18.03.17 Mitochondrial inheritance

Question 1

What are some of the functional properties of mitochondria?

Answer 1

1. Sub-cellular double-membrane organelles responsible for producing cellular energy in the form of ATP via the oxidative phosphorylation pathway (OXPHOS).
2. Location for important biochemical pathways: tricarboxylic acid (TCA) cycle, and parts of urea cycle.
3. Regulation of apoptosis, cytosolic Ca concentration and Fe-S cluster biogenesis.

Question 2

What are the properties of mtDNA, inc. size, no. genes etc.

Answer 2

16.6kb circular dsDNA molecule

encoding for 37 genes:

Question 3

How many copies of the mt genome are present in each cell?

Answer 3

Each cell can contain up to several thousand copies of the mt genome (depends on energy demand of tissues).

Question 4

Which genes do mtDNA encode for?

Answer 4

13 polypeptides of the OXPHOS system,
2 ribosomal RNAs
22 tRNAs (see Fig. 2).

Question 5

Describe some of the properties of mt genes, including relative mutation frequency and inheritance.

Answer 5

1. No introns, very few non-coding bases, termination codons are created post-transcriptionally by polyadenylation.
2. Higher mutation frequency (~10x) than nDNA (close proximity of the mtDNA mutations to OXPHOS on inner mt membrane = increased susceptibility to damage through leakage of reactive oxygen species).
3. MtDNA is almost exclusively transmitted through the maternal line.

Question 6

What is meant by the terms heteroplasmy and homoplasmy?

Answer 6

The mtDNA can all be identical (homoplasmy). Alternatively there can be a mixture of two or more mt genotypes (heteroplasmy).

In these cases there is typically a threshold level where the mutation becomes clinically significant i.e. 50-60% for deleted mtDNA molecules; >90% point mutations in tRNA. However, a threshold level of <25% was detected in an individual affected with m.5545C→T (Sacconi et al., 2008).

Question 7

Which three major classes of disease does mt dysfunction play a role in?

Answer 7

1. Primary mt disease
2. Neurodegeneration
3. Cancer

As well as contributing to the decline in tissue integrity with age.

Question 8

Give a brief overview of mt disease.

Answer 8

1. Incidence <1/6500
2. ~1 in 200 asymptomatic carriers
3. Clinically heterogeneous group of disorders which arise as a result of dysfunction of the mt respiratory chain

Question 9

What is the cellular effect of dysfucntion of the mt respiratory chain?

Answer 9

If a cell cannot produce enough ATP via OXPHOS, they may shunt pyruvate to lactate causing systemic lactic acidosis

Question 10

Describe the clinical manifestations of mitochondrial disease, including age of onset.

Answer 10

Often unobserved; some disease causing mutations affect all copies of the mt genome (homoplasmic mutation); others only present in a proportion of mt (heteroplasmic mutation).

Many involve multiple organ systems and frequently effect tissues with high metabolic demand e.g. nervous system, skeletal muscle or heart (Fig. 4).

Other tissues include β-cells of pancreas (diabetes); inner hair cells of cochlea (deafness); renal tubules (kidney dysfunction).

Some only affect a single organ (e.g. the eye in LHON, see table below).

May present at any age, recent advances have shown that many mtDNA disorders present in childhood, while many nuclear genetic mt disorders present in adult life.

Question 11

Give two examples of primary mitochondrial disease caused by deletions in the mt genome.

Answer 11

All three have several deleted genes, are heteroplasmic and are usually sporadic.

1. Pearson syndrome: pancytopoenia, anaemia, lactic acidosis, pancreatic failure, infancy onset.
2. Kearns-Sayre syndrome: progressive myopathy, deafness, opthalmoplegia, cardiomyopathy, adult onset
3. CPEO (chronic progressive external opthalmoplegia) Opthalmoplegia, ptosis, impaired eye movement

Question 12

Give three examples of primary mitochondrial disease caused by maternally-inherited heteroplasmic (only) point mutations in the mt genome.

Answer 12

1. MELAS (Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) point mutations in MT-TL1
2. MERRF (Myoclonic epilepsy and ragged-red fibres) - ME, myopathy, cerebellar ataxia. PM in MT-TK.
3. NARP (Neurogenic weakness, ataxia and retinitis pigmentosa) sensory neuropathy, ataxia, RP. PM in MT-ATP6
4. MIDD (Maternally-inherited diabetes and deafness): Diabetes, deafness. PM in MT-TL1

Question 13

What is an example of a disease caused by sporadic heteroplasmic point mutations in the mt genome?

