Association of Butyrylcholinesterase-K Allele and Apolipoprotein E ɛ 4 Allele with Cognitive Decline in Dementia with Lewy Bodies and Alzheimer’s Disease

Abstract

Background:

A common polymorphism of the butyrylcholinesterase gene, the K-variant (BCHE-K) is associated with reduced butyrylcholinesterase (BuChE) activity. Insufficient studies exist regarding the frequency and role of BCHE-K in dementias.

Objective:

To determine the association of BCHE-K and APOE ɛ4 with diagnosis and rate of cognitive decline in dementia with Lewy bodies (DLB) and Alzheimer’s disease (AD) patients.

Methods:

Genomic DNA from 368 subjects (108 AD, 174 DLB, and 86 controls) from two routine clinical cohort studies in Norway; DemVest and TrønderBrain, were genotyped for BCHE-K and APOE ɛ4. The mild dementia DemVest subjects received annual Mini-Mental State Examination assessments for five years.

Results:

BCHE-K frequency was lower in DLB (33.9% ; p < 0.01) than in control subjects (51.2%), and was numerically lower in AD as well (38.9% ; p = 0.11). More rapid cognitive decline was associated with the APOE ɛ4 genotype, but not with the BCHE-K genotype. In an exploratory analysis of patients who completed all five follow-up visits, there was greater cognitive decline in BCHE-K carriers in the presence of the APOE ɛ4 allele than in the absence of these polymorphisms.

Conclusion:

BCHE-K is associated with a reduced risk for AD and DLB whereas APOE ɛ4 is associated with more rapid cognitive decline. The greater cognitive decline in individuals with both APOE ɛ4 and BCHE-K alleles require prospective confirmation in well-controlled trials.

Keywords

Apolipoprotein E genotype butyrylcholinesterase-K cognition dementia Mini-Mental State Examination

INTRODUCTION

Cholinergic disturbances play an important role in several types of dementia, including Alzheimer’sdisease (AD) and dementia with Lewy bodies (DLB) [1]. The cholinergic enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) catalyze the hydrolysis of the neurotransmitter, acetylcholine (ACh) [2]. Synaptic membrane-bound AChE is more important in regulating cholinergic neurotransmission, while BuChE plays a more important role in the control of extracellular ACh levels that modulate the functional properties of glial cells [3 –5]. In the cerebrospinal fluid (CSF) of patients with mild AD, BuChE activity is associated with CSF markers of astroglial reactivity and function such as glial fibrillary acidic protein (GFAP) and S100B [3]. Higher BuChE activity is associated with higher levels of these astroglial reactivity markers, with lower levels of proinflammatory cytokines and higher complement factor levels [3].

The butyrylcholinesterase gene (BCHE) is located in the long q-arm of chromosome 3 [6, 7] and over 40 mutations have been identified. The BCHE-K variant is the most common polymorphism of BCHE, which is resulted from substitution of an alanine residue at codon 539 to threonine (A539T). Other mutations of BCHE gene includes the atypical, J, H, and silent variants. Studies have shown that the distribution and catalytic properties of this enzyme in humans depend on the type of BCHE variant present [8 –10]. For instance, BCHE-K produces a BuChE protein variant with approximately 30% lower enzymatic activity compared to the wild type [11].

The activity of BuChE in CSF is reduced in APOE ɛ4 carriers with mild AD [12]. This reduction in APOE ɛ4 carriers is more marked in those who are also BCHE-K carriers, and this group of patients in particular exhibited a BCHE-K allele-dose dependent reduction in BuChE activity. In contrast, APOE ɛ4 non-carriers show higher and similar CSF BuChE enzymatic activity regardless of BCHE-K carrier status [12]. Interestingly, there is a BCHE-K allele-dose dependent decrease in the markers of glial reactivity (GFAP and S100B), which are found mainly in APOE ɛ4 carriers [3].

