Diagnostic Utility of Cerebrospinal Fluid α -Synuclein in Creutzfeldt-Jakob Disease: A Systematic Review and Meta-Analysis

Abstract

Background:

Creutzfeldt-Jakob disease (CJD) can be difficult to distinguish clinically from some non-prion neurological diseases. Previous studies have reported markedly increased levels of α-synuclein in cerebrospinal fluid (CSF) of CJD patients, indicating that it is a potential diagnostic biomarker.

Objective:

The aim of this study was to assess the diagnostic power of CSF α-synuclein in discriminating CJD from non-prion disorders.

Methods:

The Ovid MEDLINE, Cochrane, and Embase databases were searched for articles published on or before February 25, 2022, using the search term (prion diseases OR Creutzfeldt-Jakob syndrome) AND (synuclein OR α-synuclein). The difference in CSF α-synuclein levels between CJD and non-prion diseases was calculated using random-effects models (I² > 50%) or fixed-effects models (I² < 50%) in terms of standardized mean difference (SMD) and 95% confidence interval (CI). The publication bias was estimated using funnel plots and the Egger’s test.

Results:

Ten studies were included in this study. The concentrations of CSF α-synuclein were significantly higher in CJD patients compared to total non-prion controls (SMD = 1.98, 95% CI 1.60 to 2.36, p < 0.00001), tauopathies (SMD = 1.34, 95% CI 0.99 to 1.68, p < 0.00001), synucleinopathies (SMD = 1.78, 95% CI 1.11 to 2.44, p < 0.00001), or Alzheimer’s (SMD = 1.14, 95% CI 0.95 to 1.33, p < 0.00001). CSF α-synuclein could distinguish CJD from non-prion diseases with overall sensitivity of 89% (95% CI 80–95%), specificity of 92% (95% CI 86–95%), and AUC of 0.96 (95% CI: 0.94–0.97).

Conclusion:

CSF α-synuclein has excellent diagnostic value in discriminating CJD from non-prion neurological diseases. Given the high heterogeneity among the included studies, further studies are needed to confirm its clinical utility.

Keywords

Creutzfeldt-Jakob disease CSF biomarkers meta-analysis systematic review

INTRODUCTION

Creutzfeldt-Jakob disease (CJD) is a rapidly progressive and fatal neurodegenerative disease characterized by aggregation of misfolded prion protein Scrapie (PrP^Sc) in the brain. Clinical diagnosis of CJD is often challenging due to the phenotypic overlap with other neurological diseases, especially tauopathies and synucleinopathies. Histopathological examination of brain tissues is the diagnostic gold standard but is rarely undertaken due to the little prognostic benefit and the risk of secondary iatrogenic transmission. Cerebrospinal fluid (CSF) proteins are less invasive and reproducible diagnostic markers of CJD. However, the diagnostic accuracy of 14-3-3 protein and total tau protein (t-tau) are not satisfactory [1, 2]. Thus, there is considerable interest in discovering more effective biomarkers in order to improve CJD diagnosis. Among the potential novel candidates, CSF α-synuclein has shown close to optimal accuracy [3, 4].

α-synuclein is the major component of presynaptic proteins and aberrantly expressed in synucleinopathies [5]. Some studies have shown that the loss of synapses is also one of the primary neuropathological features in prion diseases [6 –8]. Furthermore, CSF α-synuclein levels are higher in CJD patients compared to those with non-prion diseases, indicating its potential as a reliable diagnostic biomarker for CJD. Elevated CSF α-synuclein has also been associated with disease duration [9]. The underlying mechanisms were not well-characteristic, but it might be related to the severe neuronal damage. Regarding diagnostic performance, there are reports that the diagnostic accuracy of CSF α-synuclein is even higher than that of t-tau and 14-3-3 protein and shows a close to optimal accuracy in using as the first discriminatory test to select samples for second-generation prion RT-QuIC analysis [3 , 11]. However, due to differences in sample size and assay type, the clinical utility of CSF α-synuclein is ambiguous. A recent meta-analysis comprehensively evaluated the diagnostic accuracy of CSF biomarkers in patients with CJD but did not include α-synuclein [12]. In fact, no systematic review or meta-analysis has analyzed the diagnostic value of this biomarker.

The aim of this systematic review and meta-analysis was to evaluate the diagnostic performance of CSF α-synuclein in differentiating CJD from non-prion neurological diseases.

