Identification of Different Allelic Variants of PrP Gene Effective on Scrapie in Naeinian Goats Breed

Document Type : Genetics & breeding

Authors

• Mojtaba Najafi ¹
• Seyed Hasan hafezian ²
• Mohammad ALi Rouhi ³
• mohsen godarzi ³

¹ Sari Agricultural Sciences and Natural Resources University

² Animal Science Department, Faculty of Animal Sciences and Fisheries, Sari Agricultural Sciences and Natural Resources University, Sari, Iran.

³ SANRU

10.22067/ijasr.v8i3.49027

Abstract

Introduction The accumulation of improperly folded forms of host-encoded cellular prion protein (PrPc) in the central nervous system (CNS) lead to a fatal neurodegenerative disease in sheep and goats, namely Scrapie. The application of genetic breeding programs to eradicate transmissible spongiform encephalopathies in goats is an important aim for reasons of animal welfare as well as human food safety and food security. Scrapie and scrapie-like diseases are associated with polymorphisms and mutations of the gene coding for PrP, a host neuronal membrane glycoprotein which is found in an aggregated form (scrapie-associated fibrils) in extracts of brain tissues from all mammals affected by these diseases. The different allelic forms of PrP gene have been shown to make animals variably susceptible to this disease. It has been established that presence of abnormal forms of the prion protein (PrP) is associated with scrapie. Several amino acid polymorphisms caprine PrP encoding genes have been reported to be associated with scrapie susceptibility. Sheep exposed to Scrapie have been shown to gain highest scrapie resistance in the presence of Q171R polymorphism and maximum scrapie susceptibility in the presence of A136V polymorphism. Based on A136V, R154H and Q171R/H polymorphisms and their five alleles (ARQ, VRQ, AHQ, ARR and ARH) sheep and goats can be classified into five groups (R1-R5). The most resistant genotype (R1) is ARR/ARR and the most susceptible genotypes (R5) are VRQ/VRQ, VRQ/ARQ, VRQ/ARH and VRQ/AHQ. Additionally, other caprine PrP polymorphisms I142M, H143R, N146S/D, R154H, R211Q and Q222K have been shown to be associated with low scrapie risk. Although scrapie is an animal health issue, its presence has not been investigated in Iranian goat breeds, where sheep and goats are major livestock species. Based on the positive impact of PrP genetics on sheep scrapie in Europe in the past decade, we have established caprine PrP gene variation in 120 Naeinian goats from the Isfahan, Iran to evaluation of genetic variation in this important region of PrP gene.
Material and Methods The DNA was extracted from 120 blood samples of Naeinian goats from Isfahan province using modified salting out method. After amplification of the desired fragment by polymerase chain reaction (PCR), genotyping of samples was carried out by PCR-SSCP Analysis (Single-Strand conformation Polymorphism). In the following, direct sequencing method was used to confirm the genotyping results. Obtained sequences were analyzed by Chromas Pro and BioEdit. In order to evaluate the amino acid polymorphisms caprine PrP encoding gene was used from Expasy server site. In addition, to evaluate the Hardy-Weinberg equilibrium, we used the chi square test in SAS 9.1 software. All statistical tests were considered significant with a level of P≤0.05.
Results and Discussion The results of the present study showed that nine binding patterns were observed at PrP locus in studied goat population. The results of direct sequencing were confirmed the PCR-SSCP analysis results. Genetic analysis on protein sequences revealed an amino acid polymorphism in codon 186 (T- M or K) and two silent polymorphisms in codons 138 and 143. Based on codons 136, 154 and 171, all goats showed ARQ haplotype and there is no variation in these three codons. Additionally, the results of Hardy-Weinberg test confirmed that this population was not compatible with the HWE (P< 0.05).
Conclusion It is noticed that all of polymorphisms in exon 3 of PrP gene are important and can be used to improve the breeding programs. According to three codon system (codons 136, 154 and 171), all of the studied goats had shown ARQ haplotype and based on previous studies, Naeinian goats probably have categorized in low resistance group (R3). Although, in this study was identified the allelic different forms in the mentioned region, association study between these polymorphisms, especially in 186 codon, with scrapie disease need to more investigation. Due to lack of information and knowledge about genetic variation in this gene and association of its polymorphisms with Scrapie disease could cause suggest an appropriate strategy for increase of resistance in Iranian goat breeds, therefore, applying the appropriate strategy in the selection and breeding programs could be effective to reduce the risk of the disease and consequences events.
Material and Methods: The DNA was extracted from 120 blood samples of Naeinian goats from Esfahan province using modified salting out method. After amplification of the desired fragment by polymerase chain reaction (PCR), genotyping of samples was carried out by PCR-SSCP Analysis. In the following, direct sequencing method was used to confirm the genotyping results. Then, obtained sequences were analyzed by bioinformatics softwars, for example, Chromas Pro, BioEdit. In order to evaluation of amino acid polymorphisms caprine PrP encoding gene was used from Expasy server site.
Results: The results of the present study showed that nine binding patterns were observed at PrP locus in studied goat population. The results of direct sequencing was confirmed the PCR-SSCP analysis results. Genetic analysis revealed an amino acid polymorphism in codon 186 (T- M or K) and two silent polymorphism in codons 138 and 143.
Conclusion: it is noticed that all of polymorphisms in exon 3 of PrP gene are important and can be used to improve the breeding programs. According to three codon system (codons 136, 154 and 171), all of the studied goats had shown ARQ haplotype which offer Naeinian goats were classified in low resistance group (R3).

