| Background: Premature coronary heart disease(PCHD)is a relatively early onset type of coronary artery atherosclerosis with possible higher hereditary potential and more familial clustering.Epigenetic investigation against PCHD is helpful for better understanding on the interaction of genetic background and environment factors.It also can shed a light on the discovery of susceptible gene markers and,furthermore,the development of novel diagnostic and therapeutic sites.The mode of “integrative disease and syndrome”in traditional Chinese medicine(TCM)study is in accordance with the idea of epigenetics for it combines the principles of partial and comprehensive perspective,as well as the congenital and environmental perspective towards diseases.Therefore,a comprehensive epigenetic study will provide valuable information for the biomedical understanding of PCHD of the “blood stasis”syndrome in TCM.Objective: A literature review on the epigenetic studies investigating TCM syndromes of PCHD in terms of DNA methylation and hydroxymethylation was made to achieve a comprehensive understanding of the area.Quantative or descriptive literature analysis was considered to applied according to the characteristics of included literature.The method of epigenome-wide study(EWAS)was applied by a high throughout Me DIP DNA methylation array in order to screen differential methylated gene among PCHD with the “blood stasis” syndrome and other statuses.Bioinfomatic analysis was carried out to identify the relevant biological function and pathways.We also aimed to investigate the association among DNA methylation,DNA hydroxymethylation and m RNA expression mapping results of PCHD of the “blood stasis” syndrome.Methods:(1)Due to the insufficient amount of literature and the heterogeneity on study subjective,goal,and method,only a descriptive analysis was justifiable to be made after a comprehensive literature retrieval.(2)peripheral blood mononuclear cells(PBMCs)from patients with PCHD of “blood stasis” syndrome,PCHD of“non-blood-stasis” syndrome,“blood stasis” syndrome of non-PCHD and healthy controls were collected,respectively.Me DIP DNA methylation array was applied to comprehensively test the epigenomic methylation level in promoter regions.Differential enrichment peaks(DEPs)by comparison from every 2 groups and the corresponding differential genes were obtained.GO function and KEGG pathway analysis were used to map the differential genes.(3)Correlation analysis were carried out by seeking the common differential genes of every comparison obtained from the array data of DNA methylation,DNA hydroxymethylation and m RNA expression,respectively.Results:(1)Existing literature has obtained differential DNA methylation gene data from CHD of “blood stasis” syndrome,as well as the differential DNA hydroxymethylation gene data from PCHD of “blood stasis” syndrome.Some candidate genes have gone through further recheck experiments.There's a lack of high-throughput DNA methylation study on PCHD of “blood stasis” syndrome,and the association study among DNA methylation,hydroxymethylation and m RNA expression too.(2)Me DIP array identified 683 up-regulated differential enrichment peaks(DEPs)and 286 down-regulated DEPs in comparison between PCHD of “blood stasis” syndrome and healthy controls;442 up-regulated DEPs and 355down-regulated DEPs were identified in comparison between PCHD of“non-blood-stasis” syndrome and healthy controls;472 up-regulated DEPs and 540down-regulated DEPs were identified in comparison between “blood stasis”syndrome of non-PCHD and healthy controls;697 up-regulated DEPs and 286down-regulated DEPs were identified in comparison between PCHD of “blood stasis”syndrome and PCHD of “non-blood-stasis” syndrome;376 up-regulated DEPs and380 down-regulated DEPs were found in comparison between PCHD of“non-blood-stasis” syndrome and “blood stasis” syndrome of non-PCHD.