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Genomics > W4 - The Mitochondrial Genome > Flashcards

W4 - The Mitochondrial Genome Flashcards

1
Q

What is the Mitochondrial structure and function?

A

Central role in energy metabolism. The energy in our food is oxidised and used as substrates to generate ATP oxidative phosphorylation. This occurs in the inner membrane of the mitochondria.

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2
Q

What does the mitochondrial genome (mtDNA) look like?

A
  • 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
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3
Q

What do Mitochondrial genome encode for?

A

Proteins of oxidative phosphorylation.
There are complexes 2 to 5.
Complex 2 is the only subunit where only nuclear-DNA is encoded.

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4
Q

What does the non-coding region contain?

A

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)

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5
Q

What is the mtDNA packed into?

A
  • mtDNA is packaged into structures called nucleoids
  • One or two copies of mtDNA per nucleoid
  • Transcription factor A (TFAM) acts as histone protein
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6
Q

What are the exceptions to the “universal” genetic code?

A
  • 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)
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7
Q

What are mtDNA haplogroups?

A

Different variants of mitochondria are known as mtDNA haplogroups. They are purely maternally inherited - variant depending on ethnic group and are subdivided discreetly due to no recombination happening.

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8
Q

How does mitochondrial DNA replicate?

A
  • 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!!
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9
Q

What is the machinery used for DNA replication?

A

Contains mechanisms required for DNA processing and repair, dNTP pool and others with unclear functions.

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10
Q

What is the Mitochondrial DNA polymerase?

A
  • Polymerase gamma (Pol gamma)
  • Heterotrimer protein:
  • One catalytic subunit (POLgA)
  • Two accessory subunits (POLgB)
  • POLgA contain 3’ – 5’ exonuclease domain to proofread newly synthesized DNA
  • POLgB enhances interactions with DNA template and increases activity and processivity of POLgA.
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11
Q

What is Mitochondrial DNA helicase TWINKLE?

A
  • TWINKLE
  • Hexamer – six TWINKLE subunits
  • Unwinds double stranded mtDNA template to allow replication by Pol(y in Greek)
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12
Q

What is the Mitochondrial single stranded binding protein?

A
  • Binds to single stranded DNA
  • Protects against nucleases
  • Prevents secondary structure formation
  • Enhances mtDNA synthesis by stimulating TWINKLE helicase activity
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13
Q

Where does DNA replication start?

A

In the non-coding region.
mtDNA replication starts in Origin of heavy strand (OH).

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14
Q

What are the steps of Strand displacement model of mtDNA replication?

A

1) Replication of heavy strand begins at O(little H at the bottom) - starts at origin of heavy strands.

2) Replication of light strand begins at O (little L at the bottom)

3) Replication of both Strands completed

4) Segregation of daughter molecules

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15
Q

What does Strand displacement model of mtDNA
replication imply?

A

Parental heavy strand displaced and coated with mtSSBP
TWINKLE helicase unwinds mtDNA
Mitochondrial RNA Polymerase (POLRMT) synthesizes RNA primer using light strand as template
POLg uses RNA primer to replicate DNA at OH

Heavy strand replication passes OL
Stem loop structure is formed preventing mtSSBP binding
Mitochondrial RNA Polymerase (POLRMT) synthesizes RNA primer using heavy strand as template
POLg uses RNA primer to replicate light strand DNA at OL

Synthesis proceeds until both strands are fully replicated

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16
Q

What happens after replication?

A

Daughter molecules are segregated.

After mtDNA replication, the new daughter molecules are mechanically linked via a hemicatenane structure, which requires Top3alpha.

Role of Top3a: In proteins with a deficient Top3a, there is concatenation of mitochondrial DNA, so they are not able to segregate.

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17
Q

What is Mitochondrial disease?

A

Often affects the vital organs:
Heart, skeletal muscles, Metabolic: liver & pancreas, Nervous system: brain & peripheral nerves.

  • 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
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18
Q

What are some Mitochondrial syndromes?

