| Background and Objective IgA nephropathy is one of the common primary glomerulonephritis,which is a primary glomerulonephritisdeposited by a large number of IgA particles and IgA-based immune complex particles in the glomerular mesangial region.IgA nephropathy is the most important cause of end-stage renal disease,and its incidence varies greatly in different regions and races.Meanwhile,there is obvious family clustering.For the pathogenesis of IgA nephropathy,IgA nephropathy is associated with immune response to disease,associated with a variety of pathogenic factors,but its etiology and pathogenesis is not completely clear,it is usually considered that it was associated with immune disorders,inflammation,infection,genetic factors,with thedevelopment of molecular biology technology,researches at domestic and abroad confirmed that the cause and progression of IgA nephropathy is associated with genetic factors.This research is divided into two parts,the first part focus on the immune inflammatory cytokine related gene single nucleotide polymorphism,select the main sites of IL1B and IL10 genes,to explore the relationship between the susceptibility and development of candidate gene SNP and IgA nephropathy,the second part focus on miRNAs which may associated with IgA nephropathy reported in previous literature,and analyze the correlation ofmiRNA level and IgA nephropathy susceptibility,IgA nephropathy clinical manifestation and renal function,so as to explore the effect of miRNA on the IgA nephropathy susceptibility and progression,through the research of the above two parts,the purpose of this study is to find out the data of the incidence and development of IgA nephropathy in the residents,and to provide reference for the early diagnosis and intervention of IgA nephropathy.Methods 1)This study adopts frequency matched case-control study,184 patients who were diagnosed with IgA nephropathy in the First Affiliated Hospital of Baotou medical school of Inner Mongolia Autonomous Region was recruited as case group from February 2012 to October 2014,237 people who received physical examination at our hospital at the same time were selected as the control group,which was matching according to the frequency of gender and age group of the case group.The medical records,clinical data acquisition and basic information including age,gender,occupation,education,marital status,systolic blood pressure,diastolic blood pressure,serum IgA(slgA),serum creatinine(Scr),blood urea nitrogen(BUN)were collected fromthe case group and the control group,and the difference between casesand control group were compared.2)5ml peripheral venous blood was collected by EDTA-Na2 anticoagulant tube,the sequenom MassARRAY platformand matrix assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS)method was used to detect the polymorphism of IL1?-511C>T,IL1?-31T>C,IL1?+3953T>C,IL10-592A>C,IL10-819C>T,IL10-1082A>G.The allele frequency and genotype frequency distribution of the 6 loci in the case group and the control group were analyzed.,single factor and multi factor non-conditional Logistic regressionwere conducted to analyze the relationship between polymorphisms of candidate gene and susceptibility to IgA nephropathy patients.The correlation between polymorphisms of candidate gene loci and major clinical manifestations(hypertension,renal insufficiency,massive proteinuria,gross hematuria)was analyzed.The linkage disequilibrium between 3 loci of IL1B and 3 loci of IL10 was analyzed by SHEsis software.Haplotype construction was performed to analyze the relationship between haplotype and the risk of IgA nephropathy.Based on the results of genetic polymorphism analysis,we further analyzed the interaction between genes and genes related to the pathogenesis of IgA nephropathy,and the effect of interaction on susceptibility.3)100ml midpiece urine were collected from healthy control and patients,using Trizol kit to extract total RNA urine samplesby kit method,the expression of miR-141,miR-146a,miR-155,miR-192,miR-200a,miR-200b,miR-200c,miR-205,miR-4299 miRNA was detected by RT-qPCR.Two independent sample