Primary Research On The Effect Of DNA Methylation On The Expression Of The Fetal Hemoglobin

Objective:Illuminate the effect of methylation status of ?-globin gene(HBG)on the expression of the HbF of ?-thalassemia minor;In order to screen out the biological pathways and genes were related to the HbF,we used the bioinformatics analysis of the all methylated sites of whole genome.Finally offer new target and basic theory for ?-thalassemia.Methods:Collected the 301 samples(2ml of peripheral anticoagulation)of patients who were diagnosed with ?-thalassemia minor,then hydrosulfite transformation after extracting DNA,PCR augmenting the target fragments,quantify pyrosequencing the methylation of the promoter of the HBG,analyzing the correlate between different condition of methylation of the HBG and the expression of HbF.Collected the 8 samples of cord blood of preterms and 8 samples of cord blood of term infants,Magnetic activated cell sorting beads(MACS)were used to classify nucleated red blood cells,then hydrosulfite transformation after extracting DNA,used Illumina Infinium? Human Methylation 850 Bead Chip to detection all methylation sites of whole genome.The primary data was beneficiated with minfi of R software,then filtrated the different methylation sites and different methylation genes with IMA of R software.(The screening criteria was p<0.05,|beta.difference |>0.14).GO analysis and KEGG analysis were used for finding traget genes and signaling pathway.Results: On account of the high homology of HBG1 and HBG2,the CpG sites were coincident,includes five CpG sites.On the quantitative analysis of five sites of the degree of methylation and expression of HbF,there were no significant correlation all.On qualitative analysis,five sites present a highly methylated or moderate degree of methylation,no low methylation.According to the HbF expression in 295 cases of specimens after the grouping,found that there were no significant difference during 4 groups(p>0.05);After grouping 295 samples according to the methylation degree,it was found that only the HbF of site+49 were significant different(p<0.05),the HbF of other 4 sites were meaningless(p>0.05).The results of llumina 850 K methylated gene chips: The changes in the term group were as follows compared with the preterm group: Total 4749 different methylation sites and 2600 corresponding genes were filtrated.4749 different methylation sites included 4359 hypomethylation sites and 390 hypermethylation sites.The results of GO analysis: Wnt signaling pathway which gathering the 4genes(ABL2 ?DAB2?ANKRD6? MLLT3),all were hypomethylation genes;The results of KEGG analysis: Nothch signaling pathway which was related to hemopoiesis enrichment factor was 2.29 gathering the 14 genes,there were 11 hypomethylation genes(CREBBP?PSEN2?KAT2B?LFNG?CTBP2?MAML3?DTX3L?NUMB?NOTCH1?HDAC1?RBPJ),3 hypermethylation genes:(NCOR2?MAML2 ? PTCRA).Conclusion:The methylation of HBG promoter region of?-thalassemia minor were hypermethylation?moderate methylation,which inhibition the expression of HbF,but there was no significant correlation between the methylation of ? globin gene(HBG)promoter region of ?-thalassemia minor and the expression of HbF quantificationally.The study of gene chips finded the new pathway:Wnt signaling pathway and Notch signaling pathway may be relative to the regulate and control of HBG,which provided a new target for research on mechanism of expression of HBG.