MITOCHONDRIAL DISEASES

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.

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

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

MITOCHONDRIA LIFE CYCLE :FUSION, FISSION AND AUTOPHAGY

• 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.

Unravelling the genetics of

mitochondria

MITOCHONDRIAL DNA

• Circular, double stranded, and composed of

heavy and light chains or strands

• Contains 16,569 bp

• Encodes 13 proteins

22 tRNA

2rRNA

MITOCHONDRIAL VS NUCLEARGENOME

• Mitochondrial genome has

. smaller number of genes

. higher copy number

. less effective repair mechanisms

. higher mutation rates

Peculiarities of Mitochondrial Genetics

Maternal inheritance

high copy number

heteroplasmy

bottleneck and segregation

threshold

MATERNAL INHERITANCE

MATERNAL MODE OF INHERITANCE

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 )

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

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

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

PATHOPHYSIOLOGY

• Primary mitochondrial disease:

Diseases involving defects of oxidative phosphorylation.

• Tissues with high aerobic demands such as

brain tissue,

heart muscle ,

skeletal muscle

usually more severely affected.

• Mitochondrial disease can arise through :

1. defect in mtDNA

2. defect in nuclear-encoded mitochondrial

protein

mtDNA and disease

• Mutation creates two distinct classes of mtDNA variants :

- single base pair variants - mtDNA rearrangements (deletions and

insertions)

CLINICAL SYNDROMES OF mtDNA mutations

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

APPROACH TO MITOCHONDRIAL DISORDERS

• 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

• Examples

– Mental retardation and diabetes mellitus

– Migraine and hypotonia

– Gastrointestinal dysmotility and stroke

– Hypothyroidism and cardiomyopathy

– Dysautonomia and deafness

– Depression and renal tubular acidosis

• Family history, intermittent disease, biochemical data

(lactic acidosis, elevated Krebs cycle intermediates)

can all increase suspicion of mitochondiral disease

• Mitochondrial disease affects tissues most

highly dependent on ATP production

– Nerves

– Muscles

– Endocrine

– Kidney

• 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)

SYSTEM CLINICAL MANIFESTATIONS

Cardiovascular heart failure arrhythmias sudden death left ventricular myocardial noncompaction

Pulmonary dyspnea orthopnea respiratory failure respiratory acidosis

neurologic encephalopathy ataxias movement disorders seizure disorder mental retardation stroke like episodes migraine

endocrine diabetes mellitus diabetes insipidus hypothyroidism hypoparathyroidism ACTH deficiency

Ocular optic atrophy external opthalmoplegia ptosis retinitis pigmentosa cataract

Musculoskeletal myopathy•Skeletal muscle : ocular>axial/proximal>bulbar>distal•Smooth muscle : dysphagia•Cardiac : cardiomyopathy myalgias

Renal renal tubular defects benign renal cysts focal segmental glomerulosclerosis nephritic syndrome

Hematological anemia leukopenia thrombocytopenia eosinophilia

Gastrointestinal malabsorption villous atrophy pseudo-obstruction

LABORATORY EVALUATION

– 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

• 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 / cerebellumhigh signal change on T2WI, particularly

occipital generalized leukoencephalopathy.

Cerebellar atrophy (pediatrics)

• 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

• 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

MUSCLE BIOPSY

– 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.

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.

IMMUNOHISTOCHEMISTRY

– Abnormal protein accumulation in ragged red fibers:

• Desmin

• αβcrystallin,

• Heat shock proteins,

• Dysferlin,

• Emerin,

• Caveolin.

• ELECTRON MICROSCOPY:

– Usually not specific or sensitive in adults with non-

diagnostic histochemistry results ,

– Ultrastructure may be only evidence of

mitochondrial pathology in 6%

MOLECULAR GENETICS

• 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.

– If a recognized point mutation is not identified,

the entire mitochondrial genome may be

sequenced.

PRINCIPLES OF TREATMENT

Treat Underlying Neurologic Issues

– Seizures(antiepileptic drugs)

– Spasticity- baclofen, botulinium toxin

– Dystonia- diazepam, botulinium toxin, trihexyphenidyl

– headache –

acute: nonsteroidal anti-inflammatory drugs and

acetaminophen; avoid aspirin and triptans in MELAS,

chronic: amitriptyline, calcium blockers, riboflavin, coenzyme

Q10,

• 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).

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.

MITOCHONDRIAL GENETIC DISORDERS

REARRANGEMENTS POINT MUTATIONS

CPEO MELAS

Kearns-Sayre syndrome MERRF

Pearson marrow pancreas syndrome CPEO

Diabetes and deafness Myopathy

Cardiomyopathy

NARP

LHON

Nuclear genetic disorder

• Autosomal dominant progressive ophthalmoplegia• Mitochondrial neurogastrointestinal

enecephalomyopathy• Leigh syndrome• Cardioenecephalomyopathy• Optic atrophy and ataxia• Tubulopathy, encephalopathy and liver failure

MELAS(Mitochondrial myopathy, Encephalopathy, Lactic Acidosis and Stroke

like episodes)

– 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

– 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

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

Normal: Mild SDH staining of amedium sized perimysial vessel.

Increased SDH staining of amedium sized perimysial vesselin a MELAS patient.

MUSCLE BIOPSY – SDH STAIN

Scattered "ragged red" muscle fibers: Gomori trichrome

Scattered abnormal, vacuolated fibers with clear rim: H & E

KEARNS-SAYRE SYNDROME (KSS)

– 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

– 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)

MERRF(Myoclonic Epilepsy with

Ragged Red Fibres)

– 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

DIAGNOSISSerum 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

Leber’s hereditary optic neuropathy (LHON)

– 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

LEIGH’S SYNDROME

– 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

– Progressive psychomotor deterioration,

respiratory failure

– MRI leukodystropy, changes in basal ganglia and

brain stem

Neuropathy, Ataxia, Retinitis Pigmentosa(NARP)

– Onset :childhood

– Moderate heteroplasmy for T8993G/C in ATPase

6gene (same as Leigh but lower mutant load)

– Polyneuropathy, cerebellar ataxia, retinitis

pigmentosa

– Muscle biopsy -normal

Mitochondrial, Neurogastrointestinal encephalomyopathy(MNGIE)

– 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

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

• Association with neurodegenerative disorders

– Parkinson disease

– Alzheimer disease

– Huntington disease

– Friedreich ataxia

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