The Mitochondrial Genome

Question 1

Describe features of the mitochondrial genome and how it differs from the nuclear genome

Answer 1

• contains multiple copies of genome
• double stranded circular
• no introns, but does have non-coding region used for altering gene expression
• only 13 OXPHOS proteins coded by mtDNA
• 37 mtDNA derived genes

Question 2

Explain the process of mitochondrial DNA replication

Answer 2

STRAND DISPLACEMENT:
- twinkle unwinds helix
- replication begins at heavy origin
- using light strnd template
- heavy strand replication at light origin
- causes parent strand displacement
- heavy strand coated in mtSSB preventing sticking
- light chain formed using heavy template
- fully replicated strands
- nucleoids segregate

Question 3

Describe features and causes of mitochondrial diseases

Answer 3

Features:
- rare monogenic
- OXPHOS disorders
- heart brain muscle liver most impacted
- heterogenous (effetc multiple organs)
- start at any age and varied onset/severity
- Leigh Syndrome
- basal ganglia bi-lateral lesions
- accumulation of red fibres
- pseudo-intestinal obstruction
- ruptures of eye vessels
- exercise intolerance
- varied progressions between individuals / poorly characterised

Causes:
damaged mitochondria = reduced/ disrupted OXPHOS

Question 4

Describe diagnostic signs and tests for mitochondrial diseases

Answer 4

diagnostics
- blood : * Blood/CSF lactic acid >2.1 mM
* Lactic acid/pyruvate ratio
* Amino Acids (e.g. alanine)
* Organic acids
- urine : buildup of pyruvate causes conversion to lactate
(lack specificity, but non invasive)

• neuroimaging
• MRI & MRS
• atrophy of cerebellum, brainstem pons
• XS lactate buildup

muscle histology
- detect OXPHOS defect
- specific assays
* Haematoxylin and eosin (H&E)
* Gomori trichrome (ragged red fibres)
* SDH (SDH-rich or ragged blue fibres) succinate dehydrogenase
* COX (COX-negative fibres)
* Combined COX/SDH
- genetic screening
- NGS

Question 5

Describe the genetics of mitochondrial disease, including inheritance patterns and the phenomenon of heteroplasmy

Answer 5

• potentially sex-linked?
• mtDNA 8969 mutation G>A
• 8993 T>G
• alternating penetrance (unaffected older generations)
  HETROPLASMY: 2 or more mtDNA variants
• disease only manifests if >80% of mtDNA is of pathogenic variant (subject to specific mutation) increasing importance of geentic screening
• inhertiance of mutation load is random
  HOMOPLASMY: harmful when only variation is pathogenic. maternal lineage therefore all children of affected homoplasmic patient will manifest disease

Question 6

Describe the process of mitochondrial replacement therapy

Answer 6

MRT:
- oocyte donation
- removal of faulty mtDNA replacement with donor
- nuclear insert of patient DNA
- IVF once egg is reconstructed

Question 7

suggest how NGS can be used in prognosis and diagnosis of mitochondrial genetic disease.

Answer 7

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)
- off-target reads in whole-exome sequencing