Mitochondrial Disorders

Question 1

What is the structure of mitochondria

Answer 1

Double membrane - inner and outer, between them is the inter membrane space

The inner membrane contains the mitochondrial matrix, where the metabolic reactions occur

The membrane has OXPHOS proteins embedded in it

It originates from bacterial ancestor as evidenced from its circular DNA and its architecture

Question 2

Discuss mitochondrial organisation

Answer 2

Mitochondria form networks and are organised differently depending on cell type

They can be interconnected forming networks that are dynamic (constantly moving)
Individual mitochondria can separate, divide and fuse together (fission and fusion)

In cardiac muscles they form distinct groups
This suggests they can group for specific functions

They are transported on cytoskeletal microtubules
Facilitated by dynein and kinesin which bind to mitochondria by adaptor protein Milton and Miro

This is found on the surface of mitochondrial cells

Movement is important in cells such as neurones

Question 3

Describe how mitochondria generate ATP

Answer 3

Generation of ATP is via oxidative phosphorylation (OXPHOS)

Nutrients are oxidised to produce acetyl-CoA
Processed by the TCA/Krebs/citric acid cycle

Reducing equivalents are produced serving as electron donors in the respiratory chain
NADH and FADH2

This are oxidised in the respiratory chain and result in production of ATP

Question 4

Describe mitochondrial function

Answer 4

Central hubs of metabolism - anabolic synthesis of nucleotides required for DNA replication and growth
Important target for cancer therapy

Calcium homeostasis critical for muscle contraction

Important for AA production e.g. glutamate - neurotransmitter

Innate immunity - protein complexes on the outside of the mitochondrial membrane
Mitochondrial antiviral signalling complex

Question 5

Describe the mitochondrial genome

Answer 5

Circular molecule (16.6kb) - multicopy genome (10-100,000 copies per cell)

37 genes - 13 oxidative phosphorylation protein subunits, 22 transfer RNAs, 2 ribosomal RNAs
No introns

Maternally inherited (some recent evidence of paternal inheritance controversial)

Question 6

Discuss mitochondrial diseases

Answer 6

Rare monogenic diseases - largest group of inherited metabolic disorders

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) and start at any age

Wide severity spectrum e.g. adult-onset hearing loss, fatal cardiomyopathy in infancy

Question 7

List some mitochondrial diseases

Answer 7

Leigh syndrome - most common mitochondrial disease presentation (>80 genes)
LHON - Lebar’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 Neuropathy Gastro-Intestinal disease Encephalopathy

Question 8

What are the hallmarks of mitochondrial disease

Answer 8

Muscle biopsy - mitochondrial myopathy, staining reveals mitochondria staining and accumulation around the edges of the muscle cell ‘ragged red fibre’

Bilateral lesions in the basal ganglia - diagnostic for leigh syndrome

Also symptoms include hypertrophic cardiomyopathy, anaemia, pseudo-intestinal obstruction, enlarged blood vessels in the eye

Question 9

How do you diagnose mitochondrial disease

Answer 9

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

Question 10

What are the clinical features of mitochondrial disease

Answer 10

Neurological components - imaging such as MRI and MRS

MRI can show bilateral lesions and atrophy of cerebellum, pons, brain stem and white matter disorders

Progressive - start in one part of the body and then develop into others (rate of progression can vary)

Few individuals may share the same variants - thus not well characterised

Question 11

What are low invasive biochemical investigations for mitochondrial disease

Answer 11

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

Question 12

What stains can be used for histochemical staining of mitchondria

Answer 12

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

Also, high resolution respirometry measures oxygen consumption

Question 13

How can you analyse mitochondrial using electrophoresis

Answer 13

Each of the enzymes are comprised of many distinct subunits

Analysis can be performed with blue-native gel electrophoresis

Deficiency of a single protein can affect the overall organisation/assembly of the complexes

One complex may be split into many fragments because it is missing one component

Question 14

Discuss mtDNA disease inhertiance

Answer 14

mtDNA mutations cause OXPHOS disorders, including myopathy

Mitochondrial diseases can also be AR, AD, X-linked and de-novo as most mitochondrial disorders are mainly caused by mutations in nuclear genes

mtDNA inheritance is via the maternal lineage - and can look like recessive inheritance

Homoplasmy - females carrying these mutations will pass it onto all children
Heteroplasmy -two or more different variants can be present
>80% of mutant mtDNA = disease manifestation

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

Question 15

Discuss mitochondrial bottleneck during oogenesis

Answer 15

During early stages of development there are a group of cells specified to become primordial germ cells and differentiate into oocytes

There’s a reduction in the amount of mitochondria as well as asymmetric division of mitochondria as they differentiate and divide

Thus each oocyte gets varying levels of mutant and non-mutant mtDNA

Question 16

Discuss mtDNA genome sequencing

Answer 16

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)
Heteroplasmy in blood can decrease with age
Detect SNVs, single or multiple deletions, duplications

Quantitative PCR for mtDNA depletion (caused by mutations in nuclear genes affecting mtDNA replication/maintenance)

Question 17

Discuss exome MIT

Answer 17

68 Adults were studied, with varying phenotypes
Most were investigated prior, and had their DNA sequences
However, they did not have a genetic diagnosis

They were classified using the Bernier criteria

DNA samples were collected - blood, muscle biopsy, fibroblast

WES performed and analysed
Alignment using BWA and variant calling with SAMTOOLS
Formulated a list of variants which were filtered/prioritised

Sanger sequencing of patient and relative were used for validation as well as experimental validation for novel disease genes

Question 18

What is the Bernier criteria

Answer 18

Diagnostic criteria for respiratory chain disorders in adults and children

It allows you to prescribe the patient a likely diagnosis

Definite - 2 major or 1 major and 2 minor criteria
Probable - 1 major and 1 minor or 3 minor criteria
Possible - 1 major or 2 minor (of which one must be clinical)

Criteria
Clinical, histological, enzymology, functional, molecular and metabolic

Question 19

What is Mitocarta

Answer 19

A research study to identify all mitochondrial localised proteins

Several criteria used

Isolated mitochondria from humans and tissues and performed mass-spec quantitative methods Presence of N-terminal mitochondrial targeting sequence (MTS)

Localised to mitochondria by immunofluorescent of GFP-tagged protein studies

Presence of mitochondrial domain in protein sequence

Coexpressed with the PGC1-alpha (mitochondrial biogenesis TF)

> 1150 known mitochondrial proteins (5% of all nuclear genes)
20% don’t have a known function

Question 20

Discuss mtDNA mutations identified by NGS

Answer 20

Prior clinical mtDNA mutations were overlooked but found via exome sequencing

This was despite the fact that only nuclear DNA was looked for - no baits for mtDNA

This could be because as mtDNA is so abundant, there was a lot of off-target capture of mtDNA

This means there may be questions regarding the authenticity/accuracy of this data

This could also mean that it may have been sequencing pseudogenes, but was verified by mini-sequencing and sanger on mtDNA itself

Question 21

What is the significance of POLG

Answer 21

Pre-NGS diagnostic centred around DNA sequencing of mtDNA and a handful of nuclear genes commonly involved in these disorders

An example is polymerase gamma - POLG is involved in replicating mtDNA
Has 100s of mutations reported