| BackgroundKawasaki disease(KD)is an unexplained systemic vasculitis syndrome.It was first reported by the Japanese scholar Kawasaki in 1967.It is mainly characterized by fever,skin and mucous membrane damage,and lymphadenopathy.Its main hazard is coronary artery abnormality,such as coronary artery dilatation,coronary artery aneurysm,coronary artery stenosis,thrombosis,myocardial infarction,etc.,can lead to sudden death.About20?25%of untreated children can result in coronary artery abnormality.Most of the cases occurs in children under 2 years of age.The majority of the patients occurred in Asia.The incidence of children aged less than 5 years old with Kawasaki disease in Japan was 264.8 per 100,000 in 2012.The domestic report in Beijing was 40.9 to 55.1 per 100,000 from 2000 to 2004.The incidence rate of KD children under 5 years old in in Shanghai were 68.8 to 107.3 per 100000 from 2013 to 2017.Moreover,epidemiology evidence has found that the incidence of KD has been increasing in recent years,and it has become one of the most common acquired heart diseases in children.The etiology and pathogenesis of KD are not clear nowdays.It is believed that KD is triggered by exogenous antigens on the basis of certain genetic predisposition,resulting in immune disorders.The incidence rate of Asian children and the incidence rate of siblings is higher.This suggests that KD has genetic susceptibility.Integrin a 2(ITGA2)is an important collagen receptor,which is highly expressed in platelets and epithelial cells,and plays an important role in the adhesion process of platelets and fibro collagen.The adhesion process will lead to the activation of platelets and the formation of thrombus,which plays an important role in inflammatory response,coronary heart disease,atherosclerosis,and ischemic stroke and so on.However,the relationship between single nucleotide polymorphisms(SNP)of ITGA2 gene and KD coronary artery disease has not been reported in Chinese Han population,which provides a theoretical basis for further understanding the distribution of ITGA2 gene in Chinese population.Studies on its pathogens are good for early diagnosis and treatment.Providing theoretical basis for the treatment of Kawasaki disease with coronary artery lesions is good for gene-targeted therapy and prognosis improvement.MicroRNAs(miRNAs)are a group of highly conserved non coding RNAs with about 20 nucleotides,which widely exist in plants,microorganisms and mammals.Studies have found that in the occurrence and development of cardiovascular disease,tumor and other diseases,miRNA can degrade the mRNA of the transcribed target gene or inhibit the protein encoded by the target gene by combining with the partial sequence of the 3'UTR region of the target gene.As a potential biomarker,plasma miRNA has become a research hotspot in the diagnosis and treatment of diseases.Since miRNA expression is relatively stable in tissues and blood,it is not easy to be digested by nuclease,and miRNA expression is highly specific in different diseases,different tissues and different cells,the research of plasma miRNA in early diagnosis of diseases and specific drug development has become a hot spot.The role of plasma miRNA in the pathogenesis of Kawasaki disease and its target has not been fully understood.Therefore,this study aims to screen out the significant differential expression of plasma miRNA in Kawasaki disease,detect and verify the differential expression of miRNA through gene chip sequencing and stem loop RT-PCR,analyze its function,study its pathogenesis and find new biomarkers Prevention,early diagnosis and treatment of Kawasaki disease with coronary artery injury are important contents,which can provide theoretical basis for early diagnosis and gene targeted therapy of Kawasaki disease with coronary artery injury and improve prognosisThe diagnosis of KD mainly depends on the clinical manifestations.Early treatment and diagnosis of KD are the key to the prognosis of KD.The treatment of Kawasaki disease is intravenous administration of IVIG and oral aspirin.After standard treatment,the incidence of coronary artery abnormality due to Kawasaki disease can be decreased to about 5%.Aspirin has anti-inflammatory effect,antiplatelet effect and shortens fever time.Although meta-analysis showed that aspirin treatment did not affect the formation of coronary artery aneurysms in KD,it is the initial treatment for KD.It was first reported that patients with Kawasaki disease were treated with IVIG since 1983.After wards,the randomized controlled study confirmed that IVIG could reduce the risk of coronary artery aneurysms in KD,and the best effect of IVIG was given on the first 7-10 days during the onset of the disease.However,patients with persistent fever,systemic inflammatory signs and/or coronary artery abnormalities should also be treated 10 days later of illness onset.However,there are some studies that showed that IVIG was more likely to be unresponsive when the course of disease is less than 4 days of illness onset.In 2017,American Heart Association(AHA)scientific statement on diagnosis,treatment and long-term management of KD suggested that KD should be treated as soon in the course of illness as the diagnosis can be established.And the diagnosis could be made by experienced pediatrician as early as the third day of illness onset.Although the mechanism of IVIG remains unclear,IVIG has systemic anti-inflammatory effect,which can reduce fever