Mitochondrial Disorders - Comprehensive Notes

Mitochondrial Disorders

mtDNA Genome Contents

• mtDNA contains genes for:
  • tRNAs
  • rRNAs
  • Cytochrome oxidase subunits
  • NADH-dehydrogenase subunits
  • ATPase subunits
• mtDNA genes are located on both strands.
• The functions of all human mtDNA Open Reading Frames (ORFs) are assigned.
• Nuclear DNA also contains mitochondrial genetic information, including genes for:
  • DNA polymerase
  • Replication factors
  • RNA polymerase
  • Transcription factors
  • Ribosomal proteins
  • Translation factors
  • Aminoacyl-tRNA synthetase
  • Additional cytochrome oxidase, NADH, and ATPase subunits.
• Most mitochondrial (and chloroplast) proteins are coded by nuclear genes.

Transcription of mtDNA

• mRNAs from the mtDNA are synthesized and translated in the mitochondria.
• Gene products encoded by nuclear genes are transported from the cytoplasm to the mitochondria.
• Mammalian and vertebrate mtDNAs are transcribed as a single large RNA molecule (polycistronic) which is then cleaved to produce mRNAs, tRNAs, and rRNAs before processing.
• Most mtDNA genes are separated by tRNAs that signal transcription termination.

Translation of mtDNA

• Mitochondria mRNAs do not have a 5' cap (yeast and plant mt mRNAs have a leader sequence).
• Specialized mtDNA-specific initiation factors (IFs), elongation factors (EFs), and release factors (RFs) are utilized for translation.
• AUG is the start codon (binds with fMet-tRNA like bacteria).

Mitochondria & Oxidative Phosphorylation

• Oxidative phosphorylation (OXPHOS) involves several complexes (I-V) located in the inner mitochondrial membrane.
• These complexes facilitate the transfer of electrons and protons, generating ATP.
• Complex I: NADH dehydrogenase
• Complex II: Succinate dehydrogenase
• Complex III: Cytochrome bc1 complex
• Complex IV: Cytochrome c oxidase
• Complex V: ATP synthase.
• The process involves the TCA cycle (carbohydrate metabolism) and beta-oxidation (fatty acid metabolism) which feeds into the OXPHOS complexes.

Mitochondrial Genome

• The mitochondrial genome is a circular chromosome of 16.6 kb.
• Most cells contain at least 1000 mtDNA molecules distributed among hundreds of individual mitochondria.
• Mature oocytes have more than 100,000 copies of mtDNA.

mtDNA Composition

• 16.6 kb in size (compared to 3 billion in the nuclear genome).
• Contains 37 genes:
  • 22 tRNAs
  • 2 rRNAs
  • 13 proteins (all subunits of the electron transport chain (ETC)).
• mtDNA is entirely devoted to energy metabolism.

Human Mitochondrial Genome

• Key genes include 12S rRNA, 16S rRNA, Cytochrome b (Cyt b), and genes encoding NADH dehydrogenase subunits (ND1-ND6, ND4L), Cytochrome Oxidase subunits (COI, COII, COIII), and ATPase subunits (ATPase6, ATPase8).
• Mutations in these genes can cause diseases such as Leber Hereditary Optic Neuropathy (LHON) and Myoclonic Epilepsy with Ragged Red Fibers (MERRF).
• Control region of mtDNA includes the D-loop.

Mitochondrial Diseases

• Different rearrangements and point mutations identified in mtDNA can cause human disease, often involving the central nervous and musculoskeletal systems (e.g., myoclonic epilepsy with ragged-red fibers- MERRF).
• Mitochondrial diseases have a distinctive pattern of inheritance due to three unusual features of mitochondria: replicative segregation, homoplasmy and heteroplasmy, and maternal inheritance.

Replicative Segregation

• Absence of tightly controlled segregation.
• At cell division, multiple copies of mtDNA in each mitochondrion replicate and sort randomly among newly synthesized mitochondria.
• Mitochondria are distributed randomly between the two daughter cells.
• This process is known as replicative segregation.

