Int J Clin Exp Med 2019;12(1):1269-1275 www.ijcem.com /ISSN:1940-5901/IJCEM0073744 Original Article Association between the ADAMT33 variant and carotid artery intima-media thickness in the Chinese Han population Xiaolin Zhang, Liwen Liu, Ruoxi Gu, Xiaozeng Wang Cardiovascular Research Institute and Department of Cardiology, The General Hospital of Northern Theater Command, 83 Wen Hua Road, Shenyang, Liaoning Province, China Received January 31, 2018; Accepted October 8, 2018; Epub January 15, 2019; Published January 30, 2019 Abstract: Background: The ADAM33 with a disintegrin domain and a metalloprotease domain attaches an important role in regulating smooth vascular cell migration and proteolysis. In the present study, we investigated the associa- tion between ADAM33 variants and carotid artery intima-media thickness (CIMT) in the Chinese Han population. Methods: In a community population (n=620), CIMT was determined using the ultrasound to detect the carotid artery intima-media thickness. We screened the ADAM33 variations using PCR-direct sequencing method and in- vestigated the relationship between ADAM33 variations and CIMT in Chinese Northern Han population. Results: The ADAM33 expression was increased in the atherosclerotic carotid artery from CIMT patients compared with the normal subjects by the immunohistochemical staining. Furthermore, ADAM33 rs514174 was closely related to the increased risk of CIMT patients (OR=1.43, 95% CI: 1.08-1.89, P≤0.05). In addition, the rs514174 TT genotype of ADAM33 was significantly associated with the increased ADAM33 mRNA expression in patients with CIMT (P<0.05). Conclusion: Our study provides the further support for the ADAM33 rs514174 variant as a direct risk factor for CIMT. The ADAM33 rs514174 variant attaches an important role for the early identification occurrence of CIMT. Keywords: ADAM33, carotid intima-media thickness, carotid plaque, polymorphism Introduction pathogenic mechanisms between CAD and stroke and susceptibility to CIMT [4, 5]. Atherosclerosis, leading to the occurrence of coronary artery disease (CAD) and stroke, is a CIMT is a reliable marker for early atherosclero- major cause of morbidity and mortality in the sis and can predict future cardiovascular events industrialized countries [1]. However, the patho- such as CAD and stroke. Moreover, CIMT is usu- genesis of atherosclerosis is poorly understood ally used as an intermediate phenotype for the and new methods are required to predict the atherosclerosis in the clinical studies. CIMT has existing atherosclerosis risk at present [2, 3]. the significant heritability and several genetic variants related to risk of CAD and stroke have During the process of atherosclerosis, the been subsequently reported to be associated extracellular matrix remodeling attaches an with the interindividual variability in the athero- important role in the atherosclerotic plaque sclerotic process of CIMT [6]. Thus, the mecha- development, instability and rupture. Carotid nism that these genetic variants influencing the intima-media thickness (CIMT), as a reliable atherosclerosis process of CIMT may be similar marker can early predicting the occurrence of to that which will contribute to alter the suscep- future atherosclerosis events [1, 4]. From the tibility to CAD and stroke [7]. recent genome-wide association mapping stud- ies, several genetic variants known to be relat- The ADAMs containing a disintegrin and metal- ed to the risk of CAD and stroke have also been loprotease domain are the transmembrane and found to be associated with the occurrence of secretory proteins belonging to the zinc prote- CIMT risk, suggesting that there may be similar ase superfamily. ADAM33, as a member of the ADAMT33 and CIMT ADAMs family locates on chromosome 20 p13 DNA isolation and genotype and contains the 22 exons [8]. ADAM33 palys an important role in the inflammation and vas- We used the modified salt-extraction method culature regenerative processes, which plays to isolate the DNA from the 5 ml whole blood the key roles in the process and development from the 620 subjects. Following the manufac- of atherosclerosis. For example, ADAM33 was turer’s instruction (Tiangen Biotech Co. Ltd., reported to be expressed in the atherosclerotic Beijing, China) genomic DNA were collected plaques and there was a significant association from the blood leukocyte pellets [12]. The between ADAM33 rs574174 variant and the ADAM33 forward and reverse primer such as atherosclerosis severity in a cohort of CAD pa- 5’-CTCTCCAGATGCTGGCATCG-3’ and 5’-TGTTT- tients in the Caucasian population [9, 10]. TAAGGAACATCACA-3’ were designed by the However, the association between the ADAM33 Primer 5.0 software. Through the direct PCR sequencing (http://www.sangon.com/) the variants and CIMT in carotid plaques patients ADAM33 rs514174 variant was determined. was unknown in the Chinese Han population. The resulting fragments were separated on the Therefore, we performed a case-control study 2% agarose