and reduce acute reactants.Possible mechanisms included regulating the level and production of cytokines,increasing the activity of T-cell inhibitor,the effect on the complement system,inducing the inhibitory Fc? receptor on macrophages,down regulating antibody synthesis and providing anti idiotypic antibodies.Despite the fact that IVIG can prevent KD coronary artery disease,the timing and effectiveness of IVIG still need to be further determined.The timing and effectiveness of IVIG still need to be further determined that IVIG treatment is the earlier,the better.Objective1.To investigate the association between ITGA2 single nucleotide polymorphism and coronary artery abnormalities due to KD in Chinese Han children.2.To investigate the association between microRNAs and coronary artery abnormalities due to KD in children3.Retrospective analysis of large sample clinical data to study the effect of high dose IVIG on the outcome of coronary artery abnormalities due to KD.Method1.Using case-control method,we selected the core gene locus of platelet activation pathway,and used NCBI dbSNP database and SNP info information to screen the research SNPs that meet the following criteria:(1)in the Chinese Han offspring reported in HapMap data,the allele frequency?5%and not included in the existing genome-wide association study of Kawasaki disease;(2)Located in the gene regulatory region or affecting the activity of transcription factor binding sites or microRNA binding sites in promoters or changing the amino acids in exons.Chi square test was used to compare the demographic characteristics and SNP genotype distribution differences in the case and control;odds ratio(or)and 95%confidence interval(CI)were calculated by single factor and multi factor Logistic regression analysis to evaluate the correlation strength.2.Real time quantitative PCR(RT-PCR)was used to detect the differential expression of miRNA in serum of KD group before and after IVIg treatment and normal control group.MiRNA with significant differential expression was selected for further verification in a larger sample of clinical data.The role and mechanism of key miRNA in the occurrence and development of KD coronary artery disease3.The purpose of this study is to explore the effect of high-dose IVIG treatment time on the mid-term outcome of Kawasaki disease:retrospective cohort study was conducted to analyze the data of eligible KD children from January 2015 to December 2017.The end point of follow-up was December 2018.The study was registered in China clinical trial registration center with registration number of chictr180015800.Primary outcome of this study was the initial diagnosis of coronary artery disease by ultrasound and the results of 12-month follow-up.The secondary outcomes were inflammatory markers before and after IVIG treatment.The area,sensitivity and specificity under ROC curve were used as indicators to evaluate the best time for IVIG to treat KD.T-test and chi square test were used to compare the oral characteristics,inflammatory indicators and the composition ratio of coronary artery lesions in each group.Univariate and multivariate logistic regression were used to analyze the risk factors of KD combined with CALs.Results1.The association of integrin ?2 gene polymorphisms with KD.818 cases were selected in KD group and 1401 healthy cases in control group.There was no significant difference in sex and age between the two groups.Platelet activation pathway on ITGA2 gene polymorphism rs1126643 C>T were chosen.The HWE of ITGA2/rs1126643 in the study population is 0.9626>0.05.The distribution of genotypes in the study population is consistent with HWE,which indicates that this study is a representative population.According to the presence or absence of CAAs,KD was divided into two subgroups,including 116 cases(14.18%)of CAAs.Multivariate logistic regression analysis showed that the difference of ITGA2/rs1126643 between the case group and the control group was statistically significant.Compared with the healthy control group,we found that ITGA2/rs1126643 TT genotype was statistically significant.Compared with rs1126643 CC genotype,after age and gender adjustment,the risk of rs1126643 TT genotype carriers was reduced by 38%(adjusted OR=0.62,95%CI=0.43-0.88,p=0.0078),TTvsCT+CC(adjusted OR=0.63,95%CI=0.44-0.889,p=0.0093).Further stratified analysis showed that when rs1126643 CC/CT was used as the control genotype,TT genotype had more significant protective effect on female children and non CAAs at the age of?60 months.2.The association of micro RNA with coronary artery abnormality due to KD.In the study of miRNA and coronary artery disease in KD.We selected 5 KD patients before and after IVIg treatment.The results of microarray showed that the level of miR-126 in KD patients before treatment was significantly lower than that after IVIg treatment(more than 2 times).The samples were further expanded to 10 KDchildren before and after high-dose gamma globulin treatment,and 5 healthy children.Real time quantitative PCR was used to detect and verify the differential expression of miRNA.Quantitative PCR showed that the differentially expressed miRNAs were low expressed in KD children,but the candidate miRNAs were detected in healthy children and KD patients receiving IVIG treatment.The statistical results showed that among the differentially expressed miRNAs,miR-126 was significantly differentially expressed in Kawasaki disease,with