|Year : 2020 | Volume
| Issue : 3 | Page : 134-140
The association of serum clusterin levels and Clusterin rs11136000 polymorphisms with Alzheimer disease in a Turkish cohort
Gamze Guven1, Ebru Ozer1, Basar Bilgic2, Hasmet Hanagasi2, Hakan Gurvit2, Ebba Lohmann3, Nihan Erginel-Unaltuna1
1 Department of Genetics, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul, TR, Turkey
2 Department of Neurology, Istanbul Faculty of Medicine, Behavioural Neurology and Movement Disorders Unit, Istanbul University, Istanbul, TR, Turkey
3 Department of Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, University of Tübingen; DZNE, German Center for Neurodegenerative Diseases, Tübingen, GER, Germany
|Date of Submission||25-Oct-2019|
|Date of Decision||10-Apr-2020|
|Date of Acceptance||11-Apr-2020|
|Date of Web Publication||16-Sep-2020|
Department of Genetics, Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul
Source of Support: None, Conflict of Interest: None
Objectives: Several large-scale genome association studies have shown that variants in the “Clusterin”' (CLU) gene are important risk factors for Alzheimer's disease (AD). It has also been shown that plasma CLU levels were elevated in patients with AD and associated with disease severity and progression. In this study, we aimed to investigate whether the CLU rs11136000 polymorphism was associated with AD in our cohort of Turkish patients. We also evaluated the association of serum CLU levels and rs11136000 genotypes between patients and controls. Materials and Methods: Genotyping was performed in 327 patients who were diagnosed as having AD (mean age: 67.2 ± 10.8 years) and 344 controls (mean age: 57.7 ± 13.1 years). The rs11136000 genotypes were determined using quantitative real-time polymerase chain reaction with hydrolysis probes. Serum CLU levels were assessed in 25 patients with AD and 10 controls using enzyme-linked immunosorbent assay. Results: Our results showed no significant difference in genotype and allele frequencies of CLU rs11136000 polymorphisms between patients with AD and controls. Serum CLU levels in patients with AD did not differ from those of the controls. Furthermore, serum CLU levels showed no major difference between carriers of CC and TT + CT genotypes in the controls and patients with AD. Conclusion: Our results suggest that the CLU rs11136000 polymorphism is not associated with AD in our Turkish patients, and rs11136000 genotypes may not have an effect on serum CLU levels.
Keywords: Alzheimer's disease, Clusterin, polymorphism, serum, Turkey
|How to cite this article:|
Guven G, Ozer E, Bilgic B, Hanagasi H, Gurvit H, Lohmann E, Erginel-Unaltuna N. The association of serum clusterin levels and Clusterin rs11136000 polymorphisms with Alzheimer disease in a Turkish cohort. Neurol Sci Neurophysiol 2020;37:134-40
|How to cite this URL:|
Guven G, Ozer E, Bilgic B, Hanagasi H, Gurvit H, Lohmann E, Erginel-Unaltuna N. The association of serum clusterin levels and Clusterin rs11136000 polymorphisms with Alzheimer disease in a Turkish cohort. Neurol Sci Neurophysiol [serial online] 2020 [cited 2020 Sep 28];37:134-40. Available from: http://www.nsnjournal.org/text.asp?2020/37/3/134/295181
| Introduction|| |
Alzheimer's disease (AD) is an irreversible and progressive neurodegenerative disease characterized by decreased daily living activities and impaired cognitive abilities. Various factors have been identified in the pathogenesis of the disease including genetic factors. Numerous large-scale genome-wide association studies have identified several susceptibility genes associated with AD. Since the discovery of the cholesterol-related gene “Apolipoprotein E” (APOE) epsilon 4 (ε4) allele, as the major risk variant for AD, it has been assumed that other genes involved in the metabolism of cholesterol may also be risk factors for the AD. Among these genes, “Clusterin” (CLU), also known as “Apolipoprotein J,” has been strongly associated with an increased risk of AD.
CLU is a lipoprotein and multifunctional chaperone protein expressed in almost all mammalian tissues most abundantly in the brain. CLU specifically binds soluble amyloid β (Aβ) in cerebrospinal fluid (CSF) and is involved in the transport of Aβ across the blood–brain barrier.,, It is also involved in the aggregation, deposition, and toxicity of Aβ. Studies have shown that CLU protein levels in CSF and plasma were increased in patients with AD,,,, and plasma CLU levels were associated with disease severity and rapid cognitive decline.
