel in the intron 1, two nonsynonymous single nucleotide polymorphisms (SNPs), octapeptide repeats in the coding region and a 14-bp indel in the 3'-untranslated region in 178 animals representing Japanese Brown, Kuchinoshima feral, Mishima, Japanese Shorthorn and Holstein. In 64 Japanese Brown cattle, three indel sites were polymorphic. All of the six sites were monomorphic in Kuchinoshima. The 23-bp and 12-bp indel sites were polymorphic in Mishima cattle. The 23-bp and 14-bp indel sites were polymorphic in Japanese Shorthorn cattle. Both SNP sites were monomorphic in all cattle examined in this study. At the 23-bp indel site, the genotype frequencies of Japanese Brown and Holstein breeds were similar to that of BSE affected cattle. We estimated 12 different haplotypes from these genotypic data. A '23-12-K6S14+' haplotype was the major haplotype in all populations, whose frequencies ranged from 0.50 to 1.00.     

Frequencies of PRNP gene polymorphisms in Vietnamese dairy cattle for potential association with BSE

Summary Since 2004, significant associations between bovine spongiform encephalopathy (BSE) susceptibility in cattle and frequencies of insertion/deletion (ins/del; indel) polymorphisms within the bovine prion protein gene (PRNP) have been reported. In this study, we investigated the frequencies of indel polymorphisms within two variable sites, a 23-bp indel polymorphism in the promoter region (23indel) and a 12-bp indel polymorphism in intron 1 region (12indel), in the PRNP in 206 Vietnamese dairy cattle and seven Japanese BSE-affected cattle. In Vietnamese dairy cattle, the frequency distributions of del allele and del/del genotypic polymorphisms in the 23indel site, which are thought to be associated with BSE susceptibility, were significantly higher, whereas the frequencies of del allelic and del/del genotypic polymorphisms in the 12indel site, which have been reported to confer BSE susceptibility, were significantly lower. We have provided evidence that Vietnamese dairy cattle have a unique genetic background in the PRNP gene in comparison with cattle or sires previously reported in other countries.     

Sequence variations of the bovine prion protein gene (PRNP) in native Korean Hanwoo cattle

Bovine spongiform encephalopathy (BSE) is one of the fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs) caused by infectious prion proteins. Genetic variations correlated with susceptibility or resistance to TSE in humans and sheep have not been reported for bovine strains including those from Holstein, Jersey, and Japanese Black cattle. Here, we investigated bovine prion protein gene (PRNP) variations in Hanwoo cattle [Bos (B.) taurus coreanae], a native breed in Korea. We identified mutations and polymorphisms in the coding region of PRNP, determined their frequency, and evaluated their significance. We identified four synonymous polymorphisms and two non-synonymous mutations in PRNP, but found no novel polymorphisms. The sequence and number of octapeptide repeats were completely conserved, and the haplotype frequency of the coding region was similar to that of other B. taurus strains. When we examined the 23-bp and 12-bp insertion/deletion (indel) polymorphisms in the non-coding region of PRNP, Hanwoo cattle had a lower deletion allele and 23-bp del/12-bp del haplotype frequency than healthy and BSE-affected animals of other strains. Thus, Hanwoo are seemingly less susceptible to BSE than other strains due to the 23-bp and 12-bp indel polymorphisms.     

Variation in the coding region of the prion protein gene in Slovak cattle

This study was conducted to investigate the presence of single nucleotide polymorphisms (SNPs) in the coding region of the bovine prion protein (PrP) gene among healthy and bovine spongiform encephalopathy (BSE-) affected cattle in Slovakia. Denaturing gradient gel electrophoresis (DGGE) and single-strand conformation polymorphism (SSCP) followed by DNA sequencing were used to identify SNPs and variations in octapeptide repeats. Altogether three single nucleotide polymorphisms (g234a, c339t and c576t) and variations in the number of octapeptide repeat units (5 or 6) were found in the analysed part of the prion protein gene. All single nucleotide polymorphisms were silent, causing no amino acid changes. Significant differences (P < 0.05) in the genotype distribution of g234a polymorphism were observed when the homozygous genotype with a mutated allele (caa/caa) was compared to the heterozygous genotype -/cag among healthy and BSE-affected cattle. The homozygous genotype caa/caa was characteristic of the group of BSE-affected cattle. Additionally, the homozygous genotype caa/caa was significant for the group of Simmental crossbreeds among healthy cattle. The allele and genotype distribution of the other polymorphisms was not significantly different among groups of healthy and BSE-affected cattle. The possible influence of a silent mutation on expression of the gene is not clearly determined and needs further investigations.     

