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Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) S18Y polymorphism in Alzheimer's disease
© Zetterberg et al; licensee BioMed Central Ltd. 2010
- Received: 23 November 2009
- Accepted: 19 March 2010
- Published: 19 March 2010
Alzheimer's disease (AD) is characterized by protein aggregates, i.e. senile plaques and neurofibrillary tangles. The ubiquitin-proteasome system has been proposed a role in proteolytic removal of these protein aggregates. Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme with important functions in recycling of ubiquitin. The S18Y polymorphism of the UCHL1 gene confers protection against Parkinson's disease. In this study, the genotype and allele frequencies of the UCHL1 S18Y polymorphism were investigated in 452 AD patients and 234 control subjects, recruited from four memory clinics in Sweden. Using a binary logistic regression model including UCHL1 allele A and APOE ε4 allele positivity, age and sex as covariates with AD diagnosis as dependent variable, an adjusted OR of 0.82 ([95% CI 0.55-1.24], P = 0.35) was obtained for a positive UCHL1 allele A carrier status. The present study thus do not support a protective effect of the UCHL1 S18Y polymorphism against AD.
- Senile Plaque
- Binary Logistic Regression Model
- UCHL1 Gene
- S18Y Polymorphism
- Neuropathological Score
Aggregation of aberrant proteins is a hallmark of several neurodegenerative diseases, including Alzheimer's (AD), Huntington's (HD) and Parkinson's (PD) diseases. In this context, the ubiquitin-proteasome system (UPS) has been ascribed a central role in preventing the formation of pathological protein aggregates by proteolytic removal of defect proteins . Proteins destined for degradation by the UPS are labelled with a 76-amino acid peptide, ubiquitin, through a series of conjugation steps by the E1, E2 and E3 enzymes respectively. There are also two classes of de-ubiquitinating enzymes; the ubiquitin-specific processing proteases (UBPC) and the ubiquitin carboxy-terminal hydrolase family (UHC). For recent reviews on the UPS, see [2, 3]. Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is an isoform of the UHC group, expressed mainly in neurons and testis/ovary. However, UCHL1 has also been found in cells of the human diffuse neuroendocrine system and is expressed is several forms of cancer [4–6]. UCHL1 has an important role in recycling of ubiquitin through hydrolysis of peptide-ubiquitin bonds and processing of ubiquitin precursors, but it also possesses ubiquitin ligase activity . Moreover, it has been shown that UCHL1 associates with monoubiquitin and elongates its half-life, thus ensuring stability of ubiquitin within neurons .
Several genetic variants of the UCHL1 gene have been described; both those leading to gain-of-function and those resulting in loss-of-function. The I93 M mutant results in 50% reduced activity , whereas the S18Y variant exhibits increased hydrolytic activity . Other studies showed comparable hydrolase activity of the UCHL1 S18Y variant when compared to the wild type enzyme, but a reduction in ligase activity . It has also been demonstrated that the UCHL1 S18Y polymorphism has a specific ability to act as a potent antioxidant in neuronal cell culture systems .
A number of reports have demonstrated a protective effect of the S18Y polymorphism against sporadic PD in different populations [13–16], although conflicting data exist . As for AD, data on UCHL1 genotype frequencies and its effect on risk of AD is scarce and conflicting [18, 19]. The purpose of this study was to investigate the UCHL1 S18Y polymorphism in AD patients and controls in the Swedish population. Given that we, for subsets of the participants, have previously collected data on levels of CSF biochemical markers and neuropathological scores for AD, associations between the UCHL1 S18Y polymorphism and these variables could be investigated in addition to the genetic risk analysis.
The study population consisted of 452 patients with AD and 234 subjects without dementia, all of Swedish nationality. The AD patients and control subjects were recruited and diagnosed from four memory clinics in Sweden; Malmö (371 patients, 48 controls), Huddinge (52 patients, 70 controls), Göteborg/Mölndal (29 patients, 95 controls) and Linköping (21 controls). The patients were invited to participate at their first visit to the respective clinic, where they also gave their informed consent. The control subjects were recruited by advertisement in the local newspapers or at senior citizen organization meetings. A few of these controls were spouses or unrelated friends of the patients. The study was approved by the local Ethical Commissions at the respective academic center and the tenets of the Declaration of Helsinki were followed. Parts of the AD and the control groups in this work have also been included in previous studies on other polymorphisms [20, 21].
