基于5hmC-Seal测序技术检测糖尿病肾病患者血浆游离DNA中5-羟甲基胞嘧啶的变化

To detect changes of 5-hydroxymethylcytosine in plasma cell-free DNA in patients with diabetic nephropathy based on 5hmC-Seal sequencing technology

目的通过生物信息学方法筛选血浆游离DNA来源的糖尿病肾病中的关键差异通路和羟甲基化修饰基因,为寻找诊断糖尿病肾病的生物标志物及阐明其潜在的分子机制奠定基础。方法利用5hmC-Seal测序方法对糖尿病和糖尿病肾病患者血浆游离DNA的5-羟甲基胞嘧啶进行富集,并通过测序得到全基因组5-羟甲基胞嘧啶,对两组患者进行差异分析得到差异羟甲基化基因,再利用差异基因的羟甲基化水平进行基因集变异分析,最后再对富集到的差异通路里的差异基因通过MCODE插件筛选关键基因,并进行单基因和多基因诊断疾病的准确性分析。结果基因集变异分析得到在糖尿病和糖尿病肾病两组患者中的差异通路:神经活性配体受体相互作用通路、肾脏上皮通路、趋化因子信号通路和已有文献报道与糖尿病肾病发病机制相关的Wnt信号通路,Wnt信号通路中富集到18个差异羟甲基化基因,通过MCODE筛选到关键模块基因有11个,分别为CTNNB1、GSK3B、CREBBP、CUL1、NFATC1、CCND3、WNT11、PRKACB、DKK1、SMAD3、CCND1,最后进行ROC分析有9个基因的诊断效能较好(AUC值≥0.8):CTNNB1、GSK3B、CREBBP、CUL1、NFATC1、WNT11、PRKACB、DKK1、CCND1。结论本研究羟甲基化测序结果表明,Wnt信号通路在糖尿病肾病发生发展中的关键作用,CTNNB1、GSK3B、CREBBP、CUL1、NFATC1、WNT11、PRKACB、DKK1、CCND1有可能成为诊断糖尿病肾病的生物标志物。

Objective To screen the key differential pathways and hydroxymethylation-modified genes in plasma cell-free DNA derived diabetic nephropathy by bioinformatics methods,to lay the foundation for finding biomarkers of diagnosis in diabetic nephropathy and elucidating the underlying molecular mechanism.Methods 5-hydroxymethylcytosine in plasma cell-free DNA of patients with diabetes and diabetic nephropathy were enriched by 5hmC-Seal sequencing method,and whole genome 5-hydroxymethylcytosine were obtained by sequencing.The differential analysis of the two groups in patients were carried out to obtain the differentially hydroxymethylated genes,and then the hydroxymethylation levels of differentially genes were used.Gene set variation analysis was performed,and the differential genes in enriched differential pathways were screened for the key genes through MCODE plug-in,and the accuracy of single-gene and multi-gene diagnosis of diseases were analyzed.Results Gene set variation analysis revealed the differential pathways in the two groups of patients with diabetes and diabetic nephropathy,including neuroactive ligand receptor interaction,renal cell carcinoma,chemokine signaling pathway,and Wnt signaling pathway related to the pathogenesis of diabetic nephropathy reported in the literature.18 differentially hydroxymethylated genes were enriched in Wnt signaling pathway,and 11 key module genes were screened by MCODE,namely CTNNB1,GSK3B,CREBBP,CUL1,NFATC1,CCND3,WNT11,PRKACB,DKK1,SMAD3,CCND1.Finally,9 genes had good diagnostic performance(AUC value≥0.8)by ROC analysis,including CTNNB1,GSK3B,CREBBP,CUL1,NFATC1,WNT11,PRKACB,DKK1,CCND1.Conclusion The results of hydroxymethylation sequencing in this study indicate that Wnt signaling pathway plays a key role in development of diabetic nephropathy,CTNNB1,GSK3B,CREBBP,CUL1,NFATC1,WNT11,PRKACB,DKK1,CCND1 may become biomarkers for the diagnosis of diabetic nephropathy.