Analysis of association between butyrylcholinesterase K variant and apolipoprotein E genotypes in Alzheimer's disease

Neuroscience Letters 371 (2004) 142–146

Analysis of association between butyrylcholinesterase K variant and apolipoprotein E genotypes in Alzheimer’s disease Asad Vaisi Raygania,b,∗ , Mahine Zahraia , Akbar Soltanzadehc , Mahmood Doostia , Ebrahim Javadia , Tayebeh Pourmotabbedd a

b

Department of Medical Biochemistry, Tehran University of Medical Sciences, Tehran, Iran Department of Medical Biochemistry, Kermanshah University of Medical Sciences, Kermanshah, Iran c Department of Neurology, Tehran University of Medical Sciences, Tehran, Iran d Department of Molecular Sciences, Health Science Center, University of Tennessee, USA Received 3 July 2004; received in revised form 23 August 2004; accepted 24 August 2004

Abstract Recent studies indicate that there is a synergic association between butyrylcholinesterase-K variant (BChE-K) and apolipoproteinE-␧4 (ApoE-␧4) to promote risk for Alzheimer’s disease (AD). Most subsequently replicative studies have been unable to confirm these finding. We attempted to replicate this finding in 105 AD cases and age and sex matched 129 controls from Tehran population, Iran. The BChE genotype of patients were found to be significantly different from controls (χ2 = 12.2, d.f. = 2, p = 0.002). The frequency of BChE-K allele was also found to differ significantly in cases compared to controls [24% versus 12% (χ2 = 20.6, d.f. = 2, p < 0.001)] leading to an increased risk of AD in subjects with this allele (OR = 2.5, 95% CI = 1.64–3.8, p = 0.001). This risk was found to increase from (OR = 2.37, 95% CI = 1.3–4.2, p = 0.006) in subjects less than 75 years old to (OR = 3.16, 95% CI = 1.41–7.1, p = 0.001) in subjects 75 years and older. But, the ApoE-␧4 allele association risk was found to decrease from (OR = 9.5, 95% CI = 3.74–24.1, p = 0.001) in subjects <75 years to (OR = 1.36, 95% CI = 0.49–4.1, p = 0.58) in those subjects 75 years and older. Furthermore, we found a very strong synergic association between BChE-K and ApoE-␧4 OR = 19.1 (95% CI = 428–85.45, p < 0.001). In spite of this, synergism decreased from OR = 36.2 (95% CI = 4.4–296, p = 0.001) in subjects <75 year olds to OR = 6.2 (95% CI = 0.9–72.4, p = 0.06) in subjects ≥75 years. We have found that BChE-K and ApoE-␧4 alleles act synergistically to increase the risk of the late-onset AD, particularly in age group <75 years in Tehran, Iran. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; Butyrylcholinesterase K; Variants; Apolipoprotein E; Iranian; Aging; Genetic polymorphism; Tehran population

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder leading to dementia. Although the exact molecular mechanisms leading to AD are not fully understood, the ␧4 allele of apolipoprotein E (ApoE-␧4) has consistently been shown to be associated with non-familial late onset of AD. However, the possession of ApoE-␧4 allele is neither necessary nor sufficient for the disease initiation/progression [36]. ∗

Corresponding author. Tel.: +98 21 6112739; fax: +98 21 8953004. E-mail address: [email protected] (A.V. Raygani).

0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.08.057

This has prompted a search for other genetic factors that may confer susceptibility to AD. One of the candidates identified to date is the K variant of butyrylcholinesterase gene (BChEK) [25–27]. BChE is a hydrolytic enzyme expressed in most human tissues including regions of hippocampus and amygdala [7,11,30]. Although the exact physiological function of BChE is unknown [12], it has been reported that BChE is associated with AD pathology and its expression is substantially increased in AD brains [12,32,34]. BChE is also associated with the neurofibrillay tangles and amyloid plaques

