Neuroscience Letters 593 (2015) 56–60

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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet

Research article

CYP2J2 rs890293 polymorphism is associated with susceptibility to Alzheimer’s disease in the Chinese Han population Huacheng Yan a,b , Yanying Kong a,c , Baoxia He d , Mukun Huang a , Jian Li a , Jiaqiang Zheng a , Lei Liang a , Jianjun Bi a , Shujin Zhao a , Lei Shi a,∗ a

Department of Pharmacy, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou 510010, China Laboratory of Molecular Biology, Center for Disease Control and Prevention of Guangzhou Military Command, Guangzhou 510507, China c Department of Pharmacy, Guangzhou First People’s Hospital, Guangzhou 510180, China d Department of Pharmacy, Henan Cancer Hospital, Zhengzhou 450003, China b

h i g h l i g h t s • The CYP2J2 rs890293 T allele and GT + TT genotype are associated with risk of LOAD. • The involvement of CYP2J2 in LOAD is independent of ApoE-4. • AD pathogenesis involves CYP epoxygenase pathway and arachidonic acid metabolism.

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Article history: Received 10 February 2015 Received in revised form 9 March 2015 Accepted 16 March 2015 Available online 18 March 2015 Keywords: Alzheimer’s disease Apolipoprotein E Cytochrome P450 2J2 Epoxyeicosatrienoic acids Single nucleotide polymorphism

a b s t r a c t Alzheimer’s disease (AD) is a common neurodegenerative disorder characterized by progressive cognitive dysfunction and memory loss. Increasing evidence indicates that inflammation in the brain is a powerful factor in AD progression. Epoxyeicosatrienoic acids, the biologically active derivatives of arachidonic acid, synthesized by cytochrome P450 (CYP) epoxygenases, have been proven to have powerful anti-inflammatory effects. The aim of this study was to examine whether polymorphism in CYP2J2, encoding one of the most common CYP epoxygenase isoforms, is associated with late-onset AD (LOAD). This case-control study genotyped 672 representatives of the Chinese Han population, including 321 LOAD patients and 351 healthy controls matched for age and gender, for the functional rs890293 polymorphism within CYP2J2 by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS). The CYP2J2 rs890293 T allele and GT + TT genotype were significantly associated with an increased risk of LOAD. Further data stratification according to the presence of the apolipoprotein E (APOE) e4 allele confirmed a strong association between CYP2J2 rs890293 and LOAD, and indicated that the involvement of CYP2J2 in LOAD was independent of ApoE-4. Our study demonstrated that CYP2J2 rs890293 is a possible predisposing genetic factor for progression of LOAD. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Alzheimer’s disease (AD) is the most common form of dementia in the elderly population, characterized by progressive deterioration in cognitive function and behavior [31]. One in 10 people over the age of 65 and nearly half of those over the age of 85 suffer from

Abbreviations: AA, arachidonic acid; A␤, ␤-amyloid; AD, Alzheimer’s disease; ApoE, apolipoprotein E; CIs, confidence intervals; CYP, cytochrome P450; EETs, epoxyeicosatrienoic acids; HWE, Hardy–Weinberg equilibrium; LOAD, late-onset AD; MMSE, mini-mental state examination; ORs, odds ratios; SNPs, single nucleotide polymorphisms. ∗ Corresponding author. Tel.: +86 20 88653436; fax: +86 20 88653436. E-mail address: lucyshi622. [email protected] (L. Shi). http://dx.doi.org/10.1016/j.neulet.2015.03.024 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.

AD [29]. Currently, the prevalence of AD is more than 35 million people worldwide; out of them, 6 million are in China [37]. Despite significant advances in AD research over the past three decades, the cause of AD remains incompletely understood and no effective treatment is available to stop or reverse AD progression. AD patients usually die within 3–9 years after the diagnosis [24,29], which makes AD the fourth leading cause of death after cancer, cardiovascular disease, and stroke in developed countries. Pathological characteristics of AD include extracellular deposits of ␤-amyloid (A␤) protein outside of the brain neurons and formation of senile plaques, hyperphosphorylation of tau proteins in the neurons of the frontal cortex, formation of neurofibrillary tangles, loss of cortical neurons, vascular amyloidosis, inflammatory infiltration, and oxidative stress [4]. Accordingly, there are multiple

