PATH30001 > lecture 29 > Flashcards

lecture 29 Flashcards

(23 cards)

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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?

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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?

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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?

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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?

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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?

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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?

Study These Flashcards
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?

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

currently in slope of enlightenment

23
Q

Summary?

Study These Flashcards
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
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