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1.1 Review of literature
To detect point mutation at codon 211 of PRNP gene, a study was conducted on 536 Korean native cattle for the presence of point mutation at codon 211 of the PRNP gene using genomic DNA (Kim et al 2017). To evaluate the Familial BSE, specifically E211K, the putative mutations in both Korean dairy cattle (Holstein) and Korean native cattle (Hanwoo) were identified as well the direct sequencing of codon 211 and the adjacent regions of the bovine prion protein (PRNP) gene 384 Hanwoo and 152 Holstein cattle was carried out. No mutation of E211K was identified in either of the Korean cattle. From the above analysis it was concluded that E211K is a postulated mutation; hence, larger sample size and further detection in other countries was suggested.

To analyze the relationship between growth traits of healthy cattle and these indels, which could benefit for breeding through marker-assisted selection (MAS) and healthy cattle selection this study was conducted (Yang et al 2017). In the recent study the 23-bp indel and the 12-bp indel of PRNP in healthy individuals of different breeds were identified .The 1558 healthy Chinese cattle sampled were obtained from 6 indigenous breeds and two loci of PRNP were genotyped in these cattle. In order to evaluate the relationship between PRNP polymorphisms and phenotypic performance, phenotypic records for growth were utilized, which confirmed the presence of these two indel polymorphisms loci in the studied breeds. It was revealed from association analysis that the 23-bp indel was significantly contributing to the heart girth and body length in 18-months-old Nanyang cattle. There was a Significant relation between the 12-bp indel to the growth traits in three cattle breeds, such as the daily gained weight in 12-months-old Nanyang cattle, the body weight in Xia’nan cattle and the cannon circumference and rump length in Ji’an cattle. These results suggest that these two indels may affect bovine growth traits, which can be utilized for breeding through marker-assisted selection and healthy cattle selection.

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Yaman et al 2017 investigated amino acid and octarepeat polymorphisms of the PRNP coding region (exon 3) in Anatolian, Murrah, and Murrah × Anatolian (M × A) crossbred water buffaloes, and compare these breeds with other breeds of water buffalo, some American bison (Bison bison), and the native breeds of cattle. The PRNP gene of water buffalo was detected for the presence of one non-synonymous and three synonymous single nucleotide polymorphisms (SNP).the Triplet base changes (G/A/C) were observed at position 126; but coding to proline (P) was predicted in each case. An A/G substitution was detected at positions 234 and 285, without any amino acid replacements. Substitution of Glycine with serine (G108S) was resulted by G/A substitution at position 322 and at codon 211 (E211K) and the substitution of lysine with glutamic acid related to the atypical or hereditary BSE, was not detected. The coding sequence of interest was investigated for the detection of octarepeat polymorphisms. Octarepeat orders were observed in two forms as either R1 + R2+R3+R4+R5 +R6, or R1+R2+RN1+R4+R5+R6. It was found that all animals have six octarepeats. because of having both sequences had the PRNP6 octarepeat allele, The octarepeat genotype was regarded as wild-type PRNP6/6. Nucleotide and predicted amino acid sequences of the octarepeats.

