The Predictive Study Of PON Polymorphism In Diabetic Nephropathies

The Human Genome Project (HGP) has been completed. Thesescientific achievements facilitate the humanbeing to understandthemselves well and discover the mystery of life science., but alsocontribute to the interaction mechanism on the level of genome , showthe genetic essence of most human serious disease. Then the method ofdisease diagnosis and treatment is established on the gene level.Most human serious diseases, such as tumor, cadio-brain-vasculardisease and diabetes,diabetic nephropathy, are all associated withgenetic factors. Each individual is decided by the number of suscepticgenes, more susceptic genes, more high of the risk of having disease.The construction of human genome maps, including genetic map,physical map, DNA sequeence map and single nucleotide polymorphism(SNP) map has been completed.The SNP map combines linkage study with linkage disequilibrium,and integrates the DNA sequence map, physical map and genetic map atthe DNA level. It makes a great progress in mapping the susceptic genesof polygenic disease.Diabetic nephropathy (DN) is a serious complication of diabetes.Itsearly diagnosis is very important to treatment and prognosis of DN,however,early diagnosis of DN have no effective method by thenow .DN has been related to artherosclerosis (AS) which attributes toperoxidation and the shortage of antioxidase. It was reported fromabroad, the level of HDL and PON is obvious low to normal in advancedstage DN which explains PON polymorphisms is possible predict factorsof DN. The aim of research is to confirm the possibility that PONmay be the estimate factor of DN earlier period examine bypredisposing genes PON sieving in DN.1. Object1.1 Sample datasSimple type 2 diabetes mellitus(T2 DM)154,which are patientsin hospital from JiLin province people's hospital(2003.3-8).All matchtype 2 diabeteses of 1997 ADA diagnosis standard, expel the myocardialinfarction, the cerebral disease, diabetic nephropathy, retinopathy;Male77,female77, age 43～76 years old, average age (64.5 ±10.3) yearsold.Type 2 diabetic nephropathy (DN) 145, which are patients inhospital from JiLin province people's hospital (2003.3-8). Male 75,female70, age 42~ 77 years old, average age (64.7 ±11.23) years olddiabetic nephropathy diagnostic criteria match the WHO kidney diseasediagnosis standard, and was expel by CT heart and Cerebral vesselsdisease.Control group 97, those who is health examination of theout-patient from JiLin province people's hospital;by history, healthexamination, laboratory and electrocardiography, diabetes are expelcerebrovascular disease, myocardial infarction, nephropathy, retinopathy,male 50, female 47, age 42-75 years old, mean age(62.4±10.9)yearsold.1.2 Blood sample collect and disposalAfter permission, take peripheral vein blood 5ml, -20℃ DNAkeep to extract genome DNA.2. To sure The SNPs of candidate gene and the candidate geneLand (http://www.ncbi.nlm.nih.gov/), strike Map Viewer, inputdisease name, then caging all districts with male chromosome. Thescope of the male district is generally between the 10-20 cMs.2.1 To sure the SNPs with RFLP of candidate geneBy humanbeing genome data bank, to sure all knowned gene inMale district of related chromosome with diseases and expressedsequence tag, in the chromosome district of the 10-20 cMs, contain the100-200 genes and ESTs equally. Browse each gene and the SNPsdiagram on the ESTs, to chose imply SNPs with the restrict enzyme, toconstruct RFLP-SNPs map.2.2 The grade of heterozygosis accreditAfter make selection the SNPs order, using firstly 20 DNA sampleswhich no the blood relationship do the genotype analysis, only choosethose SNPs which heterozygosis degrade more than 10% to do genotypeanalysis.3.Primer designAfter the SNPs genetic mark to be examine are sured, take theadvantage of the SNPs data