Supplementary Appendix

Transcription

1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Mok T, Jaunmuktane Z, Joiner S, et al. Variant Creutzfeldt Jakob disease in a patient with heterozygosity at PRNP codon 129. N Engl J Med 2017;376: DOI: /NEJMc

2 Supplementary material to: Pathologically confirmed variant CJD in a prion protein gene codon 129 heterozygous patient Tze How Mok MRCP 1,2, Zane Jaunmuktane FRCPath 3, Susan Joiner MSc 1, Tracy Campbell BSc 1, Catherine Morgan MD 4, Benjamin Wakerley MD 4, Farhad Golestani MD 4, Peter Rudge FRCP 1,2, Simon Mead MD 1,2, Hans Rolf Jäger MD 5, Jonathan D F Wadsworth PhD 1, Sebastian Brandner FRCPath 1,3 and John Collinge FRS 1,2 1 MRC Prion Unit and Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK 2 National Prion Clinic, National Hospital for Neurology and Neurosurgery, UCL Hospitals NHS Foundation Trust, Queen Square, London, WC1N 3BG, UK 3 Division of Neuropathology, National Hospital for Neurology and Neurosurgery, UCL Hospitals NHS Foundation Trust, Queen Square, London, WC1N 3BG, UK 4 Gloucestershire Hospitals NHS Foundation Trust, Great Western Road, Gloucester, GL1 3NN, UK 5 Neuroradiological Academic Unit, Department of Brain Repair & Rehabilitation, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK Correspondence to Professor John Collinge at MRC Prion Unit jc@prion.ucl.ac.uk tel fax Keywords: prion, Creutzfeldt-Jakob disease, prion protein, bovine spongiform encephalopathy Contents Summary... 3 Introduction... 4 Case History... 5 Discussion... 8 Competing interests... 9 Figure Figure Reference List

3 2

4 Summary The epizootic prion disease of cattle, bovine spongiform encephalopathy (BSE), infected humans causing variant Creutzfeldt-Jakob disease (vcjd) and provoked an animal and public health crisis. While the incidence of vcjd has been in decline in the UK since its peak in 2000, all definite diagnoses have been in patients with a particular prion protein genotype (MM) at polymorphic codon 129, where either methionine (M) or valine (V) is encoded. Experience of other acquired human prion diseases and animal models argued that further cases might occur in the other PRNP genotypes (VV and MV) with longer incubation periods and perhaps different disease phenotypes. Here, we report the first definite vcjd patient with an MV genotype. The patient met diagnostic criteria of probable sporadic CJD but neuropathology and molecular analysis of prion strain type confirmed vcjd. 3

5 Introduction Prions are transmissible agents which cause lethal neurodegenerative diseases of humans and other animals and which are composed of assemblies of misfolded host-encoded cellular prion protein (PrP) 1. Human prion diseases have genetic, sporadic or acquired etiologies. The acquired forms, arising from environmental exposure to prions, include kuru of the Eastern Highlands Province of Papua New Guinea, transmitted following incubation periods that can exceed 50 years by ingestion of infected tissues at mortuary feasts 2 ; iatrogenic CJD, transmitted by contaminated neurosurgical instruments or the use of cadaver-derived medical and surgical products 3 ; and vcjd, which arose from exposure to BSE prions 4-6. First recognised in UK young adults in 1995, vcjd reached peak incidence in 2000, and has subsequently declined, with 223 deaths by Clinical features which may help distinguish vcjd from sporadic CJD include: young age of clinical onset; distal pain, numbness and paraesthesia; chorea; early cerebellar ataxia and psychiatric features. On investigation, a diagnosis of vcjd is supported by presence of the pulvinar sign in the thalamus on magnetic resonance brain imaging, a positive blood test (direct detection assay) 7 or tonsil biopsy demonstrating disease-related PrP 8. Histology and immunocytochemical study of autopsy or biopsy tissues reveals florid PrP amyloid plaques in the brain, abnormal PrP immunoreactivity in peripheral lymphoreticular tissues and immunoblot of brain tissue demonstrates a characteristic type of protease-resistant PrP (PrP Sc ) 5, Transmission of vcjd infection by blood and blood products is recognised and screen of archived surgical tissues has estimated that 1 in 2000 of the UK population may have subclinical infection 12. Genetic factors, notably a common single nucleotide polymorphism at codon 129 of PRNP are known to determine susceptibility, incubation period and the clinical phenotype of sporadic and acquired prion diseases 2, In the UK population, genotype frequencies are estimated at MM 42%, MV 46%, VV 12% 17. To date, all definite cases of vcjd in the UK and other countries were found to have the MM genotype at codon 129, one of the strongest known associations of a common polymorphism with any disease 17. One patient with a suspected diagnosis of vcjd and MV genotype, died without tonsil biopsy or confirmatory autopsy 18, and transmission of vcjd infection via blood transfusion was detected post mortem in the spleen of a patient with the MV genotype 19. In kuru, the MV genotype is associated with particularly long incubation times and relative resistance to disease 2, 20. In iatrogenic CJD in the UK, long incubation periods are determined by both MV and MM genotypes 15. Transgenic mice expressing human PrP with different codon 129 genotypes are differentially susceptible to vcjd and BSE prions and show multiple pathological phenotypes 4

