Prion protein gene mutation detection using long-read Nanopore sequencing

Abstract

Prion diseases are fatal neurodegenerative conditions that affect humans and animals. Rapid and accurate sequencing of the prion gene PRNP is paramount to human prion disease diagnosis and for animal surveillance programmes. Current methods for PRNP genotyping involve sequencing of small fragments within the protein-coding region. The contribution of variants in the non-coding regions of PRNP including large structural changes is poorly understood. Here we use long-range PCR and Nanopore sequencing to sequence the full length of PRNP, including its regulatory region, in 25 samples from blood and brain of individuals with various prion diseases. Nanopore sequencing detected the same variants as identified by Sanger sequencing, including repeat expansions/contractions. Nanopore identifies additional single-nucleotide variants in the non-coding regions of PRNP, but no novel structural variants were discovered. Finally, we explore somatic mosaicism of PRNP s octapeptide repeat region, which is a hypothetical cause of sporadic prion disease. While we find changes consistent with somatic mutations, we demonstrate that they may have been generated by the PCR. Our study illustrates the accuracy of Nanopore sequencing for rapid and field prion disease diagnosis and highlights the need for single-molecule sequencing methods for the detection of somatic mutations.

Genetic counseling for prion disease: Updates and best practices

“…Ideally a multidisciplinary team, including a neurologist, a genetic counselor, and a social worker will have the opportunity to manage genetic prion disease cases together. Key preparation will include understanding the penetrance 33 should be employed. Bearing in mind this limitation of NGS, in the event that prion disease is part of a broad differential diagnosis of a neurodegenerative disease and there is a family history of a similar condition, a large NGS dementia panel may be considered as a first step to rule out conditions such as familial Alzheimer's disease or frontotemporal dementia.…”