Lecture 29 - Mitochondrial Diseases 2 Flashcards Preview

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Flashcards in Lecture 29 - Mitochondrial Diseases 2 Deck (30):

Where are the complexes in the inner mitochondrial membrane encoded?

Both nuclear DNA and mitochondrial DNA


How prevalent are OXPHOS disorders?

1:5000 live births

(also see the figure 1:6500, but this is probably too conservative)

However, there are some ethnicities which have much greater rates: e.g. NSM Lebanese


Describe the features of the mtDNA

• Double stranded
• Circular
• 37 genes


List the unique features of mtDNA

• Maternally inherited
• Multiple copies (within each cell, as opposed to one from mum, one from dad with nuclear DNA)
• High mutation rate
• Heteroplasmy
• Threshold effect
• mtDNA bottleneck
• Tissue-specific segregation / selection


Describe mtDNA heteroplasmy, the bottleneck effect and the threshold effect

What is the clinical implication of this?

1. Heteroplasmy: different versions of the DNA within the same cell (due to high mutation rate)
2. Bottleneck effect: small number of mother's mitochondria go into each oocyte; randomly selected
3. Threshold effect: for disease to be seen, the mutant load of mtDNA must be above a given threshold

• 20% mutant mtDNA
• 80% normal mtDNA

In egg development, there will be different eggs with different amounts of the mutant mtDNA

Certain oocytes may have a greater or lesser proportion of the mutant mtDNA

Possible to get zygotes with:
• 80% mutant load → severe disease
• 60% mutant load → mild disease
• 20% mutant load → no disease

In summary:
Due to heteroplasmy, the bottleneck effect and the threshold effect, it is possible to have very different outcomes


What is the most common OXPHOS disease in childhood?
Give some features of the disease
What is the main pathological feature?

Leigh Disease

• Age of onset: 6 months
• Progressive neurodegeneration
• Motor and/or intellectual regression
• Focal Spongiform lesions = demyelination, gliosis


How many people carry pathogenic mtDNA mutation?
How many people are diagnosed with mtDNA disease?
Why is there this discrepancy?

mtDNA mutations: 1/200

mtDNA disease: 1/10000

This is probably because of mutant load: the mutation rate is below threshold

NB Some communities have higher rates of the mutation in the population, such as the NSW Lebanese


What proportion of the mtDNA genes can have pathogenic mutations?

35 of the 37 mtDNA genes


What are the different genes that can be mutated resulting in OXPHOS disease?

How are these genes inherited?

What proportion of OXPHOS diseases do these mutations account for?

1. Mutation in mtDNA genes

2. Mutation in subunits encoded by nuclear genes

3. Mutation in OXPHOS biogenesis genes
Involved in:
• Insertion of Iron / Sodium
• Assembly of complexes
• Insertion of protein into membrane

4. mtDNA replication genes

5. mtDNA expression genes

6. Genes involved in membrane dynamics

7. RNA transport, nucleotide transport and synthesis genes

These genes can be inherited in all different ways:
• Autosomal recessive (most)
• Autosomal dominant
• X-linked

These mutations only account for 50% of OXPHOS diseases
For 50% of cases, the molecular basis is not known


In what ways have OXPHOS disease genes been identified?

Mitochondrial genome discovered in 1988, whereafter it was sequenced:

• mtDNA sequencing

• Candidate studies

• Linkage studies

• Targeted exome sequencing

• Whole exome sequencing

About 15 genes were discovered each year


Mutations in which genes can cause to Leigh syndrome?

Up to 50 genes that can lead to Leigh disease:
• 30 Autosomal genes
• 12 mtDNA genes
• 2 X-linked genes

Even though there is this heterogeneity, the disease presents very similar in all the cases


What are the challenges of OXPHOS molecular diagnosis?

How can this be (partially) overcome?
What are the drawbacks?

1. Large number of candidate genes

2. Mostly private mutations; i.e. no hotspots like in other disorders

3. Common mutations only in a few genes

4. Genotype / Phenotype correlation often poor
• Phenotype can not guide the genotype investigations

5. If performing sequential testing, there are so many genes to look at, and it requires expert guidance

→ NextGen sequencing is a good option
• Allows much greater volume of DNA to be sequenced
• However, doesn't have same sensitivity or specificity of Sanger sequencing


Describe the changes to the cost of sequencing of the genome

2001: $100M

2013: $8000


Compare Sanger and NextGen sequencing

What can NextGen technology allow us to sequence?

• 1 target DNA
• Average of all DNA molecules
• around 800 bp per run

• Thousands of DNAs at a time (MitoExome: all of the known mitochondrial proteins)
• Single molecule DNA sequence
• 800 million bp per run
• Candidate gene approach, but at a much larger scale

Can sequence:
• 10, 40, up to 1000 genes (i.e. whole MitoExome)
• Whole exome (20,000 known genes)
• Whole Genome


What is massively parallel sequencing?

aka NextGen sequencing


How big is the human genome?
How many genes?