H. Yan et al. / Neuroscience Letters 593 (2015) 56–60

theories about AD pathogenesis, which consider the involvement of protein oligomerization [14], gene mutations [3,5], synaptic inactivation [25], mitochondrial dysfunction [16], inflammation [23], and abnormal metabolism of cholesterol [19]. However, until now, the etiology and pathogenesis of AD are still not clear, and many epidemiological studies argue that AD develops as a result of complex interactions between multiple genetic and environmental factors [4]. With the advances in molecular biology and the rapid development of the human genome project, the discovery of genes underlying AD susceptibility and the elucidation of pathogenic mechanisms underlying AD at the genome level have become the focus of medical research. To date, several genetic features have been identified as being involved in AD development. Early-onset familial AD has been associated with mutations in the genes encoding the amyloid precursor protein and presenilin (PS)-1 and PS-2, while the e4 allele of the apolipoprotein E gene (APOE) has been linked to lateonset AD (LOAD) [4]. In addition, genome-wide association studies have revealed that several single nucleotide polymorphisms (SNPs) within genes such as CLU, PICALM, CR1, SORL1, TREM2, CD33, CD2AP, BIN1, EPHA1, MS4A4A, and CYP46A1 are closely related to the risk of developing LOAD [15,26]. However, these genetic abnormalities could not fully account for AD progression, indicating that other genes may be involved in LOAD aetiology. Increasing evidence indicates that the onset and development of AD are often accompanied by chronic inflammation, which not only plays critical roles in neuronal death, but also drives AD pathogenesis and progression [2,12]. Recent studies have demonstrated that cytochrome P450 (CYP) epoxygenases, which convert arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs), exert powerful anti-inflammatory effects [7]. Furthermore, a post-mortem study has shown that the protein and mRNA levels of CYP epoxygenases in the brain of AD patients are significantly lower than those in the brains of healthy individuals [30]. These observations led us to hypothesize that the decrease in expression or loss of function of CYP epoxygenases in the brain may play an important role in the pathogenesis and progression of AD. In humans, CYP2J2 is one of the predominant CYP epoxygenase isoforms; it is abundantly expressed in the heart and is also present in many regions of the brain, especially in the cerebral cortex and hippocampus [10], which correlates with A␤ distribution in the brains of AD patients [34]. CYP2J2 is located on human chromosome 1p31.3–31.2 [22]; currently, 799 CYP2J2 polymorphisms have been identified (http://www.ncbi.nlm.nih.gov/snp, access date: 3 August, 2014). One of the most relevant polymorphisms in terms of frequency and functional importance, rs890293 (G/T), is located at −50 bp in the proximal CYP2J2 promoter region [36]. It has been shown that rs890293 is associated with susceptibility to various diseases, including coronary artery disease [32], atherosclerosis [18], myocardial infarction [21], and essential hypertension [1,28]. In addition, carriers of the CYP2J2 rs890293 T allele have reduced levels of CYP2J2 mRNA in the heart, possibly due to its interference with a putative binding site for the transcription factor Sp1 [32]. In this case-control study, we examined the possible association of the rs890293 SNP in CYP2J2 with susceptibility to LOAD in the Chinese Han population and further stratified the distribution of rs890293 alleles according to the presence of the APOE e4 allele.

2. Patients and methods 2.1. Subjects Informed consent was obtained from each participant or his/her legal guardians, and the protocol of this study was approved by the

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Ethics Committee of Guangzhou General Hospital of Guangzhou Military Command. Patients diagnosed with LOAD were recruited from the Home for the Aged Guangzhou, Guangzhou Brain Hospital, and Guangzhou General Hospital of Guangzhou Military Command from 2010 to 2013. In total, 321 LOAD patients and 351 healthy controls, matched for age and gender, were enrolled in this study. All participants were unrelated and belonged to the Chinese Han population. Clinical diagnosis of probable AD was made according to the revised criteria of National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer’s Disease and Related Disorders Association (NINCDS/ADRDA), as described previously [8]. Other inclusion criteria were the absence of advanced, severe, progressive, or unstable infectious, metabolic, immunologic, endocrinological, hepatic, hematological, pulmonary, cardiovascular, gastrointestinal, and/or urological diseases. Blood glucose (GLU), triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured using a 7100 Automatic Biochemical Analyser (Hitachi Ltd., Tokyo, Japan). A mini-mental state examination (MMSE) was performed on all participants by means of a questionnaire.