Mutant mtDNA

• When a mutation arises in the mtDNA, it is initially present in only one mtDNA molecule within a mitochondrion.
• Through replicative segregation, a mitochondrion containing a mutant mtDNA will acquire multiple copies of the mutant molecule.
• With cell division, a cell containing a mixture of normal and mutant mtDNAs can distribute varying proportions of mutant and wild-type mitochondrial DNA to its daughter cells.

Homoplasmy and Heteroplasmy

• One daughter cell may receive mitochondria containing only a pure population of normal mtDNA or a pure population of mutant mtDNA (homoplasmy).
• Alternatively, the daughter cell may receive a mixture of mitochondria, some with and some without mutation (heteroplasmy).
• The phenotypic expression of a mutation in mtDNA depends on the relative proportions of normal and mutant mtDNA in the cells making up different tissues, leading to reduced penetrance, variable expression, and pleiotropy.

Maternal Inheritance

• Nuclear DNA is inherited from all ancestors, while mitochondrial DNA is inherited from a single maternal lineage.
• Sperm mitochondria are generally eliminated from the embryo, ensuring mtDNA is inherited from the mother.
• Maternal inheritance in the presence of heteroplasmy is associated with additional features:
  • The number of mtDNA molecules within developing oocytes is reduced before being amplified to the quantities seen in mature oocytes. This is the mitochondrial genetic bottleneck.

Mutant mtDNA and Offspring

• Mothers with a high proportion of mutant mtDNA molecules are more likely to produce eggs with a higher proportion of mutant mtDNA and are therefore more likely to have clinically affected offspring.
• An exception occurs when the mother is heteroplasmic for a deletion mutation in her mtDNA; deleted mtDNA molecules are generally not transmitted to their children.

Leber Hereditary Optic Neuropathy (LHON)

• Pedigree shows maternal inheritance.

Examples of Mitochondrial Diseases

• MELAS (Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-like episodes)
• MERRF (Myoclonic Epilepsy with Ragged Red Fibres)
• Leber Hereditary Optic Neuropathy (LHON)
• External Ophthalmoplegia
• Kearns-Sayre syndrome
• Chronic progressive external ophthalmoplegia
• NARP (Neurogenic weakness Ataxia with Retinitis Pigmentosa)

Specific Disease Examples

• MELAS: Associated with tRNALeu mutation [MTTL1*MELAS3243G].
• MERRF: Associated with tRNALys mutation [MTTK*MERRF8344G].
• LHON: Caused by mutations in ND1, ND2, ND4, ND5, ND6, CO1, ATP6, CO3, and CYTB genes.
• Kearns-Sayre syndrome: Caused by a deletion of 4.9kb affecting the region between ATP8 and ND5.
• NARP: Caused by ATP6 gene mutation.

Clinical Presentation of Mitochondrial Diseases

• Tissues most highly dependent on ATP production are primarily affected:
  • Nerves
  • Muscles
  • Endocrine system
  • Kidney
• Low energy-requiring tissues are rarely directly affected but may be secondarily:
  • Lung
  • Connective tissue
• Symptoms can be intermittent:
  • Increased energy demand (illness, exercise)
  • Decreased energy supply (fasting)

Nuclear Encoded Mitochondrial Disease

• Caused by mutations in genes coding for mitochondrial proteins that are transported to the mitochondria after expression.
• These proteins have a signature localization sequence (~30 amino acids) that targets them to particular receptors on mitochondrial membranes.
• After binding the receptor, these proteins are transported inside the mitochondria where they function in OX-PHOS, iron metabolism, mitochondrial DNA replication, and integrity.

Protein and Substrate Defects

• Protein import defects: low levels of Pyruvate Dehydrogenase activity.
• Substrate Transport Defects: affect fatty acid oxidation (e.g., CACT gene - carnitine-acylcarnitine translocase mutations affect transport of long-chain fatty acids).
• Substrate utilization defects: Carnitine palmitoyl transferase (CPT2) mutations impair the oxidation of long-chain fatty acids.
• Iron Transport Defects: Fredreich’s Ataxia is caused by a mutation in frataxin, leading to excess iron accumulation in the mitochondria.
• Electron Transport Chain Defects: Mutations in genes coding enzymes that work in Complex I, II, and III of the OXPHOS system.

