The Mitochondrial Genome

Question 1

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

Answer 1

• 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)

Question 2

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

Answer 2

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

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

Question 3

What type of proteins does the mitochondrial genome encode?

Answer 3

Encodes proteins of Oxidative Phosphorylation

Question 4

What does the non coding region (NCR) contain?

Answer 4

Contains regulatory sequences for replication and transcription

Question 5

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

Answer 5

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

Question 6

What is the mtDNA packaged into?

Answer 6

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

Question 7

What are the exceptions to the universal genetic code?

Answer 7

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)

Question 8

What does the mitochondria require?

Answer 8

Both Nuclear and mtDNA encoded proteins

Question 9

What does mtDNA encode for?

Answer 9

• Encodes for 13 proteins of OXPHOS
• But OXPHOS requires >100 proteins

Question 10

What is required for mtDNA to make OXPHOS proteins?

Answer 10

It must be:
- Replicated
- Transcribed
- Translated

Question 11

What do Nuclear genes encode for?

Answer 11

• 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

Question 12

What are the three requirements for Mitochondria DNA replication machinery?

Answer 12

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

Question 13

Describe the mitochondrial DNA Polymerase

Answer 13

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

Question 14

Describe the Mitochondrial DNA helicase TWINKLE

Answer 14

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

Question 15

Describe the Mitochondrial single stranded binding protein (mtSSBP)

Answer 15

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

Question 16

Explain the process of mitochondrial DNA replication (PART 1)

Answer 16

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

Question 17

Explain the process of mitochondrial DNA replication (PART 2)

Answer 17

2. Replication of heavy strand begins
3. replication of light strand begins at OL
4. replication of both strands completed
5. Segregation of daughter molecules

Question 18

Explain the process of mitochondrial DNA replication (PART 3)

Answer 18

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

Question 19

Explain the process of mitochondrial DNA replication (PART 4)

Answer 19

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

Question 20

Explain the process of mitochondrial DNA replication (PART 5)

Answer 20

14. Synthesis proceeds until both strands are fully replicated
15. After replication daughter molecules are segregated

Question 21

What are some mitochondrial diseases?

Answer 21

• 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

Question 22

What are some mitochondrial syndromes?

Answer 22

• 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

Question 23

What are the types of diagnosis for mitochondrial disease?

Answer 23

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

Question 24

What are the low invasive biochemical investigations?

Answer 24

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

Question 25

What are the muscle history investigations?

Answer 25

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

Question 26

In summary, How can diagnosis be carried out?

Answer 26

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)

Question 27

What type of disorders does mtDNA mutations cause?

Answer 27

Oxidative Phosphorylation disorders

Question 28

How are mtDNA diseases inherited? (VD)

Answer 28

Maternally inherited

Question 29

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

Answer 29

- Homoplasmy: Healthy mtDNA - Heteroplasmy: Mutant mtDNA

Question 30

What does Heteroplasmy levels determine? (VD)

Answer 30

Determines disease manifestation

Question 31

What does Heteroplasmy levels affect?

Answer 31

- Affects penetrance and severity of disease - Variable penetrance also in homoplasmic mutations LHON: 50% Males affected and 10% females affected

Question 32

What is mtDNA genome sequencing?

Answer 32

- 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

Question 33

How can mtDNA mutations be identified?

Answer 33

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.

Question 34

What can mutations in mtDNA replication machinery cause?

Answer 34

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

Question 35

What does dominant mutations in TWINKLE cause?

Answer 35

mtDNA deletions and late onset mitochondrial myopathy

Question 36

What are the NGS Sequencing approaches for nuclear genes

Answer 36

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

Question 37

Describe the genetic counselling for mtDNA mutations

Answer 37

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)

Question 38

How do you prevent the transmission of mtDNA mutations?

Answer 38

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

Question 39

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

Answer 39

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