Germline substitutions in the endothelial cell tyrosine kinase receptor TIE2/TEK cause a rare inherited form of venous anomalies, mucocutaneous venous malformations (VMCM)1-4. We now identified a somatic 2nd hit causing loss-of-function of the receptor in a resected VMCM. We assessed for whether such localized, tissue-specific events play a role in the etiology of the far more common sporadic VM. Eight somatic TIE2 mutations were identified in lesions from 28 out of 57 patients (49.1%), not detected in their blood or in control tissues. The somatic mutations included a frequent L914F change, and a series of double-mutations that occurred in cis, all of which show ligand-independent hyperphosphorylation in vitro. When overexpressed in HUVECs, L914F showed abnormal localization and response to ligand, differing from wild-type and the common inherited R849W mutant, suggesting they may have distinct effects. The presence of the same mutations in multifocal VMs in two patients, suggests a common origin for the abnormal endothelial cells in the distant sites. In conclusion, these data illustrate that a sporadic disease may be explained by somatic changes in a gene causing rare, inherited forms, and pinpoint TIE2 pathways as potential therapeutic targets for VM.

Mutations in the angiopoietin receptor TIE2/TEK have been identified as the cause for autosomal dominantly inherited cutaneomucosal venous malformation (VMCM). Thus far, two specific germline substitutions (R849W and Y897S), located in the kinase domain of TIE2, have been reported in five families. The mutations result in a 4-fold increase in ligand-independent phosphorylation of the receptor. Here, we report 12 new families with TEK mutations. Although the phenotype is primarily characterized by small multifocal cutaneous vascular malformations, many affected members also have mucosal lesions. In addition, cardiac malformations are observed in some families. Six of the identified mutations are novel, with three located in the tyrosine kinase domain, two in the kinase insert domain, and another in the carboxy-terminal tail. The remaining six are R849W substitutions. Overexpression of the novel mutants resulted in ligand-independent hyperphosphorylation of the receptor, suggesting this is a general feature of VMCM-causative TIE2 mutations. Moreover, variation in the level of activation demonstrates, for the first time, that widely differing levels of chronic TIE2 hyperphosphorylation are tolerated in the heterozygous state, and are compatible with normal endothelial cell function except in the context of highly localized areas of lesion-pathogenesis.

PTHR1-signaling pathway is critical for the regulation of endochondral ossification. Thus, abnormalities in genes belonging to this pathway could potentially participate in the pathogenesis of Ollier disease/Maffucci syndrome, two developmental disorders defined by the presence of multiple enchondromas. In agreement, a functionally deleterious mutation in PTHR1 (p.R150C) was identified in enchondromas from two of six unrelated patients with enchondromatosis. However, neither the p.R150C mutation (26 tumors) nor any other mutation in the PTHR1 gene (11 patients) could be identified in another study. To further define the role of PTHR1-signaling pathway in Ollier disease and Maffucci syndrome, we analyzed the coding sequences of four genes (PTHR1, IHH, PTHrP and GNAS1) in leucocyte and/or tumor DNA from 61 and 23 patients affected with Ollier disease or Maffucci syndrome, respectively. We identified three previously undescribed missense mutations in PTHR1 in patients with Ollier disease at the heterozygous state. Two mutations (p.G121E, p.A122T) were present only in enchondromas, and one (p.R255H) in both enchondroma and leukocyte DNA. Assessment of receptor function demonstrated that these three mutations impair PTHR1 function by reducing either the affinity of the receptor for PTH or the receptor expression at the cell surface. These mutations were not found in DNA from 222 controls. Including our data, PTHR1 functionally deleterious mutations have now been identified in five out 31 enchondromas from Ollier patients. These findings provide further support for the idea that heterozygous mutations in PTHR1 that impair receptor function participate in the pathogenesis of Ollier disease in some patients.

Maffucci syndrome (MS) is a rare congenital disorder characterized by multiple central cartilaginous tumors (enchondromas) in association with cutaneous spindle cell hemangiomas. These patients have a high incidence of malignant transformation. No familial case is known and the etiopathogenic cause remains unknown. In enchondromatosis (Ollier disease, OD), which is comprised of enchondromas only, 4 mutations in the PTHR1 gene have been identified in 4 patients; 3 were somatic and 1 was germline. No PTHR1 mutations have been detected in MS, whereas somatic IDH1 and, more rarely, IDH2 mutations have been observed in 77% of patients with MS and 81% of patients with OD. These genetic alterations are shared with other tumors, including glioma, leukemia and carcinoma. To search for underlying somatic genomic causes, we screened MS tissues using Affymetrix SNP-chips. We looked for CNVs, LOH and uniparental isodisomy (UPID) by performing pairwise analyses between allelic intensities in tumoral DNA versus the corresponding blood-extracted DNA. While common chromosomal anomalies were absent in constitutional DNA, several shared CNVs were identified in MS-associated tumors. The most frequently encountered somatic alterations were localized in 2p22.3, 2q24.3 and 14q11.2, implicating these chromosomal rearrangements in the formation of enchondromas and spindle cell hemangiomas in MS. In one chondrosarcoma specimen, large amplifications and/or deletions were observed in chromosomes 3, 6, 9, 10, 12, 13, and 19. Some of these genetic changes have been reported in other chondrosarcomas suggesting an etiopathogenic role. No LOH/UPID was observed in any Maffucci tissue. Our findings identify frequent somatic chromosomal rearrangements on 2p22.3, 2q24.3 and 14q11.2, which may unmask mutations leading to the lesions pathognomonic of MS.

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