METHODS

Search strategy

The Ovid MEDLINE, Cochrane, and Embase databases were searched for articles published on or before February 25, 2022 using the following terms: (prion diseases OR Creutzfeldt-Jakob syndrome) AND (synuclein OR α-synuclein). The full list of search terms is shown in the Supplementary Material. There were no language or document type restrictions. The reference lists of all relevant original articles and reviews were manually searched for additional studies. The initial search results were uploaded to Endnote X9. Following the removal of duplicate articles, two authors independently selected potential studies based on titles and abstracts. The full-text papers were then evaluated separately according to the eligibility criteria. Any discrepancies were resolved with a third person.

Inclusion and exclusion criteria

The inclusion criteria for the studies were as follows: 1) diagnosis of sporadic CJD (sCJD) or genetic CJD (gCJD) using established criteria [13 –16], 2) comparison between CJD and non-prion diseases, 3) analysis of CSF α-synuclein levels in CJD and control subjects, and 4) conducted on human subjects.

Studies that measured CSF α-synuclein from post-mortem samples or plasma, or did not have complete α-synuclein data, or were reviews, conference abstracts or case reports were excluded. In addition, studies that included iatrogenic CJD patients were also excluded.

Data extraction

Two researchers extracted the data using a custom-designed data extraction form. Patient characteristics, sample sizes, and mean CSF α-synuclein level and standard deviations (SD) were the primary variables. In case the CSF α-synuclein data was in the form of stratified data or presented as median and inter-quartile (IQR), relevant mathematical formulas were used for conversion [17]. Other variables were author and year of publication, gender distribution, average age, diagnostic criteria, cut-off value, and the sensitivity and specificity of α-synuclein.

Quality assessment

The quality of the selected studies was assessed using the Newcastle-Ottawa Quality Assessment Scale (NOS). Three major domains were scored: 1) selection (0–4 points); 2) exposure (0–3 points), and 3) comparability (0–2 points). Higher scores indicated better methodological quality. All studies in this meta-analysis had a score of seven or above, indicating high quality. Two authors separately assessed the studies quality, and any disagreement was settled via a third person.

Data analysis

Statistical analyses were performed using Review Manager (RevMan 5.4) and Stata 16.0. Due to the variability caused by experimental assays and different laboratories, standardized mean difference (SMD) was used to compare the mean CSF α-synuclein between CJD and non-prion diseases. Heterogeneity across studies was assessed by Chi square test and Higgins I² index. Random-effect model was selected to calculate the pooled mean effect size if the heterogeneity was statistically significant (p < 0.05 and I² > 50%). Otherwise, the fixed-effect model was used. Sensitivity analyses were performed by removing the data of one study at a time to assess whether the results of the meta-analysis were significantly affected by a single study. The publishing bias was assessed using funnel plot and Egger’s test. Based on the random effects model, the overall sensitivity, specificity, positive likelihood ratio (PLR) and negative likelihood ratio (NLR), and the corresponding 95% CIs were pooled. In addition, the area under the curve (AUC) was calculated to assess the diagnostic accuracy. p-value < 0.05 was considered statistically significant.

RESULTS

A total of 1,638 studies were initially retrieved (429 from Ovid MEDLINE, 1 from Cochrane, 1,208 from Embase), of which 1,292 remained after removal of duplicates. After excluding 1,253 papers on the basis of title and abstract, the full-text of the remaining 39 studies were reviewed, and 10 articles were finally included. In addition, eight articles were included in the diagnostic analysis. The detailed flow diagram is shown in Fig. 1. The data extracted from these studies are summarized in Supplementary Table 1.

Fig. 1

Flow chart of the search and selection process.

Concentration of CSF α-synuclein in CJD patients

The data of sCJD and gCJD patients were pooled into the CJD group. The CSF α-synuclein levels were significantly higher in the CJD patients compared to the non-prion disease patients (SMD = 1.98, 95% CI 1.60 to 2.36, p < 0.00001, 10 studies, n = 992, Fig. 2A), but the heterogeneity was very high among these studies (p < 0.00001, I² = 92%). Sensitivity analysis indicated the robustness of our results. The funnel plot shown in Fig. 2B and Egger’s test did not produce a significant result (t= 1.53, p = 0.156), which meant that there was no publication bias.

Fig. 2

A) Forest plot showing the standardized mean difference (SMD) in CSF α-synuclein concentrations between CJD patients versus non-prion subjects. B) Funnel plot of estimated publication bias.

