Research On The Pathogenesis Of PSTPIP1-associated Inflammatory Diseases And A Heterzyogous N-terminal Truncation Mutation Of NFKBIA Results In An Impaired NF-κB Dependent Inflammatory Response

PART Ⅰ RESEARCH ON THE PATHOGENESIS OF PSTPIP1-ASSOCIATED INFLAMMATORY DISEASESObjective:Mutations in the PSTPIP1 could cause rare autoinflammatory diseases.At present,there are only dozens of cases have been reported and patients with PSTPIP1-associated inflammatory diseases(PAID)have varied phenotype.Heterozygous mutations in the PSTPIP1 gene cause the dominant inherited pyogenic arthritis,pyoderma gangrenosum,and acne(PAPA)syndrome,which as typically caused by missense mutation p.A230 T and p.E250 Qin PSTPIP1.While the missense mutation p.E250 K and p.E257 K cause the PSTPIP1-associated myeloid-related proteinemia inflammatory(PAMI)syndrome with fever,rashes and hypercalprotectinemia and hyperzincemia with neutropenia.In this study,we analyzed the clinical,immunological features and treatment of three patients with PAID and compared them with the previous reports.Methods: Three patients with PAID diagnosed by our clinical center were enrolled this study.The clinical manifestation and treatment were collected for each patient.The plasma and peripheral blood mononuclear cells(PBMCs)were collected and their PBMCs were stimulated with LPS and ATP and the supernatant was collected.The plasma calprotectin levels were detected by ELISA,and the concentrations of inflammatory factors in plasma and supernatant were measured by Biolegend Human Inflammation Panel.At the same time,we constructed the overexpressed THP-1 cell lines of PSTPIP1 p.N236 K,which is one of novel mutation of PAMI syndrome,and the expression levels of inflammatory factors for WT and Mutant THP-1 of PSTPIP1 were measured by the same method.Flow cytometry was used to detect apoptosis-related molecules of neutrophils in patient with PAMI syndrome(PSTPIP1 p.N236K).Results:Two patients presented recurrent fever with rashes,the level of plasmas calprotectin and serum zinc were significantly elevated.The concentrations of inflammasome-related cytokines(IL-1β,IL-18),and IL-6,TNF-α were higher than the healthy control.In plasma of first patient and second patient,these concentrations were higher in the PBMCs supernatant after stimulation of second patient but not in it of first patient.Genetic testing revealed heterozygous mutations in PSTPIP1,c.708C>G,p.N236K(first patient,not previously reported)and c.769G>A,p.E257K(second patient,previously reported as PAMI syndrome).The third patient presented a typical PAPA syndrome phenotype,including pyogenic arthritis,pyoderma gangrenosum,and acne.His plasma calprotectin level was also elevated than normal control but lower than first and second patients,while his serum zinc level was normal.Those cytokines concentrations as mentioned before in the PBMCs supernatant after stimulation were increased than normal control.Genetic testing revealed a heterozygous mutation in PSTPIP1,(third patient,c.748G>A,p.E250K),which had been reported as PAMI syndrome.The concentrations of IL-1β,IL-18 of THP-1supernatant after stimulation were higher in the novel mutation of p.N236 K in PSTPIP1 than it of WT.At the same time,the expression levels of active-caspase 3,Fas and Fas L were higher in the neutrophil of first patient than them of healthy donors.Conclusion: The clinical phenotypes are varied.The novel mutation of p.N236 K in PSTPIP1 could cause PAMI syndrome,the mutation of p.E250 K could cause PAMI syndrome reported previously,as well as PAPA syndrome discovered in this study.This study extends the phenotype of the spectrum.In view of the heterogeneity of the clinical phenotype of the mutation in PSTPIP1 and the limitation of classifying by mutation site,we suggest the term of PAID as a new class that encompass all syndromes with PSTPIP1 mutation.At the same time,our study indicated that inflammasome hyper-activation may be involved in the pathogenesis of PAID,and the cause of neutrophil deficiency of PAMI syndrome might be related to the increase of neutrophil apoptosis.PART Ⅱ A HETERZYOGOUS N-TERMINAL TRUNCATION MUTATION OF NFKBIA RESULTS IN AN IMPAIRED NF-κB DEPENDENT INFLAMMATORY RESPONSEObjective:Primary immunodeficiency caused by NFKBIA gene mutation is extremely rare.To date,there are 19 patients with NFKBIA mutation have been reported with various clinical manifestation.Missense and nonsense mutations could lead to impair the phosphorylation of 32 or36 serine of IκBα and then IκBα degradation would be inhibited,leading to NF-κB activation obstacles.The clinical manifestations include recurrent infections,anhidrotic ectodermal dysplasia with immunodeficiency(EDA-ID).This study reports the clinical manifestation and immunophenotype of a Chinese patient with NFKBIA mutation,and compares her phenotype with previous reports.Methods:One patient with NFKBIA truncated mutation diagnosed by our clinical center were enrolled this study.The peripheral blood mononuclear cells(PBMCs)were collected and her PBMCs were stimulated with LPS,IL-1β and IFN-γ and the supernatant was collected.The concentrations of inflammatory factors in supernatant were measured by Biolegend Human Inflammation Panel.The NF-κB p65 phosphorylation level was detected by immunofluorescene.At the same time,the clinical manifestation and treatment were collected for the patient and we also reviewed all the previously reported patients with NFKBIA mutation.Results:The patient presented recurrent infections with EDA-ID,including recurrent fever,fungal infection,chronic diarrhea with thinning hair,no eyebrows,growth retardation.Laboratory exams showed that the levels of Ig A and Ig M,and CD4+ T cells count were decreased,while the CD8+ T cells count was increased.The concentrations of NF-κB pathway related cytokines(IL-1β,IL-6,IL-8,IL-12,IL-18,TNF-α,IFN-γ)were decreased significantly in the supernatant of the PBMCs stimulation.The immunofluorescene indicated that the NF-κB p65 phosphorylation was impaired.Genetic testing revealed a heterozygous truncated mutation in NFKBIA(c.40G>T,p.E14X).According to the review,we found that patients with NFKBIA mutation had varied phenotype,all patients presented ID but some patients did not have EDA,and truncation mutations in NFKBIA had less damage to NF-κB pathway activation than missense mutations.Conclusion:The clinical phenotypes in patients with NFKBIA mutation are diverse.We realized that the disease caused by NFKBIA mutation cannot be classified according to the mutation type.At the same time,we must be vigilant for PID with early onset to achieve early diagnosis and treatment.