This document provides information on mitochondria and mitochondrial DNA. It discusses: 1) The structure and functions of mitochondria, including that they contain DNA (mtDNA) and encode proteins involved in oxidative phosphorylation. 2) The properties of mtDNA, including that it is circular, double-stranded, and encodes 37 genes including tRNAs and rRNAs. 3) Mitochondrial mutations and diseases, noting that mutations in mtDNA or nuclear genes can cause disorders by disrupting oxidative phosphorylation or mtDNA maintenance mechanisms. Common mtDNA disorders discussed include MELAS, MERRF, and LHON. 4) Methods for diagnosing mitochondrial disorders, including biochemical tests, imaging,

1. DR.N.E.NANDHINI
I YEAR PG.

2. MITOCHONDRIA
 Cellular organelle of eukaryotic cells.
 Two layers with internal matrix mitosol.
 Components of ETC and oxidative phosporylation
present in inner membrane.
 FUNCTIONS:
 1) cell respiration
 2) lipids synthesis
 3) storage and transport of ATP
 4) brown fat mitochondria
 5) elongation of fatty acids.

3. MITOCHONDRIAL DNA(mtDNA)
 Small circular chromosome in mitochondria.
 mtDNA containing regions- nucleoids.
 2-3 or upto 6.
 circumference : 5 microns.
 Either attached to membrane or not.

4. STRUCTURE
 Circular.
 Double stranded,
 Varies by nucleotide composition.
 H STRAND- Heavy strand.
 guanine rich.
 L STRAND- Light strand.
 cytosine rich.

5. GENES ENCODED
 37 genes.
 13- proteins for respiratory chain.
 22- tRNA
 2- rRNA.
 H-Strand encodes 28 genes.
 L- Strand encodes 9 genes.

7. RESPIRATORY PROTEINS IN mtDNA
COMPLEXES TOTAL
SUBUNITS
ENCODED BY
mtDNA
NADH dehydrogenase >25 7
Succinate
dehydrogenase
4 0
Ubiquinone cyto-
oxidase
9 1
Cyt –c-oxidase 13 3
ATP Synthase 12 2
>63 13

8. GENERAL PROPERTIES
 Rapid rate of denaturation.
 Shorter.
 More guanine and cytosine.
 Has DNA Polymerase.
 Flow of DNA from nucleus to mitochondria and
viceversa- PROMISCUS DNA.

9. TRANSCRIPTION
 Transcribes continuously and produce polycistronic
RNA.
 H-Strand- clockwise direction.
 L- Strand- anticlockwise direction.
 ATPase 6,8 and ND 4,4L - Overlapping genes.
 tRNA acquire specific L shape get recognised and
cleaved.

10. REPLICATION
 Unidirectional.
 D- LOOP- Initiation of replication.
 DNA Polymerase gamma complex.
 3 units.
 POL G and 2 subunits POL G 2.

12. DIFFERENCE BETWEEN UNIVERSAL GENETIC
CODE AND mt GENETIC CODE
UNIVERSAL
GENETIC CODE
MITOCHONDRIAL
GENETIC CODE
NO OF tRNA 55 22
TERMINATION
CODON
UAA, UGA, UAG UAA, UAG,AGA,AGG
UGA TERMINATION
CODON
TRYPTOPHAN
AGA,AGG ARGININE TERMINATION
CODON

13. MITOCHONDRIAL MUTATIONS
 Diseases due to mutation show distinctive pattern of
inheritance due to
1) REPLICATIVE SEGREGATION
2) HOMOPLASMY AND
HETEROPLASMY.
3) MATERNAL INHERITANCE

14. REPLICATIVE SEGREGATION
 During cell division, multiple copies of mtDNA in
mitochondria replicate and sort randomly between the
new cells.
 These new mitochondria are then randomly distribute to
daughter cells.
 Lack of tightly controlled segregation is a unique feature.
.
.

15. HOMOPLASMY AND HETEROPLASMY
 HOMOPLASMY: The daughter cell may receive
mitochondria with either pure population of normal
mtDNA or mutant mtDNA.
 HETEROPLASMY: The daughter cell will receive
mitochondria as a mixture of both normal and mutant
DNA.

