Comprehensive Notes on Mitochondrial Disease

Mitochondrial Disease Notes

Mitochondrial Function

  • Mitochondria are essential for:
    • ATP generation via oxidative phosphorylation (OXPHOS).
    • Calcium buffering
    • Iron-sulfur (Fe-S) cluster biogenesis.
    • Regulating cell death through the release of pro-apoptotic molecules like cytochrome c (forms apoptosome), AIF (induces nuclear chromatin condensation), and SMAC/DIABLO (inhibits XIAP, allowing caspase-3 activation).
  • Mitochondria exist as a network within the cell.
  • They are the 'engine' of the cell.
  • Mitochondrial proteins:
    • Approximately 1,200 are nuclear-encoded and imported into the mitochondria.
    • 13 are mtDNA-encoded.

Energy Generation

  • Human energy expenditure:
    • 100kcal/hour=116W100 kcal/hour = 116W
    • Oxygen consumption: 380380 Litres/day
    • ATP usage: 65kg65kg ATP/day
  • Mitochondrial inner membrane surface area: 14,000m214,000 m^2 (muscle creatinine-guanidine (MCG) = 17,718m217,718 m^2)
  • Brain energy usage: 2% of weight, 20% of energy use.
  • Diseases affecting mitochondrial function significantly impact neurological function.

Cell Metabolism: 3 Main Stages

  • Glycolysis: Occurs in the cytosol, producing pyruvate and ATP through substrate-level phosphorylation.
  • Pyruvate Oxidation: Pyruvate is converted to Acetyl CoA in the mitochondria.
  • Citric Acid Cycle (TCA Cycle): Acetyl CoA enters the cycle in the mitochondria.
  • Oxidative Phosphorylation (Electron Transport and Chemiosmosis):
    • Takes place in the mitochondria.
    • Involves redox reactions (transfer of electrons).
    • Generates ATP through oxidative phosphorylation.
  • Redox Reactions: OIL RIG (Oxidation Is Loss, Reduction Is Gain).

Oxidative Phosphorylation (OXPHOS)

  • Chemiosmotic Theory (Peter Mitchell, Nobel Prize 1978).
  • Performed by 4 electron transport chain enzyme complexes (I-IV) and ATPase (CV).
  • NADH and FADH2 (from the TCA cycle & glycolysis) are used to generate a mitochondrial membrane potential (Δψm)(\Delta \psi m).
  • Δψm\Delta \psi m drives F1Fo-ATP synthase (CV) to generate ATP.
  • ATP Yield:
    • 1 glucose molecule theoretically yields 38 ATP molecules: 2 from glycolysis, 2 from the TCA cycle, and 34 from OXPHOS.
    • In reality, the yield is typically 26-28 ATP molecules from OXPHOS.
    • TOTAL = 30 or 32 ATP.

OXPHOS Complexes and Genetics

  • OXPHOS complexes are made from both nuclear-encoded genes (nDNA) and mtDNA-encoded genes.
  • Disease-causing mutations have been identified in all 13 mtDNA genes.
  • Mutations also occur in many different nDNA-encoded genes.
  • Complexes I, III, and IV can form a ‘supercomplex/respirasome’ (SC), important for packaging, stability, and electron transfer between complexes.

Mitochondrial Genetics: Human mtDNA

  • Size: 16,569 base pairs (bp).
  • Contains 13 polypeptide genes:
    • 7 for complex I (pink).
    • 1 for complex III (orange).
    • 3 for complex IV (blue).
    • 2 for complex V (yellow).
  • 22 tRNA genes.
  • 2 rRNA genes (green).
  • D-loop: non-coding region, contains OH, PH, and PL.
  • Inheritance: Maternally inherited.
  • Copy number: Multiple copies per cell.
  • Heteroplasmy: Presence of multiple mtDNA variants within a cell.
    • Homoplasmy: 100% identical mtDNA.
  • Disease threshold: The percentage of mutated mtDNA required to cause disease symptoms.

