Detection of Prion Protein Seeding Activity in Tear Fluids
May 11, 2023
N Engl J Med 2023; 388:1816-1817
DOI: 10.1056/NEJMc2214647
TO THE EDITOR:
The development of the real-time quaking-induced conversion (RT-QuIC) assay has been an innovation in the diagnosis of prion diseases.1 This assay enables the detection of abnormally folded prion protein scrapie (which causes Creutzfeldt–Jakob disease [CJD] and other prion diseases) in brain, cerebrospinal fluid, and other tissues.2,3 We modified our detection protocol by using a recombinant prion protein containing the human PRNPsequence with the E200K mutation as a substrate (see the Supplementary Appendix, available with the full text of this letter at NEJM.org). On the basis of the theory that misfolded prion protein may be detectable in tissues other than brain and cerebrospinal fluid, we seeded the reaction with tear fluid obtained from patients with CJD, healthy carriers of the PRNPmutation, and controls (Figure 1).
We initially studied tear samples obtained from 13 patients with prion disease (of whom 9 had sporadic CJD and 4 had genetically determined prion disease [1 each with P102L and D178-N and 2 with T183A]), from 5 healthy carriers of the PRNP mutation (3 with P102L and 1 each with D178N and 5-OPRI), and from 26 controls (healthy donors and patients with other neurologic diseases). The test was positive in 8 of 9 patients with sporadic CJD, in 3 of 4 patients with genetic prion disease, and in 4 of 5 healthy carriers of PRNP; the test results were negative in all the controls. These results were validated in an analysis of tear fluid in consecutively acquired samples obtained from patients, which showed seeding activity in 5 of 6 patients with prion disease and in 2 of 3 healthy mutation carriers; testing was negative in samples obtained from 68 controls with various neurologic disorders (Tables S1 through S3 in the Supplementary Appendix).
Our study showed that the modified RT-QuIC assay can detect prion disease in tear fluid obtained from most patients with prion disease. In total, the modified assay was positive in 16 of 19 patients (84%) and had sensitivity for both sporadic and genetic forms of CJD that was similar to the data we have obtained from cerebrospinal fluid. Samples of cerebrospinal fluid that were tested with RT-QuIC showed an earlier and more intense signal response than tear fluid. An additional finding was the detection of seeding activity in healthy PRNP carriers, which indicates possible diagnosis at the asymptomatic disease stage4; alternatively, seeding may present in this group because of the intrinsic instability of the mutated protein. Although our findings are based on limited data, the seeding response was similar in symptomatic and asymptomatic patients, a result that favors the hypothesis of mutational instability (Figure 1B, 1C, and 1D). Additional longitudinal studies will be needed to test this theory.
The advantage of noninvasive tear-fluid testing is that it does not require special extraction and sample-handling protocols. The test may be used in situations in which cerebrospinal fluid cannot be obtained or repeated sampling is necessary. However, this assay will not replace the standard examination of cerebrospinal fluid in the diagnosis of prion disease, because the latter is more informative with respect to potential differential diagnoses in progressive neurologic disorders.5
Matthias Schmitz, Ph.D. Susana Silva Correia, Ph.D. Peter Hermann, M.D. Fabian Maass, M.D. Stefan Goebel, M.D. Timothy Bunck, M.D. Angela Correia, M.Sc. University Medical Center, Göttingen, Germany
Paul Lingor, M.D. Klinikum Rechts der Isar, Munich, Germany
Andre Fischer, Ph.D. German Center for Neurodegenerative Diseases, Göttingen, Germany
Inga Zerr, M.D. University Medical Center, Göttingen, Germany ingazerr@med.uni-goettingen.de
Disclosure forms provided by the authors are available with the full text of this letter at NEJM.org.
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Supplementary Appendix
Supplement to: Schmitz M, Silva Correia S, Hermann P, et al. Detection of prion protein seeding activity in tear fluids. N Engl J Med 2023;388:1816-7. DOI: 10.1056/NEJMc2214647
This appendix has been provided by the authors to give readers additional information about the work.
