摘要
目的 收集睡眠呼吸暂停综合征(OSAS)合并高血压和原发性高血压(EH)患者进行全基因组甲基化测序寻找差异甲基化位点.在高血压人群,进一步探讨OSAS合并糖尿病与葡萄糖转运蛋白4基因[溶质载体家族2A4(SLC2A4)基因]DNA甲基化的关系.方法 全基因组甲基化阶段:从2010年1月至3月,连续招募年龄30～60岁、肾功能和肝功能正常的高血压患者,完善多导睡眠监测,中重度OSAS合并高血压患者作为试验组(12例),年龄、体重指数匹配的非OSAS EH患者作为对照组(12例),通过Microarray高通量基因表达检测外周血甲基化情况.单基因验证阶段:全基因组甲基化检测和京都基因和基因组数据库(KEGG)富集通路发现2型糖尿病通路有四个差异基因,选取其中的SLC2A4基因,进一步扩大样本量在高血压人群收集OSAS合并糖尿病(40例)、OSAS非糖尿病(66例)、非OSAS非糖尿病(39例)三组患者,验证单基因甲基化差异位点.结果 全基因组研究中,OSAS合并高血压相比较EH共鉴定出516个差异甲基化位点(低甲基化361个,高甲基化155个),所有选择的差异甲基化位点均存在显著性差异(P＜0.05和|Beta值差异|＞0.14).通过基因本体(GO)、KEGG富集进行功能学基因富集分析.与非OSAS非糖尿病组相比,OSAS非糖尿病组中SLC2A4基因存在高甲基化位点:CpG2-11、CpG2-13、CpG2_15、CpG1_18、CpG2_21.22、CpG2_23.24 和 CpG22_27;OSAS 合并糖尿病组高低甲基化位点并存,低甲基化位点为 CpG1_2、CpG1_5、CpG2_6、CPG17、CPG110.11、CPG117、CPG118.19、CPG214、CPG218、CPG225、CPG227,高甲基化位点为CpG2-13、CpG2_15和CpG2_20.结论 OSAS合并高血压较EH,存在差异甲基化位点,为后续甲基化研究提供差异甲基化基因数据库.选取2型糖尿病通路中的SLC2A4基因进行单基因验证,证实该基因甲基化差异参与OSAS糖尿病共病.
Abstract
Objectives Patients with obstructive sleep apnea syndrome(OSAS)combined with hypertension and es-sential hypertension were collected for genome wide methylation researchto look for differentially methylated sites.In the hypertensive population,the relationship between DNA methylation of glucose transporter 4(GLUT4)[sol-ute carrier family-2A4(SLC2A4)]gene and OASA combined with diabetes mellituswas further explored.Methods In the genome-wide methylation stage,from January to March 2010,consecutive hypertensive patients aged thirty to sixty years with normal renal and hepatic functions were recruited to complete polysomnography moni-toring.Among them,12 patients with moderate to severe OSAS combined with hypertension were selected as the ex-perimental group,and 12 patients with age-and body mass index-matched non-OSAS essential hypertension were selected as the control group.The peripheral blood methylation was tested by Microarray high-throughput gene ex-pression assay.In the single-gene validation stage,genome-wide methylation detection and Kyoto Encyclopedia of Genes and Genomes(KEGG)-enriched pathway revealed four differential genes in the type 2 diabetes pathway,in which SLC2A4 was selected,and the sample size was further expanded to collect three groups of patients with OSAS combined with diabetes mellitus(40 cases),OSAS nondiabetics(66 cases),non-OSAS nondiabetics(39 ca-ses)in the hypertension population,to verify the single gene methylation difference.Results Genome-wide study involving OSAS combined with hypertension compared to primary hypertension identified a total of 516 differentially methylated sites(361 hypomethylated and 155 hypermethylated),and all selected differentially methylated sites were significantly different with P＜0.05 and|Beta difference|＞0.14.Functionalistic gene enrichment analysis was performed by Gene Ontology(GO)and KEGG enrichment.Compared with the non-OSAS nondiabetics group,there were hypermethylated sites in SLC2A4 gene in the OSAS nondiabetics group:CpG2-11,CpG2-13,CpG2_15,CpG1_18,CpG2_21.22,CpG2_23.24,and CpG22_27;whereas,in the OSAS combined with diabetes mellitus group,high and low methylation sites co-existed(low methylation sites:CpG1_2,CpG1_5,CpG2_6,CPG17,CPG110.11,CPG117,CPG118.19,CPG214,CPG218,CPG225,CPG227,and hypermethylated sites:CpG2-13,CpG2_15,and CpG2_20).Conclusion Compared with essential hypertension,OSAS combined with hyperten-sion has differential methylation sites,which provides a database of differential methylation genes for subsequent methylation studies.The single gene verification of SLC2A4 gene in the type 2 diabetes pathway confirmed that the gene methylation difference was involved in OSAS diabetes comorbidity.
基金项目
新疆维吾尔自治区人民医院院内科研项目(20160201)
NETL
NSTL
万方数据