Clinical And Immunological Analysis Of 20 Patients With X-linked Lymphoproliferative Syndrome And Establishment Of A Method For Detecting XIAP Protein By Whole Blood

PARTⅠCLINICAL AND IMMUNOLOGICAL ANALYSIS OF 20 PATIENTS WITH X-LINKED LYMPHOPROLIFERATIVE SYNDROMEObjective:To analyze and compare the clinical,genetical and immunological characteristics of patients with XLP-1 and XLP-2 for providing precise diagnosis and treatment.Methods:The clinical data and routine laboratory testing were collected from patients’medical records.SH2D1A and XIAP mutations were investigated by next generation sequencing and sanger sequencing.SAP and XIAP Protein expression were evaluated by flow cytometry and western blotting.In addition,we studied the lymphocyte subsets by cytometric immunophenotyping.Result:In this study,thirteen patients were confirmed as XLP-1(SAP deficiency)and seven patients were confirmed as XLP-2(XIAP deficiency).All the patients were males from nonconsanguineous families.Of our 20patients,19 presented with disease symptoms at a very early age.The mean ages at onset in SAP-deficient patients and XIAP-deficient patients were3.05 and 3.02 years,respectively.The mean ages at diagnosis in SAP-deficient patients and XIAP-deficient patients were 7.86 and 3.65 years,respectively.One XIAP-and three SAP-deficient patients died,while 3 of 7and 4 of 13,respectively,developed hemophagocytic lymphohistiocytosis(HLH).Epstein-Barr virus(EBV)infection was significantly more common in SAP-deficient(76.9%)than XIAP-deficient(28.6%)patients,as was hypogammaglobulinemia(76.9%vs.33.3%).None of the seven XIAP-deficient patients had colitis or lymphoma.Five of the thirteen patients had R55X mutations suggesting that R55X mutation was the hotspot mutation in China.Three novel mutations in XIAP were documented-c.224A>G,c.951G>A,and del Exon 4.Nine SAP-deficient patients and five XIAP-deficient patients showed markedly deficient SAP and XIAP expression,respectively,in lymphocytes.Significantly reduced levels of switched memory B cells were observed in six SAP-deficient patients with persistent hypogammaglobulinemia.Among five XIAP-deficient patients,two with HLH showed a significant decrease in all B cell subsets,a decrease in NK cells and a significant increase in CD8~+T cells.Six SAP-deficient patients,progressively developed different degrees of bronchiectasis,severe pulmonary consolidation or atelectasis,which may be related to their long time from onset to genetic diagnosis and during that period no regular IVIG treatment was performed.1 of 13 SAP-deficient patients and 1 of 7 XIAP-deficient patients received HSCT treatment and are now alive and well.Conclusion:We firstly summarized the clinical features,genetical and immunological characteristics of Chinese XIAP-deficient patients and reported the differences and similarities between SAP deficiency and XIAP deficiency in China.Acute visceral hemorrhage caused by HLH is the most common cause of death in both two types of XLP.None of the seven XIAP-deficient patients had colitis,whether they will develop colitis in the future warrant further investigation.In children with recurrent respiratory infections and persistent hypogammaglobulinemia,regular IVIG therapy should be performed as soon as possible.HSCT is the only curative therapy for XLP and,this therapy should be urgently considered.PART Ⅱ ESTABLISHMENT OF A METHOD FOR DETECTING XIAP PROTEIN BY WHOLE BLOODObjective: A method for detecting XIAP protein using the lysed whole blood was established and applied to patients with XIAP deficiency.Methods: 300μl peripheral whole blood was collected in a vacutainer containing Ethylene Diamine Tetraacetic Acid(EDTA)or Heparin as anticoagulant,then whole blood was lysed by red blood cell lysate.Nonspecific site was blocked with serum full Ig G antibody from homologous species of anti-human XIAP antibody and an isotype control antibody group was set up.The expression of XIAP protein was detected by flow cytometry after incubated with anti-human XIAP antibody.This method was applied to detect applied in both healthy control and XIAP-deficient patient.Results: The method of detecting XIAP protein using whole blood was successfully established.For healthy control or patient with XIAP deficiency,the results detected by whole blood or PBMCs samples were both reliable.Conclusion: We raised a method to detect XIAP protein using lysed whole blood,which requires less blood collection thus cause less damage to the patient.In addition,this method is less time-consuming and the result is more accurate.