LPL methylation of peripheral blood leukocytes Prophet of atherothrombotic stroke

Background: Lipoprotein lipase (LPL) is a glycoprotein enzyme playing a pivotal role in energy and lipoprotein metabolism. It hydrolyzes triglyceride-rich lipoprotein and produces fatty acids as well as monoacylglycerol. Multiple risk factors including age, sex, hypertension, smoking and hyperlipidemia contribute to ischemic stroke (IS). Hyperlipidemia is an extremely pivotal risk factor for ischemic stroke especially atherothrombotic stroke (ATS) in that it can lead to intracranial or extracranial vessels’ atherosclerosis analogous to atherosclerosis of coronary artery. However, potential epigenetic mechanisms contributing to IS are not been explored thoroughly. The aim of this study was to illuminate relationship among individuals’ peripheral blood leukocytes methylation level of LPL-promoter-CpG dinucleotides, serum lipid and ATS of Chinese Han population in Hunan Province. Methods: Peripheral blood samples were collected from acute atherothrombotic stroke patients and healthy people, and methylation level of cytosine–phosphate–guanine (CpG) island in LPL promoter region was measured by qPCR and pyrosequencing. Biochemical and anthropometric variables have also been taken into consideration. Results: Integral LPL-promoter-CpG dinucleotides methylation level of case group is evidently higher than control group (38.8±8.4% percentile vs 25.7±6.6% percentile, P value=0.000). DNA methylation at the CpG9~16 locus and CpG20~21 locus was related to ATS after adjusting for gender, previous history of diabetes and hypertension, smoking, drinking, body mass index, and blood lipid levels(CpG9 49.3±24.9, 31.3±13.6, P value =0.02; CpG10 61.3±24.5, 34.0±18.4, P value =0.002; CpG11 71.3±17.3, 32.0±21.1, P value =0.000; CpG12 75.3±17.7,

CpG20 52.0±13.2, 27.3±20.9, P value =0.001; CpG21 55.3±11.9, 34.0±22.3, P value =0.004). There is no statistically significant connection between either carotid atherosclerosis or gender and methylation level. Besides, we found a negative association between LPL methylation status and HDL-C levels, whereas LPL gene methylation was linked with LDL-C levels and age positively.

Conclusion:
We illustrate that epigenetic modification of LPL methylation may have an influence on lipids metabolism and occurrence of ATS, which may be a promising indicator for occurrence risk of ischemic stroke. Whether these findings are valid need further warrant and future prospective.

Background
Epigenetics is a kind of biological phenomenon bringing about heritable alteration while void of DNA sequence change [1]. There are mainly three forms of epigenetic modifications: DNA modification, histone post-translational modifications (PTMs) and chromatin remodeling. Extrinsic factors especially lifestyle and environment can influence the epigenome, for example, exposure to cigarette smoke, nutrients of foods, stress exposure in early life and pollutants exposure in the environment [1].DNA methylation is a well-studied and stable epigenetic mark, occurring at position 5' of the cytosine pyrimidine ring, especially under the circumstance of guanine residues locating next to cytosine residues-CpG dinucleotides, a potential methylation site [2].
Stroke is ranking second among causes of death and adult disability all around the world [3]. Ischemic stroke(IS) accounts for 41-79% of all strokes (ischemic stroke, intracerebral hemorrhage and subarachnoid hemorrhage) statistically [4]. IS is resulted from arteries' occlusion or stenosis (intracranial or extracranial). It is associated with lifestyle features like smoking, high body-mass index and dietary habits and underlying diseases including hypertension, diabetes and hyperlipidemia. Beyond that, genes and epigenetics are increasingly playing a pivotal role in the pathophysiology[5].
Lipoprotein lipase (LPL) is a rate-limiting enzyme catalyzing triglyceride-rich lipoproteins (TLRs) hydrolysis and decreasing levels of chylomicrons (CM), low-density lipoproteins (LDL) and very low-density lipoproteins (VLDL) [6]. It has been proved that LPL plays a role in hypertriglyceridemia and atherosclerotic diseases such as coronary heart disease and atherothrombotic stroke [7], but specific mechanism has not been fully explored yet. We investigate connections of DNA methylation with ATS and correlated atherosclerotic sorts in ATS patients and focus on CpG loci locating in LPL promoter region. To our knowledge, this is the first article to illuminate the relationship between LPL gene methylation and ATS via detecting all of the CpG methylation loci locating in LPL promoter region and display that LPL gene can be an underlying indicator to evaluate occurrence risk of ATS, thus early intervention of gene methylation reversal treatment could be a promising treatment target to reduce ATS risk.

