The Research Of Gene Polymorphism And MicroRNA Combined Control Of Children's Warfarin Individualized Drugs

PART ? Warfarin-related genes polymorphisms in Han population in ChinaResearch BackgroundWarfarin is the most commonly prescribed oral anticoagulant for the treatment and prevention of thromboembolic events.Challenges related to the use of warfarin pertain to its low therapeutic index and the large interindividual variability inmaintenance dose requirement.Although variation in the initial and maintenance dose is known to depend on body,weight,age,and dietary vitamin K intake as well as concomitant medications,there is growing evidence for a strong hereditary component.Over the last decade,a large body of evidence has accumulated showing that polymorphisms in three genes influence warfarin maintenance dose in adults:cytochrome P450 2C9(CYP2C9)responsible for the metabolism of S warfarin,vitamin K epoxide reductase complex subunit 1(VKORC1)as the molecular target of warfarin,and cytochrome P450 4F2(CYP4F2)as the primary vitamin K1 oxidase in the liver.Several meta-analyses have already shown the impact of CYP2C9,VKORC1,and CYP4F2 on warfarin maintenance dose in adults.The Clinical Pharmacogenetics implementation Consortium published dosing guidelines in 2017,and recommended the use of pharmacogenetic testing to determine initial warfarin doses in adults.The expression and function of proteins(including drug-metabolizing enzymes)continue to develop during infancy and childhood.Different proteins develop at different speeds and patterns.The relationship between phenotype and genotype between adults and children is often different,so it is difficult to combine Studies in adults are inferred to children.International pharmacogenetic studies report the effect of genotypes on optimal maintenance doses of warfarin in children.Studies have shown that CYP2C9 and VKORC1 gene polymorphisms are significantly associated with maintenance doses of warfarin in children.However,the clinical application of warfarin in pediatrics is short and the evidence-based basis is limited.At present,children's anticoagulation therapy is inferred based on adult studies.The current research on children's warfarin has the following problems:1.Children's warfarin research in the world Very few;2.No Chinese children's research;3.The effects of different research genotypes are large,and some of the research genes are less effective than clinical factors such as age;4.The age of each study is large,and there may be differences in the role of genes of different ages;5.Except for VKORC1 and CYP2C9,other genotypes are rarely involved in epigenetics and have not been reported.There is currently no research on warfarin drug gene polymorphism in Chinese Han children.To this end,this study conducted a large-scale genetic polymorphism study of warfarin drug genes CYP2C9 and VKORC1 in Chinese Han children,providing a basis for individualized treatment of warfarin in Chinese children.ObjectiveWarfarin is the most widely used oral anticoagulant in the clinic.Its treatment window is narrow,and children's warfarin doses vary widely among individuals,which can easily cause bleeding or embolism.CYP2C9 and VKORC1 gene polymorphisms can significantly affect warfarin dose.This project aims to study the distribution of gene polymorphisms of warfarin drug genes CYP2C9 and VKORC1 in Chinese Han children,and to provide evidence for individualized warfarin treatment in Chinese children.MethodsA total of 2622 Chinese Han children who were treated at the Guangzhou Women and Children's Medical Center from January 2016 to December 2018 were collected,including 1710 males and 912 females,including 1388 Kawasaki disease and 1238 non-Kawasaki disease(among them Fever 82 children with non-Kawasaki disease,32 other diseases,and 1124 healthy controls).The age is 1 to 176 months,and the median age is 25 months.Collect general clinical data such as age,gender,weight,height,and type of disease.The study was approved by the hospital's ethics committee(approval number:20140730026),and all children's families signed informed consent.All children who were enrolled were given 3 mL of peripheral venous blood and placed in a sodium citrate anticoagulation tube,and stored in a refrigerator at 4?.DNA genes were extracted with a commercial Omega kit for 5 days,and then stored in a refrigerator at-20?.After all the children of the owner have been collected,the DNA products are amplified by PCR reaction uniformly.The PCR amplification and sequence analysis of VKORC1rs9923 231,CYP2C9*3rs1057910,and