Summary
The development of in vitro misfolded protein amplification systems, such as the real-time quakinginduced conversion (RT-QuIC) assay has been a major innovation in prion disease diagnosis. It enables the detection of abnormally folded prion protein scrapie (PrPSc) in tissues (e.g. brain tissue) and body fluids, such as cerebrospinal fluid (CSF) from prion disease patients. Meanwhile, the CSF RT-QuIC has been established in routine diagnostics in prion diseases worldwide. We modified our RT-QuIC protocol by using a novel recombinant PrP substrate exhibiting a higher seeding efficiency through the introduction of the E200K PRNP mutation in the full-length human PrP sequence and tested tear fluids from prion disease patients and controls. Using tear fluid, the assay was positive in 8 out of 9 sporadic CJD patients, 3 out of 4 genetic prion disease patients, and none of 26 control individuals. The results were validated in a consecutively acquired cohort, being positive in 5 out of 6 prion disease patients and negative in all 68 controls across the full spectrum of various neurological disorders. Of interest, the test was positive in tear fluids from 6 out of 8 asymptomatic PRNP mutation carriers (HMC). Our study demonstrates that PrP seeding activity can be detected in prion disease patients using noninvasively obtained TFs. The test opens new avenues for early diagnosis and potentially for follow-up studies.
snip...
Discussion
For the development of a solid and reproducible test, there are several questions to be addressed, such as a robust substrate, optimal running conditions for test sensitivity and specificity and the best biomatrix to be studied. We hypothesized that rec PrP substrates with prion disease mutations in the RT-QuIC may facilitate the seeding conversion process of PrPC, synthesized a recombinant protein carrying the E200K mutations, and used it for further studies.
Various matrices have been discussed for biomarker assessment in CJD. CSF with its ability to reflect central nervous processes more accurately than peripheral fluids such as blood components17 is most often used for biomarker research. Invasiveness and the potential risk due to lumbar puncture present a major drawback of this method, especially concerning the need for longitudinal sample collection, as needed for research on reliable disease progression markers in clinical trials. PrP amplification assays have shown poor utility for detection of PrPSc in blood18 and urine.19 In this regard, new approaches using minimal-invasively accessible biomaterial, such as skin punch biopsy or olfactory mucosa offer great potential.6,20-22 In search for a non-invasively accessible biofluid, we decided to evaluate the potential applicability of the methodology for tear fluids for various reasons: similar to CSF, tear fluids are low in protein content, in contrast to serum or plasma, and might therefore have less preanalytical problems compared to blood or tissues. TF is very easy to collect, even less invasive than blood. The collection of tears requires no medical specialist, no advanced training and no technical equipment. The collection of tears is risk-free and can be done at different time points for follow up studies. With its close connection to the brainstem by the autonomic nerve system, it might also reflect CNS processes for some part. Tear fluids have already been used to detect relevant biomarkers for neurodegeneration like neurofilament light chain or alpha-synuclein in Parkinson’s disease.23,24
Major findings of this study are the detection of seeding activity in TF in sporadic CJD and genetic prion diseases. Although the numbers are still low in this exploratory study, we see abnormal seeding activity in a majority of patients using our modified protocol. An important observation is the detection of seeding activity in TF in healthy mutation carriers. Very limited information is available in the literature on PrP seeding activity in these individuals. Some studies have shown that the chance of detecting a CSF positivity in mutation carriers is very low when Ha 90-231 or bank vole recombinant PrP is used.25 Seeding activity has been reported in single E200K and P102L PRNP mutation carriers at presymptomatic stage in the CSF, one of whom converted shortly after but the other remained asymptomatic after several years of follow up.26 One possible interpretation is that pathological PrP seeding activity precedes clinical onset and increase of biomarkers of neurodegeneration as suggested by Mok et al.26, and potentially indicates the earliest disease stage. Detectable seeding activity may also be primarily present in individuals with a pathogenic PRNP mutation due to potential intrinsic instability of the mutated protein. Although based on very limited data, the seeding response was similar in symptomatic and non-symptomatic mutation carriers.
It is not clear yet if the detection of seeding activity in TF in prion disease has implications for public health. Because of the extremely high sensitivity in the femtomolar detection range, RT-QuIC has been applied to various peripheral tissues before.6,8,27,28 Others used sensitive genetically modified mice models and reported low levels of infectivity in various organs outside the brain.29 Although these data are clearly a matter of concern, the interpretation needs to take into account the highly artificial experimental settings, use of genetically modified animals and highly effective inoculation routes. Surveillance studies have not revealed any evidence from human to human transmission except for what is already known in iatrogenic cases and failed to identify disease clusters.13,30 Despite that, continuous vigilance and further research is needed for solid and substantiated risk estimation.
