Session 3: Mitochondrial Inheritance And Disease Flashcards
what is a primary mitochondrial disease?
Disease due to a mtDNA mutation- affects the mitochondrial respiratory chain and OXPHOS (Oxidative phosphorylation)
what is the pathogenesis of mitochondrial disease?
Disease is due to
- low energy production
- ROS generation - reactive oxygen species
- lactic acidosis
what is the incidence and age of onset of mitochondrial disease?
Mitochondrial diseases can be present at birth, butcan also occur at any age.
Incidence:
12.5 per 100,000 in adults
4.7 per 100,000 in children
What is the phenotype of mitochondrial disease?
can affect any tissue
most affected are those with the highest energy demand e.g. brain and muscle
CNS
encephalopathy
seizures
dementia
stroke-like episode
ataxia
depression
EYE
opthalmoplegia
cataracts
ptsosis
optic atrophy
CARDIAC
hypertrophic cardiomyopathy
dilated cardiomyopathy
GI
dysphagia
psuedo obstruction
constipation
hepatic failure
ENDOCRINE
diabetes
hypothyrpidism
gonadal failure
PNS
myopathy
neuropathy
HEARING
sensironeural deafness
describe the mitochondrial genome
replicates independently of nDNA
circular- 16.6Kb long and
codes 37 genes- 13 mitochondrial peptides, 22tRNAs and 2rRNAs
no introns and so splicing is not a mechanism of disease.
maternally transmitted
each cell has 100-100,000’s of copeis of the MT genome
can be heterplasmic or homoplasmic
The level of mutant mtDA can vary within individuals from the same family and between tissues in the same individual due to the mitochondrial bottleneck where WT and Mut mt is randonly distributed to daughter cells - this can be uneven resultin in cell lines with higher or lower levels of heteroplasmy
why may a mt variant not be detected in blood?
some variants are lost from blood due to rapid mitotic division.
e.g. m.3243A>G
- most common mtDNA mut and found in MELAS and MIDD
- this makes genetic testing difficult and need to carefully consider the choice of tissue
what is the function of the mitochondria?
Produces energy for the cell via the oxidative phopshorylation pathway (OXPHOS)
also involved in ca2+ signalling, cellular metabolism, haem and steroid synthesis
why is there a high mutation rate in mitochondria?
- inefficient damage repair
- proximity of the DNA to OXPHOS resulting in damage from ROS
- high replicative rate
what is OXPHOS?
the OXPHOS pathway transfers electrons through e- transport carriers to create an electrochemical gradient across the MM which is used to drive the production of ATP from ADP
What is the heteroplasmy threshold for mitochondrial disease.
For mtDNA variants to manifest with a phenotype there is a threshold level of mutant that needs to be present
~40-60% for mtDNA deletions
~90% for point mutations in mtDNA tRNAs
what are the 3 mitochondrial deletion diseases and there characteristics?
- de novo
- heteroplasmic
- several genes deleted
Pearsons- anemia, pancytopenia, lacic acidosis, pancreatic failure (onset in infancy)
Kearns-Sayre- myopathy deafness, opthalmoplegia, cardiomyopathy (adult onset)
CPEO- opthalmoplegia, ptosis, imparied eye movement
what are secondary mitochondrial disease?
due to nDNA defects and involves genes that have a direct effect on the function of the mitchondria
Give an example of nDNA mutation affecting mtDNA maintenance and expression
direct effects e.g. POLG, POL2 (specific mtDNA polymerase and TWNK
indirect effect e.g. SLC25A4 and RRM2B- affect nucleoside transport, synthesis and salvage
- mtDNA needs a supply of dNTPs for replication, im mitotically active tissue this is provided by import from the cytoplasm. In non-mitotically active tissue the dNTPS are obtained from the salvage pathway- these differences result in tissue specific phenotype
Give an example of nDNA mutation resulting in mt dysfunction without affecting mtDNA maintenance or function?
Mutations in the assembly factors of the respiratory chain e.g SURF mutations in MELAS
mutations in genes involved in mt dynamics e.g. fusion and fission which in turn perturbs the number and distribution of mitochondria
e.g. MFN2 in CMT (fusion)
OPA1 in optic atrophy (fission)
what are the mtDNA point mutation diseases?
maternally inherited (except AID- sporadic)
heteroplasmic
most common mut is m.3243A>G in MEALS and MIID
MELAS- myopahty, encephalopathy, acidosis and stroke like episodes
MERRF- myoclonic epilepsy and ragged red fibres
NARP- neurogenic weakness, ataxia and retinitis pigmentosa
MIDD- maternally inherited diabetes and deafness
AID - amnio glycoside induced deafness
LHON- leber hereditary optic neuropathy
How is mt disease associated with neurodegenerative disorders?
Parkinsons- mtDNA deletions are observed in neurons of parkinsons patients and mutations in genes involved in mitochondrial function are mutated in early onset familal parkinsons
alzheimers - respiratory chain dysfunction due to mtDNA mut found in neurons of patients
How can mt be used as a cancer biomarker?
