Transcript A Mosaic Activating Mutation in AKT1 Associated With The

A Mosaic Activating Mutation
in AKT1 Associated With The
Proteus Syndrome
Lindhurst, et al.
Jake Bowling
Proteus Syndrome
• Joseph Merrick- Elephant Man
• Characterized by patchy/segmental overgrowth &
hyperplasia of multiple tissues, notably skin, and high
susceptibility to tumors.
• <1 in a million
Evidence For Mosaicism
• Has never been reported to recur in a
family
• Has been reported in discordant twins
• Wide variety of severities
• ~20 years
Hypothesis
• Proteus is caused by a somatic
mutation that is lethal when constitutive,
resulting in a mosaic disorder.
• Earlier mutation = more severe disease
• Approach- Sequence genomes of
affected tissues, searching for common
mutations
Initial Sequencing
• 4 tissue labels- “affected”, “unaffected”, “unknown”
and control.
• 11 cutaneous biopsies from 6 Proteus patients (7
affected, 4 unaffected)
• Found over 200,000 variants compared to human
reference sequence
• Only one was significant (present in 3.6-51% of
reads) - AKT1G>A , predicting a substitution of lysine
for glutamine at amino acid 17 (missense).
• AKT1 codes for a protein kinase, part of a signaling
pathway.
Validation
• Assay using restriction enzyme digestion (E.
coli), PCR, electropherography.
• Mutation negative in all 27 controls tested.
• 97 affected samples from 29 Proteus
patients- 75 mutation positive (p-value <.001)
• 26 of 29 patients mutation positive (only 6
reads from the 3)
• A) Shows enzyme
restriction
methodology inc.
forward & reverse
primers,
endonuclease site.
• C) Shows prevalence
of mutant allele in the
29 patients (ranges
1% to 47%)
Functionality
• Western Blot used antibodies
specific to phosphorylation at
Ser473 and Thr308
• Cells grown serum-free show
higher phosphorylation
(activation) in mutant cells than
wild-type cells (p-value <.005).
• Bottom Line: Mutation causes
activation / upregulation.
• PI3K-AKT-mTOR pathwaylarge signaling pathway,
overactivation limits apoptosis.
Conclusions
• Definitive- This mutation causes Proteus
• Not exclusive
• 2 of 38 proteus blood smears mutation
positive- supports mutation being detrimental
to hematopoeisis and makes diagnosis with
blood challenging.
• Mice data supports
Cancer
• Catalogue of Somatic Mutations in
Cancer database
• 116 of 7942 samples contained
mutation (breast, thyroid, urinary, lung,
endometrial)
• AKT upregulation - tumor susceptibility