Detection of 14-3-3 protein in the CSF of genetic Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) is the most prevalent human prion disease, a group of fatal neurodegenerative illnesses that can manifest as sporadic, inherited, and infectious disorders. The clinical diagnosis of CJD is based on the evaluation of progressive dementia with pyramidal and cerebellar signs, myoclonus, and periodic EEG discharges.¹ Variable phenotypes are not rare among patients with CJD,² and since the clinical symptoms may overlap with other neurologic diseases, definite diagnosis of CJD requires a histologic examination of the affected brain tissue.³ Cases suspected to be genetic are regularly tested for the presence of known mutations in the PrP gene(PRNP), and if positive, characteristic clinical symptoms will suffice to establish a definite CJD diagnosis.³ This diagnostic approach should be regarded with caution since detection of a mutation in PRNP in the presence of confusing neurologic symptoms may result in diagnosis of mutation carriers as CJD patients while they actually suffer from a less severe and even treatable neurologic disease.

The detection of the 14-3-3 protein, a protein involved in signal transduction,⁴ in the CSF of patients with CJD, has been suggested recently as a tool for a noninvasive premortem diagnostic test for CJD.⁵ This protein was detected in the CSF of patients with sporadic CJD as well as in a few patients with genetic CJD.^6-8 Although the 14-3-3 protein was also present in the CSF of patients with additional acute neurologic conditions, it was not detected in the CSF of patients with other dementing disorders.⁵

Since a commercial antibody for the 14-3-3 is available, we tested the presence of the 14-3-3 protein in the CSF of a relatively large group of patients with genetic E200K CJD, two E200K carriers suffering from other neurologic conditions, one case of D178N CJD, as well as in the CSF of controls with assorted neurologic disorders.

Materials and methods. Patient population. The presence of 14-3-3 protein was tested in CSF samples collected from 24 patients diagnosed as having CJD and from 48 patients suffering from other neurologic diseases. Mutations in the PrP gene were determined by analyzing PrP polymerase chain reaction products.⁹

CSF specimens storage. Most of the CSF samples were of deceased patients, and were stored under assorted freezing conditions (recommended-70°C).

CSF 14-3-3 immunoassay. The immunoassay was performed under conditions described by Hsich et al.⁵ The α 14-3-3β antisera were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Statistical analysis. The 95% confidence intervals (95% CI) were calculated using the normal approximation for the binomial distribution.¹⁰

Results. The α 14-3-3 β antisera reacted with a 30-kD band in CSF samples from sporadic and genetic E200K or D178N CJD, whereas no such band was detected in CSF from patients suffering from other dementias or diverse neurologic diseases, including non-CJD E200K carriers(figure).

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Figure Immunostaining for anti-14-3-3 β polyclonal rabbit antibody following CSF separation on SDS-PAGE. Lanes 2-5: CSF samples of different patients with E200K CJD; lanes 1,9,10: CSF of patients with other dementias; lanes 6-7: patients with non-CJD E200K; lane 8: CSF of a patient with viral encephalitis.

The intensity of the 30-kD band was not necessarily the same for each patient and did not correlate with the stage of the disease. Interestingly, the intensity of the band did not differ between two CSF samples collected from the same patient at different time points before death. Whether the 14-3-3 profile changes during long intervals of the disease course remains to be established.

Table 1 summarizes the results of the 14-3-3 β assay for CJD patients and for non-CJD E200K mutation carriers. Twenty-three of the 24 CSF samples from CJD patients were positive for 14-3-3 β. Seven CSF samples were collected from patients with sporadic CJD while the remaining 17 samples were obtained from patients with genetic CJD. Of the genetic cases, 16 patients carried the E200K mutation (two of them homozygous for the mutation) and one the D178N mutation. The only CSF sample negative for the presence of the 14-3-3 protein was red, probably resulting from mixing of the CSF with hemolyzed blood cells during its collection.

The results of the 14-3-3 assay for the CSF samples obtained from patients suffering from other neurologic disorders are summarized intable 2. While most of them were negative for the 14-3-3 protein, two samples obtained from patients with cerebrovascular accident(CVA) and five samples from patients with diagnosed viral encephalitis were positive. Similar results were described previously.⁵ Interestingly, the 14-3-3 immunoassay was also positive in a patient with diagnosed amyotrophic lateral sclerosis and dementia as well as in a patient with neuropathy. Both patients died and, therefore, it is not possible to reevaluate the diagnosis provided in these cases.

The sensitivity of the 14-3-3 immunoassay as a marker for CJD was 95.8%(95% CI 76.9-99.8%) in the genetic and sporadic cases, and 93.75% (95% CI 67.7-99.7%) in genetic E200K CJD. The specificity was 94.4% in other dementias (95% CI 70.1-99.6%).

Most of the CSF samples were collected and stored within the last 8 years by physicians and technicians working in our department. For various reasons not all the samples were stored frozen at -70°C, the ideal conditions for protein stability. The fact that the storage conditions were not crucial suggests that 14-3-3 protein was very stable.

Discussion. In this study, we tested a relatively large group of E200K CJD cases for the presence of 14-3-3 in the CSF. The 14-3-3 protein was detected in the CSF of all patients with CJD (except one with irregular color). Contrary to other reports,⁸ the 14-3-3 test was also positive for one patient with D178N CJD. We also tested the presence of the 14-3-3 protein in the CSF of two neurologic patients who did not have CJD but who were carriers of the E200K mutation and found it to be negative. It is important to investigate the point in time when a carrier of a PrP mutation becomes positive for the presence of the 14-3-3 in CSF and whether this event correlates with disease outbreak. However, for ethical considerations, it is difficult to collect repeated CSF samples from healthy carriers of the E200K mutation. Tg mice, which have been developed in the last years to mimic human prion diseases, may be used to elucidate this issue.

That the 14-3-3 assay was positive in CJD cases as well as in patients with strokes or viral encephalitis but negative in Alzheimer's disease suggests that the presence of the 14-3-3 protein in the CSF is a result of an acute brain disease rather than slow chronic brain damage. Acute phases of diverse neurologic disorders may share common pathologic processes.

The high sensitivity and specificity of the 14-3-3 assay indicates this test may be reliable for premortem diagnosis of CJD and in some cases even substitute for brain histologic examination. In individuals carrying the E200K mutation, this assay may help in avoiding misdiagnosis of E200K carriers as CJD patients.