The BCHE-K variant has also been associated with reduced phosphorylation of tau in patients with dementia [13]. In confirmed late-onset (>65 years of age) AD, a greater load, both of overall neuritic plaques and of cholinesterase-positive neuritic plaques, has been demonstrated in the temporal cortex of BCHE-Kcarriers, who were <80 years of age than of all other patients [14]. Interestingly, a mutation in the 5’untranslated region of the BCHE gene (rs509208) in a genome-wide association studies investigation of the Alzheimer’s Disease Neuroimaging Initiative (ADNI) mild cognitive impairment (MCI)/mild AD cohort was shown to be an independent and substantial contributor to cortical fibrillar Aβ burden in humans [15]. Together, APOE and BCHE loci explained 15% of the variance in cortical Aβ levels (APOE 10.7% ,BCHE 4.3%) [15].

Nonetheless, the results of studies regarding the frequency of BCHE-K carriers in dementia are inconsistent, likely due to variation by geographical region and small sample sizes. The allele is generally found in up to one third of Caucasians and Asians [16, 17]. Although some studies have found evidence of a protective effect of the BCHE-K allele on the likelihood of developing AD [18 –20], other studies have suggested that the BCHE-K allele increases the risk of developing AD in the presence of an APOE ɛ4 allele [21 –24] while decreasing the risk in non- APOE ɛ4 carriers [25, 26]. A high frequency of BCHE-K has been observed in DLB [27]. In addition, significantly higher frequencies of BCHE-K, APOE ɛ4 alone and both alleles together have been seen in mild to moderate DLB compared to patients with mild to moderate Parkinson’s disease with dementia (PDD) [28], particularly in patients aged <75 years.

The APOE ɛ4 allele is associated with a more rapid rate of cognitive decline and hippocampal atrophy before the clinically symptomatic phase of dementia, in amnestic MCI and in mild AD [29, 30]. The influence of APOE ɛ4 on the rate of progression of the disease diminishes with advanced age and progression of AD [28, 31]. Studies suggest that the BCHE-K variant [32, 33] is associated with slower cognitive decline. In longitudinal studies in amnestic MCI, mild to moderate PDD and mild AD individuals with BCHE-K without an APOE ɛ4 allele generally had the slowest cognitive decline, in contrast to the most rapid decline in those with both BCHE-K and APOE ɛ4 alleles [21 , 34]. However, in moderate AD the rates of decline in these genotype groups seems to be reversed [34].

Few studies have examined the role of APOE ɛ4 on cognitive decline in DLB and none in a routine clinical setting, where patients are receiving standard of care treatment. Similarly, the combined effect of APOE ɛ4 and BCHE-K genotypes on cognitive decline in dementia, again in a routine clinical setting where patients may be receiving cholinesterase inhibitors and other treatment has not been examined.

In the present study, we wished to examine the distribution of BCHE-K in a relatively large DLB cohort, and compare the findings with AD and healthy controls. We also explored the association of BCHE-K and APOE ɛ4 genotypes with the rate of cognitive decline in a subgroup of patients in a routine clinical setting, who were followed for five years.

MATERIALS AND METHODS

Patient cohorts

The study cohorts consisted of 368 ethnic Norwegians, recruited as a part of two large ongoing studies in Norway. 108 patients were diagnosed with AD, 174 with DLB, and 86 were healthy controls.

In the DemVest study, AD and DLB patients were recruited from all dementia clinics in the region of western Norway [35] during the period from 2005–2007 and the recruitment of DLB was subsequently continued. Additional DLB patients and the control subjects were included from the TrønderBrain study, recruited through hospitals, outpatient clinics, nursing homes, or from local care authorities in central and southwestern parts of Norway during the period from 2003–2006 [36, 37]. The study designs differed somewhat. In contrast to the probable or possible dementia cases in TrønderBrain, DemVest included only patients with mild dementia, i.e., Mini-Mental State Examination (MMSE) score 20 or higher or a Clinical Dementia Rating score of 1, and the patients were followed annually. The controls subjects included caregivers not genetically related to the patients, and other elderly volunteers recruited from societies for retired people in the same area, all without first-degree relatives with dementia. All were healthy for their age, and when tested were found to be cognitively normal.

Written, informed consent was obtained from all patients or their proxies, and from the control individuals. Both studies were approved by their respective regional committee for medical research ethics, which included permission to send project materialabroad.

