Identical mitochondrial somatic mutations unique to chronic periodontitis and coronary artery disease.

CONTEXT: The inflammatory processes involved in chronic periodontitis and coronary artery diseases (CADs) are similar and produce reactive oxygen species that may result in similar somatic mutations in mitochondrial deoxyribonucleic acid (mtDNA). AIMS: The aims of the present study were to identify somatic mtDNA mutations in periodontal and cardiac tissues from subjects undergoing coronary artery bypass surgery and determine what fraction was identical and unique to these tissues. SETTINGS AND
DESIGN: The study population consisted of 30 chronic periodontitis subjects who underwent coronary artery surgery after an angiogram had indicated CAD.
MATERIALS AND METHODS: Gingival tissue samples were taken from the site with deepest probing depth; coronary artery tissue samples were taken during the coronary artery bypass grafting procedures, and blood samples were drawn during this surgical procedure. These samples were stored under aseptic conditions and later transported for mtDNA analysis. STATISTICAL ANALYSIS USED: Complete mtDNA sequences were obtained and aligned with the revised Cambridge reference sequence (NC_012920) using sequence analysis and auto assembler tools.
RESULTS: Among the complete mtDNA sequences, a total of 162 variations were spread across the whole mitochondrial genome and present only in the coronary artery and the gingival tissue samples but not in the blood samples. Among the 162 variations, 12 were novel and four of the 12 novel variations were found in mitochondrial NADH dehydrogenase subunit 5 complex I gene (33.3%).
CONCLUSIONS: Analysis of mtDNA mutations indicated 162 variants unique to periodontitis and CAD. Of these, 12 were novel and may have resulted from destructive oxidative forces common to these two diseases.

INTRODUCTION

Periodontal diseases are highly prevalent inflammatory disorders that cause tissue damage and loss of teeth as a result of complex interactions between pathogenic bacteria and the host's immune response.[123] Common forms of periodontal diseases have been associated with cardiovascular diseases, stroke, adverse pregnancy outcomes, pulmonary diseases, and diabetes,[34] but causal relations have not been established.[2] Despite their different etiologies, periodontitis, and cardiovascular diseases may share similar pathogenic mechanisms[5] and common risk factors. Several short-term studies have reported that treatment of periodontal diseases reduces the concentrations of inflammatory markers in blood.[6789]

Mitochondria are unique organelles that constantly metabolize oxygen and produce reactive oxygen species (ROS) as a by-product.[8] ROS have been implicated in the connective tissue damage in inflammatory diseases such as periodontitis, neuronal degeneration, cardiovascular disease and aging.[89101112] One possibility is that periodontitis and atherosclerosis are associated with ROS production by polymorphonuclear leukocytes during inflammation. Overproduction of ROS increases oxidative stress resulting in the production of free oxygen radical groups that damage lipids, proteins, and deoxyribonucleic acid (DNA) molecules[10] in the affected tissues of both diseases.[1112]

The aims of the present study were to identify somatic mitochondrial DNA (mtDNA) mutations in periodontal and coronary artery tissues from subjects undergoing coronary artery bypass surgery, and determine what fraction was identical and unique to these tissues.

MATERIALS AND METHODS

This study was planned and conceived by the Department of Periodontics, SVS Institute of Dental Sciences, Mahabubnagar and the patient pool was selected from Yashoda Super Specialty Hospital (YSSH), Hyderabad. The study samples were collected during the period of August 2011 to May 2012.

Sample size calculation and study population

A minimum sample size of 29 subjects was required to identify a minimum of 15 novel mutations across three tissues (gingiva, blood and coronary artery tissue) at α = 0.05 with expected variance of 0.8 for having ß = 0.1.[11] The study population consisted of 30 chronic periodontitis subjects who were advised to undergo cardiac surgeries for coronary artery disease (CAD) after diagnosis from an angiogram. Patients were diagnosed as having chronic periodontitis when there was a clinical evidence of probing depth or CAL ≥5 mm in at least 2 sites in each of the 4 quadrants with 30% or more of the dentition showing bleeding on probing (BOP) at baseline examination. Approval from the Institutional Ethical Committee, SVSIDS (SVS number 109007) and Institutional Review Board, YSSH were obtained for the required clinical research. The nature and purpose of the study were explained to the subjects, and written consent was taken.

Sample collection

Gingival tissue

Subjects were screened for BOP (Ainamo and Bay's Index)[6] and measurement of pocket depths was done. The gingival samples were taken from sites having probing pocket depth ≥5 mm. An internal bevel incision was made at the site of the pocket, 0.5 mm from the gingival margin along with the pocket lining and the obtained sample was fixed in 70% alcohol and stored at −4°C.

Coronary artery tissue

A coronary artery tissue sample was collected from the atherosclerotic block in CAD patients. Also, a blood sample was also collected from the subject's forearm during the cardiac surgery. The cardiac sample was fixed in 70% alcohol and stored along with the blood at −4°C and +4°C respectively.

