In the current study, novel amino acid substitutions were detected in the native Ethiopian sheep breed’s PRNP gene. These substitutions (H99L, N103N, A116T, and A116E) are in a proteinase K resistant region of the prion protein. Variants at codon 99 and 103 are localized closer to the peptide cleavage sites. Variants A116T and A116E are placed in the hydrophobic palindrome region (A116GAAAAG) which is described as the critical motif in the process of conversion of PrPC to PrPSc and its propagation . A previous work by Yang et al., 2014 reported PrP with L141R154 haplotype displayed extended beta sheet in the N-terminal palindromic region than the other variants at these codons .The study also predicted possible correlation between conformational change in ovine PrP upon mutation and different forms of scrapie .Hence, due to physicochemical properties variation among variants, substitutions might influence the conformational plasticity of prion protein and could potentially trigger the emergence of new scrapie forms.
As in previous studies, non synonymous and synonymous substitutions i.e. G127G, S138S, R231R, L237L, G126A, 126GA, 127GV, 127GA, 142IT, N146S, N146NS were also identified in the population under study [10-12]. In the present study, the variant at codon 127 in particular was highly polymorphic. Amino acids at codon 126 and 127 are localized in the highly conserved glycine rich motif GAVVG126G127LGGYMLG.This residue was reported to antagonize prion disease development by blocking amyloidal fibril formation . A recent work by Vitale et al.,2019 reported polymorphism at this position and implicated its importance in the normal cellular function of prion protein .The sequence variation in palindrome residue (PrP 113-120) and glycine repeat regions (PrP 124-128) synergistically may affect cellular prion protein conformational flexibility.
So far, there are evidences that support the link between scrapie susceptibility and prolonged incubation period and host PRNP genotypes. Known polymorphic alleles at codon 136A/V, 154R/H, and 171R/H/Q are implicated in classical scrapie resistance or susceptibility . A recent work by Cassmann et al., 2019 reported, 171Q/K genotypes are associated to scrapie resistance/susceptibility . In atypical scrapie, AHQ, ARQ and ARR genotypes along with homozygosity for phenylalanine at codon 141 are susceptible haplotypes. ARR genotype which is a central protective haplotype in classical scrapie is not protective in atypical scrapie [9, 23].
In the current study, a significant proportion of the population under study were potentially less resistant to classical and atypical scrapie (fall under scrapie resistance category groups 2 and 3). The haplotype, A136L141R154Q171, was predominant in the population under study. In the present study, the frequency of the ARQ allele was lower in Menz breed than the other two breeds. On the contrary, ARQ was the most prevalent allele in Afar breed. The observed allele frequency variation might be due to the geographic barriers favoring inbreeding and later results in genetic distinction among breeds.
In countries such as Canada and Czech Republic, where scrapie was once apparently prevalent, the resistant haplotype, ARR, became dominant over the previously reported VRQ and ARQ haplotype [4, 24, 25]. Genetic based eradication programs are one of the reasons why ARR haplotype is prevalent in such areas [26, 27]. A study by Migliore S. et al.,2017, speculated positive selection on scrapie resistance goat PRNP variants in the places where there was scrapie epidemics. Similarly, in India, China, Israel, Iran, and Turkey where scrapie case has never been epidemic, the predominant allele was ARQ [29-35]. Studies from Tunisia and Algeria reported ARQ, ARR, AHQ, ARH, and VRQ as major alleles. In the same studies, additional polymorphisms which were also previously reported in Spanish and Italian sheep breeds were identified [12, 36-38]. ARQ was recorded at a significantly higher frequency in scrapie-affected Spanish sheep. At the same time, this genotype was dominant in both healthy and scrapie affected sheep breeds . Similarly, ARQ haplotype was predominant in three Sicilian autochthonous sheep breeds. However, ARR haplotye was also reported at high frequency than other common scrapie associated haplotypes .In rare Greek sheep breeds where scrapie has not yet reported, ARQ haplotype was detected in 50% of the sampled population . Despite the long standing selective breeding programs in European countries, there has been no legalized selective breeding practices for scrapie in Bulgaria. A study reported ARQ and ARR as the predominant haplotypes among selected Bulgarian sheep flocks . The high frequency of ARR could be due to the importation of resistant sheep from elsewhere.
Earlier experimental studies linked specific alleles to survival rate after scrapie infection along with the ARQ genotype. T112ARQ , AT137RQ, AC151RQ, and ARQK176 haplotypes were identified as potential protective variants [9, 41, 42]. In the present study, these haplotypes were not detected. Rather, the wild type variants i.e. M112, M137, R151, and N176 were highly prevalent. Due to the absence of the resistant genotypes and potential protective alleles such as the aforementioned variants, the population under study was genetically less resistant to scrapie.
In recent years community-based breeding programs have been practiced in Ethiopia [15, 16]. Hence, higher genetic diversity i.e. Heterozygosity and scrapie resistant genotypes would be expected. In the present study however, homozygous genotypes were relatively dominant over heterozygous genotypes. This could be due the limited practice of extensive crossbreeding. Inadequate crossbreeding may compromise efficient animal production as well . However, genetic diversity in native Ethiopian sheep breed’s PRNP was still detected when compared to other breeds worldwide. Therefore, the balance between production improvement through selective breeding and preservation of native breeds should be maintained.