WARBURG EFFECT
tumours preferentially use glycolysis to produce ATP and injury of the respiratory chain is a key event in carcinogenesis
mt genome could be used as an early biomarker of cancer as mutation does not appear to be restricted to certain cancer types.
What approach is used for diagnosis of mitochondrial disease?
considers phenotype and family history and often requires several approaches including biochemistry, immunohistochemistry, nuclear and mitochondrial DNA testing
what are the requirements and usage of histochemistry in mitochondrial disease diagnosis?
Needs muscle biopsy
can stain for ragged red fibres using a gromi trichome stain (sub sarcolemma collection of MT)
can test for the activity of specific mitochondrial enzymes
- SDH (succinate dehydrogenase) loss is indicative of a complex II deficiency
- COX (oxidase) subunitss encoded by both nuclear and mitchondrial DNA
Normal IHC does not exclude a diagnosis and can see age related mitchondrial defects so need to consider this in older patients
what are the requirements and usage of biochemistry in mitochondrial disease diagnosis?
requires muscle biopsy- want a sample enriched for mt
can measure rates of flux, substrate oxidation and AT|P generation and can measure the level of activity of each XOPHOS substrate seperately
Need a lot of sample (50-100mg) may not detect subtle OXPHOS deficiencies especially when mosaic and only a few muscle fibres are affected
How are mt DNA rearrangements detected?
- includes sinlge dels and dups and multiple mt DAN deletions
- long range PCR is the primary method for detection and may also detect age related mt deletions
the presence of multiple mtDNA deletions may indicate that there is a deletion in a nDNA gene involved in mtDNA maintenance e.g. POLG, ANT1, TWNK
How are mt point mutations detected?
common mt mutations can be detected by sequencing
- technique need to be sensitive to detect low heteroplasmy (mosaicism)
What is mutation searching for mitchondrial disease?
real time PCR can be used to detect mt copy number- an increase in mtDNA CN suggests that the moelcualr effect is likely to be located in the mitochondrial genome, hence whole mtDNA genome sequencing may be considered
need to consider heteroplasmy and use a technique that is sensitive to low level mutation
85-90% of patients with a suspected primary mitochondrial disease do not have a pathogenic mutation detected in the mdt genome highlighting the importance of considering an nDNA mutation- screen genes involved in OXPHOS or mt genome maintenance
NGS is proving pivital in mt disease diagnosis and >250 nDNA genes have now been shown to cause mitochondrial disease
What are the considerations for mt disease testing (sample type)?
Muscle is best but may not be practical or desired by the patient
- however failure make a diagnosis in blood does not mean the patient does not have mt disease, just that the mutation is not detectable in blood
- many labs include caveat on their reports to this affect and request a muscle sample if a diagnosis is still strongly suspected.
Urine can be used for some mutations e.g. m.3243A>G in MELAS as levels in urine correspond with the level in blood.
Give examples of the importance of tissue type in mt disease diagnosis
CPEO- mtDNA deletions are only detected in skeletal muscle
KSS- mtDNA deletions can be detected in all tissue so blood can be tested
LHON- pateints have homoplasmic mutations and these are inherited by all mother offspring but only 50% of males and 10% of female offspring express the phenotpye demonstrating the importance of other genetic modifiers in the expression of mitchondrial disease
what are the considerations for prenatal diagnosis for mitochondrial disease?
mtDNA heteroplasmy can make interpretation of prenatal results complicated
- a heteroplasmic point mutation can be transmitted in variable amounts
- the level of heteroplasmy in a CVS sample may not accurately reflect the heteroplasmy in the fetus
- if a mutation is detected it is not possible to predict the severity of the disease or if the baby will be affected at all.
What are the options for presenting transmission of mitochondrial disease?
oocyte donation/nuclear transfer
where the nuclear genome from the oocyte of an affected mother in transfered into a donor enucleated oocyte with healthy mtDNA
3 parent babies
What are the treatments for mitochondrial disease?
No effective treatment so emphasis is on prevention by PGD/PND
germline therapies are being developed to limit the carry over of mitochondrial DNA
-It has been demonstrated to be technically possible to transmit <3% of maternal mtDNA with spindle chromosomal transfer in primates and pronuclear transfer in preimplantation human embryos
therapies targeted at correcting mtDNA mutations are difficult to develop as they need to be specific and be able to cross the mitochondrial double membrane- 1 way that has been developed to overcome this is to engineer allotypic expression of mt genes where the WT gene is expressed in the nucleus but includes a mitochondrial targeting sequence
When was mitochondrial donation licenced in the UK?
Feb 2015- house of lords voted to allow British researchers to pursue investigation into fertility treatment using mtDNA replacement therapy. Backed by the HEFA who granted licences for experimental use of this technique in humans
2018- HEFA grants a licence for mitochondrial donation to the wellcome centre for mitochondrial research. UK becomes the first country to to leaglly offer the procedure to a patient
what is the mitochondrial bottleneck?
