泛發(fā)性膿皰型銀屑病患者DNA甲基化相關研究及臨床分析
發(fā)布時間：2018-09-01 16:37
【摘要】：泛發(fā)性膿皰型銀屑病(generalized pustular psoriasis,GPP)是一種系統(tǒng)性炎癥性皮膚病,嚴重時可威脅生命。GPP發(fā)病率較低,但發(fā)病時常常伴有高熱及全身不適,嚴重時可威脅生命。因而GPP相關的病因及發(fā)病機制研究一直為皮膚科領域的熱點。目前為止,隨著關于GPP遺傳背景研究的開展,IL36RN和CARD14兩個基因逐漸被認為是GPP的致病易感基因。但是一些臨床現(xiàn)象仍然無法解釋,如大多數(shù)單獨發(fā)生的GPP是由IL36RN基因的純合或復合雜合突變引起的,但人群中也有正常個體攜帶IL36RN基因突變;為何感染、妊娠和特殊藥物應用等狀態(tài)能誘發(fā)GPP的發(fā)作。因此易感基因遺傳方式雖然解釋了一部分GPP病人的發(fā)病,但無法全面揭示性別、年齡及誘因等非遺傳方式對疾病發(fā)生發(fā)展的影響,例如為何感染、妊娠和特殊藥物應用等狀態(tài)能誘發(fā)GPP的發(fā)作。表觀遺傳學是指在研究基因的核苷酸序列未發(fā)生改變的情況下,對基因的表達進行調(diào)控。這種調(diào)節(jié)可遺傳給后代并為可逆性的基因表達調(diào)節(jié)。有研究表明,表觀遺傳調(diào)控機制在系統(tǒng)紅斑狼瘡等自身免疫性疾病中發(fā)揮重要的作用,但有關GPP的表觀遺傳學的研究較少。我們課題組在之前的前期實驗中發(fā)現(xiàn)GPP患者外周血單個核細胞中存在異常的DNA甲基化狀態(tài)改變,這表明DNA甲基化可能是參與GPP發(fā)病的重要機制之一。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)為甲基供體,將甲基(-CH3)添加至胞嘧啶第五位碳原子上,生成5-甲基胞嘧啶(5mC)的生物學過程。因此,本課題擬首次對GPP患者及正常人的外周血單個核細胞(peripheral blood mononuclear cell,PBMC)進行全基因組DNA甲基化測序及相關研究,以全面揭示甲基化改變在GPP的發(fā)生發(fā)展中的作用,填補相關領域的空白。第一部分泛發(fā)性膿皰型銀屑病患者外周血單個核細胞DNA甲基化測序目的研究GPP患者及正常健康人PBMC的全基因組DNA甲基化狀態(tài),探討DNA甲基化在GPP患者中的表達差異。方法提取3例GPP患者和3例正常健康人的PBMC中的基因組DNA,并采用Illumina Human Methylation 450K BeadChip芯片技術對其進行DNA甲基化測序。結果通過Illumina Human Methylation 450K BeadChip芯片測序,我們得到了足夠測序深度和分辨率的全基因組甲基化圖譜。甲基化發(fā)生異常變化的差異甲基化區(qū)域達到6328個。和正常健康人的PBMC相比,GPP的PBMC全基因組DNA呈現(xiàn)異常甲基化狀態(tài),其中甲基化程度升高位點5921個,甲基化程度降低位點407個。2例同為妊娠狀態(tài)下誘導的GPP患者,在主要成分分析中分布更為接近。GO分析顯示甲基化明顯異常改變的基因類型包括免疫細胞遷移、炎癥反應及信號傳導等。結論GPP患者PBMC中基因組DNA較正常健康人發(fā)生異常的甲基化狀態(tài),其中眾多甲基化水平差異表達的基因與病理生理過程相關,功能涉及多個方面,信號通路極其復雜;妊娠可能通過改變機體內(nèi)甲基化水平的狀態(tài)影響疾病的發(fā)生。第二部分泛發(fā)性膿皰型銀屑病患者外周血單個核細胞PDCD1基因調(diào)控序列甲基化水平及調(diào)控因子研究目的選擇細胞程序性死亡受體1(programmed cell death 1,PDCD1)基因?qū)PP患者PBMC的DNA甲基化測序結果進行驗證。同時研究GPP患者的PBMC的甲基化轉移酶(DNA methyltransferase,DNMT)及甲基化CpG結合蛋白(Methyl-CpG binding proteins,MBD)的表達。方法選取9例GPP患者及10例健康正常人的PBMC為研究對象,采用飛行質(zhì)譜法對PDCD1基因甲基化進行測序,以驗證甲基化芯片的結果。