A

Leigh syndrome Most common mitochondrial disease presentation (>80 genes)

LHON Leber’s Hereditary Optic Neuroretinopathy

KSS Kearns-Sayre Syndrome

MELAS Mitochondrial Encephalomyopathy Lactic Acidosis Stroke like episodes

MERFF Myoclonus Epilepsy Red Ragged Fibres

NARP Neurogenic muscle weakness Ataxia
Retinitis Pigmentosa

MINGIE Mitochondrial myopathy NeuropathyGastroIntestinaldiseaseEncephalopathy

19
Q

What are the Hallmarks of mitochondrial disease?

A

In a brain MRI, you would be able to see abnormal, bilateral lesions in the basal ganglia of the brain 🧠. Classic feature of disease.

Muscle biopsy - ragged red fibres

Hypotrophic cardiomyopathy
Pseudo Intestinal obstruction
Sideroblastic anemia
Enlargement of vessels in the eye.

20
Q

What are the basic steps of diagnosis of mitochondrial disease?

A
  • Clinical signs
  • Blood and tissue histochemical and analyte measurements
  • Neuroimaging
  • Enzymatic assays of OXPHOS in tissue samples and cultured cells
  • DNA analysis
21
Q

What are some Clinical features of mitochondrial disease?

A

Patients may have symptoms in one part of the body, but then develop symptoms in another part later on. It is progressive and the rates vary. This is caused by hundreds of different genes. Heterogeneity between patients and the disease caused has been found not only in patients with different genes, but also for patients with mutation in the same gene. Even for the same mutation.

22
Q

What does a low invasive biochemical investigation consist of?

A
  • Blood/CSF lactic acid >2.1 mM
  • Lactic acid/pyruvate ratio
  • Amino Acids (e.g. alanine)
  • Organic acids
23
Q

What is Muscle histology?

A
  • Haematoxylin and eosin (H&E)
  • Gomori trichrome (ragged red fibres)
  • SDH (SDH-rich or ragged blue fibres)
  • COX (COX-negative fibres)
  • Combined COX/SDH
24
Q

How is Spectophotometry used?

A

Spectrophotometry of OXPHOS activity
* Complex I
* Complex II
* Complex III
* Complex I + IV
* Complex I + III
* Complex II + III
* High resolution respirometry measures oxygen consumption

25
Q

How is electrophoresis used?

A

Blue-native gel electrophoresis of OXPHOS
complexes

26
Q

How is electron micrography used?

A

Electron micrography of abnormal muscle
mitochondria
The shape of the mitochondria would change and become large and unusual swollen.
Can also be seen with paracrystalline inclusions (PCI)

27
Q

How do genetics of mitochondrial diseases work?

A

Next Gen sequencing is a game changer for mitochondrial disease diagnosis.

There is a point mutation on gene m8969 converting G to A. Eg. Only 2 children suffer from the disease but no predecessors do. How?

28
Q

What are the different cellular populations of Mitochondrial DNA?

A

A single cell can contain thousands of DNA copies.
Homoplasmy - The presence of just one single mitochondrial variant. This could be healthy or pathogenic.
Heteroplasmy - When there are two or more variants of mitochondrial DNA present. Someone could equally have two healthy variants as opposed to two problematic ones.

29
Q

What do Heteroplasmy levels determine?

A

Heteroplasmy levels determine disease
manifestation
Relative quantity of pathogenic patients in mitochondria determines if the disease manifests or not (>80%)

Referring back to the former example, it shows that the 2 children had high levels of the variant as opposed to low levels of the variant by other family members.

30
Q

How does mtDNA mutations inheritance work?

A

Heteroplasmic mutations = Inheritance of mutation load is random

Homoplasmic mutations = Females carrying homoplasmic mutation transmit to all children.

Mitochondrial diseases are maternally inherited. But can also be transmitted through autosomal recessive and autosomal dominant modes of inheritance and X-linked inheritance and de novo mutations. Most are caused by mutations in nuclear genes.

31
Q

How does Heteroplasmy affect penetrance and severity of the disease?

A
  • m8993T>G
  • > 90% = leigh syndrome
  • 60-75% = NARP
  • <60% asymptomatic
  • Variable penetrance also in homoplasmic mutations
  • LHON
  • 50% males affected
  • 10% females affected
32
Q

What is the mitochondrial bottleneck during
oogenesis?

