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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