MOLECULAR MEDICINE REPORTS 13: 4791-4799, 2016 Integration of gene expression and DNA methylation profiles provides a molecular subtype for risk assessment in atherosclerosis SHENG-CHAO MA1,2*, HUI-PING ZHANG3*, FAN-QI KONG2, HUI ZHANG2, CHENG YANG2, YANG-YANG HE2, YAN-HUA WANG2, AN-NING YANG2, JU TIAN2, XIAO-LING YANG2, MING-HAO ZHANG2, HUA XU2, YI-DENG JIANG2 and ZHENG YU1 1Department of Physiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, Sichuan 610041; 2Department of Pathophysiology, Basic Medical School, Ningxia Medical University; 3Department of Prenatal Diagnosis Center, General Hospital of Ningxia Medical University, Yinchuan, Ningxia Hui 750004, P.R. China Received April 27, 2015; Accepted February 29, 2016 DOI: 10.3892/mmr.2016.5120 Abstract. The aim of the present study was to identify an effec- panel (TIMP1, ABCA1, and ACAT1) may serve as a valuable tive method for detecting early-phase atherosclerosis (AS), as biomarker for the early detection of AS. well as to provide useful DNA methylation profiles to serve as biomarkers for the detection of AS. A total of 300 individuals Introduction (150 AS patients and 150 healthy subjects) were recruited for peripheral blood DNA methylation analyses at 12 gene Atherosclerosis (AS) of the carotid arteries is a major cause promoter loci using nested methylation‑specific polymerase of stroke and transient ischemic attack, which remains the chain reaction in a test set. Based on the test set, the promoter leading cause of mortality in China and is currently the most methylation of TIMP metallopeptidase inhibitor 1 (TIMP1), common cause of mortality worldwide (1,2). The symptoms ATP binding cassette subfamily A member 1 (ABCA1), and of AS are not always detected by patients until complications acetyl-CoA acetyltransferase 1 (ACAT1) were determined to arise and the therapeutic methods to treat AS are poor; there- be candidate biomarkers; demonstrating the highest sensitivity fore, it is particularly important to establish an appropriate, (88%) and specificity (90%). The biomarkers that were candi- rigorous and efficient detection strategy for early‑phase AS, in dates for early AS detection were validated in an independent order to limit disease progression before it results in clinical validation set (n=100). In the validation set, the combination consequences (3). There are two prevalent methods for diag- of TIMP1, ABCA1 and ACAT1 methylation achieved sensi- nosing AS: Doppler ultrasound and computed tomographic tivity, specificity and coincidence rate values of 88, 70 and angiography (CTA), which have become the standard methods 79%, respectively. In the current pilot study, the patterns of for diagnosing AS (4,5). However, neither of these methods DNA methylation of AS-associated genes were observed identify the early asymptomatic pathological changes of AS to be significantly altered in the peripheral blood of AS and ultrasound only assesses plaque in the carotid artery. patients. Thus, the AS‑specific methylation of the three‑gene Therefore, it is paramount to develop non-invasive methods for diagnosing high-risk, asymptomatic individuals before the onset of clinical events or symptoms (6,7). Furthermore, data supporting the routine use of Doppler ultrasound to screen for carotid stenosis in an asymptomatic population is considered to be weak, as there is a low overall prevalence of treatable Correspondence to: Professor Yi-Deng Jiang, Department of Pathophysiology, Basic Medical School, Ningxia Medical University, AS in the general asymptomatic population (8). It is widely Shengli Street, Yinchuan, Ningxia Hui 750004, P.R. China hypothesized that the next generation of screening tests will E-mail: [email protected] be based on molecular biomarkers (9). DNA methylation, a stable epigenetic mark, is a Professor Zheng Yu, Department of Physiology, West China School non-traditional, heritable factor involved in gene transcrip- of Preclinical and Forensic Medicine, Sichuan University, Chengdu, tion regulation (10). DNA methylation occurs at position 5' of Sichuan 610041, P.R. China E-mail: [email protected] CpG dinucleotides; certain regions of DNA are rich in CpG and are termed CpG islands (GCIs), which predominantly *Contributed equally locate in the promoter region (11). Changes in patterns of DNA methylation may lead to inappropriate gene expres- Key words: DNA methylation profiles, CpG islands, atherosclerosis sion, and contribute to the development and progression of disease (12). In addition, previous studies have demonstrated aberrant DNA methylation in AS (13). Alterations in DNA 4792 MA et al: DNA METHYLATION PROFILES AS A BIOMARKER FOR ATHEROSCLEROSIS methylation have been identified as an early event in AS; adhesion molecule 