statistical difference(p<0.05),indicating that the screening results were reliable.Previous studies showed that the expression of miR-126 in the blood of patients after treatment increased.The above data showed that miR-126 had a positive effect on vascular endothelium.The target gene of miRNA-126 was predicted by using the authoritative database target scan online software.Double luciferase reporter gene experiment confirmed that miR-126 combined with target gene spredl(p<0.05),the difference was statistically significant.These results indicate that miR-126 can affect the fluorescence activity of spredl gene by binding to 3'UTR.Western blot was used to verify that the target gene spredl of miR-126 mimics was down regulated,and the protein levels of VEGF,FGF-1,ras-p21 and mapk-p38 were increased by 1.67,1.93,1.76 and 1.86 times,respectively.After adding miR-126 inhibitor,the protein level of spredl in miR-126 inhibitor group was 1.51 times higher than that in cell group,At the same time,the protein levels of VEGF,FGF-1,ras-p21 and mapk-p38 were reduced to 0.54,0.64,0.59 and 0.63 respectively by the negative regulator spredl,which confirmed that miR-126 could promote the growth of vascular endothelial cells.The results showed that mimics could increase cell viability and change cell cycle.The proportion of cells in G2 phase was significantly increased,while the proportion of cells in S phase was significantly decreased.However,the response of HCAEC transfected with inhibitor was opposite.The cell viability and the proportion of G2 phase cells decreased significantly,while the proportion of S phase cells remained unchanged.These results indicate that miR-126 in KD children can indeed play a positive role in HCAEC.After treatment,the expression of miR-126 in KD children's blood is increased,and the condition is improved.2.The timing of IVIG treatment on the prognosis of coronary artery leisions due to KD.This group met the inclusion criteria of KD and followed up 1281 cases in 12 months.The results of 12-month follow-up showed that the best days for IVIG to predict KD coronary artery disease were 7.5 days,and the area under ROC was 0.80(95%CI,0.76-0.84,P<0.001).816(56.9%)were male,the median age was 12 months(11-36 months),141(11%)were patients with IVIG resistant KD.The median time of IVIg treatment was 6 days.At the time of diagnosis,339 patients(26.5%)had coronary artery lesions(CALs),94 patients(7.3%)had coronary artery aneurysms(CAAs).After 12 months of follow-up,there were still CALs in 39 cases(3.0%)and CAAs in 23 cases(1.8%).According to the time of IVIG treatment,all patients were divided into four groups:the first group received IVIG treatment within the fourth day,the second group was 5-7 days,the third group was 8-10 days,and the fourth group was more than 10 days after the onset.77 cases in the first group,817 cases in the second group,249 cases in the third group and 138 cases in the fourth group.There was a significant difference between the four groups(P=0.001).At the time of diagnosis,CALs was 14.1%,19.5%,35.3%and 58.7%in group 1 to 4,and CAAs was 2.6%,3.5%,8.4%and 30.4%in group 4.After 12 months follow-up,the incidence of CALs and CAAs in groups 3 and 4 were higher than those in groups 1 and 2(p<0.001).There was no significant difference between CALs and CAAs in the first and second groups.Compared with the third group,the difference between the two groups was statistically significant(P<0.05).At 1 month,the relative risk of CALS and CAAS in KD was or 2.1(95%CI 1.4-3.0),or 3.9(95%CI 1.9-8.2),respectively.At 12 months,the relative risk of CALS and CAAS in KD was or 5.3(95%CI 2.0-13.9),or 13.5(95%CI 2.9-14.1),respectively.For the logistic regression analysis of multiple factors influencing KD with CALs,The age,gender,duration of fever,duration of IVIG,nonresponsiveness of IVIG,percentage of leukocytes,neutrophils and C-reactive protein were selected as independent variables for logistic regression analysis.The results showed that the duration of IVIG treatment and IVIG-resistant KD were independently risk factors related to CALs(p?0.001).Conclusion1.ITGA2 C807T(rs1126643)gene polymorphisms in the core gene locus of platelet activation pathway may be related to the genetic susceptibility of KD with coronary artery lesions in Chinese population.These findings highlight an opportunity to design larger,multi-center prospective trials regarding the interplay between ITGA2 and coronary artery abnormality due to KD.2.The expression of miR-126 decreased significantly in KD and increased significantly after IVIg treatment.Moreover,it can increase the expression of VEGF and FGF-1 by directly regulating the target gene spredl,thus promoting the activation of angiogenesis.It is suggested that miR-126 and its target gene spredl may be related to the occurrence and development of coronary artery lesions in Kawasaki disease,and may become a potential new target for early diagnosis and treatment of Kawasaki disease.3.This study shows that IVIG can effectively prevent coronary artery disease within 7 days of illness onset.IVIG treatment did not increase the incidence of coronary artery disease and the incidence of failure to use IVIG within 4 days.The high risk factors of KD combined with CALs were the days of IVIG use and IVIG resistance.Therefore,we suggest that high dose IVIG should be used as early as possible in the treatment of acute KD. |
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