Genetics of Mitochondrial disorders

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  • 1. Mitochondrial DNA and Disease
  • 2. Introduction
    • Mitochondria are present in all human cells.
    • Provide ATP by oxidative phosphorylation (OXPHOS).
    • Role in other biochemical pathways: fatty acid b-oxidation, Krebs citric acid cycle, parts of the urea cycle.
    • The role of mitochondria DNA (mtDNA) in disease is now an important area of research.
    • More than 100 mutations have been liked to human disease.
    • Mitochondrial disorders may also be due to inherited defects of the nuclear genome.
  • 3. MtDNA: Structure
    • Small, circular, double stranded molecule of 16,493 bases encoding 2 rRNAs, 22 tRNAs, and 13 proteins.
    • These proteins are all part of the OXPHOS system.
    • MtDNA is dependent on the nucleus for:
    • Proteins involved in its transcription, translation, replication, and repair, all of which take place in the mitochondrion.
    • Production of all mitochondrial proteins other than those encoded by mtDNA.
  • 4. OXPHOS System
    • The OXPHOS system comprises the four respiratory chain complexes (I to IV) and ATPase (complex V).
    • These are embedded in the inner mitochondrial membrane and consist of 85 subunits, 13 being encoded by mtDNA.
    • The OXPHOS system is responsible for ATP production and also is an important source of superoxide radicals.
    • Defects of the OXPHOS system result in failure of energy metabolism and increased free radical mediated damage.
  • 5. Respiratory chain and OXPHOS system
  • 6. Human mtDNA
  • 7. MtDNA: Inheritance
    • MtDNA is inherited through the female line.
    • Paternal mtDNA from the sperm is either not incorporated into the fertilized ovum, or its replication is suppressed and the mtDNA degraded.
    • The mutation is transmitted from mother to all offspring and subsequently by her daughters.
    • MtDNA mutations often coexist with wild type (normal) mtDNA: heteroplasmic form.
    • The proportion of mutant to wild type vary between individuals and between tissues of the same individual.
  • 8. MtDNA: Inheritance
    • The mutant load correlates with the degree of biochemical defect, but this is tissue dependent and recessive.
    • 5% to 20% of wild-type mtDNA can compensate at the biochemical level for certain mutations.
    • Mitochondria are randomly segregated at cell division resulting in varying proportion of wild-type and mutant mtDNA in daughter cells.
    • A high mutant load in the oocyte population would result in a high proportion of offspring at risk of developing disease.
  • 9. Mitochondrial Diseases: Overview
    • The clinical expression of disorders of the mitochondrial respiratory chain is very broad and can include any system.
    • Brain and muscle are most commonly affected, as these tissues are probably the most dependent on OXPHOS.
    • Common features: proximal myopathy, external ophthalmoplegia, seizures, dementia, ataxia, deafness, retinitis pigmentosa, stroke and diabetes mellitus.
    • The genotype- phenotype correlation is not exact.
    • Estimated prevalence of mutation is 16/100,000.
  • 10. Chronic Progressive External Ophthalmoplegia (CPEO) and Kearns-Sayre Syndrome
    • Onset: any time from adolescence to late adulthood, usually before the age of 30.
    • Ptosis: Symmetric or asymmetric, slowly progressive, usually nonfatigable.
    • External ophthalmoplegia: diplopia is uncommon.
    • Salt and pepper pigmentary retinopathy may be seen.
    • Proximal myopathy may be present.
    • Disability is not significant and life span is usually normal.
  • 11. Biochemical assay of respiratory chain function
    • Mitochondria are often enlarged with abnormal cristae.
    • Vacuolated matrix and paracrystalline inclusions.
    • However, EM findings are not specific.
    EM
    • Features of mitochondrial myopathy.
    • RRF, COX negative, SDH positive fibres may occur in inflammatory myopathy, old age.
    • 10% of pts with mtDNA mutation have normal biopsy.
    Muscle Biopsy
    • May be elevated at rest; significant and sustained increase with exercise
    S. Lactate
    • Normal or mild nonspecific myopathic features
    EMG
    • Usually normal
    CK Diagnosis of CPEO
  • 12. SDH staining of muscle biopsy
  • 13. CPEO: Genetic analysis
    • 70% have the mtDNA deletions; affecting same segment of mtDNA molecule in all tissues, but the proportion varies.
    • It is extremely rare to find the deletions in blood. DNA confirmation therefore requires a tissue sample- muscle.
    • The proportion of deleted mtDNA is stable over time.
    • One important mtDNA deletion is the 4977 base pair deletion (common deletion) which spans the region from the ATPase 8 gene to the ND5 gene.
    • This occurs in 30% of patients with CPEO or KSS.
    • mtDNA deletions: occur in 80% of patients with KSS.
  • 14. KSS
    • Defined by CPEO and pigmentary retinopathy with either complete heart block, CSF protein > 1 g/L, or ataxia.
    • Onset: before age of 20, although late onset is also seen.
    • Clinical Features:
    • Ptosis: asymmetric in 58% or unilateral in 8%.