6 including that of sporadic CJD as well as vcjd Consequently, there has been considerable speculation about whether further waves of vcjd might occur in the other (MV and VV) genotypes, associated with longer incubation times and perhaps more variable clinicopathological phenotypes. We now report the first definite diagnosis of vcjd in an MV individual. Case History A 36-year old right handed male scaffolder was referred to the UK National Prion Clinic (NPC) in August 2015 with a history of personality change, cognitive decline, ataxia and myoclonus. It was difficult to determine the onset of his illness precisely as he had persistent low mood following the death of his fiancée from an unrelated cause four years previously. In October 2014 he became uncharacteristically irascible, argumentative and failed to complete a series of projects at work. In the subsequent four months, he became progressively abulic, developed intermittent postural instability, gait ataxia, and myoclonus in his upper limbs. He also had blurred vision and dizziness but no limb pain or chorea. Five months after the change in his personality, his episodic memory deteriorated; his speech became slurred and the ataxia worsened to the extent that he required walking aids. In May 2015, he exhibited disinhibited behaviour, developed a craving for sweet foods and a delusion that he was in another place to where he was located (reduplicative para-amnesia). When assessed by the NPC in August 2015, the Mini Mental State Examination (MMSE) was 25/30, losing points on orientation for time, memory and construction of intersecting pentagons. On bedside cognitive testing using an instrument specifically designed for patients with prion disease 25, he performed poorly in verbal and visual recognition memory, phonological processing, calculation, spelling, information processing, copying sequential hand postures (Luria) and verbal fluency. Performance on object naming, praxis, letter cancellation, reading fragmented letters and irregular words was normal. Cranial nerve examination revealed abnormal convergence, broken (saccadic) visual pursuit and cerebellar dysarthria. In the limbs he had multifocal myoclonus, generalised hyperreflexia, sustained ankle clonus, extensor plantar responses and gait ataxia. Bilateral grasp reflexes were present. Appreciation of pinprick, temperature and joint position were intact but vibration sense was diminished below the knees. 5

7 There was no significant past medical history apart from adenoidectomy in childhood. He had never had a blood transfusion, neurosurgery, donated blood or received growth hormone. In the family history his parents and sister were well. A paternal uncle had dementia and died in his sixties. The patient was born in the UK where he had always lived and always ate a normal omnivorous diet. PRNP sequencing showed no mutations and the genotype at codon 129 was methionine-valine heterozygous (MV). Magnetic resonance imaging (MRI) of his brain showed restricted diffusion in the basal ganglia, hypothalamus, insular cortex and medial thalami but not the pulvinar (see figure, printed correspondence). The electro-encephalogram (EEG) was encephalopathic, without any evidence of periodic complexes. The cerebrospinal fluid (CSF) was acellular, with normal protein and glucose levels; protein was not detected, s100b was 0.39 (<0.41ng/mL) and the real-time quakinginduced conversion assay 26 was negative (National CJD Research and Surveillance Unit). vcjd blood test (Direct Detection Assay) 7 was negative. The clinical features and investigations met the criteria for a diagnosis of probable sporadic CJD 27. The patient was discharged to a local care home, and was reviewed on three occasions over the next twelve weeks by the NPC. During this time, he started to confabulate, developed visual hallucinations, optic ataxia, restricted up gaze, upper limb apraxia, prosopagnosia and progressive daytime somnolence. He became chair bound by early January 2016, and bedbound by mid-february Subsequently, there was a rapid deterioration in his condition with the emergence of severe agitation and dysphagia. He died in February The fresh whole brain weight was 1490g. Histological examination with H&E stain showed frequent amyloid plaques, including florid plaques and cluster plaques in cerebral and cerebellar cortex and microplaques in the basal ganglia (Figure 1). In the neuropil in all areas, there was microvacuolar degeneration, which was most severe in the caudate nucleus and putamen, less intense in the thalamus and mild in the cerebral and cerebellar cortex. Immunostaining for abnormal prion protein labelled amyloid plaques and showed a distinct widespread stellate pericellular and perivascular labelling in the cerebral and molecular layer of the cerebellar cortex. In the basal ganglia and thalamus there was a prominent linear prion protein deposition along neuronal processes. The spleen contained only minute amounts of abnormal prion protein in lymphoid tissue, whilst no prion 6