gel stained with ethidium bromide to determine the association between the and visualized under ultraviolet light. All the ADAM33 variations and CIMT to investigate subjects genotypes were retyped twice by the whether ADAM33 rs514174 variation was asso- independent investigators who did not know ciated with CIMT. the subjects’ identities and phenotypes. Methods RNA extraction and real-time quantitative PCR (RT-qPCR) amplification Study population The Ficoll gradient density centrifugation meth- od was used for the peripheral blood mononu- 620 consecutively recruited subjects aged clear cell (PBMC) separation. PBMCs were 50-75 years from the General Hospital of resuspended in RPMI medium with 10% fetal Northern Theater Command outpatients were bovine serum. Total RNA was extracted from included in our study. We collected the carotid PBMCs using Trizol reagent (Invitrogen, USA) artery intima-media thickness measurements and reverse-transcribed into cDNA using the by the ultrasound, blood samples were collect- appropriate reagents, including random hexam- ed through the selected cases, and the full ers, Superscript II, and dNTPs (TaKaRa Bio, clinical data for each individual were gathered Japan). Using the ABI Prism 7300 Sequence by the full-time staff. This study conformed well Detection System (Applied Biosystems) the RT- with the principles outlined in the Declaration qPCR was performed in a final volume of 20 µl of Helsinki. Signed informed consent for par- with the SYBRPremix Ex Taq II (TaKaRa Bio, ticipation in the study was obtained from all Japan). ADAM33 forward and reverse primers individuals. were the 5’-TCTCATGAGCCCAGCAGCCA-3’ and Ultrasound imaging the 5’-CAAGCTGCCTGCAGGTGCTG-3’. β-Actin forward and reverse primers were 5’-AGCGA- Carotid artery ultrasound as a valuable data to GCATCCCCCAAAGTT-3’ and 5’-GGGCACGAAGG- investigate the value of CIMT was performed CTCATCATT-3’. Furthermore, PCR reactions we- using a portable ultrasound machine (HI VISION re duplicated for each sample and mean valu- Preirus, Hitachi Medical Systems, Japan). Opti- es were used for the further analysis. We used mized images of left and right carotid artery the relative quantification to evaluate the tar- intima-media thickness were selected and fro- get expression of ADAM33 gene, and the hou- zen at the end of diastollic heart. The CIMT of sekeeping gene expression of β-actin was used the common carotid artery was measured by as an internal control to normalize the target the ultrasonic software at both the near and far ADAM33 mRNA expression. walls of the left and right sides in an area Carotid arterial tissue free of atherosclerotic plaque. The ultrasound parameters were captured and analyzed using 10 consecutive patients with the internal carot- semi-automated software analysis for each id stenosis (≥70%) treated with carotid endar- carotid region. The instrument and methods terectomy at the General Hospital of Shenyang used were as previously described [11]. Military Region. Carotid plaques were removed 1270 Int J Clin Exp Med 2019;12(1):1269-1275 ADAMT33 and CIMT Table 1. The Characteristics of subjects between the U test to compare among the different patients with CIMT and control subjects groups, and then multiple groups compari- Control Subjects Patients with sion by one-way ANOVA were assessed. Variables P (n=310) CIMT (n=310) The mRNA expression level between CIMT Mean age (years) 64.7±9.6 65.5±10.3 >0.01 patients and control subjects were also analysed by ANOVA. The distribution of Male/Female 193/117 189/121 Match genotype and allele frequencies between BMI (kg/m2) 26.1±2.5 26.8±3.2 >0.01 CIMT patients and control groups were DM (%) 38 (12.2) 78 (25.1) <0.01 analysed by the Hardy-Weinberg equilibri- Hypertension (%) 65 (20.9) 180 (58.1) <0.01 um. The CIMT patients were evaluated by Smokers (%) 56 (18.1) 152 (49.0) 0.01 the P values, 95% confidence intervals TG (mmol/l) 2.11±0.98 2.33±1.27 <0.01 (95% CIs) and odds ratios (ORs). The popu- TC (mmol/l) 4.33±1.05 4.75±1.09 <0.01 lation size of 620 individuals is sufficient LDL (mmol/l) 2.28±0.56 2.63±0.52 <0.01 to detect a 2% variance explained by a HDL (mmol/l) 1.20±0.28 1.53±0.31 <0.01 single variant on CIMT at α=0.05 and HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipo- power of 80%. protein cholesterol; TC, total cholesterol; TG, triglyceride. BMI, body mass index; DM, diabetes mellitus. Continuous data are expressed Results as the mean ± SD. P<0.05 versus control group. Baseline characteristics from the patients using the carotid endarterec- The baseline characteristics of the study par- tomy and snap-frozen on dry ice and stored at ticipants were shown in Table 1. CIMT mea- -80°C. Carotid artery tissues containing the surements and DNA for genotyping analysis atherosclerotic lesions and apparently normal were available for all the collected subjects. mural areas were collected from the athero- Genotype frequencies were in Hardy-Weinberg sclerosis patients (6 males, 4 females; aged equilibrium.