Clinical Analysis Of 2724 Cases Of Kawasaki Disease In A Single Center And Study On Prediction And Key Genes Of Intravenous Immunoglobulin Non-responsive Kawasaki Disease

Kawasaki disease(KD)is an acute febrile extraneous disease mainly occurring in children under 5 years old.It is characterized by systemic immune vasculitis and mainly affects small and medium blood vessels.Coronary artery lesions(CALs)are the main hazard of KD,which has replaced rheumatic fever as the most common cause of acquired heart disease in children and is one of the risk factors for ischemic heart disease in adults.High-dose intravenous immunoglobulin(IVIG)is the standard treatment for KD,but about 10-20%of children do not respond to initial treatment with IVIG,and the risk of coronary artery disease(CALs)in KD patients without IVIG response is significantly increased.In severe cases,coronary artery aneurysm(CAA)may occur,coronary artery stenosis,atresia,myocardial infarction and other complications,which seriously affect the prognosis of KD patients.Timely screening of high-risk patients who may develop IVIG nonresponse,and early treatment with more intensive therapy or other adjuvant therapy can reduce the risk of CALs.At present,the pathogenesis of IVIG non-responsive KD is not clear.Although clinical prediction models have been established in many countries and regions,none of the models can be effectively applied to different regions and populations.Remedial treatment for IVIG non-responsive KD patients can not quickly and efficiently reduce the incidence of vascular inflammation and CALs.Therefore,it is of great significance for improving the success rate of IVIG treatment and prognosis of children with KD to clarify the pathogenesis of IVIG non-responsive KD,find new effective treatment approaches,establish prediction models in this region and timely predict early intervention of patients with IVIG non-responsive KD.Part I:Clinical Features of Kawasaki disease in Children’s Hospital of Soochow University From 2011 Through 2020Objective:To understand the epidemiology and clinical features of Kawasaki disease(KD)in Suzhou from 2011 through 2020,and to study the clinical features of IVIG non-responsive KD.Methods:Through clinical retrospective analysis,the clinical and epidemiological data of hospitalized children diagnosed with KD in Children’s Hospital of Soochow University from 2011 through 2020 were analyzed.SPSS 25.0 software was used for statistical analysis,and P<0.05 was considered to be statistically significant.Results:1)From January 2011 to December 2020,a total of 2724 patients were diagnosed with KD,including 1658 males(60.9%)and 1066 females(39.1%),with a male to female ratio of 1.6:1.The median age was 20 months,including 305 patients(11.2%)≤6 months old,792 patients(29.1%)≤1 year old,and 231 patients(8.5%)≥5 years old.There were 575 patients(21.1%)with incomplete KD,758 patients(27.8%)with CALs,and 175 patients(6.4%)with no response to IVIG.2)Main clinical manifestations:There were 2371 patients(87%)with conjunctival congestion,2226 patients(81.7%)with lip chap,2163 patients(79.4%)with cervical lymph nodes enlargement,1985 patients(72.9%)with rash,1876 patients(68.9%)with red berry tongue,Among the other clinical manifestations,there were 645 patients(23.7%)with perianal desquamation,and 424 patients(15.6%)with changes in the site of BCG vaccination.3)798 cases(29.3%)occurred in spring,783 cases(28.7%)in summer,631 cases(23.1%)in winter,and 512 cases(18.8%)in autumn.From 2011 to 2019,the number of cases basically followed the trend of spring>summer>winter>autumn,but in 2020,winter>summer>autumn>spring.4)Among the patients without response to IVIG,there were 107 males(61.2%)and 68 females(38.9%),with a male to female ratio of 1.6:1,with an average age of 26.1 months and a median age of 21 months.There was no difference in gender,age and sensitive group.The number of patients with IVIG nonresponse increased year by year,fluctuated slightly from 2015 to 2018,and peaked in 2020(9.4%).The incidence of hand and foot swelling,finger and toe peeling,skin rash and changes in BCG vaccination site in patients without IVIG response was significantly higher than that in patients with IVIG sensitivity(P≤0.05),the incidence of CALs in patients without IVIG response was higher,and patients without IVIG response were more likely to be KD(P=0.011).Conclusion:1)This study approximately reflected the epidemiological characteristics of KD in Suzhou:male patients were more common,children aged 1-5 years were the most affected group,the peak of incidence was in spring and summer,and the annual incidence of KD increased year by year.The peak of incidence was in spring and summer,and the annual incidence of KD increased year by year;2)The incidence of non-responsive KD of IVIG is on the rise.Patients with non-responsive KD of IVIG are more likely to have swelling of hands and feet,peeling of fingers and toes,rash and changes at the site of BCG inoculation.Non-responsive KD of IVIG is more likely to be complete KD,and the risk of coronary