“Possible Role Of Mitochondria DNA Mutations In Chronic Periodontitis”- Guest lecture as a part of AP State Periodontists Meet, 13/4/2010, Sri Sai College of Dental Surgery, Vikarabad, India.

1. Dr R Viswa Chandra MDS;DNB
Reader
Department of Periodontics
SVS Institute of Dental Sciences
Mahabubnagar AP

2. BACKGROUND
• Periodontal diseases are inflammatory disorders that
give rise to tissue damage and loss, as a result of
complex interactions between pathogenic bacteria and
the host’s immune response.
• There is an increasing body of evidence available to
implicate reactive oxygen species (ROS) in the
pathogenesis of a variety of inflammatory disorders, of
which periodontal disease is no exception.

3. • In the human periodontium, the neutrophils are the
primary producers of ROS namely hydrogen peroxide
(H2O2), hypochlorous acid (HOCl) and singlet oxygen
(1O2).
• This phenomenon is called as the “neutrophil mediated
tissue injury*” wherein it has been proved that the
neutrophil has the potential to destroy
1. Gingival epithelium
2. Glycosaminoglycans of the gingival connective tissue
3. mtDNA of the Gingival and periodontal tissues.
*Deguchi et al. J Periodontal Res 1990; 25:293-299.

4. What is mtDNA?
• Although most DNA is packaged in chromosomes within
the nucleus, mitochondria also have a small amount of
their own DNA. This genetic material is known as
mitochondrial DNA or mtDNA.
• Mitochondrial DNA contains 37 genes, all of which are
essential for normal mitochondrial function. Thirteen of
these genes provide instructions for making enzymes
involved in oxidative phosphorylation (OXPHOS). The
remaining genes provide instructions for making
molecules called transfer RNAs (tRNAs) and ribosomal
RNAs (rRNAs).

5. 1.ATP synthase: ATP6, ATP8
2.Cytochrome c oxidase: CO1, CO2, CO3, CYB
3.NADH dehydrogenase: ND1, ND2, ND3, ND4, ND4L, ND5, ND6
4.12S, 16S: RNR1, RNR2
5.tRNA: TA, TC, TD, TE, TF, TG, TH, TI, TK, TL1, TL2, TM, TN, TP, TQ, TR, TS1,
TS2, TT, TV, TW, TY, 1X

6. mtDNA vs Nuclear DNA
• In each mitochondrion, an identical loop of DNA about
16,000 base pairs long containing 37 genes. In contrast,
nuclear DNA consists of three billion base pairs and an
estimated 70,000 genes.
• The only extra chromosomal DNA in human and animal
cells and is influenced by local cells.
• Replicates faster than nuclear DNA without efficient DNA
repair systems and is continually exposed to high levels
of ROS generated from the electron transport chain of
mitochondria.
• Studies seem to suggest that Mitochondrial DNA
damage is more extensive and persists longer than
nuclear DNA damage in humans.

7. INHERITANCE PATTERNS
The offspring inherit the Mothers mtDNA.
Mechanisms for this include simple dilution (an egg contains 100,000 to
1,000,000 mtDNA molecules, whereas a sperm contains only 100 to 1000)

8. Activation of
neutrophils
ROS production
Damage to mtDNA
Aberrant /increased ROS
production
TISSUE DAMAGE
•Collagen
•GAG

9. ASSESSMENT OF mtDNA AND MITOCHONDRIAL FUNCTION
Analysis of complete
mitochondrial genome
Analysis of Mitochondrial
morphology
Cytofluorometric Measurement of
Mitochondrial Membrane
Potential
Cytofluorometric Measurement of
ROS
Respirometry
Western blot analysis

10. STUDY PROTOCOL & RESULTS
CP Group C Group
N 30 30
Age 35.6±11.45 35.4±10.9
GI 2.3±0.66* 1.22±0.78
PI 2.06±0.64* 1.01±0.69
PD 5.39±1.01* 2.1±0.91
CAL 6.35±1.00* 0.00±0.00
Gingival sample during periodontal
procedures on which the following
tests were performed:
•Analysis of complete
mitochondrial genome
•Cytofluorometric Measurement
of Mitochondrial Membrane
Potential
•Cytofluorometric Measurement
of ROS
•Respirometry
•Western blot analysis

