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CSF α-synuclein does not differentiate between parkinsonian disorders
Neurobiology of Aging 33 (2012) 430.e1– 430.e3 www.elsevier.com/locate/neuaging
Negative results
CSF ␣-synuclein does not differentiate between parkinsonian disorders M.B. Aertsa, R.A.J. Esselinka, W.F. Abdoa,b, B.R. Bloema, M.M. Verbeeka,c,* a
Department of Neurology, Parkinson Center Nijmegen (ParC), and Alzheimer Centre Nijmegen, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Centre, the Netherlands b Department of Intensive Care, Parkinson Center Nijmegen (ParC), and Alzheimer Centre Nijmegen, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Centre, the Netherlands c Department of Laboratory Medicine, Parkinson Center Nijmegen (ParC), and Alzheimer Centre Nijmegen, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Centre, the Netherlands Received 26 April, 2010; received in revised form 23 September 2010; accepted 2 December 2010
Abstract Differentiating between Parkinson’s disease (PD) and atypical Parkinsonism (AP) is clinically relevant but challenging. A timely and correct diagnosis might result in better targeted treatment strategies, adequate patient counseling, and early recognition of disease-specific complications. We aimed to investigate whether cerebrospinal fluid (CSF) concentrations of ␣-synuclein are of additional diagnostic value. We examined 142 consecutive patients with parkinsonism, mean disease duration 39.7 mo (Parkinson’s disease (PD), n ⫽ 58; MSA, n ⫽ 47; dementia with Lewy bodies (DLB), n ⫽ 3; VaP, n ⫽ 22; progressive supranuclear palsy (PSP), n ⫽ 10; CBD, n ⫽ 2). Gold standard was the clinical diagnosis established after 2 years of clinical follow-up. CSF concentrations of ␣-synuclein, blood pigments and the erythrocyte count were determined. No differences between CSF ␣-synuclein concentrations of patients with PD with the reference values from our laboratory were observed. We neither found significant differences between patients with PD and AP nor between AP subgroups. Adjustment for age, disease severity or presence of erythrocytes or blood pigments in CSF did not alter these results. Our results imply that CSF ␣-synuclein is currently unsuitable as biomarker to differentiate between PD and AP. © 2012 Elsevier Inc. All rights reserved.
1. Introduction The differentiation between idiopathic PD and atypical Parkinsonism (AP) is important but can be challenging. A timely and correct diagnosis might result in better targeted treatment strategies, adequate patient counseling and—perhaps most importantly— early recognition of disease-specific complications. It would be helpful to identify new biomarkers that facilitate the differential diagnosis in patients with early stages of parkinsonism. Interestingly, a recent study showed markedly lower levels of ␣-synuclein in the cerebrospinal fluid (CSF) of patients with PD as compared with controls (Hong et al., 2010). However, the potential use of ␣-synuclein as a biomarker for the differentiation between different forms of parkinsonism has not yet
* Corresponding author. Tel.: 0031 24 3615192; fax: 0031 24 3668754. E-mail address: [email protected]. (M.M. Verbeek). 0197-4580/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2010.12.001
been studied rigorously. Here, we aimed to investigate whether CSF concentrations of ␣-synuclein might facilitate this diagnostic challenge. 2. Methods We recruited 142 consecutive outpatients with a hypokinetic-rigid syndrome and an uncertain clinical diagnosis between January 2003 and December 2006. All patients received a lumbar puncture and a cerebral MRI scan (see Supplementary methods). CSF concentrations of ␣-synuclein and blood pigments and the total erythrocyte count were determined (see Supplementary methods). The method of ␣-synuclein analysis and its validation has been published previously (van Geel et al., 2008). Prior to data analysis, data were log-transformed to obtain a normal distribution for ␣-synuclein.