8 protein could be reliably detected in the appendix and mesenteric lymph nodes. Immunoblot analyses of patient brain homogenates (frontal cortex, parietal cortex, thalamus and cerebellum) demonstrated the presence of type 4 PrP Sc by the London classification scheme (Figure 2) pathognomonic of vcjd 5. 7

9 Discussion The clinical picture in vcjd classically begins with psychiatric disturbance and limb pain followed by ataxia, accompanied by myoclonus and chorea in many patients, with relatively late cognitive decline as compared to classical CJD. Our patient s history was complicated by a prolonged depression related to bereavement but he did have early personality alteration followed by ataxia. However, the cognitive decline developed early in the evolution of his illness and he never had chorea or limb pain. He was not classified as probable or possible vcjd according to established epidemiological diagnostic criteria 28. Crucially, the MRI did not show the pulvinar sign, found in 91% of patients with definite vcjd 28, but rather features typical of sporadic CJD. These MRI features have been described in a small number of usually older vcjd patients 29, 30. In a review of 106 pathologically proven vcjd cases, 12 were not classified as probable or possible vcjd; 8/12 of these showed the pulvinar sign but lacked diagnostic clinical features, and the remaining 4/12 had suboptimal MRI investigation 28. While the clinical and investigation features in our patient were not typical of vcjd, the pathology in the CNS and appearances on immunoblot were indistinguishable from previous cases and allowed a diagnosis of definite vcjd. CSF examination for the protein, and the real-time quaking-induced conversion assay are known to have low sensitivity in vcjd, so these negative results were not surprising 26. Experimental transmission of BSE and vcjd prions to transgenic mice expressing human PrP demonstrated powerful effects of the PRNP codon 129 polymorphism in determining susceptibility, incubation time and pathological phenotype. These effects relate to both the importance of homologous protein interactions in prion propagation 14 and the preferential propagation of different prion strains by PrP with different primary structures via conformational selection 31. Transgenic mice expressing human PrP M129 faithfully reproduce the neuropathology and molecular strain type of vcjd when challenged with BSE or vcjd prions. However, BSE transmission to these transgenic mice, in addition to producing a vcjd-like phenotype, can also result in a distinct molecular phenotype that is indistinguishable from a type of sporadic CJD 21. Propagation of the BSE prion strain seems to require expression of human PrP M129. Mice expressing only human PrP V129 develop a distinct neuropathological phenotype and molecular strain type 22. Such animal models suggested that primary and secondary human infection with BSE-derived prions may result in sporadic CJD-like or novel phenotypes in addition to vcjd, depending on the PrP genotype of the prion source and the recipient 22. Mice modelling the human PrP MV genotype show multiple phenotypes on challenge with BSE and vcjd prions 23. The current case demonstrates that the classical neuropathological and molecular strain features of vcjd are permissible in MV humans, 8

