Establishment Of IKBKB Point Mutation Mouse Model And Preliminary Study Of Immunological Phenotype And Neutrophil Function And Clinical And Immunological Characteristics Of A Case With IFIH1 Mutation

PARTⅠ ESTABLISHMENT OF IKBKB POINT MUTATION MOUSE MODEL AND PRELIMINARY STUDY OF IMMUNOLOGICAL PHENOTYPE AND NEUTROPHIL FUNCTIONObjective: According to the homozygous IKBKB gene c.1183T> C,p.Y395 H mutation from a patient with combined immunodeficciency previously,we construct the homologous site-specific mutant mouse model by using CRISPR / Cas9 technology.The effects of this mutation on neutrophil function was investigated,hoping to provide an experimental clue of the pathogenesis of the disease.Methods: CRISPR/Cas9 technology was used to construct the corresponding point mutation mouse model.Genomic DNA was genotyped by PCR and confirmed by Sanger sequencing.Both splenocytes and thymocytes were isolated by using density gradient centrifugation,and then the mRNA and protein expressions of members of IKKs family were detected by Real-time PCR and western blot respectively.The MOE(molecular operating environment)software was used to analyze the PDB structure of the protein and to establish the 3D model.Then proportion of the Treg cells was analyzed by flow cytometry.The neutrophils were isolated from the bone marrow and luminol chemiluminescence was used to detect the production of neutrophils reactive oxygen species(ROS)through NADPH pathway and extracellular traps(NETs)formation.Results: The results of gene Sanger sequencing showed that point-mutant stable genotype mice were successfully constructed.The WT mice and homozygous mutants were then used for the following study.In addition,the mRNA expressions of IKBKB in splenocytes and thymocytes from mutant mice were comparable to that in wild-type cells,whereas the IKKβ was obviously decreased.Furthermore,the protein structure analysis showed that the conformational structure of mutant IKKβ protein obviously changed.And for the Treg cells proportion,the mutants were decreased evidently.As compared with wild-type mice,there was no statistical difference in extracellular ROS production by bone marrow neutrophils of mutant mice,but the peak of intracellular ROS production in mutant mice showed significant decrease.Moreover,the basal level of neutrophils extracellular traps from bone marrow of mutant mice was significantly lower than that of wild type,and the trends of NETs change after stimulation was also lower than that of wild type mice.Analysis of the protein structure showed that the spatial configuration of the mutant IKKβ protein was significantly changed.Conclusion: The IKBKB Y397 H homozygous mutation causes the abundance of IKKβ protein reduced and Treg cells decreased obviously,moreover partially impaired respiratory bursts in the bone marrow neutrophil manily through intracellular pathway,and possibly leading to reduced bactericidal function of the neutrophils,these results may be related to mutations that cause changes in protein structure,which reduces its stability and affects on the differentiation,development,and functioning of immune cells,but how this mutation causes abnormal NFкB pathway function and how adaptive cells and neutrophils function impaired are under investigation by our colleques.This study provides the first-hand experimental data for the impact of IKBKB mutant from a genuine human mutation on adaptive immune and innate immune cell homeostasis regulation and its possible pathogenic mechanism.PART Ⅱ CLINICAL AND IMMUNOLOGICAL CHARACTERISTICS OF A CASE WITH IFIH1 MUTATIONObjevtive: In April 2019,our hospital has diagnose and treat a patient who was found a novol IFIH1 gene mutation in a high-throughput sequencing report,the child’s clinical manifestations were fever,cough,multiple respiratory virus infections,backward growth,which is inconsistent with the primary immunodeficiency disease(PID)caused by loss-of-function of this gene or gain-of-function which results in Aicardi-Goutières syndrome(AGS).We conducted pathogenicity prediction and sequence conservation analysis,combined the clinical symptoms and immunological phenotypes to discuss how to verify the immunological function of different mutations in the IFIH1 gene,and finally to avoid misdiagnosis of PID patients.Methods: A child with IFIH1 gene mutation was taken as the research object.The patient’s medical history,immune-related high-throughput sequencing results were collected and analyzed,and the outcome was followed.Peripheral blood was collected from the child and peripheral blood mononuclear cells(PBMCs)were extracted.We use flow cytometry to detect lymphocyte subsets,Treg cells subsets,low density neutrophils(LDN),and the level of type Ⅲ interferon(IFN-γ)of T cells which were stimulated by ionomycin,at the same time,ELISA were used to detect type Ⅰinterferon(IFN-α and IFN-γ)levels.Results: Immune-related high-throughput sequencing and Sanger sequencing of the c DNA confirmed the presence of compound heterozygous mutations in exon 15 of the IFIH1 gene(c.2808-2A> G from his mother,c.2836G>A from his father),the production of type Ⅰ and type Ⅲ interferons were reduced,the proportion of Treg cells was normal,and the LDN cells increased obviously.Conclusion: This study reported a case of primary immunodeficiency disease of a novol mutation in the IFIH1 gene with an atypical clinical phenotype.For the Conflicting Aicardi–Goutières syndrome(AGS)caused by IFIH1 gene gain-of-function and severe respiratory infections caused by IFIH1 gene loss-of-function,in-depth immune function evaluation should be conducted to find out the etiology,and the key factors of the pathways in type I interferon secretion are necessarily needed to be tested to identify whether the mutation sites cause gain-of-function or loss-of-function,and the relevant cases would be diagnosed in time and accurately and treated as soon as possible.