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PATH30001 > lecture 29 > Flashcards

lecture 29 Flashcards

1
Q

What are mitochondrial oxidative phosphorylation disorders?

A
  • mitochondria are the ‘powerplants’ of the cell
  • 5 individual functional enzyme (OXPHOS) complexes
  • complex subunits encoded both by mitochondrial (mtDNA) and nuclear DNA
  • OXPHOS disorders (or mitochondrial diseases) affect 1:5000 live births.
  • present at any age, affect any organ and with any symptom with variable severity
  • inherited maternally through mtDNA, X-linked, autosomal recessive, autosomal dominant fashions
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2
Q

How many subunits in the complexes are respectively encoded for by mtDNA and nDNA?

A

Complex I:

  • 7 mtDNA
  • 37 nDNA

Complex II:

  • 0 mtDNA
  • 4 nDNA

Complex III:

  • 1 mtDNA
  • 10 nDNA

Complex IV:

  • 3 mtDNA
  • 11 nDNA

Complex V:

  • 2 mtDNA
  • 12 nDNA
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3
Q

What does mitochondrial DNA look like?

A
  • double stranded circular DNA
  • found in mitochondria
  • inherited from mother
  • 16.5 kB
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4
Q

What are unique features of mtDNA?

A
  • maternal inheritance
  • multiple copies
  • high copies
  • high mutation rate
  • heteroplasmy
  • threshold effect
  • mtDNA bottleneck
  • tissue-specific segregation/selection
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5
Q

What is leigh disease?

A
  • the most common mitochondrial disease of childhood
  • typically healthy until ~6 months
  • progressive, episodic neurodegenerative disorder
  • motor and or intellectual refression with signs of brainstem dysfunction
  • focal symmetric spongiform lesions in CNS
    → demyelination, gliosis, capillary proliferation
  • usually appears after an insult e.g. viral insult
  • usually don’t live past 18 months of age
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6
Q

How common are OXPHOS disorders?

A

Childhood-onset OXPHOS disorders

  • skladal et al, Brain 2003
  • 6.2/100,000 births
  • 71.4/100,000 in NSW lebanese
  • 10 or 20 founder mutations in certain populations

adult-onset oxphos disorders
- 9.2/100,000

total minimum birth prevalence:

  • 6.2 + 9.2 = 15.4/100,000 or 1/6500 births
  • probably quite an under estimation: probably 1/5000 is more accurate

~ 1/200 people carry pathogenic mtDNA mutaions but only ~1/10,000 diagnosed with mtDNA disease

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7
Q

What are the genes that can cause an OXPHOS disorder?

A
  • 35 of 37 mtDNA genes: tRNA, subunit, rRNA, deletions and duplications
  • 29 or ~80 nuclear subunits
  • 39 oxphos biogenesis genes
  • 6 mtDNA replication genes: POLG, POLG2, C10orf2, MPV17, MGME1, DNA2
  • 10 RNA transport, nucleotide transport, synthesis genes
  • 24 mtDNA expression genes
  • 12 membrane dynamics

120 nuclear gene defects

  • 104 autosomal recessive
  • 15 autosomal dominant
  • 6 x-linked
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8
Q

How have OXPHOS genes been identified?

A
  • mtDNA sequencing
  • candidate
  • linkage, candidate
  • MMCT, linkage, candifate
  • targeted exome
  • whole exome
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9
Q

Whaat causes leigh syndrome?

A
  • 30 autosomal genes
  • 12 mt DNA genes
  • 2 x-linked genes
  • clearly very complex
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10
Q

What are challenges of OXPHOS molecular diagnosis?

A
  • large number of candidate genes
  • mostly private mutations
  • not really hotspots
  • common mutations in only a few genes
  • genotype/phenotype correlation often poor
  • molecular diagnosis may require sequential testing of many genes and currently needs expert guidance
  • can ‘next generation sequencing’ allow us to sequency 100, 1000 or 20,000 genes in suspected patients faster and cheaper?
  • yes but sensitivity and specificty for medical genetic testing are still being established
  • next gen sequencing is being used for other conditions with similarly large numbers of causative genes, including inherited forms of deafness, blindness, epilepsy, cardiomyopathy, X-linked mental retardation, neuromuscular diseases etc
  • sequencing is starting to become more affordable
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11
Q

What is the difference between sanger and nextgen sequencing technologies?

A

sanger sequencing

  • 1 target DNA
  • average of all DNA molecules
  • ~800 bp per run

nextgen

  • thousands of DNAs at a time
  • single molecule DNA sequences
  • > 800,000,000 bp per run

human genome

  • 3gb (3 x 10^9)
  • 20,000 genes
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12
Q

What are the flavours of nextgen sequencing?