2.2. Genotyping Genomic DNA was isolated from peripheral blood leukocytes, according to the standard procedures by using commercial DNA isolation kits (Tiangen, Beijing, China). We performed genotyping for CYP2J2 rs890293 and APOE rs429358 and rs7412 polymorphisms by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) by using the Sequenom MassARRAY system (Sequenom Inc., San Diego, CA, USA). Primers used for genotyping were designed manually: rs890293 forward, 5 -ACGTTGGATGTTTTCTGAGACCGGTGCGTG-3 and reverse, 5 -ACGTTGGATGTAGGAGAGTCCGAGGATGGA-3 ; rs429358 forward, 5 -ACGTTGGATGGCTGGGCGCGGACATGGAG3 and reverse 5 -ACGTTGGATGTGCACCTCGCCGTGGTACT-3 ; rs7412 forward, 5 -ACGTTGGATGTAAGCGGCTCCTCCGCGAT-3 and reverse, 5 -ACGTTGGATGGCCCCGGCCTGGTACACTG-3 . Genotyping was conducted using the iPLEX system, as previously described [13,35] and consisted of the following steps: PCR was performed in a reaction mixture volume of 5 ␮L, which included 1.9 ␮L water, 0.5 ␮L 10 × PCR buffer (with 20 mM MgCl2 ), 0.4 ␮L 25 mM MgCl2 , 0.1 ␮L 25 mM dNTP, 0.1 ␮L HotStarTaq DNA polymerase, 1 ␮L of each primer, and 1 ␮L DNA template. Amplification was performed as follows: 94 ◦ C for 15 min for initial denaturation, followed by 45 cycles each consisting of 94 ◦ C for 20 s, 56 ◦ C for 30 s, and 72 ◦ C for 1 min, and a final extension at 72 ◦ C for 3 min. Following PCR, 0.5 U of shrimp alkaline phosphatase (SAP, Sequenom, San Diego, CA, USA) was added to 5 ␮L of PCR product to dephosphorylate residual dNTPs. The mixtures were incubated at 37 ◦ C for 45 min, followed by enzyme inactivation at 85 ◦ C for 5 min. A primer extension reaction was performed in a total volume of 9 ␮L containing 7 ␮L of SAP-treated PCR product and 2 ␮L of iPLEX reaction mix (extension primers, the enzyme, mass-modified ddNTPs, and buffer; Sequenom). Thermal cycling conditions included a denaturation step at 94 ◦ C for 30 s, followed by 40 cycles, each consisting of 94 ◦ C for 5 s and 5 cycles of 52 ◦ C for 5 s and 80 ◦ C for 5 s, and a final extension step at 72 ◦ C for 3 min. Prior to the analysis, ion exchange resin (SpectroCLEAN; Sequenom) was added directly to the products of the primer extension reaction to remove Na+ , K+ , and Mg2+ ions. The primer extension products were spotted using SpectroCHIP (MassARRAY Nanodispenser, Samsung, Korea) and detected using a MassARRAY Compact Mass spectrometer (Sequenom) and the Sequenom real-time detection software (TYPER4.0 software).

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Table 1 Demographic and clinical characteristics of the study population. Characteristics

Patients (n = 321)

Age (years) Gender (male), n (%) GLU (mmol/L) TG (mmol/L) TC (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) MMSE (scores) APOE e4, n (%)

Controls (n = 351) 80.38 ± 6.82 119 (33.9) 5.81 ± 1.86 1.03 ± 0.64 4.05 ± 1.17 1.47 ± 0.39 2.92 ± 0.88 28.17 ± 4.86 55 (15.7)

79.657.91 107 (33.3) 6.02 ± 2.14 1.67 ± 0.85 4.94 ± 1.06 1.22 ± 0.36 3.03 ± 1.01 16.12 ± 5.62 116 (36.1)

t(␹2 )

P-value

−1.624 0.024 1.039 4.093 2.462 −1.527 0.076 6.628 37.024

0.165 0.876 0.258

CYP2J2 rs890293 polymorphism is associated with susceptibility to Alzheimer's disease in the Chinese Han population.

Alzheimer's disease (AD) is a common neurodegenerative disorder characterized by progressive cognitive dysfunction and memory loss. Increasing evidenc...
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