Diagnostik der Zukunft: Wissen wir mit Proteomik und Genomik wirklich mehr?

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1 Diagnostik der Zukunft: Wissen wir mit Proteomik und Genomik wirklich mehr? Prof. Dr. J. Wiltfang Klinik für Psychiatrie und Psychotherapie (Direktor: Prof. Dr. J. Wiltfang), Rheinische Kliniken, Universität Duisburg-Essen

2 A. Alzheimer Amyloid cascade hypothesis of Alzheimer s Dementia extracellular soluble oligomers APP β secretase γ secretase Aβ peptides 1-40 & 1-42 amyloidogenic intermediates intracellular Tau & ptau neurodegeneration Aβ1-40 Aβ1-42 dementia MCI Amyloid fibrills neurofibrillary tangles neuoinflammation β amyloid plaques 2

3 BMB Liquor-Blut des KND Status: Liquor Blut, Baseline München LMU Erlangen Berlin Göttingen Bonn Freiburg Heidelberg Hamburg Düsseldorf Leipzig München TU International grösste, prospektive Liquor-Blut-DNA BMB für MCI & frühe Demenzen, Monitoring der Pränalytik, optimierte SOPs; Zum Vergleich: ADNI (USA): ca. 400 CSF Mannheim Homburg 3 3 Frankfurt 2 17 Gesamt

4 CSF-NDD: Predictive diagnostics of incipient AD? ApoE ε4 negative ApoE ε4 positive Total Tau (CSF, pg/ml) Aβ42/40 Ratio (CSF) Appr. 40% of the MCI patients show already the AD-indicative CSF profile and they are enriched in ApoE e4 (p<0.025) J. Wiltfang et al., Competence Net Dementias, unpublished data 4

5 CSF dementia biomarker and time to AD (Aβ1-42, total-tau, & phosho-tau181) 1.0 CSF normal: low risk for AD Probability of no AD CSF abnormal: high risk for AD Time (months) Hansson (2007) Lancet Neurology 5

6 The German CND Study (n=380) patients categorized according to the NDD data NDD status N CSF normal 153 CSF abnormal Test Mann-Whitney < p Aβ CSF abnormal CSF normal Groups analyzed Multiplexing of Aβ peptides in plasma (INNO-BIA assay) Median 25%-75% 10%-90% Wiltfang et al., oral presentation, AD conference, Washington 06/2007 6

7 Subset of the German CND Study (n=184) Group Disease group CSF criteria Clinical/Neuropsych. N MCI-AD-NDD+ MCI of AD type Aβ Ratio < 0.11 and ptau > 70 D-AD-NDD+ Early AD AβRatio < 0.11 and ptau > 70 MCI-O-NDD- D-O-NDD- MCI of non-ad type Early other dementia Aβ Ratio > 0.11 and ptau < 50 Aβ Ratio > 0.11 and ptau < 50 CDR = 0.5 and evidence of MCI-AD CDR > 0.5 and evidence of D-AD CDR = 0.5 and evidence of MCI-O CDR > 0.5 and evidence of D-O selection of patients according to clinical data supported by corresponding neurochemical dementia diagnostics (NDD) findings Wiltfang et al., oral presentation, AD conference, Washington 06/2007 7

8 Subset of the German CND Study Test Kruskal-Wallis p 0.12 Aβ Groups analyzed D-AD-NDD+ D-O-NDD- MCI-AD-NDD+ MCI-O-NDD- Median 25%-75% 10%-90% Wiltfang et al., oral presentation, AD conference, Washington 06/2007 8

9 Towards blood-based NDD?? Ray et al., Nature Medicine, 2007:published online

10 Blood-NDD: Accuracy for early and incipient AD claimed to be close to 90% Ray et al., Nature Medicine, 2007:published online

11 Summary: Neurochemical Dementia Diagnostics (CSF-NDD) CSF-NDD developed from former exclusion diagnostics to positive diagnostics of dementias Dementia biomarkers validated in independent multicenter studies: total-tau, phospho-tau, Aβ1-42 Diagnostic relevance for AD: phospho-tau > total-tau and ratio Aβ1-42/Aβ1-40 > Aβ1-42 A multiparametric diagnostic approach is superior to the use of single neurochemical dementia markers: diagnostic specificity & sensivitity for early AD 80-90%. CSF-NDD does enter international diagnostic guidelines Predictive CSF-based NDD of incipient AD To be validated: blood-based NDD Lewczuk et al., Wiltfang: Electrophoresis, 2004 Wiltfang et al.: Annals of Neurolgy, 2003 Wiltfang et al.:j. Neurochemistry, 2002 Wiltfang et al.: J. Biological Chemistriy, 2001 Itoh, Wiltfang et al.: Annals of Neurology, 2001 Otto et al., Wiltfang: Neurology, 2000 Bibl, et al., Wiltfang: Molecular Psychiatry, 2007 Bibl, et al., Wiltfang: Brain,