11. C E G
Among the 264 variations, 14 were novel mutations, which were not present in the control
group, neither in world population databases nor in blood samples of chronic periodontitis
groups.
Patient No
Nucleotide
Position
Reference
Base Change
(Blood)
Base Change
(Tissue)
Gene
Amino acid
change
Mitomap/
Significance
Frequency
CP12,CP22,CP26,CP30 189 A A G D-loop -
Leukemia
(Associated) 4
CP22,CP26,CP30 1243 T T C 12S rRNA -
Hear Impairment
(Associated) 3
CP17 2120 G G A 16S rRNA - Novel 1
CP1 2233 A ins 16S rRNA - Novel 1
CP7 4234 A A T ND1 Thr to Ser Novel 1
CP6,CP12,CP22,CP28,CP30 5640 G G A ND2 Ala to Thr AD;PD (Associated) 5
CP5 6965 T T G COI Thr (Syn) Novel 1
CP25 7302 T T C COI Leu (Syn) Novel 1
CP15 7796 A A G COII IIe to Val Novel 1
CP22 8115 G G G/A COII Gly to Glu Novel 1
CP10 8346 C C T tRNA Lys - Novel 1
CP25 8538 T T C ATPase6 Asn (Syn) Novel 1
CP11,CP13,CP14,CP16,CP19,CP20,CP24 12308 A A G tRNA Leu -
CPEO/Stroke/CM
(unclear) 7
CP10 12498 C C T ND5 Phe (Syn) Novel 1
CP3 13203 A A G ND5 Ala (Syn) Novel 1
CP19 13866 A A G ND5 Lys (Syn) Novel 1
CP4 13950 C C T ND5 Pro (Syn) Novel 1
CP17 13971 C C T ND5 Ser (Syn) Novel 1
CP22 14693 A A G tRNA Glu -
Hearing loss
(Associated) 1
CP27 15924 A A G tRNA Thr - LIMM (Associated) 1
CP7 15928 G G A tRNA Thr -
Multiple Sclerosis
(Associated) 1
CP5,CP9,CP13,CP14,CP20,CP28 16189 T T C D-loop -
Type 2 Diabetes;
Cardiomyopathy 6

12. 489
10400
14783
15043
M5
M35 M
16129
1888
M3
709
3921
12477
14323
M5a
M2b
M25
M2
12007
M18
8701
9540
10398
10873
I1
U
16126
4580
482
246
198
12498
15942
16318T
152
182
195
522-523d
1453
2831T
3630
5744
6647
9899
13254
14766
16169+C
16183
16189
16320
199
12561
482
5432
10670
15924
16093
15928
16304
195A
522-523d
15431
15259 250
573+CC
4529T
8251
10034
15924
16129
16391
12705
16223
6734
16311
2755
3384
7759
9449
13215
195
12285
16274
8594
10754
14544
16304
16524
R
U1a
11467
12308
12372
285
2218
4991
6026
7581
13104
14070
14364
15148
15954C
16183C
16189
16249
16051 U2
U2b
146
2706
5186T
12106
13194
15049
M30d
M30
M35a
447G
1780
8502
11083
15670
16274
16319
R8
5510
R6
508
3720
5390
5426
6045
6152
10876
13020
13734
15907
16129C
16362
16189
U2e
R8a
N
W
195
204
207
1243
3505
5460
8251
8994
11947
15884C
16292
16519
1406
13263
15784
W3
Evolutionary analysis of these patients showed diverse mtDNA
haplogroup background.
Gopalakrishna P, Govindaraj P, Vanniarajan A, Chandra RV, Reddy AA, Singh L , Thangaraj K.
International Poster Journal. 2008, Volume 10, Issue No 04.

13. A B
C D
Mitochondria with
abnormal cristae were
observed in two CP
patients, of these one
have swollen cristae and
another have less cristae.
Bizarre shaped
mitochondria were
observed in five CP
patients (Figure C).
Vacuolated and outer
membrane damaged
mitochondria were seen
in six and three
respectively (Figure D), in
which one patient shows
vacuolation with
concentric mitochondria
(Figure B).
Gopalakrishna P, Govindaraj P, Vanniarajan A, Chandra RV, Reddy AA, Singh L , Thangaraj K.
Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1777, Supplement 1: S77-S78.

14. *
Analysis of Mitochondrial Membrane Potential (ΔΨm)
Mitochondrial membrane potential is generated
by mitochondrial electron transport chain, which
drives a proton flow from matrix through inner
mitochondrial membrane to cytoplasm, thus
creating an electrochemical gradient. This
gradient is in turn responsible for the formation
of ATP.
CP* group has about 4-fold decrease in the
mitochondrial membrane potential compared to
C group (p>0.01). It is interesting to note that
the CP group which have either novel or/and
diseases associated mutations or both are
showing more decreased level of ΔΨm)
compared to remaining CP patients.

