The Preliminary Study Of Pathogenesis In Autosomal Dominant Hyper IGE Syndrome With STAT3Mutations

Part One: Clinical features, STAT3Gene Mutations and Th17CellAnalysis in Patients with Hyper-IgE SyndromeObjective: To study the clinical features, the spectrum ofheterozygous STAT3mutations, and the quantified Th17cell numbers innine patients with hyper-IgE syndrome（HIES）phenotype in mainlandChina.Methods: We diagnosed patients with HIES from nine families on thebasis of a National Institutes of Health （NIH） score of≥40points,sequenced the STAT3gene, and quantified Th17cells in peripheral bloodof seven patients by flowcytometry.Results: All nine patients had eczema, recurrent pneumonia,extremely high serum IgE levels and eosinophilia with the range of NIHscores45-77points. STAT3hot mutations V637M or R382W/Q wereidentified in five patients. We identified two novel heterozygous missensemutations （T620S and R609G） located in Src homology2（SH2） domain in2patients, respectively. In two other studied patients, we were unable to find STAT3mutations. Quantified Th17cell numbers were markedlydecreased or absent （0%⁰.28%of CD4+T cells）in six STAT3-mutantpatients and almost normal（0.53%of CD4+T cells） in one wild-typeSTAT3patient compared with healthy controls（0.40%-2.25%of CD4+Tcells）.Conclusion: Heterozygous STAT3mutations, including two novelmissense mutations, were identified in seven of nine unrelated HIESfamilies in mainland China. Not all HIES patients whose NIH scores above40points carry STAT3mutations. Those whose Th17cell numbersstrikingly decreased probably had AD-HIES with STAT3mutations. Part Two: Construction of the Eukaryotic Expression Vector of NovelSTAT3Mutation T620SObjective: To construct eukaryotic expression vector of novel STAT3mutation T620S and transfect it into COS7cells for further study of thefunction of mutated STAT3protein.Methods: Primers were designed according to the sequence of humanSTAT3mRNA （NM₁39276.2） in Genbank, the sites of restrictionenzymes were added to the terminations of the primers. The DNA of thevector of pAcGFP1-N1-STAT3-WT was used as the template to amplify the target gene segment, which was cloned to T-vector. After verification bydouble enzyme digestion and sequencing, the recombinational T-vector andpAcGFP1-N1vector were double enzyme digested. The two target genesegments undergoing enzyme digestion were purified by gel extraction.The eukaryotic expression vector pAcGFP1-N1-STAT3-T620S wasconstructed after the processes of linkage and transformation. Doubleenzyme digestion and sequencing were performed to identify thecorrectness of the vector. The vector was transfected into COS7cells toobserve the green fluorescence intensity （STAT3expression） and thetransfection efficiency was detected by flowcytometry.Results: Both the results of double enzyme digestion and sequencingshowed that the eukaryotic expression vector pAcGFP1-N1-STAT3-T620Swas successfully constructed, the expression of green fluorescence wasobserved in COS7cells. The transfection efficiency was47.02%byflowcytometry.Conclusion: The eukaryotic expression vector of STAT3novelmutation T620S was successfully constructed and expressed in COS7cellsby transient transfection, which laid the foundation for studying thefunction of STAT3protein. Part Three: The Impacts of the Mutations in STAT3Hot Domains onthe Function of STAT3ProteinObjective: To investigate the changes of the molecular activation andmovements in AD-HIES related STAT3hot mutations and the novelmutation （T620S） and the impacts of the changes in the pathogenesis ofAD-HIEMethods: The human STAT3wild-type plasmid （WT-STAT3-GFP）and three mutant plasmids （R382W-STAT3-GFP, V637M-STAT3-GFPand T620S-STAT3-GFP） were transiently transfected into COS7cellswhich did not have endogenous STAT3by Lipofectamine2000. Eachtransfected plasmid was divided into two groups: unstimulated group andstimulated group. The lipofectamine2000group and the vectorpAcGFP1-N1group were the control groups. The cytoplasmic and nuclearprotein was extracted after the cells underwent20minutes treatment byEGF （100ng/ml）36hours post-transfection. The expression quantities ofSTAT3and phosphorylated STAT3in cytoplasm and nucleus of each groupwere detected by Western blot and the differences between groups werecompared. The accumulation and nuclear translocation of STAT3werecontinuously observed by the laser scanning confocal microscope withinone hour after the stimulation of EGF （100ng/ml）24hourspost-transfection. The differences between groups were compared. Thenucleus was dyed by DAPI at the timing of one hour after the stimulation of EGF to observe the accumulation of STAT3in the nucleus.Results: Analysis of the performed Western blots, demonstrated theexpression of the STAT3-GFP fusion protein （120KD） in wild-type STAT3and the three mutant STAT3groups in both cytoplasm and nucleus ofCOS7cells, with or without the stimulation of EGF. However, theexpression of T620S mutation was less and two bands at120KD and92KDwere seen. The phosphorylated STAT3was expressed in wild-type STAT3and R382W mutant STAT3in the both cytoplasm and nucleus of COS7cells with EGF stimulation. The pSTAT3was expressed less in the V637Mmutation and nearly deficient in the T620S mutation. Observations fromthe laser scanning confocal microscope, showed that accumulation andnuclear translocation occurred rapidly after EGF stimulation inWT-STAT3-GFP and prominent accumulation in the nucleus was seenwithin one hour. The nuclear export started around2.5hours afterstimulation and was almost complete after7hours. The time ofaccumulation and nuclear translocation was later and the expressionquantity was less in R382W-STAT3-GFP in comparison withWT-STAT3-GFP. Its export finished within4hours. A small amount ofaccumulation was seen in cytoplasm in V637M-STAT3-GFP. Littleaccumulation was observed at the timing of one hour around nuclearmembrane and the nuclear translocation was not obviously seen. Theaccumulation of STAT3in T620S-STAT3-GFP was barely seen within one hour. The results of the observation of the laser scanning confocalmicroscope were mainly in accordance with the results of western blot.Conclusion: There was an immediate correlation betweenphosphorylation and nuclear translocation of STAT3, the nucleartranslocation was limited while the phosphorylation was deficient. Theimpacts of the mutations V637M and T620S located in the SH2domain onthe phosphorylation, accumulation and nuclear translocation of STAT3wasmore remarkable in comparison to the R382W mutation in DNA-bindingdomain. As in the same domain, the impacts of T620S mutation were moresignificant than V637M mutation on the function of STAT3protein. Thepathogenesis of AD-HIES can be further illustrated by intensive study inthe impacts of different mutations in different domains on the function ofSTAT3protein, thus leading to new treatment modalities for AD-HIES.