泛发性脓疱型银屑病患者DNA甲基化相关研究及临床分析

发布时间：2018-09-01 16:37

【摘要】：泛发性脓疱型银屑病(generalized pustular psoriasis,GPP)是一种系统性炎症性皮肤病,严重时可威胁生命。GPP发病率较低,但发病时常常伴有高热及全身不适,严重时可威胁生命。因而GPP相关的病因及发病机制研究一直为皮肤科领域的热点。目前为止,随着关于GPP遗传背景研究的开展,IL36RN和CARD14两个基因逐渐被认为是GPP的致病易感基因。但是一些临床现象仍然无法解释,如大多数单独发生的GPP是由IL36RN基因的纯合或复合杂合突变引起的,但人群中也有正常个体携带IL36RN基因突变;为何感染、妊娠和特殊药物应用等状态能诱发GPP的发作。因此易感基因遗传方式虽然解释了一部分GPP病人的发病,但无法全面揭示性别、年龄及诱因等非遗传方式对疾病发生发展的影响,例如为何感染、妊娠和特殊药物应用等状态能诱发GPP的发作。表观遗传学是指在研究基因的核苷酸序列未发生改变的情况下,对基因的表达进行调控。这种调节可遗传给后代并为可逆性的基因表达调节。有研究表明,表观遗传调控机制在系统红斑狼疮等自身免疫性疾病中发挥重要的作用,但有关GPP的表观遗传学的研究较少。我们课题组在之前的前期实验中发现GPP患者外周血单个核细胞中存在异常的DNA甲基化状态改变,这表明DNA甲基化可能是参与GPP发病的重要机制之一。DNA甲基化是指在甲基化酶的催化作用下,以S-腺苷甲硫氨酸(S-adenosylmethione,SAM)为甲基供体,将甲基(-CH3)添加至胞嘧啶第五位碳原子上,生成5-甲基胞嘧啶(5mC)的生物学过程。因此,本课题拟首次对GPP患者及正常人的外周血单个核细胞(peripheral blood mononuclear cell,PBMC)进行全基因组DNA甲基化测序及相关研究,以全面揭示甲基化改变在GPP的发生发展中的作用,填补相关领域的空白。第一部分泛发性脓疱型银屑病患者外周血单个核细胞DNA甲基化测序目的研究GPP患者及正常健康人PBMC的全基因组DNA甲基化状态,探讨DNA甲基化在GPP患者中的表达差异。方法提取3例GPP患者和3例正常健康人的PBMC中的基因组DNA,并采用Illumina Human Methylation 450K BeadChip芯片技术对其进行DNA甲基化测序。结果通过Illumina Human Methylation 450K BeadChip芯片测序,我们得到了足够测序深度和分辨率的全基因组甲基化图谱。甲基化发生异常变化的差异甲基化区域达到6328个。和正常健康人的PBMC相比,GPP的PBMC全基因组DNA呈现异常甲基化状态,其中甲基化程度升高位点5921个,甲基化程度降低位点407个。2例同为妊娠状态下诱导的GPP患者,在主要成分分析中分布更为接近。GO分析显示甲基化明显异常改变的基因类型包括免疫细胞迁移、炎症反应及信号传导等。结论GPP患者PBMC中基因组DNA较正常健康人发生异常的甲基化状态,其中众多甲基化水平差异表达的基因与病理生理过程相关,功能涉及多个方面,信号通路极其复杂;妊娠可能通过改变机体内甲基化水平的状态影响疾病的发生。第二部分泛发性脓疱型银屑病患者外周血单个核细胞PDCD1基因调控序列甲基化水平及调控因子研究目的选择细胞程序性死亡受体1(programmed cell death 1,PDCD1)基因对GPP患者PBMC的DNA甲基化测序结果进行验证。同时研究GPP患者的PBMC的甲基化转移酶(DNA methyltransferase,DNMT)及甲基化CpG结合蛋白(Methyl-CpG binding proteins,MBD)的表达。方法选取9例GPP患者及10例健康正常人的PBMC为研究对象,采用飞行质谱法对PDCD1基因甲基化进行测序,以验证甲基化芯片的结果。同时采用Trizol法提取9例GPP患者及10例健康正常人的PBMC中的总RNA,应用Real-time PCR的方法检测PDCD1、DNMT及MBD蛋白的mRNA表达水平。结果与正常健康人PBMC相比,GPP患者的PBMC中的PDCD1基因呈高甲基化状态。PDCD1的mRNA表达水平显著降低,且与甲基化水平呈负相关。与正常对照组相比,GPP患者的PBMC中DNMT3a、DNMT3b、MBD1、MBD2、MBD4及MBD5较对照组水平升高,而DNMT1、MBD3水平较对照组降低。结论GPP患者的PBMC中基因PDCD1的甲基化飞行质谱检测结果与全基因组DNA甲基化芯片测序结果一致,且PDCD1 mRNA表达水平异常;GPP患者体内甲基化相关调控基因异常表达,这为甲基化在GPP发病中的作用及机制提供了重要的依据,为GPP的治疗提供了新的线索第三部分泛发性脓疱型银屑病患者外周血单个核细胞PDCD1下游因子相关表达研究目的为了进一步明确PDCD1在GPP发生发展中扮演的角色,本部分对PDCD1的配体及信号通路中NF-κ B在GPP患者的PBMC中的表达量进行研究,并且检测了下游IL-17、IL-22及IL-4的含量。方法采用Trizol法提取9例GPP患者及10例健康正常人的PBMC中的总RNA,应用Real-time PCR检测PDCD1的配体细胞程序性死亡受体配体1(programmed cell death ligand 1,PDL1)及细胞程序性死亡受体配体2(programmed cell death ligand 2,PDL2)的 mRNA 表达水平。采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ElISA)对 23 例 GPP 患者及 24 例健康正常人的外周血血清进行IL-17、IL-22及IL-4的含量检测。结果与正常健康人相比,GPP患者的PBMC中PDL1及PDL2表达上调,且IL-17、IL-22及IL-4的浓度显著上升。结论PDCD1及其配体途径在GPP患者体内异常表达,可能在GPP的发生发展中扮演重要的角色。第四部分IL36RN基因突变与泛发性脓疱型银屑病患者临床表现及治疗的联系目的研究GPP患者中IL36RN突变与长期随访的临床表现、复发频率及阿维A治疗疗效的关系。方法这项回顾性的研究纳入61例GPP患者及48例寻常型银屑病患者。并按照IL36RN突变类型将GPP患者分为纯合突变组(homozygous mutation group,HOMG)25 例,杂合突变组(heterozygous mutation group,HEMG)7 例及无突变组(non-mutation group,NMG)29 例。结果HOMG中21例患者初始表现为诱因诱发的GPP,其中13例在脓疱发作期后转化为红皮病;HEMG中的5例患者及NMG中的23例患者起病及脓疱发作期后均表现为PV。大多数患者对阿维A治疗反应较好。随访过程中部分患者在阿维A维持剂量(10-30mg/d)下轻度复发(0-2次每年)。IL36RN突变与疾病的发病年龄及甲下脓疱相关,对阿维A的疗效无明显影响。结论IL36RN突变不是影响阿维A疗效的主要因素。诱因可能对GPP的发生、临床表现及疾病的转归起到重要的作用。低剂量的阿维A可能对GPP的复发有一定控制作用。
