摘要目的 采用基因芯片技术筛选大骨节病DNA差异甲基化位点和基因,探讨DNA甲基化与大骨节病发病机制的关系.方法 收集12例大骨节病患者(病例组)、12例健康人(对照组)外周血血样,提取血液DNA,利用450K芯片技术检测病例组和对照组DNA的差异甲基化情况,并进行对照分析,按校正后P值和甲基化差异分值结合GenomeStudio软件筛选差异甲基化基因及位点,对筛选出的差异甲基化基因进一步采用亚硫酸氢钠处理后测序法(BSP)进行验证.结果 共分析484 948个位点,病例组和对照组差异显著的位点共93个,其中高甲基化位点34个,低甲基化位点59个.位点所对应的基因共50个,43个基因无文献报道.聚类分析结果显示,在免疫反应、抗原处理、磷和磷酸代谢及磷酸化作用、金属离子结合等过程中存在大量甲基化差异基因.利用BSP法验证,人类白细胞抗原(HLA)-DRB1基因在病例组与对照组间的甲基化率(48％比70％)无显著性差异(x2=3.688,P＞0.05).结论 大骨节病人群外周血DNA存在不同于正常人群的高或低差异甲基化位点,BSP验证HLA-DRB1基因位点与甲基化芯片结果未发现显著性差异.

abstractsObjective We used the DNA methylation microarrays to investigate the differential methylation genes and loci sites in Kashin-Beck disease (KBD),to study the relationship between DNA methylation and KBD pathogenesis.Methods Totally 12 KBD adults and 12 healthy adults were selected and peripheral blood samples were collected and DNA was extracted.Illumina 450K bead-chip was applied to detect methylation status in KBD and healthy controls.Aberrant hyper-methylated sites were filtrated according to the P value after correction and methylation differences,together with GenomeStudio soft.Screened genes were validated using bisulfite sequencing polymerase chain reaction (BSP) technology.Results A total of 484 948 loci sites were analyzed and compared,93 differential methylated loci were found by comparing KBD and normal people,including 34 hypermethylated sites and 59 hypomethylated sites.There were 50 genes corresponding to the loci,43 genes not reported in literature.According to gene ontology analysis,the genes were involved in the immune response,antigen processing,phosphate and phosphoric acid metabolism and phosphorylation and the process of metal ions in combination.However,in the verification test using BSP method,there was no significant difference in methylation rate in human leukocyte antigen (HLA)-DRB1 between the case and the control group (48％ vs 70％,x2 =3.688,P ＞ 0.05).Conclusions The high and low differentially methylated sites in peripheral blood DNA of KBD patients are significantly different from those of the health control.HLA-DRB1 locus is not significantly different between the BSP verification test and methylation chip.