Marjorie J. Lindhurst

Mosaic overgrowth with fibroadipose hyperplasia is caused by somatic activating mutations in PIK3CA

The phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway is critical for cellular growth and metabolism. Correspondingly, loss of function of PTEN, a negative regulator of PI3K, or activating mutations in AKT1, AKT2 or AKT3 have been found in distinct disorders featuring overgrowth or hypoglycemia. We performed exome sequencing of DNA from unaffected and affected cells from an individual with an unclassified syndrome of congenital progressive segmental overgrowth of fibrous and adipose tissue and bone and identified the cancer-associated mutation encoding p.His1047Leu in PIK3CA, the gene that encodes the p110α catalytic subunit of PI3K, only in affected cells. Sequencing of PIK3CA in ten additional individuals with overlapping syndromes identified either the p.His1047Leu alteration or a second cancer-associated alteration, p.His1047Arg, in nine cases. Affected dermal fibroblasts showed enhanced basal and epidermal growth factor (EGF)-stimulated phosphatidylinositol 3,4,5-trisphosphate (PIP3) generation and concomitant activation of downstream signaling relative to their unaffected counterparts. Our findings characterize a distinct overgrowth syndrome, biochemically demonstrate activation of PI3K signaling and thereby identify a rational therapeutic target.

PIK3CA-related overgrowth spectrum (PROS): Diagnostic and testing eligibility criteria, differential diagnosis, and evaluation

Somatic activating mutations in the phosphatidylinositol‐3‐kinase/AKT/mTOR pathway underlie heterogeneous segmental overgrowth phenotypes. Because of the extreme differences among patients, we sought to characterize the phenotypic spectrum associated with different genotypes and mutation burdens, including a better understanding of associated complications and natural history. Historically, the clinical diagnoses in patients with PIK3CA activating mutations have included Fibroadipose hyperplasia or Overgrowth (FAO), Hemihyperplasia Multiple Lipomatosis (HHML), Congenital Lipomatous Overgrowth, Vascular Malformations, Epidermal Nevi, Scoliosis/Skeletal and Spinal (CLOVES) syndrome, macrodactyly, Fibroadipose Infiltrating Lipomatosis, and the related megalencephaly syndromes, Megalencephaly‐Capillary Malformation (MCAP or M‐CM) and Dysplastic Megalencephaly (DMEG). A workshop was convened at the National Institutes of Health (NIH) to discuss and develop a consensus document regarding diagnosis and treatment of patients with PIK3CA‐associated somatic overgrowth disorders. Participants in the workshop included a group of researchers from several institutions who have been studying these disorders and have published their findings, as well as representatives from patient‐advocacy and support groups. The umbrella term of “PIK3CA‐Related Overgrowth Spectrum (PROS)” was agreed upon to encompass both the known and emerging clinical entities associated with somatic PIK3CA mutations including, macrodactyly, FAO, HHML, CLOVES, and related megalencephaly conditions. Key clinical diagnostic features and criteria for testing were proposed, and testing approaches summarized. Preliminary recommendations for a uniform approach to assessment of overgrowth and molecular diagnostic testing were determined. Future areas to address include the surgical management of overgrowth tissue and vascular anomalies, the optimal approach to thrombosis risk, and the testing of potential pharmacologic therapies.

Knockout of Slc25a19 causes mitochondrial thiamine pyrophosphate depletion, embryonic lethality, CNS malformations, and anemia

