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,
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.
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.
11.
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.
19.
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
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).
43.
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
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