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Keywords:

  • Alzheimer's disease;
  • apolipoprotein E;
  • cholesterol;
  • clusterin;
  • polymorphism

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Background:  Previous papers have reported that clusterin (CLU, also called apolipoprotein J) maintains amyloid β-peptide (Aβ) solubility and protects against Aβ neurotoxicity. Recently, two large genome-wide association studies (GWAS) identified that a specific single nucleotide polymorphism (SNP) on the gene has been reported to modify the risk for Alzheimer's disease (AD). The present study aimed to investigate whether common single nucleotide polymorphisms (SNP) of the CLU gene are associated with AD.

Methods:  Six SNP, genotyped using TaqMan technology, were analyzed using a case–control study design. Furthermore, an analysis of the cases divided according to apolipoprotein E (APO E) status was also carried out. Our case–control dataset consisted of 180 AD patients and 130 age-matched controls.

Results:  The present study failed to detect any association between the SNP of the CLU gene and AD. Although rs7982 and rs1532277 showed marginal association in the APO E4 negative group, the linkage disequilibrium analysis results suggest this to be a false positive.

Conclusion:  The negative associations were mainly the result of our small sample size. Larger genetic studies in different ethnics and future meta-analysis are needed to clarify the relationship between the CLU gene and AD.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

The pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid plaques. Familial AD has been associated with mutations in three genes: (i) the amyloid precursor protein (APP); (ii) presenilin-1; and (iii) presenilin-2 genes. The three genes alter production of amyloid β-peptide (Aβ), the principal component of β-amyloid in senile plaques. Apolipoprotein E (APO E) is an established genetic risk factor for familial and sporadic AD.

A previous paper reported that clusterin (CLU, also called apolipoprotein J), a ubiquitous multifunctional glycoprotein, maintains Aβ solubility.1,2 CLU has been also found to protect against Aβ neurotoxicity.3,4 It has been revealed that the protein level of CLU was increased in the hippocampus and the frontal cortex in AD brain.5 Thus, CLU plays a pivotal role in the pathogenesis of the disease.6,7 Recently, two large genome-wide association studies (GWAS) identified that a specific single nucleotide polymorphism (SNP) on the gene has been reported to modify the risk for AD.8,9 Although several replication papers have been issued recently, common SNP on the gene were not fully studied.

In the present study, we investigated whether six SNP covering the entire gene region are associated with Japanese sporadic AD. CLU influences Aβ metabolism with APO E in vivo.10 Synergetic effects of CLU and APO E on AD were also clarified.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

DNA was extracted from white blood cells using a standard method. The sporadic Japanese AD cases (n= 180, male : female = 79:101) were obtained from the inpatients and outpatients of the hospitals where the authors work. The mean age of the AD group (67.4 years, SD 6.2) was not significantly different from that of the control group (64.4 years, SD 6.7). All the AD cases were diagnosed according to the National Institute of Neurological and Communicational Disorders and Stroke/Alzheimer's Disease and Related Disorders Association criteria and none had a familial history of AD. The control group (n= 130, male : female = 63:67) was obtained from healthy volunteers with no history of dementia or other neuropsychiatric diseases. The purpose and significance of the present study were explained in detail to each patient and his/her family, and all subjects provided their informed consent. The study protocol was approved by the Ethics Committee of the Juntendo University School of Medicine. Information on the single nucleotide polymorphisms (SNP) was obtained from the SNP database (dbSNP) established by the National Center for Biotechnology Information. The chosen SNP were validated, according to the dbSNP and had minor allele frequencies (MAF) greater than 5%. SNP of the CLU gene were genotyped using TaqMan technology on an ABI7500 system (Applied Biosystems, Foster City, CA, USA). All probes and primers were designed by the Assay-by-Design TM service of Applied Biosystems. A standard polymerase chain reaction (PCR) was carried out using the TaqMan Universal PCR Master Mix reagent kit (Applied Biosystems) in a 10 µL volume. Hardy-Weinberg equilibrium (HWE) tests were carried out for all SNP for both cases and controls. APO E genotypes for all the samples were determined according to a previous report.11 Statistical analysis of the AD cases divided into the APO E4 positive and APO E4 negative subgroups was also carried out. Differences in the genotypic frequencies were evaluated using a case–control study design and applying the Fisher's exact probabilities test (a P-value of <0.05 was considered statistically significant). A logistic regression analysis was carried out to estimate the relationship between onset of AD, APO E status and the six SNP using spss software version 17.0 for Windows (SPSS, Chicago, IL, USA). A P-value of <0.05 was considered statistically significant. Linkage disequilibrium (LD) between the SNP, as well as a haplotype analysis was carried out using SNPAlyse version 5 (DYNACOM, Yokohama, Japan). LD, denoted as D′, was calculated from the haplotype frequency using the expectation–maximization algorithm. SNP were considered to be in LD if D′ was greater than 0.75.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Our sample set had the power to detect an odds ratio of at least 1.90, assuming a significance level of 0.05, power of 0.80, and an exposure frequency of 0.30 in the controls. The six SNP on the gene were genotyped in our Japanese samples and were found to be in HWE. The genotypic frequencies of each SNP for our cases and controls are shown in Table 1. There was no significant difference in the genotypic distribution for any polymorphism of the CLU gene. Although the APO E4 positive subgroup failed to show any genetic association between the SNP and the disease, rs7982 and rs1532277 showed a marginal association in the APO E4 negative group (Table 2).

