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 pathway- large 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