A.V. Raygani et al. / Neuroscience Letters 371 (2004) 142–146

[13]. There are at least seven different genetic variation of BChE, among which the K allele is the most common one and it accounts for 12–15% allele frequency in the Caucasian population [3,18]. In this allele, the G at position 1615 in the BChE gene has been mutated to A resulting in Ala-539 to Thr change in the protein molecule. This mutation reduced the activity of the enzyme by 30%. Despite the fact that inhibitors of cholinesterases including BChE, and acetylcholinesterase are routinely used for treatment of AD [1,8,14], no direct association between the BChE-K and AD has been found to date and the subject is still controversial. As mentioned above, apo-lipoprotein ␧4 allele (ApoE-␧4) is also implicated as a risk factor associated with AD. Association studies with ApoE-␧4 allele, BChE-K gene, and AD have revealed conflicting results. A few studies have demonstrated a positive association between ApoE-␧4 allele, BChEK gene, and the disease [10,25,26,30,31,33,37,40], whereas others have shown no link between the presence of these genes and the disease [2,4,5,6,16,20–22,23,24,35]. It is possible that this discrepancy is due to differences in frequency of BChE-K variant in different ethnic population. In this study we assessed the relationship between BChE-K and ApoE-␧4 allele with AD in Tehran’s population. Contrary to the previous reports, we have found that BChE-K and ApoE-␧4 alleles act synergistically to increase the risk of the late-onset AD, particularly in age group <75 years, in a genetically homogenous population in Tehran, Iran. The target population for the case–control analysis consisted of 105 AD cases (60 females and 45 males with a mean age of 75 ± 10 years) with no reported family history of AD or dementia, and 129 unrelated controls (45 males with a mean ago of 73.5 ± 11.5 years). Ethical approval for this study was obtained from the Research Ethics committee, Tehran University of Medical Sciences. Informed written consent was obtained from each subject or from his/her legal guardian before collecting blood samples. Cases were ascertained, diagnosed, and certified by neurospecialists from Shariati hospital and Rozbeh neurology and psychiatry center at Tehran University of Medical Sciences, using the NINCDS-ADRDA clinical diagnostic criteria ([28], DSM-IV). A CT scan or MRI was performed to aid diagnosis. A patient with a family history of dementia (greater than one first-degree relative with any form of dementia) was excluded from the analysis. Volunteer control group consisted of matched age/sex and ethnic background subjects. They were selected based on medical history and physical examination. The controls exhibited no signs of dementia and were spouses of clinically ascertained AD or dementia patients. Subjects’ cognitive function was assessed using Mini Mental State Examination (MMSE) [9]. DNA was extracted from peripheral blood leukocytes [15,19]. BChE gene was detected by polymerase chain reaction (PCR) method using mismatch oligonucleotide primers, forward 5 -ATATTT TAC AGG AAATAT TGA TGT A-3 and reverse 5 -ATTAGAGACCCACACAACTT-3 as previously

143

Table 1 Overall distribution of BChE genotypes Cases

WT/WT WT/K K/K

Controls

No

%

No

%

61 37 7

58.1 35.2 6.7

99 29 1

76.7 22.5 0.8

(χ2 = 12.2, d.f. = 2, p = 0.002). WT/WT: wild type of BChE genotype.

described [18]. The PCR products were digested with Mae III restriction endonuclease and subjected to electrophoresis on a 12% polyacrylamide gel. The gels were stained with ethidium bromide and photographed. The BChE-K allele was identified as a 115 bp PCR product. The presence of ApoE gene was identified by PCR-RFLP, as described [38]. The SPSS statistical software package version 11.5 was used for the statistical analyses. Genotype and allele frequencies of BChE and ApoE were compared between AD cases and controls using χ2 -test. Odds ratios (OR) as estimates of relative risk for disease were calculated and 95% confidence intervals obtained by SPSS logistic regression. The interaction between the BChE-K and ApoE-␧4 allele was determined using logistic regression model. Two-tailed Student’s t-test was also used to compare quantitative data. Statistical significance was assumed at the p < 0.05 level. Distribution of the BChE genotype was found to differ significantly in AD subjects compared to control (χ2 = 12.2, d.f. = 2, p = 0.002) (Table 1). The distribution of BChE genotypes in AD subjects less than 75 years old (χ2 = 6.5, d.f. = 2, p = 0.046), and greater than 75 years old (χ2 = 8.3, d.f. = 2, p = 0.016) differed significantly when compared with the control groups. Whereas the genotype distribution of BChE in controls did not differ between these age groups (χ2 = 3.2, d.f. = 2, p = 0.3) (Table 2). Table 3 shows the distribution of the ApoE genotypes in subjects (AD and control) according to age. The distribution of the ApoE genotype in AD was found to differ significantly from that of the control group (χ2 = 19.8, d.f. = 3, p < 0.001). The difference in the distribution of ApoE genotypes was significant only in the AD subjects with less than 75 years old (χ2 = 26.9, d.f. = 3, p < 0.001). But, in the controls the two age group difference was not significant (χ2 = 0.06, d.f. = 3, p = 0.8). Table 2 Distribution of BChE genotypes in subjects aged between <75 years and ≥75 years old <75 years∗ Case