15. a
b
c
B
C
*
AAnalysis of ROS
Mitochondria are a significant source of ROS
producer in the cells which although plays an
important role as signaling molecules for various
cellular processes are also responsible for various
diseased conditions including ageing.
ROS analysis revealed that the CP group
increased by 18% ROS production when compared
to the C group

16. HSP 27
HSP 60
HSP 70
Actin
Complex IV
Absence of these proteins may result in improper import of cytosolic proteins
which can be responsible for defective respiratory complex synthesis.

17. Respiration
0
50
100
150
200
250
1 2 3 4 5 6 7
Time
O2conc.inpg/ml
Flux control
-2
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7
Time
O2con.inpg/ml
Respirometry analysis
Small changes in cellular respiration, minor alterations in respiratory control
ratios and minor differences in the respiratory effects of inhibitors may indicate
significant mitochondrial defects, severe injuries of mitochondrial problems of
mtDNA or decisive alterations in the stake of mitochondrial signaling cascade

18. DISCUSSION
CHANGING TRENDS ON THE ROLE OF GENETICS IN PERIODONTAL
DISEASE PATHOGENESIS

19. Ever since the basic and peculiar molecular characteristics
of the mitochondrial genetic system were discovered, the
number of mutations in mtDNA and its associated
diseases has grown spectacularly and generated a
group of conditions in what today could be called as
“MITOCHONDRIAL MEDICINE”
Miquel, 1998 and Chinnery et al ., 2000

20. • In addition to specific mutations, mtDNA can suffer other
types of damage such as the loss of part of the same
(deletions) or the addition of a new DNA fragment
(duplications) that, as in the previous cases, affects the
biogenesis of the Oxphos system and, therefore, ATP
synthesis.
• This type of mutations is often spontaneous, probably
caused by damage in nuclear genes that control mtDNA
replication.

21. • Interest in their study has grown enormously due to the
large number of patients diagnosed with these disorders
and to the fact that they appear at any life period. In
addition, many of these mutations are transmitted
through the maternal line which means that an
individual’s diagnosis can have implications for many
generations in one family.
• Since the first diseases caused by mtDNA damage were
described in 1988, over 150 mutations, 100 deletions
and around 50 specific mutations that are associated
with human diseases were found.

22. • Recent studies seem to suggest that oxidative
mitochondrial injury in the periodontal tissues may lead
to potential pro-inflammatory cytokine formation by the
affected cells.
• Inflamed gingival fibroblast from the persons with adult
periodontitis was highly susceptible to mitochondria and
caspase-dependent apoptosis induced by butyric acid
compared to the healthy gingival fibroblasts*.
Kurita-Ochiai T, Seto S, Suzuki N, Yamamoto M, Otsuka K, Abe K, Ochiai K. Butyric acid induces
apoptosis in inflamed fibroblasts. J Dent Res. 2008 Jan;87(1):51-5.

23. • Mitochondrial mutations are known to be associated with
a large number of diseases and Periodontitis can now be
added to this list.
• Very few studies on mtDNA mutations in periodontal
diseases.
• The only major study* suggested that 5-kb deletion of
mtDNA was observed in gingival tissue of the patient
with chronic periodontitis.
*Canakçi CF, Tatar A, Canakçi V, Cicek Y, Oztas S, Orbak R. New evidence of premature oxidative DNA damage:
mitochondrial DNA deletion in gingival tissue of patients with periodontitis. J Periodontol 2006; 77:1894-1900.

24. FUTURE DIRECTIONS
• EPIGENETICS- mtDNA mutations are evidences on how
local cells can cause direct mutations in DNA.
“Cells are not slaves to DNA
It can be the other way round too”
Strategies to combat ROS production and/or use anti-
mitochondrial antibodies to combat excessive ROS
production.
• A BETTER WAY TO EXPLAIN PERIODONTAL
MEDICINE?
• MATERNAL LINE OF INHERITANCE?

25. CONCLUSION
In conclusion, initial evidence seems to suggest that
mitochondrial DNA mutations may have a significant role
in the initiation and progression of periodontal disease.
The role of this vicious cycle where oxidative stress
leads to mitochondrial DNA mutations which can lead to
abnormalities and further oxidative stress in the
periodontal tissues is an exciting possibility which
requires further study.