All AD patients underwent clinical examination, neuropsychological evaluations including Mini-Mental State Examinaton (MMSE), computed tomography (CT) or magnetic resonance imaging (MRI) of the brain and routine blood analyses. The healthy elderly controls were classified as non-demented on the basis of a structured interview performed by a research nurse that included information on health history, lifestyle-related variables and psychosocial situation. Only controls with an MMSE score of at least 26 were included in the study. In addition, 65 years was set as the youngest age of controls for participation in the study.
Patients were clinically diagnosed with probable late-onset AD according to the NINCDS-ADRDA criteria  by a dementia investigation team that included specialists in geriatric medicine and psychiatry. To avoid inclusion of cases with familial Alzheimer's disease (FAD) only patients 60 years of age or older were included in the study. In addition, all patients were questioned about their family history regarding AD and those with suspect heredity were excluded. Individuals with significant psychiatric or somatic diseases other than AD were excluded. Data on CSF biomarkers, including the concentrations of total-tau (T-tau), phospho-tau181 (P-tau181) and Aβ1-42 was available for 260 of the AD patients and 117 of the controls. Neuropathological diagnosis and scores based on senile plaques and neurofibrillary tangles were available for 65 of the AD patients and 80 of the controls. Genomic DNA was extracted from whole blood samples and brain tissue using standard methods. APOE (gene map locus 19q13.2) genotyping had previously been performed by minisequencing, as described in detail .
The UCHL1 (gene map locus 4p14; [Entrez gene ID: 7345]) S18Y, 54C>A polymorphism (rs5030732) was analyzed using the Dynamic Allele Specific Amplification (DASH) tehnology as described earlier . The PCR were carried out with HotStarTaq DNA Polymerase® (QIAGEN, Hilden, Germany) in a final volume of 25 μl, containing 5-20 ng of template DNA. Optimal conditions were: 1 mM MgCl2, 0.2 mM dNTPs, 0.02 U Taq polymerase, 0.16 pmol/μl of the forward biotinylated primer (5'Biotin-GCCGCCTTGTCTCCTCTCAGCAG3') and 0.78 pmol/μl of the reverse primer (5'GTCACTGGCCTGCGACCCC3'), (Invitrogen, Paisley, UK) in 1 × PCR buffer (Roche, Mannheim, Germany). The cycling profile was: 15 min at 95°C, then 39 cycles: 30 sec at 95°C, 45 sec at 60°C, 45 sec at 72°C and finally 10 min at 72°C. To identify UCHL1 alleles the probe 5'GACAGAAACGCACTTGT-Rox3' (MWG Biotech, London, United Kingdom) was used. The accuracy of the DASH method was verified by DNA sequencing of 15 individuals representing the different UCHL1 genotypes (five of each genotype).
Primary analyses compared differences between the AD patients and control subjects regarding age, sex, MMSE score, biochemical markers, neuropathological scores, genotype and allele frequencies using Fisher's exact χ2 test, t-test and Mann-Whitney U test. Secondary analysis of UCHL1 allele A-carrier status was performed with a binary logistic regression model with diagnosis (AD versus control) as dependent variable and allele positivity, age, sex and APOE ε4 allele status as independent variables. Significance was set at P < 0.05. SPSS 16.0 (SPSS Inc, Chicago, Il) was used as statistic software.