bank to searches each sequence of DNA ofSNP, then design PCR primer sequence of DNA with SNPs andsynthesize by the sequence biology ( Dalian) limited company.4.Genotyping4.1 Withdraw DNA from human genome5ml peripheral vein peripheral vein anticoagulated blood was taken,Use trace DNA kit (Ning Bo ) to withdraw genome DNA.4.1.1 Step(1)Take the blood sample 100 uL, add to the 1.5 mL centrifugetube, then add 100 uL 1×TE buffer shaking evenly;(2)vibrationsimultaneously, add agent B1 200 uL;shaking evenly;(3)vibrationsimultaneously, add agent B2 200 uL;bene thoroughly(4)centrifuge by14000rpm, 1min;(5)add agent B3 400 uL, then centrifuge by14000rpm, 1min;(6)Repeat the step (5)once(;7)The empty columncentrifuging(14000rpm, 2min), then move into the clean 1.5 mL tocentrifuge tube, and addthe agent B4 100 uL s(14000rpm, 1min);now 100 uL solution in centrifuge tube, where there are genome DNAwithdrawed.4.1.2 The examination of the content and purity of DNAMeasure the Absorption of DNA sample in the OD260nm and theOD280nm, DNA purity =OD260nm/OD280nm.4.2 PCR amplificationPCR reaction system 50 uL:DNA 1.0uL, water 38.75uL, primer(200umol/L)0.5uL, Taq enzyme (5u/uL)0.25uL, 10 × PCRbuffer(contain Mg2+)5.0uL, dNTP Mixture (2.5umol/L)4.0uL.PCR reaction condition : 94 ℃ force-apomorphosis, 94 ℃apomorphosis 0.5min → 61 ℃ anneal → 72 ℃ extend, Total 35circulations, the last 72℃ extend 8 minutes.4.3 RFLPAdd corresponding restriction enzyme and buffer in the reactionsystem after PCR, put them in incubator 37℃ for 5 hours. To judgegene type by electrophoresis and cleavage map4.3.1 Enzyme reaction systermTake the PCR product 18 uLs,add restriction enzyme 5ul, 10×buffer2uL, aqueous bath 37℃ 16h, aqueous bath 65℃ 20min.4.4 Electrophoresis image5.Statistics analysis5.1 Hardy-Weinberg(H-W)Balance examination5.2 Allel and genotype frequenciesTake chi square test;Risk factor analysis using Logistic analysis.Result一,Genotype judge1,PON1 gene (192) PCR product is 996bp frag, if Gln→Argvariation(A→G, GAA→CGA), after RFLP 68bp and 31bp frage wereproduce. Homozygote RR:68bp, 31bp;homozygote QQ:99bp;heterozygote QR: 99bp,68bp,31bp.2,PON1 gene(55) PCR product is 170bp frag, after RFLP,homozygote(200umol/L)0.5uL, Taq enzyme (5u/uL)0.25uL, 10 × PCRbuffer(contain Mg2+)5.0uL, dNTP Mixture (2.5umol/L)4.0uL.PCR reaction condition : 94 ℃ force-apomorphosis, 94 ℃apomorphosis 0.5min → 61 ℃ anneal → 72 ℃ extend, Total 35circulations, the last 72℃ extend 8 minutes.4.3 RFLPAdd corresponding restriction enzyme and buffer in the reactionsystem after PCR, put them in incubator 37℃ for 5 hours. To judgegene type by electrophoresis and cleavage map4.3.1 Enzyme reaction systermTake the PCR product 18 uLs,add restriction enzyme 5ul, 10×buffer2uL, aqueous bath 37℃ 16h, aqueous bath 65℃ 20min.4.4 Electrophoresis image5.Statistics analysis5.1 Hardy-Weinberg(H-W)Balance examination5.2 Allel and genotype frequenciesTake chi square test;Risk factor analysis using Logistic analysis.Result一,Genotype judge1,PON1 gene (192) PCR product is 996bp frag, if Gln→Argvariation(A→G, GAA→CGA), after RFLP 68bp and 31bp frage wereproduce. Homozygote RR:68bp, 31bp;homozygote QQ:99bp;heterozygote QR: 99bp,68bp,31bp.2,PON1 gene(55) PCR product is 170bp frag, after RFLP,homozygote(G148A),PON(C311S)polymorphism. A allel and S allel are riskfactor of type 2 DN(A,OR=2.05,95%CI:1.17-3.29,P<0.05)(S:OR=2.29,95%CI:1.23-4.26,P<0.05).ConclusionThe results show:1, In Chinese, there are PON1(Q192R), PON1(L155M),PON2( G148A), PON2( C311S), PON3( A99A) polymorphism.2, PON2( G148A) polymorphism and DM, DN are related.3, PON2( C311S) polymorphism and DM, DN are related.4, PON2(C311S) polymorphism expresses in DN more obviously.5, The polymorphism of PON2 (G148A) and PON2 (C311S) maybe prognostic factors in patients with DN.6, There are no relationships between PON1 (Q192R), PON1(L55M), PON3 (A99A) and DN.