Answer 13

Leigh/Leigh like: Encephalopathy, lactic acidosis. PM in MT-ATP6.

Question 14

Which two diseases are caused by maternally-inherited heteroplasmic or homoplasmic point mutations in the mt genome?

Answer 14

1. AID (aminoglycide-induced deafness: deafness. PM in MT-RNR1
2. LHON (Leber hereditary optic neuropathy): Sub-acute bilateral visual failure and optic atrophy. Point mutations in MT-ND1, MT-ND4, MT-ND6.

Question 15

> 1200 proteins in the mt are encoded by nDNA genes, in which defects can affect mtDNA maintenance and expression leading to mtDNA depeletion and multiple deletions. Give examples.

Answer 15

1. POLG, POLG2, PEO1 - all have direct effects
2. Indirect effects include disruption of nucleoside transport, salvage and synthesis e.g. SLC25A4 (encodes ANT the ATP/ADP translocator) and RRM2B- Since mtDNA continually replicates independently of nDNA, it requires a continual supply of dNTP’s. In mitotically active tissue, these are largely provided by import from the cytoplasm. However, in post-mitotic tissue, the mt is dependent on the salvage pathway again resulting in tissue specific phenotypes.

Question 16

Give examples of genes that influence mt dysfunction without affecting its expression or maintenance.

Answer 16

1. Components and assembly factors of the respiratory chain: e.g. SURF1 mutations in Leigh syndrome, MEGDEL syndrome
2. Factors involved in mt dynamics including fusion and fission which in turn perturb…
    The number and distribution of mt.
    Communication within and between mt.
    Quality control (excessive fission targets a mt for mitophagy).
3. Mutations in the fusion specific gene MFN2 have been implicated in CMT2A whilst mutations in the fission related gene OPA1 are associated with dominant optic atrophy (DOA) (Chen et al 2014).
4. Components involved in other functions of the mt, i.e. metabolic (such as steroid synthesis), apoptotic, autophagy (perturbation may play a role in HD).

Question 17

Give examples of mtDNA mutations seen in neurodegenerative disorders.

Answer 17

1. Parkinson disease – number of genes important in early onset familial form encode proteins essential for mt function e.g. PINK1 and parkin; mtDNA deletions observed in neurons in PD patients.
2. Respiratory chain dysfunction caused by mtDNA mutations reported in neurons of patients affected by Alzheimer disease and multiple sclerosis.

Question 18

How is mtDNA implicated in ageing?

Answer 18

Causality remains unclear – study of mutator mouse (homozygous knock-in mutation affecting highly conserved residue of mt polymerase-γ) shows 3-5 fold increase in frequency of mt point mutations and increased incidence of large-scale rearrangements (a linear form of mtDNA). After 25 wks it manifests signs of premature aging; however it does not show oxidative stress or ROS-induced damage. There is evidence of increased caspase 3, a marker of apoptosis, in a variety of tissues suggesting the increased mutation burden could be inducing apoptosis.

Greaves, 2012 suggests that clonal expansion of mtDNA mutations that occur throughout life, due to either oxidative damage or errors of the mtDNA polymerase, cause cellular dysfunction rather than accumulations of new mutations with age.

Question 19

What is the Warburg effect?

Answer 19

Warburg effect – tumour cells preferentially utilise glycolysis to produce ATP and injury to the respiratory machinery is a key event in carcinogenesis (Warburg, 1956).

Question 20

How might the mtDNA be useful as an early biomarker of cancer?

Answer 20

1. MtDNA offers a number of advantages for early cancer detection, somatic mutations in mt are common in human cancers and unlike nuclear genes do not appear to be restricted to cancer type (Maitra et al., 2004).
2. Secondly due to the high copy number of mtDNA, detection is easier in clinical samples where the quantity of DNA may be restricted. It has been reported that mutations occur early in multistep tumour progression (Jeronimo et al., 2001).

Question 21

Which pathology techniques can be used to diagnose mt disease?

Answer 21

1. Histochemistry
2. Biochemistry
3. Molecular genetics

Question 22

Describe the histochemistry tests that can be used in mt disease diagnosis?

Answer 22

Need muscle biopsy.

1. Gomori trichrome stain - ragged red fibre (subsarcolemmal collection of mt).
2. Test for specific mt enzymes:
   a SDH (succinate dehydrogenase) - indicative of complex II deficiency.
   b) COX (cytochrome c oxidase) - subunits encoded by both mt and nuclear genomes.

Normal histochemistry does not exclude mt disorder.
May also see normal age-related defects.

Question 23

Describe the biochemical tests that can be used in mt disease diagnosis?