10 although the scanty deposition of prion protein in peripheral lymphoreticular tissue was unusual, as has been seen in a recent MM case 32. In conclusion, we describe the first definite case of vcjd in a PRNP codon 129 heterozygote. This patient had pathognomonic neuropathology and molecular strain type but a clinical presentation and neuroradiological features more typical of scjd. Additional cases will be needed to draw firm conclusions about any consistent change in phenotype of human BSE prion infection. It remains to be seen if this patient marks the start of a second wave of vcjd in the UK in the commonest MV genotype, mirroring the long incubation periods seen in MV individuals developing kuru, the major example of an epidemic human prion disease 2. There is considerable uncertainty in the predicted number and timing of future cases. It is possible there may be no, or only a few, further cases. However, if MV individuals have broadly similar susceptibility to BSE prion infection to MM individuals but simply much longer incubation periods, a few hundred cases might be expected over the years ahead. This case emphasizes the importance of autopsy in all cases of prion disease, in the UK, elsewhere in Europe and worldwide, in an attempt to determine the prevalence of BSE prionrelated disease in man, the range of possible clinical and pathological phenotypes, and the relative efficiencies of different routes of secondary transmission. The paucity of LRS involvement in this patient also has implications for studies aiming to estimate the prevalence of subclinical infection in the UK population by anonymous screening of surgical appendix tissue 12, 32. Competing interests J.C. is a Director and J.C. and J.D.F.W. are shareholders of D-Gen Limited, an academic spin-out company working in the field of prion disease diagnosis, decontamination, and therapeutics. D-Gen owns the ICSM35 antibody used in this study. The other authors declare no potential conflict of interest. 9

11 Figure 1. Neuropathology of vcjd with PRNP MV genotype. A, microvacuolar degeneration and frequent florid plaques throughout the cortex (H&E). B, immunostaining for abnormal prion protein (ICSM35 antibody) highlights amyloid plaques and shows widespread stellate pericellular deposits. C, Focal abnormal prion protein deposits are seen in the spleen. Scale bar: 50µm 10

12 Figure 2. Immunoblotting of MV vcjd. Panels A-C are immunoblots of brain homogenate analysed with anti-prp monoclonal antibody 3F4 and high sensitivity enhanced chemiluminescence to characterize protease-resistant prion protein 33. (A) Equivalent aliquots of 10% (weight to volume) brain homogenate (frontal cortex) from a normal human control case, a reference vcjd case or the patient analysed before (-) or after (+) digestion with proteinase K (PK). (B) PK-digested 10% (weight to volume) brain homogenate (frontal cortex) from the patient or reference cases of sporadic CJD (scjd) and vcjd. The provenance of the brain sample is designated above each lane. For the reference cases the PrP Sc type (types 2, 3 or 4 designated T2, T3 or T4; London classification 34 ) and PRNP codon 129 genotype (M, methionine, V, valine) are shown. (C) PK-digested 10 % (weight to volume) homogenates prepared from different regions of the patient s brain; frontal cortex, parietal cortex, thalamus and cerebellum. The volumes of samples loaded for immunoblots shown in panels B and C were varied to give equivalent levels of total PrP signal intensity. 11

13 Reference List 1. Collinge J. Prion diseases of humans and animals: their causes and molecular basis. Annu Rev Neurosci 2001;24: Collinge J, Whitfield J, McKintosh E et al. Kuru in the 21st century--an acquired human prion disease with very long incubation periods. Lancet 2006;367(9528): Brown P, Preece M, Brandel JP et al. Iatrogenic Creutzfeldt-Jakob disease at the millennium. Neurology 2000;55(8): Bruce ME, Will RG, Ironside JW et al. Transmissions to mice indicate that 'new variant' CJD is caused by the BSE agent. Nature 1997;389: Collinge J, Sidle KC, Meads J, Ironside J, Hill AF. Molecular analysis of prion strain variation and the aetiology of 'new variant' CJD. Nature 1996;383(6602): Hill AF, Desbruslais M, Joiner S, Sidle KCL, Gowland I, Collinge J. The same prion strain causes vcjd and BSE. Nature 1997;389: Edgeworth JA, Farmer M, Sicilia A et al. Detection of prion infection in variant Creutzfeldt- Jakob disease: a blood-based assay. Lancet 2011;377(9764): Hill AF, Butterworth RJ, Joiner S et al. Investigation of variant Creutzfeldt-Jakob disease and other human prion diseases with tonsil biopsy samples. Lancet 1999;353(9148): Spencer MD, Knight RS, Will RG. First hundred cases of variant Creutzfeldt-Jakob disease: retrospective case note review of early psychiatric and neurological features. BMJ 2002;324(7352): Zeidler M, Sellar RJ, Collie DA et al. The pulvinar sign on magnetic resonance imaging in variant Creutzfeldt-Jakob disease. Lancet 2000;355(9213): Hill AF, Zeidler M, Ironside J, Collinge J. Diagnosis of new variant Creutzfeldt-Jakob disease by tonsil biopsy. Lancet 1997;349(9045): Gill ON, Spencer Y, Richard-Loendt A et al. Prevalent abnormal prion protein in human appendixes after bovine spongiform encephalopathy epizootic: large scale survey. BMJ 2013;347:f Collinge J, Palmer MS, Dryden AJ. Genetic predisposition to iatrogenic Creutzfeldt-Jakob disease. Lancet 1991;337: Palmer MS, Dryden AJ, Hughes JT, Collinge J. Homozygous prion protein genotype predisposes to sporadic Creutzfeldt-Jakob disease. Nature 1991;352: Rudge P, Jaumuktane Z, Adlard P et al. Iatrogenic CJD due to pituitary-derived growth hormone with genetically determined incubation times of up to 40 years. Brain 2015;138:

