Mitochondrial inheritance and disease

Question 1

what is a primary mitochondrial disease?

Answer 1

Disease due to a mtDNA mutation- affects the mitochondrial respiratory chain and OXPHOS

Question 2

what is the pathogenesis of mitochondrial disease?

Answer 2

Disease is due to

• low energy production
• ROS generation
• lactic acidosis

Question 3

what is the incidence and age of onset of mitochondrial disease?

Answer 3

onset at age

12. 5 per 100,000 in adults
13. 7 per 100,000 in children

Question 4

What is the phenotype of mitochondrial disease?

Answer 4

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

Question 5

describe the mitochondrial genome

Answer 5

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

Question 6

why may a mt variant not be detected in blood?

Answer 6

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

Question 7

what is the function of the mitochondria?

Answer 7

Produces energy for the cell via the oxidative phopshorylation pathway (OXPHOS)

also involved in ca2+ signalling, cellular metabolism, haem and steroid synthesis

Question 8

why is there a high mutation rate in mitochondria?

Answer 8

• inefficient damage repair
• proximity of the DNA to OXPHOS resulting in damage from ROS
• high replicative rate

Question 9

what is OXPHOS?

Answer 9

the OXPHOS oathway 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

Question 10

What is the heteroplasmy threshold fro mitochondrial disease.

Answer 10

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

Question 11

what are the 3 mitochondrial deletion diseases and there characteristics?

Answer 11

• 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

Question 12

what are secondary mitochondrial disease?

Answer 12

due to nDNA defects and involves genes that have a direct effect on the function of the mitchondria

Question 13

Give an example of nDNA mutation affecting mtDNA maintenance and expression

Answer 13

direct effects e.g. POLG, POL2 (specific mtDNA polymerase and TWNK

indrect 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

Question 14

Give an example of nDNA mutation resulting in mt dysfunction without affecting mtDNA maintenance or function?

Answer 14

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)

Question 15

what are the mtDNA point mutation diseases?

Answer 15

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

Question 16

How is mt disease associated with neurodegenerative disorders?

Answer 16

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

Question 17

How can mt be used as a cancer biomarker?

Answer 17

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.

Question 18

What approach is used for diagnosis of mitochondrial disease?

Answer 18

considers phenotype and family history and often requires several approaches including biochemistry, immunohistochemistry, nuclear and mitochondrial DNA testing

Question 19

what are the requirements and usage of histochemistry in mitochondrial disease diagnosis?

Answer 19

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

Question 20

what are the requirements and usage of biochemistry in mitochondrial disease diagnosis?

Answer 20

requires muscle biospy- 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

Question 21

How are mt DNA rearrangements detected?

Answer 21

• 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

Question 22

How are mt point mutations detected?

Answer 22

common mt mutations can be detected by sequencing

- technique need to be sensitive to detect low heteroplasmy (mosaicism)

Question 23

What is mutation searching for mitchondrial disease?

Answer 23

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

Question 24

What are the considerations for mt disease testing (sample type)?

Answer 24

Muscle is best but may not be practical or desired by the pateint

• 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.

Question 25

Give examples of the importance of tissue type in mt diease diagnosis

Answer 25

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

Question 26

what are the considerations for prenatal diagnosis for mitochondrial disease?

Answer 26

mtDNA heteroplasmy can mke interpretation of prenatal results complicated

• a heteroplasmic point mutation cxan 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.

Question 27

What are the options for presenting transmission of mtichondrial disease?

Answer 27

oocute 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

Question 28

What are the treatments for mitchondrial disease?

Answer 28

No effective treatment so emphasis is on prevention by PGD/PND

germline therapies are being developed to limit the carry over of mitchondrial DNA
-It has been demonstrated to be tehcnically 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 espression of mt genes whee the WT gene is expressed in the nucelaus but includes a mitochondrial targeting sequence

Question 29

When was mitochondrial donation licenced in the UK?

Answer 29

feb 2015- house of lords voted to allow British researchers to pursue investigation into fgertility treatment using mtDNA replacement therpay. 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

Question 30

what is the mitochondrial bottleneck?

Answer 30

During the production of oocytes a random selection of mt are transfered 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

Question 31

what are the reproductive options for women with a pathogenic mt DNA mutation?

Answer 31

1. oocyte donation
2. prenatal diagnosis- suitable for patients with low levels of heterpolasmy
3. PIGD- embryo sampled at cleavage or balstocystr stage- suitable for patients with low levels of heteroplasmy
4. 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