A
  • Illumina highseq and myseq
  • Salt sequencing?
  • ion torrent
  • minION - oxford nanoxpore
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13
Q

What is illumina sequencing

A
  • sequences of fluorescently labelled DNA fragments are amplified in clusters on a flowcell substrate

DNA <1 ug
sample preparation → cluster growth (0.1 - 0.5 billion) → sequencing (2 x 35 - 100 bases)
excitation and emission

image acquisition → base calling

get DNA and shear it into small fragments

  • add adaptor proteins to each end
  • these allow you to bind your DNA to a flow cell
  • amplify through bridging PCR in clusters
  • do it about 35 times
  • release from flow cell and sequencing reaction happens
  • add fluorescently activated nucleotides to the mix
  • when the right nucleotide is incorporated it excites and allows an emission
  • emission is read by the machine
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14
Q

How does illumina IGS sequencing compare to traditional sequencing in output?

A

sanger
- heterozygous appears as two peaks

illumina
- sequence read out, some have A and some have G

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15
Q

What is ion torrent?

A
  • individual molecules within single wells on a chip are sequenced in reactions that release protons for detection
  • DNA captured on a bead
  • amplified in an emulsion amplification (PCR within an oil bubble)
  • each bead added to individual wells on a chip

454 vs ion

  • 454: when there is a nucleotide added it emits light and that is what is measured
  • ion: when nucleotides are added they release protons; change in amplitude that is measure in flow cell
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16
Q

How can we use nextgen sequencing?

A
  • sequence 10, 40 or 100 genes
    → mtDNA, complex I deficiency…
  • sequence 1000 or more genes
    → MitoExome (mtDNA and all known mitochondrial proteins)
    i.e. candidate gene approach at a larger scale than sanger sequencing
  • whole exome sequencing
    → all 20,000 known genes (60Mb or 6 x 10^7 bp)
  • in each case,, we need a large bioinformatic infrastructure and software to be able to sieve through the data to find the needles in the hay stack
17
Q

What is the ‘MitoExome’ strategy?

A
  • 1034 genes, entire mtDNA, mitocarta, 1.4Mb coding exons X 44 OXPHOS patient samples, severe biochemical defects
→ hybrid in-solution selection/illumina sequence 
→ 14 exons = 40K baits 
→ ~40% on target 
→ 87% targets well-covered 
→ 145X mean coverage

known disease variants
- average of 1 per patient but only 8 of 42 were relevant to the patient’s disease

1.4 mbp covered

18
Q

Prioritisation sounds great, does it work?

A
  • non-consanguineous patients compared with 371 controls
  • looked like detecting real changes
  • unmasking the right genes
  • prioritised variants in known disease genes
  • 11 patients had prioritised variants in known disease genes
  • 10/11 validated by segregation, DNA, RNA and or protein analyses
19
Q

What was seen in the mitoexome cohort?

A
  • 2 genes had 2 patients from unrelated families
  • 6 genes had patients with biochemical profiles consistent with the known/suspected role of the gene
  • causation was proven for 4 genes via lentiviral correction studies
  • 4 genes have since had additional patients reported in the literature
  • expression of wildtype and mutant proteins showed functional defect
  • 2 genes eliminated by finding other cause in patient
20
Q

What was determined from original study?

A
  • 10 diagnoses in known disease genes: 7 nDNA and 1 mtDNA
  • 10 diagoses in 8 novel disease genes
    → CI subunit
    → CIII subunit
    → mtDNA translation (initiation)
    → mtDNA translation (elongation)
    → mito membrane lipid kinase
    → iron sulfur metabolism
    → CIII assembly factor
    → CIV assembly factor
  • 43% hd no prioritised genes, where are the missing genes?
    → missed in Mitoexome sequencing (e.g. poor coverage or exon deletions)
    → detected by not prioritised (e.g. de novo dominant, variants >0.005 freq)
    → non-targeted region (intron/regulatory) or gene
    → complex inheritance
21
Q

Is mitoexome the best nextgen strategy?

A
  • no
  • all approaches have advantages and disadvantages
  • targeting only known “disease genes” is popular in diagnostic labs
  • mitoexome includes mtDNA as well as ~1000 nuclear genes
  • whole exome includes 20,000 genes but not mtDNA
  • whole genome includes everything but is a lot of data
22
Q

What stage are we at with next gen sequencing?

A
  • technology trigger
  • peak of inflated expectations
  • trough of disillusionment
  • slope of enlightenment
  • plateau of productivity

currently in slope of enlightenment

23
Q

Summary?

A
  • OXPHOS disorders occur in ~1/5000 people
  • almost any symptom, any organ, any age of onset
  • mtDNA genetics: unique features
  • pathogenic mutations found in 35 of 37 mtDNA genes
  • currently 120 nuclear “disease” genes encoding subunits or proteins required for biogenesis, mtDNA replication and expression, mito nucleotide pool, mito membrane composition
  • diagnosis reliant on weighing evidence from multiple sources
  • Known OXPHOS disease genes appear to account for only ~50% of childhood- onset OXPHOS disorders
  • nextgen sequencing will transform diagnosis of complicated genetic diseases with locus and allelic heterogeneity such as mitochondrial and other metabolic diseases, epilepsy, deafness, mental retardation, heart disease and familial cancers

PATH30001 (34 decks)

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