Skip directly to site content Skip directly to page options Skip directly to A-Z link Skip directly to A-Z link Skip directly to A-Z link
Volume 24, Number 7—July 2018

Diagnosis of Methionine/Valine Variant Creutzfeldt-Jakob Disease by Protein Misfolding Cyclic Amplification

Article Metrics
citations of this article
EID Journal Metrics on Scopus
Daisy BougardComments to Author , Maxime Bélondrade, Charly Mayran, Lilian Bruyère-Ostells, Sylvain Lehmann, Chantal Fournier-Wirth, Richard S. Knight, Robert G. Will, and Alison J.E. Green
Author affiliations: Etablissement Français du Sang, Montpellier, France (D. Bougard, M. Bélondrade, C. Mayran, L. Bruyère-Ostells, C. Fournier-Wirth); University of Montpellier, Montpellier (S. Lehmann); University of Edinburgh, Edinburgh, Scotland, UK (R.S. Knight, R.G. Will, A.J.E. Green)

Cite This Article


A patient with a heterozygous variant of Creutzfeldt-Jakob disease (CJD) with a methionine/valine genotype at codon 129 of the prion protein gene was recently reported. Using an ultrasensitive and specific protein misfolding cyclic amplification–based assay for detecting variant CJD prions in cerebrospinal fluid, we discriminated this heterozygous case of variant CJD from cases of sporadic CJD.

Variant Creutzfeldt-Jakob disease (vCJD) is a neurodegenerative infectious disease caused by transmission of a cattle prion disease (bovine spongiform encephalopathy) to humans (1). Most vCJD cases have occurred in the United Kingdom, where an estimated 1 in 2,000 persons is potentially asymptomatically infected, although there is some uncertainty about interpretation of detection of abnormal prion protein (PrPTSE) in appendix tissues on which this incidence is based (2) (Public Health England,

Until recently, all clinical cases of vCJD for which the prion protein gene has been analyzed have been shown to be methionine homozygous at codon 129, a genotype present in almost 40% of Caucasian populations. The report of the first definite heterozygous methionine/valine vCJD patient who died in 2016 (3) underlined previous concern about a possible second wave of vCJD cases (4). The clinical features of this patient were more similar to those of patients with sporadic CJD (sCJD) than to those with vCJD. This patient had met the agreed surveillance diagnostic criteria for probable sCJD (5). However, vCJD was diagnosed during an autopsy; florid plaques were observed by histologic examination of the brain and peripheral detection of PrPTSE in lymphoid tissues. Western blot analysis of brain tissue confirmed a type 2B molecular profile of PrPTSE, which is characteristic for vCJD.

A diagnostic test to identify methionine/valine heterozygous vCJD cases is urgently needed to enable discrimination between heterozygous vCJD and sCJD and in view of the potential reservoir of methionine/valine heterozygous asymptomatic vCJD carriers in the blood donor population. We developed a highly sensitive and specific assay that accurately detects vCJD prions in blood even before the occurrence of clinical signs (6). We adapted this assay, which was based on protein misfolding cyclic amplification (PMCA) (7), for specific detection of vCJD in cerebrospinal fluid (CSF) and confirmed the ability of this assay to differentiate patients with atypical heterozygous vCJD from patients with sCJD.

The Study

We blindly analyzed 98 CSF samples provided by the National CJD Research and Surveillance Unit (Edinburgh, Scotland, UK) and the Centre Hospitalier Universitaire de Montpellier (Montpellier, France) after obtaining appropriate consent. Clinicians distributed CSF samples into blinded panels from the United Kingdom and France; 41 from patients with vCJD; 23 from patients with sCJD; 1 from a patient with genetic CJD; and 33 from patients with non-CJD, including samples from patients with Alzheimer’s disease and patients with nonneurodegenerative diseases.

CSF samples were thawed at room temperature and used directly in PMCA. We performed PMCA amplification by using brains from humanized transgenic mice as substrate for normal prion protein. After successive rounds of 160 cycles of PMCA for 15 min and sonication for 20 s, we detected PrPTSE by using Western blot after digestion with proteinase K (6).

Of the 98 CSF samples analyzed, our assay identified 40 of 41 cases of clinical vCJD, including the methionine/valine heterozygous patient, thus showing a diagnostic sensitivity of 97.6% (95% CI 87.1%–99.9%) (Table). One CSF sample from a probable case of vCJD showed a negative result. After decoding by clinicians, we retested this sample in duplicate; it showed a positive result.

