Prevalence in Britain of abnormal prion protein in human appendices before and after exposure to the cattle BSE epizootic
Prevalence in Britain of abnormal prion protein in human appendices before and after exposure to the cattle BSE epizootic
O. Noel Gill, Yvonne Spencer, Angela Richard-Loendt, Carole Kelly, David Brown, Katy Sinka, Nick Andrews, Reza Dabaghian, Marion Simmons, Philip Edwards, Peter Bellerby, David J. Everest, Mark McCall, Linda M. McCardle, Jacqueline Linehan, Simon Mead, David A. Hilton, James W. Ironside & Sebastian Brandner
Acta Neuropathologica (2020)Cite this article
Abstract
Widespread dietary exposure of the population of Britain to bovine spongiform encephalopathy (BSE) prions in the 1980s and 1990s led to the emergence of variant Creutzfeldt-Jakob Disease (vCJD) in humans. Two previous appendectomy sample surveys (Appendix-1 and -2) estimated the prevalence of abnormal prion protein (PrP) in the British population exposed to BSE to be 237 per million and 493 per million, respectively. The Appendix-3 survey was recommended to measure the prevalence of abnormal PrP in population groups thought to have been unexposed to BSE. Immunohistochemistry for abnormal PrP was performed on 29,516 samples from appendices removed between 1962 and 1979 from persons born between 1891 through 1965, and from those born after 1996 that had been operated on from 2000 through 2014. Seven appendices were positive for abnormal PrP, of which two were from the pre-BSE-exposure era and five from the post BSE-exposure period. None of the seven positive samples were from appendices removed before 1977, or in patients born after 2000 and none came from individuals diagnosed with vCJD. There was no statistical difference in the prevalence of abnormal PrP across birth and exposure cohorts. Two interpretations are possible. Either there is a low background prevalence of abnormal PrP in human lymphoid tissues that may not progress to vCJD. Alternatively, all positive specimens are attributable to BSE exposure, a finding that would necessitate human exposure having begun in the late 1970s and continuing through the late 1990s.
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Discussion The Appendix-3 Study was designed to measure the prevalence of abnormal PrP in appendices removed in operations performed before 1980 (historical), and after 2000 in those born since 1996 (new), i.e. in appendices taken from outside the population considered most at-risk of acquiring vCJD from BSE-related prions in the food chain. The overall prevalence of immunopositive samples found in these groups that were assumed to be unexposed to BSE was not lower than the prevalence in the most highly BSE-exposed cohort surveyed in the Appendix-2 Study. Examination of the available data on the appendectomy history of each human vCJD case to date showed that none of the positive appendices from this study (Appendix-3), nor the Appendix-2 Study [17], could have come from the 178 known vCJD cases in the UK.
The absence of a consistent difference between individual positive samples within Appendix-3 or between Appendix-2 and -3 is noteworthy. It might not have been possible to infer from these data alone that differences in immunostaining pattern of individual samples relate to source or strain, rather than host, or age of sample. Had we seen such differences it could have suggested different sources for the abnormal PrP detected.
One question is whether the IHC staining found in these prevalence studies was necessarily related to vCJD. The pattern of the staining observed in the positive specimens, however, is highly distinctive and consistent with that found in vCJD cases (both before and after onset of clinical symptoms). The abnormal accumulation of PrP in lymphoid tissue, as detected by immunohistochemistry, has only ever been found in humans with vCJD, and not in other human prion diseases such as sCJD [19, 20], and the transmitted forms of iatrogenic CJD [18] or Kuru [6, 10].
Two interpretations of the prevalence of abnormal PrP in different populations in Britain may be given. First, there is no significant difference in the prevalence of vCJD-related abnormal PrP between any of the appendix survey populations, i.e. there is a low background prevalence of abnormal PrP in human lymphoid tissues that may not progress to vCJD. This background prevalence is unrelated to the intensity and extent of dietary exposure to BSE. The alternative interpretation is that although there is no statistical difference in the prevalence of vCJD-related abnormal PrP across birth and exposure cohorts in the populations studied, the central estimates vary in a direction consistent with the changing intensity over time of the observed BSE epidemic in cattle. All positive specimens may therefore be attributable to BSE exposure.
This second interpretation, however, suggests that human exposure began in the late 1970s and continued through the late 1990s, albeit at a much lower rate than in the mid-1980s. Although cases of BSE were not described until 1986, back-calculation models indicate that cases could have been occurring, and infectivity possibly entering the food chain, for several years before the disease was identified in cattle [8, 46]. In addition, the origins of BSE have never been unequivocally established [40], so it could have been present at a very low prevalence for a long time prior to its amplification through the animal feed chain [46]. Cases of BSE continued to occur in animals born after the total feed ban put in place in the UK in July 1996, the reinforced feed ban in Ireland in October of the same year and the total feed ban in the rest of EU in 2001 [40], and such cases could provide one possible route of exposure for the vCJD cases identified in the post-1996 birth cohort.
