SMYD2 promoter DNA methylation is associated with abdominal aortic aneurysm (AAA) and SMYD2 expression in vascular smooth muscle cells

Bradley J Toghill,Nicolas Sylvius,Athanasios Saratzis,Matthew J Bown,Peter J Freeman

doi:10.1186/s13148-018-0460-9

Bradley J Toghill, Nicolas Sylvius + Show 3 more

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https://doi.org/10.1186/s13148-018-0460-9

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Abstract

BackgroundAbdominal aortic aneurysm (AAA) is a deadly cardiovascular disease characterised by the gradual, irreversible dilation of the abdominal aorta. AAA is a complex genetic disease but little is known about the role of epigenetics. Our objective was to determine if global DNA methylation and CpG-specific methylation at known AAA risk loci is associated with AAA, and the functional effects of methylation changes.ResultsWe assessed global methylation in peripheral blood mononuclear cell DNA from 92 individuals with AAA and 93 controls using enzyme-linked immunosorbent assays, identifying hyper-methylation in those with large AAA and a positive linear association with AAA diameter (P < 0.0001, R2 = 0.3175).We then determined CpG methylation status of regulatory regions in genes located at AAA risk loci identified in genome-wide association studies, using bisulphite next-generation sequencing (NGS) in vascular smooth muscle cells (VSMCs) taken from aortic tissues of 44 individuals (24 AAAs and 20 controls). In IL6R, 2 CpGs were hyper-methylated (P = 0.0145); in ERG, 13 CpGs were hyper-methylated (P = 0.0005); in SERPINB9, 6 CpGs were hypo-methylated (P = 0.0037) and 1 CpG was hyper-methylated (P = 0.0098); and in SMYD2, 4 CpGs were hypo-methylated (P = 0.0012).RT-qPCR was performed for each differentially methylated gene on mRNA from the same VSMCs and compared with methylation. This analysis revealed downregulation of SMYD2 and SERPINB9 in AAA, and a direct linear relationship between SMYD2 promoter methylation and SMYD2 expression (P = 0.038). Furthermore, downregulation of SMYD2 at the site of aneurysm in the aortic wall was further corroborated in 6 of the same samples used for methylation and gene expression analysis with immunohistochemistry.ConclusionsThis study is the first to assess DNA methylation in VSMCs from individuals with AAA using NGS, and provides further evidence there is an epigenetic basis to AAA. Our study shows that methylation status of the SMYD2 promoter may be linked with decreased SMYD2 expression in disease pathobiology. In support of our work, downregulated SMYD2 has previously been associated with adverse cardiovascular physiology and inflammation, which are both hallmarks of AAA. The identification of such adverse epigenetic modifications could potentially contribute towards the development of epigenetic treatment strategies in the future.

Highlights

Abdominal aortic aneurysm (AAA) is a deadly cardiovascular disease characterised by the gradual, irreversible dilation of the abdominal aorta
Global DNA methylation and homocysteine analysis in AAA patients and controls Global genomic DNA methylation was assessed in peripheral blood DNA of 185 individuals, and circulating homocysteine (HCY) was assessed in blood plasma from 137 of the same individuals using enzyme linked immunosorbent-assay (ELISA)
Global DNA methylation was significantly higher in men with large AAA (> 55 mm, n = 48, global DNA methylation 1.86% (± 0.6%)) compared to men with small AAA (30–55 mm, n = 45, global DNA methylation 0.93% (± 0.52)) and controls (< 25 mm, n = 92, global DNA methylation 0.79% (± 0.43%)) (Fig. 1a)

Summary

Introduction

Abdominal aortic aneurysm (AAA) is a deadly cardiovascular disease characterised by the gradual, irreversible dilation of the abdominal aorta. Our objective was to determine if global DNA methylation and CpG-specific methylation at known AAA risk loci is associated with AAA, and the functional effects of methylation changes. Abdominal aortic aneurysm (AAA) is a degenerative cardiovascular disease and a global health concern. AAA is responsible for between 2 and 4% of deaths in white males over the age of 65 [1,2,3]. Progressive aneurysm growth may lead to rupture, characterised by internal aortic haemorrhage, which results in death in approximately 80% of cases. It is well known that AAA is multifactorial with known risk factors that contribute towards aneurysm development (smoking, male sex, increased age, white European ancestry, atherosclerosis and hyperlipidaemia) [5, 6]. 10 genomic risk loci have been identified through the conduct of genome-wide association studies (GWASs) [9,10,11,12,13,14,15,16]

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