Our assay also showed high analytical specificity; 0 of 57 potentially cross-reacting CSF specimens from patients with sCJD, gCJD, Alzheimer's disease, and other nonneurodegenerative diseases showed a positive result (specificity 100% [95% CI 93.7%–100%]) (Table). The case-patient with methionine/valine heterozygous vCJD was specifically discriminated from the 12 methionine/valine heterozygous neuropathologically confirmed sCJD case-patients tested.


We then compared by using Western blot the PrPTSE molecular signature obtained for the clinical vCJD amplified samples from classical methionine homozygous cases and the new methionine/valine heterozygous vCJD case with that of the reference brain sample from a patient with vCJD (Figure). As expected, the profile obtained after PMCA amplification of the CSF from the methionine/valine heterozygous vCJD patient was similar to those obtained for methionine homozygous vCJD patients. The characteristic type 2 mobility and clear predominance of the diglycosylated isoform was obtained for all vCJD patients before or after amplification.


We report a specific detection method that enables clinical diagnosis of a heterozygous methionine/valine heterozygous vCJD patient. This patient was the first definite heterozygous patient described since the start of the vCJD epidemic in the United Kingdom in 1996 (3). Clinical diagnosis was difficult because clinical signs and symptoms, particularly cerebral appearance by magnetic resonance imaging, were suggestive of sCJD (3). The vCJD blood test (direct detection assay) developed by the Medical Research Council Prion Unit (London, UK) (8) showed a negative result for this case-patient. We found characteristic vCJD prion protein amplification in the CSF, which led to a specific diagnosis of vCJD because sCJD samples did not show positive results by PMCA. This result also demonstrates the possibility of amplifying methionine/valine heterozygous vCJD prion protein by PMCA with a substrate from humanized transgenic mice that overexpress homozygous methionine prion protein (9). However, PMCA analysis should be performed in a Biosafety Level 3 laboratory and requires highly experienced personnel.

Iatrogenic transmission of vCJD by blood transfusion has been documented in 3 recipients of nonleukodepleted erythrocyte concentrates from blood donors during development of disease (10). One additional probable case of vCJD transmission by blood transfusion was identified during an autopsy of a methionine/valine heterozygous patient who died from a nonneurologic disorder and in whom vCJD prion protein was detected in the spleen (11). The presence of infectivity in blood of the definite methionine/valine heterozygous vCJD patient involved in our study is uncertain and requires further investigation.

From a clinical point of view, prion amplification technologies, such as PMCA and real-time quaking-induced conversion (RT-QuIC), have already shown their sensitive detection of disease-related prion protein in biologic fluids (6,1214). Independent studies have shown that detection of prion protein seeding activity in CSF by RT-QuIC might have a specificity of 99%–100% for diagnosis of sCJD (13,15). Application of RT-QuIC and PMCA for CSF samples might represent a suitable strategy for premortem discrimination between sCJD and vCJD including methionine/valine heterozygous case-patients, particularly for cases with a heterozygous codon 129 genotype in which clinical distinction between sCJD and vCJD is problematic.

Dr. Bougard is a research scientist in charge of the Prion Group at Etablissement Français du Sang of Montpellier, France. Her primary research interests include development of innovative tools for the prevention of transfusion risk associated with nonconventional agents.



We thank Maria-Teresa Alvarez and Jacques-Damien Arnaud for providing helpful assistance in the L3 facility at the Etablissement Confiné d’Expérimentation–Centre d'Elevage et de Conditionnement Expérimental des Modèles Animaux of the University of Montpellier.

This study was supported by Etablissement Français du Sang and the UK Department of Health Policy Research Programme (grant PRST061400008).



