Related Studies And Clinical Analysis Of DNA Methylation In Patients With Generalized Pustular Psoriasis

Generalized pustular psoriasis(GPP)is a systemic inflammatory skin disease which may be life-threatening in severe case.GPP has low morbidity,but it frequently accompanies with high fever and general malaise in the case of disease onset,which may even be life-threatening in severe case.Therefore,research on GPP-related etiology and pathogenesis has always been the hotspot in Dermatology.With the carrying out of GPP genetic background research at present,IL36RN and CARD 14 genes have been gradually recognized to be the susceptibility genes of GPP.However,some clinical phenomena remain to be further explained.For instance,a majority of sporadic GPP cases are induced by homozygous or compound heterozygous mutations of IL36RN gene,but there are also normal individuals carrying IL36RN gene mutation among population.Why conditions such as infection,gestation and use of special drugs can induce GPP.It is indicated in research that epigenetic regulatory mechanism plays an important role in autoimmune diseases,such as systemic lupus erythematosus,but epigenetic research regarding GPP is lacking.Our research group discovered in our previous experiments that abnormal changes in DNA methylation state could be seen in peripheral blood mononuclear cells of GPP patients,indicating that DNA methylation might account for one of the important mechanisms participating in GPP pathogenesis.DNA methylation is referred to as the biological process that treats S-adenosylmethione(SAM)as the methyl donor,adds methyl(-CH3)into the fifthcarbon atom of cytosine to produce 5-methylcytosine(5mC)under the catalysis of methylase.Modified state of DNA methylation may be affected by environmental factors and changes with age,which may explain the association of environmental factors with the genesis and development of disease.DNA methylation state may have reversible and rapid changes when exposing to a certain external environment or a certain stimulating factor.Consequently,this project proposed to carry out genome wide DNA methylation sequencing of peripheral blood mononuclear cells in GPP patients and normal subjects for the first time,so as to comprehensively unveil the role of methylation in the genesis and development of GPP and to fill in the blank in relevant field.Part I DNA Methylation Sequencing of Peripheral Blood Mononuclear Cell in Patients with GPPObjective To investigate the genome wide DNA methylation state of peripheral blood mononuclear cells in GPP patients and normal subjects,with an aim to probe into the role of DNA methylation in the pathogenesis of GPP as well as its underlying mechanism.Methods Genome DNA was extracted from peripheral blood mononuclear cells of 3 GPP patients and 3 normal subjects,and DNA methylation sequencing was carried out using Illimina Human Methylation 450K BeadChip chip.Results Genome wide methylation pattern with enough sequencing depth and resolution was obtained using Illimina Human Methylation 450K BeadChip chip sequencing.6311 differential methylation regions showed abnormal changes of methylation.Compared with peripheral blood mononuclear cells from normal subjects,genome wide DNA in peripheral blood mononuclear cells from patients with GPP displayed abnormal methylation state,among which,390 sites had elevated methylation levels,while 5921 sites had reduced methylation levels.PCA analysis revealed closer distribution in two gestation-induced GPP patients.GO analysis suggested that genotypes with distinctly abnormal methylation changes included immune cell migration,inflammatory response and signal transduction.Conclusion Compared with normal subjects,peripheral blood mononuclear cells from patients with GPP display abnormal methylation state,among which,numerous genes with differentially expressed methylation levels are related to pathological and physiological processes.Meanwhile,it involves multiple functions and its signal pathway is extremely complicated.Pregnancy may affect the occurrence of disease through altering methylation level in the body.Part II Analysis of PDCD1 Gene Regulatory Sequence Methylation Level and Expression of Regulating Factor in Peripheral Blood