Blood sample processing[13]

To the blood sample, 4 volumes of reagent A (Tris HCl + sucrose + MgCl2 + Triton X) was added in a polypropylene tube. It was mixed gently till the solution became clear. It was centrifuged at 2500 rpm for 5 min to obtain a pellet, free from red blood cells (RBCs). The supernatant containing lysed RBCs was then discarded carefully. The pellet was disturbed thoroughly and half the volume (as that of blood sample) of reagent B (Tris HCL + NaCl + Na-EDTA) was added and mixed thoroughly and gently by inverting for 3–4 min till the solution became viscous. Then reagent C (1/4th volume of reagent B) was added, mixed gently for 3–4 min and then centrifuged at 2500–3000 rpm for 7–8 min to separate into 3 layers viz., aqueous layer, protein layer and solvent layer. An equal volume of chloroform was added to the supernatant, mixed gently for a minute and centrifuged at 2500 rpm for 5 min. The aqueous phase was transferred to a fresh tube and 2 volumes of chilled absolute alcohol was added and mixed gently to precipitate the DNA. The DNA lump was spooled into a fresh Eppendorf tube and washed twice with 70% alcohol and a short spin was given to remove the alcohol. The pellet was dried properly and ensured that whole alcohol was dried off. The pellet was dissolved in 50–100 µl of TE (Tris HCl + EDTA) and incubated at 55°C for 45 min to enhance the dissolution. The DNA samples were stored at −4°C.

Coronary artery and gingival tissue processing[13]

The tissue was minced into small pieces with a sterile scalpel and any adherent adipose tissue was separated out. The tissue pieces were transferred into an autoclaved porcelain mortar and pestle and frozen by adding liquid nitrogen. The tissues were ground thoroughly with the pestle with frequent additions of liquid nitrogen. Then, the tissue homogenate was transferred to a sterile 35 ml tube, and the liquid nitrogen was allowed to evaporate. Lysis buffer-ST (Tris HCl + EDTA + NaCl, 1.0 ml for every 500 mg of tissue) was added along with 150 mg/ml proteinase-K (and SDS) to bring the final concentration to 2%. After incubation for 12–16 h, the lysate was transferred to an autoclaved 100 ml conical flask and an equal volume (~10 ml) of Tris-saturated phenol (pH 8.0) was added and mixed gently for 10 min. The lysate was transferred into 50 ml centrifuge tube and centrifuged for 10 min at 10,000 rpm at 15°C. The supernatant was collected into a 100 ml conical flask and half of the volume of Tris-saturated phenol and chloroform: Isoamyl alcohol (24:1) was added and mixed gently for 10 min. The lysate was transferred to the centrifuge tube and centrifuged for 10 min at 10,000 rpm at 15°C. The supernatant was collected into a 100 ml conical flask and equal volume (~10 ml) of chloroform: Isoamyl alcohol (24:1) was added, mixed gently for 10 min, and transferred into the centrifuge tube and centrifuged for 10 min at 10,000 rpm at 15°C. After centrifuging, the lysate was collected into a fresh 50 ml tube, and 1/20th volume of 3M sodium acetate (pH 5.5) was added with equal volumes of isopropyl alcohol. The DNA was precipitated by slowly swirling the tube, thrice with 70% ethanol to remove excess salt, vacuum dried and dissolved in appropriate volume of TE.

Deoxyribonucleic acid preparation for polymerase chain reaction

Samples were prepared using a commercially available kit PrepMan® (Applied Biosystems, Foster City, CA) to provide the highest yield, concentration, and purity of mtDNA from the blood, coronary artery and gingival tissue samples.[13]

Analysis of complete mitochondrial genome

mtDNA was extracted from the blood, coronary artery and gingival tissue samples using the standard protocol.[14] Complete mitochondrial genome was amplified using 24 sets of overlapping primers.[15] The amplicons were directly sequenced using the ABI BigDye Terminator cycle sequencing kit, (Applied Biosystems, Foster City, CA, USA) and analyzed using ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Complete mtDNA sequences were aligned with the revised Cambridge reference sequence (NC_012920)[15] using sequence analysis and auto assembler tools. All the mismatched nucleotide sequences were carefully noted, compared with controls and searched in the human mitochondrial genome databases such as MITOMAP (http://www.mitomap.org) and mitochondrial database (mtDB) (http://www.genpat.uu.se/mtDB) for their significance. Each mtDNA haplogroup was constructed to find out if anyone haplogroup was associated with both diseases.

RESULTS

The demographics of subjects with chronic periodontitis are shown in Table 1. The age ranged between 39 and 84 years with the mean age being 59.08 ± 20.54. The pocket depths ranged between 3 and 7 mm and CAL ranged between 3 and 8 mm with mean being 4.4 ± 1.98 and 5.02 ± 1.34, respectively. The bleeding index showed a range from 94% to 100% with the mean being 94.2 ± 1.82.