In this study, our aim was to investigate the association between AD and the intronic CLU rs11136000 (NC_000008.11:g.27607002T>C)polymorphism, which has been previously shown to have a significant association with AD, in a cohort of Turkish patients. To evaluate the effects of this polymorphism on peripheral CLU levels, we compared the serum CLU levels among rs11136000 genotype carriers.
| Materials and Methods|| |
Patients and controls
The study population comprised 327 patients who were diagnosed as having AD and 344 controls with no history of any major neurologic, psychiatric, or systemic disease. All participants underwent detailed clinical and neuropsychological examinations, neuroimaging in most cases. The diagnosis of AD was based on the National Institute of Neurological and Communicative Disorders and Stroke and Alzheimer's disease. The Mini-Mental State Examination test (MMSE) and Geriatric Depression Scale (GDS) were used to evaluate the global cognitive status and the presence of depressive symptoms in participants.
The study was approved by the Ethics Committee of the university (Date: October, 17, 2016, No: 1209). A neurologist took the necessary clinical information after obtaining informed consent from the patients or their legally authorized representatives.
Genomic DNA was extracted from peripheral blood leukocytes according to the standard procedures. Genotyping of CLU and APOE was performed using real-time polymerase chain reaction with labeled hydrolysis probes. Ten milliliters of reaction mixture consisted of 50 ng genomic DNA, 10 μM of each primer, 1 μM of each probe, and 5 μL of ×2 LC480 Probes Master. Amplification was performed using a LightCycler 480 real-time PCR instrument (Roche Diagnostics, Germany). Cycling conditions were 2 min at 50°C; 10 min at 95°C; 45 cycles of 95°C for 10 s; 56°C for 30 s; and 72°C for 1 s. The endpoint analysis was assessed using the LightCycler 480 genotyping software.
Measurement of serum clusterin levels
Serum samples were collected for the measurement of CLU levels. Only 35 individuals were available for serum sampling; therefore, CLU levels were measured in this randomly selected group. Peripheral blood samples were collected, centrifuged at 1400 g for 10 min, and serum samples were stored at −80°C. CLU levels in serum samples were determined using a Human CLU ELISA kit (BioVendor, Heidelberg, Germany) in duplicate according to the manufacturer's protocol.
Genotype and allelic distributions were compared using Pearson's Chi-square test. The Hardy–Weinberg equilibrium (HWE) was computed to the expected genotype distribution. The independent t-test was used to compare continuous variables and expressed as means and standard deviation, whereas categorical variables were compared using the Chi-square test. Maximum likelihood estimates of odds ratios (OR) and associated 95% confidence intervals (CIs) were calculated for dominant, recessive, and overdominant comparison models through logistic regression analysis adjusted for age, sex, and APOE ε4 carrier status (carrier and noncarrier) as covariates. The association between rs11136000 genotypes and lipid parameters was determined through univariate analysis of variance using age, sex, and APOE ε4 carrier status as covariates. Correlations between serum CLU levels and demographic and biochemical characteristics were tested using Spearman's rho. All statistical analyses were performed using the Statistical Package for the Social Sciences version 21.0 software (IBM Corp, Armok, NY, USA). The results were considered statistically significant with P < 0.05.
| Results|| |
Demographic and biochemical characteristics of the study group
The clinical data and lipid parameters of all patients and controls are summarized in [Table 1]. As shown in the [Table 1], APOE ε4 allele carriers were significantly more frequent (P < 0.001) in the AD group. In addition, the total MMSE score was found to be significantly decreased, as expected in patients with AD. Furthermore, the GDS scores of the patient group were significantly increased.
|Table 1: Descriptive characteristics and lipid parameters of patients and controls|
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There was also a statistically significant difference (P < 0.001) in terms of age between the AD (mean age: 67.2 ± 10.8 years) and controls (mean age: 57.7 ± 13.1 years). Lipid parameters showed no statistically significant difference between the two groups.