Demographic, clinical and genetic characteristics of patients with AD and controls
AD patients (n = 452)
Controls (n = 234)
Sex, f/m (%)
n = 452
n = 216
76.2 ± 6.4 (60-102)
75.9 ± 7.1 (65-94)
Mean ± SD (range)
n = 341
n = 216
MMSE score, Median (25th -- 75th percentiles)
22 (19-25) n = 367
29 (29-30) n = 113
T-tau (pg/ml), Mean ± SD
621 ± 319 n = 260
376 ± 162 n = 117
P-tau181 (pg/ml), Mean ± SD
76.9 ± 31.2 n = 260
63.3 ± 19.9 n = 91
Aβ1-42 (pg/ml), Mean ± SD
409 ± 100 n = 260
720 ± 200 n = 117
SPs and NFTs score, Median (25th -- 75thpercentiles)
7 (6-8.5) n = 65
1 (0-3) n = 80
APOE ε4 allele-carriers n (%)
number of ε4 alleles:
n = 452
n = 216
UCHL1 genotype and allele frequencies in patients with AD and controls
frequencies, n (%)
AD patients (n = 452)
Controls (n = 234)
UCHL1 allele frequencies, n (%) †
UCHL1 allele A-carrier n (%)
UCHL1 allele C-carrier n (%)
Binary logistic regression of AD diagnosis versus UCHL1 allele A- and APOE allele ε4-carrier status, age and gender.
Variable, n = 557
UCHL1, allele A positivity
APOE, allele ε4 positivity
CSF biomarkers and neuropathological score in relation to UCHL1 allele A-carrier status in AD patients and control subjects
UCHL1allele A-carrier status
T-tau (pg/ml), Mean ± SD
640 ± 368
613 ± 297
367 ± 175
381 ± 156
P-tau181 (pg/ml), Mean ± SD
80.6 ± 35.1
75.3 ± 29.4
58.8 ± 16.7
65.7 ± 21.1
Aβ1-42 (pg/ml), Mean ± SD
406 ± 91.5
410 ± 104
749 ± 195
703 ± 202
SPs and NFTs score
Median (25th -- 75th percentiles)
Aggregation of proteins is a major feature of several neurodegenerative disorders, including Alzheimer's disease (AD). Several lines of evidence suggest that the ubiquitin-proteasome system (UPS) is involved in the pathogenesis of AD . Accumulation of ubiquitin in senile plaques and neurofibrillary tangles [25–28], changes in proteasome subunit composition in AD  and an association of AD with polymorphic variants of UBQLN1, encoding for ubiquilin which is a ubiquitin-like protein , are some of the signs pointing towards a role of the UPS in AD. Of special interest is the finding that UCHL1 is oxidized in AD and that it is down-regulated in affected brain areas of AD patients [31, 32]. Also interesting is the finding that exogenous UCHL1 ameliorated β-amyloid-induced synaptic and memory dysfunction in an AD mouse model .
The S18Y polymorphism of the UCHL1 gene is associated with lower incidence of Parkinson's disease [13–16]. The mechanism for this protective effect is not known, but it may be at least partially explained by the increased antioxidative capacity demonstrated in neuronal cells expressing the UCHL1 S18Y variant . Little is known about its effect on AD prevalence. A Chinese study has demonstrated lower frequencies of the A allele and the AA genotype in female AD patients as compared to female controls . However, a genetic study in a Colombian population could not find an association between UCHL1 genotypes and AD . The number of genome-wide association studies in the AD field has increased rapidly; none of these has reported the UCHL1 S18Y polymorphism among the significant SNPs found however [34, 35].
The allele and genotype frequencies of the UCHL1 S18Y polymorphism seen in this study are in accordance with previous results in Swedish populations . The present study do not support a role of the UCHL1 S18Y polymorphism AD however.
This work was supported by grants from the Swedish Research Council, the Royal Swedish Academy of Sciences, the Sahlgrenska University Hospital, the Göteborg Medical Society, Swedish Brain Power, Hjalmar Svenssons forskningsfond, Stiftelsen för Gamla Tjänarinnor and Alzheimerfonden.
