Introduction

Alzheimer’s disease (AD) is the most common form of dementia in the elderly, accounting for 50% of all dementia1. It has been documented that genetic factors, along with environments, extremely contributes to the pathogenesis of AD2,3. Clusterin gene (CLU), also known as apolipoprotein J (ApoJ), is currently the third most associated risk gene according to Alzgene database (http://www.alzgene.org/). It is located in chromosome 8p21–p12 which is a chromosomal region of interest in AD4 and it may explain around 9% of the late-onset AD (LOAD) attributable risk5,6. Many large genome-wide association studies (GWAS) have identified that rs2279590, rs11136000, rs9331888, rs7012010, rs7982 and rs1532278 in CLU was substantially associated with AD risk in individuals of Caucasian ancestry and other populations7,8,9,10,11. Several independent candidate gene studies have then replicated and confirmed these results in various Caucasian populations or other populations, although the strongest associated variant sometimes differed12,13,14,15,16,17,18,19,20,21,22,23. Our group previously reported that rs9331949 and rs9331888 variation in the CLU gene played significant role in sporadic LOAD in the Han Chinese population24,25,26,57. However, no evidence supported that CLU genotypes impact the Aβ burden or tau in CSF in our study. More evidence may be needed to explain the interactions between CLU and Aβ burden in CSF.

Genetically, multiple variations within CLU, such as rs2279590, rs11136000, rs9331888, rs7012010, rs9331949, rs7982 and rs1532278, have been identified to be associated with the risk of AD in multi-center, large scale GWAS, meta-analysis or replication studies. Among these loci, rs11136000 and rs9331888 were mostly investigated. Moreover, we performed linkage disequilibrium (LD) analysis and discovered that rs7982, rs11136000, rs1532278 and rs9331888 were in LD. The haplotypes (GCCG, ATTC) were related to the levels of amyloid deposition. Thus the haplotype-based analysis validated that CLU genotypes were related to the levels of amyloid deposition. The results presented here are not only correlative, but also support that CLU modulates the alteration of the biomarkers of Aβ markers to influence the risk of AD in vivo.

To date, continuous variable phenotypic analysis is now widely used to elucidate the specific role of genetics of multiple diseases. Distincted from the previous two categorical variable analysis (case vs control), the phenotypic analysis can not only be more sensitive to the association between genetic mutation and AD, but also provide more intuitively to explain the specific genetic effects on brain structure and function58. To date, numbers of GWAS–validated or GWAS-promising candidate loci have been certificated that they influence imaging and clinical features in AD40,59,60,61.

The advantage of our study is the method we use. Imaging genetics is an emergent transdisciplinary research field, in which genetic risk is assessed with imaging measures as quantitative traits (QTs) or continuous phenotypes. QT association studies have increased statistical power and decreased sample size requirements, thus imaging genetics studies have advantages over traditional case-control designs62,63. Although the differences across phenotypes with the same SNP might reflect power differences due to sample size differences, our findings that CLU modulates the alteration of the biomarkers of Aβ markers to influence the risk of AD in vivo were also supported that by animal studies from 10 years ago linking CLU/APOJ to amyloid deposition. Hence, the important role of this paper is that it confirmed the results of animal studies with in vivo neuroimaging data. However, the neuroimaging data were available only in a subset of participants in some QT analyses, e.g., half of participants with MRI information, 70% with FDG-PET, and 55% with AV45. Therefore, the QT analysis had a reduced sample size in some cases. Besides, the ADNI data was restricted to Caucasians to avoid genetics stratification across ethnicities. The 7 loci in CLU, however, have different frequencies in different races; therefore, our results cannot represent the other ethnicities, warranting the replications in other races.