For analyzing the frequency of polymorphisms associated with BSE susceptibility in Chinese buffalo and also compared the frequency of these polymorphisms in cattle with BSE, healthy buffalo and cattle, Previously study was conducted (Zhao et al 2015). The study also investigated the distributions of 12-bp and 23 indel polymorphisms in Chinese buffalo, a total of 312 animals were genotyped, with genotype, allele, and haplotype frequencies. Surprisingly low frequencies were found for the D23/D23 genotype (0.026) and D23 allele (0.050) in the 23-bp indel polymorphism site. In the 12-bp indel polymorphism site, the D12/D12 genotype was undetected, while the frequency of D12 allele was found extremely low (0.026). Four haplotypes (D23-D12, D23-I12, I23-D12 and I23-I12) were constructed by utilizing the program PHASE 2.1. The haplotype D23-D12 was found only in the Guangxi breed and the predominant I23-I12 haplotype was found in buffalo (0.928). From analysis three significant findings were obtained in buffalo: 1) the 23- and 12-bp indel polymorphisms extremely low deletion allele frequencies were identified; 2) in six octarepeats in CDS significantly low allelic frequencies were identified and 3) in buffalo CDSs the presence of S4R, A16V, P54S, G108S, V123M, S154N and F257L substitutions were identified. The McDonald–Kreitman test was performed for comparing the buffalo coding sequence to other species in order to reveal five groups (Bison bonasus, , Bos gaurus ,Bos indicus, Boselaphus tragocamelus, Syncerus caffer caffer) with significantly divergent non-synonymous substitutions from buffalo, suggesting potential divergence of the PRNP gene in buffalo and others species. These findings have revealed the evidence that PRNP gene in buffaloes have a unique genetic background in comparison with cattle
The frequencies of 23-bp indel polymorphism and 12-bp indels polymorphism in the promoter region (23indel) and in intron 1 region (12indel) was studied by Uchida et al 2014 respectively, they also studied the octapeptide repeat polymorphisms and SNPs in the bovine PRNP of cattle and water buffaloes in Vietnam, Indonesia and Thailand. In the 23 indel site of cattle of Indonesia and Thailand and water buffaloes, the frequency of the deletion allele in the 23 indel site was comparatively lower. The deletion allele frequency in the 12indel site was significantly lower in all of the cattle and buffaloes categorized in each subgroup. The deletion allele has been accounted to be related to susceptibility to classical BSE in both indel sites of the breed. Regardless of the fact that allele with 6 octapeptide repeats has been described to be most common in many breeds of cattle, in some Indonesian local cattle breeds, the frequency of the allele with 5 octapeptide repeats was significantly higher. For domestic cattle, the four SNPs reported in Indonesian local cattle have not been reported so far. This study gave the information on PRNP of livestock in Southeast Asian countries for the first time.

Galvao et al 2012 find out the genotypic profile of each animal sampled and at intron 1 (12-bp indel) of the bovine PRNP gene and the promoter region (23-bp indel) in BSE-free Caracu cattle indel polymorphisms was identified. For the two polymorphic regions examined, the Caracu cattle exhibited high allele frequency, 12ins (70 %) and 23ins (72.5 %), genotype frequencies of 50 % for 12ins/ins and 50 % for 23ins/del, and a high frequency of the 12ins–23ins haplotype (57.5 %). Of the 40 animals sampled, 15 had the 12ins–23ins/12ins–23ins diplotype.

For the analysis of both sequence variations in the coding region of PRNP and for the presence of indel polymorphisms in the non-coding region of PRNP by using a large number of samples from native breed in Korea (Hanwoo cattle) a and also analyzed the haplotype of gene variations and genetic frequency associated with BSE susceptibility and compared them to those of other cattle breeds was performed in an early studies (Choi et al 2012). Polymorphisms and mutations were identified in the coding region of PRNP, determined their frequency, and evaluated their significance. In this study four synonymous polymorphisms and two non-synonymous mutations in PRNP were identified, butno novel polymorphisms were found. It was concluded that the sequence and number of octapeptide repeats are completely conserved, and the coding region haplotype frequency was identical to that of other B. taurus strains. The 23-bp and 12-bp insertion/deletion (indel) polymorphisms in the non-coding region of PRNP were examined; as a result Hanwoo cattle had a lower 23-bp del/12-bp del haplotype frequency and deletion allele than healthy and BSE-affected animals of other strains. It was concluded by this study that Hanwoo are less susceptible to BSE as compared other strains due to the 23-bp and 12-bp indel polymorphisms.