- Oddo S: The ubiquitin-proteasome system in Alzheimer's disease. J Cell Mol Med. 2008, 12: 363-373. 10.1111/j.1582-4934.2008.00276.x.PubMedPubMed CentralView ArticleGoogle Scholar
- Petroski MD: The ubiquitin system, disease, and drug discovery. BMC Biochem. 2008, 9 (Suppl 1): S7-10.1186/1471-2091-9-S1-S7.PubMedPubMed CentralView ArticleGoogle Scholar
- Tai HC, Schuman EM: Ubiquitin, the proteasome and protein degradation in neuronal function and dysfunction. Nat Rev Neurosci. 2008, 9: 826-838. 10.1038/nrn2499.PubMedView ArticleGoogle Scholar
- Dhillon AP, Rode J, Dhillon DP, Moss E, Thompson RJ, Spiro SG, Corrin B: Neural markers in carcinoma of the lung. Br J Cancer. 1985, 51: 645-652.PubMedPubMed CentralView ArticleGoogle Scholar
- Otsuki T, Yata K, Takata-Tomokuni A, Hyodoh F, Miura Y, Sakaguchi H, Hatayama T, Hatada S, Tsujioka T, Sato Y, Murakami H, Sadahira Y, Sugihara T: Expression of protein gene product 9.5 (PGP9.5)/ubiquitin-C-terminal hydrolase 1 (UCHL-1) in human myeloma cells. Br J Haematol. 2004, 127: 292-298. 10.1111/j.1365-2141.2004.05205.x.PubMedView ArticleGoogle Scholar
- Thompson RJ, Doran JF, Jackson P, Dhillon AP, Rode J: PGP 9.5--a new marker for vertebrate neurons and neuroendocrine cells. Brain Res. 1983, 278: 224-228. 10.1016/0006-8993(83)90241-X.PubMedView ArticleGoogle Scholar
- Healy DG, Abou-Sleiman PM, Wood NW: Genetic causes of Parkinson's disease: UCHL-1. Cell Tissue Res. 2004, 318: 189-194. 10.1007/s00441-004-0917-3.PubMedView ArticleGoogle Scholar
- Osaka H, Wang YL, Takada K, Takizawa S, Setsuie R, Li H, Sato Y, Nishikawa K, Sun YJ, Sakurai M, Harada T, Hara Y, Kimura I, Chiba S, Namikawa K, Kiyama H, Noda M, Aoki S, Wada K: Ubiquitin carboxy-terminal hydrolase L1 binds to and stabilizes monoubiquitin in neuron. Hum Mol Genet. 2003, 12: 1945-1958. 10.1093/hmg/ddg211.PubMedView ArticleGoogle Scholar
- Leroy E, Boyer R, Auburger G, Leube B, Ulm G, Mezey E, Harta G, Brownstein MJ, Jonnalagada S, Chernova T, Dehejia A, Lavedan C, Gasser T, Steinbach PJ, Wilkinson KD, Polymeropoulos MH: The ubiquitin pathway in Parkinson's disease. Nature. 1998, 395: 451-452. 10.1038/26652.PubMedView ArticleGoogle Scholar
- Setsuie R, Wada K: The functions of UCH-L1 and its relation to neurodegenerative diseases. Neurochem Int. 2007, 51: 105-111. 10.1016/j.neuint.2007.05.007.PubMedView ArticleGoogle Scholar
- Liu Y, Fallon L, Lashuel HA, Liu Z, Lansbury PT: The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson's disease susceptibility. Cell. 2002, 111: 209-218. 10.1016/S0092-8674(02)01012-7.PubMedView ArticleGoogle Scholar
- Kyratzi E, Pavlaki M, Stefanis L: The S18Y polymorphic variant of UCH-L1 confers an antioxidant function to neuronal cells. Hum Mol Genet. 2008, 17: 2160-2171. 10.1093/hmg/ddn115.PubMedView ArticleGoogle Scholar
- Carmine Belin A, Westerlund M, Bergman O, Nissbrandt H, Lind C, Sydow O, Galter D: S18Y in ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) associated with decreased risk of Parkinson's disease in Sweden. Parkinsonism Relat Disord. 2007, 13: 295-298. 10.1016/j.parkreldis.2006.12.002.PubMedView ArticleGoogle Scholar