VKORC1rs9934438 genes are performed by SNaP shot method,pyrosequencing method,and direct sequencing method.The frequency of allele distribution in all children was tested by Hardy-Weinberg test.Divided into three groups of VOKRC1rs9923231,CYP2C9*3 rs1057910,VKORC1rs9934438 by gender,X2 test was used to compare the three genotype distributions;grouped by disease category,VOKRClrs9923231,CYP2C9*3rs1057910,VKORC1rs9934438 three genotype and allele distribution The comparison was made by X2 test.The relationship between gender and disease type and the genotype and allele distribution of the above genes was analyzed.ResultsIncluding 2622 Chinese Han children in this study,genetic testing showed that the distribution frequencies of TT,CT,and CC genotypes of VKORC1 rs9923231 genes were 80.32%,18.38%,and 1.30%;the distribution frequencies of VKORC1 rs9934438 genes AA,AG,and GG genotypes were respectively For 79.94%,18.76%,1.30%.The distribution frequencies of CYP2C9*3rs1057910 gene AA,AC,and CC genotypes were 92.37%,7.4%,and 0.23%,respectively.Grouped by sex,the three genes CYP2C9*3 rs1057910,VOKRC1 rs9923231,and VOKRC1 rs9934438 had no significant differences in genotype distribution between groups(all P>0.05),that is,sex for Chinese Han children VOKRC1 rs9923231,VOKRClrs9934438 And CYP2C9*3 rs1057910 genotype distribution had no effect.Divided into the Kawasaki disease group(n=1388)and the control group(n=1238)according to the type of disease,there were no significant differences in genotypes and allele distributions between VOKRC1 rs9923231,VOKRClrs9934438 and CYP2C9*3 rs105791(all P>0.05)Conclusion1.Chinese Han children warfarin gene CYP2C9*3rs 1057910 is mainly wild type,while warfarin genes VKORC1 rs9934438 and rs9923231 are mainly variants;2.Gender and disease classification have no effect on the distribution of Chinese Han children's VOKRC1 rs9923231,VOKRC1rs9934438 and CYP2C9*3 rs1057910 genotypes;3.Chinese Han children VKORC1 rs9923231,CYP2C9*3 rs1057910 and VKORC1 rs9934438 genotypes and allelic mutation rates are similar to those of overseas Chinese,but they are significantly different from Caucasians4.The polymorphism of warfarin drug genes in children is closely related to the warfarin dose,bleeding and thrombosis and other complications.Warfarin pharmacogenomics needs to be tested before children warfarin medication,which is safe and effective.Ground treatment.PART ? Correlation research of VKORC1 gene polymorphism and warfarin maintenance dosage in Kawasaki disease children with coronary artery aneurysmsResearch BackgroundCoronary aneurysms(CAA)caused by Kawasaki disease are prone to coronary artery thrombosis,stenosis or even occlusion,which can lead to ischemic cardiomyopathy,which has become the most common cause of acquired heart disease in children.The guidelines recommend that children with Kawasaki disease and coronary aneurysms require long-term oral warfarin therapy.However,children's warfarin has a narrow therapeutic window and it is difficult to achieve steady-state doses.Unlike adults,there is currently no international standard for the use of children's warfarin and a formula for predicting doses.The use of children's warfarin is based on effective clinical experience and is difficult Reach a stable dose,and prone to complications such as bleeding and thrombosis.The steady state of warfarin dose is closely related to genetics and genetics.There are differences between people and children.Adults also have different doses of drugs by more than 20 times,which limits the widespread use of warfarin in children.There is currently no formula for steady-state warfarin doses in children with Kawasaki disease coronary aneurysms in China.This article combines genetic polymorphism,micrRNA,clinical factors and other factors to explore the usage and stable dose of warfarin in children with coronary artery aneurysms associated with Kawasaki disease.Based on multiple regression analysis,the main factors affecting the stable dose of warfarin in Chinese children are obtained.An attempt was made to establish a model for calculating the dose and dose of warfarin for children,and to provide a basis for children's clinically individualized warfarin treatment.IntroductionChildren with large coronary aneurysms due to Kawasaki disease need Anticoagulant therapy with warfarin.However,it is difficult to predict the warfarin dose due to individual differences among children.Adult studies have shown that the gene polymorphisms of vitamin K cyclooxygenase reductase 1(VKORC1)and cytochrome P4502C9(CYP2C9)affect the major genes of warfarin dose.We