Novel Variations at Three Different Positions of Prion Protein Coding Gene in Ethiopian Sheep Breeds and the Resistance/Susceptible Status to Classical and Atypical Scrapie

Background: Classical Scrapie susceptibility in sheep has been linked to three polymorphisms at positions 136, 154, and 171 in the PRNP gene whereas atypical scrapie susceptibility is related to a polymorphism at position 141. Many other variants over the length of the prion protein coding gene were reported. Since infectious prion protein itself seems to be polymorphic, the identified novel PRNP gene variations may play a crucial role in fighting against the emergence of a new form of scrapie disease. Many studies conducted around the world to identify disease resistant status and new variants of PRNP gene in different breeds. However, such in-depth studies have never addressed the African continent’s sheep breeds. Therefore, genotyping native Ethiopian sheep breeds PRNP gene provides essential information to the current knowledge. This study aimed to identify potential novel variations in the Ethiopian sheep PRNP gene, thereby determine the uniqueness of the native breeds and predict scrapie status of sheep population based on the genotypes distribution. Results : Five novel variants were identified in the PRNP gene of native Ethiopian sheep. Four non-synonymous heterozygous substitutions at H99Q (CAC-->CAA), H99L (CAC-->CTA), A116E (GCA-->GAA), A116T (GCA-->ACA), and one synonymous N103N (AAC-->AAT) variants were detected. In addition to the novel variations, polymorphisms at 126,127,138,142,146,231, and 237 positions were also identified. The haplotype ARR was observed only in Menz and Afar breeds with frequencies 0.02 and 0.05 respectively. However, neither ARR/ARR nor VRQ/VRQ genotypes were identified in all of the breeds. Conclusion: Two of the novel variations at position 99 and 103 that are placed

Background Prion diseases are a collective name for infectious neurodegenerative diseases caused by the misfolding of Prion protein [1].The misfolded protein (PrP Sc ) has a different structural dynamic than cellular prion (PrP c ). Specific motifs of prion protein were identified in relation to the conversion of PrP c to PrP Sc [ 2].
Though the exact underlined disease mechanism is not yet known, transmission and susceptibility of prion diseases have genetic bases. Previous studies identified three polymorphisms (A136V, R154H, and Q171R/H) related to scrapie resistance/susceptibility status in sheep PRNP (prion protein coding gene). ARQ represents susceptible widespread wild type allele; ARR and VRQ are the most resistant and highly susceptible genotypes respectively [7-10]. In atypical scrapie susceptibility is higher in individuals with AHQ, AHQ/ARQ and ARR genotypes along with homozygosity for phenylalanine at position 141 [11]. An earlier study linked genotypes such as AC 151 RQ to prolonged incubation period after scrapie exposure [10]. Together with that, several studies reported polymorphisms such as G126A, G126G, G127G, G127V, G127A, and S138S [3][4][5][6] in sheep prion protein with or 4 without direct effect to susceptibility to scrapie.
In the past several years there were efforts to genetically characterize local breeds of many countries in identifying new variants and determining resistance/susceptible haplotypes against scrapie. Based on the findings, measures were taken to control and reduce transmission of transmissible spongiform encephalopathy horizontally and vertically [12][13][14]. However, there are limited studies that addressed Sub-Saharan African countries such as Ethiopia where livestock is the main economic source and a large proportion of the population depends on livestock products. On the other hand, the ever-increasing animal product demand enhances crossbreeding programs for the last few decades through the importation of exotic animals and the distribution of crossbred F1 in different parts of Ethiopia. Such practices are the potential factors in changing the genetic structure of the population and may introduce new disease susceptible phenotypes [15,16].
Taking in to account the public health, economic and scientific merits genotyping native Ethiopian sheep breeds PRNP gene provides essential information to the current knowledge [17]. This study aimed to identify novel variations in the Ethiopian sheep PRNP gene, thereby determine the uniqueness of the native breeds and predict scrapie status of sheep population based on the genotypes distribution.

