2. Introduction
• Mitochondria are crucial to the flow of energy in
cells.
• Mitochondria presumably originated as parasites
that formed a symbiotic relationship with
eukaryotic cells more than 2 billion years ago, in
response to an increase in atmospheric oxygen.
3.
4. Primary cellular functions of mitochondria
Supply energy to cell in form of ATP
Generate and regulate reactive oxygen
species
Buffer cytosolic calcium ions
Regulate apoptosis through the
mitochondrial permeability transition pore
5. Serves as a cellular site for the following
metabolic pathways –
Electron transport chain
Tricarboxylic acid cycle or Krebs cycle
Beta oxidation of fatty acids
Gluconeogenesis
Urea Synthesis
8. • Mitochondria can’t be synthesized
de novo, so new mitochondria must arise from
existing mitochondria.
• At any point of time, mitochondria are in a
dynamic flux between fission and fusion.
11. MITOCHONDRIAL DNA
• Circular, double stranded, and composed of
heavy and light chains or strands
• Contains 16,569 bp
• Encodes 13 proteins
22 tRNA
2rRNA
12. MITOCHONDRIAL VS NUCLEAR
GENOME
• Mitochondrial genome has
. smaller number of genes
. higher copy number
. less effective repair mechanisms
. higher mutation rates
13. Peculiarities of Mitochondrial Genetics
Maternal inheritance
high copy number
heteroplasmy
bottleneck and segregation
threshold
16. HETEROPLASMY
• Cells contain hundreds of mitochondria , and
each mitochondria contains hundreds of mtDNA.
So cells contain thousands of copies of mtDNA.
• For the most parts ,their sequence will be
identical (homoplasmy )
17.
18. Mutation arises in mtDNA
Mixed population of wild- type and mutant mtDNA within a single cell
( heteroplasmy )
Heteroplasmic cells divide and the mtDNA is distributed randomly to
daughter cells resulting in skewed populations of wild type or mutant
mtDNA
Random mitotic segregation of mtDNA causes varying proportions of
mutant mtDNA In daughter cells
Degree of heteroplasmy determines clinical phenotype
19. BOTTLENECK AND SEGREGATION
• Of the 1,50,000 mtDNA molecules in human oocytes
,only a small proportion of mtDNA is transmitted
during oogenesis and subsequently to embryo.
• Important implications in
high intrafamilial clinical variation
changing phenotype over time
20. THRESHOLD
• For heteroplasmic mtDNA mutations
• Cell can compensate for reduced wild-type mtDNA
until a certain threshold is met
- function of cell become compromised
• Disease occurs when enough cells in a tissue are
affected
• Threshold depends on specific mutation and cell types
Ex : neurons have a lower threshold for disease state
22. • Mitochondrial disease can arise through :
1. defect in mtDNA
2. defect in nuclear-encoded mitochondrial
protein
23. mtDNA and disease
• Mutation creates two distinct classes of
mtDNA variants :
- single base pair variants
- mtDNA rearrangements (deletions and
insertions)
25. mtDNA vs Nuclear DNA mutations
Feature
mtDNA mutations Nuclear DNA mutations
Mode of
inheritance
Maternal
Mendelian
Age of onset
Adults
Infancy / childhood
Severity of
disease
Less
More
Lactic acidosis
More common
Not seen
27. • Idiopathic, chronic, intermittent or progressive
illness involving at least two different high-energy
requiring tissues
– Neuron (brain, esp. basal ganglia, special senses
and autonomic neuron)
– Muscle (skeletal, cardiac, or smooth)
– Endocrine gland
– Renal tubule
28. • Examples
– Mental retardation and diabetes mellitus
– Migraine and hypotonia
– Gastrointestinal dysmotility and stroke
– Hypothyroidism and cardiomyopathy
– Dysautonomia and deafness
– Depression and renal tubular acidosis
29. • Family history, intermittent disease, biochemical data
(lactic acidosis, elevated Krebs cycle intermediates)
can all increase suspicion of mitochondiral disease
30. • Mitochondrial disease affects tissues most
highly dependent on ATP production
– Nerves
– Muscles
– Endocrine
– Kidney
31. • Low energy-requiring tissues are rarely directly
affected, but may be involved secondarily
– Lung
– Connective tissue
• Symptoms can be intermittent
– Increased energy demand (illness, exercise)
– Decreased energy supply (fasting)
36. – Serum CK level: mildly elevated in mitochondrial
myopathies but are often normal,High-CPEO and
ptosis;Very high in limb weakness
– Lactate level: fasting blood lactate conc >3mm/l support
the diagnosis
– CSF lactate: fasting conce>1.5mm/l
• Normal level can be seen in NARP
• Elevated with short exercise
37. • Electrocardiography and echocardiography
– cardiac involvement
– (cardiomyopathy or atrioventricular conduction defects).
• Neuroimaging :
– suspected CNS disease.
• CT: basal ganglia calcification +/ diffuse atrophy
• MRI: focal atrophy of the cortex / cerebellum
high signal change on T2WI, particularly occipital
generalized leukoencephalopathy.
Cerebellar atrophy (pediatrics)
38. • Neurophysiologic studies:
– indicated in individuals with limb weakness,
sensory symptoms, or areflexia.
– Electromyography (EMG) is often normal but may
show myopathic features.
– Nerve conduction velocity (NCV)
may be normal or may show a predominantly
axonal sensorimotor polyneuropathy
39. • Electroencephalography (EEG)
– Indicated in suspected encephalopathy / seizures.
Encephalopathy: generalized slow wave activity on
the EEG.
Seizures : Generalized or focal spike and wave discharges
may be seen
41. – More specific test of
mitochondrial myopathies
– analyzed for histologic or histochemical evidence
of mitochondrial disease.