3 x 10^9
20,000 genes


List some NextGen sequencing technologies

• Illumina
• Ion torrent
• MinION (3rd generation)


Describe Illumina sequencing

"Sequencing of fluorescently labelled DNA fragments"


-- Target preparation --

1. DNA sheared into small fragments

2. Addition of adapters to each end of the DNA fragment

3. Adapters bind DNA to a flow cell covered with oligonucleotides through hybridisation

-- Cluster generation --

4. Amplification of different pieces of DNA that have bound to flow cell
• Through bridge formation

5. Repeat 35 times
• End up with many copies of the DNA fragment tethered to the flow cell (single stranded)

-- Sequencing --

6. Sequential addition of fluorescently labelled nucleotides

7. When the correct nucleotide is incorporated, it becomes excited, and allows an emission specific for the nucleotide that was added

8. Emission read by the machine


Describe Ion torrent sequencing

Chip with half a billion wells
Beads contained in the wells

1. DNA sheared into small fragments captured on a bead

2. DNA fragments on bead amplified by PCR in an emulsion

3. Bead in well

-- Sequencing --

4. Addition of complimentary nucleotides
• A given nucleotide will give off a proton when incorporated

5. Subtle chemical change detected on a machine
• Based on which nucleotide (A, C, G, or T) is incorporated, there will be a different signal


Compare whole genome and whole exome sequencing

Whole exome:
• All 20,000 known genes sequenced

Whole genome:
• 3 x 10^9 bp sequenced


How much is 3Gb?

Why is it such a challenge to find mutations in the whole genome?

(3 x 10^9)

This is similar to the number of letter in the Harry Potter series if it had 5000 volumes instead of 7

Mutations are like:
• Change in letter
• Change in word
• Change in a page
within these 5000 volumes


Describe the MitoExome Project
• The cohort
• Method
• Stats of results
• General overview of results

Over 1000 genes sequenced:
including all the known mitochondrial encoding genes

Within 44 OXPHOS patients
• Had severe diseases, determined to by OXPHOS disorders
• Diverse group
• Defects in the various complexes (Complex I, Complex IV or a combination)

• Hybrid in-solution selection of the candidate genes
• Illumina sequencing

• Average 1 change per patient (i.e. gene not 'normal')
• However only 8 of the mutations in the 42 patients were relevant to the disease
• The rest are probably 'normal variation'
• 10 of the patients identified to have known disease genes
• 10 more patients identified with 8 novel disease genes
• 43% of patients had no prioritised genes, i.e. the mutated genes causing their disease are still unknown → where could they be?

Nuclear DNA:
• Over 1000 genes sequenced
• More than 1 million bases sequenced
• Good depth (i.e. fidelity of sequencing?)
• 29 rare mutations identified (i.e. classified as rare from the '1000 Genomes Project')
• 17 rare, protein modifying mutations

• All 37 genes sequenced
• Good coverage
• Only 3 rare mutations found
• 0 rare, protein modifying mutations found


What is Prioritisation, in the context of the MitoExome Study?

Describe the 'Prioritisation' observed in the MitoExome study

Prioritisation: an indication that one is looking at the correct genes in the disease group
Ie Looking at mutations in genes causing OXPHOS diseases

MitoExome study:
In the 31 subjects study, there was prioritisation observed compared to controls in:
• Known or candidate genes
• Known disease genes
• Candidate disease genes

This means that in the subjects of the study, there were many more known disease genes etc observed than in the control group

The study was unmasking the correct genes


What is good about NextGen sequencing?

1. Price
• Has come down markedly since 2007

2. Quantity
• Allows sequencing of a much greater volume of genetic material


What was observed in the various candidate genes in the MitoExome cohort?

1. Two of the candidate genes were mutated in two different patients from different families. These two individuals had the same clinical phenotype
• MTFMT (2)

2. Six mutated genes in patients, that are associated with a specific
biochemical phenotype. In these six patients, the biochemical phenotype matched this
• C1orf31
• C6orf10
• MTFMT (2)
• UQCR10

3. Four of these genes were then proved to be the cause of the disease
• Through lentiviral correction studies

4. Four of the candidate genes have since had additional patients reported in the literature
• Confirmation of these genes as causing OXPHOS disease

5. Two of the genes have been eliminated, because other disease causing mutations in these patients were found


What did lentiviral correction studies show in the context of the candidate gene in the MitoExome study?

Describe this process

These studies showed Causation of the mutated gene in the disease process

1. WT version of gene put into lentiviral vector

2. Viral particle put into fibroblast cells in patient

3. See whether the enzyme defect is corrected
a. Correction → the gene plays a causative role in the disease
b. No correction → no causation displayed

• Two patients, both with a defective Complex I, but with different mutations
• Two lentiviral vectors, each with either the WT of the gene mutated in either Patient #1 or Patient #2
• Lentiviral vector was able to restore Complex I function only in the patient that had the corresponding mutation
→ Confirmation of causation


What about the causal genes in those 43% of patient in the MitoExome study whose mutations are still unidentified?

These mutations could be:

1. In genes that were missed with the MitoExome sequencing (since only 1000 genes that were covered)
For example:
• Poor coverage
• Exon deletions

2. Detected but not prioritised

3. Non-targeted region
For example:
• Intron
• Regulatory region
• Unidentified genes in the mitochondrial genome

4. Complex inheritance
• Epigenetics etc


What are the advantages and disadvantages of the various sequencing approaches:
• MitoExome
• Whole exome
• Whole genome?

• mtDNA + 1000 nuclear genes
• ? still missing genes

Whole exome:
• 20,000 genes
• Excludes mtDNA

Whole genome:
• Sequencing of everything
• However, too much data


Describe the Gartner Hype cycle in the context of NextGen sequencing

1. Technology trigger

2. Exponential rise

3. Peak of inflated expectations
• Everyone was very excited

4. Trough of disillusionment
• Patients weren't being diagnosed as well and as quickly as was thought possible
• Expensive
• Bio-informatic issues

5. Slope of enlightenment
• Less expensive
• Technology sufficiently utilised to put together good protocols in terms of analysing the data

6. Plateau of productivity

At the moment with NextGen, we are on the 'Slope of enlightenment'


What proportion of childhood-onset OXPHOS disorders have had their causation identified?

Only about 50%