Since there are few reports available on the frequency of the BCHE-K variant from the general population, none of which are from Norway, we also studied the distribution of the BCHE-K variant in 1,721 cognitively normal controls and 189 patients with AD, from the DemGene study [38, 39].

Diagnostic procedures and cognitive rating

In both studies, a clinical diagnosis of AD and DLB was made by experienced and trained research clinicians, according to consensus criteria and standardized diagnostic rating scales as described previously [35, 36]. Cognition was assessed using the MMSE, and the diagnoses were reviewed regularly by the research team.

All DLB patients from the DemVest study and about 20% of DLB patients from the TrønderBrain, study had a dopamine transporter single photon emission computed tomography (SPECT) scan using ¹²³I-Ioflupane to support the diagnosis. Autopsy confirmation for the diagnosis was available for 36 patients in the DemVest study and two patients in TrønderBrain.

DemVest is a prospective longitudinal study, and annual assessment with MMSE for up to five years is available for around 173 patients. According to the protocol and international recommendations, clinicians were recommended to start treatment with a cholinesterase inhibitor in patients with AD and DLB, and nearly all patients with AD and DLB were treated with a cholinesterase inhibitor from baseline. Longitudinal follow-up data are not available for TrønderBrain patients.

DNA extraction from frozen blood

Blood samples were collected and handled according to standardized procedures, aliquoted, frozen, transported and stored at –80°C at Karolinska Institutet, Stockholm, Sweden. After thawing, approximately 3 mL of whole blood was used for DNA extraction using the Gentra Puregene blood kit (cat no. 158389) from Qiagen according to the manufacturer’s instructions.

BCHE-K and APOE genotyping

BCHE-K genotyping was performed with purified DNA using the TaqMan SNP genotyping assay kit (cat no. 4351379, Applied Biosystems), and confirmed in whole blood using the TaqMan Sample-to-SNP assay kit (cat no. 4403087, Applied Biosystems), according to the manufacturer’s instructions, using a StepOne Plus thermal cycler (Applied Biosystems) as described previously [12] APOE genotyping was performed as described [36].

In DemGene subjects, genome-wide genotyping was performed with their DNA samples as described in HumanOmniExpress BeadChip Kit (Illumina),followed by single nucleotide polymorphism (SNP) calling. Further then, the SNP for BCHE-K variant was imputed using the methods as described in [38].

Statistical tests

Chi-square and Fisher exact tests were used to compare the genotype frequency and gender differences between the groups. The age, disease duration, and MMSE between the groups were compared using one-way analysis of variance (ANOVA). Longitudinal analyses were performed with a linear mixed effects model, adjusting for covariates using R-program for statistical computing. A model with random intercept and slope and Independently, Identically Distributed (IID) residuals was used to produce a fit to the data. Further explorative analyses were performed using repeated-measures ANOVA.

RESULTS

Demographic

Table 1 shows the demographic and clinical characteristics of the study groups. There was a significant difference in age, gender, disease duration, and baseline MMSE scores between the groups. The percentage of females was significantly higher in AD than DLB (p < 0.001) and controls (p < 0.01). Similarly, baseline MMSE was higher in AD patients (p < 0.001) compared to overall DLB patients from both study centers. However, this difference was due to the lower MMSE scores observed among DLB subjects from the TrønderBrain study, which showed a significantly lower MMSE than the DLB subjects from DemVest (p < 0.01), as expected due to design differences.Similarly, the duration of disease was longer among DLB patients in the TrønderBrain study than those included in DemVest (p < 0.01). The duration of DLB was also longer than duration of AD (p < 0.001), again due to the DLB patients from the TrønderBrain study (Table 1).

Distribution of BCHE-K and APOE ɛ4 genotypes

The percentage of subjects carrying one or two K allele (BCHE-K carriers) was significantly higher in the control group (51.2%) than the DLB group (33.9% ; χ² = 7.1, p < 0.01), and a non- significant trend compared to the AD group (38.9% ; χ² = 2.9, p = 0.11). The cohort groups followed the Hardy Weinberg Equilibrium and no ascertainment bias was observed in both control (χ² = 0.08) and dementia groups (AD, χ² = 0.63; DLB, χ² = 0.57), which was calculated using the Hardy Weinberg equilibrium calculator including analysis for ascertainment bias [40]. The proportion with at least one K-variant did not differ significantly between DLB patients from DemVest (30.3%) and TrønderBrain (38.7%).