Keywords

• Different Allelic Forms
• Direct Sequencing
• Naeinian goat
• PCR-SSCP

20.1001.1.20083106.1395.8.3.9.2

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References

1- Agrimi, U., M. Conte, L. Morelli, M. Di Bari, G. Di Guardo, C. Ligios, G. Antonucci, G. Aufiero, N. Pozzato, and F. Mutinelli. 2003. Animal transmissible spongiform encephalopathies and genetics. Veterinary research communications, 27(1):31-38.

2- Animal Health Australia. 2001. the National Animal Health Information System (NAHIS) Scrapie. http://www. Aahc.Com. au/nahis/disease/ dislist asp.

3- Arsac, J. N., O. Andreoletti, J. M. Bilheude, C. Lacroux, S. L. Benestad and T. Baron. 2007. Similar biochemical signatures and prion protein genotypes in atypical scrapie and Nor98 cases, France and Norway. Emerging infectious diseases, 13(1):58.

4- Babar, M., A. Farid, B. Benkel, J. Ahmad, I. Sajid, M. Imran, T. Hussain and A. Nadeem. 2008. Genetic variability at seven codons of the prion protein gene in nine Pakistani sheep breeds. Journal of genetics, 87(2):187.

5- Baneh, H., M. Najafi and G. Rahimi. 2012. Genetic parameter estimates for early growth traits in Naeini goat. Animal Production Science, 52(11):1046-1051.

6- Baylis, M. and W. Goldmann. 2004. The genetics of scrapie in sheep and goats. Current molecular medicine 4(4):385-396.

7- Belt, P. B., I. H. Muileman, B. E. Schreuder, J. Bos-de Ruijter, A. L. Gielkens and M. A. Smits. 1995. Identification of five allelic variants of the sheep PrP gene and their association with natural scrapie. Journal of General Virology, 76(3):509-517.

8- Benestad, S., P. Sarradin, B. Thu, J. Schönheit, M. Tranulis and B. Bratberg. 2003. Cases of scrapie with unusual features in Norway and designation of a new type, Nor98. The Veterinary Record, 153(7):202-208.

9- Billinis, C., C. H. Panagiotidis, V. Psychas, S. Argyroudis, A. Nicolaou, S. Leontides, O. Papadopoulos and T. Sklaviadis. 2002. Prion protein gene polymorphisms in natural goat scrapie. Journal of General Virology, 83(3):713-721.

10- Bossers, A., B. E. Schreuder, I. H. Muileman, P. B. Belt and M. A. Smits. 1996. PrP genotype contributes to determining survival times of sheep with natural scrapie. Journal of General Virology, 77(10):2669-2673.

11- Buschmann, A., G. Lühken, J. Schultz, G. Erhardt and M. H. Groschup. 2004. Neuronal accumulation of abnormal prion protein in sheep carrying a scrapie-resistant genotype (PrPARR/ARR). Journal of General Virology, 85(9):2727-2733.

12- Byun, S., Q. Fang, H. Zhou and J. Hickford. 2009. An effective method for silver-staining DNA in large numbers of polyacrylamide gels. Analytical Biochemistry, 385(1):174-175.

13- Capucchio, M., F. Guarda, M. Isaia, S. Caracappa and V. d. Marco. 1998. Natural occurrence of scrapie in goats in Italy. Veterinary Record, 143(16):452-453.

