Decreased mitochondrial DNA (mtDNA) copy number determined by real-time PCR in patients with gestational diabetes mellitus (GDM)

Abstract

Women with gestational diabetes mellitus (GDM) are at risk of subsequent development of overt diabetes, mainly non-insulin-dependent mellitus (NIDDM), later in life. Although susceptibility to GDM has been suggested to have genetic determinant, the precise genetic basis for GDM is yet unclear. There are some reports that mtDNA content is specifically decreased determine by competitive PCR in adult, but not fetal, pancreatic islets of the Goto-Kakizaki (GK) rat, a genetic model of non-insulin-dependent diabetes. In terms of alterations of the mtDNA content, low birth weight babies were born from mothers whose mtDNA content was decreased. The object of this study is to determine mtDNA copy number using real-time PCR in patients with GDM. Prospective observational study with retrospective analysis. Peripheral blood samples were collected from 68 patients with GDM (age range, 28–33) who were under the inclusion criteria for GDM and from 79 controls. After mtDNA-specific probe and 28S rRNA-specific probe were produced, 5’ and 3’ ends were labeled with FAM reporter and TAMRA quencher. PCR was performed independently for mtDNA and 28S rRNA, respectively. This reaction was performed in triplicate using DNA Engine Opticon 2 (MJ Research). This program measures the detection threshold value (CT), which is defined as the PCR cycles at which a statistically significant increase in reporter fluorescence can be detected above the base line. CT is an inverse proportion to log of target DNA copy number. For the quantification of mtDNA content, a comparative analysis was performed by the amplification of endogenous control (nuclear DNA, 28S rRNA). Between the GDM and control groups, the ratio of mtDNA/28S rRNA was compared using CT of mtDNA of real-time PCR and that of 28S rRNA. All data was expressed as mean±SD. Statistical analysis was performed using Student’s t-test. Statistical significance was set at p < 0.01. In our study, the mean average GDM patient age was 31.7±4.3 years and that of the control group was 31.0 years. The ratio of mtDNA/28S rRNA was 1.205304±0.830688 in GDM patients and 1.797466±1.13551 in control group (p = 0.0004), respectively. In this study, we have used real-time PCR to examine mtDNA copy number in GDM patients and control group that showed decreased in GDM patients compared to control group. Considerable interest has been focused on the quantification of mtDNA in some mtDNA diseases since this severity of clinical symptoms is thought to be related to increased content of abnormal mtDNA. There was also report on decreased mtDNA content in adult pancreatic islets of the GK rat. In terms of decreased mtDNA content in the occurrence of diabetes mellitus, we have applied real-time PCR to analyze mtDNA copy number in GDM and control group. Our result presented here indicated that mtDNA copy number of GDM patients was decreased. Using the Student’s t-test to compare between GDM patients and control group, it was found that there are significant differences in two groups. It can be speculated that metabolic dysfunction may induce mtDNA damage leading to decrease in mitochondrial volume. Our study eventually demonstrated that quantification of mtDNA using real-time PCR is clinically feasible in screening patients with GDM and will further contribute to making an active treatment.