t tests were used to compare the difference in the level of miRNA expression between patients with IgA nephropathy and normal controls.Spearman rank correlation analysis was conducted to analyze correlation between the expression of IgA in patients and clinical manifestations,between the expression of IgA in patients and katafuchi score,in order to analysis the relationship between miRNA expression levels and susceptibility and progression of IgA nephropathy.Results The first part 1)There was no significant difference in the demographic characteristics between the case group and the control group in terms of age,sex,education level,residence,marital status,occupation and so on(P>0.05),which was comparable between the two groups.2)Hardy-Weinberg balance test was conducted on 6 SNPs sites of the candidate gene,the results showed that in the control group,except IL10-511C>T and IL1?-31T>C(P<0.01),the genotype distribution of other loci were accord with Hardy-Weinberg genetic equilibrium(P value of 0.878,0.999,0.838,0.576),it shows that the gene frequency distribution in this study has a good representation.3)There was no significant difference in the distribution of allele frequency of IL10-590C>T and IL10-819C>T between the two groups two(P>0.05),The difference in the distribution of allele frequency of IL 1?-511C>T,IL1?-31T>C,IL1?+3953T>C,IL10-1082A>G between the two groups has statistical significancef(P<0.05).4)There was no significant difference in the distribution of genotype frequency of IL1?-31T>C?IL10-592A>C and IL10-819C>T between the two groups(P>0.05),the difference in the distribution of genotype frequency of IL1?-511 C>T,IL1?-31T>C,IL1?+3953T>C,IL10-1082A>G between the two groups has statistical significancef(P<0.05).5)The single factor Non-conditional Logistic regression analysis showed that,compared with CC genotype of IL1?-511C>T,CT+TT gene type can increase the risk of IgA nephropathy(OR=1.945,95%CI= 1.205-3.139);compared with TT genotype of IL1B-31T>C,TC+CC gene type can increase the risk of IgA nephropathy(OR=1.5 90,95%CI=1.014-2.493);compared with TT genotype of IL1B+3953T>C,TC+CC gene type can increase the risk of IgA nephropathy(OR=1.905,95%CI=1.180-3.076);compared with AA genotype of IL10-1082A>G,AG+GG gene typecan increase the disease risk(OR=1.678,95%CI=1.137-2.477).The two loci of IL10-592A>C and IL 10-819C>T were not found to be associated with the pathogenesis of IgA nephropathy(P>0.05).6)Multivariate Logistic regression analysis showed that L1B-511C>T,IL1?-31T>C,IL1 P+3953T>C,IL10-1082A>G four loci were associated with the genetic susceptibility of IgA nephropathy(P<0.05).The CT+TT genotype of IL1B-511C>T(OR=1.968,95%CI=1.200-3.226)and TC+TT gene type of IL1?-31T>C(OR=1.611,95%CI=1.010-2.569)and TC+TT gene type of IL1?+3953T>C(OR=1.924,95%CI=1.173-3.148),AG+GG gene type of IL10-1082A>G(OR=1.640,95%CI=1.094-2.457)can increase the risk of IgA nephropathy.7)Different genotypes of IL1?-511C>T,IL1?-31T>C,IL1?+3953T>C,IL10-592A>C,IL10-1082A>G are not associated with hypertension,renal insufficiency,proteinuria and hematuria(P>0.05);IL10-819C>T gene polymorphism is associated with hypertension in patients with IgA nephropathy,carry the CT+TT genotype had a lower risk of hypertension(OR=0.472,95%CI=0.226-0.985)but not associated with renal insufficiency,proteinuria and hematuria(P>0.05).8)IL1B-511C>T,-31T>C,+3953T>C three gene loci were ont found in linkage disequilibrium(D'<0.5,R2<0.8),the difference was statistically significant in the case group and the control group between CTC and CCT,TTC,TCC four haplotypes(P<0.05),IL10-590 T,-819C>T C,gene-1082A>G three loci were not found linkage disequilibrium(D'<0.5,R2<0.8).The three haplotypes of ACA,ACG and CTG were statistically significant between the case group and the control group(P<0.05).9)The interaction between IL1?-511C>T CT+TT and IL1?-31T>C TT(OR=0.30695%CI =0.130-0.719),IL1?-511C>T CT+TT and IL10-1082A>G AG+GG(OR=2.116,95%CI=1.063-4.210),IL1?-31T>C TC+CC and IL1?+3953T>C TC+CC(OR=2.685,95%CI=1.026-7.026),IL1?-31T>C TC+CC and IL10-1082A>G AA(OR=0.245,95%CI=0.128-0.468),IL1?