The MTDNA T16189C Polymorphism Of Non-diabetes Mellitus Severe Coronary Heart Disease Patients In Chongqing District By Nested-PCR-RFLP

ObjectiveA nested-polymerase chain reaction-restriction fragment lengthpolymorphism (nested-PCR-RFLP) method was developed to detect theserum mtDNA T16189C variant. The established method was used toanalyze the mtDNA T16189C polymorphism of non-diabetes mellitussevere coronary heart disease (CHD) patients in Chongqing district.Methods1. The method of extracting serum DNA was developed. External andinternal primers were designed according to the sequence of mtDNAT16189C. The internal primers were used for conventional PCR and theexternal and internal primers for nested-PCR respectively. The sensitivity ofconventional PCR and nested-PCR was compared for detecting serummtDNA and the mtDNA T16189C was confirmed by RFLP and DNAsequencing. The methodologies of nested-PCR-RFLP were evaluated.2. Serum and the associated clinical characters of82non-DM severe CHD patients were collected, and50non-DM and non-CHD volunteerswere enrolled as control. Serum mtDNA was extracted, and the levels wereassessed by conventional PCR and nested-PCR among different groups.Serum DNA was extracted and mtDNA T16189C polymorphism wasanalyzed by the established method. The mtDNA T16189C variate wasdetected by the nested-PCR-RFLP and the dependency was evaluatedbetween serum mtDNA content and/or mtDNA T16189C variate rate andnon-DM severe CHD.Results1. Extraction of serum DNA and evaluation of developednested-PCR-RFLP method:(1) established the method of extracting serummtDNA and the optimum reaction system of PCR and restriction enzymeMnlI successfully. The mtDNA was confirmed by nested-PCR and themtDNA T16189C variant was confirmed by RFLP and DNA sequencingsuccessfully.(2) The nested-PCR was fast and uncomplicated for mtDNAT16189C variant with an optimal sensitivity and a high degree of specificity.2. Relationship between the serum mtDNA content or mtDNAT16189C polymorphism with non-DM severe CHD patients:（1）ThemtDNA content in non-DM severe CHD was significantly lower thanhealthy controls (χ2=9.979, P<0.05; OR＝0.318,95%CI：0.153～0.660).Moreover, the women, the younger, the nomal LDL-c and the non-smoking were significantly higher in non-diabetes mellitus severe CHD group thanthat in control group(P<0.05), but there were no significant differencesabout the pressure, HDL-c, TC, impaired glucose tolerance（IGT）（P>0.05）; Based on multivariate non-conditional logisticregression analysis, smoking was the main factors reducing the serummtDNA content(OR＝3.70；95%CI：1.285～10.870).（2）The mtDNAT16189C variant in the IGF patients was more prevalent than in healthycontrols, but there was no statistically significant(P>0.05); and the variant insevere CHD patients accompanying IGTwas significantly higher thanthat innormal blood glucose CHD and healthy controls(P<0.05), but there was nodifference between normal blood glucose CHD patients and healthycontrols(P>0.05); the variant in severe CHD patients accompanyinghypertension was significantly higher than that in normal pressure andhealthy controls(P<0.05), but there were no differences between normalpressure CHD patients and healthy controls(P>0.05); and there were nosignificant differences about the sex,age, smoking, LDL-c,HDL-c,TC.Based on multivariate non-conditional logistic regression analysis,hypertension was the main factors increasing the serum mtDNA T16189Cvariate （OR＝3.265；95%CI：1.061～10.06）（3）The mtDNAT16189C variate rate in highcontent group was higher thanthat inthe low ofnon-DM severe CHD (χ2=5.612, P<0.05; OR＝3.154,95%CI：0.197～8.307). ConclusionsEstablished the method for extracting serum DNA and thenested-PCR-RFLP for T16189C variate successfully. Nested-PCR-RFLPprovides a rapid serum genotyping method which could be used as a widevariety of epidemiological studies. The mtDNA content in non-DM severeCHD was significantly lower than healthy controls. Moreover, the content inwomen, the younger, the nomal LDL-c and the non-smoking weresignificantly higher in non-DM severe CHD group than that in control group,and smoking was the main factors reducing the serum mtDNA content.There were associations between mtDNA T16189C variate andhypertension/IGT, and hypertension was the main factor inducing the serummtDNAT16189C variate. The mtDNAT16189C variate rate in high contentgroup was higher than that in the low of non-DM severe CHD.