artery damage of non-responsive KD of IVIG is higher.Part Ⅱ:Nomogram for prediction of intravenous immunoglobulin non-responsive Kawasaki disease in SuzhouObjective:To screen the predictors of IVIG non-responsive KD in Suzhou,China,and develop a Nomogram for the prediction of IVIG non-responsive KD in Suzhou,China.Methods:Clinical and laboratory data of children with Kawasaki disease who received IVIG during their hospitalization in Children’s Hospital of soochow University were retrospectively analyzed.Non-response to IVIG was defined as persistent fever 36 hours after completion of initial treatment with IVIG or refever after fever abated.Baseline variables were analyzed using the minimum absolute contraction and selection operator(LASSO)to determine predictors of IVIG nonresponse.These predictors were then incorporated to construct a predictive Nomogram,and the calibration curve and area under ROC characteristic curve(AUC)were used to evaluate the model performance,Decision Curve Analysis(DCA)was used to evaluate clinical efficacy..The prediction efficiency of the prediction model is verified by using the test set data and compared with the developed model.Results:From January 2011 to December 2020,2742 kawasaki disease patients were admitted to Children’s Hospital affiliated to Soochow University.Among 2537 patients who met the inclusion criteria of this study,118 patients(118/2537,4.65%)showed IVIG non-response.Age,C-reactive protein(CRP),total white blood cell(WBC),hemoglobin(Hb),neutrophil ratio(N%),platelet(PLT),serum alkaline phosphatase(ALP),albumin(ALB)and incomplete Kawasaki disease(iKD)were screened as predictive factors for IVIG nonresponse by LASSO regression analysis.Based on the Nomogram constructed by these predictive factors,the prediction ability of the Nomogram prediction model based on these nine factors on the training set is 0.73,0.780 sensitivity and 0.616 specificity.In the test set data,the AUC of the new model was 0.768,the sensitivity was 0.833,and the specificity was 0.670.Compared with the developed model,the new model has good predictive value,with AUC=0.77,sensitivity 0.91 and specificity 0.64.Conclusions:Age,C-reactive protein(CRP),total white blood cell(WBC),hemoglobin(Hb),neutrophil ratio(N%),platelet(PLT),serum alkaline phosphatase(ALP),albumin(ALB)and incomplete Kawasaki disease(iKD)were included to establish Nomogram to predict IVIG non-responsive KD in suzhou,this new model is a useful tool to predict IVIG non-responsive KD in suzhou.Part III:Bioinformatics analysis of key genes in intravenous immunoglobulin non-responsive Kawasaki diseaseObjective:Key genes related to IVIG non-responsive KD were screened to find potential diagnostic markers and therapeutic targets,providing molecular theoretical basis for timely diagnosis of IVIG non-responsive KD and improving the success rate of KD treatment.Methods:GSE16797 microarray dataset which were from the Gene Expression Omnibus(GEO)was analyzed to screen differentially expressed genes(DEGs)related to sensitivity to IVIG therapy.Analysis of Gene ontology(GO),Kyoto City Encyclopedia of Gene and Genome(KEGGK)pathway enrichment analysis,protein-protein interaction(PIP)network screening of key genes,ROC curve of prediction efficiency of key genes,RT-QPCR verification of differentially expressed genes,KD transcriptome data from our hospital were used to verify the expression of key genes before and after IVIG treatment.Results:Compared with the IVIG sensitive and non-responsive groups,1154 differentially expressed genes were screened,including 701 up-regulated genes and 453 down-regulated genes in the IVIG non-responsive group.Cut the difference of gene GO analysis mainly from the biological process of involving the antigen receptor mediated signaling pathway positive adjustment,T cell differentiation,cell adhesion,T cell receptors signaling pathways,such as genetic variations raised GO analysis obtained the biological process mainly involves neutrophil activation and neutrophil degranulation,neutrophils mediated immune response,etc.,PPI network was used to screen out 10 key genes:MYC、HGF、CCR7、GNAQ、MMP9、ARRB2、MAPK1、CD28、LCK and CD2.The key genes showed certain accuracy in predicting IVIG non-response,and AUC were:0.972,0.889,0.889,0.861,0.833,0.833,0.833,0.806,0.778,among which,the AUC of MYC,HGF,CCR7 and GNAQ was higher than 0.85,indicating high diagnostic value.The relative expression levels of MMP9(P=0.0325)and GNAQ(P=0.0102)in IVIG non-responsive group were higher than those in IVIG sensitive group.MMP9,GNAQ and ARRB2 were differentially expressed before and after IVIG treatment,and their expressions were upregulated in the acute stage,while they were significantly down-regulated after gamma globulin infusion.Conclusions:There are differences in gene expression between IVIG non-responsive KD and IVIG sensitive KD in KD acute stage.MMP9 and GNAQ are potential functional key genes related to IVIG reactivity,which may be potential biomarkers for predicting IVIG non-responsive KD and potential targets for treating IVIG non-responsive KD.