Funding
The study was funded by the Robert Koch-Institute through funds from the Federal Ministry of Health; grant no. 1369-341
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May 11, 2023
N Engl J Med 2023; 388:1816-1817
DOI: 10.1056/NEJMc2214647
TO THE EDITOR:
The development of the real-time quaking-induced conversion (RT-QuIC) assay has been an innovation in the diagnosis of prion diseases.1 This assay enables the detection of abnormally folded prion protein scrapie (which causes Creutzfeldt–Jakob disease [CJD] and other prion diseases) in brain, cerebrospinal fluid, and other tissues.2,3 We modified our detection protocol by using a recombinant prion protein containing the human PRNPsequence with the E200K mutation as a substrate (see the Supplementary Appendix, available with the full text of this letter at NEJM.org). On the basis of the theory that misfolded prion protein may be detectable in tissues other than brain and cerebrospinal fluid, we seeded the reaction with tear fluid obtained from patients with CJD, healthy carriers of the PRNPmutation, and controls (Figure 1).
We initially studied tear samples obtained from 13 patients with prion disease (of whom 9 had sporadic CJD and 4 had genetically determined prion disease [1 each with P102L and D178-N and 2 with T183A]), from 5 healthy carriers of the PRNP mutation (3 with P102L and 1 each with D178N and 5-OPRI), and from 26 controls (healthy donors and patients with other neurologic diseases). The test was positive in 8 of 9 patients with sporadic CJD, in 3 of 4 patients with genetic prion disease, and in 4 of 5 healthy carriers of PRNP; the test results were negative in all the controls. These results were validated in an analysis of tear fluid in consecutively acquired samples obtained from patients, which showed seeding activity in 5 of 6 patients with prion disease and in 2 of 3 healthy mutation carriers; testing was negative in samples obtained from 68 controls with various neurologic disorders (Tables S1 through S3 in the Supplementary Appendix).
Our study showed that the modified RT-QuIC assay can detect prion disease in tear fluid obtained from most patients with prion disease. In total, the modified assay was positive in 16 of 19 patients (84%) and had sensitivity for both sporadic and genetic forms of CJD that was similar to the data we have obtained from cerebrospinal fluid. Samples of cerebrospinal fluid that were tested with RT-QuIC showed an earlier and more intense signal response than tear fluid. An additional finding was the detection of seeding activity in healthy PRNP carriers, which indicates possible diagnosis at the asymptomatic disease stage4; alternatively, seeding may present in this group because of the intrinsic instability of the mutated protein. Although our findings are based on limited data, the seeding response was similar in symptomatic and asymptomatic patients, a result that favors the hypothesis of mutational instability (Figure 1B, 1C, and 1D). Additional longitudinal studies will be needed to test this theory.
The advantage of noninvasive tear-fluid testing is that it does not require special extraction and sample-handling protocols. The test may be used in situations in which cerebrospinal fluid cannot be obtained or repeated sampling is necessary. However, this assay will not replace the standard examination of cerebrospinal fluid in the diagnosis of prion disease, because the latter is more informative with respect to potential differential diagnoses in progressive neurologic disorders.5
Matthias Schmitz, Ph.D. Susana Silva Correia, Ph.D. Peter Hermann, M.D. Fabian Maass, M.D. Stefan Goebel, M.D. Timothy Bunck, M.D. Angela Correia, M.Sc. University Medical Center, Göttingen, Germany
Paul Lingor, M.D. Klinikum Rechts der Isar, Munich, Germany
Andre Fischer, Ph.D. German Center for Neurodegenerative Diseases, Göttingen, Germany
Inga Zerr, M.D. University Medical Center, Göttingen, Germany ingazerr@med.uni-goettingen.de
Disclosure forms provided by the authors are available with the full text of this letter at NEJM.org.
snip...
Supplementary Appendix
Supplement to: Schmitz M, Silva Correia S, Hermann P, et al. Detection of prion protein seeding activity in tear fluids. N Engl J Med 2023;388:1816-7. DOI: 10.1056/NEJMc2214647
This appendix has been provided by the authors to give readers additional information about the work.