In summary, our results showed that four loci (rs11136000, rs1532278, rs2279590, rs7982) showed significant associations with the Aβ deposition at the baseline level while genotypes of rs9331888 (P = 0.042) increased Aβ deposition. Besides, rs9331888 was significantly associated with baseline volume of left hippocampus (P = 0.014). We then further validated the association with Aβ deposition in the AD, mild cognitive impairment (MCI), normal control (NC) sub-groups. The results in sub-groups confirmed the association between CLU genotypes and Aβ deposition further. Moreover, our findings are also supported by animal studies from 10 years ago linking CLU/APOJ to amyloid deposition. These findings further supported the hypothesis that CLU genetic variations modulate the alteration of the biomarkers of Aβ markers to influence the risk of AD. These findings raise the possibility that the biological effects of CLU may be relatively confined to neuroimaging trait and hence may offer clues to the mechanisms through which particular genetic variants might influence AD risk.

Methods

ADNI dataset

The data in this study were obtained from Alzheimer’s Disease Neuroimaging Initiative (ADNI)64. ADNI is a large, multicenter, longitudinal neuroimaging study, launched in 2003 by the National Institute on Aging, the National Institute of Biomedical Imaging and Bioengineering, the Food and Drug Administration, private pharmaceutical companies, and nonprofit organizations65. The initial goal of ADNI is to recruit 800 subjects. However, it has been followed by ADNI-GO and ADNI-2. Thus these three protocols have covered more than 1500 adults who are 55 to 90 years old to participate in the research, including cognitively normal (CN) older individuals, mild cognitive impairment (MCI), and early dementia patients with due to AD66. The study was approved by the institutional review boards of all participating centers (Ocean University of China, Qingdao Municipal Hospital, Nan**g First Hospital, Memory and Aging Center in University of California, and ADNI) and written informed consent was obtained from all participants or authorized representatives. In addition, the methods were carried out in accordance with the approved guidelines.

Participants

Participants were screened and enrolled according to criteria demonstrated in the ADNI study protocol (http://www.adni-info.org/scientists/adnistudyprocedures.aspx). We restricted the participants to whose genotype data of CLU SNPs were available and comprised 812 individuals. Baseline and longitudinal data including structural MRI and PET results were collected and all participants underwent a battery of clinical tests including Clinical Dementia Rating scale sum of boxes (CDRSB), Alzheimer’s disease Assessment Scale (ADAS-cog), Mini-Mental State Exam (MMSE), Rey Auditory Verbal Learning Test (RAVLT) and Functional Activities Questionnaire (FAQ) at baseline. According to the National Institute of Neurological and Communication Disorders/Alzheimer’s Disease and Related Disorders Association criteria for probable AD (NINCDS-ADRDA: probable AD), participants of AD were included if with a MMSE score between 20 and 26, a global Clinical Dementia Rating (CDR) of 0.5 or 1.0 and a CDRSB of 1.0 to 9.0. Amnestic MCI subjects achieved a MMSE score of 24 to 30 as well as a CDR score of 0.5 while the cognitively normal control individuals with a CDR score of 0. Furthermore, in this study, subjects with any serious neurological disease except for possible AD, any history of brain lesions or trauma, or psychoactive medication use (including antidepressants, neuroleptics, chronic anxiolytics, or sedative hypnotics) were excluded. In order to avoid population stratification effects which can lead to spurious genetic associations, we performed the principal component analysis (PCA). We assigned genotype-determined ancestry by comparing ADNI patients and populations form HapMap Phase 3 data and only individuals clustering with European HapMap samples were retained in our study.

SNP selection and Genoty**

Seven AD associated SNPs were selected for analysis.They have been validated to associate with AD in ethnically distinct populations7,8,9,10,11,21,28,67: rs2279590, rs11136000, rs9331888, rs7012010, rs9331949, rs7982, rs1532278. CLU genotypes were extracted from the ADNI GWAS PLINK format data68. We performed the quality control (QC) procedures using PLINK software. The inclusion criteria were as follows: minimum call rates >90%, minimum minor allele frequencies (MAF) > 0.01, Hardy-Weinberg equilibrium test P > 0.001.

PET measure-Aβ deposition

PET imaging data with amyloid tracer, florbetapir (AV-45), were obtained from UC Berkeley-AV45 analysis dataset on website (http://adni.loni.usc.edu/data-samples/access-data/). This institute used a native-space MRI scan for each subject which is segmented with Freesurfer (version 4.5.0) to define cortical grey matter regions of interest (ROI) (frontal, anterior/posterior cingulate, lateral parietal, lateral temporal) that make up a summary cortical ROI69,70. Notebly, the whole cerebellum was defined as reference region. Each florbetapir scan was applied to the corresponding MRI and mean florbetapir uptake within the cortical and reference region was calculated. Finally, SUVRs were created by averaging across the 4 cortical regions and dividing the cortical summary ROI by the whole cerebellum.

