A Mosaic Activating Mutation in AKT1 Associated With The Proteus Syndrome Lindhurst, et al.

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A Mosaic Activating Mutation in AKT1 Associated
With The Proteus SyndromeLindhurst, et al.

• Jake Bowling

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Proteus Syndrome

• Joseph Merrick- Elephant Man
• Characterized by patchy/segmental overgrowth
  hyperplasia of multiple tissues, notably skin,
  and high susceptibility to tumors.
• lt1 in a million

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Evidence For Mosaicism

• Has never been reported to recur in a family
• Has been reported in discordant twins
• Wide variety of severities
• 20 years

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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

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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) - AKT1GgtA , predicting a substitution of
  lysine for glutamine at amino acid 17 (missense).
  
• AKT1 codes for a protein kinase, part of a
  signaling pathway.

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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 lt.001)
• 26 of 29 patients mutation positive (only 6 reads
  from the 3)

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• 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)

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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 lt.005).
• Bottom Line Mutation causes activation /
  upregulation.
• PI3K-AKT-mTOR pathway- large signaling pathway,
  overactivation limits apoptosis.

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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

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Cancer

• Catalogue of Somatic Mutations in Cancer database
• 116 of 7942 samples contained mutation (breast,
  thyroid, urinary, lung, endometrial)
• AKT upregulation - tumor susceptibility