Answer 23

Need muscle biopsy; prepare enriched sample enriched for mt.

Can measure rates of flux, substrate oxidation and ATP production; can measure activity of each complex separately.

Question 24

Describe the molecular genetics tests that can be used detect MtDNA rearrangements?

Answer 24

Single deletions, duplications and multiple mtDNA deletions.

1. long-range PCR is typical front line test - (may detect normal age-related deletions).
2. Southern blot - PvuII or BamHI digest and hybridise with total mtDNA.

Multiple mtDNA deletions may be due to nuclear gene mutations (POLG, ANT1 or Twinkle).

Question 25

What are the limitations to the biochemical tests for mt disease?

Answer 25

Often large quantities of muscle (~50-100mg of tissue) are required and may fail to detect subtle OXPHOS deficiencies especially when only a few muscle fibres are affected e.g. mild mosaic deficiencies.

Question 26

Describe the molecular genetics tests that can be used detect common MtDNA point mutations?

Answer 26

1. restriction digest.
2. sequencing, pyrosequencing etc.

Consideration of technique sensitivity.

Question 27

Describe the molecular genetics tests that can be used to performed mutation scanning in MtDNA?

Answer 27

Molecular effect is likely to be located in mtDNA genome hence whole mt genome sequencing should be considered.
 need to consider heteroplasmy
80-95% of patients with clinically suspected primary mt disease do not have detectable pathogenic mtDNA mutation highlighting importance of screening of nuclear genes (nuclear encoded complex subunits and nuclear encoded mt genome maintenance genes).

Next -generation sequencing strategies are proving pivotal in discovery of new disease genes and the diagnosis of clinically affected patients; pathogenic variants in >250 genes have now been shown to cause mitochondrial disease.

Best strategy targeted WES.

Question 28

What are the considerations for testing for mt disease?

Answer 28

Although the vast majority of testing is performed on blood, muscle is the best tissue to test, however this may not always practical or desired by the patient.

When testing blood it must be remembered that a negative result does not reflect an overall negative, just that mutations were not detected in blood. Many labs put caveats in reports highlighting this and requesting further tissue types if a diagnosis is strongly suspected.

Urine can be used as an alternative to muscle (especially for m.3243A>G) as levels in urine correlate with levels in muscle.

Question 29

Give examples of why it is important to test the correct tissue type for detection of mitochondrial disease?

Answer 29

1. CPEO - mtDNA deletions are confined to skeletal muscle only – muscle biopsy sample required.
2. KSS - deletions are usually present in all tissues - blood leukocytes or muscle biopsy can be used.
3. Most LHON patients have homoplasmic mutations; although all offspring inherit the mutation only 50% of males and 10% of females develop impaired vision. This indicates nuclear genetic factors are important in the expression of mt disease.

Question 30

Discuss prenatal testing for mitochondrial disorders.

Answer 30

MtDNA heteroplasmy increases the difficulty in interpreting prenatal genetic testing.

A heteroplasmic mtDNA point mutation may be transmitted in variable amounts.

The percentage level of mutant mtDNA in a CVS biopsy may not reflect that in other foetal tissues, and the level may change during development and throughout life.

If prenatal testing indicated a mt mutation, it may not be possible to reliably predict how severely the baby would be affected, or even if the baby was affected at all.

Preventing transmission - ooctye donation or nuclear transfer where the nuclear genome from the oocyte or embryo of an affected woman is transplanted into a donor enucleated oocyte or embryo with healthy mt (see Craven et al., 2011 Hum Mol Gen for a good review on recent progress in this field).

Question 31

What are the treatments available for patients with mitochondrial disease?

Answer 31

No available therapy to date to treat mt disorders hence focus is on prevention – prenatal and pre-implantation diagnosis (PND and PGD).

Development of germline therapies to limit the carry-over of mutant mtDNA. It has been shown that it is technically possible to transmit <3% maternal mtDNA with spindle–chromosomal complex transfer in non-human primates (Tachibana et al., 2012), and with pronuclear transfer in pre-implantation human embryos (Craven et al., 2010 and Fig. 5).

Therapies aimed at correcting primary mtDNA defects have the added complication of targeting the mt and penetrating the double membrane. Recent approaches have focused on allotypic expression of mt genes where the WT gene is expressed within the nucleus and engineered to contain a mitochondrial targeting sequence (this another therapeutic strategies reviewed by Farrar et al., 2013).

Question 32

Describe the forms of mitochondrial transfer which can be used in ART procedures.

Answer 32

1. Pronuclear transfer

2. Maternal spindle transfer