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SGUL - Genomics > The Mitochondrial Genome > Flashcards

The Mitochondrial Genome Flashcards

1
Q

What are some features of the mitochondrial genome? (mtDNA)

A
  • Double stranded circular molecule (16.6kb) (15000x smaller than chromosome 1)
  • Consists of the heavy and light strand
  • Multicopy genome (10-100,000 copies per cell)
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2
Q

What are some features of the mitochondrial genome? (mtDNA) (PART 2)

A

37 genes:
- 13 oxidative phosphorylation protein subunits
- 22 transfer RNAs
- 2 ribosomal RNAs

  • no introns
  • D-loop is a non coding region
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3
Q

What type of proteins does the mitochondrial genome encode?

A

Encodes proteins of Oxidative Phosphorylation

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4
Q

What does the non coding region (NCR) contain?

A

Contains regulatory sequences for replication and transcription

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5
Q

Where does the replication and transcription for the non coding regions start?

A
  • mtDNA replication starts in the origin of heavy strand (OH)
  • Transcription starts at heavy strand promoter (HSP) and Light Strand Promoter (LSP)
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6
Q

What is the mtDNA packaged into?

A
  • mtDNA is packaged into structures called nucleoids
  • One or two copies of mtDNA per nucleoid
  • Transcription factor A (TFAM) acts as a Histone protein
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7
Q

What are the exceptions to the universal genetic code?

A

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

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8
Q

What does the mitochondria require?

A

Both Nuclear and mtDNA encoded proteins

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9
Q

What does mtDNA encode for?

A
  • Encodes for 13 proteins of OXPHOS
  • But OXPHOS requires >100 proteins
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10
Q

What is required for mtDNA to make OXPHOS proteins?

A

It must be:
- Replicated
- Transcribed
- Translated

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11
Q

What do Nuclear genes encode for?

A
  • Encodes all the proteins involved in Replication, Transcription and translation of mtDNA
  • These proteins are then all imported into the mitochondria
  • in total >1000 proteins but only 13 made by mtDNA
  • All the others are made by nuclear genes
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12
Q

What are the three requirements for Mitochondria DNA replication machinery?

A
  • Mitochondrial DNA polymerase
  • Mitochondrial DNA helicase TWINKLE
  • Mitochondrial single stranded binding protein (mtSSBP)
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13
Q

Describe the mitochondrial DNA Polymerase

A

Polymerase gamma (Polg)
- A Heterotrimer protein which has one catalytic subunit (POLgA) and two accessory subunits (POLgB)

  • POLgA contain 3’-5’ exonuclease domain to proofread newly synthesised DNA
  • POLgB enhances interactions with DNA template and increases activity and processivity of POLgA
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14
Q

Describe the Mitochondrial DNA helicase TWINKLE

A
  • The twinkle is a hexamer: Made of 6 twinkle subunits
  • Unwinds double stranded MTDNA template to allow replication by Polg
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15
Q

Describe the Mitochondrial single stranded binding protein (mtSSBP)

A

Binds to the single stranded DNA
- Protects against nucleases
- Prevents secondary structure formation
- Enhances mtDNA synthesis by stimulating TWINKLE helicase activity

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16
Q

Explain the process of mitochondrial DNA replication (PART 1)

A

DNA replication starts in the non coding region (NCR)
- mtDNA replication starts in origin of heavy strand (OH)

17
Q

Explain the process of mitochondrial DNA replication (PART 2)

A
  1. Replication of heavy strand begins
  2. replication of light strand begins at OL
  3. replication of both strands completed
  4. Segregation of daughter molecules
18
Q

Explain the process of mitochondrial DNA replication (PART 3)

A
  1. Parental heavy strand displaced and coated with mtSSBP
  2. TWINKLE helicase unwinds mtDNA
  3. Mitochondrial RNA Polymerase (POLRMT) synthesizes RNA primer using light strand as template
  4. POLg uses RNA primer to replicate DNA at OH
19
Q

Explain the process of mitochondrial DNA replication (PART 4)

A
  1. Heavy strand replication passes OL
  2. Stem loop structure is formed preventing mtSSBP binding
  3. Mitochondrial RNA Polymerase (POLRMT) synthesizes RNA primer using heavy strand as template
  4. POLg uses RNA primer to replicate light strand DNA at OL
20
Q

Explain the process of mitochondrial DNA replication (PART 5)

A
  1. Synthesis proceeds until both strands are fully replicated
  2. After replication daughter molecules are segregated
21
Q

What are some mitochondrial diseases?