SLC25A19 mutations cause Amish lethal microcephaly (MCPHA), which markedly retards brain development and leads to α-ketoglutaric aciduria. Previous data suggested that SLC25A19, also called DNC, is a mitochondrial deoxyribonucleotide transporter. We generated a knockout mouse model of Slc25a19. These animals had 100% prenatal lethality by embryonic day 12. Affected embryos at embryonic day 10.5 have a neural-tube closure defect with ruffling of the neural fold ridges, a yolk sac erythropoietic failure, and elevated α-ketoglutarate in the amniotic fluid. We found that these animals have normal mitochondrial ribo- and deoxyribonucleoside triphosphate levels, suggesting that transport of these molecules is not the primary role of SLC25A19. We identified thiamine pyrophosphate (ThPP) transport as a candidate function of SLC25A19 through homology searching and confirmed it by using transport assays of the recombinant reconstituted protein. The mitochondria of Slc25a19−/− and MCPHA cells have undetectable and markedly reduced ThPP content, respectively. The reduction of ThPP levels causes dysfunction of the α-ketoglutarate dehydrogenase complex, which explains the high levels of this organic acid in MCPHA and suggests that mitochondrial ThPP transport is important for CNS development.

Clinical delineation and natural history of the PIK3CA -related overgrowth spectrum

Somatic mutations in the phosphatidylinositol/AKT/mTOR pathway cause segmental overgrowth disorders. Diagnostic descriptors associated with PIK3CA mutations include fibroadipose overgrowth (FAO), Hemihyperplasia multiple Lipomatosis (HHML), Congenital Lipomatous Overgrowth, Vascular malformations, Epidermal nevi, Scoliosis/skeletal and spinal (CLOVES) syndrome, macrodactyly, and the megalencephaly syndrome, Megalencephaly‐Capillary malformation (MCAP) syndrome. We set out to refine the understanding of the clinical spectrum and natural history of these phenotypes, and now describe 35 patients with segmental overgrowth and somatic PIK3CA mutations. The phenotypic data show that these previously described disease entities have considerable overlap, and represent a spectrum. While this spectrum overlaps with Proteus syndrome (sporadic, mosaic, and progressive) it can be distinguished by the absence of cerebriform connective tissue nevi and a distinct natural history. Vascular malformations were found in 15/35 (43%) and epidermal nevi in 4/35 (11%) patients, lower than in Proteus syndrome. Unlike Proteus syndrome, 31/35 (89%) patients with PIK3CA mutations had congenital overgrowth, and in 35/35 patients this was asymmetric and disproportionate. Overgrowth was mild with little postnatal progression in most, while in others it was severe and progressive requiring multiple surgeries. Novel findings include: adipose dysregulation present in all patients, unilateral overgrowth that is predominantly left‐sided, overgrowth that affects the lower extremities more than the upper extremities and progresses in a distal to proximal pattern, and in the most severely affected patients is associated with marked paucity of adipose tissue in unaffected areas. While the current data are consistent with some genotype–phenotype correlation, this cannot yet be confirmed.

Mosaic Activating Mutations in FGFR1 Cause Encephalocraniocutaneous Lipomatosis

Encephalocraniocutaneous lipomatosis (ECCL) is a sporadic condition characterized by ocular, cutaneous, and central nervous system anomalies. Key clinical features include a well-demarcated hairless fatty nevus on the scalp, benign ocular tumors, and central nervous system lipomas. Seizures, spasticity, and intellectual disability can be present, although affected individuals without seizures and with normal intellect have also been reported. Given the patchy and asymmetric nature of the malformations, ECCL has been hypothesized to be due to a post-zygotic, mosaic mutation. Despite phenotypic overlap with several other disorders associated with mutations in the RAS-MAPK and PI3K-AKT pathways, the molecular etiology of ECCL remains unknown. Using exome sequencing of DNA from multiple affected tissues from five unrelated individuals with ECCL, we identified two mosaic mutations, c.1638C>A (p.Asn546Lys) and c.1966A>G (p.Lys656Glu) within the tyrosine kinase domain of FGFR1, in two affected individuals each. These two residues are the most commonly mutated residues in FGFR1 in human cancers and are associated primarily with CNS tumors. Targeted resequencing of FGFR1 in multiple tissues from an independent cohort of individuals with ECCL identified one additional individual with a c.1638C>A (p.Asn546Lys) mutation in FGFR1. Functional studies of ECCL fibroblast cell lines show increased levels of phosphorylated FGFRs and phosphorylated FRS2, a direct substrate of FGFR1, as well as constitutive activation of RAS-MAPK signaling. In addition to identifying the molecular etiology of ECCL, our results support the emerging overlap between mosaic developmental disorders and tumorigenesis.

