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Flashcards in lecture 29 Deck (23):
1

What are mitochondrial oxidative phosphorylation disorders?

- 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

2

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

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

3

What does mitochondrial DNA look like?

- double stranded circular DNA
- found in mitochondria
- inherited from mother
- 16.5 kB

4

What are unique features of mtDNA?

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

5

What is leigh disease?

- 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

6

How common are OXPHOS disorders?

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

7

What are the genes that can cause an OXPHOS disorder?

- 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

8

How have OXPHOS genes been identified?

- mtDNA sequencing
- candidate
- linkage, candidate
- MMCT, linkage, candifate
- targeted exome
- whole exome

9

Whaat causes leigh syndrome?

- 30 autosomal genes
- 12 mt DNA genes
- 2 x-linked genes
- clearly very complex

10

What are challenges of OXPHOS molecular diagnosis?

- 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

11

What is the difference between sanger and nextgen sequencing technologies?

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

12

What are the flavours of nextgen sequencing?

- Illumina highseq and myseq
- Salt sequencing?
- ion torrent
- minION - oxford nanoxpore

13

What is illumina sequencing

- 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

14

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

sanger
- heterozygous appears as two peaks

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

15

What is ion torrent?

- 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

16

How can we use nextgen sequencing?

- 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

What is the 'MitoExome' strategy?

- 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

Prioritisation sounds great, does it work?

- 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

What was seen in the mitoexome cohort?

- 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

What was determined from original study?

- 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

Is mitoexome the best nextgen strategy?

- 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

What stage are we at with next gen sequencing?

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

currently in slope of enlightenment

23

Summary?

- 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