A

This answers why in families the heteroplasmy variants differ so much.
During early development, there were a group of cells that specialised to become primordial germ cells. These then differentiate to become primary oocytes - this means there is a reduction of mitochondria when it divides as well as asymetric division itself. This leads to different levels of inheritance.

33
Q

What are the features of the mtDNA genome sequencing?

A
  • Blood, urine, fibroblasts, tissue
  • Next-generation sequencing of mtDNA
  • 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)
34
Q

What are the nuclear genetic causes of OXPHOS diseases?

A

Mutations in any mitochondrial DNA subunits of Oxidative Phosphorylation complexes can lead to disease.

Requires mitochondrialDNA replication with transcription, translation. Mutations in proteins involved in these steps can lead to secondary mitochondrial disease.

35
Q

How do mutations in mtDNA replication machinery
cause secondary mutations in mtDNA

A
  • mtDNA deletions
  • mtDNA depletion
  • Occurs in post-mitotic tissues:
  • Brain
  • Muscle
  • Heart
  • Liver
36
Q

What did they find out about dominant mutations in TWINKLE?

A

Dominant mutations in TWINKLE (one of the replicating proteins) cause mtDNA
deletions and late-onset mitochondrial myopathy.

  • Multiple mtDNA deletions in muscle, brain and heart
  • Progressive external ophthalmoplegia (PEO), muscle weakness, exercise intolerance
  • No cure and lack of treatments
  • Lack of biomarkers for diganosis and as outcome measures in clinical trials
  • Reasons for tissue-specificity unknown
37
Q

What is the candidate gene sequencing approach prior NGS?

A

Sanger sequencing can be performed on known genes known to cause clinically recognisable syndromes. This would be laborious and the diagnostic yield would be very low. Since Next Gen sequencing had been on the rise, there are nearly 400 known gene mutations that causes mitochondrial disease.

38
Q

What are Monogenic defects associated with
mitochondrial disease

A

> 300 mitochondrial disease genes
Genes coloured blue are mtDNA genes
All others are nuclear genes

39
Q

What is the NGS sequencing approach for nuclear genes?

A
  • Sequencing panels of disease specific genes
  • Fewer genes but better sequence coverage. Good for clinical practice but new disease genes overlooked.
  • Whole exome sequencing
  • All coding genes but some important regions not well sequenced
  • Optimised Whole exome sequencing for capture of all known disease associated regions
  • Costs more
  • Whole genome sequencing
  • Sequencing everything but even more expensive and data analysis complex
40
Q

What does Genetic counselling for mtDNA mutations consist of?

A
  • Homoplasmic or heteroplasmic?
  • Prognosis depends on mutation, heteroplasmy levels, variable penetrance of
    homoplasmic mutations and therefore difficult to predict
  • Recurrance risks
  • Strictly maternally inherited (new evidence suggests paternal inheritance
    possible)
  • Homoplasmic mutations passed to all children
  • Heteroplasmic mutations passed in variable amounts
  • Low for de novo mutations (risk for potential germline mosaicism)
41
Q

How would you prevent transmission of mtDNA mutations?

A
  • Oocyte donation
  • Prenatal diagnosis
  • Preimplantation genetic diagnosis (PGD)
  • Mitochondrial replacement therapy
42
Q

Why is neural imaging so important?

A

MRI and MRS (Magnetic Resonance Spectroscopy) can be used for disease identification.

Neural imaging is important.

MRI - Can be used to see bilateral lesions of the basal ganglia. Atrophes of the cerebellum, brainstems and white matter disease.

MRS - Can be used to investigate specific metabolites in the brain like lactate.

43
Q

What is the mitochondrial replacement therapy for mtDNA disease?

A

The aim is to remove the mitochondrial DNA of the mother that has a pathogenic variant to an oocyte of the donor. The children born will have the nuclear DNA of the mother and the father and the mitochondrial DNA from the donor.

Aka - 3 parent babies.

Genomics (20 decks)

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