studied the effects of VKORC1,CYP2C9 genotypes,and microRNA on the dose of warfarin in children with giant coronary aneurysms caused by Kawasaki disease.Combined with clinical factors such as gender,age,weight,and body surface area,we evaluated the Chinese children's Chinese by multiple regression The main influencing factors of stable dose of warfarin are trying to establish a dose model of warfarin usage in children to provide basis for warfarin treatment in Chinese children.Methods1.Inclusion criteria? Patients with KD GCAA or coronary artery dilatation and thrombosis within a short period of time diagnosed in our hospital;? Good drug compliance and long-term follow-up;? Oral stable dose of warfarin? 2 months,the target internationalization ratio(INR)of the same period is stable Between 1.5 and 2.5? 2 months;?Liver function,renal function,electrolytes,blood routine,urine routine indexes are all within the normal range.2.Exclusion criteria:There may be other CAA diseases,such as multiple vasculitis and Chronic EB virus infection.3.Polymorphisms of CYP2C9*2(rs1799853),CYP2C9*3(rs1057910),and VKORC1(rs9923231)gene loci were selected for detection and group analysis.CYP2C9 is further divided into AA,AC,and CC types,and VKORC1 rs9923231 Subdivided into TT type,TC type and CC type.The aforementioned genetic tests were performed before and after warfarin administration,and were divided into gene tests before and after warfarin treatment.4.Using the detection of drug gene polymorphism in warfarin cases,collect 3 mL of peripheral venous blood from children(hexamethylene diamine tetraacetic acid anticoagulant),and use TaqMan real-time polymerase chain reaction for VKORC1(rs 9923231)and CYP2C9*3(rs 1057910)for genotyping,the operation was performed according to the kit instructions.According to the sequence design of CYP2C9*2,CYP2C9*3,and VOKRC1,*1 wild type was used as the reference sequence,and the PCR method was used for amplification.5.Record the clinical and genetic data of the patient.A linear regression analysis was performed to assess the effect of clinical and warfarin gene polymorphisms on the maintenance dose of warfarin.Results1.Comparison between genotypesThe warfarin dose of patients with CT or CC genotype(rs 9923231)of VKORC1 was higher than that of patients with TT genotype(0.130±0.033 vs.0,103 ±0.030mg/kg·d,p<0.05).Only 3 patients carried the CYP2C9*3 allele(rs 1057910)and their warfarin dose was lower than the wild CYP2C9*1/*1 genotype(0.127 ±0.039 vs.0.107 ± 0.032 mg/kg·d,p>0.05).Warfarin doses of patients with VKORC1 CC/CT and CYP2C9*1/*1 genotype are higher than those of VKORC1 TT and CYP2C9*1/*1 genotype(0.127 ± 0.336 vs.0.104±0.292 mg/kg·d,p<0.05).No significant differences were found between the others.Patients with the VKOCR1 TT genotype reached the target INR earlier than those with the CT or CC genotype(10.38±8.88 vs.27.89±15.70 days,p<0.01).CYP2C9*1/patient*1 and*1/*1 reached the target INR at the same time(13.23± 12.47 vs.25.67 ± 2.08 days,p>0.01).The frequencies of miRNA133a-2-191G>A genotypes of GG,GA,and AA were 94.6%,3.2%,and 1.2%,respectively.Patients with three genotypes of miRNA133a-2-191G>A had no steady-state doses of warfarin Significantly affected.2.Effects of clinical and genetic variables on warfarin doseIn univariate regression analysis,warfarin dose was significantly associated with genotypes of age,weight,height,BSA and VKORC1.The INR of the CYP2C9 and miRNA13 3a-2-191 G>A genotype was not statistically significant.Candidate variables are entered into the final multiple regression analysis.Body weight and VKORC1 genotype accounted for 44.2%of the total variation in the final model.Compared to the VKORC1 genotype,body weight had a greater effect on warfarin dose(33.0%and 11.2%,respectively).There was a negative correlation between body weight and warfarin dose(r=-0.58,p<0.01).3.Prediction of warfarin dose model for childrenThe prediction algorithm for warfarin dose in South China is as follows:Warfarin daily dose(mg/kg)=0.133-0.002*weight(kg)+ 0.026*VKORC1(VKORC1:CC/CT type is 1;TT type is 0)Conclusions1.Weight and VKORC1 genotype mainly determine the warfarin dose of children with Kawasaki disease in China.2.CYP2C9 genotype and miRNA133a-2-191G>A genotype had no significant effect on warfarin dose of Kawasaki disease in Chinese children.3.A child warfarin dose model based on body weight and VKORC1 genotype can help accurately predict the child warfarin dose required.4.The effectiveness of this warfarin dose model must be validated in populations of other races with different indications.