It was observed in a study conducted by Imran et al 2012 revealed that both H-type BSE and L-type BSE are basically sporadic prion disorders. However, in the prion protein gene (PRNP),H-type BSE has recently been associated with E211K polymorphism. Two polymorphisms in the bovine PRNP are also associated with susceptibility to classical BSE: a 23 bp insertion/deletion (indel) in the PRNP promoter region and a 12 bp indel in the first intron. There is no information available regarding BSE susceptibility in Pakistani cattle.. This study was aimed to achieve this information. A total of 236 cattle from 7 breeds and 281 buffaloes from 5 breeds were screened for E211K polymorphism and 23 bp and 12 bp indels employing triplex PCR. The E211K polymorphism was not observed in any of the animals studied. The 23 bp insertion allele was underrepresented in studied cattle breeds while the 12 bp insertion allele was overrepresented. Both 23 bp and 12 bp insertion alleles were overrepresented in studied buffalo breeds. Almost 90% of alleles were insertion alleles across all studied buffalo breeds. The average frequency of 23 bp and 12 bp insertion alleles across all studied cattle breeds was found to be 0.1822 and 0.9407, respectively. There were significant differences between Pakistani and worldwide cattle were observed in terms of allele, genotype and haplotype frequencies of 23 bp and 12 bp indels. The higher observed frequency of 12 bp insertion allele suggests that Pakistani cattle are relatively more resistant to classical BSE as compared to European cattle. However, the key risk factor for classical BSE is the dietary exposure of cattle to contaminated feedstuffs.

An earlier study was conducted and a possible association of both studied PRNP promoter indel polymorphisms with susceptibility to BSE in Polish Holsteins was identified (Gurgul et al 2011). This study showed a significantly higher frequency of alleles deletion of 23 and 12 bp indel polymorphisms in diseased than in control animals (P = 0.0242 and P = 0.0024, respectively). The 23 bp indel polymorphism genotype distribution did not differ significantly between the selected groups; however, increased frequency of 23 bp del/del genotype was observed in BSE-affected cattle. A significant difference was of genotypic distribution of 12 bp indel polymorphism between the diseased and healthy animals (P = 0.0066), with approximately two times higher frequency of 12 del/del genotype in BSE-affected animals.A significant relation between the investigated PRNP indel polymorphisms (23 and 12 bp indels), and susceptibility of Polish Holstein-Friesian cattle to classical BSE (P.05) was found. The deletion variants of both polymorphisms were significantly associated to increased susceptibility, whereas insertion variants were found protective against BSE.

studied the sire and dam groups of Polish Holstein– Friesian cattle to determine the frequency of two putative PRNP polymorphic loci at allele, genotype, and Haplotype study was conducted by Czarnic et al 2011. The results obtained were compared with the frequencies in BSE-free andBSE affected German Holstein cattle populations. Allele frequencies at the 23 bp indel promoter polymorphism were 0.622 (del) and 0.378 (ins), with 0.613 and 0.387 in sires and 0.633 and 0.366 in dams. Allele frequencies at the 12 bp indel intron polymorphism were 0.527 (del) and 0.473 (ins), with 0.529 and 0.471 in sires and 0.543 and 0.456 in dams. Four haplotypes were identified in this population that is (23–12del, 23–12ins, 23del–12ins, and 23ins–12del). Haplotype 23–12del occurred most frequently in both sire and dam groups. A similar genetic pattern for the 23 bp indel polymorphism was observed by the comparative analysis of Polish Holstein–Friesian and German Holstein populations and 12 bp indels polymorphism was significantly different.

Zhu et al 2011 conducted as a study on the four main beef cattle breeds (Hereford, Simmental, Black Angus, and Mongolian) from North China and investigated the polymorphism distributions of 23- and 12-bp indels of the PRNP gene. The 23-bp insertion / 12-bp insertion was the major haplotype in Mongolian cattle,, whereas in Simmental ,Hereford, and Black Angus cattle, the 23-bp deletion / 12-bp deletion was the major haplotype. It was concluded from the current study that Mongolian cattle could be more resistant to BSE in comparison with other three cattle breeds, due toits relatively low frequencies of deletion genotypes and alleles of 23- and 12-bp indel polymorphisms. As a result,this race will be productive for selective breeding to improve resistance against BSE in this area.