[Abstract]:Generalized pustular psoriasis (GPP) is a systemic inflammatory skin disease, which can threaten life in severe cases. The incidence of GPP is low, but it is often accompanied by high fever and general discomfort, and can threaten life in severe cases. Therefore, the etiology and pathogenesis of GPP has been a hot spot in dermatology. Up to now, with the development of genetic background research on GPP, IL36RN and CARD14 genes have gradually been considered as pathogenic susceptible genes of GPP. However, some clinical phenomena can not be explained. For example, most of GPP occurring alone is caused by homozygous or compound heterozygous mutation of IL36RN gene, but there are normal individuals in the population carrying IL36RN gene. Because of mutation, why infection, pregnancy and special drug use can induce the onset of GPP. Therefore, although the genetic mode of susceptibility gene can explain the pathogenesis of some GPP patients, it can not fully reveal the influence of non-genetic mode such as sex, age and inducement on the occurrence and development of disease, such as why infection, pregnancy and special drug use and so on. Epigenetics is the regulation of gene expression without altering the nucleotide sequence of a gene. This regulation can be passed on to offspring and regulates reversible gene expression. Studies have shown that epigenetic regulation plays a role in autoimmune diseases such as systemic lupus erythematosus. Our team found abnormal changes in DNA methylation in peripheral blood mononuclear cells of patients with GPP in previous experiments, suggesting that DNA methylation may be one of the important mechanisms involved in the pathogenesis of GPP.DNA methylation refers to the catalysis of methylase. In this study, S-adenosylmethione (SAM) was used as a methyl donor to add methyl (-CH3) to the fifth carbon atom of cytosine to produce 5-methylcytosine (5mC). Therefore, the peripheral blood mononuclear cell (PBMC) of patients with GPP and normal persons was first studied. Genomic DNA methylation sequencing and related research, in order to fully reveal the role of methylation changes in the occurrence and development of GPP, fill the gap in related fields. Methods Genomic DNA was extracted from PBMCs of 3 patients with GPP and 3 normal controls and sequenced by Illumina Human Methylation 450K Bead Chip chip. Genome-wide methylation maps with enough depth and resolution were sequenced. There were 6328 differentially methylated regions with abnormal changes in methylation. Compared with normal healthy PBMCs, the genome-wide DNA of GPP PBMCs showed abnormal methylation status, including 5921 sites with increased methylation and 407 sites with decreased methylation. GO analysis showed that the genomic DNA in PBMC of GPP patients had abnormal methylation status, including immunocyte migration, inflammation and signal transduction. Genes expressed are related to pathophysiological processes, and their functions involve many aspects. Signal pathways are extremely complex. Pregnancy may affect the occurrence of diseases by altering the level of methylation in the body. Part II Methylation level and regulatory factors of PDCD1 gene in peripheral blood mononuclear cells of patients with generalized pustular psoriasis Objective To select the programmed cell death 