牛朊蛋白基因多态性对疯牛病影响的研究进展

朊蛋白(prion protein,PRNP)是近年来已证明的人和部分哺乳动物传染性海绵状脑病(transmissible spongiform encephalopathy,TSE)的主要根源,该蛋白编码基因的多态性显著影响了人和动物对TSE的易感性或抗病性。牛传染性海绵状脑病俗称"疯牛病"。作者分析了疯牛病的起源、监测和预防措施;简要介绍了牛PRNP基因的结构与功能;系统分析了牛科动物PRNP基因非编码区多态性与抗病性作用;总结了牛科动物PRNP基因启动子区域内23 bp插入/缺失和第1内含子区域内12 bp插入/缺失对疯牛病易感性的影响,为牛的抗病分子育种提供指导。     

BSE‐associated polymorphisms in the prion protein gene: an investigation

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.     

Prion protein gene polymorphism and blood lymphocyte profile in cows naturally infected with bovine leukemia virus

The polymorphic loci of the bovine prion protein (PRNP) gene, comprising 23-bp insertion/deletion (23-bp indel) within the promoter sequence and 12-bp insertion/deletion (12-bp indel) within the intron 1 sequence, are located in regions which play a key role in gene expression. The objective of this study was to determine whether the 23-bp and 12-bp insertion/deletion polymorphism within the PRNP gene leads to significant differences in the blood lymphocyte profile and to investigate changes in the composition of these cells in cattle naturally infected with Bovine Leukemia Virus. An analysis of the effect of the bovine PRNP gene polymorphism on the blood lymphocyte profile revealed considerable differences between animals with the 23-bp indel genotypes, and small and statistically non-significant differences between those with the 12-bp indel genotypes. 23-bp del/del homozygotes had a significantly lower percentage of T lymphocytes with the phenotypes CD2 (P < 0.01), CD8 (P < 0.01) and WC1-N2 (P < 0.05), and a higher ratio of CD4 to CD8 T lymphocytes, compared to animals with the 23-bp ins/ins genotype. The obtained results indicate that the 23-bp indel polymorphism, in contrast to the 12-bp indel polymorphism, has a significant effect on changes in the blood lymphocyte profile. The size of blood lymphocyte subpopulations was also found to change under the influence of enzootic bovine leukosis. The direction of those changes in EBL-positive animals is consistent with that observed in 23-bp del/del homozygotes, which may testify to the adverse effect of this genotype on immunological efficiency.     

杂交牛(大额牛×云南黄牛)朊蛋白基因 的分子克隆及其序列分析

朊蛋白(prion protein,PRNP)基因编码朊蛋白,是引起疯牛病的主效基因。本研究利用PCR方法首次从杂交牛(大额牛×云南黄牛)基因组中扩增了PRNP基因,GenBank登录号为HQ875337。PCR产物直接双向测序表明,该序列包含杂交牛PRNP基因795 bp的开放阅读框(ORF),编码264个氨基酸前体蛋白。生物信息学分析结果发现,该蛋白包含1个信号肽、3个α螺旋、2个β折叠、6个八肽重复序列、1个疏水区域、1个二硫键和1个糖基磷脂酰肌醇锚定位点。与已报道的其他牛PRNP基因进行序列比对分析,核苷酸和氨基酸的同源性均在97%以上。     

Frequencies of bovine PrP gene polymorphisms in Holstein and Japanese Black bulls in Japan

We screened for Japanese Black and Holstein bull sire samples to detect single nucleotide polymorphisms (SNPs) involving animo‐acid substitutions in the bovine prion gene in the entire coding region of the PRNP gene. Although three silent SNPs were found, we could not detect any SNP with animo‐acid substitution. We also examined the polymorphism of the octapeptide repeat number in these samples. There was no homozygous bull with repeat number 5. The frequency of heterozygous (6/5) bulls was 8% in the Japanese Black bull and 4% in the Holstein bull, respectively. The bull samples used in this study contain popular elite sires, so it appears that the polymorphisms of prion protein (PrP) are rather difficult to find in these two breeds in Japan, except for polymorphism of the octapeptide repeat number.     

