The Mitochondrial Genome

Question 1

What is the mitochondrial genome (mtDNA)?

Answer 1

The Mitochondrial genome (mtDNA)
Double stranded circular molecule (16.6kb) (15,000x smaller than chromosome 1)
Consists of the heavy and light strand
Multicopy genome (10-100,000 copies per cell)
37 genes
- 13 oxidative phosphorylation protein subunits
- 22 transfer RNAs
- 2 ribosomal RNAs
No introns
D-loop is a non coding region where replication and transcription are initiated
Maternally inherited, no recombination

Question 2

Why do we have a mitochondrial genome?

Answer 2

The Mitochondrial genome encodes proteins of oxidative phosphorylation
Complex 1-5
And we can see the subunits that are encoded by the mitochondrial DNA; 7 for complex 1
Most of the subunits are nuclear encoded
Complex 2 is the only OXPHOS subunit which is entirely nuclear encoded

Question 3

Where does transcription start in the mitochondrial DNA?

Answer 3

Non-coding region (NCR) of mtDNA contains regulatory sequences for replication and transcription
mtDNA replication starts in Origin of heavy strand (OH).
Transcription starts at Heavy strand promoter (HSP) and Light strand promoter (LSP)
The Origin of Light chain (OL) is then also important for mitochondrial DNA replication

Question 4

How is mtDNA packaged?

Answer 4

mtDNA is packaged into structures called nucleoids
One or two copies of mtDNA per nucleoid
Within these nucleoid structures a protein called transcription factor A (TFAM) acts as histone protein

Question 5

What are the exceptions to the universal genetic code?

Answer 5

Genetic code in vertebrate mitochondria
AUA and AUG code for methionine (AUA codes for isoleucine in nuclear DNA)
UGA codes for tryptophan (stop codon in nuclear DNA)
AGA and AGG are stop codons (not arginine)

Question 6

What are mtDNA haplogroups?

Answer 6

Different variants of mtDNA are known as mitochondrial haplogroups
Because mtDNA are maternally inherited, different variants are restricted to different ethnic groups
And because mitochondrial DNA does not recombine as mutations have occurred over time this divides the mtDNA into different haplogroups
We see the oldest haplogroups in Africa as L0, 1, 2 and 3

Question 7

How does mitochondria need both nuclear and mtDNA encoded proteins?

Answer 7

mtDNA encodes 13 proteins of OXPHOS
- But OXPHOS requires >100 proteins
To make the 13 OXPHOS proteins mtDNA must be:
- Replicated
- Transcribed
- Translated
All proteins involved in replication, transcription and translation of mtDNA are encoded by nuclear genes and imported into mitochondria
In total >1000 mitochondrial proteins but only 13 made by mtDNA, all others made by nuclear genes!!

Question 8

What does mitochondrial DNA polymerase (and its different versions look like)?

Answer 8

Polymerase gamma (Polγ)
- Heterotrimer protein
· One catalytic subunit (POLγA)
· Two accessory subunits (POLγB)
POLγA contain 3’ – 5’ exonuclease domain required to proofread newly synthesized DNA
POLγB enhances interactions with DNA template and increases activity and processivity of POLγA.

Question 9

What is TWINKLE?

Answer 9

Mitochondrial DNA helicase TWINKLE
TWINKLE:
- Hexamer – six TWINKLE subunits
Unwinds double stranded mtDNA template to allow replication by Polγ

Question 10

What is the mtSSBP and its role?

Answer 10

Mitochondrial single stranded binding protein (mtSSBP)
Binds to single stranded DNA
- Protects against nucleases
- Prevents secondary structure formation
- Enhances mtDNA synthesis by stimulating TWINKLE helicase activity

Question 11

Where does replication start?

Answer 11

DNA replication starts in the non-coding region (NCR)

mtDNA replication starts in Origin of heavy strand (OH).

Question 12

What is the strand displacement model of mtDNA replication?

Answer 12

This is an overview of the most widely accepted model of mitochondrial DNA replication
So replication begins at the OH
Followed by replication at the OL
This leads to the completion of the replication of both strands and eventually segregation of the daughter molecules

Question 13

Recall the steps of the strand displacement model of mtDNA replication.

Answer 13

Parental heavy strand displaced and coated with mtSSBP
TWINKLE helicase unwinds mtDNA
Mitochondrial RNA Polymerase (POLRMT) synthesizes RNA primer using light strand as template
POLγ uses RNA primer to replicate DNA at OH
Heavy strand replication passes OL
Stem loop structure is formed preventing mtSSBP from binding this allows access for POLRMT
Mitochondrial RNA Polymerase (POLRMT) synthesizes RNA primer using heavy strand as template
POLγ uses RNA primer to replicate light strand DNA at OL
Synthesis proceeds until both strands are fully replicated
After replication daughter molecules are segregated
This process of segregation requires the protein topoisomerase 3-alpha (Top3-a)
This allows the DNA molecules to separate

Question 14

What are mitochondrial diseases and their characteristics?

Answer 14

Mitochondrial diseases are a group of rare monogenic diseases
- Affect between 1:2000 – 1:4000 individuals
Oxidative phosphorylation disorders are most common form of mitochondrial disease
- Affect highly metabolic organs abundant in mitochondria
Can affect one (isolated) or several organ systems (multisystem).
Start at any age
Wide severity spectrum e.g.
- Adult-onset hearing loss
- Fatal cardiomyopathy in infancy

Question 15

How can we diagnose mitochondrial diseases?