Additionally, it has been demonstrated that sheep are susceptible to BSE [30, 44], and the disease can transmit between sheep under field conditions [31]. It has never been isolated from commercial sheep populations, but it has been observed in goats [43]. Sheep-passaged BSE can demonstrate increased ‘virulence’ on subsequent inter-species transmission [38, 44], and can cause disease indistinguishable from vCJD in transgenic mouse models [32, 38]. A non-bovine route of exposure is therefore hypothetically possible.
Neither interpretation, on its own, is entirely satisfactory and it is possible to speculate about a combination of both. There could be ‘background’ prevalence in all groups plus some additional prevalence associated with BSE in the most highly exposed population. Detailed appraisal of the histological findings, however, showed no consistent differences between the positive samples that might have indicated two or more different sources. A large study of a population entirely unexposed to BSE prions would be necessary to determine whether a background prevalence exists, and such a study would pose additional challenges to those faced when implementing the Appendix-3 survey.
Whichever interpretation is preferred, the contrast between the prevalence of abnormal PrP and the number of clinical vCJD cases seen to date (mid-2020) strongly suggests that possibly none of those in whom abnormal PrP is detected through an ante-mortem lymphoid tissue survey will develop any symptoms of prion disease.
New research proposals have been sought that utilise some of the archived additional slides and cuts of formalin fixed tissues from each positive appendix [Department of Health Policy Research Programme—Research call on vCJD 2016: https://clahrcprojects.co.uk/news/department-health-policy-research-programme-invitation-applications] (accessed March 2020). In response, laboratory investigations are underway to elucidate the nature of the immunopositive samples. One approach is using in vitro conversion models to amplify the abnormal prion prior to conducting Western blotting analysis and transmission studies in mice (Green A; personal communication). Another is attempting discrimination of vCJD infected from uninfected fixed tissues through DNA methylation array “profiling” (Mead S; personal communication).
A variety of risk management measures remain in place to limit the risks of person-to-person transmission of prions by blood transfusion or by re-use of surgical instruments in the general population. Whichever way the Appendix-3 Study is interpreted, the prevalence range of prion infection remains a concern, and maintenance of the full range of precautionary measures is a judgement that would need to be balanced against the costs and benefits of these risk reduction measures [1, 37, 42]. More specifically, it is reasonable to assume that the highest prevalence of asymptomatic infection is in the cohort that had greatest exposure to BSE and which contains all known clinical cases of vCJD, the 1961 to 1985 birth cohort [36]. The findings of the Appendix-3 Study, however, challenge the assumption that a specific cut-off date defines a low-risk population, i.e. those born after 1996. Therefore, the difference between the interpretations of the Appendix-3 prevalence has practical implications for risk management.
The discovery that not only PrP but also other proteopathic seeds such as amyloid-β can be iatrogenically transmitted between humans has in the last few years received significant attention [33]. Whilst experimental transmission of amyloid-β had been demonstrated for some years [12, 13, 34], the observation of human transmission of amyloid-β through contaminated human growth hormone [28, 33, 41] has prompted additional studies examining the potential transmission through other human-derived products such as dura mater transplants [14, 35], intravascular embolization material [3], and through surgical instruments [29]. The transmissibility into susceptible animals of amyloid-β contained in human growth hormone preparations has provided further evidence of the historic role of this product in the development of cerebral amyloid angiopathy (CAA), a potentially lethal vascular disease, in affected individuals [26, 39]. These observational and experimental studies have put new emphasis on the necessity of adequate surveillance of relevant human diseases, sensitive detection of proteopathic seeds other than PrP [27] and their effective decontamination, for example on surgical instruments and medical devices.
In conclusion, the Appendix-3 Study has not produced a clear answer to the question of whether the presence of abnormal PrP, as detected by IHC, in the British population is limited to those exposed to the BSE epizootic. The results raise the possibility of abnormal prion exposure both before the presumed BSE epizootic and after 1996 when exposure to BSE-related prions in the food chain in Britain was considered “extremely low”.
Pract Neurol 2010; 10: 250–251
Variant CJD: where has it gone, or has it?
Bob Will
The feared large scale epidemic of variant Creutzfeldt–Jakob disease (vCJD) has thankfully not materialised. The number of cases identified annually in the UK has been in decline since 1999 although there could still be a tail to the outbreak lasting for many years (fi gure). Internationally, the trend in the number of vCJD cases is also in decline and bovine spongiform encephalopathy (BSE) is now a rare disease, even in the UK. One explanation is that the measures introduced to control these diseases were effective; indeed, it is of interest that, to date, no case of vCJD in the UK was born after 1989 when the specified bovine offal ban was introduced whereas there have been three cases born after this date in other European countries where legislative measures to minimise human exposure to BSE were introduced some years later. However, BSE and vCJD control measures are very costly and there will be pressure in the coming years to withdraw or amend relevant legislation and guidance. An important question is whether there are continuing realistic concerns about public health in relation to vCJD and not simply, as has been suggested in the press, scaremongering by some researchers keen to maintain funding.
