Clinical And Immune Characteristics Of 2 Patients With Congenital Keratosis And The Mechanism Of WASp Promoting Negative Regulation Of B Cells Mediated By BCR-FCγRⅡB-pSHIP Through CD19 And ITIM Pathways

PART I ANALYSIS OF CLINICAL AND IMMUNOLOGICAL CHARACTERISTICS AND GENE MUTATION OF 2 CHILDREN WITH CONGENITAL KERATOSISBackground:Dyskeratosis Congenita(DC)is a syndrome resulting from defective telomere maintenance.Immunodeficiency associated with DC can cause significant morbidity and lead to premature mortality,but the immunological characteristics and molecular hallmark of DC patients,especially young patients,have not been described in detail.Objective:To analyze the clinical data,telomere relative length,gene mutation site information and detailed immunological phenotype of 2 patients with congenital keratosis in our hospital,in order to provide information for early diagnosis,immune evaluation and targeted immunotherapy of DC patients.Methods:We summarize the clinical data of two juvenile patients with DC.Gene mutations were identified by whole exome and direct sequencing.Swiss-PdbViewer was used to predict the pathogenicity of the identified mutations.The relative telomere length was determined by QPCR,and a comprehensive analysis of lymphocyte subsets and CD57 expression was performed by flow cytometry.Results:Both patients showed typical features of DC without severe infection.In addition,patient 1(P1)was diagnosed with Hoyeraal-Hreidars son syndrome due to cerebellar hypoplasia.Gene sequencing showed P1 had a compound heterozygous mutation(c.204G>T and c.178-245del)in PARN and P2 had a novel hemizygous mutation in DKC1(c.1051A>G).Lymphocyte subset analysis showed B and NK cytopenia,an inverted CD4:CD8 ratio,and decreased naive CD4 and CD8 cells.A significant increase in CD21low B cells and skewed numbers of helper T cells(Th),regulatory T cells(Treg),follicular regulatory T cells(Tfr),and follicular helper T cells(Tfh)were also detected.Short telomere lengths,increased CD57 expression,and an expansion of CD8 effector memory T cells re-expressing CD45RA(TEMRA)were also found in both patients.Conclusion:Unique immunologic abnormalities,CD8 T cell senescence and shortened telomere together as a hallmark,occur in young DC patients before progression to severe disease.PART II THE MECHANISM OF WASP PROMOTING NEGATIVE REGULATION OF B CELLS MEDIATED BY BCR-FCΓRIIB-PSHIP THROUGH CD19 AND ITIM PATHWAYSBackground:Wiskott-Aldrich syndrome(WAS),namely eczema,thrombocytopenia and immunodeficiency syndrome,is an X-linked primary immunodeficiency.In addition to the typical clinical manifestations,it has been found that WAS patients have a high incidence of autoimmune diseases(42-70%),but the detailed mechanism is still not fully elucidated.Recent studies have clarified that the BCR-FcyRⅡB signaling pathway maintains the immune balance by negatively regulating the activation of B cell signals.In addition,previous studies have found that WASp defects cause damage to the negative regulation of BCR-FcγRⅡB-pSHIP mediated B cell signals,which is more damaged than the activation signal,leading the body in a state of relatively strong activation and causing to autoimmunity.Objective:To explore the mechanism of WASp defect leading to this negative regulation damage and provide scientific target information for the treatment of autoimmunity in children with WAS.Methods:We analyzed the expression of upstream SHIP molecules in the B cell signaling pathway mediated by BCR-FcγRⅡB-pSHIP in WAS patients and was-ko mice by using flow cytometry,TIRF and western-blot。Results:We found that compared with the control group,WASp defects impaired phosphorylation of Lyn,a molecule directly related to SHIP recruitment and activation at ITIM.Furthermore,although the defects did not affect the expression of FcyRⅡB in rest state,it does affect the phosphorylation of FcyRⅡB.In addition,we also found that as an important co-receptor of BCR,the phosphorylation of CD 19 was also decreased in the case of WASp defects,which was involved in the recruitment of Lyn,Fyn and other signal molecules.Conclusion:The results showed that WASp defects affected the negative regulation of BCR-FcγRⅡB-pSHIP mediated B cell signaling by decreasing the activation of CD 19,CD32b and Lyn,which means that WASp promoting negative regulation of B cells mediated by BCR-FcγRⅡB-pSHIP through CD 19 and ITIM pathways.