Nuclear Disease Genes of the Mitochondrial OXPHOS System

  • OXPHOS complexes are large.
  • Require assembly factors to help build the mature complex.
  • Mutations in assembly factor genes also cause OXPHOS deficiencies and disease.

Mitochondrial Disease

  • Definition: "Any symptom, any organ, any age, any mode of inheritance".
  • Incidence: Affects approximately 1 in 4,300 live births.
  • Severity: Often severe in children and frequently fatal.
  • Treatment: Lacks effective treatments and therapies.
  • Manifestations:
    • Neurological: Encephalopathy, myoclonic seizures, stroke-like episodes.
    • Muscular: Myopathy (skeletal and cardiac).
    • Combination: Encephalomyopathy, dystonia, ataxia.

Mitochondrial Disease Symptoms

  • Encephalopathy: Disorder or disease of the brain.
  • Neuropathies: Affecting nerves.
  • ‘Stroke-like’ episodes: Ischemia (loss of blood flow).
    • Symptoms: Convulsions, visual abnormalities, numbness, hemiplegia (weakness on one side of the body), and aphasia (language impairment).
  • Myoclonic epilepsy: Seizures involving uncontrolled muscle contractions or twitching.
  • Ataxia: Loss of muscle coordination (neurological).
  • Dystonia: Sustained muscle contractions (twisting, spasms, etc., neurological).
  • Myopathy: Disease of the muscle, resulting in weakness.
  • Deafness and Blindness.
  • Lactic acidosis: Anaerobic glucose metabolism, increase in blood lactate, and decrease in pH.

Examples of Mitochondrial Disease Symptoms

  • Ataxia: (loss of coordination)
    • Mutation in nuclear gene: POLG – mitochondrial DNA polymerase (reduced mtDNA copy number and increase in mtDNA mutations).
  • Dystonia: (twisting, spasms).
  • Myoclonic epilepsy: (seizures, muscle contractions).

Lactic Acidosis

  • Glycolysis requires NAD+NAD^+.
  • NADHNADH is oxidized to NAD+NAD^+ by complex I.
  • ATP generation by OXPHOS.
  • If an OXPHOS defect is present, NADHNADH accumulates.
  • Excess NADHNADH is oxidized to NAD+NAD^+ by the reduction of pyruvate to lactate.
  • Lactate is generated as a byproduct of NADHNADH oxidation.
  • Excess lactate production leads to lactic acidosis.

Mitochondrial Fatty Acid Oxidation

  • Mitochondria oxidize fatty acids.
  • Most naturally occurring fatty acids have an even number of carbon atoms.
  • Oxidation occurs at the β-carbon (C3), hence β-oxidation.

Fatty Acid Metabolism

  • Triacylglycerols (triglycerides) are our fat storage form.
  • They have a glycerol backbone with 3 fatty acids.
  • Most triacylglycerols are “mixed,” meaning the 3 fatty acids differ in chain length & number of double bonds.
  • Lipases hydrolyze triacylglycerols, releasing 1 fatty acid at a time.
  • Free fatty acids are bound to albumin in the bloodstream.
  • Plasma membrane fatty acid transporters such as CD36 facilitate uptake.
  • Fatty acids are ‘activated’ (esterified) with Coenzyme A by Acyl-CoA Synthases (ER & outer mitochondrial membrane) to form acyl-CoA.

Mitochondrial Fatty Acid β-Oxidation

  • Involves four reactions:
    1. dehydrogenation
    2. hydration
    3. dehydrogenation
    4. thiolysis
  • Can result in ketogenesis.

Defects in Mitochondrial Fatty Acid β-Oxidation

  • Can cause disease.
  • Hypoketotic hypoglycemia: low blood sugar levels.
    • Symptoms: sweating, shaking, dizziness, confusion. Severe: unconsciousness, seizures, coma, death.
  • Rhabdomyolysis: muscle breakdown; release of K+K^+, PO43−PO_4^{3-}, creatine kinase, causing kidney damage.

Prevalence and Characteristics of FAO Defects

  • Much lower prevalence than OXPHOS disorders (1/10,000 to 1/100,000).
  • The heart can use different substrates for ATP generation but prefers FAO.
  • During fasting, the liver can generate ketone bodies from acetyl-CoA (hydroxybutyrate, acetoacetate, acetone).
  • Ketone bodies are converted back to acetyl-CoA (e.g., in the brain) and metabolized via the TCA cycle.
  • FAO deficiencies also reduce ketone formation (causing neurological defects during fasting).

Treating Mitochondrial Disease

  • No Cure for Mitochondrial Disease
  • Some symptoms can be treated, but efficacy varies between patients:
    • ‘Mito Cocktail’:
      • Ubiquinone (CoQ10) – electron carrier and antioxidant
      • L-carnitine – transports long-chain fatty acids across the mitochondrial inner membrane
      • Thiamine (B1) – PDH complex cofactor
      • Riboflavin (B2) – cofactors for OXPHOS complexes I and II
      • Folic Acid: to treat associated folate deficiency
      • Other vitamins and minerals
    • No strong evidence that it is beneficial for treating patients
  • Diet:
    • Avoid fasting (including overnight).
    • Fat (for OXPHOS disorders):
      • Ketogenic diet with variable results.
      • Increase medium-chain triglycerides (MCT), 8-10 carbon length. Easy transport into mitochondria.
    • Fatty acid oxidation in other organelles, e.g., peroxisomes.
    • Dichloroacetate (DCA): activates PDH complex to alleviate lactic acidosis. Some toxicity, now less commonly used.

Emerging Therapies

  • Synthetic antioxidant idebenone (ubiquinone/CoQ10) for Leber Hereditary Optic Neuropathy (LHON).
  • Increase mitochondrial biogenesis (more mitochondrial mass).
    • Bezafibrate (used to treat hyperlipidemia).
  • Increase NAD+NAD^+ levels with Acipimox (used to treat hyperlipidemia).
  • Reduce toxic ROS and use of KH17, elamipretide, EPI-743.

Mitochondrial Replacement Therapy (MRT)

  • For mtDNA mutations (Three-parent IVF).
  • Involves embryo stage - pronuclei transfer.
  • Two eggs - one from the mother (with faulty mitochondrial DNA) and another from the donor (with healthy mitochondrial DNA) - are fertilized with sperm from the father.
  • These form two pronuclei, which contain most of the genetic material.
  • Nuclei from the donor egg are removed - leaving egg cells with healthy mitochondria
  • Nuclei from the mother are put in the donor egg

Ethical Issues with MRT

  • Designer babies?
    • mtDNA does not determine physical traits (and regulation to stop misuse).
  • 3 Genetic parents?
    • Only 0.001% mtDNA.
  • May only change 1 base pair mutation!
  • Destruction of embryos.
  • Rights of the child (donor oocyte?).

Scientific Issues with MRT

  • Safety?
  • Mitochondria with mtDNA mutations can be transferred with pronuclei. Will this result in mtDNA disease or worse?
  • MRT in human stem cell lines can transfer low levels of mutant mtDNA (<2%), and this can repopulate cells following differentiation into skin, heart, etc.

MRT Legislation

  • Legislation passed in the UK in Oct 2015.
  • 2017: First license issued by the U.K. Human Fertilisation and Embryology Authority (HFEA) to Newcastle University for MRT
  • 25 women a year for five years
  • 2018: 2 women undergo procedure (MERRF syndrome).
  • 2023: May, at least 5 children born using MRT.
  • June 2018: Australia Senate Community Affairs References Committee recommendation to move towards legalizing mitochondrial donation.
  • Feb 2021: Public Consultation Process
  • March 2021: the Mitochondrial Donation Law Reform (Maeve’s Law) Bill 2021 introduced to Parliament by Health Minister Hunt.

MRT Implementation in Other Countries

  • 1st baby born by MRT in Mexico Sept 2016.
  • 36 yo mother, mtDNA 8993T>G (Leigh Syndrome), multiple pregnancy losses and death of offspring.
  • Mother has heteroplasmic mutation: 23% hair follicles, 24% blood, and 33% urine.
  • Spindle Transfer between oocytes, then fertilized with father’s sperm.
  • Embryo with 5.7% mutation.
  • Healthy male baby born (and healthy at 7 mo).
  • Boy’s mutation: 2.36% urine, 9.23% in foreskin.
  • Medical follow-ups but no testing of mtDNA mutant load unless necessary. No MRT Legislation!
  • June 2018: MRT performed in Ukraine, 1 baby born with 3 more pregnancies.
  • Clinical trial in Greece to test MRT as a fertility treatment for women who have had unsuccessful IVF – Jan 2019: one woman pregnant – Run by a Spanish clinic in Greece (currently illegal in Spain).

Patient Study #1

  • 15 months:
    • hypertrophic cardiomyopathy
    • Lactic acidosis (5-10 mM) (normal=2 mM, severe>4 mM)
  • Birth: Benign congenital hypotonia
  • 11 years:
    • Pigmentary retinopathy
  • Teens – present:
    • Kyphoscoliosis & Osteoporosis
    • Mild intellectual disability
    • Delayed puberty
  • P2 has very low levels of NDUFAF1, a complex I assembly factor!
  • P2 also has a complex I enzyme deficiency.
  • Patient fibroblast studies (unknown nuclear gene mutations).

Patient study #1 – NDUFAF1 Mutation

  • Maternal allele: Nucleotide c.1001 A>C (T207P)
  • Patient heterozygous with mutations in both alleles of exon 3
  • Paternal allele: Nucleotide c.1140 A>G (K253R)
  • Mutations likely to affect NDUFAF1 stability
  • Threonine: polar
  • Proline: cyclic structure, causes kinks in protein
  • Both + charge, different structure

Patient study #1 – BN-PAGE Analysis

  • Non-ionic detergent (solubilization from membrane)
  • Coomassie Blue G (provides negative charge)
  • Neutral pH and 4°C
  • Complexes remain intact in their native form
  • 36% residual complex I

Patient study #1 – Summary

  • Nuclear Gene Mutation (OXPHOS associated gene)
  • Heterozygous NDUFAF1 mutations
  • Decrease in NDUFAF1 protein levels.
  • Reduced steady-state levels of Complex I
  • Defect in Complex I assembly: mtDNA-encoded subunits ND2 and ND1 are not assembled correctly and degraded
  • OXPHOS Complex I deficiency causing severe Mitochondrial Disease
  • Nuclear gene OXPHOS defects generally present early in life with more severe symptoms

Patient Study #2

  • Female patient, currently 19 years of age.
  • Developed jerking movements at age 15.
  • Progressed to dementia, myoclonic epilepsy, ataxia
  • Metabolic Disorder?
  • Measure blood lactate
  • Lactate dehydrogenase (LDH)
  • Patient blood lactate very high: 8 mM (normal ~2 mM)

Patient Study #2 – Muscle Biopsy

  • Co-stain: Cytochrome oxidase (COX, Complex IV) – brown; Succinate dehydrogenase (SDH, Complex II), blue
  • Blue cells, no COX activity
  • Gomori Trichrome Stain
  • Ragged Red Fibres
  • Accumulation of abnormal mitochondria (compensation)

Pedigree Analysis

  • A pedigree is a family tree that describes the interrelationships of parents and children across generations
  • Inheritance patterns of particular traits can be traced and described using pedigrees

Patient Study #2 - Myopathy in the family

  • Maternal Inheritance Pattern – mtDNA mutation
  • Clues: only passed from mothers to offspring Can have affected males, but fathers have no affected offspring

Patient Study #2 – Family History

P1P2P3
age195342
symptomsrandom jerking at 15myoclonus at ~30hearing loss in 20s
Dementia√XX
Myoclonus√√X
Ataxia√√X
Hypoventilation√XX
Hearing loss√√√
Lactatehighnormalnormal
Conditionseveremediummild
Genetics%tRNALys mutation High (93%)Medium (75%)Low (63%)
  • Same mutation in different family members but with different mutant mtDNA load and clinical symptoms

Patient Study #2 – mtDNA Protein Translation

  • Cells
  • SDS-PAGE analysis
  • Cyclohexamide 2h 35S-Met
  • 13 proteins encoded by mtDNA
  • Radiolabel newly translated proteins (very low amount)
  • 35S-Methionine labelling of mtDNA-encoded translation products
  • Cycloheximide blocks cytosolic translation

Patient Study #2 – mtDNA

  • tRNALys mutation 8344T>G
  • Commonly associated with Myoclonic Epilepsy with Ragged-Red Fibres (MERRF)
  • Is % of mutation different in family members?
    • Heteroplasmy: amount of mt v’s wt
  • How does the % of mutation affect the symptoms?
    • Is muscle function affected to different degrees?

Patient Study #2 – Summary

  • mtDNA Mutation
  • tRNALys mutation 8344T>G
  • Commonly associated with Myoclonic Epilepsy with Ragged-Red Fibres (MERRF)
  • Defect in activity of all OXPHOS complexes
  • Different % of mtDNA mutation (heteroplasmy) in different family members associated with different disease severity
  • Note that mtDNA disease can present early in life with severe disease, or later in life with milder symptoms!

Patient study #3

  • 8 months
    • Following a fever developed Hypoketotic hypoglycemia (low blood sugar levels)
    • Fell unconsciousness, treated with intravenous (IV) drip and made a full recovery
  • 10 years
    • Signs of rhabdomyolysis: muscle pain and reduced kidney function
    • No other symptoms
  • 32 years
    • Developed arrhythmia (detected with electrocardiogram ECG)
    • Cardiomyopathy (detected with echocardiography)

Patient study #3 Blood spot analysis

  • Tandem mass spectrometry * internal standards (C2, C3, C4, C8, C12, and C16).
    1. free carnitine
    2. Acetylcarnitine
    3. tetradecenolylcarnitine (C14:1)
    4. Tetradecanoylcarnitine (C14:0)
    5. Hexadecenoylcarnitine (C16:1)
    6. Palmitoylcarnitine (C16:0)
    7. Linoleylcarnitine (C18:2)
    8. oleylcarnitine (C18:1)
    9. stearoylcarnitine (C18:0)
  • Acylcarnitine profile indicates VLCAD deficiency!

Patient study #3 Genetic analysis

  • Heterozygous mutations:
    • Maternal mutation: deletion of bp 842–3 in exon 8 (Frameshift)
    • Paternal mutation: G11A - splice site after exon 1; decreased mRNA
    • Loss of VLCAD expression and activity
  • VLCAD Deficiency
  • Treatment
    • Diet high in carbohydrate, low in long-chain fatty acids (e.g., fish, meat, avocado, olive oil)
    • Supplement with medium chain fatty acids (e.g., coconut oil, dairy)

Mitochondrial Disease Summary

  • Wide array of symptoms which can present at any age
  • Range of severity (lethal in early childhood – late onset adult disease)
  • Patient Study #1: Nuclear mutation in OXPHOS associated gene
    • Heterozygous (autosomal recessive)
    • Severe symptoms in early childhood
  • Patient Study #2: mtDNA mutation
    • heteroplasmy (% mutant) associated with disease severity
    • Presents later in life with milder symptoms (but can also be severe in early life)
  • Patient Study #3: Nuclear mutation in fatty acid β-oxidation (FAO) gene
    • Heterozygous (autosomal recessive)
    • Usually milder symptoms, can be managed by diet

Assessment Information

  • Opens Thursday 8th May 9 am 2025
  • Due Wednesday 14th May 8 pm 2025
  • Preparation
    • Lectures>Week 8 Mitochondrial Disease Case Study Folder
    • Review Mito Disease Case Study Notes
    • Review inheritance pattern examples