All probable and definite cases of vCJD internationally, in which genetic testing has been carried out (199/219), have been methionine (MM) homozygous at codon 129 of the prion protein gene. This is clearly a susceptibility factor for the development of clinical disease. However, two out of three prion protein positive appendices in a screening study of 12 674 routine appendix specimens were valine/valine (VV), and a clinically unaffected recipient of a vCJD implicated blood transfusion with disease associated prion protein in their spleen was a heterozygote (MV). This implies that there may be a population of individuals who are subclinically infected but who may never develop clinical disease within their lifespan, perhaps explaining, in part, the mismatch between the extensive human exposure to BSE in the food chain and the relatively limited number of clinical cases so far observed.
This has important implications for the risk of secondary transmission from person to person—for example, through contaminated surgical instruments or blood transfusion. According to one estimate, extrapolating from the estimated prevalence of subclinical infection, 1 in 10 000 blood donations have been derived from individuals who are infected with vCJD. The fact of transfusion transmission of vCJD is now established with three cases of vCJD developing symptoms 5–8 years after having received a blood transfusion from people who donated the blood 1.5–3.5 years before themselves developing the condition (in addition to the subclinical infection referred to above).1 Concerns have been heightened by the discovery of disease associated prion protein in the spleen of a person with haemophilia who had received factor VIII derived from a pool containing a vCJD donation.2 Furthermore, there is a signifi cant population who have been exposed to plasma products potentially contaminated with the vCJD agent but, to date, there is no evidence that clinical cases of vCJD have been caused by exposure to such treatments. One unresolved question is why there have not been more cases of vCJD linked to blood transfusion or plasma products and there is a pressing need to obtain more precise information on the prevalence of infection in the general population.
The recent identification of a possible clinical case of vCJD in an individual with an MV genotype3 reinforces the long held concern that there may be further waves of vCJD cases in individuals with a non-MM codon 129 genotype. Mathematical models suggest that the number of MV and VV cases will be limited and not exceed the primary MM outbreak, but predicting infectious outbreaks is an imprecise science, as may be inferred from the recent swine fl u epidemic which never materialised, at least not to the extent predicted. The adage that ‘Essentially, all models are wrong but some are useful’ (George Edwin Pelham Box, 2007), reinforces the need for caution in predicting the future course of the vCJD outbreak. There is also the possibility that the phenotype of vCJD may be influenced by the genetic background. It is reassuring therefore that the recent possible MV case was identified on the basis of the clinical features as this may indicate that any further such cases will also be recognised as vCJD.
However, there is clearly still a continuing need to look for new phenotypes of human prion disease. Novel forms of animal prion diseases have been identified in recent years, including atypical scrapie and the rare H and L forms of atypical BSE, probably as a result of the extensive abattoir testing of animal populations. Atypical BSE has been transmitted to a range of laboratory animals, and in a primate model the incubation period was shorter than with conventional BSE and the clinical and pathological phenotype different.4
The incubation period in human prion disease can extend to decades and there are continuing concerns and uncertainties that indicate that there are good reasons to continue research and surveillance in vCJD, despite the clear decline in the primary outbreak of vCJD.
Competing interests BW holds research grants in relation to CJD research and surveillance. Provenance and peer review Commissioned; not externally peer reviewed.
REFERENCES
1. Hewitt PE, Llewelyn CA, Mackenzie J, et al. Creutzfeldt–Jakob disease and blood transfusion: results of the UK Transfusion Medicine Epidemiological Review study. Vox Sang 2006;91:221–30.
2. Peden A, McCardle L, Head MW, et al. Variant CJD infection in the spleen of a neurologically asymptomatic UK adult patient with haemophilia. Haemophilia 2010;16:296–304.
3. Kaski D, Mead S, Hyare H, et al. Variant CJD in an individual heterozygous for PRNP codon 129. Lancet 2009;374:2128.
4. Comoy EE, Casalone C, Lescoutra-Etchegaray N, et al. Atypical BSE (BASE) transmitted from asymptomatic aging cattle to a primate. PLoS One 2008;3:e3017.
SATURDAY, JUNE 23, 2018
Diagnosis of Methionine/Valine Variant Creutzfeldt-Jakob Disease by Protein Misfolding Cyclic Amplification
Volume 24, Number 7—July 2018
Dispatch
Diagnosis of Methionine/Valine Variant Creutzfeldt-Jakob Disease by Protein Misfolding Cyclic Amplification
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)
Abstract
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.
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.
Conclusions
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 (6,12–14). 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.
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