Allele and genotype frequencies of the Clusterin rs11136000 polymorphism
For allele and genotype analyses, patients with AD were divided into two groups according to age of onset: late-onset AD (LOAD; age of onset ≥65 years, mean age: 76.5 ± 4.9 years, n = 154) and early-onset AD (EOAD; age of onset <65 years, mean age: 58.9 ± 7.5 years, n = 173). The AD groups were compared with their age matched controls. The demographic and clinical characteristics of these groups are given in [Supplementary Table S1]. The allele and genotype frequencies were compared between LOAD, EOAD, and all patients with AD versus controls age ≥70 years, controls age <70 years, and all controls, respectively. The distribution of genotypes was in HWE in all groups. The allele and genotype distributions of CLU rs11136000 polymorphisms in patients and controls within each study group are shown in [Table 2]. There was no statistically significant difference in allele and genotype frequencies in any of the groups. We also tested the association of CLU rs11136000 genotypes using dominant, recessive, and overdominant models, which were adjusted for age, sex, and APOE ε4 carrier status [Table 2]. However, no significant AD risk was observed for CLU rs11136000 genotypes in different genetic models.
|Table 2: Genotype and allele frequencies of CLU rs11136000 polymorphism in patients with Alzheimer's disease and controls in different groups|
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Association of Clusterin rs11136000 polymorphism with lipid parameters
[Table 3] shows the estimated marginal mean values of the lipid parameters according to rs11136000 genotypes among the patients and controls adjusted for age, sex, and APOE ε4 carrier status. There were no statistically significant associations between the CLU rs11136000 genotypes and lipid parameters.
|Table 3: Estimated marginal mean values±standard deviation of lipid parameters according to CLU rs11136000 genotypes|
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Serum Clusterin levels
Serum CLU levels were available for 25 patients with AD and 10 controls. The demographic and clinical characteristics of these patients and controls are given in [Supplementary Table S2]. As shown in [Figure 1], the mean serum CLU level in patients with AD was 59.4 μg/mL (range: 43.2–85.5), which was similar to that of the controls (mean: 61.9 μg/mL, range: 46.1–84.2 μg/mL). To assess whether serum CLU levels were influenced by CLU rs11136000 genotypes, we examined CLU levels in the carriers of CC versus TT + CT genotypes in controls and patients with AD [Figure 2]a and [Figure 2]b. In patients with AD, the CC carriers (mean: 57.5 μg/mL, range: 47.2–67.5 μg/mL n=10) tended to have lower serum CLU levels compared with TT + CT carriers (mean: 60.7 μg/mL, range: 43.2–85.5 μg/mL n=15). Likewise, in the control group, serum CLU levels of CC carriers (mean: 57.1 μg/mL, range: 49.3–65.7 μg/mL, n = 4) were lower than the levels for TT + CT carriers (mean: 65.1 μg/mL, range: 46.1–84.2 μg/mL, n = 6) carriers. Furthermore, the presence of the APOE ε4 alleles had no effect on serum CLU levels. The correlation analysis showed that there was no significant correlation between serum CLU levels and age, age at onset, MMSE, GDS, and lipid parameters.
|Figure 1: Serum clusterin levels in patients with Alzheimer's disease and controls. The mean is indicated together with the standard deviation (Alzheimer's disease, n = 23; Controls, n = 10)|
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|Figure 2: Serum clusterin levels based on Clusterin rs11136000 genotypes. (a) Alzheimer's disease (CC n = 10, TT + CT n = 15). (b) Controls (CC n = 4, TT + CT n = 6). The mean is indicated together with the standard deviation|
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| Discussion|| |
In the first step of our study, we investigated whether the CLU rs11136000 polymorphism was associated with the presence of AD in our cohort of Turkish patients with AD. In addition, we examined the distribution of rs11136000 genotypes in LOAD and EOAD groups and their age-matched controls.
Two different large-scale genome-wide association studies have shown that the CLU rs11136000 intronic polymorphism is significantly associated with LOAD in Caucasian populations., Both studies suggested that the T allele of rs11136000 had a protective role, and carriers of this allele had a reduced risk of AD (OR <1), whereas the C allele, which is carried by ~88% of Caucasians,, was considered the risk allele and conferred a 1.16-fold increased risk for developing AD. Thereafter, several studies investigating the association between the CLU rs11136000 polymorphism and AD were conducted in numerous populations, including Caucasian, Asian, African, and Hispanic. However, the results of these studies were inconsistent, indicating that the risk associated with the rs11136000 polymorphism might vary among the populations of different ethnic and geographic origins. A recent meta-analysis of 74,248 samples confirmed the association of the T allele with reduced AD risk in Caucasians but not in a Chinese population (OR = 0.864, 95% CI: 0.842–0.888; P < 0.001). The T allele frequency varies between 0.33 and 0.43 in Caucasian populations. Consistent with these findings, in our study, the T allele frequency was found as 0.34 in the overall study group. Although the T allele frequency in our study was similar to that in other studies, we found no statistically significant difference in allelic and genotypic distributions between the groups. Our results were consistent with the two previous studies performed in Turkish patients with AD, which also found no significant associations., However, it should not be ruled out because these studies were conducted in small-scale study groups, and therefore, further studies in larger cohorts are necessary to confirm the results reported in the Turkish cohort.