VaP (n ⫽ 22)
(p (p (p (p NS NS NS NS 25.0 (18.0–42.0) Normalb Normalc Normald 30.5 (19.5–38.0) 2.5 (0.5–23.5) 0.003 (0.01) 0.014 (0.05)
PSP/CBD (n ⫽ 12)
Controls (n ⫽ 57)
p-valuea
Fifty-eight patients had a final clinical diagnosis of PD, 47 of multisystem atrophy (MSA) (25 possible, 20 probable and 2 definite MSA), 3 of DLB (2 possible, 1 probable DLB), 22 of vascular parkinsonism (VaP), 10 of PSP (6 possible, 3 probable and 1 definite PSP) and 2 of corticobasal degeneration (CBD). Clinical and demographical data of the subjects are reported in Table 1 of the Supplementary Materials. We observed no significant differences in CSF ␣-synuclein concentrations between patients with PD and our reference values. We neither found differences in the ␣-synuclein concentrations between the PD and AP groups, nor between the different subgroups of AP (Table 1). Furthermore, we observed no differences in ␣-synuclein concentration when the group of ␣-synucleinopathies (PD, MSA and DLB) was compared with the group of tauopathies (PSP and CBD). Additional adjustments for age, disease severity or the presence of erythrocytes or blood pigments in the CSF did not change these results. Analyzing patients with only probable or definite diagnosis, and/or analyzing only patients who completed the entire follow up duration did not change our results. More details of the analyses are presented in the Supplementary results.
⫽ ⫽ ⫽ ⫽
0.79) 0.50) 0.29) 0.55)
3. Results
24.0 (23.0–34.0) 21 (0–23) 0 (0) 0 (0) 25.0 (17.0–32.0) 3 (0–62) 0.046 (0.10) 0.029 (0.07) 26.0 (20.5–32.5) 1 (0–16) 0.017 (0.05) 0.031 (0.13)
␣-synuclein (ng/mL) Total erythrocyte count (no./L) Hemoglobin (mol/L) Bilirubin (mol/L)
MSA (n ⫽ 47) PD (n ⫽ 58) CSF parameters
Table 1 CSF parameters by diagnostic group
Our study is the first to assess the value of CSF ␣-synuclein concentrations in differentiating between PD and AP. Despite the large sample-size, we found no differences in ␣-synuclein concentrations between different patient groups. Furthermore, we could not replicate the previously reported decrease in CSF ␣-synuclein concentration in patients with PD as compared with controls (Hong et al., 2010). These findings suggest that ␣-synuclein has no value as biomarker for the differential diagnosis of parkinsonism. The comparability previous research investigating CSF ␣-synuclein levels is hindered by different assays used for the detection of ␣-synuclein and differences in demographic data of included patients en controls. Consequently, the data on CSF ␣-synuclein concentrations in previous studies have been conflicting. Furthermore, if any, the observed differences were small, (Hong et al., 2010; Mollenhauer et al., 2008; Tokuda et al., 2006) with profound overlap between the different patients groups. In our specialized movement disorder center, referral and selection bias may have prompted the inclusion of an atypical patient population which may have led to more diagnostic uncertainty. Furthermore, the clinical diagnosis was confirmed neuropathologically in only 3 cases, and therefore susceptible to misclassification. However, our gold standard diagnosis was based on careful clinical assessment at baseline and after 2 y of clinical follow-up, including response to treatment and baseline cerebral MRI in all patients.
DLB (n ⫽ 3)
4. Discussion
Data represent mean and standard deviation, or median and p25–p75 range in case of non-normally distributed data. PD, idiopathic Parkinson’s disease; MSA, multiple system atrophy; DLB, dementia with Lewy bodies; VaP, vascular Parkinsonism; PSP, progressive supranuclear palsy; CBD, corticobasal degeneration; NS, non significant. a p-value for differences using 1-way ANOVA or the Kruskal–Wallis test to identify between-group differences. b Normal ⱕ 50/L; c Normal ⱕ 0.25 mol/L; d Normal ⱕ 0.50 mol/L.
