Variants in the Control Region of Mitochondrial Genome Associated with type 2 Diabetes in a Cohort of Mexican Mestizos.

BACKGROUND: According to the International Diabetes Federation, Mexico is seventh place in the prevalence of type 2 diabetes (T2D) worldwide. Mitochondrial DNA variant association studies in multifactorial diseases like T2D are scarce in Mexican populations. AIM OF THE STUDY: The objective of this study was to analyze the association between 18 variants in the mtDNA control region and T2D and related metabolic traits in a Mexican mestizo population from Mexico City. METHODS: This study included 1001 participants divided into 477 cases with T2D and 524 healthy controls aged between 42 and 62 years and 18 mtDNA variants with frequencies >15%. RESULTS: Association analyses matched by age and sex showed differences in the distribution between cases and controls for variants m.315_316insC (p = 1.18 × 10-6), m.489T>C (p = 0.009), m.16362T>C (p = 0.001), and m.16519T>C (p = 0.004). The associations between T2D and variants m.315_316ins (OR = 6.13, CI = 3.42-10.97, p = 1.97 × 10-6), m.489T>C (OR = 1.45, CI = 1.00-2.11, p = 0.006), m.16362T>C (OR = 2.17, CI = 1.57-3.00, p = 0.001), and m.16519T>C (OR = 1.69, CI = 1.23-2.33, p = 0.006) were significant after performing logistic regression models adjusted for age, sex, and diastolic blood pressure. Metabolic traits in the control group through linear regressions, adjusted for age, sex and BMI, and corrected for multiple comparisons showed nominal association between glucose and variants m.263A>G (p <0.050), m.16183A>C (p <0.010), m.16189T>C (p <0.020), and m.16223C>T (p <0.024); triglycerides, and cholesterol and variant m.309_310insC (p <0.010 and p <0.050 respectively); urea, and creatinine, and variant m.315_316insC (p <0.007, and p <0.004 respectively); diastolic blood pressure and variants m.235A>G (p <0.016), m.263A>G (p <0.013), m.315_316insC (p <0.043), and m.16111C>T (p <0.022). CONCLUSION: These results demonstrate a strong association between variant m.315_316insC and T2D and a nominal association with T2D traits.

A New Approach to Identify the Methylation Sites in the Control Region of Mitochondrial DNA.

Mitochondrial DNA (mtDNA) methylation has the potential to be used as a biomarker of human development or disease. However, mtDNA methylation procedures are costly and time-consuming. Therefore, we developed a new approach based on an RT-PCR assay for the base site identification of methylated cytosine in the control region of mtDNA through a simple, fast, specific, and low-cost strategy. Total DNA was purified, and methylation was determined by RT-PCR bisulfite sequencing. This procedure included the DNA purification, bisulfite treatment and RT-PCR amplification of the control region divided into three subregions with specific primers. Sequences obtained with and without the bisulfite treatment were compared to identify the methylated cytosine dinucleotides. Furthermore, the efficiency of C to U conversion of cytosines was assessed by including a negative control. Interestingly, mtDNA methylation was observed mainly within non-Cphosphate- G (non-CpG) dinucleotides and mostly in the regions containing regulatory elements, such as OH or CSBI, CSBII, and CSBIII. This new approach will promote the generation of new information regarding mtDNA methylation patterns in samples from patients with different pathologies or that are exposed to a toxic environment in diverse human populations.