In addition, in our study, the relationship between the CLU rs11136000 polymorphism and different lipid parameters was also investigated, but no significant association was found. Apart from our study, only one other research group has investigated the association between rs11136000 genotypes and lipid parameters. Aghajanpour-Mir et al. showed that the CC genotype of the rs11136000 polymorphism was associated with decreased high-density lipoprotein (HDL) cholesterol levels in Iranian patients with mild cognitive impairment (MCI) compared with controls and proposed that low HDL levels in CC carriers could increase the risk of developing MCI through altering glucose metabolism. It has been known that higher levels of HDL are associated with decreased AD risk  and low levels are associated with increased risk., However, we found unexpectedly high HDL levels in patients with AD with the CC risk genotype. The association between HDL levels and rs11136000 genotypes needs to be confirmed by further replicative studies because this finding was statistically nonsignificant.
Another goal of this study was to investigate the relationship between serum CLU levels and the presence of AD. High plasma CLU levels in patients with AD were reported to be associated with brain atrophy, disease severity, and rapid clinical progression. Subsequently, several studies have shown elevated levels of plasma CLU both in patients with MCI ,,, and AD.,, These results suggest to the authors that high levels of plasma CLU in patients with AD and MCI may reflect a protective response to the neurodegenerative process.,, By contrast, Schürmann et al., Silajdžić et al. and Jongbloed et al. found that plasma CLU levels were not significantly changed in patients with AD. Moreover, Ijsselstijn et al. showed no statistically significant difference in serum CLU levels between presymptomatic patients with AD and controls, suggesting that serum CLU was not an early, presymptomatic biomarker for AD. Consistently, in our study, serum CLU levels of patients with AD were not different from those of controls. Due to the discrepancies between studies concerning plasma or serum CLU levels, additional studies measuring CLU levels in serum/plasma together with CSF will be needed to elucidate the exact role of CLU in the pathogenesis of AD.
Our study also investigated whether serum CLU levels were influenced by the rs11136000 genotypes. We observed no major effect of the rs11136000 genotypes on serum CLU levels either in patients with AD or in controls. In agreement with our data, Thambisetty et al. showed that rs11136000 genotypes did not influence plasma CLU levels in patients with AD and controls. Furthermore, Cai et al. found no significant association of rs11136000 genotypes with plasma CLU levels in patients with MCI. However, there are also some studies that showed significant associations. In the study by Schürmann et al., lower levels of plasma CLU level were observed in the CC carriers among healthy controls, whereas no statistically significant difference was found in patients with AD. On the contrary, Mullan et al. showed that plasma CLU levels were lower in TT genotype carriers in the control group. The conflicting results from different studies may be due to the fact that association between CLU expression and AD is independent of genetic variation in the CLU gene  or rs11136000 may not be the polymorphism that actually causes expression changes in the CLU protein., However, we cannot exclude the effect of other CLU polymorphisms on CLU expression because this study only deals with the rs11136000 polymorphism. Different studies with a larger sample sizes should be performed to clarify the potential effects of the rs11136000 polymorphism on CLU expression levels.
| Conclusion|| |
Our results suggest that the CLU rs11136000 polymorphism may not be associated with AD risk in our cohort of Turkish patients, and rs11136000 genotypes may not have an effect on serum CLU levels. However, the relatively small sample size is a limitation of our study, which means that the results of this study should be interpreted carefully.
The authors thank all the patients and their families. Meltem Pak helped with obtaining the samples.
Financial support and sponsorship
This work was supported by the Research Fund of Istanbul University (Project No: TAB-2017-23218).
Conflicts of interestb
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]