Association of mitochondrial DNA copy number and T16189C,G10398A,C5178A polymorphisms with type 2 diabetes in Bangladeshi population.

Abstract

The significant role of mitochondria in cellular metabolism is well-established, and there is substantial evidence linking mitochondrial dysfunction to the development of type 2 diabetes (T2D), a metabolic disorder. Mitochondrial DNA (mtDNA) variations are known to contribute to mitochondrial functional impairments and are crucial in determining susceptibility to T2D. This research aims to investigate the connection between three specific mtDNA variations (C5178A, G10398A, and T16189C) and the occurrence of T2D in the Bangladeshi population. Additionally, the study explores how mtDNA variants found in the hypervariable region I (HVI) of D-loop, haplogroups of mitochondria, and the mtDNA copy number are associated with T2D in this population. The study involves 300 random individuals from Bangladesh, with an equal split of 150 healthy individuals and 150 patients diagnosed with T2D. Mitochondrial DNA was extracted separately and was validated/sequence confirmed by Sanger sequencing after PCR amplification using primers against the hypervariable region II (HVII). Restriction enzymes, AluI, DdeI and MnI were used to digest at polymorphic sites after PCR amplification of specific DNA sequences. The PCR amplified products were then sequenced to verify the cutting site. FP: 5′- ACCAGTCTTGTAAACCGGAG-3′ and RP: 5′-GTGGGCTATTTAGGCTTTAT-3′ were used to amplify HVI of mtDNA. Haplogrep2 and Phylotree17 tools were employed to determine the haplogroups. MtDNA copy number was measured using primers of mitochondrial tRNALeu (UUR) gene (FP: 5′-CACCCAAGAACAGGGTTTG-3′ and 5′-TGGCCATGGGTATGTTGTTA-3’) and nuclear β2-microglobulin gene (FP:5′-TGCTGTCTCCATGTTTGATGTATCT-3′ and RP: 5′- TCTCTGCTCCCCACCTCTAAGT-3’). The G10398A polymorphism did not show any significant association with T2D when considering all participants in the study. However, among male participants, the G10398A polymorphism could potentially serve as a protective marker against T2D (OR = 0.30, 95% CI: 0.13–0.67, p < 0.01). On the other hand, the mtDNA C5178A polymorphism demonstrated a notable association with an increased risk of T2D in the study participants as a whole (OR = 3.36, 95% CI: 1.06–10.62, p = 0.02). Furthermore, the presence of the 5178A allele was linked to a heightened risk of T2D in male participants (OR = 9.01, 95% CI: 1.12–72.73, p = 0.01 but this association was not observed in female participants. The variant T16189C within the hypervariable region I was not found to be significantly associated with T2D. According to the results of logistic regression analysis, two variants, G16048A (with an odds ratio of 0.12 and a p-value of 0.04) and G16129A (with an odds ratio of 0.42 and a p-value of 0.007), were identified as having a protective effect against T2D. Furthermore, when adjusting for age and gender, G16129A, along with two new variants, C16294T and T16325C, were found to be protective against T2D. Among the 19 major haplogroups identified, haplogroups A and H were significantly associated with an increased risk of T2D, even after making adjustments. The study also divided the participants into four groups based on their mtDNA copy numbers (lower, medium, upper, and higher, designated as LCN, MCN, UCN, and HCN, respectively). Mitochondrial DNA copy number was significantly lower in patients with T2D compared to healthy individuals only in HCN group. Additionally, six mtDNA variants were significantly linked to mtDNA copy numbers in the participants. In conclusion, the study suggests that certain haplogroups and newly discovered mtDNA variants are notably associated with T2D, while a decrease in mtDNA copy numbers, although not statistically 16 significant, was observed in T2D patients. However, larger-scale studies are needed to further establish the associations of these novel variants and haplogroups with type 2 diabetes