1 (PDCD1) gene for DNA methylation sequencing of PBMC from patients with GPP and to study the expression of DNA methyltransferase (DNMT) and methyl-CpG binding proteins (MBD) in PBMC from patients with GPP. The methylation of PDCD1 gene in PBMC of GPP patients and 10 healthy controls was sequenced by flight mass spectrometry to verify the results of methylation chip. Total RNA was extracted from PBMC of 9 GPP patients and 10 healthy controls by Trizol method, and the expression of PDCD1, DNMT and MBD protein was detected by Real-time PCR. Results Compared with normal PBMC, the expression of PDCD1 gene in PBMC of GPP patients was significantly lower and negatively correlated with the level of methylation. Compared with normal control group, the levels of DNMT3a, DNMT3b, MBD1, MBD2, MBD4 and MBD5 in PBMC of GPP patients were higher than those in control group, while the levels of DNMT1 and MBD3 were relatively higher. Conclusion The methylation of PDCD1 gene in PBMC of GPP patients is consistent with that of whole genome DNA methylation chip, and the expression of PDCD1 mRNA is abnormal. The abnormal expression of methylation-related regulatory genes in GPP patients provides an important basis for the role and mechanism of methylation in the pathogenesis of GPP. To further clarify the role of PDCD1 in the pathogenesis and development of GPP, this part describes the expression of NF-kappa B in PBMC of patients with generalized pustular psoriasis. Methods Total RNA was extracted from PBMC of 9 GPP patients and 10 healthy controls by Trizol method, and programmed cell death ligand 1 (PDL1) and programmed cell death ligand 2 (PDL1) of PDCD 1 were detected by Real-time PCR. The levels of IL-17, IL-22 and IL-4 in peripheral blood serum of 23 patients with GPP and 24 healthy controls were detected by enzyme linked immunosorbent assay (ElISA). Conclusion The abnormal expression of PDCD1 and its ligand pathway may play an important role in the development of GPP. Part IV The relationship between IL36RN gene mutation and clinical manifestations and treatment of generalized pustular psoriasis Objective To study the relationship between IL36RN mutation and long-term follow-up in patients with GPP. Methods The retrospective study included 61 patients with GPP and 48 patients with psoriasis vulgaris. GPP patients were divided into homozygous mutation group (HOMG) 25 cases and heterozygous mutation group (HEMG) 7 cases. Results Twenty-one patients in HOMG initially presented as predisposing GPP, 13 of them developed erythroderma after impetigo attack, 5 patients in HEMG and 23 patients in NMG showed PV after onset and impetigo attack. The IL36RN mutation was associated with the age of onset and subthyroid pustules, but had no significant effect on the efficacy of Avian A. Conclusion The IL36RN mutation is not the main factor affecting the efficacy of Avian A. The predisposing factors may play an important role in the occurrence of GPP, clinical manifestations and prognosis of the disease. The low dose of AVI A may have a certain effect on the recurrence of GPP.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R758.63

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