| Objective: The pathogenesis of atherosclerosis (AS) is a complicated procedure with multiple factor participating. Up to now, there are still many details and mechanisms that need to be illuminated. In the past years, epigenetic abnormal-regulation of gene expression was found to play an important role in the atherosclerosis pathogenesis. Accumulated data shows that the whole genome displays hypomethylation, while the CpG islands in promoter region of certain genes unveil hypermethylation during the initiation and development of atherosclerosis.Abnormality of LDL-R plays an important role in the occurance of AS. There are many studies about the genetic mutation of LDL-R, which shows the definite causality with familial hypercholesterolemia, as well as the atherosclerosis. However, there has been no reports on the abnormality of methyl-modification in LDL-R gene's promoter region in artherosclerosis, as well as on the relationship with atherosclerosis.Epigenetic study showed that methylation degree of estrogen receptor—αgene in atheromatous plaque of As patients significantly increased, while the expression of this gene was down-regulation. Kortelaine, who detected ER with monoclonal antibody, had found that the expression of ER-αgene declined in premenopausal female patients, which hinted that the correlation between the silence of ER-αgene and the formation of atherosclerosis. But which causative agent leading to the hypermethylation of ER-αgene have not been reported.High-homocysteinemia (HHcy) have been referred to an independent risk factor for atherosclerosis. There are many reports about the pathogenic mechanism of HHcy, most of them focus on oxidative stress injuries. At the later period of 1990's, P.E Newman raised an idea that abberant methylation pattern in genome resulted from HHcy may promote the formation of atherosclerosis. The mechanism is that the homocysteine as an product of methionine cycle in the one-carbon metabolism has negative feedback effect on the synthesis of s-adenosylmethionine, which is the only methyl-donator for DNA methylation modification. So, HHcy induced abnormal methylation modification may involve in the abberant methylation patterns in atherosclerosis-related genes and its pathogenesis.Thus, this study was designed to investigate the alteration of methylation patterns in LDL-R and ER-αgenes and the relationship between the abberant methylation in ER-αgene and the level of total plasma homocysteine (tHcy) in atherosclerosis patients, in order to illustrate the epigenomic mechanisms in the pathogenesis of As.This study includes two parts, the first one is: the methylation status of LDL-R gene's promoter region in atherosclerosis patients. And the second one is: the methylation status in ER-αgene's promoter region, the Hcy level, and the AS degree in atherosclerosis patients, and their relationships.Methods: 61 atherosclerosis patients and 28 healthy control subjects were selected for LDLR gene research, the patients was proved to have atherosclerotic plaque and the healthy was not under the carotid colorized ultrasound examination. Peripheral blood samples were taken for DNA preparation. The methylation status of CpG islands in LDLR gene promoter region was measured by using a modified coordinative method with nested-PCR, Touchdown-PCR and methylation-sensitive- single-strand conformation analysis (MS-SSCA)82 cases including 54 atherosclerosis patients and 28 healthy control subjects were selected for ER-αgene research, the carotid intima-media thickness (CIMT) and atherosclerotic plaques formation were recorded under carotid colorized ultrasound examination. Carotid plaque score was calculated using Crouse's methods. 3ml venous blood was taken in the early morning with starvation, 2ml was used to extract genome DNA and 1ml was used to detect the level of total plasma homocysteine (tHcy).The methylation status of CpG islands in ER-αgene promoter region were analyzed by nested-methylation-specific PCR (nMSP). tHcy was examined by fluorescent-biochemical method. Using spearman rank correlation to analyse the relatinship between the degree of methylation in ER-αgene and the level of tHcy.Results: Three types of methylation patterns were detected in promoter region of LDL-R gene in peripheral blood genome of atheroselerosis patients: the full-methylation, half-methylation and none-methylation. 2 cases were full-methylation and 13 cases were half-methylation, the methylation frequency was 24.59% in atherosclerosis patients. While in 28 healthy control, only 1 half-methylation sample was found, the methylation frequency was 3.57%, much lower than the one in atherosclerosis patients (p＜0.05) Hypermethylation of ER-αgene promoter region was found in 38 cases of atherosclerosis patients, the methylation frequency was 70.4%. While only 28.6% methylation frequency was detected in healthy controls, just 8 of 28 samples were found hypermethylation, much lower than the ones in atherosclerosis group (p＜0.05) Meanwhile, the level of tHcy in atherosclerosis group was significantly higher than that in control group (p＜0.05) . The spearman rank correlation analysis explored an obviously correlation between the degree of methylation in ER-αgene and the level of tHcy(r=0.809), and the severity of atherosclerotic lesion was also heightened along with the increment of plasma level of tHcy.Conclusion: An significantly higher methylation degree in promoter region of LDLR gene was found in peripheral blood genome of atherosclerosis patients compared with the controlled healthy subjects, which may suggest an involvement of aberrant methylation of LDLR gene in initiation and development of AS. The modified coordinative method with nested-PCR, Touchdown-PCR and MS-SSCA manifests merits of convenience, cheap and high efficiency, which remarkably lowered the requirement for the DNA template and increases the sensitivity and specificity.Hyermethylation of CpG islands in ER-αgene promoter region was found in high frequency in atherosclerosis patients, which is positive correlated with the increased level of plasma tHcy and the severity of atherosclerotic lesion. This clinical data further extended our in vitro experimental result that HHcy can lead to the hypermethylation of ER-αgene, and hence to promote the occurrence and development of AS.
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