Furthermore, the data of Alzheimer’s disease (AD), frontotemporal dementia, progressive supranuclear palsy, and corticobasal degeneration patients were grouped into a tauopathies group. As shown in Fig. 3, CSF α-synuclein levels were significantly higher in the CJD versus the tauopathies group (SMD = 1.34, 95% CI 0.99 to 1.68, p < 0.00001, 6 studies, n = 566, Fig. 3), although there was considerable heterogeneity (p = 0.02, I² = 62%). The sensitivity analysis did not identify any study that significantly affected the result, which indicated the robustness of our results. Egger’s test also did not show a significant result (t= 1.38, p = 0.239), indicating there was no publication bias.

Fig. 3

Forest plot comparing mean CSF α-synuclein concentrations of CJD versus tauopathies groups.

CSF α-synuclein levels were also compared between the CJD and synucleinopathies (including Parkinson’s disease (PD) and dementia with Lewy bodies (DLB)) groups and were found to be significantly higher in the former (SMD = 1.78, 95% CI 1.11 to 2.44, p < 0.00001, 6 studies, n = 596, Fig. 4). The heterogeneity was very high (p < 0.00001, I² = 88%). Sensitivity analysis showed the robustness of our results, and Egger’s test indicated lack of publication bias (t= 1.32, p = 0.258).

Fig. 4

Forest plot comparing mean CSF α-synuclein concentrations of CJD versus synucleinopathies groups.

Finally, CJD patients had higher CSF α-synuclein compared to the AD patients as well (SMD = 1.14, 95% CI 0.95 to 1.33, p < 0.00001, 6 studies, n = 566, Fig. 5). The studies included were homogeneous (p = 0.15, I² = 38%). Sensitivity analysis showed that the result was robust, and no publication bias was detected by Egger’s test (t= 0.94, p = 0.401).

Fig. 5

Forest plot comparing mean CSF α-synuclein concentrations of CJD versus AD groups.

Concentration of CSF α-synuclein in gCJD patients

The CSF α-synuclein levels were significantly higher in the gCJD patients compared to the non-prion disease patients (SMD = 1.91, 95% CI 0.75 to 3.08, p = 0.001, Fig. 6A), tauopathies (SMD = 1.45, 95% CI 0.69 to 2.20, p < 0.0001, Fig. 6B), synucleinopathies (SMD = 1.39, 95% CI 0.59 to 2.18, p = 0.001, Fig. 6C), and AD (SMD = 1.43, 95% CI 0.64 to 2.22, p < 0.0001, Fig. 6D), but the heterogeneity was very high among these studies (p < 0.00001, I² = 97.4%; p = 0.021, I² = 81.2%; p = 0.051, I² = 73.7%; p = 0.018, I² = 82.2%, respectively).

Fig. 6

Forest plot comparing mean CSF α-synuclein concentrations of gCJD versus non-prion subjects (A), tauopathies groups (B), synucleinopathies groups (C), and AD groups (D).

Diagnostic results of included studies

The results of sensitivity, specificity and other diagnostic results are shown in Fig. 7. High CSF α-synuclein differentiated CJD from non-prion diseases with a sensitivity of 89% (95% CI: 80–95%) and specificity of 92% (95% CI: 86–95%). PLR and NLR were 10.80 (95% CI: 5.79–20.13) and 0.12 (95% CI: 0.06–0.24) respectively. The ROC curve indicated that the AUC was 0.96 (95% CI: 0.94–0.97). The heterogeneity of the data was very high. Deeks’ funnel plot asymmetry test was performed to assess the publication bias and the result showed evidence of asymmetry (p = 0.098).

Fig. 7

Coupled forest plot demonstrating (A) pooled sensitivity (left) and specificity (right), (B) positive likelihood ratio (left) and negative likelihood ratio (right). The plots display diagnostic probabilities with 95% CIs. Pooled estimates for specificity and sensitivity value and positive and negative likelihood ratio are depicted as rhombus symbols. C) Hierarchical summary receiver operating characteristics (HSROC) curve of diagnostic performance of CSF α-synuclein for differentiating CJD patients from non-prion subjects, with 95% confidence region and 95% prediction region. The confidence region consists of the most likely values of true summary sensitivity and specificity. It represents the precision with which the summary points are estimated. The prediction region predicts the true sensitivity and specificity of a future study. The size of this region reflects the variation between studies. The circles indicate estimates for each study, with size proportional to study weight.

DISCUSSION

In this study, we investigated the diagnostic power of CSF α-synuclein in discriminating between CJD and non-prion diseases. CSF α-synuclein concentration was significantly higher in the CJD group compared to the overall non-prion, tauopathies, synucleinopathies, and AD groups, and was able to differentiate CJD from non-prion diseases with high sensitivity and specificity. Thus, CSF α-synuclein is a potential useful biomarker for CJD.

