TSE GENETICS IN GOATS. LOOKING OUTSIDE THE PRNP OPEN READING FRAME: MOLECULAR CHARACTERIZATION OF THE PRNP REGULATORY REGIONS AND ASSESSMENT OF THE ROLE OF THE SPRN GENE

Susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy of small ruminants, is strongly influenced by polymorphisms of the prion protein gene (PRNP) and breeding programs to increase scrapie resistance in sheep populations have been implemented. A similar approach is not applied in goats yet but it would be desirable. The knowledge of genetic factors, outside the PRNP gene, involved in the modulation of the response to scrapie in goats, could aid to overcome some limitations of future selection programs based solely on PRNP genetics. The main aim of this research project was therefore to look for TSE genetic factors outside the goat PRNP open reading frame by the molecular characterization of the PRNP gene regulatory regions and the assessment of the role of the Shadow of the Prion Protein gene (SPRN), a recently discovered member of the prion family. In the PRNP regulatory regions, 29 novel polymorphisms were identified within a 3 Kb fragment encompassing the PRNP transcription start site in goats belonging to nine goat breeds coming from Italy, Greece and Spain. Haplotype analysis, performed on the study goats using bioinformatics, predicted 13 haplotypes based on ten polymorphic sites. Most of the polymorphisms with an influence on the presence/absence of transcription factor binding sites were found in intron 1. Functional studies have also demonstrated that a conserved region in the PRNP intron I is able to bind different transcription factors, among which the binding of ELK1 has been assessed by EMSA experiments. Furthermore, the first association study investigating the role of the goat SPRN gene in scrapie occurrence was carried out in the frame of the project. The SPRN genes of goats from several scrapie outbreaks were analysed in order to detect SPRN polymorphisms and to look for association between SPRN alleles/genotypes and the occurrence of scrapie by a case-control study. A significant association with susceptibility for classical scrapie was found for a polymorphism causing the insertion of five nucleotides (602_606insCTCCC) in the 3’UTR. Bioinformatics analyses suggested the hypothesis that the identified indel may contribute to modulation of susceptibility to the disease via gene expression regulation by a miRNA-mediated post-transcriptional mechanism. In conclusion, the results presented in this Doctoral thesis provide novel knowledge on genetic factors outside the caprine PRNP open reading frame investigating their influence in the modulation of scrapie susceptibility. These data could offer a significant contribution to the control of classical scrapie in goats by future breeding programs as well as further research opportunities on other aspects related to the function and pathogenicity of the prion protein.