Seven cattle breeds for investigating the distributions of indel polymorphism in the promoter (23-bp), intron 1 (12-bp), open reading frame (24- bp), and coding region of bovine PRNP were examined by Zhao et al 2010. These polymorphisms include a 23-bp insertion/deletion (indel) in the promoter region, a 12- bp indel in intron 1, an octapeptide repeat or 24-bp indel in the open reading frame, and a single nucleotide polymorphism (SNP) in the coding region. The frequency distributions of genotypes, alleles, and haplotypes at these indel sites in 349 native Chinese cattle and sequence variants in 50 samples were investigated authors. Their results reveals that frequencies of the 12-bp deletion allele and the 23-bp deletion / 12-bp deletion haplotype was low in cattle of southern China , which have been supposed to be relevant to BSE susceptibility. Interestingly, in the 12-bp indel polymorphism, a significant difference was observed between BSE-affected cattle and healthy Chinese cattle. A total of 14 SNPs were discovered in the coding region of PRNP in Chinese cattle. Three of these SNPs were associated with amino acid changes (K3T, P54S, and S154N). The E211K substitution that was recently reported in the US atypical BSE case was not observed in this study.

Qin et al 2010 investigated the polymorphisms of PRNP gene in 2 Chinese indigenous cattle breeds of northeast China focusing on the polymorphisms of PRNP gene in SNP in exon 3, 23-bp indel in promoter region, 12-bp indel in intron 1. By analyzing genomic DNA at PRNP locus in eighty-six animals from Yanbian and Chinese Red Steppes were genotyped. In the PRNP gene exon 3 of the 2 cattle breeds examined, total 4 single nucleotide polymorphism (SNP) sites were identified. One silent nucleotide substitutions (A234G) and three were non-synonymous mutations that resulted in the amino acid exchanges (K119N, S154N, and M177V) were observed. In Yanbian with a very low frequency (0.0147), the two amino acid mutations of S154N and M177V were detected and they seems to be missing in Chinese Red Steppes. The average gene heterozygosity (He), effective allele numbers (Ne), Shannon’s information index (I) and polymorphism information content (PIC) were 0.3088, 1.5013, 0.3814 and 0.2000 in Yanbian, respectively, being relatively higher than that of Chinese Red Steppes (0.2885, 1.4985, 0.3462 and 0.1873, respectively). In both Yanbian and Chinese Red Steppes breeds, three different genotypes were identified in 23-bp indel and 12-bp indel loci. Based 23- and 12-bp indels, four haplotypes was constructed in the 2 Chinese cattle breeds, of which the 23-bp (-)/12-bp (-) was main haplotypes accounting for more than 50% of the total in both Yanbian and Chinese Red Steppes breeds. These results might be useful in understanding the genetic characteristics of PRNP gene in Chinese indigenous cattle breeds.

An earlier study was performed for comparative analysis of the 12-bp intron region, 23-bp promoter region, the octapeptide region, and relevant PRNP polymorphisms for Japanese breeds, Asian native cattle populations, purebred mythun (Bos frontalis), and a composite population of mythun 9 cattle by Shimogiri et al 2010. As a result, the 23-bp indel site in the promoter was identified as polymorphic in all populations except Japanese Shorthorn. The deletion allele (23-) was a major allele, with frequencies of 0.63–1.00 in all populations except mythun (0.18). In all populations, a 12-bp indel site in intron 1 was polymorphic. In all populations except the Mongolian native cattle (0.49), the insertion allele (12) was a major allele, with frequencies of 0.55–0.93. In all Asian native cattle , a 14-bp indel site in the 30 UTR was polymorphic, but not in all Japanese breeds show polymorphic sites, mythun, and MCC. In all populations the insertion allele (14) was a major allele and at the 23-bp indel site, a deletion (23-) allele was identified as a major allele in all populations except mythun. At the 12-bp indel site, an insertion (12?) allele was a major allele in all populations. In all Asian native cattle, the 14-bp indel site was polymorphic. The six-repeat allele in the octapeptide repeat region, was a major allele in all populations. The genotypes 5/5 and 4/6 were detected in Japanese Black and Mongolian cattle and in mythun, respectively. In Asian native cattle and mythun, two non synonymous single nucleotide polymorphisms (SNPs) (K3Tand S154N) were detected. Haplotype analysis using the genotypes of the six sites estimated 33 different haplotypes and in all populations,the haplotype 23- 12- K 6 S 14 was found .