Identification of leptin gene polymorphisms associated with carcass traits and fatty acid composition in Japanese Black cattle

Previous studies have indicated that some leptin gene polymorphisms were associated with economically important traits in cattle breeds. However, polymorphisms in the leptin gene have not been reported thus far in Japanese Black cattle. Here, we aimed to identify the leptin gene polymorphisms which are associated with carcass traits and fatty acid composition in Japanese Black cattle. We sequenced the full‐length coding sequence of leptin gene for eight Japanese Black cattle. Sequence comparison revealed eight single nucleotide polymorphisms (SNPs). Three of these were predicted to cause amino acid substitutions: Y7F, R25C and A80V. Then, we genotyped these SNPs in two populations (JB1 with 560 animals and JB2 with 450 animals) and investigated the effects on the traits. Y7F in JB1 and A80V in JB2 were excluded from statistical analysis because the minor allele frequencies were low (< 0.1). Association analysis revealed that Y7F had a significant effect on the dressed carcass weight in JB2; R25C had a significant effect on C18:0 and C14:1 in JB1 and JB2, respectively; and A80V had a significant effect on C16:0, C16:1, C18:1, monounsaturated fatty acid and saturated fatty acid in JB1. The results suggested that these SNPs could be used as an effective marker for the improvement of Japanese Black cattle.     

Detection of prion gene promoter and intron1 indel polymorphisms in Anatolian water buffalo (Bubalus bubalis)

Bovine spongiform encephalopathy (BSE) is a fatal disease caused by miss folded prion protein. Studies in the cattle, comparing genetic data from BSE diseased and healthy animals have shown that indel polymorphisms in the promoter and intron 1 of PRNP gene were associated with disease susceptibility. Several studies were conducted to find out allele and genotypic frequencies of indel polymorphisms in promoter and intron 1 of the cattle PRNP gene. Unlike domestic cattle and bison, no indel polymorphisms of the PRNP promoter and intron 1 were examined in any population of the water buffalo (Bubalus bubalis). Aim of this study was to analyse frequencies of allele, genotype, and haplotype of the indel polymorphisms (23 bp indel in promoter and 12 bp indel in intron 1) in prion protein coding gene (PRNP) of water buffalo. Therefore a PCR based procedure, previously used in cattle to detect indel polymorphisms of PRNP promoter and intron 1 locus, was applied to 106 Anatolian water buffalo DNAs. Our results have revealed high frequency of in variants and in23/in12 haplotype for PRNP promoter and intron 1 indel polymorphisms in water buffalo. The results of the study have demonstrated that frequencies of allele, genotype, and haplotype of the indel polymorphisms in PRNP gene of the Anatolian water buffalo are significantly different those from cattle and bison PRNP indel polymorphisms.     

Nucleotide and octapeptide-repeat variations of the prion protein coding gene (<Emphasis Type="Italic">PRNP</Emphasis>) in Anatolian,Murrah, and crossbred water buffaloes

Resistance to bovine spongiform encephalopathy (BSE) that is significantly associated with insertion/deletion (indel) polymorphisms at two loci (putative promoter and intron 1) on the prion protein gene (PRNP) in cattle has been well documented. Studies suggest that the insertion alleles are related to BSE resistance. Until recently, BSE has never been reported in water buffaloes (unlike cattle). Previous studies have demonstrated that the PRNP gene in water buffalo consists mostly of insertion alleles at both loci; nevertheless, whether or not water buffaloes are genetically resistant to BSE and the role of indel polymorphisms in their resistance status is not clear. We examined the coding region of PRNP to determine the nucleotide and octapeptide-repeat (octarepeats) variations of Anatolian, Murrah and Murrah × Anatolian (M?×?A) water buffaloes. Three synonymous single nucleotide polymorphisms (SNP) at positions 126, 234, and 285, and a non-synonymous SNP at position 322 (G108S) were detected. Triplet G/A/T base substitutions were observed at position 126 and two additional genotypes, T/A and T/G, at this position were determined. We also found six octarepeats that indicated the presence of the wild-type PRNP6 allele in the coding region. To the best of our knowledge, this is the first report of the T/A and T/G genotypes in water buffaloes.     

