ECON 546 Lecture Notes - Lecture 16: Tuberous Sclerosis, Akt3, Fibroblast
27 Jun 2018
School
Department
Course
Professor
BIOL 568 L16: 1/3/18
1
L16: Genetic Mosaicism in rare and common disease
o Genetic mosaicism: caused by mutation that occurs at some point after fertilisation – post-
zygotic mutations.
o All cells that derive from the mutation also have the mutation.
o Organism composed of both wt and mutation.
o Some only affect somatic tissue – will not be transmitted to next generation.
o Some only affect germline cells, some both
Most studies are of disorders of skin – patterns highly suggestive of mosaicism, easy to visualise
Mosaicism in human skin:
1. Type 1: two normal alleles. In some cells, one of the alleles acquires mutation.
2. Type 2: one germ line mutation – in all cells. Usually are LOF alleles that can be inherited.
− The other allele may acquire post-zygotic mutation.
− Cancer – ‘double hit’ hypothesis e.g. BRCA.
A recent field of research in genomics:
o Various mosaic syndromes involving the skin have been clinically recognised for years
o Hypothesis: lethal mutations surviving only by mosaicism – if in all cells, not compatible with
life
o Most of their underlying genetic causes have been identified by NGS since 2011
Idea that all the body’s cells have the same DNA is false.
Post-zygotic mutations are frequent:
o Approx. 1016 are required to generate an adult human composed of 1014 cells
o Mutation rates:
− 10-8 to 10-7 detectable mutations per nucleotide (at a mutant allele fraction >5%)
− Data from one tissue (blood)
o Genetic mosaicism is the norm rather than the exception – just an issue of how many and
where
Challenges – even for monogenic syndromes:
1. Clinical heterogeneity
a. Timing and cell lineage affected by the mutation
b. Clinical recognition and syndrome delineation are difficult
2. Access to affected tissue
a. Mutations are often not present in blood
b. Required to prove causality
3. Technological challenges
a. Identifying mosaic mutations among germline variants and sequencing errors remains
challenging
Exome sequencing in mosaic disorders:
1. Require affected tissue (fresh and uncultured).
− E.g. affected skin DNA. Must have something to compare it to e.g. blood sample in
order to identify mutations. However, if testing something that is absent in blood – will
not work.
2. Pair- or trio-based “deep” exome sequencing
− Skin sample and blood samples from parents – mutations present in child and absent
in both parents.
3. Detection threshold: 5-10% of mutant alleles
BIOL 568 L16: 1/3/18
2
Deep-targeted sequencing:
o Deep sequencing of candidate gene(s)
o Systematic analysis of non-reference alleles
o Detection threshold: ~1% of mutant alleles
o E.g. PIK3CA: One postzygotic mutation in less than 2% of reads. Sequencing depth – all
covered by at least 10, 000 reads.
Mosaic disorders of the P13K-AKT-mTOR pathway
AKT1 in Proteus syndrome:
o Took pairs of samples – affected specimen and blood samples.
o Characterised by somatic overgrowth
o All patients had post-zygotic mutation in AKT1 gene – same AA change at AA17 (v rare)
o Tested different tissue – mosaicism in varying proportion of cells. Depending on what
region is tested there will be different levels of mosaicism.
Lessons from Proteus syndrome:
o Power of NGS sequencing for detecting mosaicism
o Mutation characteristics:
− Hot spot mutations
− GOF (activating) of AKT1 – pathway involved in growth and cell division
− Known somatic driver mutations in sporadic cancer – cancer is an accumulation of
many mutations.
− Variable levels depending on the tissue tested
− Often not detectable in blood
− Supports the hypothesis of lethal mutations surviving by mosaicism, never found in
germline state.
P13K genes in mosaic overgrowth:
o Activating mutations ! overgrowth in many syndromes
o Early mutations – severe phenotype
o Later – less severe, additional finger etc.
o Phenotypes with brain overgrowth
PIK3R2 (another gene in pathway) in malformations of cortical development
o De novo germline and mosaic mutations in megalencephaly and polymicrogyria (MPPH
syndrome)
o Mosaic mutations in bilateral perisylvian polymicrogyria w normal head size i.e. somatic cells.
MTOR in hemimegalencephaly:
o Absent in blood samples
PIK3CA, MTOR et al. in focal cortical dysplasia:
o Mosaic mutations in MTOR, PIK3CA and AKT3
o Germline mutations in PTEN, TSC2 and DEPDC5
o Seizures
o Treat by surgically removing abnormal brain malformations
o Identified mutations in brain samples
Document Summary
Most studies are of disorders of skin patterns highly suggestive of mosaicism, easy to visualise. Mosaicism in human skin: type 1: two normal alleles. In some cells, one of the alleles acquires mutation: type 2: one germ line mutation in all cells. Usually are lof alleles that can be inherited. The other allele may acquire post-zygotic mutation. Cancer double hit" hypothesis e. g. brca. Idea that all the body"s cells have the same dna is false. 1016 are required to generate an adult human composed of 1014 cells: mutation rates: 10-8 to 10-7 detectable mutations per nucleotide (at a mutant allele fraction >5%) Data from one tissue (blood: genetic mosaicism is the norm rather than the exception just an issue of how many and where. Exome sequencing in mosaic disorders: require affected tissue (fresh and uncultured). Must have something to compare it to e. g. blood sample in order to identify mutations.