CSF Protein

CSF samples were collected and transported to the ADNI Biomarker Core laboratory at the University of Pennsylvania Medical Center in dry ice. Preparation of aliquots (0.5 ml) from the collected samples was conducted after thawing (1 h) at room temperature and gentle mixing. The aliquots were stored in bar code–labeled polypropylene vials at −80° C environment. The CSF proteins, including Aβ1-42, Total-tau and Phosphorylated tau181p, were calculated using the multiplex xMAP Luminex platform (Luminex Corp, Austin, TX) with Innogenetics (INNO-BIA AlzBio3; Ghent, Belgium; for research use-only reagents) immunoassay kit–based reagents. Additional analysis details and quality control procedures are showed at site (http://adni.loni.ucla.edu).The measurements of CSF biomarker for this article were cross-sectional from the baseline evaluation. Finally, a total of 501 individuals with genetic and other information were included in CSF analysis from the ADNI sites.

MRI structure

Our study used UCSF FreeSurfer datasets to conduct association test of CLU genotypes with brain structure. The cerebral image segmentation and analysis were performed with the FreeSurfer version 5.1 (http://surfer.nmr.mgh.harvard.edu/) based on the 2010 Desikan-Killany atlas71. We obtained data from motion correction and averaging of multiple volumetric T1 weighted images (when more than one is available), removal of non-brain tissue using a hybrid watershed/surface deformation procedure, automated Talairach transformation, segmentation of the subcortical white matter and deep gray matter volumetric structures (including hippocampus, amygdala, caudate, putamen, ventricles)72, intensity normalization, tessellation of the gray matter white matter boundary, automated topology correction, and surface deformation following intensity gradients to optimally place the gray/white as well as gray/cerebrospinal fluid borders at the location where the greatest shift in intensity defines the transition to the other tissue class. The technical details of these procedures are described in prior publications73.

PET measure-Glucose metabolism

FDG analysis data were from UC Berkeley and Lawrence Berkeley National Laboratory on the website (http://adni.loni.usc.edu/data-samples/access-data/)74. In this laboratory, five regions (left and right angular gyrus, bilateral posterior cingulate, left and right temporal gyrus) were treated as metaROIs (regions of interest) to analysis. Firstly, we downloaded the PET data from LONI (http://loni.usc.edu/). Then these images were spatially normalized in SPM to the MNI PET template. The mean counts from the metaROIs for each subject’s FDG scans at each time point were extracted and the intensity values were computed with SPM subroutines. Finally, the mean of the top 50% of voxels within a hand-drawn pons/cerebellar vermis region which was hand-drawn on a T1 template in MNI space was extracted. In addition, each metaROI mean was normalized by dividing it by pons/vermis reference region mean75.

Statistical Analysis

Differences in continuous variables were examined using one-way analysis of variance (ANOVA), and categorical data were tested using χ2 test. ADNI sample were stratified into three groups (CN, MCI and AD) to detect the effects of CLU genetic variations on neuroimaging phenotypes in the three clinical stages respectively. Moreover, we used a multiple linear regression model which considered age, gender, education, and ApoE ε4 status as covariates to estimate coefficients for testing possible correlation between various phenotypes and CLU genotypes. All statistical analyses were performed by R 3.12 and PLINK 8 (http://pngu.mgh.harvard.edu/wpurcell/plink/). To control multiple hypothesis testing, we used the false discovery rate (FDR) for correction76 and statistical significance was defined for FDR-corrected P < 0.05.

Additional Information

How to cite this article: Tan, L. et al. Effect of CLU genetic variants on cerebrospinal fluid and neuroimaging markers in healthy, mild cognitive impairment and Alzheimer’s disease cohorts. Sci. Rep. 6, 26027; doi: 10.1038/srep26027 (2016).