Novel Mitochondrial DNA Variations Associated with Coronary Artery Disease in Type 2 Diabetes from an Asian Indian Population

This work was carried out in collaboration between all authors. Author MG conceived and designed the experiments and wrote the first draft of the manuscript. Authors PS and JV performed the experiments. Author UKH identified patients. Authors MG, PS and SKK analyzed the data. Author SP managed the literature searches. Author QH provided input on manuscript writing. All authors read and approved the final manuscript. ABSTRACT Background: Mitochondrial dysfunction leading to insulin resistance may contribute to metabolic and cardiovascular abnormalities and subsequent increase in coronary artery disease. Since mitochondria are involved in generation of ROS, we aimed to investigate the association of mtDNA mutations with T2DM and CAD in our population. Methods: We analyzed the complete mtDNA of South Indian subjects which included patients with angiographically documented CAD [n = 120], subjects with Type 2 Diabetes Mellitus and CAD [n = 150] and healthy control subjects without clinical manifestations of atherosclerotic disease and Type 2 Diabetes [n = 100]. We detected the association of common variants of the mitochondrial genes with both T2DM and CAD, which raises the possibility of a shared mitochondrial genetic background of these metabolic disorders in our population. Results: The complete mitochondrial analysis of the control group revealed several sequence variations but did not show any novel mutations. Mitochondrial analysis of individuals with CAD and T2DM revealed a total of 36 novel variations. Mutations were more prevalent in NADH Dehydrogenase [ND] genes that encode mitochondrial enzyme Complex I . Among the 20 novel mutations in the ND genes, 17 were missense, 2 synonymous and 1 frame shift variant were observed. In Cytochrome b [Cytb] gene, 7 variations observed were novel that included 5 missense mutations in cytochrome c oxidase [CO2] were novel mutations including 1 missense mutation and 1 synonymous mutation. In rRNA genes, we identified 1 novel variant in 12s RNA and 3 in 16s rRNA. Among the CAD patient group without T2DM, 3 novel variants in ND region were identified of which 2 were synonymous and one was missense. The variants observed are not reported to have any disease association so far by any studies. Conclusions: Presence of pathogenic known and novel mutations suggests mtDNA variations have a role in the pathophysiology of CAD associated with T2DM in our population.


INTRODUCTION
The conventional risk factors associated with heart disease and stroke are unhealthy diet, physical inactivity, tobacco use and harmful consumption of alcohol. The effects of behavioral risk factor in individuals may manifest as raised blood pressure, raised blood glucose, raised blood lipids, overweight and obesity leading to metabolic syndrome [1]. Diabetes, hypertension and dyslipidemia and hereditary factors are major risk factors for coronary artery disease [CAD], which are associated with high oxidative stress and are responsible for morbidity and mortality in Asia, particularly in India. Factors that promote atherosclerosis are chronic overproduction of mitochondrial reactive oxygen species leading to increased oxidation of lowdensity lipoprotein and dysfunction of endothelial cells, as well as, destruction of pancreatic β-cells.
The frequency of CAD varies in different ethnic populations. Asian Indians are estimated to have higher risk than the people of other ethnic origin, irrespective of gender, region, or socio-economic group [2]. A recent paper showed that population origins and ancestry are important determinants of both T2DM and CAD [3]. According to the Global Burden of Disease study ischemic heart disease is the leading cause of global mortality. It is classified as 1.4 million deaths in the developed world and 5.7 million deaths in developing regions [4]. Our earlier studies on the gene polymorphisms, involved in lipid pathway have shown that the patients, who have CAD with T2DM, show a significant association with genotype when compared to non-diabetic controls [5,6]. Clinical studies globally show a worse outcome of CAD in subjects with T2DM versus non-diabetic patients. There is strong and consistent evidence that oxidative stress is crucially involved in the development of atherosclerotic vascular disease leading to CAD. Perturbations in cardiac mitochondrial metabolism associated with T2DM may be expected to attenuate myocardial tolerance to ischemia. In humans, this is supported by the fact that incidence of heart failure is increased in diabetic subjects far in excess of non-diabetic individuals with similar infarct sizes [7]. Mitochondrial DNA mutations are known to enhance ROS production [8]. Role of mitochondrial DNA damage in progressive diseases of oxidative phosphorylation has been associated with accumulation of mitochondrial DNA mutations, especially deletions [9]. The molecular disruption in insulin signaling following cardiac restricted depletion of insulin receptors results in age-dependent impairment in mitochondrial oxidative phosphorylation capacity and augmentation in oxidative stress [10]. A study described a clear association between homoplasmic mitochondrial tRNA mutation and a metabolic syndrome that was characterized by hypertension, hypomagnesaemia and hypercholesterolaemia which enhance the risk of CAD [11]. Another frequent mtDNA mutation which was associated with enhanced risk of diabetes was A3234G, in the tRNA Leu gene [12,13]. Earlier a family study showed association of tRNA Thr 15927G>A mutation in CAD/T2DM belonging to the Eastern Asian population [14]. Recent study by Santulli et al. [15] have shown that mutations in the type 2 ryanodine receptor (RyR2) which is a Ca 2+ release channel on the endoplasmic reticulum (ER) of cardiomyocytes and pancreatic β cells play a crucial role in the regulation of insulin secretion and glucose homeostasis. Given the central role of mitochondria in energy and ROS production, mtDNA is an obvious candidate for genetic susceptibility studies in the atherosclerotic process. In this paper we therefore examined the mitochondrial DNA variations among CAD patients with and without type 2 diabetes comparing them with healthy controls.

