Development of a single-allele sequencing tool to study of PRNP variants in Chile

Introduction: Prion diseases are fatal neurodegenerative disorders caused by the conversion of the cellular prion protein (PrPC) into its pathological form. PrPC is codify by PRNP gene, in which several genetic variants have been described, some of them described as pathological. In Chile, genetic research on prion diseases is focused on the detection of the genetic Creutzfeldt-Jakob disease (CJD) related to E200K mutation. However, despite the number of families affected with prion diseases, no other mutation has been reported.  

Objective: For this reason, we aimed to develop a single-allele sequencing tool to identify mutations and polymorphisms present in PRNP gene in the chilean population. 

Methods: Blood samples were collected from healthy volunteers, and genomic DNA was extracted. We used PCR to amplify the PRNP gene, followed by plasmid cloning for subsequent allele nanopore-based plasmid sequencing. 

Results: PRNP gene from donors was successfully amplified using this technique. We able to clone and sequence each allele separately. The M129V polymorphism was detected in the population studied, while the E200K mutation as well as other mutations reported in the literature were not found in the sample from healthy donors. 

Conclusion and Discussion: We established a consistent protocol for genetic studies in Chile. Although the E200K mutation was not detected in the studied population, we were able to detect M129V polymorphism. These preliminary findings reinforce the need to expand genetic analysis to the entire PRNP gene and to generate population-level data that improve the detection and understanding of these pathologies.