During the production of oocytes a random selection of mt are transferred to each oocyte- can be uneven distribution between mut and WT
- oocyte maturation is associated with the rapid replication of the mtDNA population
the restriction-amplification event can lead to random shift of mtDNA load between generations and is responsible for the variation in heteroplasmy levels between affected offspring and mothers
what are the reproductive options for women with a pathogenic mt DNA mutation?
- oocyte donation
- prenatal diagnosis- suitable for patients with low levels of heterpolasmy
- PIGD- embryo sampled at cleavage or balstocystr stage- suitable for patients with low levels of heteroplasmy
- mitochondrial donation- fertilised egg and removal of nuclear genome and transfered to a enucleated feritlised donor oocyte with mt DNA remaining- sutiable for homolasmic or high heterplasmy patients
How large is the mt genome, how many genes does it code for and how many protein coding genes?
16.6kb
37 genes
13: all proteins involved in OxPhos. Remainder are 2 rRNAs and 22 tRNAs.
What is unusual about mitochondrial termination codons?
They are added post-transcriptionally
Why does mtDNA have a higher mutation rate(10x) than nuclear DNA?
It is located closer to the sites of reactive oxygen species generation, so more oxidative damage occurs.
What three classes of disease are related to mitochondrial dysfunction?
Primary mt disease
Neurodegeneration
Cancer
What is the carrier rate for mitochondrial disease?
1/200
What is the required clinical threshold for deleted mtDNA molcules
50-60%
What is the required clinical threshold for point mutation in tRNA genes?
> 90%
Which organ systems are frequently affected? Why?
Renal tubules
Skeletal muscle
Cardiac muscle
Neurons
Cochlear hair cells
They are highly post-mitotically active and have high energy demands
What is the difference in age at presentation between mtDNA mutations and nDNA mutations?
mtDNA mutations tend to appear in childhood; nDNA mutations in adulthood.
Name the common mtDNA disorders. State whether they show disease in heteroplasmic or homoplasmic forms, and name their common mutation.
Deletion disorders:
KSS - Several deleted genes, heteroplasmic
CPOE - Several deleted genes, heteroplasmic
Pearson - Several deleted genes, heteroplasmic
Point mutation disorders:
LHON - MT-ND1 (m.3460G>A), ND4 (m.11778G>A), ND6 (c.14484T>C), hetero and homoplasmic
MELAS - MT-TL1 (m.3243A>G) Heteroplasmic
MERFF - MT-TK (m.8344A>G) Heteroplasmic
MIDD - MT-TL1 (m.3243A>G), Heteroplasmic
AID - MT-RNR1 (m.1555A>G), hetero and homoplasmic
Leigh syndrome - MT-ATP6 (m.8993T>C)
NARP - MT-ATP6 (m.8993T>G) Heteroplasmic
For each mt referral, state whether the variant can be detected in blood or muscle.
KSS - Muscle
Pearson - Blood
CPOE - Muscle
LHON - Blood
MELAS - Muscle
MERFF - Blood or Muscle
AID -Blood
MIDD - Muscle
Leigh - Blood
NARP - Blood
Name some nuclear genes associated with mitochondrial DNA expression and maintenance.
How do they cause disease?
POLG, POLG2, SLC25A4
They reduce the amount of ATP available to the mitochondria, through lack of nucleoside salvage and supply, and force the cells to enter lactic acidosis.
Name some nuclear genes that indirectly cause mitochondrial disease
How do they cause disease?
SURF1 causes Leigh syndrome - affects assembly factors of the respiratory chain.
MFN2 causes CMT2A by affecting fusion.
OPA1 causes LHON by affecting fission
How can mtDNA mutations relate to ageing?
Multiple mutations accumulate over the lifetime of a person and can cause cellular dysfunction.
How can mtDNA act as a cancer biomarker?
Increased expression of mitochondrial genes is indicative of increased cellular activity. Tumours are ATP-hungry and mutations affecting the respiratory machinery is a key event in carcinogenesis.
What histochemical methods can be used for mitochondrial diagnosis?
Stain for enzymes in the OxPhos pathway, or for ragged red fibres. Need muscle biopsy.
What biochemical methods can be used for mitochondrial diagnosis?
Can test rates of oxidation and ATP production.
Need muscle biopsy
What genetic methods can be used for mitochondrial diagnosis?
Mitochondrial rearrangements
LR_PCR, Southern blot
Common point mutations
Restriction digest if mutation affects restriction site
Sequencing
Novel mutations
Sequencing
RT-PCR
What needs to be taken into account when testing?
Tissue type being screened
Predicted level of heteroplasmy/homoplasmy
Sensitivity of test
Age of patient
What alternative tissue can be used as an alternative to muscle?
Urine - especially for m.3243A>G in MELAS/MIDD.
What considerations need to be taken into account when performing prenatal diagnosis?
CVS cells may not reflect foetus.
Cannot predict accurately how severely the child will be affected