– Respiratory chain complex studies are carried out
on skeletal muscle or skin fibroblasts.
– Ragged red fibers (RRFs) are seen on muscle
biopsy.
– Presence of more than 2% RRFs in skeletal muscle
biopsy is taken as one of the criteria for the
diagnosis of mitochondrial disease.
42. Distinctive features of muscle biopsy in
mithochondrial myopathies :
• Succinate dehydrogenase (SDH) stain:
Increased staining of muscle fibers
Most sensitive & specific stain for
mitochondrial proliferation in muscle
fibers
• Cytochrome oxidase (COX) stain:
– Absent or reduced staining of muscle
fibers:
Reduced COX activity.
– May be diffuse or in scattered fibers.
44. – Abnormal protein accumulation in ragged red fibers:
• Desmin
• αβcrystallin,
• Heat shock proteins,
• Dysferlin,
• Emerin,
• Caveolin.
45. • ELECTRON MICROSCOPY:
– Usually not specific or sensitive in adults with nondiagnostic histochemistry results ,
– Ultrastructure may be only evidence of
mitochondrial pathology in 6%
47. • Testing carried out on genomic DNA
– Blood (suspected nuclear DNA mutations and
some mtDNA mutations)
– Muscle(suspected mtDNA mutations)
– Southern blot analysis may reveal a pathogenic
mtDNA rearrangement. The deletion or
duplication breakpoint may then be mapped by
mtDNA sequencing.
48. – If a recognized point mutation is not
identified, the entire mitochondrial genome may
be sequenced.
51. • Nutritional:
– Identify and treat deficiencies in vitamins (vitamins A, B12,
E, D, folate for red blood cells), minerals (iron, zinc,
selenium, calcium, magnesium), and protein calorie
(albumin).
52. 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). Shivering is
metabolically expensive and should be avoided.
• Avoid unaccustomed strenuous exercise, especially in the
fasting state or with a concomitant illness.
• Avoid prolonged (greater than 12 hours) fasting.
57. – Most common mitochondrial encephalomyopathy
– Maternally inherited point mutation
– A3243G point mutation in tRNA-80%
– Onset in majority patients is before the age of 20 yrs
58. – Seizures: partial or generalized, may be first sign
– Stroke like episodes, do not conform to a vascular
distribution
– Hemiparesis, hemianopia and cortical blindness
– Associated condition, hearing loss, diabetes mellitus,
growth hormone deficiency
– Fatal outcome
59. Diagnosis of MELAS
CSF protein
Increased but <100mg/dl
Muscle biopsy
•Ragged red fibres
•SDH positive fibres
•COX positive fibres
CSF lactate
Increased
Imaging
•Grey and white matter involvement
•Basal ganglia calcification
•Focal lesion which mimic infraction are present in occipito-parietal
Genetics
80% have A3243G mutation in tRNA leucine
60. MUSCLE BIOPSY – SDH STAIN
Normal: Mild SDH staining of a
medium sized perimysial vessel.
Increased SDH staining of a
medium sized perimysial vessel
in a MELAS patient.
63. – Multiorgan disorder
– Triad-onset before 20yrs,CPEO ,pigmentary
retinopathy
– Plus one or more of following: complete heart
block, cerebellar ataxia, or increased CSF protein
100mg/dl
64. – Common 5-kb mtDNA deletion,
deletion/duplications, A3243G
– KSS/CPEO-like phenotype can be caused by
nuclear mutations in genes for mtDNA
maintenance (ANT1, Twinkle and POLG)
66. – Onset : childhood to middle adult
– Point mutation A8344G of tRNA lysine
– Characteristic : myoclonic epilepsy
cerebellar ataxia
progressive muscle weakness
– Others: dementia, peripheral neuropathy, optic
atrophy, hearing loss and diabetes mellitus
– Lipomas-cervical, symmetrical
67. Serum CPK
Normal or increased
Lactate
(serum and CSF)
Elevated
EMG
Myopathic
EEG
May be abnormal, non specific
Muscle biopsy
•Ragged red fibres
•SDH positive fibres
•COX negative fibres
Genetics
•A8344G mutation
•Base pair substution-T8356C, G8363A
69. – Onset in early 20s
– Maternally inherited
– Three mutation all are located within mtDNA
complex I genes
• G11778A mutation in ND4
• G3460A mutation
• T14484C mutation in ND6
– Characterized by acute and subacute bilateral
painless visual loss
– Visual loss is severe and permanent
– Dystonia or striatal degeneration
71. – Subacute necrotising encephalomyopathy
– Onset: infancy and early childhood
– Most commonly caused by high mutant loads
(>95%) of T8993G/C
– Point mutations in ATP synthase gene, affects
complex V
– Other causes include complex I def
(NDUFV1), complex IV def (SURF1), PDHC
defenciency
72. – Progressive psychomotor deterioration,
respiratory failure
– MRI leukodystropy, changes in basal ganglia and
brain stem
76. – Adolescent
– Autosomal recessive
– Mutation in thymidine phosphorylase in Ch 22
– Thymidine phosphorylase activity is reduced and
plasma thymidine levels are elevated
– Peripheral neuropathy,CPEO,gastrointestinal
dysmotility
77. Toxin induced MtDNA myopathy
– Exogenous cause of mtDNA abnormalities is HIV infection
and antiretroviral therapy
– Zidovudine induced myopathy patient presents with
myalagia,weakness , atrophy of thigh and calf muscle
– S.CK- raised
– EMG-myopathic
– Muscle biopsy-ragged red fibres with minimal
inflammation
78. • Association with neurodegenerative disorders
– Parkinson disease
– Alzheimer disease
– Huntington disease
– Friedreich ataxia