    • Dysconjugate eye movements: in 35%
    • Diplopia: transient/ persistent diplopia in 36%.
    • Ophthalmoplegia: may be severe; in 62% of patients, gaze is limited to less than 10% of normal in any direction.
    • Proximal weakness may be seen.
  • 15. MELAS: Clinical Features
    • Variable course
    • Pigmentary Retinopathy
    • Myopathy
    • Diabetes
    • Recurrent lactic acidosis with nausea and vomiting
    • Migraine with aura
    • Ataxia
    • Seizures: focal/ generalised
    • Dementia
    • Recurrent stroke like episodes: especially hemianopia, hemiplegia. Deficits may be transient or permanent
    • Deafness
    • Growth retardation
    • Onset: usually adolescence, may occur later
  • 16. MELAS: Diagnosis
    • 80% have A3243G mutation in the tRNA for leucine.
    • 4 different mutations in the tRNA leucine (UUR) gene
    Genetics Cerebral vessel histology show SDH reactivity indicating angiopathy as a significant component in the pathogenesis of stroke.
    • Ragged red fibres
    • SDH positive fibres
    • COX fibres are usually strongly positive
    Muscle biopsy
    • Invariably elevated in encephalopathy
    CSF lactate
    • Grey and white matter involvement
    • Not conforming to vascular territories
    • Parieto-occiptital predominance
    • Calcification: especially in globus pallidus, but can occur in striatum, thalamus, internal capsule
    Imaging
  • 17. MERRF: Clinical Features
    • Growth retardation
    • Neuropathy
    Lipomas are an interesting phenomenon. In some patients, this may be the only manifestation of MERRF.
    • Cervical lipomas: multiple, symmetric
    • Optic atophy
    • Ptosis
    • Ophthalmoplegia
    • Dementia
    • Deafness
    • Myopathy: proximal, mild
    • Ataxia
    • Seizures: myoclonus which may be stimulus sensitive, drop attacks, focal and generalized seizures.
  • 18. MERRF: Diagnosis
    • A8344G mutation is present in 80% of patients
    • Mutations in the tRNA lysine gene
    • Single base changes in tRNA for leucine, serine
    Genetics Cerebral vessel histology show SDH reactivity indicating angiopathy as a major component in the pathogenesis.
    • Ragged red fibres
    • SDH positive fibres
    • COX negative fibres
    Muscle biopsy
    • may be elevated
    Lactate (serum, CSF)
    • may be abnormal, but findings are nonspecific
    EEG
  • 19. NARP: Clinical Features Diagnosis
    • T8893G mutation in the ATPase 6 gene is the commonest mutation. This is heteroplasmic: at 70%, it causes NARP, at > 90%, causes Leigh syndrome.
    • T8993C mutation
    Genetics
    • Abnormal ATPase activity
    • Low blood citrulline level (surrogate marker)
    Biochemical
    • Normal
    Muscle biopsy Mental retardation Migraine Seizures Dementia R etinitis pigmentosa A taxias M yopathy N eurogenic muscle weakness
  • 20. Mutations in MtDNA COX gene mutations Myopathy and myoglobinuria mtDNA deletions Recurrent myoglobinuria, exercise intolerance Mutations in mtDNA COX genes Sideroblastic Anemia Mutations in mtDNA COX genes Motor Neuron disease mtDNA mutations Pure Myopathy mtDNA mutations, rRNA mutations Cardiomyopathy (dilated/hypertrophic) Mutation in tRNA for serine Sensorineural hearing loss 1% to 2% of NIDDM is due to A3243G mutation. It may be the only manifestation or may have associated sensorineural deafness. NIDDM Additional Phenotypes
  • 21. Leber Hereditary Optic Neuropathy
    • Maternally inherited disorder characterized by acute or subacute bilateral sequential painless visual failure.
    • The most common disease caused by mtDNA mutations, with a prevalence of 11.82 per 100,000 population.
    • The mean age of onset is in the early 20s, and 90% of patients are affected by age 45; 85% are men.
    • In most cases, the visual loss is severe and permanent, although this is dependent on the underlying mutation.
    • Only 15% of women who carry one of the primary mutations are clinically affected.
  • 22. Leber Hereditary Optic Neuropathy
    • Three primary mtDNA mutations; all are located within mtDNA complex I genes.
    • G11778A mutation in ND4 : 50% to 70% of patients.
    • G3460A mutation: 15% to 25% of patients.
    • T14484C mutation in ND6: relatively uncommon but is associated with visual recovery in 70%.
    • Mutations are homoplasmic and detectable in blood.
    • Complex I defect in muscle and platelets is known.
    • Additional features like dystonia or striatal degeneration occur- due to mutations in mtDNA complex I genes.
  • 23. Onset: adolescence; most patients are dead by 40 yrs of age. Myoneuro-gastrointestinal encephalopathy (MNGIE)
    • Autosomal recessive
    • Multiple deletions and depletion of mtDNA.
    • Mutations in thymidine phosphorylase in Ch 22.
    • Thymidine phosphorylase activity is reduced and plasma thymidine levels are elevated.
