The Correlation Analysis Between ?-defensins Genes Polymorphsims And IgA Nephropathy

Part I.Determination of the expression of gene DEFA1/3 and DEFA4Objective: To detect the expression level of m RNA of DEFA1/3 and DEFA4 gene in Ig AN,and to explore the association between the m RNA and Ig AN.Methods: Respectively collected the fasting venous blood of IgAN patients and healthy controls,then collected the blood plasma and extracting the whole blood RNA,the level of RNA was detected by dye method,which is one of Quantitative PCR,and the plasma HNP1–3 measured by ELISA.The result was statistically analyzed.Results: The expression level of mRNA(1.18 ±0.55)of DEFA1/3 in the Ig AN group was significantly higher than the healthy control group(0.84 ± 0.63,P<0.01);But,the expression level of m RNA of DEFA4 in the two groups(0.022 ± 0.03 vs 0.018 ± 0.02)with no significant difference(P>0.05).what?s more,There was no significant difference between the level of m RNA of DEFA1/3 and DEFA4 with sex,age,and pathological Lee classification(P>0.05).In addition,the concentration of HNP1-3 in group with Ig AN parents(51.45 ± 8.32)was higher than that of the healthy control group(44.33 ± 7.36,P<0.001).Conclusion: The expression level of m RNA of DEFA1/3 in parents with Ig AN of Han nationality in China was higher than healthy controls,while DEFA4 m RNA expression level was no significant difference between Ig AN patients and controls.There was no significant correlation between the expression level of m RNA of DEFA1/3 and the pathological grade of Lee,neither nor the DEFA4.In addition,the concentration of HNP1-3 in group with Ig AN parents was higher than that of the healthy control group.Part II.The correlation between DEFAs gene polymorphisms and Ig A nephropathy and its phenotypeObjective: not only to study de relationship between DEFAs genes and IgA nephropathy,but also explore the correlation between the frequency and genotype rate of DEFAs genes SNPs and Ig A nephropathy.Methods: 134 patients with IgAN and 136 healthy subjects from the First Affiliated Hospital of Wangnan Medical college,were divided into two groups.We determinated the sequences of SNPs rs2615787,rs6984215,rs2738048 and rs2738048 by Sanger sequencing,and the automatic biochemical analyzer was used for the determination of urea,creatinine and blood uric acid.Results: the level of urea?creatinine and blood uric acid namely were significantly higher in patients with Ig A nephropathy(6.72±3.12 mmol/L,113.35±64.70 umol/L,364.7±103.59 umol/L)compared to healthy control group(4.20±1.05 mmol/L,57.20±10.47,283.66±61.01 umol/L;P<0.01,P<0.001,P<0.001).The distribution of the frequency of SNPs rs2615787,rs6984215 and rs2738048 genotype in the two populations were statistically different(P<0.01).The frequency of SNP rs2615787 C allele was 20.9% in the Ig A nephropathy group and 35.3% in the healthy control group,and the difference was statistically significant(P<0.001).The C allele frequency of SNP rs6984215(24.6%)and SNP rs2738048(25.4%)were lower in patients with Ig A nephropathy compared to healthy control group(33.1%?34.6%,P<0.05).However,there was no significant difference between Ig A nephropathy patients(14.6%)and healthy controls(19.1%,P>0.05)of A allele frequency of SNP rs2738081.Conclusion: DEFAs genes may be the susceptible genes of IgA nephropathy.There may be a correlation between the SNPs rs2615787,rs6984215,rs2738048 and Ig AN susceptibility of DEFAs genes,but no correlation with pathological Lee classification.Part III.Study the DEFAs genes polymorphic by luciferase reporter gene systemObjective: To construct several Luciferase reporter gene vectors and transcriptional factor overexpression vectors that containingdifferent alleles of rs2615787 and rs2738048,study the effect of different alleles of SNP rs2615787 and rs2738048 on the transcription of luciferase in 293 cells,evaluate the regulation of the alleles of rs2615787 and rs2738048 on the promoter transcription activity of DEFAs genes.Methods: rs2738048 T,rs2738048 C,rs2615787 A,rs2615787 C PCR products were generated by PCR,and PCR products were cloned into the reporter gene PGL3 promoter.In addition,constructing overexpression vectors contains HSTF or CTF,based on the groups of PGL3 promoter+ PCDNA-HSTF-NC?PGL3-rs2615787 A + PCDNA-HSTF?PGL3-rs2615787 A+ PCDNA-HSTF-NC?PGL3-rs2615787 C+ PCDNA-HSTF?PGL3-rs2615787 C+ PCDNA-HSTF-NC?PGL3 promoter+ PCDNA-CTF-NC ? PGL3-rs2738048 T+ PCDNA –CTF ? PGL3-rs2738048 T+ PCDNA-CTF-NC?PGL3-s2738048 C+ PCDNA –CTF?PGL3-rs2738048 C+ PCDNA-CTF-NC,transfecting reporter gene vectors and over expression vectors into 293 tool cells with Hai Shen luciferase.After 48 hours of transfection,the relative activity of luciferase was detected.Results: both the Luciferase reporter gene vectors and transcriptional factor overexpression vectors were successfully constructed confirmed by restriction Enzyme digestion and sequencing.All the vectors can be expressed in 293 cell,The T and C alleles of rs2738048 not only can change the relative activity of luciferase,but also combined with the transcription factor CTH.However,the reporter vectors of PGL3-rs2738048 T,PGL3-rs2738048 C have different relative luciferase activities.The former is obviously lower than the latter.The reporter vectors of PGL3-rs2615787 A and PGL3-rs2615787 C can also be expressed in 293 cells,but have no significant effect on the relative activity of luciferase.Conclusion: the reporter vectors of PGL3-rs2738048 T,PGL3-rs2738048 C,PGL3-rs2615787 A,PGL3-rs2615787 C,PCDNA –HSTF ? PCDNA –CTF will successfully constructed.The C allele of SNP rs2738048,compared with T allele,could inhance the transcription activity of DEFAs genes.While the rs261575 alleles(A/C)have no significant effect on the relative activity of luciferase.