Title: Development of methods to retrospectively analyse fixed appendix tissue from prevalence studies
Funder: Department of Health and Social Care

Abstract: Background Following the widespread dietary exposure of the UK and other populations to bovine spongiform encephalopathy (BSE) prions and their human transmission as variant Creutzfeldt-Jakob disease (vCJD), a key question has been the prevalence of infection in the UK population. This information is crucial to guide measures to protect the public from onward transmission of infection. In particular, concern has focussed on the subclinical infection of blood and lymphoreticular (LRS) tissues outside of the central nervous system. Six studies have looked at the prevalence of abnormal prion protein (PrP) deposition as a surrogate of prion infection in LRS tissue, including the Appendix studies I-III, but the picture remains frustratingly unclear and inconsistent with working models of the BSE/vCJD outbreak. Understandably, a lack of clarity has led to challenges about the assumption of a perfect association between PrP deposition in LRS tissue and vCJD prion infection. Aims The primary aim of our proposal is to develop methodologies which allow the retrospective analysis of LRS tissue and support/refute a diagnosis of vCJD prion infection. A secondary potential outcome is the development of biomarkers that might be used ante mortem in the diagnosis and monitoring of human prion disease. Research Plan and Methods Epigenetics refers to the alteration of gene expression by stable modification of DNA and/or chromatin structures, including for example, methylation of cytosine sites. These covalent changes are dynamic, functional and disease specific and are known to be resistant to change during sample storage and tissue fixation. We will exploit the recent development of highly multiplexed DNA methylation arrays and next generation sequencing to identify a set of genomic sites with an altered methylation status that correlates with the diagnosis of prion infection. We will confirm that this profile is unaltered by tissue fixation, is specific to vCJD, and use machine learning techniques to develop a diagnostic classifier that will be validated in blinded studies. Such approaches are currently revolutionising the tissue diagnosis of cancers, rendering possible more personalized treatments. Published and in house data strongly support the likelihood that these approaches will also be applicable in prion disease. Our target is to develop a classification tool which has over 80% positive and negative predictive value in a blinded test set. In parallel with these epigenomic approaches we will also optimise methodologies for protein extraction from fixed tissue. The fixation process leads to covalent protein cross linking, principally at lysine residues, in this way fixation is known to destroy prions. In published work we have shown that it is probably futile to test for prion infection by transmission to laboratory animals and we believe that further negative animal experiments will not be meaningful. However, detection of prion seeds by highly sensitive cyclical amplification technologies may be possible and carefully controlled experiments should be attempted. Research Team The MRC Prion Unit Human Genetics Programme led by Professor Simon Mead is a world-leader in genomic approaches to human prion diseases. Also contributing will be senior investigators at the Prion Unit and Division of Neuropathology. Potential impact/influence on the relevant policy field, users and wider stakeholders If we develop strong supportive evidence for vCJD infection of Appendix I-III tissue this should lead to confidence in expensive protection measures already being undertaken, and stimulate the further development of assays to exclude vCJD carriers from blood or organ donation and identify those who need enhanced precautions for some surgical procedures. If we can show evidence against vCJD infection, this could justify a review of measures currently being taken, in conjunction with the absence of epidemiological evidence of secondary transmission.

Lay abstract: Prions are infectious pathogens or &ldquo;germs&rdquo; that cause diseases of the brain. They are made from abnormally shaped and clumped together forms of a protein called &ldquo;prion protein&rdquo;. Prions spread by changing the normal prion protein to the abnormally shaped form. The devastating epidemic of bovine spongiform encephalopathy (BSE) (often known as &ldquo;mad cow disease&rdquo;) in the 1980s-1990s was caused by the use of material from dead cattle in animal food. Through BSE-contaminated food, the condition spread to humans as variant Creutzfeldt-Jakob disease (vCJD), which is a dementia condition. Patients with vCJD deteriorate fast and have abnormal muscle movements. The condition predominantly affects young people; to date, 178 people in the UK have died of the disease since 1995. We also know that the condition has been spread between humans by blood transfusion. Consequently, measures have been taken to minimise the risk of spread between people. In order to plan such protective measures it is crucial to know how many people in the population are infected with vCJD. This has been extremely difficult to work out with confidence because there are no suitable and convenient tests for prions. Signs of vCJD prion infection in appendix tissue removed at surgery for suspected appendicitis can be found under the microscope, but despite many years of expensive research, these studies remain inconclusive. In this proposal we look to develop techniques that will enable increased confidence that an appendix sample is infected with vCJD. We propose to develop a &ldquo;genetic fingerprint&rdquo; or a characteristic genetic &ldquo;profile&rdquo; that indicates prion infection. Techniques like this have proven to be very successful in diagnosis of cancer and viral infections. In this way we should get a confident answer to the question about how many people are infected with vCJD, and therefore be able to improve our strategy for protection of the public from further spread of prions.

Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: NEUROLOGICAL