Animal selection
Whole blood was taken from genetically unrelated 97 female native sheep breeds (Washera N = 39, Menz N = 35, and Afar N = 23) that are concentrated in the respective regional breeding center. Washera sheep breed is localized in west-east Gojam and AgewAwi zones of Amhara region (11°00'0.00" N 36°39'59.99" E). This breed is commonly known by its short fat tail, short-hair and large body size. They are reared for commercial mutton production. Menz sheep breeds are one of the most common sheep breeds in Ethiopia distributed in Menz, North Shewa zones of Amhara region (10°15'00.0" N39°30'00.0"E). Their characteristic feature is a fatty short tail, well developed wooly undercoat with unique spiral horns. They are adapted to a cold environment and known for the production of meat and wool. Afar sheep named after Afar region. They are well adapted to harsh environments. They are a good source of fatty meat. Afar breeds from Amibara Woreda at a geographic coordinates 1033254, 0629012 were included in the study. Information on the phenotypes of the breeds was taken from Ethiopian Biodiversity booklet, 2018.

DNA extraction and Polymerase Chain Reaction
Genomic DNA was isolated from the EDTA treated blood using a commercial kit (Geneaid). PCR (Polymerase chain reaction) was carried out to amplify the coding region of the PRNP using forward (TCTGCAAGAAGCGACCAAAAC) and reverse (CACAGGAGGGGAAGAAAAGAGG) primers (NM_001009481.1). PCR mixture containing 200 µM of each dNTP, 2 mmol MgCl2, 5 pmol of each primer, 0,05 U Taq polymerase, 10 X PCR buffer (Thermo Fisher Scientific Inc., USA), 10-50 ng of genomic DNA and ddH2O to a final volume of 12,5 ul was used for PCR reaction. 2.5 ul of PCR product was used for further analysis.

Sequencing and bioinformatics
After incubation with 1 U Exo-SAP, a chain termination reaction was performed with BigDye™ terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc., USA).
At the final stage, all samples were purified by ethanol precipitation method and 6 sequenced by Applied Biosystems 3500 genetic analyzer (Thermo Fisher Scientific Inc, USA). Chromatograms were checked with FinchTV and aligned using Mega v7.0 software. Hardy-Weinberg equilibrium state for the multilocus allele was calculated using Popgene 32.

Results
In the present study, novel variants were detected in the three native Ethiopian  In the current study, the highly susceptible haplotypes i.e. haplotypes categorized under groups 4 and 5 were not detected. Instead, ARQ was the dominant allele in all breeds. The haplotype ARR was observed only in Menz and Afar breeds with frequencies 0.02 and 0.05 respectively. ARQ/ARQ and ARH/ARH genotypes appeared in Menz breeds with 0.29 and 0.17 frequencies respectively while ARQ/ARQ 7 genotype was dominant in Washera (0.68) and Afar (0.78) breeds. Neither the highly susceptible, VRQ, nor highly resistant, ARR, genotypes were observed in this study.
In all of the samples analyzed; homozygous Leucine at position 141 was detected

Discussion
In the current study, novel amino acid substitutions at a novel and previously   [25][26][27][28][29]. Atypical scrapie associated haplotype, ALRQ, was also prominent in the above listed countries where scrapie infection was not epidemic [25,28]. Despite there have been cross-breeding practices especially community-based breeding programs in small ruminants in recent years [15,16], in the present study frequency of homozygous genotypes were relatively dominant over heterozygous

Consent for publication-Not applicable
Availability of data and materials-The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Competing interest-The authors declare no conflict of interest.

Funding-No funding
Authors Contribution-ET involved in data collection, performed laboratory activities and wrote the manuscript. YY engaged in PCR and sequence optimization.
CU formulated concept and organized the study. All authors have read and approved the manuscript.  Figure 1 Heterozygosity at nucleotide 296 and 297 resulted in two heterozygous amino acid substituti Figure 2 Polymorphisms at cleavage site, palindrome and highly conserved hydrophobic region of PrP.

Figure 3
Amino acid substitutions over the length of the sequenced region.