Characteristics of Human Mitochondrial Diseases

• Are maternally inherited: only offspring of affected mothers are affected.
• Show deficiency in mitochondrial function.
• Are caused by a mutation in a mitochondrial gene.
• Example: myoclonic epilepsy and ragged red fiber disease (MERRF).
• Deafness, dementia, seizures can occur.
• Result from point mutations in mitochondrial tRNA.

Optic Neuropathies

• Include:
  • Leber hereditary optic neuropathy
  • Optic neuritis
  • Anterior ischaemic optic neuropathy (AION)
• Specific types:
  • Retrobulbar neuritis
  • Papillitis
  • Neuroretinitis

Anatomy of the Optic Nerve

• The optic nerve is the second of twelve paired cranial nerves but is considered part of the central nervous system.
• Composed of retinal ganglion cell axons and Portort cells
• Most axons of the optic nerve terminate in the lateral geniculate nucleus, relaying information to the visual cortex.
• Its diameter increases from about 1.6 mm within the eye to 3.5 mm in the orbit and 4.5 mm within the cranial space.

Signs of Optic Nerve Dysfunction

• Reduced visual acuity
• Diminished light brightness sensitivity
• Dyschromatopsia (color vision deficiency)
• Afferent pupillary conduction defect

Visual Field Defects

• Central scotoma
• Centrocaecal scotoma
• Papillomacular bundle defects
• Altitudinal defects
• Nerve fiber bundle defects

Optic Disc Changes

• Retrobulbar neuritis - Normal.
• Early compression.
• Papilloedema - Swelling.
• Papillitis and neuroretinitis - Swelling.
• Optic nerve sheath meningioma - Optico-ciliary shunts.
• Occasionally optic nerve glioma - Optico-ciliary shunts.
• Postneuritic - Atrophy.
• Compression - Atrophy.
• AION - Atrophy.
• Hereditary optic atrophies - Atrophy.

Special Investigations for Optic Nerve Issues

• MRI with orbital fat-suppression techniques in T1-weighted images
• Assessment of electrical activity of visual cortex created by retinal stimulation (Visually evoked potential)

Classification of Optic Neuritis

• Retrobulbar neuritis (normal disc):
  • Demyelination (most common)
  • Sinus-related (ethmoiditis)
  • Lyme disease
• Papillitis (hyperaemia and oedema):
  • Viral infections and immunization in children (bilateral)
  • Demyelination (uncommon)
  • Syphilis
• Neuroretinitis (papillitis and macular star):
  • Cat-scratch fever
  • Lyme disease
  • Syphilis

Leber Hereditary Optic Neuropathy (LHON)

• Degeneration of the retinal ganglion cells and their axons.
• Asymptomatic until visual blurring develops.

Specific Mutations in LHON

• Leber’s Hereditary Optic Neuropathy (LHON) – frequency ~1/30,000, making it the most common mitochondrial disease.
• Bilateral loss of vision caused by degeneration of the optic nerve.
• Susceptibility of retinal ganglion cells.
• Specific mutations in genes encoding components of Complex I of the oxidative phosphorylation system.
• Three most common primary mutations:
  • G11778A in ND4 gene (69%, most severe)
  • G3460A in ND1 gene (13%, less severe)
  • T14484C in ND6 gene (14%, least severe)
• Other 5% are considered secondary mutations.

Penetrance and Modifying Factors in LHON

• Penetrance is incomplete – 50-60% in males and 10-20% in females.
• Factors influencing penetrance:
  • Mutational load, tissue distribution, and threshold effect: a threshold level of ~60% mutant mtDNA may be required.
  • mtDNA background - haplogroups.
  • Nuclear genes – likely to be X-linked.
  • Environmental factors – ?alcohol, tobacco.
• General guidelines:
  • More mutant mtDNA; more likely to be penetrant!
  • More mutant mtDNA; more likely to be transmitted!

Specific Mutations and Syndromes

• MILS (Maternally-Inherited Leigh Syndrome):
  • Progressive neurologic disease with motor and intellectual developmental delay.
  • Brainstem and/or basal ganglia disease.
  • Characteristic neuropathological features.
  • Onset 3-12 months, death by 2-3 years.
  • Associated with various mtDNA genes, including ATP6.
• NARP (Neurogenic Muscle Weakness, Ataxia, and Retinitis Pigmentosa):
  • Peripheral neuropathy.
  • Cerebral and cerebellar atrophy.
  • Variable ocular manifestations.
  • Later childhood or adult onset.
  • Mutations in ATP6 gene.