The association between PRNP haplotypes and BSE disease by tagging the haplotypes d within and across the PRNP locus using a set of htSNPs was exmined by Maddroch et al 2010 . Association with disease status of 18 of the haplotype tagging SNPs, as well as the 23 and 12-bp indels, were independently tested. In the sample set, three of the htSNPs were monomorphic. Therefore, from the haplotype analysis they were excluded. The alleles at these two indels were strongly linked and the haplotype defined by the insertion allele of the 23-bp indel, and the deletion allele of 12-bp indel (ID) were not observed, while the deletion/insertion (DI) haplotype was at low frequency in both the cases and controls. A combination of Illumina goldengate assay, sequencing and PCR amplification was used to genotype 18 htSNPs and 2 indels in 95 BSE case and 134 control animals. A haplotype within the region of high LD was found to be associated with BSE unaffected animals (p-value = 0.000114). A PRNP haplotype association with classical BSE incidence has been identified. This result suggests that a genetic determinant in or near PRNP may influence classical BSE incidence in cattle.

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 was analyzed by Oztabac et al 2009. Indel polymorphisms of PRNP promoter and intron 1 locus, was detected 106 Anatolian water buffalo DNAs by PCR based procedure. This study showed 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.

The polymorphisms in German cattle; 43 cattle with bovine spongiform encephalopathy (BSE) and 48 healthy animals from six different German cattle breeds were studied previously (Sander et al 2004). All three exons as well as the promoter region of the PRNP gene were investigated in contrast to previous studies. In the bovine PRNP gene sequence variants could have an impact on the expression level of the prion protein or amino acid sequence and thus on susceptibility to BSE. In this study a total of 60 polymorphisms in the PRNP gene of German cattle were identified. Out of these 60 polymorphisms, 36 polymorphisms were newly identified, while 24 of these polymorphisms had been reported previously. No novel polymorphism affecting the amino acid sequence of the prion protein was detected. However, a 23-bp insertion/deletion polymorphism were investigated in the putative PRNP promoter region that reveals a significant association with BSE susceptibility in the animals under study.

Msalya et al 2009 examined the bovine PRNP indels polymorphism of 23bp, 12bp and 14-bp, octapeptide repeats and two SNPs (K3T and S154N) in Japanese cattle breed. Genotyped for six bovine PRNP polymorphic sites including a 23bp indel in the promoter region, a 12bp indel in the intron 1, two no synonymous 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 indels sites were polymorphic. All of the six sites were mono morphic 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 identical to that of BSE affected cattle. We estimated 12 different haplotypes from these genotypic data. A ’23-12-K6S14+’ haplotype was most important haplotype in all breed; frequencies ranged of this haplotype were from 0.50 to 1.00.

