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Abstract

Background: The Proteus syndrome is characterized by the overgrowth of skin, connective tissue, brain, and other tissues. It has been hypothesized that the syndrome is caused by somatic mosaicism for a mutation that is lethal in the nonmosaic state.

Methods: We performed exome sequencing of DNA from biopsy samples obtained from patients with the Proteus syndrome and compared the resultant DNA sequences with those of unaffected tissues obtained from the same patients. We confirmed and extended an observed association, using a custom restriction-enzyme assay to analyze the DNA in 158 samples from 29 patients with the Proteus syndrome. We then assayed activation of the AKT protein in affected tissues, using phosphorylation-specific antibodies on Western blots.

Results: Of 29 patients with the Proteus syndrome, 26 had a somatic activating mutation (c.49G→A, p.Glu17Lys) in the oncogene AKT1, encoding the AKT1 kinase, an enzyme known to mediate processes such as cell proliferation and apoptosis. Tissues and cell lines from patients with the Proteus syndrome harbored admixtures of mutant alleles that ranged from 1% to approximately 50%. Mutant cell lines showed greater AKT phosphorylation than did control cell lines. A pair of single-cell clones that were established from the same starting culture and differed with respect to their mutation status had different levels of AKT phosphorylation.

Conclusions: The Proteus syndrome is caused by a somatic activating mutation in AKT1, proving the hypothesis of somatic mosaicism and implicating activation of the PI3K-AKT pathway in the characteristic clinical findings of overgrowth and tumor susceptibility in this disorder. (Funded by the Intramural Research Program of the National Human Genome Research Institute.).

Figures

Figure 1
Figure 1. Clinical Manifestations of the Proteus Syndrome in a 12-Year-Old Boy
Panel A shows severe orthopedic manifestations, including scoliosis, overgrowth with a resultant discrepancy in leg length, and valgus deformity and distortion of the skeleton, in Patient 53. Panels B and C show the characteristic cerebriform connective-tissue nevus and overgrowth and distortion of the hands and feet. Cutaneous vascular anomalies are present on the dorsum of the foot.
Figure 2
Figure 2. Assay for the Detection of the AKT1 Mutation
Panel A shows a diagram of the modified polymerase-chain-reaction–restriction-enzyme assay that is used to detect the c.49G→A mutation in AKT1. The assay uses a restriction endonuclease that is derived from an Escherichia coli strain carrying the gene MboII from Moraxella bovis. The genomic sequence is shown above the bar; forward and reverse primers are below. The reverse primer was modified at position 28 (shown in red, chr14:105,246,548), creating an MboII restriction-enzyme site in the presence of the mutation (blue symbol and letters). The MboII consensus site is shown below the reverse primer. Panel B shows electropherograms for four samples from patients with the Proteus syndrome with varying levels of the mutant allele. Red arrows indicate the mutant allele, and blue arrows indicate the wild-type peak. The height (H) and area (A) for each peak are shown below the electropherograms and are expressed in relative fluorescence units (RFU). The numbers at the top of each electropherogram are base pairs. Ratios (provided as percentages) are the areas of the mutant peaks divided by the combined areas of the mutant and wild-type peaks. CCTN denotes cerebriform connective-tissue nevus, and ITB iliotibial band. Panel C shows the results of the MboII assay of samples from patients with the Proteus syndrome and from control subjects. The numbers on the y axis are the individual code numbers for patients for whom cell-line or tissue samples were available for testing. Grouped into single rows at the bottom of the graph are blood samples from 38 patients with the Proteus syndrome (PS), cell and tissue samples from 27 control subjects who did not have the Proteus syndrome, and blood samples from 48 control subjects. Multiple samples with the same value are represented by lines radiating from the data point, with each radiating line representing one sample.
Figure 3
Figure 3. Immunoblot Analyses of AKT Phosphorylation
To compare signals obtained from Western blot luminescence images, lysates were collected and separated on a 10% TRIS–glycine gel. AKT proteins were visualized with the use of antibodies that recognize phosphorylation at Ser473 (Panel A) or Thr308 (Panel B), and each set of lysates was also hybridized to total AKT (pan-AKT) and β-actin antibodies to assess loading variation. Lysates from a control cell line obtained from subjects without the Proteus syndrome are shown in lanes 1 and 2. Lysates from a cell line obtained from a patient with the Proteus syndrome (Patient 83 with cerebriform connective-tissue nevi [CCTN]; mutation level, 37%) are shown in lanes 3 and 4. Lysates from single-cell clones (SCC) isolated from culture established from a CCTN biopsy sample obtained from Patient 93 are shown in lanes 5 through 8, with lysates from a mutation-negative (wild-type) clone shown in lanes 5 and 6 and those from a mutation-positive clone shown in lanes 7 and 8. Lysates in lanes 1, 3, 5, and 7 were grown in serum-containing medium, indicated by a plus sign; those in lanes 2, 4, 6, and 8 were grown in serum-free medium, indicated by a minus sign, for 8 hours before harvesting. Quantitative analyses showed that in cells grown in the absence of factors that can activate the AKT pathway (i.e., in serum-free conditions), the level of phosphorylated AKT protein was higher in mutation-positive cells than in mutation-negative cells (P<0.005).

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