14 16. Cervenakova L, Goldfarb L, Garruto R, Lee HS, Gajdusek CD, Brown P. Phenotype-genotype studies in kuru: Implications for new variant Creutzfeldt-Jakob disease. Proc Natl Acad Sci USA 1999;95: Mead S, Poulter M, Uphill J et al. Genetic risk factors for variant Creutzfeldt-Jakob disease: a genome-wide association study. Lancet Neurol 2009;8(1): Kaski D, Mead S, Hyare H et al. Variant CJD in an individual heterozygous for PRNP codon 129. Lancet 2009;374(9707): Bishop MT, Diack AB, Ritchie DL, Ironside JW, Will RG, Manson JC. Prion infectivity in the spleen of a PRNP heterozygous individual with subclinical variant Creutzfeldt-Jakob disease. Brain 2013;136: Mead S, Stumpf MP, Whitfield J et al. Balancing selection at the prion protein gene consistent with prehistoric kuru-like epidemics. Science 2003;300(5619): Asante EA, Linehan J, Desbruslais M et al. BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein. EMBO J 2002;21(23): Wadsworth JD, Asante EA, Desbruslais M et al. Human prion protein with valine 129 prevents expression of variant CJD phenotype. Science 2004;306(5702): Asante EA, Linehan J, Gowland I et al. Dissociation of pathological and molecular phenotype of variant Creutzfeldt-Jakob disease in transgenic human prion protein 129 heterozygous mice. Proc Natl Acad Sci USA 2006;103(28): Bishop MT, Hart P, Aitchison L et al. Predicting susceptibility and incubation time of humanto-human transmission of vcjd. Lancet Neurol 2006;5(5): Caine D, Tinelli RJ, Hyare H et al. The cognitive profile of prion disease: a prospective clinical and imaging study. Ann Clin Transl Neurol 2015;2(5): Peden AH, McGuire LI, Appleford NE et al. Sensitive and specific detection of sporadic Creutzfeldt-Jakob disease brain prion protein using real-time quaking-induced conversion. J Gen Virol 2012;93(Pt 2): Accessed online 20th June, Heath CA, Cooper SA, Murray K et al. Validation of diagnostic criteria for variant Creutzfeldt- Jakob disease. Ann Neurol 2010;67(6): Lukic A, Mead S, Rudge P, Collinge J. Comment on validation of diagnostic criteria for variant Creutzfeldt-Jakob disease. Ann Neurol 2011;69(1): El Tawil S, Mackay G, Davidson L, Summers D, Knight R, Will R. Variant Creutzfeldt-Jakob disease in older patients. J Neurol Neurosurg Psychiatry 2015; 86(11): Collinge J, Clarke A. A general model of prion strains and their pathogenicity. Science 2007;318(5852):

15 32. Mead S, Wadsworth JD, Porter MC et al. Variant Creutzfeldt-Jakob disease with extremely low lymphoreticular deposition of prion protein. JAMA Neurol 2014;71(3): Wadsworth JD, Joiner S, Hill AF et al. Tissue distribution of protease resistant prion protein in variant CJD using a highly sensitive immuno-blotting assay. Lancet 2001;358(9277): Hill AF, Joiner S, Wadsworth JD et al. Molecular classification of sporadic Creutzfeldt-Jakob disease. Brain 2003;126(6):