Samples and Clinical Data
Blood samples were collected from 370 individuals, of whom 120 were patients diagnosed to have angiographically confirmed CAD, 150 had CAD and T2DM, while 100 age and sex matched healthy individuals were included in the study as controls. CAD patients with and without Type 2 Diabetes who were willing to participate in the study were recruited from clinics. The healthy controls were also recruited the same way. Institutional Ethics committee [KHLNo.e372/07] approval was obtained prior to collecting clinical details and samples. Medical records of the patients along with other demographic details were documented in a specified proforma. About 5 ml of intravenous blood sample from each patient was collected in an EDTA vaccutainer. Informed consent was obtained from all individual participants included in the study.

Analysis of Complete Mitochondrial DNA
Genomic DNA was isolated from all the 370 samples using standard protocol used by our group [5]. Complete mtDNA of patients and ethnically matched controls were amplified using 24 sets of overlapping primers as described earlier by us [16]. Amplicons were electrophoresed using 2% agarose gel and the cycle sequencing reaction was carried out using BigDye Terminator ready reaction kit [Applied Biosystems, Foster City, USA]. Extended products were precipitated with sodium acetate and ethanol, and dissolved in Hi-Di formamide,

In silico predictions for Novel non Synonymous Mutation
The effect of amino acid variations on protein function were predicted with PolyPhen-2 [19] prediction and PROVEAN v1.

Demographic Details
In the present study we have recruited 370 patients of which 120 had CAD, 150 of them had T2DM and CAD while the remaining 100 were healthy control group. The demographic details of the patients and their clinical details are given in Table 1. Among the 150 patients with CAD and T2DM we observed that 31% had more than two mutations, which included novel and reported variants [ Table 3]. Interestingly, the well reported A12308G mutation in tRNA Leu was observed in 15 patients with CAD and T2DM.

Phylogenetic Analysis
The mtDNA based phylogenetic analysis of the    [40]. Later it was proposed by [13] that the fatty acid-induced mitochondrial dysfunction in the pancreas leads to lipotoxicity, causing premature aging of the βcell which results in T2DM.This A12308G mutation in the tRNALeu [UUR] gene was seen in 14 patients with CAD and T2DM ie10% of our patients. This mutation accounts for more than 90% of all cases of MELAS [41] and is also associated with CPEO, stroke and cardiomyopathy [42][43][44]. The G15928A t-RNA-Thr mutation which was also seen among our patients was earlier reported to be associated with repeated pregnancy loss among Iranian women [45] and in a confirmed Parkinson disease patient [46]. This is the first study associating this mutation with CAD and T2DM. Earlier studies [28] have shown an elevated frequency of T16189C mutation present in the Dloop in patients with CAD and T2DM compared to healthy controls in Middle European Populations. We similarly found this mutation in 10% of our patients with CAD and T2DM.
Mitochondrial ribosomal mutations are considered to have disruptive potential and specific mutation in 12s rRNA like A1555G is associated with hearing loss with or without diabetes [47][48][49], 16s rRNA T3394C and A12026G mutation has been identified in T2DM patients from China [50]. We found one novel mutation in 12sr RNA C1597A and three novel mutations in 16s rRNA [ Table 2]. In the present study total mitochondrial DNA was sequenced and we identified several sequence variations; on comparing these variations with the sequence data of controls it was observed that there was only one novel variation in the patients with CAD. However, 38 novel variants were observed in the sample from CAD patients who had T2DM. We therefore propose that the variations observed in the study could be specific to T2DM patients from India, who are at a greater risk of developing complications of CAD. Our previous studies on different Indian population groups suggest that Indians are not only unique in their origin [51][52][53], but are unique in the etiology of various diseases [54][55][56][57]. Therefore, existence of novel mutations in Indian subjects is not surprising. In silico Characterization of missense Mutations: Potential pathogenicity of the missense variants found in our data was analyzed using tools like PROVEAN and Polyphen-2. In silico analysis of results of predictions is shown in Table 4.There were deleterious variants among the mutations observed which need in-depth analysis by functional assays.
This study demonstrates the high overall frequency of novel mtDNA mutations in Type 2 diabetic patients with CAD. In our patients we found that the ND region had a higher frequency of mutations compared to those with CAD without T2DM. Detailed familial studies will have to be performed in patients carrying these mutations and their relatives, in order to define the importance of the observed mtDNA defects to the clinical manifestation of T2DM leading to CAD.

CONCLUSION
Analysis of the whole mtDNA of the patients with T2DM and CAD, a complex disorder thus revealed varied range of novel mutations in the mtDNA which were not reported earlier. We report here for the first time the novel mutations in the pathogenesis of T2DM and CAD among the South Indian population which requires more in depth analysis with more number of cases and controls. Although the functional analysis could not be performed, the in silico analysis of several mutations observed in our study revealed that these mutations affect the protein coding regions, which might lead to defective mitochondrial functions and so cause serious complications in the pathogenesis of the disease.

ACKNOWLEDGMENTS
We thank the patients who participated in this study.

COMPETING INTERESTS
Authors have declared that no competing interests exist.