WT/WT WT/K K/K a ∗ ∗∗

≥75 years∗∗ Control

Case

Control

No

%

No

%

No

%

No

%

30 15 4

61.2 30.6 8.2

48 17 –a

73.8 26.2 –a

31 22 3

55.4 39.3 5.4

51 12 1

79.7 18.8 1.6

–: empty cells. (χ2 = 6.15, d.f. = 2, p = 0.046). (χ2 = 8.3, d.f. = 2, p = 0.016).

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A.V. Raygani et al. / Neuroscience Letters 371 (2004) 142–146

Table 3 Distribution of ApoE genotypes in subjects aged between <75 years and ≥75 years old <75 years∗

≥75 years∗∗

Case

␧2/␧3 ␧3/␧3 ␧/␧4 ␧4/␧4 a ∗ ∗∗ ∗∗∗

Control

All ages∗∗∗

Case

Control

Case

Control

No

%

No

%

No

%

No

%

No

%

No

%

–a

–a

21 20 8

42.9 40.8 16.3

1 56 7 1

1.5 86.2 10.8 1.5

2 46 8 –a

3.6 82.1 14.3 –

6 51 7 –a

9.4 79.7 10.9 –

2 67 28 8

1.9 63.8 26.7 7.6

7 107 14 1

5.4 82.9 10.9 0.8

–: empty cell. (χ2 = 26.9, d.f. = 3, p < 0.001). (χ2 = 1.8, d.f. = 3, p = 0.4). (χ2 = 19.8, d.f. = 3, p < 0.001).

Furthermore, The BChE-K allele frequency was found to be significantly different in AD cases when compared to controls; 24.3% versus 12%, respectively (χ2 = 20.6, d.f. = 2, p < 0.001). Computation of the OR as an estimate of relative risk for AD showed that subjects with the BChE-K variant allele were 2.5 (95% CI = 1.64–3.8, p < 0.001) times more likely to suffer from AD. OR increased from 2.37 (95% CI = 1.3–4.27, p = 0.006) to 2.64 (95% CI = 1.45–4.8, p = 0.002) in those subjects with the age greater than 74 years old. Furthermore, The BChE-K allele frequency was found to be significantly different in AD cases when compared to controls; 24.3% versus 12%, respectively (χ2 = 20.6, d.f. = 2, p < 0.001). Computation of the OR as an estimate of relative risk for AD showed that subjects with the BChE-K variant allele were 2.5 (95% CI = 1.64–3.8, p < 0.001) times more likely to suffer from AD. Division of the AD and control groups into two age groups showed that OR associated with the BCHE-K variant increased from 2.37 (95% CI = 1.3–4.27, p = 0.006) in subjects aged less than 74 years to 2.64 (95% CI = 1.45–4.8, p = 0.002) in those subjects with the age greater than 74 years old. We also used logistic regression to test any interaction between ApoE-␧4 and BChE-K in AD subjects. Using Lehmann et al. studies [27], four categories defined by the presence (+) or absence (−) of an ␧4 or K allele, logistic regression analysis (Table 4) demonstrated a strong and significant interaction between BChE-K and ApoE-␧4 in AD (χ2 = 24.66, d.f. = 1, p < 0.001), specially those with less than 75 years old (χ2 = 21.2, d.f. = 1, p < 0.001). The ApoE-␧4 and BChE-K variant increased risk of AD by 2.36 (95% CI = 1–5.3, p = 0.04) and by 1.64 (95% CI = 0.85–3.2, p = 0.13) times, respectively (Table 4). Logistic regression showed significant association with ApoE-␧4 (OR = 2.36, 95% CI = 1–5.3, p = 0.04) while using the same method, showed, no significant