荷斯坦公牛PRNP多态性与精子活力相关性及抗病性评估

为分析荷斯坦公牛朊蛋白基因(PRNP)多态性与精子活力的关系,并对其抗病性进行评估,选育抗病公牛,本实验以公牛精液为样品,研究脚基因中12bp、23 bp和24 bp 3个片段的插入/缺失多样性、基因mRNA转录水平及其与精子活力等指标的关系.结果表明,12bp和23 bp在群体中均得到3种基因型,24 bp只有2种基因型.不同基因型群体的mRNA转录水平存在显著差异,而基因型与精液产量和活力等指标存在相关性,抗病力和精子活力指标存在负相关.本研究中3个片段的插入-缺失多样性可以作为公牛选育的辅助标记.     

Bos indicus type of growth hormone receptor gene is retained in Japanese Black cattle

The growth hormone receptor (GHR) gene is responsible for growth and carcass traits, and polymorphisms associated with the variation of meat production are thought to occur in the liver‐specific promoter of the GHR gene in cattle. The aim of this study was to analyse the structure of the liver‐specific promoter of GHR in Japanese Black cattle, as the relationship between GHR polymorphism and meat production is poorly understood in this breed. Typically in European cattle, the LINE‐1 element, a family of retrotransposons, is inserted in the liver‐specific promoter. However, a short GHR promoter without the LINE‐1 sequence was found in the Japanese Black breed as in Bos indicus cattle. The frequency of the short allele was approximately 60%. In addition, 24 of 29 Holstein/Japanese Black crosses carried the short allele from their sire. The present result suggests that the short allele for GHR may be a candidate marker for improving meat production of Japanese Black cattle.     

Polymorphisms of the prion gene promoter region that influence classical bovine spongiform encephalopathy susceptibility are not applicable to other transmissible spongiform encephalopathies in cattle

Two regulatory region polymorphisms in the prion gene of cattle have been reported to have an association with resistance to classical bovine spongiform encephalopathy (BSE). However, it is not known if this association also applies to other transmissible spongiform encephalopathies (TSE) in cattle. In this report, we compare the relationship between these 2 polymorphisms and resistance in cattle affected with naturally occurring atypical BSE as well as in cattle experimentally inoculated with either scrapie, chronic wasting disease, or transmissible mink encephalopathy. Our analysis revealed no association between genotype and resistance to atypical BSE or experimentally inoculated TSE. This indicates the promoter polymorphism correlation is specific to classical BSE and that atypical BSE and experimentally inoculated TSE are bypassing the site of influence of the polymorphisms. This genetic discrepancy demonstrates that atypical BSE progresses differently in the host relative to classical BSE. These results are consistent with the notion that atypical BSE originates spontaneously in cattle.     

Milk yield, age at first calving, and the risk of BSE: an analysis at the farm level in France

The epidemic of bovine spongiform encephalopathy (BSE) in France, as in the UK, has affected dairy cattle much more than beef cattle. However, the intensification of dairy herd management as a risk factor for BSE has not to date been analyzed. For this purpose, two databases were merged: the French Milk Records database, and the French BSE database, which can be considered as being devoid of notification bias since July 2001, when systematic tests were implemented. Only pure Holstein herds were considered, which represent the vast majority of total and BSE-affected dairy herds in France. A case-control study was designed so that 20 control herds were matched to each case herd according to the location of the farm and the year of birth of the index case. Three thousand and forty five farms were included, among which 145 with a BSE case notified between July 2001 and July 2003, and 2900 controls. With respect to the risk of BSE, odds ratios for each class of milk yield and age at first calving were estimated by using conditional logistic regression models with appropriate adjustments to herd size. The two main results were the following: firstly, whereas most Holstein herds, with average production between 7000 and 10,000kg, had nearly the same BSE risk, a small category of very intensive herds, with annual milk yields above 10,000kg, were significantly more at risk than the other herds. Secondly, a very early first calving (under 26 months of age) was found to be at risk for BSE as compared to other categories, independently of the milk yield. These results are discussed in the light of the known age-dependent susceptibility to BSE.     