- Elbaz A, Levecque C, Clavel J, Vidal JS, Richard F, Correze JR, Delemotte B, Amouyel P, Alperovitch A, Chartier-Harlin MC, Tzourio C: S18Y polymorphism in the UCH-L1 gene and Parkinson's disease: evidence for an age-dependent relationship. Mov Disord. 2003, 18: 130-137. 10.1002/mds.10326.PubMedView ArticleGoogle Scholar
- Maraganore DM, Farrer MJ, Hardy JA, Lincoln SJ, McDonnell SK, Rocca WA: Case-control study of the ubiquitin carboxy-terminal hydrolase L1 gene in Parkinson's disease. Neurology. 1999, 53: 1858-1860.PubMedView ArticleGoogle Scholar
- Maraganore DM, Lesnick TG, Elbaz A, Chartier-Harlin MC, Gasser T, Kruger R, Hattori N, Mellick GD, Quattrone A, Satoh J, Toda T, Wang J, Ioannidis JP, de Andrade M, Rocca WA: UCHL1 is a Parkinson's disease susceptibility gene. Ann Neurol. 2004, 55: 512-521. 10.1002/ana.20017.PubMedView ArticleGoogle Scholar
- Hutter CM, Samii A, Factor SA, Nutt JG, Higgins DS, Bird TD, Griffith A, Roberts JW, Leis BC, Montimurro JS, Kay DM, Edwards KL, Payami H, Zabetian CP: Lack of evidence for an association between UCHL1 S18Y and Parkinson's disease. Eur J Neurol. 2008, 15: 134-139.PubMedPubMed CentralView ArticleGoogle Scholar
- Forero DA, Benitez B, Arboleda G, Yunis JJ, Pardo R, Arboleda H: Analysis of functional polymorphisms in three synaptic plasticity-related genes (BDNF, COMT AND UCHL1) in Alzheimer's disease in Colombia. Neurosci Res. 2006, 55: 334-341. 10.1016/j.neures.2006.04.006.PubMedView ArticleGoogle Scholar
- Xue S, Jia J: Genetic association between Ubiquitin Carboxy-terminal Hydrolase-L1 gene S18Y polymorphism and sporadic Alzheimer's disease in a Chinese Han population. Brain Res. 2006, 1087: 28-32. 10.1016/j.brainres.2006.02.121.PubMedView ArticleGoogle Scholar
- Andersson ME, Sjolander A, Andreasen N, Minthon L, Hansson O, Bogdanovic N, Jern C, Jood K, Wallin A, Blennow K, Zetterberg H: Kinesin gene variability may affect tau phosphorylation in early Alzheimer's disease. Int J Mol Med. 2007, 20: 233-239.PubMedGoogle Scholar
- Zetterberg M, Landgren S, Andersson ME, Palmer MS, Gustafson DR, Skoog I, Minthon L, Thelle DS, Wallin A, Bogdanovic N, Andreasen N, Blennow K, Zetterberg H: Association of complement factor H Y402H gene polymorphism with Alzheimer's disease. Am J Med Genet B Neuropsychiatr Genet. 2007, 147B (6): 720-6.View ArticleGoogle Scholar
- McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM: Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology. 1984, 34: 939-944.PubMedView ArticleGoogle Scholar
- Blennow K, Ricksten A, Prince JA, Brookes AJ, Emahazion T, Wasslavik C, Bogdanovic N, Andreasen N, Batsman S, Marcusson J, Nagga K, Wallin A, Regland B, Olofsson H, Hesse C, Davidsson P, Minthon L, Jansson A, Palmqvist L, Rymo L: No association between the alpha2-macroglobulin (A2 M) deletion and Alzheimer's disease, and no change in A2 M mRNA, protein, or protein expression. J Neural Transm. 2000, 107: 1065-1079. 10.1007/s007020070052.PubMedView ArticleGoogle Scholar
- Prince JA, Feuk L, Howell WM, Jobs M, Emahazion T, Blennow K, Brookes AJ: Robust and accurate single nucleotide polymorphism genotyping by dynamic allele-specific hybridization (DASH): design criteria and assay validation. Genome Res. 2001, 11: 152-162. 10.1101/gr.150201.PubMedPubMed CentralView ArticleGoogle Scholar