association with BChE-K (OR = 1.64, 95% CI = 0.85–3.2, p = 0.13). The odds ratio for possession of both risk factor BChE-K and ApoE ␧4 was found to be 19.1 (95% CI = 4.3–85.3, p < 0.001). The odds ratio for possession of both risk factors BChE-K and ApoE-␧4 according to the age of the subjects are presented in Tables 5 and 6. The OR associated with the BChE-K variant in the subjects less than 75 years old was 0.6 (95% CI = 0.18–2, p = 0.4) and it increased to 2.7 (95% CI = 1.1–6.3, p = 0.02) for the subjects with greater than 75 years old. Whereas, the ApoE-␧4 associated OR decreased from 4.8 (95% CI = 1.5–13.5, p = 0.006) in the younger group to 0.8 (95% CI = 0.19–3.5, p = 0.7) in the older group. The OR for possession of both risk factors, ApoE ␧4 and BChE-K, was 36.2 (95% CI = 4.4–296, p < 0.001) in the under 75 years age groups to 8 (95% CI = 0.9–72.4, p = 0.057) in those subjects of 75 years and older. These data strongly suggest that BChE-K is associated with a further increase in the risk of late-onset AD in ApoE-␧4 carriers from Tehran, Iran (χ2 = 24.66, d.f. = 1, p < 0.001), particularly in age group less than 75 years (χ2 = 21.2, d.f. = 1, p < 0.001). BChE-K and ApoE-␧4 are genetic factors that have been implicated in non-familial late onset of AD. In this study, we examined the relationship between these two AD risk factors and found that contrary to previous reports [2,4,5,6,16,20–21,23,24,35], BChE-K is a significant risk factor for late onset AD. It acts in synergy with ApoE-␧4 to promote risk for AD in Tehran’s population, particularly in subjects aged less than 75 years. This conclusion is based on the observation that distribution of BChE genotypes and the frequency of BChE-K allele in AD subjects aged 75 years and older were significantly different from control groups in Tehran’s population (χ2 = 8.3, d.f. = 2, p = 0.016).

Table 4 Carrier odds ratios in the all ages groups BChE-K

ApoE-␧4

Case

Controls

OR (95% CI)

− + − +

− − + +

45 24 16 20

86 28 13 2

Reference 1.64 (0.85–3.2, p = 0.13) 2.36 (1–5.3, p = 0.04) 19.1 (4.3–85.5, p < 0.001)

Reference (χ2 = 2.2, d.f. = 1, p = 0.13) (χ2 = 4.4, d.f. = 1, p = 0.037) (χ2 = 24.66, d.f. = 1, p < 0.001)

A.V. Raygani et al. / Neuroscience Letters 371 (2004) 142–146

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Table 5 Carrier odds ratios in the <75 years subgroup BChE-K

ApoE-␧4

Case

Controls

OR (95% CI)

− + − +

− − + +

17 16 13 15

41 4 7 1

Reference 0.6 (0.18–2, p = 0.4) 4.8 (1.5–13.2, p = 0.006) 36.2 (4.4–296, p < 0.001)

Reference (χ2 = 0.65, d.f. = 1, p = 0.4) (χ2 = 8, d.f. = 1, p = 0.005) (χ2 = 21.2, d.f. = 1, p < 0.001)

Table 6 Carrier odds ratios in the 75 years and older BChE-K

ApoE-␧4

Case

Controls

OR (95% CI)

− + − +

− − + +

16 20 3 5

45 12 6 1

Reference 2.7 (1.1–6.3, p = 0.02) 0.8 (0.19–3.5, p = 0.7) 8 (0.9–72.4, p = 0.06)