We further compared the distribution of the BCHE-Kgenotype in AD and control subjects from another large Norwegian cohort, the DemGene study. Also in this second cohort, the percentage of BCHE-K carriers was higher in the control group (41.0%), but not significantly higher than AD group (36.5% , χ² = 1.4, p < 0.26). The AD patients, from both DemVest and DemGene study had a very similar BCHE-K allele distribution (No significant difference, χ² = 0.166, p < 0.71).

The BCHE-K related demographic characteristics of the DemVest and TrønderBrain cohorts are shown in Table 2. In DLB patients from TrønderBrain, those carrying the BCHE-K polymorphism had significantly lower MMSE scores than non-carriers (p < 0.01). No other significant differences were observed between BCHE-K carriers and non-carriers in this cohort (Table 2).

As expected, there was a lower frequency of APOE ɛ4 genotype in the control (25%) than the AD(63.9% ; χ² = 26.7, p < 0.0001) and DLB groups (53.9% ; χ² = 17.4, p < 0.0001). Further details regarding the BCHE-K and APOE genotypes in the DemVest and the TrønderBrain study groups are shown in Table 3.

Detailed information on the combination of APOE ^ɛ4 +|BCHE^K + genotype among the AD, DLB and controls is provided in Table 4. There was no significant difference in baseline-MMSE between the groups of patients with different combination of the genotypes in AD and DLB patients.

Among the individuals carrying both APOE ɛ4 and BCHE-K genotype, the proportion of those aged less than 75 years in AD, DLB and control groups were 27% , 37% , and 17% respectively (Table 4).

Cognitive decline in AD and DLB patients in DemVest Cohort

Using a multiple linear mixed effect (LME) model adjusted for age, gender, and diagnosis we looked at the effect of APOE ɛ4 and BCHE-K genotypes alone, or in combination, on annual cognitive decline in the DemVest cohort, where longitudinal assessments were available (Fig. 1).

An annual decline in MMSE at a rate of 2.4 points/year was observed during the five-year follow up period. The rate of cognitive decline did not differ between BCHE-K carriers and non-carriers, but showed a significant difference between APOE ɛ4 carriers and non-carriers (p = 0.037). Further analyses based on the number of APOE ɛ4 alleles indicated that the patients with two APOE ɛ4 alleles had an overall cognitive decline of 4.2 points/year in MMSE (p = 0.007), while those with one APOE ɛ4 allele showed 3.5 points/year (p = 0.011). The LME analysis showed no statistically significant interaction of APOE ɛ4 and BCHE-K genotypes on annual cognitive decline (Fig. 1). However, as can be appreciated from Fig. 1, there appeared to be a difference in annual cognitive decline at the 5th year of follow-up between the APOE ɛ4/BCHE-K carriers and non-carriers.

To further explore this, we performed repeated-measures ANOVA, including only the patients who underwent MMSE assessment at all of the five-year follow-ups.

This analysis indicated a significant interaction between MMSE test scores and the genotype (p = 0.04). As illustrated in Fig. 2, in the absence of APOE ɛ4, the BCHE-K carriers had 1.5 point less cognitive decline on average (MMSE test score/year), compared to the APOE ɛ4 and BCHE-K carriers (p < 0.05). The characteristics of these patients are shown in Table 5.

DISCUSSION

In this study we present the frequency of theBCHE-K genotype and its clinical correlates in routineclinical practice patients with a diagnosis of DLB or AD. One of the main findings was that the BCHE-K genotype was less common among patients with DLB than in control subjects. However, the frequency of BCHE-K genotype in the control group was very high (51.2%) compared to the albeit variable frequency of 33% found in previous studies [17]. We checked this further with another Norwegian cohort (DemGene) showing 41% , which still seems higher than expected frequency of 33% in the control population of other studies [41]. This might suggest that a high BCHE-K frequency among Norwegian population, which warrants further studies.