1 (ICAM1), vascular endothelial growth Zhao et al (14) found that aberrant methylation may represent factor A (VEGFA) and nuclear factor of κ light polypep- an early biomarker for AS, and demonstrated clinical corre- tide gene enhancer in B-cells 1 (NFKB1). These genes lations with carotid intima-media thickness (14). Current contained CGIs, were expressed in peripheral blood cells, research primarily focuses on the effect of DNA methylation and were correlated with the occurrence and development of leading to AS and, to the best of our knowledge, there are AS (19-24). DNA methylation of these candidate genes was few studies that investigate the role of DNA methylation on examined in two independent test sets (total of 300 periph- the early diagnosis of AS. Detection of DNA methylation, eral blood samples) and genomic DNA was isolated from at candidate loci in AS, suggests that AS‑specific methyla- the peripheral blood mononuclear cells using the Wizard tion changes could be applied diagnostically. However, DNA Genomic DNA Purification kit (Promega Corporation, methylation affects various factors, including age, living Madison, WI, USA). An EZ DNA Methylation-GoldTM kit environment, diet and other factors limit the use of meth- (Zymo Research Corporation, Irvine, CA, USA) was used ylation changes for diagnosis. Previous studies have shown to detect the DNA methylation patterns, and integrates the that simultaneous measure various differentially methylated DNA denaturation and bisulfite conversion processes into loci may improve diagnostic use (15). Furthermore, a major one step. The DNA was modified by sodium bisulfite (Zymo limitation towards further developments of DNA methyla- Research Corporation, Irvine, CA, USA) in which unmethyl- tion in clinical use may be that the majority of studies focus ated cytosine residues were converted to thymine, whereas on single genes (16), whereas AS is associated with multiple methylated cytosine residues were retained as cytosine; this factors, including genetic and epigenetic alterations (17). difference was then utilized to specifically amplify either Measuring the methylation of individual genes was not as methylated or unmethylated DNA. NT-MSP, which consists specific or sensitive as using a panel of epigenetic markers for of a two‑step PCR amplification, was then used to detect the early detection of breast cancer (18). methylation (25). The first step of NT‑MSP uses an outer In the present study, DNA methylation was profiled in primer pair, which does not contain any CpGs. The second AS and healthy groups of 300 patients using nested methyl- step of PCR was conducted with conventional PCR primers ation-specific polymerase chain reaction (PCR; NT-MSP). serving as inner primers. The primers for NT‑MSP amplifica- Based in previous unpublished data, a biomarker panel capable tion were designed according to the bioinformatics program, of differentiating AS patients from healthy individuals, MethPrimer (www.urogene.org/methprimer/index.html) and irrespective of AS histology, was identified. The present are presented in Table I. PCR products were gel purified study provides insight into AS etiology, presents validated with an agarose gel DNA fragment recovery kit, according tissue-based diagnostic biomarkers, and supplies a framework to the manufacturer's instructions, and were sequenced by for the development of DNA methylation-based molecular Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, diagnostics for AS detection in patients. USA). To reduce mispriming and to increase efficiency, touchdown (TD) PCR was used for amplification. Samples Materials and methods were subjected to 30 cycles in a TD program (94˚C for 5 min, 94˚C for 30 sec; 56˚C for 30 sec and 72˚C for 1 min, Ethics statement. The present study was reviewed and followed by a decrease of 0.5˚C in the annealing temperature approved by the Ethics Committee of General Hospital of every second cycle). After completion of the TD program, Ningxia Medical University (Yinchuan, China). Written 20 cycles were subsequently run (94˚C for 30 sec, 56˚C informed consent was obtained from the participants before for 30 sec and 72˚C for 60 sec), terminating with a 7‑min the collection of any samples and the specimens were irrevers- extension at 72˚C. The PCR products were separated by ibly de‑identified. electrophoresis through a 2% agarose gel (Borunlaite Science & Technology, Beijing, China) containing ethidium Inclusion and exclusion criteria. Patients with AS were bromide (Tokyo Chemical Industry Co., Ltd., Shanghai, diagnosed by Doppler ultrasound examination of the carotid China) for 30 min at 100 V. The DNA bands were visual- artery. The healthy control group comprised of age‑ and ized using