14- Chelle, P. L. 1942. A case of trembling in the goat. Bull Acad Vet France, 15:294-295.

15- Dawson, M., L. Hoinville, B. Hosie and N. Hunter. 1998. Guidance on the use of PrP genotyping as an aid to the control of clinical scrapie. Veterinary Record, 142(23):623-625.

16- FAO. 2011. Statistical database, http://www.fao.org.

17- French, D. J., D. Jones, D. G. McDowell, J. A. Thomson and P. G. Debenham. 2007. Analysis of multiple single nucleotide polymorphisms closelypositioned in the ovine PRNP gene using linear fluorescent probes and melting curve analysis. BMC infectious diseases, 7(1):90.

18- Goldmann, W., T. Martin, J. Foster, S. Hughes, G. Smith, K. Hughes, M. Dawson and N. Hunter. 1996. Novel polymorphisms in thecaprine PrP gene: a codon 142 mutation associated with scrapie incubation period. Journal of General Virology, 77(11): 2885-2891.

19- Hunter, N., J. Foster, W. Goldmann, M. Stear, J. Hope and C. Bostock. 1996. Natural scrapie in a closed flock of Cheviot sheepoccurs only in specific PrP genotypes. Archives of virology, 141(5):809-824.

20- Hunter, N., W. Goldmann, G. Smith and J. Hope. 1994. The association of a codon 136 PrP gene variant with the occurrence of natural scrapie. Archives of virology, 137(1-2): 171-177.

21- Kurosaki, Y., N. Ishiguro, M. Horiuchih and M. Shinagawa. 2005. Polymorphisms of caprine PrP gene detected in Japan. Journal of veterinary medical science, 67(3): 321-323.

22- Laplanche, J., J. Chatelain, D. Westaway, S. Thomas, M. Dussaucy, J. Brugere-Picoux and J. Launay. 1993. PrP polymorphisms associated with natural scrapie discovered by denaturing gradient gel electrophoresis. Genomics, 15(1): 30-37.

23- Lauren, J., D. A. Gimbel, H. B. Nygaard, J. W. Gilbert and S. M. Strittmatter. 2009. Cellular prion protein mediates impairment of synaptic plasticity by amyloid-&bgr; oligomers. Nature, 457(7233): 1128-1132.

24- Moradi, N. 2012. the identification of polymorphisms in PrP gene by PCR-SSCP in Baluchi, Naieni and Zel sheep. . Dissertation, Sari agricultural science and natural resources University, Iran. (In Persian).

25- Najafi, M. 2012 Comparative analysis of gene structure in promoter site of alpha-s1 casein gene in naeinian goat and sheep breed. . MSc Thesis. Department of Animal Science, Sari Agricultural science and Natural Resources University, Iran, (In Persian).

26- Oesch, B., D. Westaway, M. Wälchli, M. P. McKinley, S. B. Kent, R. Aebersold, R. A. Barry, P. Tempst, D. B. Teplow and L. E. Hood. 1985. A cellular gene encodes scrapie PrP 27-30 protein. Cell, 40(4):735-746.

27- Papasavva-Stylianou, P., M. Kleanthous, P. Toumazos, P. Mavrikiou and P. Loucaides. 2007. Novel polymorphisms at codons 146 and 151 in the prion protein gene of Cyprus goats, and their association with natural scrapie. The Veterinary Journal, 173(2):459-462.

28- Rashidi, A., S. Bishop and O. Matika. 2011. Genetic parameter estimates for pre-weaning performance and reproduction traits in Markhoz goats. Small Ruminant Research, 100(2):100-106.

29- Toumazos, P. 1988. First report of ovine scrapie in Cyprus. British Veterinary Journal, 144(1):98-100.

30- Toumazos, P. 1991. Scrapie in Cyprus. British Veterinary Journal, 147(2):147-154.

31- Toumazos, P. and M. Alley. 1989. Scrapie in goats in Cyprus. New Zealand veterinary journal, 37(4):160-162.

32- Vaccari, G., M. A. Di Bari, L. Morelli, R. Nonno, B. Chiappini, G. Antonucci, S. Marcon, E. Esposito, P. Fazzi and N. Palazzini. 2006. Identification of an allelic variant of the goat PrP gene associated with resistance to scrapie. Journal of General Virology, 87(5):1395-1402.

33- Zhou, H., J. Hickford and Q. Fang. 2005. Technical Note: Determination of alleles of the ovine gene using PCR–single-strand conformational polymorphism analysis. Journal of animal science, 83(4):745-749.