The Correlation Of Gene Mutations Of Wisktt-Aldrich With Clincal Manifestation And The Specific Mutation Of WASP P460S With Bone Marrow Failure

Part one.The correlation of gene mutations of Wiskott-Aldrich syndrome with clinical manifestationObjective: To investigate the correlation of gene mutations of 28 cases with WAS gene defect with the clinical manifestation.Methods: The clinical information of 28 patients identifeid WAS defects were collectd in the Children’s Hospital of Soochow University from January 2013 to February 2018.The correlation of hot pots mutations of WAS gene and clinical manifestation in 28 patients were analyzed.Results: All patients were male.The median onset age was 1 month（range,0-83 months）.Nine mutants were identified as novel mutations among 24 mutants detected in 28 patients,including c.1234₁235dup CC,c.1093-1097 del G,c.28-30 dup C,c.436G> T,c.273 + 10₂73 + 11 dup CC,c.995₉96ins G,c.1010T> A,c.332₃33del CC and c.683C> T mutations.There were 24 cases diagnosed as classic WAS in which mutations illusstrated as missense mutation,deletion mutation,insertion mutation,splicing mutation and nonsense mutation,the mutation in 1 cases of X-linked thrombocytopenia（XLT）was missense mutation,1 case of intermittent X-linked thrombocytopenia（IXLT）was splicing mutation,2 cases of X-linked pancytopenia（XLP）were by missense mutation.Intravenous immunoglobulin（IVIG）and glucocorticoid therapy in IXLT patient were effective,only a small part of classic WAS patients（8.3%）showed transient response to IVIG and glucocorticoid therapy.Lymphoid subtypes detected by flow cytometry showed that CD3 + cells were decreased in 60.9% patients,CD19 + cells were decreased in 13.0% patients,and CD56 + 16 + cells in 4 patients were decreased,accounting for 17.3%.Of Twenty-three patients,21 patients were alive after treated with hematopoietic stem cell transplantation（HSCT）.Four patients who didn’t receive HSCT died of brain bleeding and severe infection.One patient diagnosed as IXLT got remission and survived.Conclusion: WASP gene defect is the basic criteria for the diagnosis of WAS and related diseases.HSCT is the most effective treatment for WAS,XLT,and XLP.Part two.The correlation of WASP P460 S mutation with bone marrow failureObjective: To explore the correlation between the specific mutation WASP P460 S and bone marrow failure.Methods: The next generation sequencing（NGS）was employed in two case of bone marrow failure（BMF）to find mutations of genes related to BMF and further comfirmed by Sanger sequencing.SWISS-MODEL software was used to predict protein structure change of WAS gene C1378 T mutation.WASP expression in bone marrow cells or cell lines were detected by Western blot.The WASP-WT,WASP-S272 P,and WASP-P460 S expression plasmids were constructed into PCDH-CMV-GFP-flag vector and Plu-CMVMCS-i GFP lentiviral vector,respectively.The lentiviral packaging with WASP-WT and WASP-P460 S were transfected into K562 cells.Transfected cells were treated with such drugs as MTX,CTX,and DNR at concentrations of 0,0.5,1,2 and 4μg / ml,respectively.The proliferation of transfected cells was detected by Cell Tilte-Glo reagent（CTG KIT）,apoptosis was detected by flow cytometry with Annexin V-PE / 7-AAD apoptosis kit.Cell cycle was analyzed by flow cytometry after propidium iodide staining.After transfected cells were incubated with TPA at 40 n M for 24 h,CD41 expression was detected.Western blot was employed to detect the expression of c-PARP protein.Meanwhile,the actin polymerization experiment was performed to explore the effect of WASP P460 S mutation on actin polymerization.Results: The sequencing result showed that the patients and their mothers had WASP P460 S mutation and mothers were carrier.The software showed that the WASP P460 S mutation didn’t change the structure of WAS protein（WASP）obviously.WAS protein（WASP）level expressed normally in the patient compared with normal people.K562 cells didn’t express WASP.The proliferation rate of K562-WASP-P460 S cells was significantly slower than that of K562-WASP-WT cells.Chemotherapy drugs didn’t show significant difference in both transfected cells on proliferation,apoptosis,and cell cycle arrest.The expression of CD41 didn’t show different significance on the surface of K562-WASP-P460 S cells and control which indicated that this mutation didn’t influence the differentiation of megakaryocytes.However,P460 S mutation could cause an increase in actin polymerization in mutants compared to WT.Conclusion: WASP P460 S mutation could decrease cell proliferation,increase actin polymerization in mutants compared with WT,which might be responsible for bone marrow failure.