A
  • Rare monogenic diseases: affects between 1:2000 individuals
  • Oxidative Phosphorylation disorders: Affect highly metabolic organs abundant in mitochondria
  • Can affect one or several organ systems
  • Starts at any age
22
Q

What are some mitochondrial syndromes?

A
  • Leigh syndrome: Most common mitochondrial disease presentation (>80 genes)
  • LHON: Leber’s Hereditary Optic Neuroretinopathy
  • KSS: Mitochondrial Encephalomyopathy Lactic Stroke like episodes
  • MERFF: Myoclonus Epilepsy Red Ragged Fibres
23
Q

What are the types of diagnosis for mitochondrial disease?

A
  • Clinical signs
  • Blood and tissue histochemical and analyse measurements
  • Neuro-imaging
  • Enzymatic assays of OXPHOS in tissue samples and cultured cells
  • DNA analysis
24
Q

What are the low invasive biochemical investigations?

A
  • Blood/CSF lactic acid > 2.1 mM
  • Lactic acid/Pyruvate ratio
  • Amino Acids
  • Organic acids
25
Q

What are the muscle history investigations?

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
26
Q

In summary, How can diagnosis be carried out?

A

Clinical clues
- Characteristic syndrome/ apparently unrelated symptoms
- Family history
- Multiple organ involvement
- Progressive
- Raised lactate
- COX negative fibres on muscle biopsy
- Brain MRI changes
- Genetic testing (NGS)

27
Q

What type of disorders does mtDNA mutations cause?

A

Oxidative Phosphorylation disorders

28
Q

How are mtDNA diseases inherited? (VD)

A

Maternally inherited

29
Q

What are the two types of cells in cellular populations of mitochondrial DNA?

A
  • Homoplasmy: Healthy mtDNA
  • Heteroplasmy: Mutant mtDNA
30
Q

What does Heteroplasmy levels determine? (VD)

A

Determines disease manifestation

31
Q

What does Heteroplasmy levels affect?

A
  • Affects penetrance and severity of disease
  • Variable penetrance also in homoplasmic mutations
    LHON: 50% Males affected and 10% females affected
32
Q

What is 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
  • Detect SNVs, single or multiple deletions, duplications
  • Quantitative PCR for mtDNA depletion
33
Q

How can mtDNA mutations be identified?

A

Could be identified by “off target” reads from whole exome sequencing
- This graph shows that with NGS the entire length of mtDNA is covered with several hundred short-reads. Therefore, method is highly reliable as no region is missing compared to nuclear genome.

34
Q

What can mutations in mtDNA replication machinery cause?

A
  • mtDNA deletions
  • mtDNA depletion
  • Occurs in post-mitotic tissues such as brain, muscle, heart and liver
35
Q

What does dominant mutations in TWINKLE cause?

A

mtDNA deletions and late onset mitochondrial myopathy

36
Q

What are the NGS Sequencing approaches 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

37
Q

Describe the genetic counselling for mtDNA mutations

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)

38
Q

How do you prevent the transmission of mtDNA mutations?

A
  • Oocyte donation
  • Prenatal diagnosis
  • Preimplantation genetic diagnosis (PGD)
  • Mitochondrial replacement therapy
39
Q

Describe the Mitochondrial replacement therapy for mtDNA disease (‘Three-parent babies’)

A

Making a 3 parents embryo
1. Healthy nuclear DNA removed from patients egg cell leaving behind a faulty mitochondrial DNA
2. Patients nuclear DNA transplanted into donor egg with a healthy mitochondrial DNA
3. Reconstructed egg fertilised with sperm in the lab and implanted into patient. Resulting embryo has 3 genetic parents

SGUL - Genomics (21 decks)

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