Table 1

Consolidated table showing the periodontal status of all the subjects in the current study

Analysis of the mitochondrial genome

Complete mtDNA analysis was carried out for the DNA isolated from 30 blood samples and 29 out of 30 gingival tissue samples and 28 of the 30 coronary artery tissues samples as the remaining three tissue samples did not yield enough DNA.

This study focused only on those mtDNA mutations common to coronary artery tissue samples and gingiva but not in blood. A total of 162 variations were observed across the mitochondrial genome. These variations were somatic mutations present only in the coronary artery and gingival tissues, not in the blood samples. When compared to the MITOMAP database,[11] 150 are known to occur naturally in an Asian/Indian population but 12 were novel and not reported previously. Of the 150 known variations, 23 were in the D loop (G189G and C16189), 22 were in the 12S rRNA region (C1243C), 20 were found in the ND2 region (A5640A), 44 were found in the tRNA Glu region (G14693G) and 41 were found in the tRNA Thr region (G15924G and A15928A).

Of 12 novel mutations, one mutation was (G2120A) in the 16S rRNA and one mutation (C8346T) was in the lysine-tRNA. In addition, 2 mutations were missense that included A7796G in mitochondrial cytochrome C oxidase II subunit and G8115R (heteroplasmic) which altered the amino acids from isoleucine to valine, and glycine to glutamic acid, respectively. The remaining 8 mutations were silent/synomous. Of these 8 mutations, two were in mitochondrial cytochrome c oxidase subunit I gene (T6965G; T7302C), one in mitochondrial adenosine triphosphate (ATP) synthase F0 subunit 6 (MT-ATP6) gene (T8538C), one in mitochondrial NADH dehydrogenase subunit 4 (MT-ND4) gene (A12076G) and the remaining four in mitochondrial NADH dehydrogenase subunit 5 (MT-ND5) gene (C12498T; A13866G; C13950T; C13771T). Four of the 12 novel variations were found in MT-ND5 gene. Thus, MT-ND5 gene had more novel variants compared to other genes [Figures 1 and 2].

Figure 1

Illustration showing the location-based number of all 12 novel mutations identified in this study in the human mitochondrial deoxyribonucleic acid. The mitochondrial NADH dehydrogenase subunit 5 gene had more novel variants compared to other genes. (LSP = Light strand promoter, OH = Origin of leading strand replication, OL = Origin of lagging strand replication)

Figure 2

The sequence electropherogram of mitochondrial deoxyribonucleic acid mutations observed in chronic periodontitis patients. (a) The upper panel showing (arrow) the wild-type nucleotide “G” at the position 2120 in 16 S rRNA gene, while the lower panel showing the mutant allele “A”; (b) The upper panel showing (arrow) the wild type nucleotide “A” at the position 7796 in mitochondrial cytochrome c oxidase subunit II gene, while the middle and the lower panels show the mutant allele “G” that changes amino acid from isoleucine to valine; (c) The upper panel showing (arrow) the wild-type nucleotide “G” at the position 8115 in mitochondrial cytochrome c oxidase subunit II gene, while the middle and the lower panels show the mutant allele “R” (G/A) (heteroplasmy) which changes amino acid from glycine to glutamic acid. All other 159 single nucleotide point mutations on mitochondrial deoxyribonucleic acid were likewise identified

DISCUSSION

The role of neutrophil-produced-ROS in periodontal tissue destruction was established by Chapple and Matthews.[161718] Periodontally inflamed tissues have increased ROS levels that induce the mtDNA mutations reported in this study.[111617] Because these mutations appear restricted to gingival and coronary artery tissues, they may denote somatic mutations that are caused by the chronic inflammatory process altering mitochondrial energy metabolism in these very different diseases.[11] Chronic periodontal and coronary artery tissues possessed 162 common mutations, of which 12 have not been previously reported for either disease.

There are few studies on the prevalence, impact and possible inter-tissue relationship of mtDNA mutations in gingival tissues.[1119] Mutations at the ATP synthase F0 subunit-6 gene and NADH dehydrogenase subunit-4 gene also resemble those in the present study and associate with a large number of diseases.[1819] Mutations of the MT-ND5 gene of complex I usually associate with neurological disorders,[11] but the 4 novel mutations observed in this region in this study appear silent, and were not previously reported in either periodontal or coronary artery disease. Some of the nonnovel mtDNA mutations reported here associate with leukemia, hearing loss and multiple sclerosis in other populations, but not the Indian population where these mutations are common and symptomless.[20]

It is common for apparently random mtDNA mutations to be observed in two or more unrelated tissues in humans,[21] and some may be a product of the exclusively maternal inheritance of mitochondria[2122] which was not considered. Most studies of mtDNA mutations in periodontal disease are incidence or case-control studies and only a few[1119] examined mtDNA mutations across unrelated diseased sites as in this study. In conclusion, the restriction of 12 common, unique mtDNA mutations to gingival and cardiac tissues may result from common tissue-destructive actions that could aid our understanding of how these two diseases are mechanistically related.

Financial support and sponsorship

The study was supported primarily by the authors’ institution and partly by the authors themselves.

Conflicts of interest

There are no conflicts of interest.