شناسایی واریانت‌ های آللی مختلف ژن پرایون موثر بر بیماری اسکراپی در ‌بُزهای نژاد نائینی

فرم پیچش غیر نرمال پروتئین پرایون باعث وجود بیماری های بیشماری در پستانداران از قبیل انسفالوپاتی اسفنجی گاوی (بیماری جنون گاوی) در گاو، بیماری کرتزفلد- جاکوب در انسان و بیماری اسکراپی در بز وگوسفند می شود. تمام این بیماری ها روی ساختار مغز و سیستم عصبی اثر گذاشته و تا به حال هیچ روش درمانی برای آن ها ارایه نشده است و در نهایت باعث مرگ دام می شود. با بررسی های بیشتر مشخص شد که حیوانات بر اساس تنوع ژنتیکی در ژن پرایون در گروه های مختلف از لحاظ حساسیت و مقاومت به بیماری قرار می گیرند. به ‌منظور شناسایی فرم‌های مختلف اللی ژن پرایون در بُز نژاد نائینی، از تعداد 120 راس بُز نژاد نائینی نمونه خون تهیه شد. بعد از استخراج dna با استفاده از روش نمکی بهینه یافته، قطعه مورد نظر تکثیر و با استفاده از روش های pcr-sscp و تعیین توالی تعیین ژنوتیپ شدند. در پژوهش حاضر، نتایج تعیین ژنوتیپ با روش pcr-sscp 9 الگوی باندی را نشان داد که در نهایت آنالیز ژنتیکی از نواحی کُد‌کننده ژن پرایون، یک چند‌شکلی جدید را در کدون 186 (t m or k) نشان‌داد. علاوه ‌بَر ‌این، دو جهش خاموش نیز در کدون‌های 138 و 143 مشاهده شد. با توجه به آنالیز ژنتیکی کدون‌های 136، 154 و 171 تمامی ‌بُزهای نائینی مورد‌ مطالعه دارای هاپلوتیپ arq بوده و احتمالا فراوانی بالای این هاپلوتیپ می تواند به علت همبستگی این آلل با صفات مهم و اقتصادی باشد و با توجه به پژوهش‌های پیشین احتمالاً این نژاد مقاومت کمی نسبت به بیماری اسکراپی دارد.

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

Introduction The accumulation of improperly folded forms of hostencoded 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 scrapielike 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 (scrapieassociated 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 (R1R5). 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 PCRSSCP Analysis (SingleStrand 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 HardyWeinberg 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 PCRSSCP 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 HardyWeinberg 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 PCRSSCP 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 PCRSSCP 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).