In addition, a strong synergy was found between BChEK and ApoE-␧4 allele in AD subjects from Tehran (χ2 = 24.66, d.f. = 1, p < 0.001) and this synergistic effect decreased with increasing age. The BChE-K/ApoE-␧4 associated risk was found to decrease from 36.2 (95% CI = 4.4–29.6, p < 0.001) in subjects with less than 75 year olds to 8 (95% CI = 0.9–72.4, p = 0.06) in those subjects 75 years and older. This data is in accordance with the results obtained for several other ethnic groups around the world [10,25–27,29,31,33,37]. However, several groups have not been able to find an association between the BChE-K, ApoE␧4, and the onset of AD in the populations that they were investigating [5,6,16,19–22,23,24,35]. The discrepancy observed between our data and the other investigators could be due to a lower life expectancy in Iranian population compared to that of the other nations. The controversial results among different studies may also reflect different distribution of BChE-K variant in different ethnic populations, and/or a distinct linkage of BChE-K with a nearby AD susceptibility gene, yet to be identified, in different population [17,26,39]. In conclusion we report that BChE-K variant is a significant risk factor for late onset of AD (ages >75 years), while ApoE-␧4 is consider a significant risk factor for AD subjects aged less than 75 years in the Tehran’s population. In addition, we were able to demonstrate that BChE-K synergistically increases the risk of AD in carriers of ApoE-␧4 allele particularly in age group <75 years in Tehran, Iran

Acknowledgement

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

This work was supported by grant no. 14563 from Tehran University, Medical Sciences, Tehran, Iran. [11] [12]

References [1] O. Almkvist, T. Darreh-Shori, E. Stefanova, R. Spiegel, A. Nordberg, Preserved cognitive function after 12 months of treatment with

[13]

Reference (χ2 = 5.2, d.f. = 1, p = 0.02) (χ2 = 0.9, d.f. = 1, p = 0.8) (χ2 = 4.6, d.f. = 1, p = 0.03)

rivastigmine in mild Alzheimer’s disease in comparison with untreated AD and MCI patients, Eur. J. Neurol. 11 (4) (2004) 253– 261. A. Alvarez-Arcaya, O. Combarros, J. Llorca, M. Sanchez-Guerra, J. Berciano, C. Fernandez-Viadero, N. Pena, The butyrylcholinesterase K variant is a protective factor for sporadic Alzheimer’s disease in women, Acta Neurol. Scand. 102 (6) (2000) 350– 353. C.F. Barters, F.S. Jensen, O. Lockridge, DNA mutation associated with the human BChE K-variant and its linkage to the atypical variant mutation and other polymorph sites, Am. J. Hum. Genet. 50 (1992) 1086–1130. S. Bi, Y. Zhang, J. Wu, D. Wang, Q. Zhao, Association between low-density lipoprotein receptor-related protein gene, butyrylcholinesterase gene and Alzheimer’ s disease in Chinese, Chin. Med. Sci. J. 16 (2) (2001) 71–75. N. Brindle, Y. Song, E. Rogaeva, S. Premkumar, G. Levesque, G. Yu, M. Ikeda, M. Nishimura, A. Paterson, S. Sorbi, R. Duara, L. Farrer, P. St George-Hyslop, Analysis of the butyrylcholinesterase gene and nearby chromosome 3 markers in Alzheimer disease, Hum. Mol. Genet. 7 (5) (1998) 933–935. F. Crawford, D. Fallin, Z. Suo, L. Abdullah, M. Gold, A. Gauntlett, R. Duara, M. Mullan, The butyrylcholinesterase gene is neither independently nor synergistically associated with late-onset AD in clinicand community-based populations, Neurosci. Lett. (1998) 115– 118. S. Dervish, D.L. Grantham, D.A. Hopkins, Distribution of butyrylcholinesterase in the human amygdale and hippocampal formation, J. Comp. Neural. 393 (1998) 374–390. T. Erkinjuntti, I. Skoog, R. Lane, C. Andrews, Potential long-term effects of rivastigmine on disease progression may be linked to drug effects on vascular changes in Alzheimer brains, Int. J. Clin. Pract. Nov. 57 (9) (2003) 756–760. M.F. Folstein, S.E. Folstein, P.R. McHugh, Mini-Mental state. A practical method for grading the cognitive state of patients for the clinician, J. Psychiatry Res. 12 (1975) 189– 198. E. Ghebremedhin, D.R. Thal, C. Schultz, H. Braak, Age-dependent association between butyrylcholinesterase K-variant and Alzheimer disease-related neuropathology in human brains, Neurosci. Lett. 1 (320) (2002) 25–28. E. Giacobini, Cholinergic function and Alzheimer’s disease, Int. J. Geriatr. Psychiatry 18 (Suppl. 1) (2003) 51–55. E. Giacobini, P.l. Griffini, T. Maggi, Butyrylcholinesterase. Is it important for cortical acetylcholine regulation, Neurotics Abstr. 22 (1996) 203. P. Gomez-Rooms, C. Bourse, Ultrastructural localization of butyrylcholinesterase on neurofibrillay degeneration sites in the brains of

