In the current study, novel amino acid substitutions at a novel and previously identified polymorphic site were detected in the native Ethiopian sheep PRNP protein-coding gene. These substitutions (H99L, N103N, A116T, and A116E) are spotted in a PK resistant region of the prion protein. Substitutions at amino acid 99 and 103 positions are localized more close to the signal peptide cleavage sites. Variants A116T and A116E are placed in hydrophobic palindrome region (A116GAAAAG) which was described as the critical motif in the process of conversion of PrPc to PrPSc and its propagation . Substitution with a different variant having different physicochemical properties might influence the conformational plasticity of prion protein and my further could tempt the emergence of new scrapie disease form.
Similar to previous studies, additional amino acid polymorphic sites i.e. G126A, G126A(heterozygous), G127G, G127V(heterozygous), G127A(heterozygous), S138S, I142IT, N146S, N146S(heterozygous), R231R, and L237L were identified [3–6]. In the present study, the variant at position 127 was in particular highly polymorphic. Amino acid variants at positions 126 and 127 are localized in the highly conserved Glycine repeat motif GAVVGGLGGYMLG which is reported to antagonize prion disease development by blocking amyloidal fibril formation . Recent work on the Ethiopian goat PRNP protein-coding gene reported polymorphism at this position and implied its importance in the normal cellular function of prion protein .The synergetic effect of alterations in palindrome motif PrP (113–120) and Glycine repeat regions PrP(124–128) may strongly affect cellular prion protein conformational flexibility.
The possibility of infectious disease transmission from animal to animal, animal to human and vice-versa makes genetic characterization of local breeds undeniably crucial especially in understanding and preventing transmittable disease . So far, there is strong evidence that scrapie susceptibility and prolonged incubation period are linked to different PRNP genotypes. Known polymorphic alleles at position 136A/V, 154R/H, and 176R/H/Q are implicated in classical scrapie disease susceptibility . Accordingly, the highest resistant genotype is ARR/ARR while VRQ/VRQ is the highest susceptible genotype. In atypical scrapie, AHQ, ARQ and ARR genotypes along with homozygosity for phenylalanine at position 141 are susceptible haplotypes. ARR genotype which is central protective haplotype in classical scrapie is not protective in atypical scrapie [11, 21].
In the current study, a significant proportion of the population's alleles under study were 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 is the highest frequent allele in Afar breed. The relative allele frequency variation among breeds in this study might be directly related to the geographic barriers favoring inbreeding and later results in genetic distinction among breeds.
In countries such as Canada where scrapie was once apparently high, the resistant haplotype, ARR, became dominant over the previously reported VRQ and ARQ haplotype [7, 22]. Similarly, in India and China where scrapie case was never been epidemic, the predominant allele was ARQ [23, 24]. Earlier works on sheep PRNP polymorphism from Israel, Iran, and Turkey revealed that ARQ was the dominant haplotype [25–29]. Atypical scrapie associated haplotype, ALRQ, was also prominent in the above listed countries where scrapie infection was not epidemic [25, 28]. Studies from North African countries, Tunisia and Algeria reported ARQ, ARR, AHQ, ARH, and VRQ as major alleles. Those studies also identified identical polymorphisms which were reported in Spanish and Italian sheep and this similarity can be considered as a piece of evidence that some of Mediterranean surrounding countries’ breeds had similar genetic background. If the transboundary infection had happened during the times when scrapie was epidemic in European countries, it is sound to expect a high frequent ARR and low frequent VRQ in Algeria and Tunisia [30, 31].
Earlier case-control studies linked specific alleles to survival rate after scrapie infection along with ARQ genotype. T112ARQ, AT137RQ, AC151RQ, and ARQK176 haplotypes were identified as potential protective variants [11, 32, 33]. Accordingly, the detected homozygous Methionine at position 112&137, Arginine at 151, and Asparagine at 176 in all the analyzed samples potentiates the susceptibility of the breeds to scrapie.
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 genotypes.