Methods
In this study, 15 ATS patients hospitalizing in department of neurology and mathed 15 average persons in physical examination center of Second Xiangya Hospital were recruited in 2013. People with coronary heart disease, peripheral vascular disease, aorto-arteritis, hemopathy, tuberculosis, malignant tumor, severe hepatic and renal dysfunction or application of lipid-lowering drugs within 1week before admission were excluded. Serum samples were fasting venous blood collected at early morning.The study was conducted in accordance with the guidelines laid down in the Declaration of Helsinki. All participants gave their written informed consent, which was obtained in the Ethics approval and consent to participate section. The study was reviewed and approved by the Ethics and Research Committee of Second Xiangya Hospital as well.

General clinical data comparison of the case group and control group
Compared with the control group, the case group had higher diastolic blood pressure(DBP) and LDL-C levels while lower HDL-C levels with statistically significant differences (P<0.05).Other general data such as gender, age, etc. displayed no significant differences (Table1).

2.
Comparison of LPL methylation level between the case group and control group. 3.

Comparison of LPL methylation level between individuals with and without cartiod artery atherosclerosis(CAS).
25 individuals who had undergone cartiod artery ultrasonography were divided into CAS group and non-CAS group. According to the analysis there was no obvious significance of LPL methylation level between the two group(35.8±8.7 vs 30.3±10.9, P value=0.184) as was illustrated in Table4.

4.Relationship among LPL methylation level, serum lipids and age.
A careful analysis of association between serum lipids and LPL methylation level showed great difference. In order to investigate connection of serum lipids and LPL methylation level, we conducted linear correlation analysis among HDL-C level, LDL-C level and LPL methylation level of all the participants. The results revealed remarkable negative linear correlation between HDL cholesterol level and general LPL methylation level (P value=0.001).Further analysis of CpG1-30 locus found that linear correlation at CpG9-16 locus was distinctly higher than that of other locus, and the difference was statistically significant (P value <0.05).There was an evident positive linear correlation between LDL-C level and general LPL methylation level (P value =0.004), and the linear correlation between CpG10-12, CpG15 and CpG20 locus were obviously higher than other locus with statistically significant differences (P value <0.05).Age is one of the important uncontrollable risk factors for IS.Through analyzing links of age and LPL methylation level, we found a significant positive linear correlation between age and general LPL methylation level (P value =0.011). Moreover, linear correlation at CpG 9-16 locus were higher than other sites prominently with a statistically significant difference (P value <0.05) as were shown in Table5 and Figure3-5.

5.Assciation between LPL methylation level and gender
Individuals in the two groups were further divided into four subgroups on the basis of gender. The results showed no significant difference between male and female either of the case group or the control group or the merged group(Table6). However, there existed statistically significant difference(P value <0.05) of LPL methylation level between males/female case group and control group(Table6,7).

Discussion
Epigenetics have become a crucial character in the pathophysiologic mechanism of cerebrovascular diseases [8]. It is a complex adjustive pathway regulating gene expression without altering genetic code as a response of dynamic environment and exogenous injuries [9]. DNA methylation is the most studied in epigenetics, occurring in CpG islands by adding a methyl group to cytosine to constitute 5-methylcytosine and subsequent effects are always gene silencing by way of intervening in transcription factors' combination with promoter region [10,11]. The process of DNA methylation is mediated by a group of enzymes called DNA methyltransferases(Dnmts) [11].Previous evidences suggested that DNA hypermethylation was involved in blood-brain barrier and neurovascular unit changes and poor prognosis in stroke [12] and treatment with demethylating agents manifested positive effects on ischemic brain injuries [13,14].
Intriguingly, other clinical study found that DNA hypomethylation was connected with elevated stroke risk and total mortality [15].It was also a better predictor for stroke outcome assessment than chronological age [16]. Alterations of DNA methylation levels of

Consent for publication: Written informed consent for publication of clinical details
were obtained from all of the patients included in our study. A copy of the consent form is available for review by the Editor of this journal. And we all acknowledged that the Consent for publication statement refers to consent obtained to include any individual person's data in any form (including individual details, images or videos).
Availability of data and material:The datasets analysed during the current study are available from the corresponding author on reasonable request.   Figure 1 CpG Methylation level at each locus in LPL promoter region Relationship between LPL promoter mean methylation level and LDL-C Figure 5 Relationship between LPL promoter mean methylation level and age