Analysis Of Gene Mutation And Differential DNA Methylation Profile In Patients With Myelodysplastic Syndrome

Purpose:The study aims to analyze genetic mutation and clinical characteristics and study their correlation with survival prognosis of patients with myelodysplastic syndromes（MDS）.We also analyzed the differential DNA methylation profiles of TET2/ASXL1 MDS samples and explored the role of TET2/ASXL1 cooccurring mutations in the occurrence and development of MDS.Methods:1.The clinical data of 87 patients,diagnosed with MDS in the first Hospital of Lanzhou University from June 2015 to June 2021,were retrospectively collected to analyze the characteristics of mutant genes and the relationship between mutant genes and clinical features.The complete clinical data of 108 patients with MDS were obtained from the gene expression database（GEO）.A total of 195 cases with 87 cases in our department were statistically analyzed.2.The DNA methylation sequencing data set GSE129828 was obtained from the GEO.The samples with TET2 mutation were screened by R software,and the differential methylation analysis was carried out to screen the differential methylation region（DMR）.The differentially methylated gene（DMG）were obtained by gene annotation,and the DMG was analyzed by GO and KEGG.The protein-protein interaction（PPI）network was constructed by using STRING database to screen the key genes.Results:1.Among the 87 newly diagnosed MDS patients,the median follow-up was 937（25-2008）days,and the median survival time was 599（95%CI 671-1204）days.18.4%of the patients developed acute myeloid leukemia（AML）and 44.8%died during follow-up.2.Among the 87 MDS patients,54%of the patients had at least one gene mutation,the average number of mutations was 2.The top 5 genes with higher mutation rate were ASXL1 mutation（19.5%）,SF3B1 mutation（10.3%）,TET2 mutation（9.2%）,TP53mutation（9.2%）and RUNX1 mutation（9.2%）.Comparing the clinical characteristics of different mutant genomes,it was found that compared with the non-SF3B1 mutation group,the male incidence rate,absolute neutrophil count and MDS with ring sideroblasts（MDS-RS）subtype were higher in the SF3B1 mutation group（P values were 0.038,0.04,0.005）,and the platelet count had a tendency to increase（P=0.06）.Compared with the non-TP53 mutation group,the age at diagnosis,abnormal karyotype ratio and IPSS-R score were higher in the TP53 mutation group（P values were 0.035,0.005 and 0.005）.3.Multivariate Cox regression analysis showed that age≥65 years,platelet count<100×10~9/L,RUNX1 mutation,TP53 mutation,EZH2 mutation and IPSS-R stratification were independent prognostic factors affecting the overall survival of patients with MDS.4.Of the 195 MDS patients,42（21.5%）carried TET2 mutations.81%of TET2mutated MDS patients could detect one or more co-mutant genes.Among them,genes showing comutation frequencies>10%included ASXL1 mutation（33.3%）,RUNX1mutation（26.2%）,SRSF2 mutation（19.1%）and SF3B1 mutation（16.7%）.In the context of TET2 mutation,the prognosis of patients with co-mutation,RUNX1mutation or EZH2 mutation was poor（P values were 0.032,0.025,<0.001）,and the overall survival of ASXL1 mutation tended to be shortened（P=0.08）.5.337 DMR related genes were screened.GO analysis showed that highly methylated DMG was mainly enriched in biological processes such as cell surface receptor signal pathway,cell secretion,enzyme-linked receptor protein signal pathway and so on.Hypomethylated DMG was mainly enriched in biological processes such as cell differentiation and cell development.KEGG analysis showed that hypermethylated DMG was mainly enriched in Ras signal pathway,MAPK signal pathway and other pathways.Hypomethylated DMG was mainly enriched in extracellular matrix receptor interaction,focal adhesion and other pathways.PPI network analysis identified 10 hub genes of hypermethylated and hypomethylated DMG that may be associated with TET2/ASXL1 mutant MDS,respectively.Conclusions:1.Age≥65 years,platelet count<100×10~9/L,RUNX1 mutation,TP53 mutation,EZH2 mutation and IPSS-R stratification are independent adverse prognostic factors affecting the overall survival of MDS patients.2.The most commonly co-mutated gene in patients with TET2-mutant MDS was ASXL1,which had a tendency towards poorer prognosis.The prognosis of patients with at least one mutation,RUNX1 mutation or EZH2 mutation was poor.3.There are differences in DNA methylation between TET2（+）/ASXL1（-）and TET2（+）/ASXL1（+）MDS.Functional enrichment analysis of DMGs indicated that they play vital roles in multiple cellular events such as regulation of proliferation,differentiation,maturation and apoptosis.Hub genes were selected as biomarkers by PPI network analysis.