Our data support the conclusion that the AKT1
c.49G→A variant causes the Proteus syndrome and the mosaicism hypothesis that was advanced more than 20 years ago by Happle.4,5
It has been proposed that a more circumscribed or milder manifestation of the disorder would be associated with a later occurrence of the somatic mutation in an embryo.12
Although we detected the mutation more often in affected tissues than in unaffected tissues, we did not observe an association between the proportion of mutant alleles and the overall clinical severity or specific manifestations of the phenotype. Our data do not suggest a specific stage during development at which the mutation arose in the patients who were included in our analyses.
Although many of the cell cultures derived from the affected tissues carried the mutation, the cells from which we derived these cultures may not have been the cells that caused the tissue to be abnormal. Typical cell-culture conditions support the growth of mesodermal fibroblasts. Since the Proteus phenotype can be manifested in the derivatives of all three germ layers,1,13
it is likely that lineages other than the mesoderm are affected by this mutation. We therefore microdissected sections of skin-biopsy samples (embedded in paraffin blocks) obtained from patients with the Proteus syndrome, purified DNA from the microdissected tissue, and analyzed it on restriction-enzyme digestion. The preliminary data so derived suggest that the prevalence of the mutation is higher in the upper dermis than in the epidermis or lower dermis and that the mutation is absent in glandular tissue (data not shown). Further experiments are necessary to characterize the cells in the many tissues that can be affected in this disorder.
Only 2 of the 38 peripheral-blood DNA samples that were collected from patients with the Proteus syndrome were positive for the mutation, a proportion that is significantly lower than both the proportion of apparently affected samples that were positive (75 of 97, P<0.001) and the proportion of unaffected samples that were positive (13 of 41, P = 0.004). These findings are consistent with a previous study showing that the AKT1
activating mutation is detrimental to hematopoiesis,14
and they suggest that molecular diagnosis of the Proteus syndrome with the use of peripheral-blood DNA may be challenging. Clinical molecular diagnosis on the basis of testing for the mutation in the DNA derived from peripheral blood must therefore await more sensitive methods of detection. With currently available mutation-detection methods, we recommend obtaining biopsy samples for testing.
Samples from three patients with typical Proteus syndrome (Patients 34, 46, and 52) were negative for the mutation. Clinically, we could not distinguish these patients from those with mutation-positive samples. We analyzed only two, one, and three samples, respectively, from these three patients, and we think it is likely that these samples were negative purely by chance. Alternatively, a different activating mutation in AKT1 or a mutation in a different gene may have caused the Proteus syndrome in these patients. However, full sequencing of AKT1 exons and flanking introns in these three patients showed normal sequences (data not shown).
It is interesting that the guanidine residue at position 49 of AKT1 does not include a cytidine–phosphate–guanosine (CpG) dinucleotide and is therefore not predicted to be highly mutable. Moreover, the mutation is present in a mosaic form in patients with the Proteus syndrome. We hypothesize that these two features explain the extreme rarity of this disorder.
The c.49G→A, p.Glu17Lys AKT1 variant is functionally important but uncommon in tumors. According to the Catalogue of Somatic Mutations in Cancer (COSMIC) database,15
this variant has been detected in 116 of 7942 unique cancer samples, including cancers of the breast (72 samples), thyroid (10 samples), urinary tract (9 samples), lung (6 samples), and endometrium (5 samples). It is proposed that constitutive activation of AKT1 through Ser473 and Thr308 phosphorylation underlies the oncogenic mechanism.11
We have found that the up-regulation of AKT1 phosphorylation as a result of a heterozygous mutation in AKT1
occurs in some tissues of patients with the Proteus syndrome and suggest that constitutive activation of the protein underlies the overgrowth and tumor susceptibility in these patients.
Akt1 loss and gain of function have been evaluated extensively in the mouse. The Akt1
null phenotype includes somatic and central-nervous-system growth retardation,16,17
a reduced number and caliber of lymphatic capillaries,18
reduced growth of trabecular bone, reduced formation of endochondral bone,19
dwarfism and reduced ossification of cartilage,20
and platelet dysfunction with prolonged bleeding times.21
Mice with activated forms of Akt1 have also been studied. Fukai et al.20
found that activated Akt1 in vitro stimulated cartilage calcification, a major manifestation of the Proteus syndrome. Mice that were transgenic for an activated (myristolated) form of Akt1 had skin hyperplasia, which is also a manifestation of the Proteus syndrome.22,23
Generally speaking, the mouse phenotype that results from loss of function of Akt1 is the opposite of that of the Proteus syndrome, and the phenotype that results from gain of function is strikingly similar to that of the Proteus syndrome.
Several groups have reported that patients with the Proteus syndrome had PTEN
Other investigators have argued that persons with PTEN
mutations were clinically distinct from those with the Proteus syndrome and that persons with bona fide Proteus syndrome did not have PTEN
Among patients with segmental overgrowth disorders, there is clinical overlap between those with somatic PTEN
mutations — now designated as the segmental overgrowth, lipomatosis, arteriovenous malformation, and epidermal nevus (SOLAMEN) syndrome,29
or type 2 segmental Cowden syndrome (T2SCS)30
— and those with the Proteus syndrome. AKT1 is activated by loss-of-function mutations in PTEN
which explains why patients with such mutations (those with the SOLAMEN syndrome) and patients with activating mutations in AKT1
(those with the Proteus syndrome) have overlapping but distinct clinical manifestations. The Proteus and SOLAMEN syndromes may be members of a larger family of disorders related to dysfunction in the PI3K–AKT pathway. We hypothesize that multiple disorders are caused by mutated genes encoding proteins in this pathway.
The importance of somatic genetic variation is widely appreciated in oncology, in which a broad range of variations contributes to the pathogenesis of cancer. In contrast, the Proteus syndrome is caused by a de novo somatic mutation in a single gene. Several disorders share this attribute, with the prototype being the McCune–Albright syndrome, caused by a somatic mutation in GNAS
The SOLAMEN syndrome29
and autosomal dominant polycystic kidney disease33
are caused by two mutations, each of which affects an allelic copy of the same gene. One of these mutations is inherited in the germline. Somatic acquisition of the second mutation results in disease. Germline or inherited disorders are termed simple or complex on the basis of whether they can be attributed to a single gene variant or multiple gene variants, respectively. We suggest that mosaic disorders are analogous to inherited disorders in that some of them (e.g., the Proteus and McCune–Albright syndromes) are caused by a single variant and others (e.g., many cancers) arise only after the accumulation of many somatic mutations.