M.B. Aerts et al. / Neurobiology of Aging 33 (2012) 430.e1– 430.e3
24.0 (16.0–30.5) 3.5 (0–38) 0.032 (0.06) 0.024 (0.06)
430.e2
M.B. Aerts et al. / Neurobiology of Aging 33 (2012) 430.e1– 430.e3
In conclusion, despite the aforementioned drawbacks, our results imply that CSF ␣-synuclein is currently not advocated as a diagnostic biomarker for the differentiation between PD and AP. Appendix. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neurobiolaging. 2010.12.001. References Hong, Z., Shi, M., Chung, K.A., Quinn, J.F., Peskind, E.R., Galasko, D., Jankovic, J., Zabetian, C.P., Leverenz, J.B., Baird, G., Montine, T.J., Hancock, A.M., Hwang, H., Pan, C., Bradner, J., Kang, U.J., Jensen,
430.e3
P.H., Zhang, J., 2010. DJ-1 and alpha-synuclein in human cerebrospinal fluid as biomarkers of Parkinson. Brain 133, 713–726. Mollenhauer, B., Cullen, V., Kahn, I., Krastins, B., Outeiro, T.F., Pepivani, I., Ng, J., Schulz-Schaeffer, W., Kretzschmar, H.A., McLean, P.J., Trenkwalder, C., Sarracino, D.A., Vonsattel, J.P., Locascio, J.J., ElAgnaf, O.M., Schlossmacher, M.G., 2008. Direct quantification of CSF alpha-synuclein by ELISA and first cross-sectional study in patients with neurodegeneration. Exp. Neurol. 213, 315–325. Tokuda, T., Salem, S.A., Allsop, D., Mizuno, T., Nakagawa, M., Qureshi, M.M., Locascio, J.J., Schlossmacher, M.G., El-Agnaf, O.M., 2006. Decreased alpha-synuclein in cerebrospinal fluid of aged individuals and subjects with Parkinson’s disease. Biochem. Biophys. Res. Commun. 349, 162–166. van Geel, W.J., Abdo, W.F., Melis, R., Williams, S., Bloem, B.R., Verbeek, M.M., 2008. A more efficient enzyme-linked immunosorbent assay for measurement of alpha-synuclein in cerebrospinal fluid. J. Neurosci. Methods 168, 182–185.
Negative results
CSF ␣-synuclein does not differentiate between parkinsonian disorders M.B. Aertsa, R.A.J. Esselinka, W.F. Abdoa,b, B.R. Bloema, M.M. Verbeeka,c,* a
Department of Neurology, Parkinson Center Nijmegen (ParC), and Alzheimer Centre Nijmegen, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Centre, the Netherlands b Department of Intensive Care, Parkinson Center Nijmegen (ParC), and Alzheimer Centre Nijmegen, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Centre, the Netherlands c Department of Laboratory Medicine, Parkinson Center Nijmegen (ParC), and Alzheimer Centre Nijmegen, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Centre, the Netherlands Received 26 April, 2010; received in revised form 23 September 2010; accepted 2 December 2010
Abstract Differentiating between Parkinson’s disease (PD) and atypical Parkinsonism (AP) is clinically relevant but challenging. A timely and correct diagnosis might result in better targeted treatment strategies, adequate patient counseling, and early recognition of disease-specific complications. We aimed to investigate whether cerebrospinal fluid (CSF) concentrations of ␣-synuclein are of additional diagnostic value. We examined 142 consecutive patients with parkinsonism, mean disease duration 39.7 mo (Parkinson’s disease (PD), n ⫽ 58; MSA, n ⫽ 47; dementia with Lewy bodies (DLB), n ⫽ 3; VaP, n ⫽ 22; progressive supranuclear palsy (PSP), n ⫽ 10; CBD, n ⫽ 2). Gold standard was the clinical diagnosis established after 2 years of clinical follow-up. CSF concentrations of ␣-synuclein, blood pigments and the erythrocyte count were determined. No differences between CSF ␣-synuclein concentrations of patients with PD with the reference values from our laboratory were observed. We neither found significant differences between patients with PD and AP nor between AP subgroups. Adjustment for age, disease severity or presence of erythrocytes or blood pigments in CSF did not alter these results. Our results imply that CSF ␣-synuclein is currently unsuitable as biomarker to differentiate between PD and AP. © 2012 Elsevier Inc. All rights reserved.