CSF α-synuclein levels were consistently higher in CJD versus non-prion diseases, regardless of the type of assay used or the differences of the control cohorts, indicating its potential as a first-line differential diagnostic biomarker for CJD [3 , 18–22]. Further subgroup analysis showed that CSF α-synuclein levels in CJD were also significantly higher than those in tauopathies and synucleinopathies, which were clinically overlap with CJD [4 , 18–22]. Although CSF α-synuclein levels in AD have been reported higher than those in controls, they were still remarkably lower than those in CJD and their difference even could overcome the overlap in classical neurodegenerative biomarkers (t-tau, phospho-tau, and Aβ₄₂) [19, 23]. The underlying mechanism of increased α-synuclein level in the CSF of CJD is still unclear. Aggregated and misfolded α-synuclein species are the major constituents of Lewy bodies. Lewy bodies were the pathological hallmark of synucleinopathies, such as PD and DLB, but they were uncommon in CJD, suggesting that the pathological mechanisms of the changes of α-synuclein in CJD were different from that in synucleinopathies. The differences of CSF α-synuclein changes in these two diseases also support this viewpoint. Most studies have found that CSF levels of total α-synuclein were lower in synucleinopathies compared with controls, but it was significantly higher in CJD when compared with control and non-prion diseases [24, 25]. Studies showed that α-synuclein was particularly enriched in the presynaptic terminals [26, 27], and the loss of synapses was also one of the primary neuropathological features in prion diseases [6 –8]. Thus we hypothesized that the elevation of CSF total α-synuclein in CJD may be associated with the massive and rapid neuronal synaptic destruction caused by the accumulation of PrP^Sc [28]. The α-synuclein is released from damaged neurons into the brain’s interstitial fluid and then into the CSF. CSF α-synuclein levels correlate strongly with that of t-tau, also suggesting that the variation in their concentrations reflects the same physio-pathological alterations, probably that of neuronal damage in the brain [3 , 18]. In addition, various other factors may also affect the concentration of CSF α-synuclein, such as CJD subtypes, PRNP codon 129 and 219 polymorphisms, age, and duration of disease. Further studies need to be conducted to explore the effect of these factors. It is crucial for further elucidating the mechanism of elevation of CSF α-synuclein and improving its diagnostic value.

The diagnostic accuracy of CSF α-synuclein has been reported in multiple studies, with different results. Some studies showed that the diagnostic accuracy of CSF α-synuclein was higher than that of CSF 14-3-3 protein and t-tau [4, 11], while other studies reported that the diagnostic value was relatively lower [9, 29]. This inconsistency may be related to differences in methodological factors or patient and control cohort selection. In our study, the pooled sensitivity and specificity of CSF α-synuclein were 89% and 92% respectively, which were better than that of CSF t-tau (specificity 88% and specificity 77%) and 14-3-3 protein (specificity 87% and specificity 79%) reported previously [12], indicating that it might be an optimal single CSF biomarker. Indeed, the 14-3-3 protein is detected in various neurological disorders, such as neurodegenerative diseases, inflammatory and infectious diseases, and cerebrovascular disease, while t-tau is elevated in AD and frontotemporal dementia [30 –32]. In contrast, CSF α-synuclein is seldom increased in non-prion neurological disorders. This is the important reason that the diagnostic specificity of α-synuclein is higher than the other two markers. Furthermore, the concentration of CSF α-synuclein is negatively correlated with the survival duration of CJD patients, suggesting that CSF α-synuclein is also a potential prognostic biomarker [3, 9].