In these patients who were receiving standard ofcare treatment, including cholinesterase treatment in most cases, no statistically significant associations between BCHE-K genotype and the rate of cognitive decline were observed. In contrast, we found that the APOE ɛ4 genotype was associated with more rapid cognitive decline among the patients, which is in agreement with similar longitudinal associations observed in cognitively normal elderly subjects [42, 43].

Insufficient studies regarding BCHE-K genotype frequency among patients with DLB exist. In one of the largest studies, the frequency of BCHE-K genotype in DLB and PDD was 47.4 and 34.4 % , respectively [28]. The study also showed that the proportion of patients carrying both BCHE-K and APOE ɛ4 in these two Lewy body disorders was significantly different in association with APOE ɛ4, at 28.1% and 11.5% , respectively. However, the study had no control group for comparison. In this study, BCHE-K genotype frequency was 33.9% in the DLB group, while the frequency of the combined APOE ɛ4/BCHE-K genotype was 17% in the DLB and 14% in the control group. Also considering the high frequency of the BCHE-K carriers without an APOE ɛ4 allele in the control group, a plausible explanation for the lower proportion of BCHE-K carriers without an APOE ɛ4allele among DLB patients could be due to expected differences in the phenotypic activity of BuChE-K variant protein. This protein variant is associated with 30–50% reduction in acetylcholine-degrading capacity in CSF [1], which could result in more efficient central cholinergic signaling, masking ongoing pathology in the cholinergic neural network. This might prolong the time for subjects to reach the symptom threshold for dementia. This might explain both the reported association in DLB between BCHE-K and better cognitive performance on measure of attention that implicates subcortical structures [33]. Thus, a reduced likelihood of developing dementia and/or a delay in the onset of symptoms of dementia in those with a BCHE-K allele in the absence of an APOE ɛ4 allele could be the reason for the higher BCHE-K frequency observed in healthy controls compared to DLB in our study.

In another study in patients with mild to moderate PDD, carriers of both APOE ɛ4 and BCHE-K exhibited a significantly more rapid cognitive decline than the patients carrying only one of these genotypes [28], indicating a potential interaction between the two genotypes similar to that seen in amnestic MCI [21]. In the present study, although the overall analysis did not indicate an interaction between the two genotypes, the explorative repeated-measures analysis performed on the subpopulation of the patients who completed the full 5-year follow-up period, supported the possibility that, the combined APOE ɛ4 and BCHE-K genotypes may be related to a higher rate of cognitive decline. On the other hand, in the absence of an APOE ɛ4 allele, the BCHE-K genotype may reduce the rate of cognitive decline (Fig. 2). Since this finding was based on analysis of small groups, it should currently be interpreted with caution. However, this study suggests— that over a 5-year period— standard of care therapy may have little effect on modulating the interaction of genotype on cognitive decline in patients with DLB and AD.

Moreover, the interaction between APOE ɛ4 and BuChE may be of particular interest. We have shown that in AD patients, the APOE ɛ4 genotype modulates the phenotypic expression of the BCHE-K variant, which leads to an exaggerated reduction in BuChE activity in BCHE-K carriers [12]. In the presence of APOE ɛ4 therefore, these subjects are expected to accumulate more Aβ pathology and synaptic dysfunction due to hypofunctional glial [5] and thereby reduced Aβ clearance. This is indirectly supported by 1) our finding here that among subjects aged <75, 27% of DLB and 37% of AD patients had combined APOE^ɛ4 +|BCHE^K + genotype compared to 17% among the controls (Table 4), together with 2) immunohistochemical analyses of AD brain showing about six folds accumulation of ChE-positive Aβ deposits in K-carriers patients aged less than 80 years [14, 44], and 3) enzymological analyses of cortical brain homogenate showing 37% significant increase of BuChE level in K homozygotes than wild-type homozygotes in dementia patients [45].