A novel method for rapid and reliable detection of complex vertebral malformation and bovine leukocyte adhesion deficiency in Holstein cattle

Background: Complex vertebral malformation (CVM) and bovine leukocyte adhesion deficiency (BLAD) are two autosomal recessive lethal genetic defects frequently occurring in Holstein cattle, identifiable by single nucleotide polymorphisms. The objective of this study is to develop a rapid and reliable genotyping assay to screen the active Holstein sires and determine the carrier frequency of CVM and BLAD in Chinese dairy cattle population. Results: We developed real-time PCR-based assays for discrimination of wild-type and defective alleles, so that carriers can be detected. Only one step was required after the DNA extraction from the sample and time consumption was about 2 hours. A total of 587 Chinese Holstein bulls were assayed, and fifty-six CVM-carriers and eight BLAD-carriers were identified, corresponding to heterozygote carrier frequencies of 9.54% and 1.36%, respectively. The pedigree analysis showed that most of the carriers could be traced back to the common ancestry, Osborndale Ivanhoe for BLAD and Pennstate Ivanhoe Star for CVM. Conclusions: These results demonstrate that real-time PCR is a simple, rapid and reliable assay for BLAD and CVM defective allele detection. The high frequency of the CVM allele suggests that implementing a routine testing system is necessary to gradually eradicate the deleterious gene from the Chinese Holstein population.     

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.     

High incidence of single nucleotide polymorphisms in the prion protein gene of native Brazilian Caracu cattle

Different alleles of the human and ovine prion protein gene correlate with a varying susceptibility to transmissible spongiform encephalopathies. However, the pathogenic implications of specific polymorphisms in the bovine prion protein gene (PRNP) are only poorly understood. Previous studies on the bovine PRNP gene investigated common European and North American cattle breeds. As a consequence of decades of intensive breeding for specific traits, these modern breeds represent only a small fraction of the bovine gene pool. In this study, we analysed PRNP polymorphisms in the native Brazilian Caracu breed, which developed in geographical isolation since the 16th century. A total of 10 single nucleotide polymorphisms (SNPs) were discovered in the coding region of the Caracu PRNP gene. Eight of the SNPs occurred at high frequencies in Caracu cattle (variant allele frequencies = 0.10–0.76), but were absent or only rarely observed in European and North American breeds. One of the Caracu SNPs was associated with an amino acid exchange from serine to asparagine (f = 0.17). This SNP was not detected in Holstein–Friesian, Simmental and German Gelbvieh and was only rarely detected in beef cattle (f = 0.01). We found 17 haplotypes for PRNP in the Caracu breed.     

The T allele at the g.1471620G>T in the EDG1 gene associated with high marbling in Japanese Black cattle is at a low frequency in breeds not selected for marbling

Our previous study detected a single nucleotide polymorphism (SNP), g.1471620G > T , in the 5' flanking region of the endothelial differentiation sphingolipid G-protein-coupled receptor 1 ( EDG1 ) gene, which has been considered as a positional functional candidate for the gene responsible for marbling, and showed association of the g.1471620G > T SNP with marbling in Japanese Black beef cattle. In the present study, we investigated the allele frequency distribution of the g.1471620G > T SNP among the 5 cattle breeds, Japanese Black, Japanese Brown, Japanese Short Horn, Holstein, and Brown Swiss breeds. The T allele at the g.1471620G > T SNP associated with high marbling was found at high frequency in Japanese Black breed that has been subjected to a strong selection for high marbling, while the allele was absent or at very low frequencies in the other breeds that have not been strongly selected for high marbling. Based on this finding, we hypothesized that the pressure of the strong selection for high marbling in Japanese Black breed has increased the frequency of the T allele at the g.1471620G > T SNP in the EDG1 .