  1. Will  RG, Ironside  JW, Zeidler  M, Cousens  SN, Estibeiro  K, Alperovitch  A, et al. A new variant of Creutzfeldt-Jakob disease in the UK. Lancet. 1996;347:9215. DOIPubMedGoogle Scholar
  2. Gill  ON, Spencer  Y, Richard-Loendt  A, Kelly  C, Dabaghian  R, Boyes  L, et al. Prevalent abnormal prion protein in human appendixes after bovine spongiform encephalopathy epizootic: large scale survey. BMJ. 2013;347(oct15 5):f5675. DOIPubMedGoogle Scholar
  3. Mok  T, Jaunmuktane  Z, Joiner  S, Campbell  T, Morgan  C, Wakerley  B, et al. Variant Creutzfeldt-Jakob disease in a patient with heterozygosity at PRNP codon 129. N Engl J Med. 2017;376:2924. DOIPubMedGoogle Scholar
  4. Garske  T, Ghani  AC. Uncertainty in the tail of the variant Creutzfeldt-Jakob disease epidemic in the UK. PLoS One. 2010;5:e15626. DOIPubMedGoogle Scholar
  5. Zerr  I, Kallenberg  K, Summers  DM, Romero  C, Taratuto  A, Heinemann  U, et al. Updated clinical diagnostic criteria for sporadic Creutzfeldt-Jakob disease. Brain. 2009;132:265968. DOIPubMedGoogle Scholar
  6. Bougard  D, Brandel  JP, Bélondrade  M, Béringue  V, Segarra  C, Fleury  H, et al. Detection of prions in the plasma of presymptomatic and symptomatic patients with variant Creutzfeldt-Jakob disease. Sci Transl Med. 2016;8:370ra182. DOIPubMedGoogle Scholar
  7. Saborio  GP, Permanne  B, Soto  C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature. 2001;411:8103. DOIPubMedGoogle Scholar
  8. Edgeworth  JA, Farmer  M, Sicilia  A, Tavares  P, Beck  J, Campbell  T, et al. Detection of prion infection in variant Creutzfeldt-Jakob disease: a blood-based assay. Lancet. 2011;377:48793. DOIPubMedGoogle Scholar
  9. Béringue  V, Le Dur  A, Tixador  P, Reine  F, Lepourry  L, Perret-Liaudet  A, et al. Prominent and persistent extraneural infection in human PrP transgenic mice infected with variant CJD. PLoS One. 2008;3:e1419. DOIPubMedGoogle Scholar
  10. Hewitt  PE, Llewelyn  CA, Mackenzie  J, Will  RG. Creutzfeldt-Jakob disease and blood transfusion: results of the UK Transfusion Medicine Epidemiological Review study. Vox Sang. 2006;91:22130. DOIPubMedGoogle Scholar
  11. Peden  AH, Head  MW, Ritchie  DL, Bell  JE, Ironside  JW. Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient. Lancet. 2004;364:5279. DOIPubMedGoogle Scholar
  12. Concha-Marambio  L, Pritzkow  S, Moda  F, Tagliavini  F, Ironside  JW, Schulz  PE, et al. Detection of prions in blood from patients with variant Creutzfeldt-Jakob disease. Sci Transl Med. 2016;8:370ra183. DOIPubMedGoogle Scholar
  13. McGuire  LI, Peden  AH, Orrú  CD, Wilham  JM, Appleford  NE, Mallinson  G, et al. Real time quaking-induced conversion analysis of cerebrospinal fluid in sporadic Creutzfeldt-Jakob disease. Ann Neurol. 2012;72:27885. DOIPubMedGoogle Scholar
  14. Moda  F, Gambetti  P, Notari  S, Concha-Marambio  L, Catania  M, Park  KW, et al. Prions in the urine of patients with variant Creutzfeldt-Jakob disease. N Engl J Med. 2014;371:5309. DOIPubMedGoogle Scholar
  15. Atarashi  R, Satoh  K, Sano  K, Fuse  T, Yamaguchi  N, Ishibashi  D, et al. Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion. Nat Med. 2011;17:1758. DOIPubMedGoogle Scholar




Cite This Article

DOI: 10.3201/eid2407.172105

Table of Contents – Volume 24, Number 7—July 2018

EID Search Options
presentation_01 Advanced Article Search – Search articles by author and/or keyword.
presentation_01 Articles by Country Search – Search articles by the topic country.
presentation_01 Article Type Search – Search articles by article type and issue.



Please use the form below to submit correspondence to the authors or contact them at the following address:

Daisy Bougard, Laboratoire TransDiag, Etablissement Français du Sang, 392 Ave du Professeur Jean-Louis Viala, 34084 Montpellier, France

Send To

10000 character(s) remaining.


Page created: June 18, 2018
Page updated: June 18, 2018
Page reviewed: June 18, 2018
The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.