AKT1 gene mutation levels are correlated with the type of dermatologic lesions in patients with Proteus syndrome

To the editor Proteus syndrome (PS) is characterized by progressive, mosaic, segmental overgrowth and occurs sporadically (Biesecker 2001; 2006). The mosaic nature and sporadic occurrence with lack of familial transmission led to the hypothesis that PS is caused by a post-zygotic somatic mutation, which was confirmed with the discovery of a mosaic activating c.49G>A, p.Glu17Lys AKT1 mutation (Lindhurst et al. 2011). To date, all patients who meet the clinical diagnostic criteria for PS and have been tested have this mutation (Lindhurst and Biesecker, unpublished results). While any organ or tissue can be affected, skeletal overgrowth and dermatologic lesions are the most common manifestations of PS (Turner et al. 2004; Beachkofsky et al. 2010). Cerebriform connective tissue nevi (CCTN) are a highly specific and common lesion in patients with PS (Biesecker et al. 2001; Nguyen et al. 2004). These lesions are very firm and contain deep sulci that resemble the brain, for which the lesion is named. Histology sections of CCTN show massively expanded dermis filled with thick collagen bundles (Figure 1d-f) (McCuaig et al. 2012). Epidermal nevi (EN) can be non-syndromic or occur as part of several syndromes including PS (Happle 2010). The keratinocytic EN found in PS have a rough surface, are dark in color, usually follow the lines of Blaschko, and exhibit epidermal hyperkeratosis, papillomatosis, and acanthosis (Figure 1a-c) (Nguyen et al. 2004). EN are generally noticed in the first year of life and are stable in extent whereas CCTN grow progressively after first appearing later in the first or second year (Twede et al. 2005). Figure 1 Representative examples of an EN and CCTN. Panel a shows the rough surface of the linear EN on the infraaxillary vault in patient 101. Hematoxylin and eosin stain (b, scale bar = 100 μm; c, scale bar = 50 μm) of a skin biopsy from the ... It is unknown which cells determine the formation of these lesions. Based on the histology, we hypothesized that CCTN were generated by AKT1 p.Glu17Lys in the dermis and that EN were generated by this mutation in the epidermis. To test this hypothesis, we isolated fibroblasts and keratinocytes from lesional (CCTN or EN) and non-lesional (“normal”) skin samples and measured the level of the mutant allele in each cell type. Skin samples were collected during surgical procedures or by punch biopsies from patients with PS under an IRB-approved protocol. The epidermis was separated from the dermis by treatment with dispase and keratinocyte and fibroblast cultures were established using standard protocols (Aasen and Belmonte 2010). A single keratinocyte culture was established from each biopsy. Fibroblasts were allowed to grow out of the dermal tissue until the dish was confluent. The dermal tissue was then transferred to a new dish to allow another fibroblast culture to be established from that same tissue. The number of fibroblast cultures established from a piece of dermis ranged from one to six. DNA was isolated from cultured cells harvested between passages one and four, and the mutation level was assayed using a PCR-based restriction fragment length polymorphism (RFLP) assay as described (Lindhurst et al. 2011). This assay has a lower limit of sensitivity of 0.5%. Each DNA preparation was tested at least twice and the values were averaged. In ten of the 20 samples, DNA was also isolated from the dermal tissue used to establish the fibroblast cultures after the final transfer. Seven CCTN biopsies were obtained from the feet of three patients (Table S1). The mutant allele was not detected in CCTN keratinocyte cultures whereas the mutation level from the CCTN fibroblast cultures was 9-32%. In the dermal tissue post-culturing, the mutation level was 12-21% (Figure 2). Figure 2 Percentage of AKT1 c.49G>A, p.Glu17Lys mutation in epidermal and dermal cells and tissue. One keratinocyte culture (left panel) was established from each epidermal sample. Mutation percentages are indicated by the blue circles. Multiple fibroblast ... Nine EN samples were obtained from the hand, trunk, or neck from seven patients (Table S1). The mutation level in the keratinocyte cultures was 0-44% whereas the mutation level in the fibroblast cultures was 0-38%. The mutation level in the cultured dermal tissue was 9-25%. Interestingly, the two samples with the highest levels of mutation in the keratinocytes (101EN1 and 78EN2) had the lowest mutation percentages in dermal cells or tissue. Detection of the mutant allele in the EN keratinocytes is consistent with the identification of the AKT1 p.Glu17Lys mutation in skin scrapings of PS epidermal nevi (Wieland et al. 2013). Four biopsies from two patients were obtained from tissue that appeared normal by gross examination but was from an area that was far removed from lesional tissue (designated as unaffected) or bordered a CCTN (designated as unknown) (Table S1). No evidence of the mutant allele was present in the keratinocyte cultures. The mean mutation level in the fibroblast cultures was 6-27%. We conclude that the AKT1 p.Glu17Lys activating mutation in keratinocytes is a key determinant of EN formation. Mutations in the FGFR3, PIK3CA, and RAS genes have been identified in epidermal cells of EN not associated with PS, supporting this hypothesis (Hafner et al. 2006; 2007; 2012). Histopathologically, these keratinocytic EN are strikingly similar to those found in PS and it should be noted that PIK3CA encodes the catalytic subunit of the PI3K complex which functions upstream of AKT in the same signaling pathway. In contrast, there was no correlation of the mutation levels in fibroblasts or cultured dermal tissue with the clinical lesion type. The inability to detect an AKT1 mutation in two of the EN keratinocyte cultures could be due to a sampling artifact if the mutant cells were not uniformly distributed throughout the lesion and the sample was from an area with low-level mosaicism. It seems unlikely that the lack of mutant keratinocytes in some EN indicates that these lesions form as a result of signaling from mutant cells in the dermis, since even organoid EN, such as nevus sebaceous have been shown to result from mutant cells in the epidermis. (Groesser et al. 2012) Mutant cells were found in dermal fibroblasts of both CCTN and normal-appearing skin, suggesting that the presence of mutant cells in the dermis is necessary but not sufficient to drive the formation of CCTN. The propensity of CCTNs to develop on soles and palms in PS may reflect a greater role of AKT1 in postnatal regulation of tissue architecture at these sites than elsewhere on the skin.