146

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

A.V. Raygani et al. / Neuroscience Letters 371 (2004) 142–146 aged and Alzheimer’s disease patients, Brain Res. 640 (1994) 17– 34. N.H. Greig, D.K. Lahiri, K. Sambamurti, Butyrylcholinesterase: an important new target in Alzheimer’s disease therapy, Int. Psychogeriatr. 14 (2002) 77–91. M. Gross-Bellard, A rapid procedure for extracting genomic DNA from leukocytes by phenol chloroform, Eur. J. Biochem. 36 (1973) 32–36. J.M. Grubber, A.M. Saunders, A.R. Crane-Gatherum, W.K. Scott, E.R. Martin, C.S. Haynes, P.M. Conneally, G.W. Small, A.D. Roses, J.L. Haines, M.A. Pericak-Vance, Analysis of association between Alzheimer disease and the K variant of butyrylcholinesterase (BCHE-K), Neurosci. Lett. 9 (269) (1999) 115–119. J. Hosseini, F. Firuzian, J. Feely, Ethnic differences in the frequency distribution of serum cholinesterase activity, Ir. J. Med. Sci. 100 (1997) 10–20. F.S. Jenese, L.R. Nielsen, M. Schwartz, Detection of the plasma cholinesterase K variant by PCR using amplification-created restriction site, Hum. Heard. 46 (1996) 26–31. S.W.M. Johan, G. Weitzner, R. Rozen, A rapid procedure for extracting genomic DNA from leukocytes, Nucleic Acids Res. 19 (1991) 408. P. Kehoe, H. Williams, P. Holmans, G. Wilcock, N.J. Cairns, J.M. Neal, M.J. Owen, The BChE-K variant and susceptibility to Alzheimer’s disease, J. Med. Gene (1998) 35. C.S. Ki, D.L. Na, J.W. Kim, H.J. Kim, D.K. Kim, B.K. Yoon, No association between the genes for butyrylcholinesterase K variant and apolipoprotein E4 in late-onset Alzheimer’s disease, Am. J. Med. Genet. 16;88 (2) (1999) 113–115. K.W. Kim, J.H. Jhoo, J.H. Lee, K.U. Lee, D.Y. Lee, J.C. Youn, J.Y. Youn, J.I. Woo, Neither the butyrylcholinesterase K variant nor transferrin C2 variant confers a risk for Alzheimer’s disease in Koreans, J. Neural. 108 (10) (2001) 1159–1166. S.M. Laws, B. Taddeik, C. Fisher, D. Small, R. Charnette, J. Hallmayers, W.S. Brooks, J.B.J. Kwok, P.R. Schofield, S.E. Gandy, R.N. Martins, Evidence that the butyrylcholinesterase K variant can protect against late-onset Alzheimer’s disease, Alzheimer Rep. 2 (4) (1999) 210–223. D.W. Lee, H.C. Liu, T.Y. Liu, C.W. Chi, C.J. Hong, No association between butyrylcholinesterase K-variant and Alzheimer disease in Chinese, Am. J. Med. Genet. 3 (96) (2000) 167–170. D.J. Lehmann, C. Johnston, A.D. Smith, Synergy between the genes for butyrylcholinesterase K variant and Apolipoprotein E4 in late onset confirmed Alzheimer’s disease, Hum. Mol. Genet. 6 (1997) 1933–1936. D.J. Lehmann, Z. Nagy, Z. Litchfield, C.B. Borja, A.D. Smith, Association of butyrylcholinesterase K variant with cholinesterase positive neuritis plaques in the temporal cortex in late-onset Alzheimer’s disease, Hum. Genet. 106 (2000) 447–452.