Table 1.  Genotypic frequencies of single nucleotide polymorphisms of the clusterin gene
SNPADControlsχ2P-value
  1. AD, Alzheimer's disease; SNP, single nucleotide polymorphisms.

rs10503814    
 C/C172123  
 C/T66  
 T/T000.330.57
rs2279590    
 C/C10577  
 C/T6742  
 T/T692.540.29
rs7982    
 A/A714  
 A/G6539  
 G/G106766.110.05
rs1532277    
 C/C10575  
 C/T6943  
 T/T5115.310.07
rs9331888    
 C/C5449  
 C/G8655  
 G/G34231.890.39
rs572844    
 A/A176126  
 A/G23  
 G/G202.120.47
Table 2.  Genotypic distributions of single nucleotide polymorphisms of the clusterin gene stratified by apolipoprotein E status
APO E4 positive
SNPADControlsχ2P-value
rs10503814    
 C/C7032  
 C/T41  
 T/T000.291
rs2279590    
 C/C4419  
 C/T2712  
 T/T320.210.88
rs7982    
 A/A42  
 A/G2412  
 G/G45190.160.94
rs1532277    
 C/C4418  
 C/T2713  
 T/T320.340.82
rs9331888    
 C/C1612  
 C/G4015  
 G/G1662.310.36
rs572844    
 A/A7232  
 A/G11  
 G/G201.240.77
APO E4 negative
SNPADControlsχ2P-value
  • *

    P < 0.05, statistical significant (Fisher's exact probabilities test). AD, Alzheimer's disease; APO E, apolipoprotein E, SNP, single nucleotide polymorphisms.

rs10503814    
 C/C10291  
 C/T25  
 T/T001.60.26
rs2279590    
 C/C6158  
 C/T4030  
 T/T372.70.28
rs7982    
 A/A312  
 A/G4127  
 G/G61578.030.02*
rs1532277    
 C/C6157  
 C/T4230  
 T/T296.20.04*
rs9331888    
 C/C3837  
 C/G4640  
 G/G18170.130.93
rs572844    
 A/A10494  
 A/G12  
 G/G000.440.61

We analyzed the simultaneous effects on the AD onset of APO E and the six SNP by means of multiple regression analysis (Table 3). The analysis showed that the six SNP did not correlate with the AD risk. The results only detected APO E to be associated with the risk for AD. Additionally, we did not detect any potential interaction between the six SNP flanking the studied region. We found a strong LD between rs2279590 to rs572844 (Table 4). Rs10503814 was estimated to be located on another LD block.

Table 3.  Multiple regression analysis for the effects of clusterin gene single nucleotide polymorphisms and apolipoprotein E on Alzheimer's disease
 B-valueP-valueOdds ratio95% CI
  • *

    P < 0.05: statistical significant. APO E, apolipoprotein E.

APO E−1.8720.017*0.1540.033–0.711
rs10503814−0.8430.3140.430.083–2.224
rs22795900.8940.182.4440.661–9.036
rs7982−0.2360.7280.790.208–2.994
rs1532277−0.6530.3930.5210.117–2.326
rs93318880.1030.5841.1090.766–1.605
rs5728440.760.4392.1380.312–14.641
Table 4.  Linkage disequilibrium (D' value) between single nucleotide polymorphisms
 rs10503814rs2279590rs7982rs1532277rs9331888rs572844
  • ≥0.9,

  • §

    ≥0.75,

  • <0.75.

rs10503814      
rs22795900.161     
rs7982−10.9228    
rs15322770.14450.96130.952   
rs93318881−0.8584§−0.9737−0.836§  
rs5728440.61550.6013−10.59611 

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

To date, the present study is the first to test the genetic association between CLU polymorphisms and AD in a Japanese population. From total cases analysis, two SNP, rs7982 and rs1532277, showed marginal associations with AD. Although we could not observe positive SNP from the APO E4 positive group, these two SNP showed a statistically significant association. Logistic regression analysis suggested that only APO E affects the onset of the disease. When we analyzed the haplotype of the combination of the two SNP, negative associations were confirmed for both total cases and the APO E4 negative group (data not shown). Superficial positive effects of rs7982 and rs1532277 seemed to be derived from the differences of frequencies of heterozygotes. These analyses suggested the SNP on the CLU gene were not associated with the Japanese population.

Although Tycko et al. had not been able to find positive associations between the CLU gene and AD,12 recent GWAS showed that rs11136000 on the gene was associated with AD.8,9,13 Recent replication studies also support the previous positive results.14,15 Furthermore, a positive association was confirmed with the cohort of clinically characterized and neuropathologically verified cases.16 Lambert et al. identified that another two SNP, rs2279590 and rs9331888, also affect the risk for AD.9 A negative association report with four SNP on the CLU gene has been issued previously.17 Another report suggested that common coding variability of the CLU gene did not show associations with the disease.18 The case–control samples included African-American, Hispanic and European descent/non-Hispanic populations. The two large GWAS have mainly included European Caucasian populations. Our case–control dataset consisted of a solely Japanese population. Although a very recent replication study of a Han Chinese population showed positive associations,19 one explanation for conflicting results would be ethnic difference. Compared with that, APO E4 is widely recognized as an established genetic risk factor, and the effects of CLU SNP might be small.

Another reason for our negative findings was likely to be the result of the small sample size. A replication study has also shown that small sample size might be the cause of the failure to achieve statistical significance. The average age of the current case–control study was lower than two GWAS studies. Larger genetic studies including both early and late onset AD cases would be needed in the future. The previous GWAS study showed SNP between rs3087554 to rs9331888 were in LD.8,9 Both rs7982 and rs1532277 were included in this LD. In contrast, our LD analysis in a Japanese population showed that two LD blocks lay in the CLU gene. LD analyses in different ethnic groups would help to clarify whether the CLU gene is a consistently replicated genetic risk factor for AD.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

This study was partially supported by the High Technology Research Center Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and Sportology Center, Juntendo University Graduate School of Medicine. We are grateful for the technical assistance of Ms K. Yamamoto.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES