19.03.17 Mitochondrial inheritance Flashcards
What is a mitochondria (MIT)?
- Double membraned organelle
- Produces cellular energy (ATP) via the oxidative phosphorylation pathway (OXPHOS)
- have key functions in calcium signalling, regulation of cellular mechanisms, haem and steroid synthesis and apoptosis
mtDNA - key facts
- 16.6kb circular dsDNA molecule
- It encodes 37 genes
- Contains no introns
- Packaged into protein-DNA complexes (nucleoids) that are found in inner MIT membrane
- Inefficient mtDNA repair, increased replication and exposure to reactive oxygen species, means you get a high mutation rate (10x greater then nDNA)
- mtDNA is maternally inherited
- Each cell contains 100-10,000 copies of mtDNA (more copies the higher energy demand the cell has)
- Can have multiple different copies of mtDNA (heteroplasmy) or can all be identical (homoplasmy)
- Any variants can be found in different levels of heteroplasmy across different family members of tissue types
3 main classes of MIT disease
- Primary MIT disease (variants in mtDNA) - also get secondary MIT disease where you get variants in nDNA which then indirectly affect MIT function
- Neurodegeneration
- Cancer
MIT disease - key facts
- Heterogeneous group of disorders that are caused by dysfunction of the MIT respiratory chain
- i.e. when cell can’t produce enough ATP it can change pyruvate to lactate which causes sytemic lactic acidosis
- MIT disease can be caused by nDNA or mtDNA
- Get asymptomatic carriers of pathogenic mtDNA variants - 1/1000 births get a de novo mtDNA mutation
- If heteroplasmic, must reach threshold to show phenotype
- Commonly affect tissues with high metabolic demand - nervous system, skeletal muscle or heart, beta cell sin pancreas (diabetes), cells in cochlea (deafness), and renal tubes (kidney dysfunction)
- mtDNA variants typically present in childhood, nDNA variants later in adulthood
- Testing is usually carried out on DNA extracted from blood, but can be more helpful to test affected tissues
Primary MIT disease
- Deletion syndromes - tend to get deletion of multiple genes (e.g. Pearson syndrome) - often heteroplasmic and de novo
- Point mutation syndromes - tend to be heteroplasmic and maternally inherited. E.g. MELAS, NARP, MIDD - get common mutations in each syndrome that labs screen for
Secondary MIT disease
1) Nuclear variants that affect MIT maintenance and expression
- POLG and POLG2 variants disrupt MIT specific DNA polymerase activity
- Also get variants that affect nucleoside transport, salvage and synthesis - as mtDNA replicates continually it requires a continuous supply of dNTPs.
- Anything that disrupts this supply can cause mtDNA depletion (less copies), multiple mtDNA deletions, or a mixture of the two
- This leads to both AR and AD conditions
2) MIT dysfunction without affecting MIT maintenance and expression
- Variants in components of the respiratory chain
- Variants that affect MIT dynamics (fusion and fission) - this changes number and distribution of MIT, communication within and between MIT and quality control
MIT neurodegenerative disorders
- Parkinson’s - often see mtDNA deletions in neurons of PD patients
- Alzheimer disease and MS patients often have mtDNA deletions in neurons caused by respiratory chain dysfunction
How do we diagnose and test MIT disorders?
1) Histochemistry - use gomori trichrome stain on muscle biopsy - look for ragged red fibres throughout muscle fibre
- Test for specific MIT enzymes (SDH and COX)
- Normal results do NOT rule out MIT disease
2) Biochemistry - uses muscle biopsy to look for rates of flux, substrate oxidation and production of ATP (i.e. OXPHOS deficiency).
3) Genetics
- mtDNA rearrangements - long-range PCR to detect single deletions and duplications or multiple ones (multiple deletions may be due to nDNA variants, e.g. POLG, POLG2)
- Common mtDNA point mutations - restriction digest, then seq or pyroseq
- Mutation searching - real-time PCR to look at mtDNA copy number (if increased, possibly mtDNA variant)
- Need to be careful of sensitivity of assay versus level of heteroplasmy
- Majority of suspected primary MIT patients mtDNA results are NMD - so also need to look at nuclear DNA
What tissue to test?
- Most testing is on blood
- Muscle is the best but isn’t easy to collect
- Negative test on blood DNA may just mean mutation not detectable in blood - always good to request other tissue sample for re-testing if diagnosis strongly suspected
- In some conditions (i.e. MELAS), urine is as good as muscle as mutations levels are very similar
Prenatal diagnosis for MIT variants
- Hard to interpret (due to heteroplasmy)
- CVS sample mutation level may not reflect that of actual fetal tissue (and this can even change over development)
- Can’t predict level of impairment
- Can use oocyte donation or nuclear transfer to prevent transmission
MIT disease treatment and prevention
- No available therapy so focus is on prevention (PND and PGD)
- Therapies aimed to reduced carry-over of mutant mtDNA
Options are: - Oocyte donation fetilized in vitro with fathers sperm (suitable for homoplasmic patients or high levels of heteroplasmy)
- PND - Natural fertilization and then CVS or amnio to detect level of mutant mtDNA (suitable for patients with low heteroplasmy levels)
- PGD - In vitro fertilization with patients oocyte and fathers sperm - then determine mutation level in each early embryo - implant only heathly embryos
- MIT donation - maternal nuclear genome transferred out of oocyte, and placed into donor oocyte thats had its nuclear genome removed and then fertilized in vitro with fathers sperm (suitable for homoplasmic patients or high levels of heteroplasmy)
- Also developing therapies to correct primary mtDNA defects but this is hard as they have to penetrant double membrane of MIT to get to mtDNA
Recent advances in MIT therapies
- In 2015 government allowed researchers in UK to use fertility treatment based on mtDNA replacement therapy.
- This is where nuclear genome from the oocyte/embryo of an affected woman is transplanted into a donor enucleated oocyte/embryo with healthy MIT.
MIT genetic bottleneck
- During primary oocytes production, a select number of mtDNA molecules are transferred into each oocyte
- Oocyte maturation is associated with the rapid replication of that mtDNA population
- This can change the ratio of normal/mutant mtDNA in each oocyte, thereby affecting the level of heteroplasmy between generations of the same family
- So an affected mother with a high level of heteroplasmy can have embryos with very high levels (affected), moderate levels (mildly affected) and low levels (unaffected) of mutation