16. MATERNAL INHERITANCE
 Inherited solely from mother.
 Egg – 2,00,000 molecules of mtDNA.
 Sperm- 5 molecules of mtDNA.
 Degradation in male genital tract.
 Failure to enter egg.
 Used for propelling sperm.

17. WHY MUTATIONS ARE HIGH???
 10-17 fold increased rate when compared to nuclear DNA
mutation rate.
 Due to 1) lack of protective histones.
 2) insufficient repair mechanism.
 3) close proximity to ETC so more exposure to
free radicals.

18. MITOCHONDRIAL BOTTLE NECK
 Process of restriction and subsequent amplification of
mtDNA during oogenesis is known as MITOCHONDRIAL
GENETIC BOTTLE NECK EFFECT.
 Variability in percentage of mutant mtDNA in offsprings
arises from sampling of only a subset of mtDNA during
oogenesis.

20. TYPES OF MUTATIONS
 CLASS I: Disorders of nuclear genes of mitochondria
 CLASS II: mtDNA mutations.

21. DEFECTS OF MITOCHONDRIAL
DNA
DEFECTS IN NUCLEAR DNA
AFFECTING MITOCHONDRIAL DNA
OR ENZYME COMPLEXES
PEO/multisystem with PEO
KSS
Pearson syndrome/KSS
MELAS
MERRF
MiMyCa
NARP/MILS
LHON
Diabetes, optic atrophy, deafness
Tubulopathy, diabetes, ataxia
Sideroblastic anemia
Autosomal dominant / recessive PEO
MNGIE
Leigh syndrome
Encephalopathy/cardiomyopathy
GRACILE syndrome
Hypertrophic cardiomyopathy
Myopathy
Optic atrophy, deafness, neuropathy
GRACILE, growth retardation, amino aciduria, cholestasis, iron overload, lactic acidosis,
and early death; KSS, Kearns-Sayre syndrome; LHON, Leber hereditary optic neuropathy;
MELAS, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes;
MERRF, myoclonic epilepsy with ragged-red fibers; MILS, maternally inherited Leigh
syndrome; MiMyCa, mitochondrial myopathy and cardiomyopathy; MNGIE,
myoneurogastrointestinal encephalopathy; NARP, neuropathy, ataxia, and retinitis
pigmentosa; PEO, progressive external ophthalmoplegia

22. CLASS I: DISORDERS OF NUCLEAR
GENES
 This includes
1) OXPHOS Diseases(Leigh syndrome and
paraganglioma)
2) defects in the nucleus encoded proteins for
mitochondrial integrity(progressive external ophthalmoplegia
(PEO) and mitochondrial neurogastrointestinal
encephalomyopathy (MNGIE) syndrome)
3) Mitochondrial disorders with secondary effects on
the OXPHOS system (Friedreich ataxia and hereditary spastic
paraplegia).

23. NUCLEAR DEFECTS OF OXPHOS
SYSTEM
RESPIRATORY CHAIN
COMPLEX
DISORDER FEATURES
COMPLEX I LEIGH SYNDROME b/l leisons of basal
ganglia, brain stem
spinalcord and
thalamus.
Characterised by
psychomotor
retardation, brain stem
dysfunction
COMPLEX II HEREDITARY
PARAGANGLIOMA
Benign highly vascular
tumours of
paraganglioma in hear
and neck

24. NUCLEAR DEFECTS OF OXPHOS
SYSTEM
RESPIRATORY CHAIN
COMPLEX
DISORDER FEATURES
COMPLEX III MYOPATHY,
TUBULOPATHY,
HEPATOPATHY
COMPLEX IV LEIGH SYNDROME,
HYPERTROPHIC
CARDIOMYOPATHY,
KETOACIDOTIC COMA
Ketoacidosis,
cardiomyopathy
COMPLEX V NARP SYNDROME,
LEIGH SYNDRME
Neuropathy, ataxia,
retinitis pigmentosa

25. NUCLEAR DEFECTS IN MITOCHONDRIAL
PROTEINS FOR mtDNA INTEGRITY
 The factors involved in mtDNA maintenance are all encoded by
nuclear genes, and transported into the mitochondria.
 They include those involved directly in DNA processing, such as the
mtDNA polymerase γ (POLG1), a helicase, a primase, and a ligase.
 Two human disease groups (progressive external ophthalmoplegia,
PEO and MNGIE) result from these disturbed mtDNA maintenance
mechanisms.