12 Vielen Dank für die Aufmerksamkeit 12

13 Single AβA peptides, AβA peptide ratio and total-tau Patients Alzheimer dementia (AD), n=22 non-ad dementia (nad), n=11 non-dementive mixed neuropsychiatric dieseases, i.e. disease controls (CON), n=35 CSF parameters and genotyping Aβx-42 & Aβx-40 (ELISA, The Genetics Company) ratio Aβx-42/AA 42/Aβx-4040 total-tau (ELISA, Innogenetics) ApoE-Genotypus (Innogenetics) Lewczuk P et al., Wiltfang J (2003) Neurobiology of Aging 13

14 CSF-NDD: Multiparameter Diagnostik Concentration of ttau in CSF (pg/ml ml) CON nad AD ApoE ε4+ AD ApoE ε Percentage ratio (Aβ42/40) of the Aβ peptide concentrations in CSF Lewczuk P et al., Wiltfang J (2003) Neurobiology of Aging 14

15 Comparative analysis of Receiver Operator Characteristic (ROC) for Aβx-42 and the ratio Aβx-42/AA 42/Aβx-4040 True Positive Sensitivity Aβ42 / 40 ratio Aβ Specificity False Positive The area under the curve (AUC) of the ratio (AUC=0.951) is superior to that of Aβx-42 A (AUC=0.926) Lewczuk P et al., Wiltfang J (2003) Neurobiology of Aging 15

16 Diagnostic relevance of the ratio Aβ42/40 for low- & high-aβ-load subjects CSF Aβ1-40 follows a normal distribution N=325 In low- or high-aβ-load subjects all Aβ peptide spezies 1-x are affected 60 N Aβx-40 in CSF (pg/ml) Wiltfang J et al. (2007) J Neurochem 16

17 Diagnostic relevance of the ratio Aβ42/40 for low- & high-aβ-load subjects Aß40 (pg/ml) Aß42 (pg/ml) Gesund AD Gesund AD Gesund AD Normal-Aβ-laod High-Aβ-laod Low-Aβ-laod Wiltfang J et al. (2007) J Neurochem 17

18 Diagnostic relevance of the ratio Aβ42/40 for low- & high-aβ-load subjects Aß42/Aß Gesund AD Gesund AD Gesund AD Normal-Aβ-laod High-Aβ-laod Low-Aβ-laod Wiltfang J et al. (2007) J Neurochem 18

19 Diagnostic relevance of the ratio Aβ42/40 for low- & high-aβ-load subjects CSF ptau181 (pg/ml) ) Low-Aβ-load, reduced Aβ1-42, but: normal Aβ ratio 2) High-Aβ-load, normal Aβ1-42, but: reduced Aβ ratio Median 25%-75% 10%-90% Wiltfang J et al. (2007) J Neurochem 19

20 The CSF Aβ ratio Aβ42/Aβ40 does predict incipient AD Baseline CSF analysis by urea-based Aβ-SDS-PAGE/immunoblot) of MCI (n=47) patients and contols (n=25); 4-6 years follow-up 0,24 0,22 0,20 0,18 p = p = (1) controls (n=25) (2) MCI MCI (n=25) (3) MCI AD (n=22) A 42/40 0,16 0,14 0,12 0,10 0,08 0,06 0, Mean Mean±SE Mean±SD In 19 out of 22 MCI patients (86%) incipient AD was predicted 4-6 years prior to the clinical manifestation of dementia by their baseline CSF Aβ ratio 42/40 Höglund et al., Blennow, Wiltfang: 2007, in press 20

21 Attomolar-sensitive Aβ-WIB A for blood Aβ peptides syn. Aβ syn. Aβ2-40 plasma CSF Also suprisingly stable Aβ peptide pattern in blood plasma! Lewczuk et al., Wiltfang: Electrophoresis,

22 Development of an attomolar sensitive SDS-PAGE/immunoblot for Aβ peptides Identification of the Aβ peptides by SELDI-TOF-MS, MALDI-TOF- MS, peptide synthesis & comigration 1,2 Aβ1-37 Aβ1-38 Aβ1-39 Aβ1-40 Aβ1-42 Aβ peptides Con AD 8M urea without urea The CSF Aβ peptide patterm is suprisingly stable 1 (correlation matrix of individual CSF Aβ peptides: r 1-n 0.90) Ct-truncated Aβ peptides are generated endoproteolytically 3 (studies in presenilin-1 deficient mice) 1 Wiltfang J et al., J. Neurochem., Lewczuk P, et al. Wiltfang J: Biol. Psychiatry, Wiltfang J et al., Journal of Biological Chemistry, 2001 EU Patent 22

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