- Ii K, Ito H, Tanaka K, Hirano A: Immunocytochemical co-localization of the proteasome in ubiquitinated structures in neurodegenerative diseases and the elderly. J Neuropathol Exp Neurol. 1997, 56: 125-131. 10.1097/00005072-199702000-00002.PubMedView ArticleGoogle Scholar
- Mori H, Kondo J, Ihara Y: Ubiquitin is a component of paired helical filaments in Alzheimer's disease. Science. 1987, 235: 1641-1644. 10.1126/science.3029875.PubMedView ArticleGoogle Scholar
- Perry G, Friedman R, Shaw G, Chau V: Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer disease brains. Proc Natl Acad Sci USA. 1987, 84: 3033-3036. 10.1073/pnas.84.9.3033.PubMedPubMed CentralView ArticleGoogle Scholar
- Tabaton M, Cammarata S, Mancardi G, Manetto V, Autilio-Gambetti L, Perry G, Gambetti P: Ultrastructural localization of beta-amyloid, tau, and ubiquitin epitopes in extracellular neurofibrillary tangles. Proc Natl Acad Sci USA. 1991, 88: 2098-2102. 10.1073/pnas.88.6.2098.PubMedPubMed CentralView ArticleGoogle Scholar
- Mishto M, Bellavista E, Santoro A, Stolzing A, Ligorio C, Nacmias B, Spazzafumo L, Chiappelli M, Licastro F, Sorbi S, Pession A, Ohm T, Grune T, Franceschi C: Immunoproteasome and LMP2 polymorphism in aged and Alzheimer's disease brains. Neurobiol Aging. 2006, 27: 54-66. 10.1016/j.neurobiolaging.2004.12.004.PubMedView ArticleGoogle Scholar
- Bertram L, Hiltunen M, Parkinson M, Ingelsson M, Lange C, Ramasamy K, Mullin K, Menon R, Sampson AJ, Hsiao MY, Elliott KJ, Velicelebi G, Moscarillo T, Hyman BT, Wagner SL, Becker KD, Blacker D, Tanzi RE: Family-based association between Alzheimer's disease and variants in UBQLN1. N Engl J Med. 2005, 352: 884-894. 10.1056/NEJMoa042765.PubMedView ArticleGoogle Scholar
- Castegna A, Aksenov M, Aksenova M, Thongboonkerd V, Klein JB, Pierce WM, Booze R, Markesbery WR, Butterfield DA: Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part I: creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1. Free Radic Biol Med. 2002, 33: 562-571. 10.1016/S0891-5849(02)00914-0.PubMedView ArticleGoogle Scholar
- Pasinetti GM: Use of cDNA microarray in the search for molecular markers involved in the onset of Alzheimer's disease dementia. J Neurosci Res. 2001, 65: 471-476. 10.1002/jnr.1176.PubMedView ArticleGoogle Scholar
- Gong B, Cao Z, Zheng P, Vitolo OV, Liu S, Staniszewski A, Moolman D, Zhang H, Shelanski M, Arancio O: Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induced decreases in synaptic function and contextual memory. Cell. 2006, 126: 775-788. 10.1016/j.cell.2006.06.046.PubMedView ArticleGoogle Scholar
- Beecham GW, Martin ER, Li YJ, Slifer MA, Gilbert JR, Haines JL, Pericak-Vance MA: Genome-wide association study implicates a chromosome 12 risk locus for late-onset Alzheimer disease. Am J Hum Genet. 2009, 84: 35-43. 10.1016/j.ajhg.2008.12.008.PubMedPubMed CentralView ArticleGoogle Scholar
- Harold D, Abraham R, Hollingworth P, Sims R, Gerrish A, Hamshere ML, Pahwa JS, Moskvina V, Dowzell K, Williams A, Jones N, Thomas C, Stretton A, Morgan AR, Lovestone S, Powell J, Proitsi P, Lupton MK, Brayne C, Rubinsztein DC, Gill M, Lawlor B, Lynch A, Morgan K, Brown KS, Passmore PA, Craig D, McGuinness B, Todd S, Holmes C, et al: Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer's disease. Nat Genet. 2009, 41: 1088-1093. 10.1038/ng.440.PubMedPubMed CentralView ArticleGoogle Scholar
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