基于寡核苷酸芯片的地中海贫血特异性DNA甲基化的研究
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摘要
地中海贫血是一组由于血红蛋白的珠蛋白肽链(α、β、γ)的合成抑制、失衡,引起无效造血和溶血性贫血。目前,对于这类疾病尚无有效的治疗方法,只能通过遗传筛查及产前诊断选择性的淘汰地贫患儿以控制这类疾病的发生和传播。传统诊断地中海贫血的方法实验周期长,且只能诊断临床上常见的几种基因缺失或点突变所引起的地中海贫血。因此,迫切需要寻求一种新的具有组织特异性的分子靶标,为地中海贫血的诊断提供新途径。
目前,在应用于这种高度异质性遗传病的各种分子诊断方法中,快速简便的基因芯片技术引人注目。因为,常规的基因和蛋白质组学的已不能满足临床的需要,尤其对于反复妊娠发病的或者轻型的地中海贫血患者而言,传统的分子诊断方法不能检测到基因突变或片段缺失,对于这部分病例,经典的遗传学理论无法解释其发病机制。表观遗传学的进展,为地中海贫血寻求和发现新的更简便、更精确、易于推广的诊断方法,提供了可能。
表观遗传学是与遗传学(genetic)相对应的概念,指的是基于非基因序列改变所致的基因表达水平变化,如DNA甲基化和染色质构象变化等。从目前的研究来看,X染色体剂量补偿、DNA甲基化、组蛋白密码、基因组印记、表观基因组学和人类表观基因组计划等问题都是表观遗传学研究的内容。有文献报道,在任何时候,细胞内都有处于活化状态的基因和失活状态的基因。在发育过程的不同时期发生不同类型的基因转录。有关地中海贫血在表观遗传学甲基化修饰的研究虽有报道,但既不系统又未起到指导临床诊断作用。随着DNA甲基化检测技术的发展和完善,通过寻找受甲基化调控的新位点来诊断和治疗疾病已经逐渐成为相关肿瘤和遗传病的分子诊断和治疗的手段之一。文献报道,在珠蛋白基因转录、发育调控过程中,DNA甲基化起着关键作用。以分子杂交为基础的微阵列技术应用于DNA甲基化检测中,这种方法是基于杂交的寡核苷酸微阵列,是一种在基因组中寻找新位点的方法。通过这种高通量的方法,我们可以找到基因启动子区高度甲基化的基因位点。目前为止,通过微阵列芯片技术检测单基因遗传病地中海贫血鲜有报道。受此启发,本研究拟通过DMH甲基化芯片技术分析地中海贫血中相关分子的表观遗传学甲基化修饰规律和特点,以期能寻求和发现新的诊断地中海贫血的方法,提高地中海贫血的早期诊断率和筛查准确率,为临床早期筛查和诊断地中海贫血胎儿开拓新途径。
本课题由三部分组成,即建立DNA甲基化芯片平台;应用已成熟的基因表达谱芯片进一步筛选基因组地中海贫血新的位点,利用甲基化特异PCR(MSP)验证芯片结果并探讨甲基化差异位点与地中海贫血相关性;通过Sequenom甲基化质谱测序平台对IGSF4基因启动子区测序,观察IGSF4基因在地中海贫血中的甲基化状态,并探讨其与地中海贫血发病机制的关系。
主要结果如下:
1. DNA甲基化芯片模型建立, DNA甲基化芯片数据处理系统完成。
2.通过DMH芯片及其MAS系统的功能分类共发现11条与血液病及遗传病直接相关的有甲基化变化基因,分别为CBFB、HDAC3、IL12A、PLAT、RTKN、RAD52、PTGS1、DGUOK、MRE11A和THRA(ratio>2.0)。
3.基因LARP2、HDAC3、THRA在β-地中海贫血中呈高度甲基化状态(ratio>2.0),提示LARP2、HDAC3、THRA的高度甲基化可能成为表观遗传学上调控β-珠蛋白的机制之一。
4.经MassARRAY甲基化质谱测序方法对23例地中海贫血和5例正常对照血样对比分析,结果显示,被检测的IGSF4启动子区12个CpG位点在地中海贫血中相对于正常外周血甲基化程度明显增高,呈高度甲基化状态(P<0.01),提示IGSF4基因的高度甲基化可能成为导致地中海贫血的机制之一。
5.表达谱芯片数据分析结果显示表达上调基因共有159个,下调基因共有92个。通过MAS系统分析得到与IGSF4途径相关的基因位点CSF1、CSF2、TPO、HBB、HBD、HBA和CBLC与正常外周血相比呈表达下调(ratio<0.50); CD45、AZU1和IL1B呈表达上调(ratio>2.0),提示与珠蛋白相关的基因通过表达量不同相互作用调控珠蛋白的生成和表达。
6. IGSF4基因实时定量PCR (Real-time PCR)验证结果显示,与正常外周血比,该基因在地中海贫血中呈低表达,且有显著性差异(ratio=0.18,ratio<0.50),提示IGSF4基因在地中海贫血中相对于正常外周血而言表达下调。
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.