Lack of mutation–histopathology correlation in a patient with Proteus syndrome

Proteus syndrome (PS) is characterized by progressive, disproportionate, segmental overgrowth, and tumor susceptibility caused by a somatic mosaic AKT1 activating mutation. Each individual has unique manifestations making this disorder extremely heterogeneous. We correlated three variables in 38 tissue samples from a patient who died with PS: the gross affection status, the microscopic affection status, and the mutation level. The AKT1 mutation was measured using a PCR‐based RFLP assay. Thirteen samples were grossly normal; six had detectable mutation (2–29%) although four of these six were histopathologically normal. Of the seven grossly normal samples that had no mutation, only four were histologically normal. The mutation level in the grossly abnormal samples was 3–35% and all but the right and left kidneys, skull, and left knee bone, with mutation levels of 19%, 15%, 26%, and 17%, respectively, had abnormal histopathology. The highest mutation level was in a toe bone sample whereas the lowest levels were in the soft tissue surrounding that toe, and an omental fat nodule. We also show here that PS overgrowth can be caused by cellular proliferation or by extracellular matrix expansion. Additionally, papillary thyroid carcinoma was identified, a tumor not previously associated with PS. We conclude that gross pathology and histopathology correlate poorly with mutation levels in PS, that overgrowth can be mediated by cellular proliferation or extracellular matrix expansion, and that papillary thyroid carcinoma is part of the tumor susceptibility of PS. New methods need to be developed to facilitate genotype–phenotype correlation in mosaic disorders.

A limited form of proteus syndrome with bilateral plantar cerebriform collagenomas and varicose veins secondary to a mosaic AKT1 mutation.