The distribution of CSF α-synuclein depends on the prion disease and CJD subtypes. For instance, patients with FFI and GSS have significantly lower CSF α-synuclein levels compared to patients with CJD, and similar to that of non-prion cases [4, 9]. Furthermore, the PRNP codon 129 polymorphism (methionine, M or valine, V) is also correlated with CSF α-synuclein concentration. Patients with the MV genotype have lower levels compared to those with the MM genotype. When combined with PrP^Sc (1 or 2) type, CSF α-synuclein is significantly higher in MM(V)1 and VV2 patients than the MV2K and MM(V)2C patients [4, 9]. We hypothesize that the concentration of CSF α-synuclein in different prion subtypes related to the degree and progression of neuronal injury. Previous studies have found that CSF α-synuclein concentrations were different depending on the kind of gCJD mutation. The levels of CSF α-synuclein in patients with E200K and V210I were comparable to those in patients with sCJD, while it was not detected in certain other gCJD mutations, such as G114V [33]. One possible explanation is that α-synuclein in CSF increased as a consequence of rapid and massive neuronal damage in the brain tissue, but in some mutation carriers the neuronal injury and disease progress might relatively slowly and α-synuclein is cleared from CSF. These differences could also explain the higher heterogeneity of studies included in gCJD subgroup analysis. In addition, aggregation and sequestration of α-synuclein in Lewy bodies could reduce the α-synuclein level in CSF, and there have been reports of pathologically prominent co-existent Lewy bodies in gCJD patients, but it was rare and only a few cases have been reported [34, 35]. Nevertheless, CSF α-synuclein still has a satisfactory performance in differentiating gCJD from non-prion diseases, tauopathies, synucleinopathies and AD, and no change in CSF α-synuclein levels was detected between sCJD and gCJD patients in our investigation. This may be related to the fact that only a few gCJD patients had co-existent Lewy bodies in the brain or only a few gCJD patients carried mutations with low CSF α-synuclein levels. Further studies should be conducted to compare the levels of CSF α-synuclein in gCJD patients with and without Lewy bodies and in gCJD patients with different mutations and explore the influence of different mutations and the presence of Lewy bodies on CSF α-synuclein sensitivity and specificity.

The synuclein can be divided into three species: normal synuclein, phosphorylated synuclein, and oligomeric synuclein. Amounting evidence has proven that oligomeric α-synuclein is major neurotoxic species [36, 37]. CSF oligomeric and phosphorylated α-synuclein have been reported as potential CSF biomarkers for PD and DLB [25, 38]. Therefore, investigating the diagnostic value of different species in patients with CJD is of paramount importance. However, most previous studies focused on CSF total α-synuclein in CJD, not taking into account its conformation or aggregation state, apart from one study in which phosphorylated α-synuclein was measured. In that study, although phosphorylated α-synuclein was significantly different between CJD and non-prion cases, the sensitivity and specificity were lower than total α-synuclein [21]. The diagnostic value of normal synuclein, phosphorylated synuclein and oligomeric synuclein for diagnosis of CJD need further study.

There are several limitations in this study. The studies included in this meta-analysis were highly heterogeneous. There was a lack of standard methodologies and assays, as well as pre-analytical parameters between laboratories, which resulted in the high heterogeneity of included studies and hampered clinical routine practice and its incorporation in diagnostic criteria. Although the absolute concentrations of CSF α-synuclein measured by different assays and kits varied, the levels of CSF α-synuclein were significantly higher in CJD in every study compared to non-prion cases and the correlations were excellent with each other [39]. In order to promote to clinical implementation of this biomarker, researchers around the world are working to standardize the assays and kits. Several studies showed that electrochemiluminescence-based technology outperformed the classical colorimetric system in terms of laboratory time and CSF samples volume and reproducibility [3 , 19]. Single-molecule array (Simoa), a new digital immunoassay technology, has recently been proven to be promising for measuring α-synuclein [9]. The consensus guidelines of pre-analytical treatment are also recommended [40]. CSF α-synuclein was stable for at least 6 freeze/thaw cycles and at least 12 months at –80°C, according to the guidelines, and samples with > 50 erythrocytes/μl should be excluded. CSF o-α-syn was more stable compared to t-α-syn in most of the preanalytical variables, particularly adsorption to tube surface and delayed storage processing to –80°C, while it declined significantly after multiple freeze-thaw cycles [41]. Other limitations included the small number of studies included and the lack of histopathological confirmation of CJD patients in many studies.

Conclusion

CSF α-synuclein levels were significantly higher in CJD patients compared to that in the overall non-prion disease, tauopathies, synucleinopathies, and AD groups, and discriminated between CJD and other neurological diseases with high sensitivity and specificity. Therefore, CSF α-synuclein could be used as an effective biomarker in the identification of CJD. However, the studies included were heterogeneous and further studies with standardized assays for CSF α-synuclein measurement, histopathological confirmation, patients with different subtypes of disease, and appropriately matched controls are needed to validate the results.

Footnotes

ACKNOWLEDGMENTS

This work was supported by grants from the Ministry of Science and Technology of China (2019YFC0118600), National Natural Science Foundation of China (81971011), Beijing Municipal Science and Technology Committee (D171100008217005, 7202060) and the Xuanwu Hospital Science Program for Fostering Yong Scholars (QNPY2021001).

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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