同時采用Trizol法提取9例GPP患者及10例健康正常人的PBMC中的總RNA,應用Real-time PCR的方法檢測PDCD1、DNMT及MBD蛋白的mRNA表達水平。結果與正常健康人PBMC相比,GPP患者的PBMC中的PDCD1基因呈高甲基化狀態(tài)。PDCD1的mRNA表達水平顯著降低,且與甲基化水平呈負相關。與正常對照組相比,GPP患者的PBMC中DNMT3a、DNMT3b、MBD1、MBD2、MBD4及MBD5較對照組水平升高,而DNMT1、MBD3水平較對照組降低。結論GPP患者的PBMC中基因PDCD1的甲基化飛行質(zhì)譜檢測結果與全基因組DNA甲基化芯片測序結果一致,且PDCD1 mRNA表達水平異常;GPP患者體內(nèi)甲基化相關調(diào)控基因異常表達,這為甲基化在GPP發(fā)病中的作用及機制提供了重要的依據(jù),為GPP的治療提供了新的線索第三部分泛發(fā)性膿皰型銀屑病患者外周血單個核細胞PDCD1下游因子相關表達研究目的為了進一步明確PDCD1在GPP發(fā)生發(fā)展中扮演的角色,本部分對PDCD1的配體及信號通路中NF-κ B在GPP患者的PBMC中的表達量進行研究,并且檢測了下游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中的總RNA,應用Real-time PCR檢測PDCD1的配體細胞程序性死亡受體配體1(programmed cell death ligand 1,PDL1)及細胞程序性死亡受體配體2(programmed cell death ligand 2,PDL2)的 mRNA 表達水平。采用酶聯(lián)免疫吸附試驗(enzyme linked immunosorbent assay,ElISA)對 23 例 GPP 患者及 24 例健康正常人的外周血血清進行IL-17、IL-22及IL-4的含量檢測。結果與正常健康人相比,GPP患者的PBMC中PDL1及PDL2表達上調(diào),且IL-17、IL-22及IL-4的濃度顯著上升。結論PDCD1及其配體途徑在GPP患者體內(nèi)異常表達,可能在GPP的發(fā)生發(fā)展中扮演重要的角色。第四部分IL36RN基因突變與泛發(fā)性膿皰型銀屑病患者臨床表現(xiàn)及治療的聯(lián)系目的研究GPP患者中IL36RN突變與長期隨訪的臨床表現(xiàn)、復發(fā)頻率及阿維A治療療效的關系。方法這項回顧性的研究納入61例GPP患者及48例尋常型銀屑病患者。并按照IL36RN突變類型將GPP患者分為純合突變組(homozygous mutation group,HOMG)25 例,雜合突變組(heterozygous mutation group,HEMG)7 例及無突變組(non-mutation group,NMG)29 例。結果HOMG中21例患者初始表現(xiàn)為誘因誘發(fā)的GPP,其中13例在膿皰發(fā)作期后轉化為紅皮病;HEMG中的5例患者及NMG中的23例患者起病及膿皰發(fā)作期后均表現(xiàn)為PV。大多數(shù)患者對阿維A治療反應較好。隨訪過程中部分患者在阿維A維持劑量(10-30mg/d)下輕度復發(fā)(0-2次每年)。IL36RN突變與疾病的發(fā)病年齡及甲下膿皰相關,對阿維A的療效無明顯影響。結論IL36RN突變不是影響阿維A療效的主要因素。誘因可能對GPP的發(fā)生、臨床表現(xiàn)及疾病的轉歸起到重要的作用。低劑量的阿維A可能對GPP的復發(fā)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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R758.63
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R758.63
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7 李紹波;黃sダ，
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