IMPORTANCE Proteus syndrome is an extremely rare disorder of mosaic postnatal overgrowth affecting multiple tissues including bone, soft tissue, and skin. It typically manifests in early childhood with asymmetric and progressive skeletal overgrowth that leads to severe distortion of the skeleton and disability. The genetic basis has recently been identified as a somatic activating mutation in the AKT1 gene, which encodes an enzyme mediating cell proliferation and apoptosis. OBSERVATIONS We present a 33-year-old man who developed plantar cerebriform collagenomas on the soles of both feet and varicose veins in early childhood, in the absence of any skeletal or other connective tissue abnormality. Although the patient did not meet the diagnostic criteria for Proteus syndrome, he was found to have the c.49G>A, p.Glu17Lys AKT1 mutation in lesional skin but not in his blood. CONCLUSIONS AND RELEVANCE To our knowledge, this is the mildest molecularly confirmed case of Proteus syndrome, occurring in the absence of the characteristic skeletal overgrowth. These findings extend the spectrum of Proteus syndrome pathological characteristics and suggest that somatic mutations late in development and restricted in distribution cause subtle clinical presentations that do not meet the published clinical criteria.

Somatic AKT1 mutations cause meningiomas colocalizing with a characteristic pattern of cranial hyperostosis

Somatic genetic mutations in meningiomas are associated with histologic subtypes, anatomical location, and grade. Concomitant hyperostosis occurs with some meningiomas and the pathogenesis is not well understood. Cranial hyperostosis and meningiomas are common in patients with Proteus syndrome, which is caused by a somatic activating mutation in AKT1 c.49G>A. This same mutation has also been found in 6–9% of sporadic non‐syndromic meningiomas. Sixty‐one patients with Proteus syndrome meeting clinical diagnostic criteria were evaluated at the NIH from 1997 to 2014. Of these 61, 52 had a somatic activating mutation (c.49G>A, p.Glu17Lys) in AKT1 confirmed from affected tissue samples. Photographs, physical examination and/or autopsy, X‐rays, CT, and/or MRI scan of the head were reviewed in 29/52 patients. Of the 29 patients, the most common intracranial tumor was meningioma, all co‐localizing with cranial hyperostosis, and diagnosed at younger ages than typical for isolated, non‐syndromic meningiomas. These patients had progressive cranial overgrowth that consisted primarily of diploic space expansion, and was characterized by unilateral, parasagittal, and frontal bone involvement. We hypothesize that sporadic meningothelial and transitional subtype meningiomas are a forme fruste or microform of Proteus syndrome, and activation of the AKT/PI3K pathway drives hyperostosis in both non‐syndromic, and Proteus‐related meningiomas.

Urine cell-free DNA is a biomarker for nephroblastomatosis or Wilms tumor in PIK3CA -related overgrowth spectrum (PROS)

PurposeWe set out to facilitate the molecular diagnosis of patients with PIK3CA-related overgrowth spectrum (PROS), a heterogeneous somatic disorder characterized by variable presentations of segmental overgrowth, vascular malformations, skin lesions, and nephroblastomatosis, rare precursor lesions to Wilms tumor. Molecular diagnosis of PROS is challenging due to its mosaic nature, often requiring invasive biopsies.MethodsDigital droplet polymerase chain reaction (ddPCR) was used to analyze tissues including urine, saliva, buccal cells, and blood, from eight patients with PROS. Further analyses were performed on plasma and urine cell-free DNA (cfDNA).ResultsPIK3CA variants were detected in plasma cfDNA at levels up to 0.5% in 50% of tested samples. In addition, high levels of PIK3CA variants in urine cfDNA correlated with a history of nephroblastomatosis compared with patients without renal involvement (P < 0.05).ConclusionDigital droplet PCR is a sensitive molecular tool that enables low-level variant detection of PIK3CA in various tissue types, providing an alternative diagnostic method. Furthermore, urine cfDNA is a candidate biomarker for nephroblastomatosis in PROS, which may be useful to refine screening guidelines for tumor risk in these patients.