The frequencies of the indels polymorphism in the PRNP in Vietnamese dairy cattle and Japanese BSE-affected cattle was studied by Muramatsu et al 2008. The genetic background of PRNP in Vietnamese dairy cattle and compared the frequencies of indels polymorphism in Vietnamese dairy cattle with other countries cattle was clarified in this study. The frequency distributions of insertion and deletion alleles in the 23bp indel site were 15.3% and 84.7%, in that order, for 206 Vietnamese dairy cattle were 7.1% and 92.9%, correspondingly, for 7 Japanese BSE affected cattle. The frequency distributions each of genotype (ins/ins, ins/del and del/del) in 23bp indel site were 1.5%, 27.8% and 70.7% in the Vietnamese cattle, and 0%, 14.3% and 85.7% in the Japanese BSE affected cattle. Additionally, the frequency distributions of insertion and deletion alleles in the 12bp indel site were 52.3% and 47.7% in Vietnamese dairy cattle, and 7.1% and 92.9% in the Japanese BSE affected cattle. The frequency distributions of each genotype (ins/ins, ins/del and del/del) in the 12bp indel site of these cattle were 19.5%, 65.6% and 14.9% in Vietnamese dairy cattle, and 0%, 14.3% and 85.7% in Japanese BSE affected cattle. In Vietnamese dairy cattle, the frequency distributions of deletion allele and del/del genotypic polymorphisms in 23bp indel site, which are consideration to be linked with BSE vulnerability, were significantly higher, while the frequencies of deletion allelic and del/del genotypic polymorphism in the 12bp indel site, which have been observed to confer BSE vulnerability, were significantly lower. This study proved evidence that Vietnamese dairy cattle have a distinctive genetic background in comparison with cattle previously reported in studies from other countries.

The indels polymorphism of the PRNP gene within the promoter region of the 23bp and 12bp of the intron 1 in Turkish South Anatolian red, East Anatolian red, and Turkish gray cattle was investigated by Un et al 2008. In this study collected blood samples from 150 animals of three different local breeds of the East Anatolian red, South Anatolian red, , and Turkish gray and were tested with DNA purification and PCR. In the 23bp indel polymorphism of the PRNP promoter region, the frequency of the deletion allele was higher than the insertion allele in both Anatolian red breeds. In difference, the frequency of insertion allele was higher (0.62) and the frequency of deletion allele was lower (0.38) in Turkish gray cattle. Generally, the more frequent allele in the 23bp indel polymorphism was the deletion allele in the South Anatolian cattle (frequency = 0.64), followed by the insertion allele in the Turkish gray cattle (0.62) and the deletion allele in the East Anatolian cattle (0.60). In the 12bp indel polymorphism of the PRNP intron 1, the insertion allele was more numerous in all investigated breeds (Turkish gray 0.80, East Anatolian 0.72, and South Anatolian cattle 0.69). The insertion allele in the 12bp indel, which is linked with low vulnerability to BSE, demonstrated a high frequency in all three breeds. The low vulnerability allele of the 23bp indel was recognized in Turkish gray cattle with a frequency of 0.80. Results demonstrated that local Turkish cattle have a significant genetic value for selection against BSE.

The frequencies of the identified genetic factors associated with BSE vulnerability and resistance in a varied sample proposed to represent the global population was examined by Brunelle et al 2008. This study provided a thoroughly comprehensive comparative examination of 23bp promoter region, 12bp intron 1, and significant PRNP polymorphisms for B. indicus, B. taurus, and B. indicus × B. taurus combined cattle. Variation in the frequencies of these recognized risk factors might be elucidating variation in overall resistance and vulnerability to classical BSE among the cattle groups investigated. TSE linked PRNP encoded amino acid polymorphisms were reported for B. indicus purebred and combined cattle and all had typical number of octapeptide repeats. On the other hand, variations were observed in the frequencies of the 23bp and 12bp indels polymorphism linked with two bovine PRNP transcription regulatory sites. Compared to B. taurus, B. indicus purebred and composite cattle had a drastically lower frequency of 23bp insertion alleles and homozygous genotypes. Furthermore, B. indicus purebred cattle had a drastically higher frequency of 12bp insertion alleles and homozygous genotypes in connection to both B. taurus and combined cattle. The basis of these differences can be certified to a drastically different haplotype arrangement within each breed. The frequencies of the 23bp and 12bp indels were significantly different among B. indicus and B. taurus cattle. Possible risk factors were found for the B. indicus purebred and combined cattle. Recently, no consensus exists concerning which bovine PRNP indel region is more significant with respect to classical BSE. Should one particular indel region and linked genotypes verify more influential with respect to the occurrence of classical BSE, variations concerning overall vulnerability and resistance for B. indicus and B. taurus cattle might be clarified.