[27] D.J. Lehmann, J. Williams, J. Mcbroom, A.D. Smith, Using metaanalysis to explain the diversity of results in genetic studies of lateonset Alzheimer’s disease and to identify high-risk subgroups, Neuroscience 108 (4) (2001) 541–554. [28] G. Mckhann, D. Drachman, M. Folstein, R. Katzman, D. Price, E.M. Stadlan, Clinical diagnosis of Alzheimer’s disease: report of NINCDS-ADRDA work group under the auspices of the Department of Health and Human Services task force of Alzheimer’s disease, Neurology 34 (1984) 939–944. [29] S. Mcllory, P. Crawford, V.L.A. Dynan, K.B. McGleenon, B.M. Vahidarrs, D.M. Lawson, J.T. Passmore, A.P. Butyrylcholinesterase, K variant is genetically associated with late onset Alzheimer’s disease in Northern Ireland, J. Med. Genet. 37 (2000) 182–185. [30] M. Mesulam, A. Guillozet, P. Shaw, B. Quinn, Widely spread butyrylcholinesterase can hydrolyze acetylcholine in the normal and Alzheimer brain, Neurobiol. Dis. 9 (1) (2002) 88–93. [31] P.K. Panegyres, C.D. Mamotte, S.D. Vasikaran, S. Wilton, V. Fabian, B.A. Kakulas, Butyrycholinesterase K variant and Alzheimer’s disease, J. Neurol. 246 (5) (1999) 369–370. [32] E.K. Perry, R.H. Perry, G. Blessed, Changes in brain cholinesterase in senile dementia of Alzheimer type, Neuropathol. Appl. Neurobiol. 4 (1978) 273–277. [33] C. Russ, J. Powell, S. Lovestone, C. Holmes, K variant of butyrylcholinesterase and late-onset Alzheimer’s disease, Lancet 21;351 (9106) (1998) 881. [34] J. Saez-Valero, L.R. Fodero, M. Sjogren, N. Andreasen, S. Amici, V. Gallai, H. Vanderstichele, E. Vanmechelen, L. Parnetti, K. Blennow, D.H. Small, Glycosylation of acetylcholinesterase and butyrylcholinesterase changes as a function of the duration of Alzheimer’s disease, J. Neurosci. Res. 1572 (4) (2003) 520–526. [35] A.B. Singleton, G. Smith, A.M. Gibson, R. Woodward, R.H. Perry, P.G. Ince, J.A. Edwardson, C.M. Morris, No association between the K variant of the butyrylcholinesterase gene and pathologically confirmed Alzheimer’s disease, Hum. Mol. Genet. 7 (5) (1998) 937–939. [36] W.J. Strittmattet, A.D. Roses, Apolipoprotein E and Alzheimer disease, Proc. Natl. Acad. Sic. U.S.A. 92 (47) (1995) 25–27. [37] L. Tilley, K. Morgan, J. Grainger, P. Marsters, L. Morgan, J. Lowe, J. Xuereb, C. Wischik, C. Harrington, N. Kalsheker, Evaluation of polymorphisms in the presenilin-1 gene and the butyrylcholinesterase gene as risk factors in sporadic Alzheimer’s disease, Eur. J. Hum. Genet. 7 (6) (1999) 659–666. [38] P.R. Wenham, W.H. Price, G. Blandell, Apolipoprotein E genotyping by one-stage PCR, Lancet 337 (1991) 1158–1159. [39] M. Whittaker, Cholinesterase, in: L. Beckman (Ed.), Monographs in Human Genetics, vol. 11, Karger, Basel, 1986, pp. 1–125. [40] H. Wiebusch, J. Poirier, P. Sevigny, K. Schappert, Further evidence for a synergistic association between ApoE epsilon4 and BChEK in confirmed Alzheimer’s disease, Hum. Genet. 104 (2) (1999) 158–163.