The Study Of Primary Immunodeficiency Caused By CARD10 Mutation And Its Mechanism Involved In Immune System

Background: Primary immunodeficiency disease(PID)is a kind of immune molecular or cellular defects mainly caused by single gene mutations,resulting in reduced,absent or dysregulated immune function.The clinical phenotype of PID is complex and its pathogenesis is not yet fully understood.With the continuous improvement of clinical immunology,molecular genetics and next-generation sequencing technology,new pathogenic genes of PID have been detected explosively in recent years.This disease is not rare as previously thought,and its incidence has been seriously underestimated.To date,476 PID disorders have been identified,including 485 different gene defects,but the research in this field in China is relatively backward,with only one gene was identified by Chinese scholars as corresponding authors for the first time.Early diagnosis and timely treatment will save the life of PID patients,improve their quality of life and prolong their life.Therefore,it is of great significance to detect new pathogenic genes and study the pathogenesis of PID.Purpose: The purpose of this study is to find new pathogenic genes of PID,clarify a new phenotype of PID,and provide a reference for genetic counseling of PID patients,diagnosis and treatment of clinicians,and exploration of PID related pathogenesis.Methods: From 2014 to 2020,we collected families(especially consanguineous families)that were suspected to be PID according to JMF(Jeffrey Modell foundation)standard in our department,and carefully collected the patients’ clinical history,family analysis,imaging and laboratory examination.Multicolor flow cytometry analysis of leukocyte subsets and high-throughput cytokine detection were used for immunological analysis of patients and healthy controls.The pathogenic mutations were identified by WES(whole exome sequencing)and functional analysis,and the family co-segregation was verified.HEK293 T tool cells were used to verify the pathogenicity of mutation sites.CARD10 gene knockout THP-1 cell line was constructed and used for transcriptome analysis.Patients’ PBMC(peripheral blood mononuclear cells)were stimulated with CD3/CD28 and the leukocyte subsets were analyzed by polychromatic flow cytometry.Results: Based on the JMF standard,patients with confirmed PID were finally included and performed WES,and then 2 PID patients from a consanguineous family were identified to carry homozygous missense mutation(p.R420C)of CARD10 gene by homozygous mapping,while there were no CARD10 gene mutation in a 242 control databases,Nuovo Origin or Bairui 100000 database.These two patients in this consanguineous family presented with clinical symptoms such as recurrent infection,sinusitis,asthma,bronchiectasis,autoimmune anemia and Crohn’s disease,and gastrointestinal mucosal biopsy showed early Crohn’s disease changes in the gastrointestinal tract.The expression of CARD10 m RNA and protein was decreased in the R420 C mutant.Analyses of 122 leukocyte subsets showed that the number of monocytes and dendritic cells was significantly reduced,and high-throughput inflammatory factors test showed that the levels of serum inflammatory cytokines and chemokines were significantly changed.Further transcriptome analysis of the successfully constructed CARD10 knock out THP-1 cell line suggested that CARD10 knockout may lead to abnormal antiviral pathways including EB virus infection,influenza A,novel coronavirus pneumonia,RIG-I-like receptor signaling pathway,NF-κB signaling pathway,and abnormal physiological processes such as defense response to viruses and cellular response to type I interferon.Analysis of leukocyte subsets after CD3/CD28 stimulation of PBMCs from patients with CARD10 mutations identified that CARD10 mutation may affect T cell proliferation and function.Conclusion: We report for the first time that homozygous mutations in CARD10 may cause a human primary immunodeficiency disease with an autosomal recessive inheritance pattern and that patients can present with slowly progressive autoimmunity and/or immunodeficiency.CARD10 homozygous mutations may induce primary immunodeficiency by affecting multiple immune cells and cytokines,leading to over activation of antiviral pathways.This study provides an important basis for clinical findings and genetic counseling of PID patients,and also sheds new light on the molecular mechanism by which CARD10 leads to PID.