No association between butyrylcholinesterase K-variant and Alzheimer disease in Chinese
Abstract
Increased butyrylcholinesterase (BChE) activity has been reported to be associated with the formation of amyloid plaques and neurofibrillary tangles and may consequently be involved in the pathogenesis of Alzheimer disease (AD). Because the catalytic activity of BChE-K variant is reduced by one-third compared with non-variant, we speculated that BChE-K variant has a protective effect on AD. However, Lehmann et al. [1997] reported a synergistic effect between the genes for BChE-K variant and apolipoprotein E (ApoE) ϵ4, which increases the risk for late onset AD. In the present study, we tested 89 Chinese AD patients and 101 Chinese controls and found no evidence of association between BCHE-K and AD of either early or late onset (age > 65 years). No evidence of a synergistic effect was found between the BCHE-K variant and APOE ϵ4 in this study. Our data suggest that BChE-K variant has no modifying effect on the pathogenesis of AD. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:167–169, 2000. © 2000 Wiley-Liss, Inc.
INTRODUCTION
Whether choline contributes to the pathogenesis of dementia is under vigorous survey. Several therapeutic strategies have been devised accordingly, and controversial results have been reported [Shen, 1996; Lawrence and Sahakian, 1998]. Butyrylcholinesterase (BChE) plays little part in the overall activities of choline metabolism, however, its role in dementia has been speculated due to some biochemical evidences [Gomez-Ramos and Moran, 1997]. Several variants of BChE have been described, including the usual (U), the atypical (A), the fluoride-resistant (F), the silent (S), the James (J), the Hammersmith (H), and the Kalow (K) variants [Jensen et al., 1995; Maekawa et al., 1997], etc. The most common one is the K variant, which has a point mutation at nucleotide 1615 that leads to change of the amino acid alanine 538 to threonine (GCA→ACA). We speculated that because the catalytic activity of BChE is reduced by one-third in the K variant [Bartels et al., 1992], variant-K would possess a protective effect against Alzheimer disease (AD). However, Lehmann et al. [1997] found a synergistic effect between the genes for butyrylcholinesterase (BChE) K variant and apolipoprotein E (ApoE) ϵ4 in late-onset confirmed AD—contradictory to our speculation. In this study, an amplification-created restriction polymorphism method was used to determine whether BCHE-K variant is associated with AD in a Chinese population.
MATERIALS AND METHODS
Subjects
Eighty-nine Chinese AD patients (mean age of onset 69.1 ± 9.9 years, 42 men and 47 women) and 101 Chinese controls (all more than age 65 years to exclude possible early-onset AD and reduce the likelihood of late-onset AD, mean age 73.0 ± 4.6 years, 56 men and 45 women) were enrolled in this study. All of the AD patients were recruited from the neurological clinic of Veterans General Hospital-Taipei. AD was diagnosed according to the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer and Related Disorders Association for probable AD. Control subjects were recruited either from the hospital or the community. All control subjects were older than age 65 years and were genetically unrelated to each other and to the patients. Each control subject was given clinical, mental, and neurological examinations and a mini mental-state examination (MMSE) to rule out any insidious cognitive deficit [Mckhann et al., 1984].
Laboratory Methods
DNA from peripheral blood leukocytes was isolated from EDTA-blood by routine methods. Primers K5:5′-TTT TAC AGG AAA TAT TGC TGC A-3′ coupled with K2: 5′-ATT AGA GAC CCA CAC AAC TT-3′ were designed to create an amplification PstI restriction site after polymerase chain reaction. In contrast to Jensen et al. [1996] design, non-K variant is cut into two fragments of 112 bp and 22 bp, while K variant is resistant to PstI digestion and shows a single band of 134 bp on the ethidium-bromide stained agarose gel electrophoresis. This method was selected because it is less expansive but as effective as Jensen's. Genotype assignment of APOE was made according to a previously described method [Hong et al., 1996].
Statistical Analysis
Allele frequencies of BCHE-K variant and APOE ϵ for AD patients and control subjects were estimated by counting alleles and calculating sample proportions. Comparisons of genotype frequencies and allele frequencies were made using the chi-square test. The confidence intervals of odds ratio were calculated using the equation proposed by Miettinen. A P value < 0.05 was considered statistically significant.