Answer 15

Clinical signs
Blood and tissue histochemical and analyte measurements
Neuroimaging
Enzymatic assays of OXPHOS in tissue samples and cultured cells
DNA analysis

Question 16

What are the clinical features of mitochondrial disease?

Answer 16

Quite challenging as the symptoms are so varied
Most have a neurological component
Are typically progressive- one part of the body may be affected first and then later another part may be affected
Vary in progression so have not been well characterised

Question 17

What is muscle histology and how can it be used for mitochondrial diagnosis?

Answer 17

One of the gold standard tests but is fairly invasive
Haematoxylin and eosin (H&E) staining to evaluate muscle pathology
Gomori trichrome (used to identify ragged red fibres) which in muscle cells is characterised by the accumulation of mitochondria
This accumulation of mitochondria is a cellular response to the OXPHOS defect however its not sufficient to correct the deficiency involved
SDH (SDH-rich or ragged blue fibres) shows a complex IV defect
COX (COX-negative fibres)
Combined COX/SDH

Question 18

How can electron micrography be used to diagnose mitochondrial diseases?

Answer 18

Electron micrography of abnormal muscle mitochondria
When there are defects in OXPHOS this can also affect the overall architecture of mitochondria
These changes occur as a remodelling of the lipid membrane in mitochondria
These changes can be seen using an electron microscope
They can be large or swollen and there can be observed unusual structures called paracrystalline inclusions (PCl) depicted in red

Question 19

How do cellular populations of mitochondrial DNA differ?

Answer 19

MtDNA is a multicopy genome and a single cell can contain tens of thousands of mtDNA copies
And cells from individuals might contain different mtDNA variants
The presence of just one mtDNA variant in cells is a state known as homoplasmy, a person is homoplasmic
When there are multiple mtDNA variants that is a state called heteroplasmy, not always pathogenic variants some might be fully non-pathogenic
Heteroplasmy levels determine disease manifestation
For many mitochondrial DNA mutations, a disease will manifest when at least 80% of the cells mitochondrial DNA is of a pathogenic variant
It is therefore important to quantify the amount of pathogenic variant

Question 20

What do heteroplasmy levels determine?

Answer 20

Heteroplasmy levels (mutation load) determine disease manifestation
Heteroplasmy levels affect penetrance and severity of disease
m8993T>G:
- >90% = leigh syndrome
- 60-75% = NARP
- <60% asymptomatic

Variable penetrance also in homoplasmic mutations
LHON:
- 50% males affected
- 10% females affected

Question 21

What is the difference between heteroplasmic and homoplasmic mutations?

Answer 21

Heteroplasmic mutations- inheritance of mutation load is random
Homoplasmic mutations- females carrying homoplasmic mutation transmit to all children in some disease such as LHON

Question 22

What is the mitochondrial bottleneck during oogenesis?

Answer 22

The mitochondrial genetic bottleneck is a process that describes how oocytes from an individual can have very different heteroplasmy levels of mtDNA
During the early stages of development there are a group of cells that are specified to become primordial germ cells
When these primary germ cells differentiate to become oocytes, there is a reduction in the amount of mitochondria as well as asymmetric division of the mitochondria
This results in oocytes with different heteroplasmy levels leading to different levels of risk for manifesting a disease

Question 23

What are the advantages of mtDNA genome sequencing?

Answer 23

Can be performed from blood, urine, fibroblasts, tissue
Next-generation sequencing of mtDNA advantages:
- Increased reliability and sensitivity
- More accurate detection of low level heteroplasmy
- Muscle and liver may be necessary for tissue-specific mutations not present in blood (e.g. A3243G MELAS/MIDD mutation)
- Detect SNVs, single or multiple deletions, duplications
Quantitative PCR for mtDNA depletion (caused by mutations in nuclear genes affecting mtDNA replication/maintenance)

Question 24

What is mitochondrial disease inheritance like?

Answer 24

Mitochondrial disease can also be transmitted by autosomal recessive and autosomal dominant modes of inheritance as well as X-linked inheritance
De novo mutations in mitochondrial nuclear genes can also underline mitochondrial disorders
Most mitochondrial diseases are caused by mutations in the nuclear genes

Question 25

How do nuclear genetic problems cause OXPHOS diseases?

Answer 25

Mutations in most of the nuclear encoded subunits have been linked to be the cause of an OXPHOS disease
Mutations of the proteins involved in the mtDNA replication stages can lead to secondary mitochondrial mutations

Question 26

What do dominant mutations in TWINKLE cause?

Answer 26

Dominant mutations in TWINKLE cause mtDNA deletions and late-onset mitochondrial myopathy
Multiple mtDNA deletions in muscle, brain and heart
Progressive external ophthalmoplegia (PEO), muscle weakness, exercise intolerance and ptosis (drooping eyelids)
No cure and lack of treatments
Lack of biomarkers for diagnosis and as outcome measures in clinical trials
Reasons for tissue-specificity unknown

Question 27

How would you prevent transmission of mtDNA mutations?

Answer 27

Oocyte donation
Prenatal diagnosis
Preimplantation genetic diagnosis (PGD)
Mitochondrial replacement therapy

Question 28

How can you make a three-parent embryo?

Answer 28

Mitochondrial replacement therapy for mtDNA disease
It involves:
A healthy nuclear DNA removed from patient’s egg cell, leaving behind faulty mitochondrial DNA
Patients’s nuclear DNA is transplanted into donor egg with healthy mitochondrial DNA
‘Reconstructed’ egg cell fertilised with sperm in the lab and implanted into patient
Resulting embryo has three genetic parents