Studies On DNA Methylation In Thalassemia Based On Oligonucleotide Microarray

Thalassaemia is a group of genetic abnormalities characterized the underproduction of (α,β,γ) globin chain leads to a reduced red cell life span, increased haemolysis and ineffective erythropoiesis. To date, compared to the the traditional diagnosis method of thalassemia, our group has pursued another gene-wide profiling approach for diagnosingβ-thalassemia, because of the lack of a sharply and validated screening approach for early detection. Additionally, the traditional diagnosis method of thalassemia needs long cycles of experiment, and there is very little data linking genome-wide information of its mRNA expression level. In order to control the occurrence and spread of this disease, we only depend on the genetic screening and prenatal diagnosis to make selective elimination for such anemias fetal In clinic. Therefore, an urgent need to find a new and tissue-specific molecular targets will provide a new way for prenatal diagnosis in thalassemia from epigenetic inheritance perspective.At present, To understand the molecular mechanisms on the occurrence and development of the high degree of heterogeneity of genetic diseases in genome-wide level becomes the new trends of modern biomedical research. Additionally, more and more applications of the bio-chip technology have been used by its advantages of low consumption, high-throughput in the various fields of biomedicine. Because conventional gene and proteome can't be satisfied with the need of clinic, especially for the patients of repeated pregnancy or mitis thalassemia. For this part of cases, classical theory of genetics can not explain the pathogenesy. With the development of epigenetics, the epigenetics modification offers the possibility to explore and discover more convenient, precise and easy to spread method for thalassemia.Epigenetic regulation is a process by which phenotype is modified without alterations in genotype, this heritable process is linked to epigenetic states involving DNA methylation and changes in chromatin conformation, which maintain transcriptional status throughout mitosis and DNA replication. In current study, epigenetics includes X chromosome dosage compensation, DNA methylation, histone code, genomic imprinting, and the epigenetic genomics and human genome project and other problems.As it reported, DNA methylation in chromatin play crucial role in the developmentally-regulated activation and switching of globin gene transcription. DNA microarray technology has made it possible to profile and quantify the expression of thousands of genes simultaneously based on the technology of molecular hybridization. Using this method, we found the hypermethylation in the promoter region of a gene is associated with a lower expression and plays an important role in gene silencing. Recently, the field of DNA methylation has grown ramatically and become one of the most dynamic and rapidly developing branches of molecular biology. However, there has been little study in the area when it comes to genome-wide profiling of the promoter methylation. Here, we used a genome-wide profiling method called Differential Methylation Hybridization (DMH) to search for the differential changed genes in order to establish epigenetic marks in thalassemia for early prenatal diagnosis.The study is composed of three parts for the investigation of thalassemia. That is, to establish a microarry platform of DNA methylation; using genome-wide expression profile chips to screen new differential changed genes in thalassemia, and according to the results of expression profile chips, significant genes of DNA methylation were verified by MSP, in order to investigate the relationship with thalassemia; the DNA methylation sequence of IGSF4 gene promoter region was detected by sequenom spectrometry platform and observed its methylation status in thalassemia, to explore the relationship between gene IGSF4 and thalassemia.The main results and conclusion of the study are as follows:1. The microarry platform of DNA methylation had been established, and the following data processing system had been finished.2. The differential changed genes of CBFB,HDAC3,IL12A,PLAT,RTKN,RAD52,PTGS1,DGUOK,MRE11A and THRA were showed the variation of DNA methylation through DMH chips and MAS(ratio>2.0).3. Gene LARP2 ,HDAC3 and THRA were hypermethylation inβ-thalassemia (ratio>2.0). It suggested hypermethylation of genes LARP2 ,HDAC3 and THRA may be one of the mechanisms on epigenetic regulation ofβ-globin.4. Trough Sequenom MassARRAY methylation analysis for 23 cases of thalassemia comparation with 5 cases of normal blood, the results showed hypermethylation of 12 CpG sites in the promoter region of IGSF4 (P<0.01). It suggests hypermethylation of IGSF4 inβ-thalassemia could be one of the mechanisms induced thalassemia.5. The differences in gene expression patterns for mRNA microarray display, 159 genes were upregulated and 92 genes were downregulated in both groups. Trough the MAS system and the function analysis of pathway, genes related to IGSF4 were screened out, they were CSF1,CSF2,TPO,HBB,HBD,HBA and CBLC, which were downregulated in thalassemia(ratio<0.50); the expression of genesCD45,AZU1 and IL1B were upregulated in thalassemia(ratio>2.0). It suggests genes associated with the globin gene play the role in its regulation through the differential expression levels.6. The result of Real-time PCR showed a lower level of IGSF4 inβ-thalassemia patients versus the control (ratio=0.18,ratio<0.50). It suggested the expression of IGSF4 was downregulated in thalassemia.