| Objective1. To investigate the distribution and characteristics of PAX2 gene polymorphisms in patients with Henoch-schonlein purpura (HSP) for the possible association between the gene polymorphisms of PAX2 and the genetic susceptibility of HSP and the relationship between the gene polymorphisms of PAX2 and clinical pathophyslological characteristics of HSP.2. To determine the relationship among the expression of PAX2 protein in the kidney native cells and the PAX2 genotypes and the different clinical manifestations, pathological classification of HSPN3. To detect the expression of PAX2 protein in the kidney tissue of HSPN and the correlation between it and different clinical manifestations, pathological classification of HSPN, also to study the difference of the expression of PAX2 in the kidney tissue of HSPN between the PAX2 genotypes.Methods1.By extracting DNA from 118 children with HSP (including 80 HSPN and 38 only with HSP but without renal involvement) and 100 normal healthy children in Hunan Province of China. Polymerase chain reaction (PCR) was used to amplify exon 1-12 of PAX2 gene. Finally Denaturing High Performance Liquid Chromatography (DHPLC) and DNA Sequencing Analysis were conducted for mutate screening the mutated PAX2 gene in the PCR products. 2. Applied case-control study to determine the relationship between genetic polymorphisms of PAX2 and susceptibility, clinical manifestations of HSP.3. Immunohistochemical staining was employed to determine the expression of PAX2 in the kidney tissue of 3 normal controls and 64 patients with HSPN. To make a correlation analysis on clinical manifestations and pathological classification of HSPN with the expression of PAX2. Also to analyze the expression of PAX2 between the two PAX2 genotypes.Results1. No SNP was identified with DHPLC in exon 1-7, 9, 10, 12, but two SNPs (798C>T and 909A>C) were identified in exon8 which were completely linkage to constitute two haplotypes; A SNP (1164T>A) were identified in exon 11 of PAX2 gene. The frequencies of the allele 798C/909A and 798T/909C were 93% and 7% in control group, and 89.41% and 10.59% in HSP group, respectively. There was no significant difference between the two groups (x~2=2.506, p=0.113). The frequencies of 798CC/909AA, 798CT/909AC and 798TT/909CC genotypes were 87.00%, 12.00%, 1.00% in control group, and 79.66%, 19.49%, 0.85% in HSP group, respectively. No significant difference was found between the two groups (p =0.198). The frequencies of 1164T and 1164A allele were 99.5% and 0.5% in control group, and 99.15% and 0.85% in HSP group, respectively. There was no significant difference between the two groups (x~2=0.091,p=0.662). The frequencies of 1164TT, 1164TA and 1164AA genotypes were 99.00%, 1.00%, 0 in control group, and 98.31%, 1.69%, 0 in HSP group, respectively. No significant difference was found between the two groups (p =0.661).2. No association was observed between the genotypic distribution of exon8 in PAX2 and the clinical information in patients with HSP, such as gender, age, joint or gastrointestinal phenotype (p>0.05), other than renal manifestations (p=0.039).3. The frequencies of 798CC/909AA, 798CT/909AC and 798TT/909CC genotypes were 75.00%, 25.00%, 0 in HSPN group, respectively. A significantly increased frequency of 798CT/909AC genotype was found in HSPN group compared with HSP without renal involved group (p=0.036) and control group (p=0.039). In HSPN group, it wasn't association with different serious degree or manifestations of renal damage (P>0.05).4. PAX2 re-expression was showed in almost all kidneys of HSPN children, more intense staining in distal tubules than in proximal tubules and podocytes (p<0.05), and collecting tubules not evidently. Normal control was negative.5. The correlation analysis indicated that PAX2 expression in podocytes showed positive correlation with the pathologic integral of glomerulus (p<0.05) and negative correlation with hematuria (p<0.05). That in distal tubules showed positive correlation with hematuria (p<0.05) and negative correlation with proteinuria (p<0.05). That in proximal tubules showed positive correlation with the pathologic integral of tubule (p<0.05) and negative correlation with proteinuria (p<0.05). The pathologic integral of glomerulus would be significantly increased if PAX2 staining in parietal epithelial (p<0.05).6. PAX2 expression in distal and proximal tubules was significantly increased in patients carrying 798CT/909AC genotype than 798CC/909AA genotype (p<0.05). Proteinuria was also increased in patients carrying 798CT/909AC genotype (p<0.05). Conclusions1. No genetic polymorphism in exon 1-7, 9,10, 12 of PAX2 gene in Chinese children, however the exon 8 of PAX2 gene showed two kinds of SNPs and was in complete linkage disequilibrium (798C>T+909A>C). We found a low frequency SNP (1164 T>A) in the exon 11 of PAX2 gene for the first time.2. Our results do not support a role for 798CT/909AC polymorphism in susceptibility to development of HSP, but patients carrying 798CT/909AC genotype can increase the susceptibility of renal involvement in HSP whereas this polymorphism do not influence clinical phenotype and severity of renal pathology in HSPN.3. PAX2 re-expression was showed in kidney of HSPN. There was more intense staining in tubular epithelium than in glomerulus.4. PAX2 expression in tubular epithelium was significantly increased in patients carrying 798CT/909AC genotype than 798CC/909AA genotype.5. Proteinuria was significantly increased in patients carrying 798CT/909AC genotype than 798CC/909AA genotype.6. Activation of PAX2 gene and expression in kidney may be a response to renal impairment in order to recover from damage, but do not participate in the pathogenesis of HSPN. |
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