The distribution of the 23 and 12 bp indels polymorphism of the PRNP gene in three cattle breeds (Aberdeen Angus, Charolais, and Franqueiro) was examined by Kerber et al 2007 examined, in order to arrange the way for future selection of resistant animals and significant genetic value for selection against BSE in Brazil. Except the Franqueiro herd deletion in both indels was high frequency in almost all breeds, which presented 67% of the 12bp insertion. Results demonstrated that no variations between breeds were verified for the 23bp indel while the Franqueiro was significantly different from the other groups in connection to the 12bp indel (P.001). The frequency of the 23bp deletion in healthy cattle ranges from 0.35 in the German Brown breed to 0.79 in a Japanese Holstein breed; the value for the 12 bp deletion varies from 0.14 in German Brown breed to 0.74 in Japanese Holstein breed. Hence, the data for Aberdeen Angus and Charolais (in both indels) and Franqueiro in the 23 bp deletion recline on the upper variation limit; Franqueiro presented one of low frequency for the 12 bp deletion. Franqueiro is a local breed limited to upland areas, with small numbers of animals. High frequencies of the vulnerability alleles (23 and 12 bp deletion) and haplotype (23 del/12 del) were reported in the Aberdeen Angus and Charolais herds whereas the Franqueiro presented one of the highest frequencies of resistant alleles so far explained. Data showed the require for selection in Aberdeen Angus and Charolais breeds to enhance the frequency of resistant animals in order to reduce the chance of BSE occurrences in these breeds.

Czarnic et al 2007 examined the indels polymorphisms of the bovine PRNP within the promoter region in 23bp and the 12bp in the intron 1 and 3′ untranslated region in 14bp. In this study the DNA was extracted from blood of 234 randomly tested Polish Holstein-Friesian cows and semen isolated from 47 sires used for artificial insemination (AI) in 2004. Result demonstrated that no statistically significant differences were detected in the frequency of genotypes and alleles among cows and breeding sires.

Analysis of DNA polymorphisms of the promoter region of the PRNP gene in four main German bovine breeds resulted in the identification of both SNPs and indels polymorphism as investigated by Kashkevich 2007 et al. Comparison of genotype between both healthy and affected BSE animals identified a significantly different distribution of 12bp and 23bp indels polymorphism and two SNPs within Braunvieh animals, suggesting a connection of these indels polymorphism with BSE vulnerability. The useful significance of these indels polymorphism was examined utilizing reporter gene builds in neuronal cells. A specific haplotype close to exon 1 was recognized that showed a significantly low expression level. Genotyping of nine polymorphisms within the promoter region and haplotype calculation exposed that the haplotype linked with the very low expression level was below represented in BSE affected animals of all breeds compared to healthy animals, signifying an association of reduced PRNP expression and higly elevated resistance to BSE.

