Clusterin gene (CLU), also known as apolipoprotein J (ApoJ), is a strong candidate gene for late-onset Alzheimer's disease (LOAD) according to the Alzgene database. To further characterize this association and to isolate the variants contributing to the pathogenesis of LOAD in Han Chinese, we first sequenced a small sample (n = 100) to discover variants in the promoter, exons, the 5′ and 3′ untranslated regions, and exon–intron boundaries of CLU. Follow-up genotyping of identified variants in a larger sample (n = 1592). Sequencing analysis identified 18 variants. Analysis in the larger population revealed that only the rs9331949 C allele was significantly associated with an increased risk of LOAD, even after adjusting for multiple testing (p = 0.026). Logistic analysis identified the rs9331949 polymorphism was still strongly associated with LOAD (additive model: p = 0.004, odds ratio = 1.274; dominant model: p = 0.039, odds ratio = 1.239; recessive model: p = 0.002, OR = 1.975) after adjusting for sex, age, and APOE ε4 status. Our findings implicate CLU as a susceptibility gene for LOAD in Han Chinese.
Alzheimer's disease (AD) is one of the most common neurodegenerative disorders; it is neuropathologically characterized by the formation of extracellular senile plaques from amyloid-β (Aβ) and intracellular neurofibrillary tangles containing hyperphosphorylated tau protein (Selkoe, 2001; Tan et al., 2013). It is thought to be highly heritable (heritability of 58%–79%) but genetically complex (Gatz et al., 2006). Mutations of presenilin 1 (PSEN1), presenilin 2 (PSEN2), and amyloid precursor protein (APP) genes are responsible for the familial early-onset form of the disease with Mendelian mode of inheritance (Bertram and Tanzi, 2012). However, the more common form of AD is generally considered as sporadic late-onset AD (LOAD) determined by multiple genes and environmental factors. By far, the ε4 allele of the apolipoprotein E (APOE) has been confirmed unequivocally to increase LOAD risk, but it cannot explain the full genetic variance of AD (Bekris et al., 2010). Therefore, many studies have been conducted to search for additional genes that potentially confer LOAD susceptibility (Barral et al., 2012; Ferrari et al., 2012; Komatsu et al., 2011; Lee et al., 2011;Naj et al., 2011). One of the candidate genes is clusterin (CLU, also called APOJ) on chromosome 8p21-p12, a chromosomal region of interest in LOAD (Butler et al., 2009; Wu et al., 2012; Yu and Tan, 2012). CLU is currently one of the most associated AD-risk genes on the list of top results of a continuously updated Internet site AlzGene (http://www.alzgene.org/), which provides meta-analyses for the growing list of AD candidate genes.
Clusterin or apolipoprotein J, functional similarities with APOE, is a multifunctional protein expressed at high levels in brain, ovary, testis, and liver (de Silva et al., 1990). More than 20 years ago, the first time it was reported that clusterin was associated with AD (May et al., 1990). This study done in the laboratory of Caleb Finch revealed that increased expression of clusterin was found in hippocampal samples of AD patients compared to those of age-matched controls. Since then there has been extensive research on the role of clusterin in AD pathogenesis (Gu et al., 2011; Schjeide et al., 2011; Wu et al., 2012; Yu and Tan, 2012). More and more evidence is being uncovered which points to association of clusterin with AD pathology (Wu et al., 2012; Yu and Tan, 2012). In brief, it has been revealed that the protein level of CLU was increased in the cerebrospinal fluid (CSF) and plasma of AD patients (Nilselid et al., 2006; Xing et al., 2012.), as well as the hippocampus and the frontal cortex in AD brain (Chen et al., 2012; Lidström et al., 1998). Besides, CLU mRNA is found to be significantly higher in AD affected brain areas than control brains (Oda et al., 1994). Moreover, the binding of CLU to Aβ promotes Aβ clearance through stimulation of endocytosis transport of Aβ across the blood–brain barrier (Bell et al., 2007), clears highly pathogenic Aβ42 from the central nervous system, and modulates its aggregation and deposition (Yerbury et al., 2007). In addition, CLU has been found to maintain Aβ solubility and protect against Aβ neurotoxicity (DeMattos et al., 2002; Matsubara et al., 1996). It is well known that the accumulation of Aβ in brain and Aβ toxicity in neuronal cells can contribute to the pathogenesis and progression of AD (Hardy and Selkoe, 2002; Zlokovic et al., 2005). All these observations suggest that the CLU gene could be considered a functional candidate gene for AD susceptibility.
Initially, Tycko et al. (1996) investigated the genetic association of the CLU polymorphisms and AD in a sample of African American, Hispanic, and white/non-Hispanic individuals, but found no association. Subsequently, a 2-stage genome-wide association study (GWAS) included nearly 15,000 individuals from Europe (Lambert et al., 2009), and another GWAS study involved a similar number of participants (>16,000) from Europe and the United States (Harold et al., 2009) identified that significant association between LOAD and single-nucleotide polymorphisms (SNPs) in the CLU locus. After these original observations, numerous GWASs and replication studies have examined the association between CLU polymorphisms in LOAD risk in independent cohorts, but the results have been conflicting (Carrasquillo et al., 2010; Corneveaux et al., 2010; Guerreiro et al., 2010; Kamboh et al., 2012; Yu et al., 2010). To investigate the involvement of the CLU gene in LOAD, a 2-step design study was carried out. We first sequenced in the promoter, exons, the 5′ and 3′ untranslated regions, and exon–intron boundaries of the CLU gene for mutations in a total of 50 LOAD patients and 50 controls. The identified variants were then partially selected to be analyzed in a much larger case-control sample (n = 1592), which was well matched for age, sex, and ethnic background in the Han Chinese.
