The Study Of Conditions Influencing Polymerase Chain Reaction-restriction Fragment Length Polymorphism In The Leu262 Val Polymorphism Of PARL Gene

Objective To determine the optimum experimental conditions of the Leu262Val polymorphism of PARL gene detected by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP). Method In the PCR, three pairs of primers were designed by the Primer 5.0 Software, the optimum primers were delected after contrast; to optimize PCR program by touchdown PCR(TD-PCR);4 kinds of primers'concentration (0.32umol/1,0.4μmol/L,0.6μmol/L,0.8μmol/l) were detected; in the RFLP,3 kinds of PCR product quantity(5μl,10μl,15μl) and 5 kinds of incubation time (12h,8h,4h,2h,1h) were detected when the incision enzyme quantity was unchanging, experimental results were observed from 2% agarose. Result (1)The first pair of primers was the best, PCR product was 545bp; (2) TD-PCR showed no non-specificity band and simplified the process for seeking the optimum annealing temperature in Ordinary amplification. (3)When the primers concentration was 0.4μmol/L, PCR production level was higher than others and less dimer of primers. (4)It was 10μl PCR production amount that was digested could creat a clear digested electropherogram. (5) It was 4 hours that was demanded in incubated reaction at 37℃condition. Conclution The design of primers were the key of PCR result, the optimum primer's concentration was 0.4μmol/L; TD-PCR optimized conndiitions of ordinary PCR, it was more simple and effective than the ordinary; the effective RFLP protocol:the system was 20μl including 10μl PCR reaction mixture and 10U enzyme were incubated at 37℃for 4 hours. Objective To determine the possible modulating effect of the Leu262Val polymorphism of PARL gene on the risk of type 2 diabetes Mellitus (T2DM) in Kun ming Han people. Method A case-control study was conducted in our study; the Leu262Val polymorphism of PARL gene was detected by PCR-RFLP method in 147 unrelated Kunming Han T2DM patients and 113 controls. The genotypes and allele frequencies distributions were compared among cases and controls, at the same time, the related characteristics of cases and controls were compared. Results (1) No significant differences were observed in genotype distributions and allele frequencies of the Leu262Val polymorphism between cases and controls, (χ2=2.468,,P>0.05;χ2=1.335, P>0.05);(2) The age, BMI, WHR, SBP, DBP, ALT, CRE, TG, LDL-C, FPG in cases were higher than controls and the HDL-C was significantly lower than controls (P<0.05). (3) In T2DM patients, the level of HbAlc% in C allele genotype carriers were higher than others(P<0.05). No significant differences were observed in the related clinical. characteristics among the different genetype carriers of controls (P>0.05). (4) Logistic regression analysis indicate that HDL-C (OR:0.119,95%CI: 0.025-0.366) was protective factors of BMI in T2DM patients,Conclusion The Leu262 Val polymorphism of PARL gene is not significantly associated with a risk of T2DM in Kunming Han people;The level of HbAlc% in C allele genotype carriers were higher than others, HDL-C was protective factor of BMI In T2DM patients. Objective To determine the possible modulating effect of the Leu262Val polymorphism of PARL gene on the risk of type 2 diabetic nephropathy(DN) in Kunming Han people. Methods The Leu262Val polymorphism of PARL gene was detected by PCR-RFLP method in 176 DN patients, in which including 91 microalbuminuria (DN1 group),85 macroalbuminuria (DN2 group); 102 free-DN type 2 diabetes and 113 normal controls. The genotype and allele frequencies distributions were compared among different groups, at the same time, the related characteristics of cases and controls were compared. Results (1) No significant differences were observed in genotype distributions and allele frequencies among the groups(P>0.05). (2)In T2DM patients, the levels of diabetic duration(years), SBP, DBP, BUN, CRE, UA, TC, LDL-C, HbA1c%, FA in DN2 group were higher than DN1 and DNO groups; also the levels of age, LDL-C and FPG were higher than DNO, the levels of 2hCP was lower than DNO; the levels of age, diabetic duration SBP, DBP in DN1 group were higher than DNO group.(3) No significant differences were observed in the related clinical characteristics among the different genetype carriers in DN patients (P>0.05). (4) Logistic regression analysis indicate that the diabetic duration, SBP, TG, HbAlc% were risk factors of DN in T2DM patients. Conclusion The Leu262Val polymorphism of PARL gene is not significantly associated with a risk of type 2 diabetic nephropathy(DN) in Kunming Han people,。In T2DM patients, the diabetic duration, SBP, TG, HbAlc%were risk factors of DN. Objective To explore the Association of the Leu262Val polymorphism of PARL gene and Pre-diabetes,Diabetes mellitus paitents in Wa People. Methods The Leu262Val polymorphism of PARL gene was detected by PCR-RFLP in 263 Wa people with pre-diabetes (PD),110 Wa peopl with Diabetes mellitus(DM) and 127 normal controls (NC). The genotypes and allele frequencies distributions were compared among people with pre-diabetes, Diabetes mellitus and normal controls, at the same time, the related clinical and biochemical indicators were also compared. To recruit in the survey by questionnaire, investigate the prevalence of diabetes and other possible risk factors to DM. Logistic regression analysis was used to estimate the risk markers of pre-diabetes and Diabetes mellitus.Results (1) There was no significant differences in genotypes and allele frequencies distributions among PD, DM and NC(P>0.05). (2)The female proportion and the leve of age, SBP, DBP, AST, FPG in both DM and PD were higher than NC;the leve of BUN and CRE in DM were higher than PD and DM(P<0.05). (3) There were no significant differences in the related clinical and biochemical indicators among the three groups with different genetypes (P>0.05).(4) The female, age, edible animal oil were risk factors of PD and DM in Wa people. Conclusion The Leu262Val polymorphism of PARL may not be risk factors for PD and DM in Wa People; By improving the habits of food, it is hoped to prevent the development of IGR and Diabetes mellitus. Objective To investigate the discrepancy of the Leu262Val polymorphism of PARL gene between the Han and Wa Ethnic Chinese of Yunnan province. Methods According our previous research, the genotype frequency and allele frequency of the Leu262Val polymorphism of PARL gene,at the same time, the related characteristics of cases and controls were compared were compared between 260 Han People (113 normal glucose controls and 147 DM patients) and 237 Wa people (127 normal glucose controls and 110 DM patients). Results (1) There were significant differences in genotype frequencies and allele frequencies between Han and Wa People in the whole samples (P<0.05). (2) There were significant differences in genotype frequencies and allele frequencies between Han and Wa People in the normal glucose controls(P<0.05). (3) There was no significant differences in genotype frequencies and allele frequencies between Han and Wa People in DM patients (P>0.05). (4) In the.normal glucose controls, the levels of CRE, UA, TC, HDL-C, LDL-C in Han people were higher than Wa people; the levels of ALT, AST, FPG were lower than Wa people. (5) In DM patients, the male proportion and the levels of BMI, ALT, TC, TG, LDL-C, HBA1C% in Han people were higher than Wa people; the levels of AST, HDL-C were lower than Wa people. Conclusion There may exist racial diversity with the Leu262Val polymorphism of PARL gene between the Han and Wa Chinese of Yunnan province, because the distributions of genotype frequencies and allele frequencies were different.