1. Introduction The differentiation between idiopathic PD and atypical Parkinsonism (AP) is important but can be challenging. A timely and correct diagnosis might result in better targeted treatment strategies, adequate patient counseling and—perhaps most importantly— early recognition of disease-specific complications. It would be helpful to identify new biomarkers that facilitate the differential diagnosis in patients with early stages of parkinsonism. Interestingly, a recent study showed markedly lower levels of ␣-synuclein in the cerebrospinal fluid (CSF) of patients with PD as compared with controls (Hong et al., 2010). However, the potential use of ␣-synuclein as a biomarker for the differentiation between different forms of parkinsonism has not yet
* Corresponding author. Tel.: 0031 24 3615192; fax: 0031 24 3668754. E-mail address: [email protected]. (M.M. Verbeek). 0197-4580/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2010.12.001
been studied rigorously. Here, we aimed to investigate whether CSF concentrations of ␣-synuclein might facilitate this diagnostic challenge. 2. Methods We recruited 142 consecutive outpatients with a hypokinetic-rigid syndrome and an uncertain clinical diagnosis between January 2003 and December 2006. All patients received a lumbar puncture and a cerebral MRI scan (see Supplementary methods). CSF concentrations of ␣-synuclein and blood pigments and the total erythrocyte count were determined (see Supplementary methods). The method of ␣-synuclein analysis and its validation has been published previously (van Geel et al., 2008). Prior to data analysis, data were log-transformed to obtain a normal distribution for ␣-synuclein.
VaP (n ⫽ 22)
(p (p (p (p NS NS NS NS 25.0 (18.0–42.0) Normalb Normalc Normald 30.5 (19.5–38.0) 2.5 (0.5–23.5) 0.003 (0.01) 0.014 (0.05)
PSP/CBD (n ⫽ 12)
Controls (n ⫽ 57)
p-valuea
Fifty-eight patients had a final clinical diagnosis of PD, 47 of multisystem atrophy (MSA) (25 possible, 20 probable and 2 definite MSA), 3 of DLB (2 possible, 1 probable DLB), 22 of vascular parkinsonism (VaP), 10 of PSP (6 possible, 3 probable and 1 definite PSP) and 2 of corticobasal degeneration (CBD). Clinical and demographical data of the subjects are reported in Table 1 of the Supplementary Materials. We observed no significant differences in CSF ␣-synuclein concentrations between patients with PD and our reference values. We neither found differences in the ␣-synuclein concentrations between the PD and AP groups, nor between the different subgroups of AP (Table 1). Furthermore, we observed no differences in ␣-synuclein concentration when the group of ␣-synucleinopathies (PD, MSA and DLB) was compared with the group of tauopathies (PSP and CBD). Additional adjustments for age, disease severity or the presence of erythrocytes or blood pigments in the CSF did not change these results. Analyzing patients with only probable or definite diagnosis, and/or analyzing only patients who completed the entire follow up duration did not change our results. More details of the analyses are presented in the Supplementary results.