In addition the accelerated debut of clinical symptoms due to synaptic ACh signalin deficits in carriers of both of these alleles may lead to severe cholinergic neurodegeneration and early onset of amnestic symptoms [21], and so they may show an initial robust symptomatic response to cholinesterase inhibitiontherapy in MCI and mild AD, but poor response at later stages of the disease as increasing damage to cholinergic synapses renders them non-functional [5]. This may explain our observation that patients with both the APOE ɛ4 and BCHE-K have a more rapid cognitive decline [28].

As was mentioned, we found that the frequency of BCHE-K is more common in controls than in people with DLB, suggesting that the BCHE-K variant may act as a protective factor against DLB, particularly in homozygotes. However, our finding did not support previous reports of a slower rate of decline in BCHE-K carriers, as this may only be evident in patients with early disease in those without a concomitant APOE ɛ4 allele. However, exploratory analyses suggested a possible association between the BCHE-K variant and the APOE ɛ4 allele and more rapid cognitive decline over a 5-year period despite the use of cholinesterase treatment. Future longitudinal studies on the impact of BCHE-K and other BCHE mutations on the occurrence and course of AD and DLB should control for recruitment biases; the influence of gender, age, and stage of disease; and include larger sample sizes and CSF assessments of BuChE activity, complement, markers of glial activity, and AD biomarkers.

Limitations include that the DLB patients were recruited at two clinical centers, while the AD and healthy controls were recruited at one of the centers, which might have influenced the findings. Even though the frequency of BCHE-K carriers did not show statistical differences between the centers, the frequency of BCHE-K carriers was numerically higher in DLB patients from TrønderBrain compared to the DemVest DLB patients. The rate of cognitive decline in AD is strongly influenced by gender, genotype, diagnosis, stage of dementia, and cholinesterase treatment [5, 46] and due to the small sample size, it is not possible to fully disaggregate the contribution of these factors in patients with DLB in the current study. However, the use of multiple linear mixed effect modeling allowed us to adjust for the effects of age and gender, and nearly all patients in the longitudinal study were treated with a cholinesterase inhibitor from baseline. Another potential limitation is cohort bias effect, whereby slow progressing patients are more likely to enroll in a cohort study and complete follow-up visits than fast progressing patients. This may have influenced the distribution of genotypes in the study and underestimated the proportion of individuals carrying both BCHE-K and APOE ɛ4 alleles who develop DLB and AD. In addition, most DLB patients have some degree of AD-type pathology, and it has been suggested that DLB patients with BCHE-K and APOE ɛ4 alleles may have a more AD-like phenotype [28]. Although the number of cases in the present work is relatively large for a DLB group, it is still small for this kind of analysis. The number of cases in the AD group is also small. The BCHE-K genotyping was performed using two separate SNP preparations, which adds to the strength of this study.

Diagnoses were based on clinical assessment, and thus misdiagnosis cannot be excluded. However, the most common misclassification in DLB is AD, and given the relatively similar genotype distributionmisdiagnosis is unlikely to influence the findings. Misclassification of healthy and diseased patients is unlikely given the large difference in cognition between the groups, the comprehensive and standardized diagnostic procedures, including frequent availability of results from DaTScan and autopsy in a subset from the DemVest study. The study also has the advantage of a longitudinal design for a subset of individuals, up to five years.

Footnotes

ACKNOWLEDGMENTS

The authors would like to thank the patients and their caregivers as well as the healthy individuals for participating in the study.

This study was supported by the grants from Swedish Alzheimer Foundation, Loo & Hans Osterman Foundation; KI Foundations; Olle Engkvist Byggmästare Foundation; Åke Wibergs Foundation;Åhlén-Foundation (Åhlén-stiftelsen); Gunvor and Josef Anérs Foundation; Gamla Tjänarinnor Foundation; Magnus Bergvalls Foundation; Demens Foundation (Demensfonden); Gun and Bertil Stohnes Foundation; Ragnhild & Einar Lundströms Foundation; Foundation for Sigurd & Elsa Goljes Memory. DemGene is supported by the Research Council ofNorway (225989, 223273, 237250/EU), Norwegian Health Association and KG Jebsen Foundation. Dr Aarsland has received research support and/or honoraria from Astra-Zeneca, H. Lundbeck, Novartis Pharmaceuticals and GE Health.

Authors’ disclosures available online ().

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