目前,在应用于这种高度异质性遗传病的各种分子诊断方法中,快速简便的基因芯片技术引人注目。因为,常规的基因和蛋白质组学的已不能满足临床的需要,尤其对于反复妊娠发病的或者轻型的地中海贫血患者而言,传统的分子诊断方法不能检测到基因突变或片段缺失,对于这部分病例,经典的遗传学理论无法解释其发病机制。表观遗传学的进展,为地中海贫血寻求和发现新的更简便、更精确、易于推广的诊断方法,提供了可能。
表观遗传学是与遗传学(genetic)相对应的概念,指的是基于非基因序列改变所致的基因表达水平变化,如DNA甲基化和染色质构象变化等。从目前的研究来看,X染色体剂量补偿、DNA甲基化、组蛋白密码、基因组印记、表观基因组学和人类表观基因组计划等问题都是表观遗传学研究的内容。有文献报道,在任何时候,细胞内都有处于活化状态的基因和失活状态的基因。在发育过程的不同时期发生不同类型的基因转录。有关地中海贫血在表观遗传学甲基化修饰的研究虽有报道,但既不系统又未起到指导临床诊断作用。随着DNA甲基化检测技术的发展和完善,通过寻找受甲基化调控的新位点来诊断和治疗疾病已经逐渐成为相关肿瘤和遗传病的分子诊断和治疗的手段之一。文献报道,在珠蛋白基因转录、发育调控过程中,DNA甲基化起着关键作用。以分子杂交为基础的微阵列技术应用于DNA甲基化检测中,这种方法是基于杂交的寡核苷酸微阵列,是一种在基因组中寻找新位点的方法。通过这种高通量的方法,我们可以找到基因启动子区高度甲基化的基因位点。目前为止,通过微阵列芯片技术检测单基因遗传病地中海贫血鲜有报道。受此启发,本研究拟通过DMH甲基化芯片技术分析地中海贫血中相关分子的表观遗传学甲基化修饰规律和特点,以期能寻求和发现新的诊断地中海贫血的方法,提高地中海贫血的早期诊断率和筛查准确率,为临床早期筛查和诊断地中海贫血胎儿开拓新途径。
本课题由三部分组成,即建立DNA甲基化芯片平台;应用已成熟的基因表达谱芯片进一步筛选基因组地中海贫血新的位点,利用甲基化特异PCR(MSP)验证芯片结果并探讨甲基化差异位点与地中海贫血相关性;通过Sequenom甲基化质谱测序平台对IGSF4基因启动子区测序,观察IGSF4基因在地中海贫血中的甲基化状态,并探讨其与地中海贫血发病机制的关系。
主要结果如下:
1. DNA甲基化芯片模型建立, DNA甲基化芯片数据处理系统完成。
2.通过DMH芯片及其MAS系统的功能分类共发现11条与血液病及遗传病直接相关的有甲基化变化基因,分别为CBFB、HDAC3、IL12A、PLAT、RTKN、RAD52、PTGS1、DGUOK、MRE11A和THRA(ratio>2.0)。
3.基因LARP2、HDAC3、THRA在β-地中海贫血中呈高度甲基化状态(ratio>2.0),提示LARP2、HDAC3、THRA的高度甲基化可能成为表观遗传学上调控β-珠蛋白的机制之一。
4.经MassARRAY甲基化质谱测序方法对23例地中海贫血和5例正常对照血样对比分析,结果显示,被检测的IGSF4启动子区12个CpG位点在地中海贫血中相对于正常外周血甲基化程度明显增高,呈高度甲基化状态(P<0.01),提示IGSF4基因的高度甲基化可能成为导致地中海贫血的机制之一。
5.表达谱芯片数据分析结果显示表达上调基因共有159个,下调基因共有92个。通过MAS系统分析得到与IGSF4途径相关的基因位点CSF1、CSF2、TPO、HBB、HBD、HBA和CBLC与正常外周血相比呈表达下调(ratio<0.50); CD45、AZU1和IL1B呈表达上调(ratio>2.0),提示与珠蛋白相关的基因通过表达量不同相互作用调控珠蛋白的生成和表达。
6. IGSF4基因实时定量PCR (Real-time PCR)验证结果显示,与正常外周血比,该基因在地中海贫血中呈低表达,且有显著性差异(ratio=0.18,ratio<0.50),提示IGSF4基因在地中海贫血中相对于正常外周血而言表达下调。
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.
引文
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