(3)GO function analysis showed that biological functions mainly including apoptosis/pyroptosis,antigen presenting and processing,cellular adhesion,MAPK activation,cytokine response were enriched in up-regulated methylated genes(P<0.01),while GABA synaptic signal transmission,respiratory burst,hypoxia adaption,amine catalyzing process were enriched in down-regulated methylated genes of PCHD of “blood stasis” syndrome versus healthy controls(P<0.01);biological functions mainly including glutamine receptor regulation,neurotransmitter receptor activity,signal pathway,ion transfer,receptor biological synthesis and cell-to-cell communication were enriched in up-regulated methylated genes(P<0.01),while Gram negative bacteria defense,positive regulation of TNF and its super family,Th1 cellular immune response were enriched in down-regulated methylated genes of PCHD of “blood stasis” syndrome versus PCHD of“non-blood-stasis”syndrome(P<0.01);functions including intra-cellular amide metabolism,cardiac contraction power,smooth muscle contraction regulation,peptide metabolism,negative regulation on muscle contraction,intranucleus DNA replication,positive regulation on insulin secretion,mitochondrial membrane potential,lipid binding were enriched in up-regulated methylated genes(P<0.01),while ubiquitin-dependent protein metabolic process and endoplasmic reticulum-cytosol protein transfer were enriched in down-regulated methylated genes of PCHD of “blood stasis” syndrome versus “blood-stasis” syndrome of non-PCHD(P<0.01).KEGG pathway analysis showed that biological pathways predominantly including c AMP signal transmission,PPAR signal transmission,viral myocarditis related pathways,glycine/serine/threonine metabolism,unsaturated lipid acid synthesis,glioma,alpha linolenic acid,p53 signal transmission,AMPK signal transmission were enriched in up-regulated methylated genes(P<0.05),while folic acid synthesis,acute myelocytic leukemia,cytokine-cytokine receptor interaction,MODY type diabetes and inflammatory bowel diseases were enriched in down-regulated methylated genes of PCHD of “blood stasis” syndrome versus healthy controls(P<0.05);pathways predominantly including MAPK signal transmission,ketone synthesis and degradation,estrogen receptor signal transmission,Rap1 signal transmission,c AMP signal transmission,gastric acid production,oxytocin signal transmission,renin secretion,valine/leusine/isoleusine degradation were enriched in up-regulated methylated genes(P<0.05),while phosphoinositide metabolism,thiamine,pertussis,EGFR tyrosine kinase inhibitor resistance,insulin signal transmission,allogeneic graft rejection,actin celluar skeleton regulation were enriched in down-regulated methylated genes of PCHD of“blood stasis”syndrome versus PCHD of “non-blood-stasis” syndrome(P<0.05);pathways predominantly including Gn RH signal transmission,choline metabolism,MAPK signal transmission,long-term drug synergy,amphetamine addiction,calcium reabsorption,NF-?B signal transmission,circadian regulation,transcription error in cancer,Rap1 signal transmission were enriched in up-regulated methylated genes(P<0.05),while cytokine-cytokine receptor interaction,inflammatory bowel diseases,Th17 differentiation,platelet activation,rheumatic arthritis,hematopoietic cell line and MODY diabetic pathway were enriched in down-regulated methylated genes of PCHD of “blood stasis” syndrome versus “blood-stasis” syndrome of non-PCHD(P<0.05).(4)The correlation analysis failed to identify a common differential susceptible gene among the 3 array results in comparison of PCHD of“blood stasis” syndrome versus healthy controls.However,in light of the “DNA methylation-DNA hydroxymethylation-m RNA expression” axis,several common genes(LAMP1,RRAS,HLA-B,MED22,MORF4L2,PM20D2)with change both in terms of DNA methylation and m RNA expression level may justify preference for further investigation.Conclusion:(1)Results of High throughput DNA methylation array Me DIP showed essential heterogeneity among patients with PCHD of “blood stasis” syndrome,PCHD of “non-blood-stasis” syndrome,“blood stasis” syndrome of non-PCHD and healthy controls with respect to DEP distribution,biological function,pathway.The DNA methylation characteristics of PCHD of “blood stasis” syndrome can not be illustrated by that of PCHD and “blood stasis” syndrome simply added together,which supports the consistency of the so-called “integrative TCM syndrome and disease” mode of TCM in modern times.(2)Multiple biological functions and pathways are involved in the susceptible differential methylated genes of PCHD of“blood stasis”syndrome,therefore the feature is probably to be described as an epigenetic network.(3)LAMP1,RRAS,HLA-B,MED22,MORF4L2 and PM20D2 have substantial change both in terms of DNA methylation and m RNA expression level in PCHD of “blood stagsis” syndrome and may worth preference for further investigation. |
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