26. PROGRESSIVE EXTERNAL
OPHTHALMOPLEGIA
 PEO- Two types.
 autosomal dominant trait (adPEO), or more rarely as an
autosomal recessive trait (arPEO).
 Onset- usually between 18 and 40 years of age,.
 CPEO is characterised by a progressive paralysis of the eye muscles
leading to impaired eye movement and ptosis
 The combination of arPEO, severe gastrointestinal dysmotility,
peripheral neuropathy, cachexia,diffuse leukoencephalopathy on
brain MRI) identifies the mitochondrial neurogastrointestinal
encephalomyopathy (MNGIE)

27. mtDNA MUTATIONS
 It has been identified in mtDNA:
 (1) missense mutations in the coding regions of genes
that alter the activity of an oxidative phosphorylation
protein;
 (2) point mutations in tRNA or rRNA genes that impair
mitochondrial protein synthesis;
 (3) Deletions or duplications of the mtDNA molecule.
They are generally somatic in origin, although a small
proportion is inherited, in some diseases

28. mtDNA DISORDERS
 MERRF (Myoclonic Epilepsy with Ragged Red
Fibres)
 MELAS (Myopathy, Epilepsy, Lactic acidosis,
Stroke-like episodes)
 LHON (Leber’s Hereditary Optic neuropathy)
 Kearn-Sayre syndrome (eye problems, heart
block, ataxia and loss of coordination

29. MELAS
 ONSET: Usually early adolescence.
 CLINICAL FEATURES: growth retardation, deafness
 Recurrent stroke like episodes hemianopia,
hemiplegia.
 Dementia, ataxia
 Recurrent lactic acidosis with nausea and vomitting.
 Diabetes , myopathy.

30. DIAGNOSIS
Imaging Grey and white matter involved.
Calcifications in globus pallidus
CSF Lactate elevated
Muscle Biopsy Ragged red fibres
SDH fibres positive
COX Fibres negative
Genetics 80% people has A3243G mutations
in t RNA for leucine

32. MERRF
 CLINICAL FEATURES:
SEIZURES- Focal/ generalised
ataxia , myopathy
deafness, dementia,ptosis
optic atropy
cervical lipomas

33. DIAGNOSIS
Imaging Non -specific
CSF Lactate elevated
Muscle Biopsy Ragged red fibres
SDH fibres positive
COX Fibres positive
Genetics A8344G mutations in tRNA
for lysine

34. KSS, Kearns-Sayre syndrome
 Characterised by chronic progressive external
ophthalmoplegia(CPEO) with complete heart block
 ONSET: Any time from adolescence.
 CLINICAL FEATURES: Ptosis
 external ophthalmoplegia
 proximal myopathy.

35. DIAGNOSIS
EMG Non -specific
CSF Lactate Elevated initially and
sustained elevation on
exercise
Muscle Biopsy Ragged red fibres
COX Fibres negative

36. LHON
 Leber’s Hereditary Optic neuropathy
 ONSET: Early 20’s
 Acute or subacute bilateral painless visual loss
 Severe and permanent loss
 Mutations : G1778A in ND 4 - 50-70%
 G3460A in ND 4 – 15-20%
 T4484C in ND 6 – 20-30%

37. TOXIN INDUCED mtDNA ABNORMALITY
 New exogenous cause- HIV Infection and anti retroviral
treatment.
 HIV Infection: decreases mtDNA
abnormalities in OXPHOS system
oxidative damage
 ART treatment: inhibits DNA POL G COMPLEX
associated with myopathy, lipodystropy,
hepatic failure, lactic acidosis.

38. METHODS OF DIAGNOSIS
Noninvasive screening tests
 An electrocardiogram or echocardiogram may demonstrate
cardiomyopathy and cardiac conduction defects, the most
common cardiac features of mitochondrial disorders.
 Ophthalmologic examination may disclose the presence of
retinal pigmentary abnormalities or optic atrophy. An
electroretinogram (ERG) may be indicated.