The comparison of the allelic and genotypic frequencies of 23bp in the promoter region and 12bp in the intron of the PRNP gene indels polymorphism among 449 BSE affected cattle and 431 healthy cattle was conducted by Haase et al 2007 to verify the earlier-reported connection in a large sample. In this study used to German healthy group and also investigated Swiss cattle for the first time. Swiss cattle used to be appropriate for enlarging the available animals as some breeds within Germany and Switzerland are associated. Additionally, a plenty number of confirmed BSE affected cases was available in Switzerland. Group of nine polymorphisms was used for genotyping and haplotype calculation in 127 control breeding bulls and 293 German cows tested positive for BSE. The promoter activity of the major haplotypes was identified by luciferase reporter gene experiments in neuronal cells and the connection of differentially expressed haplotypes to BSE vulnerability was examined by evaluating the representation of these haplotypes in the BSE affected and healthy groups. Allelic, genotypic and haplotypic frequencies of the two indels polymorphism were examined in 449 BSE affected cattle and 431 healthy cattle from Switzerland and Germany including all 43 German BSE affected and 16 German healthy animals from original study. Allelic and genotypic distributions similar data were compared the 23bp indel polymorphism once more demonstrated a significant association with susceptibility to BSE. On the other hand, some further breed-specific allelic and genotypic distributions were examined, mostly linked to the Brown breeds. This Study helped earlier findings that indels polymorphism in the PRNP gene promotor region have an association on vulnerability to BSE. Moreover, breed specific variations exist that needs to be accounted for when analyzing such data.

Julling et al 2006 investigated the effects of two indels polymorphism that were possibly linked with BSE in a small case control study. In this study 23bp indel in the promoter region and the 12bp indel in intron 1 influence binding sites for transcription factors (RP58 and SP1, respectively) and therefore may be associated with the expression of PRNP gene. Examined the both indels polymorphism in BSE affected and healthy animals of four different breeds: Holstein-Friesian cattle from the United Kingdom (UK Holstein) and Germany (German Holstein), German Brown and German Fleckvieh. Demonstrated that previously reported of the 23bp and 12bp indels polymorphism in the regulatory region of the bovine PRNP gene are strongly associated with BSE occurrence in cattle. Genotyping of BSE affected and healthy animals of UK Holstein, German Holstein, German Brown and German Fleckvieh breeds exposed a significant high demonstration of the deletion at both polymorphic sites in BSE affected animals (P = 2.01 × 10-3 and P = 8.66 × 10-5, correspondingly). The main effect on susceptibility is linked with 12bp indel polymorphism. Compared with non-carriers, heterozygous and homozygous carriers of the 12bp deletion have moderately higher risks of occurrence BSE, ranging from 1.32 to 4.01 and 1.74 to 3.65 in the different breeds. These values corresponding to the population characteristic risks ranging from 35% to 53%. Results demonstrate a considerable genetic PRNP gene linked part for BSE vulnerability in cattle. Though the BSE risk presented by the deletion of the 12bp indel in the regulatory region of PRNP gene is significant, the main risk cause for BSE in cattle is ecological, i.e. exposure to feeding stuffs infected with the infectious agent.

The frequencies of the BSE linked PRNP indels polymorphism for a varied group of commercial U.S. artificial insemination (AI) sires containing of 39 different breeds were examined by Seabury et al 2004 examined and evaluated them to those recently described for healthy cattle and BSE affected cattle. Forward primer sequences from previously reported primer sets targeting indels polymorphism within the putative bovine PRNP gene promoter region, intron 1, and the 30 UTR (untranslated region) were produced with unique 50 fluorescent markers and used to build up a rapid multiplexed PCR assay for identifying BSE linked indels polymorphism as well as facilitating polymorphism studies and marker-assisted collection. Significantly differences (p < 0.05 all tests) were found among the frequencies of bovine PRNP gene promoter alleles for 48 healthy German cattle previously explained and 132 commercial U.S. cattle sires. The frequency of the 23bp promoter region observed for commercial U.S. cattle sires was found strongly be similar to that recently expressed for 43 BSE affected German cattle. No significant difference (p = 0.051) was found among the distributions of promoter genotypes for healthy German cattle and our group of commercial U.S. cattle sires. Interestingly, drastically differences (p < 0.01; p < 0.02) were also observed among the frequencies and distributions of intron 1 alleles and genotypes, likewise, for BSE affected German cattle and our group of U.S. cattle sires. No significantly allele or genotype differences were found for 14-bp 3’UTR indel for any given assessment among German cattle and commercial U.S. cattle sires.