| Part1A methodological evaluation of Klotho gene G-395A, F352V,C370S polymorphism detected by ASP-PCR and FQ-PCRObjective To evaluate the method of Klotho gene G-395A,F352V,C370S polymorphism detection. Methods Klotho gene G-395A,F352V, C370S polymorphism were detected by allelic specific primer polymerase chain reaction (ASP-PCR) and real-time fluorescent quantitative polymerase chain reaction(FQ-PCR). Results (1) The results of Klotho gene G-395A polymorphism detected by ASP-PCR were unstable and less reproducible; the V allele PCR products of F352V were not found; the specific PCR amplification products of C370S were not clear and with lots of non-specific PCR products.(2) The results of Klotho gene polymorphism detected by FQ-PCR were stable and reproducible. Genotype were confirmed by gene sequenencing. Conclution FQ-PCR may be superior to ASP-PCR in Klotho gene G-395A,F352V,C370S polymorphism detection. Part2A study on the association of three SNP sites in Klotho gene with lower limb artherosclerosis in type2diabetesObjective To investigate the association of three SNP sites(G-395A,F352V, C370S) in Klotho gene with type2diabetes and type2diabetes complicated by lower limb artherosclerosis in Kunming Han chinese. Methods The three SNP genotypes of Klotho gene were detected by FQ-PCR method in237unrelated patients with type2diabetes(B group),including79not complicated by lower limb artherosclerosis(B1group),158complicated by lower limb artherosclerosis(B2group) and85normal controls(A group). The genotype frequencies,allele frequencies and clinical indexes were compared among groups. Results (1)The site of G-395A exist polymorphism and present three kinds of genotype,the frequencies of genotype and allele are following:GG69.9％,GA25.8%,AA4.3%,G82.8%,A17.2%.(2) The polymorphisms of F352V and C370S are in complete linkage disequilibrium and present two kinds of genotype respectively,the number of FV/CS genotype is found only2,the rest is all FF/CC genotype.(3) No statistical differences were observed in genotype and allele frequency of G-395A between A and B group (χ2=0.290, P=0.590;χ2=0.229, P=0.633),the GA+AA genotype in B2group was higher than those in B1group (χ2=4.456, P=0.035).(4) The GA and AA genotypes of G-395A is independent correlated with type2diabetes complicated by lower limb artherosclerosis (OR=3.199,95%CI:0.860-11.893). Conclusion (1)The G-395A,F352V,C370S sites of Klotho gene exist polymorphisms in Kunming Han chinese, the polymorphisms of F352V and C370S are in complete linkage disequilibrium and they are rare.(2) The GA and AA genotypes of G-395A might be genetic risk factors of type2diabetes complicated by lower limb artherosclerosis. Part3A study on the association of plasma Klotho level with type2diabetes and bone mineral densityObjective To investigate the association of plasma Klotho level with type2diabetes(T2DM) and bone mineral density(BMD) in Kunming Han chinese. Methods The plasma levels of Klotho were detected in220T2DM patients(T2DM group) and76normal controls(NC group) using enzyme linked immuno sorbent assay (ELISA),the related clinical and biochemical parameters were tested in these subjects. The difference were compared among groups. BMD was measured by Dual-energy X-ray absorptiometry (DEXA) made in America in159T2DM patients.According to the standard of osteoporosis recommended by WHO in1994, the subjects were divided into three groups:normal bone mass (BMD0,n=77),reduced bone mass(BMD1,n=59)and osteoporosis(OP,n=23).The correlation of bone mineral density with plasma Klotho level and its related factors in T2DM patients were analysed.Results (1) The plasma Klotho levels were lower in T2DM group than in NC group (4.92±0.47vs5.38±0.60, P<0.01),the difference has statistical significance after covariance analysis (P<0.01).(2)Multiple stepwise regression analysis showed:insulin sensitivity index(ISI) and serum creatinine (Scr) were independent determinants for plasma Klotho concentrations(B=2.743,-0.004, P<0.05,all).(3) Plasma Klotho levels were not significantly different among the three groups (4.95±0.48vs4.96±0.47vs4.91±0.49, P>0.05).(4) BMD for the first, second,third,fourth,total lumbar spine,femoral neck,trochanter and total body were not associated with plasma Klotho levels in these subjects(P>0.05,all).(5)Logistic regression analysis showed:age, diabetic duration, HDL-C and BMI were independent determinants for BMD in T2DM patients(B=0.037,0.005,-1.011-0.175, OR=1.038,1.005,0.364,0.84,respectively). Conclusion (1) the plasma Klotho level decrease in type2diabetes, it is independent correlated with ISI. Klotho maybe a monitoring indicator of the incidence of insulin resistance and type2diabetes.(2) BMD might be not associated with plasma Klotho level in T2DM patients. But age and diabetic duration are risk factors for reduced BMD and osteoporosis in T2DM patients,HDL-C and BMI are protect factors for it. |
PDF Full Text Request