DNA methylation analysis of archival lymphoreticular tissues in Creutzfeldt–Jakob disease

The exposure of the UK and other European populations to bovine spongiform encephalopathy (BSE) prions caused human variant Creutzfeldt-Jakob Disease (vCJD) and a prolonged public health crisis. Throughout, a key question has been the prevalence of vCJD prion infection in the UK population. vCJD has several distinct features including immunohistochemically detectable abnormal prion protein (PrP) in peripheral lymphoreticular system tissues (LRS eg. tonsil, appendix). Surveys have detected abnormal PrP in the LRS of the UK population, but it remains unclear if these represent carriers of vCJD infection, some other form of prion infection, or another phenomenon altogether. Concern about the infectiousness of these possible carriers has been used to justify precautionary, expensive and ongoing health protection measures. Archival appendix samples are formalin fixed and paraffin embedded, a process that makes conventional assays of prion infection challenging. Here, we sought to use methylation array technology that assays >850,000 sites where chemically stable DNA modification occurs to develop a computational method to classify tissue samples by prion disease status. We assembled nearly 450 lymphoreticular tissue samples from patients with different prion diseases following biopsy or autopsy, and non-prion disease patients following tonsillectomy and appendicectomy, either frozen or processed as formalin fixed or formalin fixed paraffin embedded. DNA was extracted, bisulphite converted and assayed using Illumina Infinium Methylation EPIC (850K) BeadChips. Data were normalised and filtered, then analysed by case-control study, t-distributed stochastic neighbour embedding plots, and random forest classification methods. We show substantial differences in DNA methylation between prion diseases cases and controls, which can be exploited to classify LRS samples with reasonable levels of accuracy (82-97%). Archival appendix samples with abnormal PrP were most similar to, and classified with, control appendix samples, rather than prion disease samples; several interpretations are compatible with these findings. ### Competing Interest Statement Prof Collinge is a director and shareholder of D-Gen Limited (London), an academic spinout company working in the field of prion disease diagnosis, decontamination, and therapeutics. None of the other authors report any conflict of interest. ### Funding Statement This study was funded by the Department of Health s Policy Research Programme and the Medical Research Council (UK). SM and JC are NIHR Senior Investigators. Some samples were acquired through funding by NIHR s Biomedical Research Centre at University College London Hospitals NHS Foundation Trust. ZJ, and SB are supported by the Department of Health s NIHR Biomedical Research Centre s funding scheme to UCLH. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: London Queen Square Research Ethics Committee gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data analysed in this report will be deposited here on publication