39. BIOCHEMICAL STUDIES
 There is no one specific screening test.
 Elevated lactate is suggestive, but not specific, for
mitochondrial disorders.
 CSF lactate may be elevated.
 Several laboratory studies such as serum lactate, pyruvate, plasma
amino acids, complete blood count, electrolytes, carnitine,
acylcarnitine profile, ammonia, and creatine phosphokinase (CPK).
 Serum CPK values are usually normal in mitochondrial disorders
except in mitochondrial depletion


40. ELECTROPHYSIOLOGIC STUDIES
 Electroencephalogram (EEG) results may be
normal, show evidence of seizures, or show
generalized slow waves consistent with an
encephalopathy.
 Polyspike and wave discharges -MELAS and
MERRF.

41. BRAIN MAGNETIC RESONANCE IMAGING
 Magnetic resonance imaging and spectroscopy are important tools in
the diagnosis of mitochondrial disorder.
 Brain atrophy is common in children with mitochondrial disease.
 Basal ganglia calcification are common in KSS and MELAS.
 Diffuse signal abnormalities of the white matter are characteristic of
KSS and myoneurogastrointestinal encephalopathy (MNGIE).

42. MUSCLE BIOPSY
 The hallmark of mitochondrial dysfunction is abnormal
mitochondrial proliferation, seen as Ragged Red Fiber
(RRF) with modified Gomori trichrome staining.
 The mitochondrial aggregates cause the contour of
musclr fibre irregular - RAGGED
 These fibers also stain strongly for succinate
dehydrogenase (SDH), and cytochrome oxidase (COX).

44. MITOCHONDRIAL DNAANALYSIS
 Genetic analysis is needed for genetic counseling.
 If the patient fits a specific phenotype (ie LHON, MERRF, MELAS) a
blood / muscle test for a point mutation may be positive.
 Mitochondrial DNA length mutations (common deletion) are best
detected by Southern blot analysis in total mtDNA extracts from
blood lymphocytes.
 In some patients the studies are negative, despite high clinical
suspicion

45. GENERAL PRINCIPLES OF
TREATMENT
 Treat Underlying Neurologic Issues
 Identify and Treat Nutritional Deficiencies
 Avoid Metabolic Stressors

46. TREAT UNDERLYING NEUROLOGIC
ISSUES
 Seizures (antiepileptic drugs, avoid valproic acid).
 Spasticity (baclofen, botulinum toxin
 Dystonia (diazepam, botulinum toxin ,trihexyphenidyl).
 Headache (acute: nonsteroidal anti-inflammatory drugs
and acetaminophen; avoid aspirin and triptans in
MELAS, chronic: amitriptyline, calcium blockers,
riboflavin, coenzyme Q10, -lipoic acid).

47. IDENTIFY AND TREAT
NUTRITIONAL DEFICIENCIES
 Identify and treat deficiencies in vitamins (vitamins A,
B12, E, D), minerals (iron, zinc, selenium, calcium,
magnesium), and protein calorie (albumin).
 Mitochondrial cocktail

48. WHAT IS "THE MITO COCKTAIL"?
 Referring to the combination of vitamins and
supplements used as therapies in the treatment and
management of mitochondrial disease and
mitochondrial dysfunction, the "Mito Cocktail" is
unique to every patient.

49. MITO COCKTAIL
 Includes
 CO Q 10- Ubiquonone, ubiquinol, ibedenone
 Vitamin B Complex
 Vitamin C, E
 L-Carnitine
 N acetyl cysteine

50. AVOID METABOLIC STRESSORS
 Extremes of heat and cold are not well tolerated. Fever
should be treated with acetaminophen (10 mg/kg every 4
hours to 15 mg/kg every 4 hours).
 Patients should avoid unaccustomed strenuous exercise.
They should not exercise in the fasted state or with a
concomitant illness.
 Avoid prolonged (greater than 12 hours) fasting

51. REFERENCES
 Cell biology and molecular biology- Ajay Paul
 Cell biology, organelle structure and functions- David
E Sadva
 Textbook of biochemistry- Devlin
 Textbook of biochemistry- Leningher