Mononuclear Cells of GPP PatientsObjective To verify DNA methylation sequencing results of peripheral blood mononuclear cells of GPP patients through PDCD1 gene.And to investigate expression of methylase and methylated CpG binding protein in peripheral blood mononuclear cells of GPP patients.Methods Peripheral blood mononuclear cells from 9 GPP patients and 10 normal subjects were selected as the objects of study,and PDCD1 gene methylation sequencing was carried out using flight mass spectrometry,so as to verify results of methylation chip.Meanwhile,total RNA was extracted from peripheral blood mononuclear cells of 9 GPP patients and 10 normal subjects using Trizol method,andPDCD1,DNMT,MBD mRNA expression level was detected with real-time PCR.Results Compared with peripheral blood mononuclear cells of normal subjects,PDCD1 gene in peripheral blood mononuclear cells of GPP patients showed hypermethylation state,and PDCD1 mRNA expression level was remarkably reduced.Compared with normal subjects,DNMT3a,DNMT3b,MBD1,MBD2,MBD4levels in peripheral blood mononuclear cells of GPP patients were elevated,while DNMT1 and MBD3 levels were decreased.Conclusion Methylation flight mass spectrometry sequencing results of gene PDCD1 are consistent with those of genome wide DNA methylation chip sequencing results,and PDCD1 mRNA expression level is abnormal;Methylation related regulatory genes are abnormally expressed in GPP patients,which has provided important foundation for the role of methylation in the pathogenesis of GPP as well as its mechanism.Part III Expression of PDCD1 Downstream Factors in Peripheral Blood Mononuclear Cells of GPP PatientsObjective To further determine the role of PDCD1 in the genesis of GPP,expression quantities of PDCD1 receptor and downstream NF-κB in peripheral blood mononuclear cells of GPP patients were studied,and contents of downstream IL-27,IL-22 and IL-4 were detected.Methods Total RNA was extracted from peripheral blood mononuclear cells of 9 GPP patients and 10 normal subjects using Trizol method,and mRNA expression levels of PDCD1 ligands PDL1 and PDL2 were detected with real-time PCR.Contents of IL-27,IL-22 and IL-4 were detected in peripheral sera from 23 GPP patients and 24 normal subjects using ELISA.Results Compared with normal subjects,expression of PDL1 and PDL2 was up-regulated in peripheral blood mononuclear cells of GPP patients,and concentrations of IL-27,IL-22 and IL-4 were notably elevated.Conclusion PDCD1 and its ligand pathway are abnormally expressed in GPP patients,which may play an important role in the genesis and development of GPP.Part IV Association of IL36RN Mutations with Clinical Features in Patients with Generalized Pustular PsoriasisObjective:To investigate the correlation between IL36RN mutations and clinical features,recurrence frequency and therapeutic response to acitretin in GPP patients during a long-term follow-up.Methods:This retrospective cohort study included 61 GPP patients and 48 patients with psoriasis vulgaris(PV).According to the results of mutation,patients were classified into three subgroups:homozygous mutation group(HOMG),heterozygous mutation group(HEMG),and non-mutation group(NMG).Results:Two genetic variants in IL36RN were identified(c.115+6 T>C and c.227C>T).The c.115+6 T>C mutation was detected in 52.5%of the patients.Initially,21 of the 25 HOMG patients were diagnosed with GPP with provocative factors,but 13 of whichdeveloped erythrodermic psoriasis after the pustular phase.Patients in the HEMG(5/7)and NMG(23/29)maintained their PV diagnosis before and after the pustular phase.Most patients responded markedly to acitretin,but patients who were prescribed an acitretin maintenance dosage(10-30 mg/d)had a mild recurrence(0-2 times per year)during the follow-up.IL36RN mutations were strongly linked with early-onset and hyponychial pustules,but not with the therapeutic efficacy of acitretin.Conclusions:Early-onset and hyponychial pustules may be specific to IL36RN mutations.IL36RN mutation alone is an insufficient indicator for acitretin therapy,but other provocative factors play important roles in disease onset,clinical manifestations and disease outcome.Low-dose maintenance therapy with acitretin might help reduce the recurrence of GPP.