Summary
The development of in vitro misfolded protein amplification systems, such as the real-time quakinginduced conversion (RT-QuIC) assay has been a major innovation in prion disease diagnosis. It enables the detection of abnormally folded prion protein scrapie (PrPSc) in tissues (e.g. brain tissue) and body fluids, such as cerebrospinal fluid (CSF) from prion disease patients. Meanwhile, the CSF RT-QuIC has been established in routine diagnostics in prion diseases worldwide. We modified our RT-QuIC protocol by using a novel recombinant PrP substrate exhibiting a higher seeding efficiency through the introduction of the E200K PRNP mutation in the full-length human PrP sequence and tested tear fluids from prion disease patients and controls. Using tear fluid, the assay was positive in 8 out of 9 sporadic CJD patients, 3 out of 4 genetic prion disease patients, and none of 26 control individuals. The results were validated in a consecutively acquired cohort, being positive in 5 out of 6 prion disease patients and negative in all 68 controls across the full spectrum of various neurological disorders. Of interest, the test was positive in tear fluids from 6 out of 8 asymptomatic PRNP mutation carriers (HMC). Our study demonstrates that PrP seeding activity can be detected in prion disease patients using noninvasively obtained TFs. The test opens new avenues for early diagnosis and potentially for follow-up studies.
snip...
Discussion
For the development of a solid and reproducible test, there are several questions to be addressed, such as a robust substrate, optimal running conditions for test sensitivity and specificity and the best biomatrix to be studied. We hypothesized that rec PrP substrates with prion disease mutations in the RT-QuIC may facilitate the seeding conversion process of PrPC, synthesized a recombinant protein carrying the E200K mutations, and used it for further studies.
Various matrices have been discussed for biomarker assessment in CJD. CSF with its ability to reflect central nervous processes more accurately than peripheral fluids such as blood components17 is most often used for biomarker research. Invasiveness and the potential risk due to lumbar puncture present a major drawback of this method, especially concerning the need for longitudinal sample collection, as needed for research on reliable disease progression markers in clinical trials. PrP amplification assays have shown poor utility for detection of PrPSc in blood18 and urine.19 In this regard, new approaches using minimal-invasively accessible biomaterial, such as skin punch biopsy or olfactory mucosa offer great potential.6,20-22 In search for a non-invasively accessible biofluid, we decided to evaluate the potential applicability of the methodology for tear fluids for various reasons: similar to CSF, tear fluids are low in protein content, in contrast to serum or plasma, and might therefore have less preanalytical problems compared to blood or tissues. TF is very easy to collect, even less invasive than blood. The collection of tears requires no medical specialist, no advanced training and no technical equipment. The collection of tears is risk-free and can be done at different time points for follow up studies. With its close connection to the brainstem by the autonomic nerve system, it might also reflect CNS processes for some part. Tear fluids have already been used to detect relevant biomarkers for neurodegeneration like neurofilament light chain or alpha-synuclein in Parkinson’s disease.23,24
Major findings of this study are the detection of seeding activity in TF in sporadic CJD and genetic prion diseases. Although the numbers are still low in this exploratory study, we see abnormal seeding activity in a majority of patients using our modified protocol. An important observation is the detection of seeding activity in TF in healthy mutation carriers. Very limited information is available in the literature on PrP seeding activity in these individuals. Some studies have shown that the chance of detecting a CSF positivity in mutation carriers is very low when Ha 90-231 or bank vole recombinant PrP is used.25 Seeding activity has been reported in single E200K and P102L PRNP mutation carriers at presymptomatic stage in the CSF, one of whom converted shortly after but the other remained asymptomatic after several years of follow up.26 One possible interpretation is that pathological PrP seeding activity precedes clinical onset and increase of biomarkers of neurodegeneration as suggested by Mok et al.26, and potentially indicates the earliest disease stage. Detectable seeding activity may also be primarily present in individuals with a pathogenic PRNP mutation due to potential intrinsic instability of the mutated protein. Although based on very limited data, the seeding response was similar in symptomatic and non-symptomatic mutation carriers.
It is not clear yet if the detection of seeding activity in TF in prion disease has implications for public health. Because of the extremely high sensitivity in the femtomolar detection range, RT-QuIC has been applied to various peripheral tissues before.6,8,27,28 Others used sensitive genetically modified mice models and reported low levels of infectivity in various organs outside the brain.29 Although these data are clearly a matter of concern, the interpretation needs to take into account the highly artificial experimental settings, use of genetically modified animals and highly effective inoculation routes. Surveillance studies have not revealed any evidence from human to human transmission except for what is already known in iatrogenic cases and failed to identify disease clusters.13,30 Despite that, continuous vigilance and further research is needed for solid and substantiated risk estimation.
Funding
The study was funded by the Robert Koch-Institute through funds from the Federal Ministry of Health; grant no. 1369-341
Only registered and activated users can see links., Click Here To Register...
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terry