⫽ ⫽ ⫽ ⫽
0.79) 0.50) 0.29) 0.55)
3. Results
24.0 (23.0–34.0) 21 (0–23) 0 (0) 0 (0) 25.0 (17.0–32.0) 3 (0–62) 0.046 (0.10) 0.029 (0.07) 26.0 (20.5–32.5) 1 (0–16) 0.017 (0.05) 0.031 (0.13)
␣-synuclein (ng/mL) Total erythrocyte count (no./L) Hemoglobin (mol/L) Bilirubin (mol/L)
MSA (n ⫽ 47) PD (n ⫽ 58) CSF parameters
Table 1 CSF parameters by diagnostic group
Our study is the first to assess the value of CSF ␣-synuclein concentrations in differentiating between PD and AP. Despite the large sample-size, we found no differences in ␣-synuclein concentrations between different patient groups. Furthermore, we could not replicate the previously reported decrease in CSF ␣-synuclein concentration in patients with PD as compared with controls (Hong et al., 2010). These findings suggest that ␣-synuclein has no value as biomarker for the differential diagnosis of parkinsonism. The comparability previous research investigating CSF ␣-synuclein levels is hindered by different assays used for the detection of ␣-synuclein and differences in demographic data of included patients en controls. Consequently, the data on CSF ␣-synuclein concentrations in previous studies have been conflicting. Furthermore, if any, the observed differences were small, (Hong et al., 2010; Mollenhauer et al., 2008; Tokuda et al., 2006) with profound overlap between the different patients groups. In our specialized movement disorder center, referral and selection bias may have prompted the inclusion of an atypical patient population which may have led to more diagnostic uncertainty. Furthermore, the clinical diagnosis was confirmed neuropathologically in only 3 cases, and therefore susceptible to misclassification. However, our gold standard diagnosis was based on careful clinical assessment at baseline and after 2 y of clinical follow-up, including response to treatment and baseline cerebral MRI in all patients.
DLB (n ⫽ 3)
4. Discussion
Data represent mean and standard deviation, or median and p25–p75 range in case of non-normally distributed data. PD, idiopathic Parkinson’s disease; MSA, multiple system atrophy; DLB, dementia with Lewy bodies; VaP, vascular Parkinsonism; PSP, progressive supranuclear palsy; CBD, corticobasal degeneration; NS, non significant. a p-value for differences using 1-way ANOVA or the Kruskal–Wallis test to identify between-group differences. b Normal ⱕ 50/L; c Normal ⱕ 0.25 mol/L; d Normal ⱕ 0.50 mol/L.
M.B. Aerts et al. / Neurobiology of Aging 33 (2012) 430.e1– 430.e3
24.0 (16.0–30.5) 3.5 (0–38) 0.032 (0.06) 0.024 (0.06)
430.e2
M.B. Aerts et al. / Neurobiology of Aging 33 (2012) 430.e1– 430.e3
In conclusion, despite the aforementioned drawbacks, our results imply that CSF ␣-synuclein is currently not advocated as a diagnostic biomarker for the differentiation between PD and AP. Appendix. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neurobiolaging. 2010.12.001. References Hong, Z., Shi, M., Chung, K.A., Quinn, J.F., Peskind, E.R., Galasko, D., Jankovic, J., Zabetian, C.P., Leverenz, J.B., Baird, G., Montine, T.J., Hancock, A.M., Hwang, H., Pan, C., Bradner, J., Kang, U.J., Jensen,
430.e3
P.H., Zhang, J., 2010. DJ-1 and alpha-synuclein in human cerebrospinal fluid as biomarkers of Parkinson. Brain 133, 713–726. Mollenhauer, B., Cullen, V., Kahn, I., Krastins, B., Outeiro, T.F., Pepivani, I., Ng, J., Schulz-Schaeffer, W., Kretzschmar, H.A., McLean, P.J., Trenkwalder, C., Sarracino, D.A., Vonsattel, J.P., Locascio, J.J., ElAgnaf, O.M., Schlossmacher, M.G., 2008. Direct quantification of CSF alpha-synuclein by ELISA and first cross-sectional study in patients with neurodegeneration. Exp. Neurol. 213, 315–325. Tokuda, T., Salem, S.A., Allsop, D., Mizuno, T., Nakagawa, M., Qureshi, M.M., Locascio, J.J., Schlossmacher, M.G., El-Agnaf, O.M., 2006. Decreased alpha-synuclein in cerebrospinal fluid of aged individuals and subjects with Parkinson’s disease. Biochem. Biophys. Res. Commun. 349, 162–166. van Geel, W.J., Abdo, W.F., Melis, R., Williams, S., Bloem, B.R., Verbeek, M.M., 2008. A more efficient enzyme-linked immunosorbent assay for measurement of alpha-synuclein in cerebrospinal fluid. J. Neurosci. Methods 168, 182–185.