RESULTS
The demographic characteristics of the subjects are shown in Table I. The allelic frequencies of BCHE-K and APOE ϵ4 are shown in Table II. No statistically significant difference in BCHE-K variant frequency was found between control group and the AD patients; when AD patients were divided into early- and late-onset subgroups, this difference was still insignificant. The odds ratio for AD patients carrying both BCHE- K and APOE ϵ4 was 2.43 (C.I. = 0.43 ∼ 13.67) in the early-onset group and 2.98 (C.I. = 0.53 ∼ 16.67) in the late-onset group. The odds ratio for AD patients carrying APOE ϵ4 and BCHE non-K was 6.09, and for BCHE-K and non-APOE ϵ4 was 1.52. No synergistic effect between BCHE-K and APOE ϵ4 in late-onset AD patients was observed as shown in Table III. The allelic frequency of BCHE-K (0.123) was similar to a previous study in a Japanese population [Maekawa et al., 1997].
| Parameters | Control group (n = 101) | AD group (n = 89) |
|---|---|---|
| Mean age | 73.0 ± 4.6 | 69.1 ± 9.9 |
| M/F | 56/45 | 42/47 |
| Age > 65 years | 101 | 69 |
| Case no. | 101 | 89 |
| Parameters | BchE-K variant allele frequency | ApoE ϵ4 allele frequency |
|---|---|---|
| Control | 0.123 | 0.069 |
| AD | 0.135 | 0.242** |
| Early onset AD | 0.14 | 0.240* |
| Late onset AD | 0.133 | 0.243** |
- * P < 0.03.
- ** P < 0.0001 (chi square with Yates' correction) versus controls.
| Subjects | Proportions of both alleles | Odds ratio | |
|---|---|---|---|
| Controls | Cases | ||
| Onset >65 years | 67/101 | 27/62 | Reference |
| Non-APOE ϵ4/non-K | |||
| Onset >65 years | 5/101 | 6/62 | 2.98 |
| APOE ϵ4/K | |||
| Onset >65 years | 9/101 | 18/62 | 4.96 |
| APOE ϵ4/non-K | |||
| Onset >75 years | 67/101 | 9/25 | Reference |
| Non-APOE ϵ4/non-K | |||
| Onset >75 years | 5/101 | 2/25 (8%) | 2.98 |
| APOE ϵ4/K | |||
| Onset >75 years | 9/101 | 8/25 (40%) | 6.61 |
| APOE ϵ4/non-K | |||
DISCUSSION
K variant is the most common variant of BChE. Its allele frequency in the present study was 0.123. The age of control group was selected to be older than age 65 years in order to exclude possible cases of early-onset AD and reduce the possibility of late-onset AD. In the K-variant of BChE catalytic activity has been shown to be reduced by one-third [Bartels et al., 1992]. Because increased BChE activity is associated with the formation of amyloid plaques and neurofibrillary tangles and may be involved in the pathogenesis of AD [Gomez-Ramos and Moran, 1997], it is reasonable to suggest that a variant of BChE with decreased activity might contribute less effect, or even provide a protective effect against AD pathogenesis. The present study found no association between BChE K-variant and patients with AD. The possibility of an ethnic difference, with the BChE- K variant playing a comparatively minor role in the pathophysiology of AD in Chinese should be considered. Another explanation for the lack of association between BChE-K and AD found in the present study may be that BChE facilitated the maturation process of amyloid plaques and neurofibrillary tangles via another pathway, i.e., the so called ‘non-classical action‘ or “non-synaptic action” [Jensen et al., 1995]. This non-classical action had been surveyed extensively in many fields, including: bioscavanger, tumorigenesis, tissue or organ development, detoxification of cocaine intoxication, and AD. If the “non-classical action” is not affected in the BChE K-variant, no association between BChE K-variant and AD will be reasonably expected. Furthermore, one BChE variant, the S variant, causes a stop codon at codon 129. No BChE activity could be detected in these cases and neither association between S variant and AD nor protective effect of S variant has been reported—it is worthy of further survey to elucidate the role of BChE in AD. The results of the present study argue against the reported association between K variant of BChE and AD, however, they do not contradict the reported biochemical evidence on the role of BChE in AD. Although, the present study failed to demonstrate a role of BChE K-variant in the development of AD, these negative findings do not rule out that other BChE mutations may be involved in the development of AD. Considering the biochemical evidence that supports the role of BChE in the pathophysiology of AD, further studies are needed to determine whether other variants of the BChE gene are associated with AD. We conclude that the BChE K-variant plays no modifying role in the pathogenesis of AD.