    Genetics
    • Features of leukodystrophy
    MRI
    • Typical features of mitochondrial myopathy
    Muscle biopsy Investigations Sensorimotor
    • Peripheral Neuropathy
    Nausea, vomiting, diarrhoea
    • GIT
    Diagnostic criteria: Peripheral neuropathy, CPEO, and gastrointestinal dysmotility.
  • 24.
    • Respiratory chain defects: Defects in complex I or II (nuclear gene)
    • Deficiency of complex IV
    Biochemical abnormalities
    • Optic atrophy
    • Respiratory abnormalities
    Onset: usually in the first few months of life or during childhood. Leigh’s Syndrome
    • Mutation of SURF gene (responsible for assembly and maintenance of complex IV).
    • ATPase 6 mutations with mutation load of >90% is seen in 20% of patients.
    • MELAS or MERRF mutations
    Genetics
    • Pyruvate dehydrogenase deficiency
    • Biotinidase deficiency
    • Lactic acidosis
    • Seizures
    • Oculomotor disturbance
    • Psychomotor retardation
    Subacute necrotising encephalomyelopathy.
  • 25. mtDNA Mutations Secondary to Nuclear Gene Mutations
    • Two such mutations have been identified.
    • These cause multiple mtDNA deletions and depletion.
    • Adenine nucleotide translocator 1 and TWINKLE gene (a DNA helicase) mutations: result in AD inheritance of CPEO, multiple mtDNA deletions or moderate depletion.
    • POLG mutations: may result in AD or AR CPEO, Alpers syndrome (an AR disorder with epilepsy, cortical blindness, and liver failure), and noted in patients who present with progressive external ophthalmoplegia and develop PD in later life.
  • 26. Nuclear Gene Mutations Of Respiratory Chain Subunits
    • Extremely rarely reported.
    • This may reflect the deleterious nature of such mutations resulting in early abortions.
    • Manifest in the neonatal period or early infancy, although late-onset patients have been identified.
    • Varied phenotypes: infantile Leigh syndrome, cardiomyopathy, seizures, liver failure, progressive ataxia (especially in late onset cases).
    • The association with paragangliomas and pheochromocytomas is very rare.
  • 27. Toxin Induced MtDNA Abnormalities
    • A newly recognized exogenous cause of mtDNA abnormalities is HIV infection and antiretroviral therapy.
    • Untreated patients have reduced mtDNA content, abnormalities of OXPHOS function, and oxidative damage.
    • Antiretroviral treatment inhibits mtDNA POLG and induces a secondary mtDNA depletion that can be associated with toxicity, including myopathy, lipodystrophy, lactic acidosis, and hepatic failure.
  • 28. Treatment of Mitochondrial disorders
    • Treatment remains unsatisfactory.
    • Supportive measures: like eye props or ptosis surgery for patients with CPEO.
    • Coenzyme Q10 supplementation.
    • Antioxidants: N-acetylcysteine and coenzyme Q10 improved OXPHOS function and reduced free radical production in cybrid cells carrying the T8993G mutation that causes NARP and Leigh syndrome.
    • Coenzyme Q10 and antioxidants have not been tested in any large trial.
    • Specific treatment: eg deafness, diabetes.
  • 29. Future research focus
    • Strategies to modify the mtDNA mutant load:
    • 1. Preferential expansion of wild-type mtDNA:
    • Skeletal muscle satellite cell expansion by vigorous exercise or toxic damage can shift heteroplasmy in favor of wild type, as mutant mtDNA is absent or at a low level in these cells.
    • 2. Suppression of mutant mtDNA expansion:
    • Importing into mitochondria of endonucleases that might selectively destroy a specific mutant sequence has been possible in vitro.
  • 30. Future research focus
    • 3. Import of normal tRNA from cytosol:
    • In cells with tRNA mutations of mtDNA.
    • Import of normal tRNAlys from cytosol to mitochondria improved OXPHOS function in cybrid cells bearing the tRNAlys A8344G mutation that causes MERRF.
    • Treatment of MNGIE:
    • Short term: Platelet transfusion as these contain thymidine phosphorylase.
    • Definitive treatment: bone marrow transplantation.
  • 31. Genetic Counselling
    • Inheritance is from mother to all offspring with subsequent passage by the daughters alone.
    • Males with mtDNA mutation can be reassured that they almost certainly will not transmit the mutation.
    • Mitochondrial diseases due to nuclear gene mutations will be transmitted by mendelian inheritance.
    • Mothers with CPEO have a low risk of transmission (4%), and the risk of more than one sibling being affected is low.
    • Options include IVF using a donor egg; nuclear transfer from a maternal egg and fertilization in a donor cytoplasm using paternal sperm is a future possibility.
  • 32. Conclusion
    • Much more remains to be discovered about the molecular pathogenesis of various mutations affecting mtDNA.
    • Attention is also focused on nuclear gene mutations causing secondary mtDNA abnormalities.
    • Thus, mitochondria are becoming an increasing focus for the biomedical sciences
    • Future research will no doubt enlighten us more about their association with disease and about normal mitochondrial function and physiology.