-31T>C TC+CC and IL10-1082A>G AG+GG(OR=0.452,95%CI=0.249-0.820),were statistically significant.No statistically significant interaction was found among the other genotypes(P>0.05).The second part 1)According to the test results of miRNA expression in urine samples,the expression level of miR-141,miR-200a,miR-200b,miR-200c and miR-429 in patients with IgA nephropathy was lower than control group(P<0.05),while the expression level of miR-146a and miR-155 in patients with IgA nephropathy was higher than control group(P<0.05),There was no difference in the level of miR-192,miR-205 expression between the two groups(P>0.05).2)The expression level of miR-141 was negatively correlated with gross hematuria(r=-0.222,P=0.002),the expression level of miR-146a and gross hematuria was negatively correlated(r=-0.170,P=0.021),while the rest of miRNA expression level were not correlated with hypertension,renal insufficiency,proteinuria,hematuria(P>0.05).3)The renal tubulointerstitium score is positively related to the expression level of miR-146a(r=0.206,P=0.005),and negative correlated with the expression level of miR-155(r=-0.204,P=0.006).The expression level of the remaining miRNA was not related to the score of the three main dimensions of the katafuchi score.(P>0.05).4)There was no correlation between the level of target miRNA expression in this study and the score of three sub items of katafuchi glomerular integral(P>0.05).5)The expression level of miR-146a and tubulointerstitial inflammatory cell infiltration score was positively correlated(r=0.196,P=0.008),and the expression level of miR-155 and renal tubulointerstitial fibrosis scores were negatively correlated(r=-0.162,P=0.028)was negatively correlated with renal tubular atrophy score(r=-0.213,P=0.004),The expression level of the remaining miRNA was not related to the score of the three main dimensions of the katafuchi Renal tubule score.(P>0.05).6)The expression of miR-192 and vascular hyalinosis integralscore are positive correlated(r=0.145,P=0.049),and the expression of miR-200c is negatively correlated with the vascular wall thickening integral score(r=-0.161,P=0.029),The expression level of the remaining miRNA was not related to the score of the two main dimensions of the katafuchi blood vessel score.(P>0.05).Conclusions 1)The CT+TT genotype of IL1?-511 C>T,TC+CC genotype of IL1?-31T>C,TC+CC genotype of IL1?+3953T>C,and IL10 genotype of IL10-1082A>G can increase the risk of nephropathy.2)The risk of high blood pressure in patients with IgA nephropathy carrying the CT+TT genotype of IL10-819C>T is low.3)There was no linkage disequilibrium between three loci of IL1? gene,but there were differences between four haplotypes of CTC,CCT,TTC,and TCCin the case group and the control group.There was no linkage disequilibrium in the four loci of the IL10 gene,but the distribution of three types of haplotypes of ACA,ACG and CTG were different in the case group and the control group.4)There was interaction effect between IL1?-511C>T CT+TT and IL1?-31T>C TT,IL1?-511C>T CT+TT and IL10-1082A>G AG+GG,IL1?-31T>C TC+CCand IL1?+3953T>C TC+CC,IL10-31T>C TC+CC and IL10-1082A>G AA,IL1?-31T>C TC+CC and IL10-1082A>G AG+GG.5)The expression levels of miR-141,miR-200a,miR-200b,miR-200c and miR-429 in IgA nephropathy patients were lower than those in the normal control group,while miR-146a and miR-155 in the patients with IgA nephropathy were higher than those in the normal control group.6)The expression level of miR-141 and miR-146a was negatively correlated with the hematuriae.7)The expression level of miR-146a and katafuchi in renal tubulointerstitial integral score and tubulointerstitial inflammatory cell infiltration score were positively correlated;the expression level of miR-155 and renal tubulointerstitial integral score,tubulointerstitial fibrosis score,renal tubular atrophy score was negatively correlated;the expression of miR-192 and vascular hyalinosis integral score was positively related;the expression of miR-200c level and thickening of the vessel wall integralscore was negatively related. |
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