Intermediate Phenotypes

• Any combination of NARP and MILS, along with other symptoms of mtDNA disease.
• Specific mutations: m.8993T>G/C.

Mutations in ATPase Subunit 6 Gene (Complex V)

• Common mutation in ATPase subunit 6 gene (complex V) – m.8993T>G or m.8993T>C.
• Minimal tissue-dependent and age-dependent variation.
• Phenotype predictability based on mutation load:
  • Up to 60% - unaffected.
  • 60-90% - NARP.
  • >90% - MILS.
• MILS/NARP are multi-system disorders with a continuum of phenotypes dependent upon Mutational Load!

Specific Mutations – m.3243A>G

• Associated with:
  • MELAS (mitochondrial encephalo-myopathy syndrome with lactic acidosis and cerebro-vascular accident episodes).
  • Maternally inherited diabetes with deafness.
  • Maternally inherited cardiomyopathy.
  • CPEO (chronic progressive external ophthalmoplegia).
• Very diverse phenotypes, including disorders of the brain, ears, eyes, skeletal muscle, cardiac muscle, and metabolism.

MT-TL1 Gene - tRNA Leucine

• Associated with specific mutations, like m.3243A>G
• Factors:
  • Mutational load - variable within families.
  • Tissue distribution – muscle in CPEO, nerves in MELAS.
  • Threshold effect - certain tissues may be more susceptible to mitochondrial dysfunction than others.
  • Modifying factors – mitochondrial, nuclear, environmental.
• Progression – sequential muscle biopsies show increased levels of mutant mtDNA.

Heteroplasmic and Homoplasmic Cells

• Heteroplasmic cells: Contain a mixture of organelle DNA.
• Homoplasmic cells: Carry only one type of organelle DNA.
• Random partitioning of organelles during cell division is the basis of mitotic segregation.

Maternal Inheritance of MERRF

• MERRF (myoclonic epilepsy and ragged red fiber disease) is maternally inherited.
• Symptoms include uncontrolled jerking, muscle weakness, deafness, heart problems, kidney problems, and progressive dementia.
• Pedigree analysis shows maternal inheritance and variations in the severity of symptoms.

Disease Phenotypes and mtDNA Ratio

• Disease phenotypes reflect the ratio of mutant-to-wild-type mtDNAs and the reliance of cell type on mitochondrial function.
• MERRF patient:
  • Heteroplasmic mitochondrial tRNA mutation.
  • Random partitioning.
  • Different tissues are affected differently.

Other Human Mitochondrial Diseases

• Kearns-Sayre Syndrome (KSS):
  • Symptoms in eyes, muscles, heart, brain.
  • Deletion mutation in mtDNA.

mtDNA Deletion Syndromes

• Three overlapping phenotypes:
  • Kearns-Sayre syndrome (KSS) – multisystem disorder, childhood onset, retinopathy, and ophthalmoplegia.
  • Pearson syndrome – sideroblastic anaemia, exocrine pancreas dysfunction, death in infancy.
  • CPEO – variable myopathy, ptosis, and ophthalmoplegia.
• No specific region of mtDNA involved; at least one tRNA gene is deleted.
• Usually flanked by short repeat sequences.
• Usually de novo, rarely inherited.
• Common deletion of 4977 base pairs.

Nuclear DNA Mutations

• Deficiency of ETC – AR Leigh syndrome.
• Deficiency of mtDNA maintenance – AD CPEO – POLG1.
• mtDNA depletion syndrome – DGUOK – SUCLA2.

Key Understanding Points

• Mitochondria, its genome, and role in energy metabolism within a cell.
• Mitochondrial inheritance - Homoplasmy and Heteroplasmy, penetrance, significance of matrilineal (maternal) inheritance.
• General categories of defects arising from mutations in mitochondrial genes (MERRF, MELAS, LHON etc.).
• General categories of defects arising from mutations in nuclear genes that code for mitochondrial proteins.
• Significance of the regulation of mitochondrial genes by nuclear proteins.