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Blue rubber bleb nevus syndrome (Bean syndrome) is a rare, severe disorder of unknown cause, characterized by numerous cutaneous and internal venous malformations; gastrointestinal lesions are pathognomonic. We discovered somatic mutations in TEK, the gene encoding TIE2, in 15 of 17 individuals with blue rubber bleb nevus syndrome. Somatic mutations were also identified in five of six individuals with sporadically occurring multifocal venous malformations. In contrast to common unifocal venous malformation, which is most often caused by the somatic L914F TIE2 mutation, multifocal forms are predominantly caused by double (cis) mutations, that is, two somatic mutations on the same allele of the gene. Mutations are identical in all lesions from a given individual. T1105N-T1106P is recurrent in blue rubber bleb nevus, whereas Y897C-R915C is recurrent in sporadically occurring multifocal venous malformation: both cause ligand-independent activation of TIE2, and increase survival, invasion, and colony formation when expressed in human umbilical vein endothelial cells., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Inherited vascular malformations are commonly autosomal dominantly inherited with high, but incomplete, penetrance; they often present as multiple lesions. We hypothesized that Knudson's two-hit model could explain this multifocality and partial penetrance. We performed a systematic analysis of inherited glomuvenous malformations (GVMs) by using multiple approaches, including a sensitive allele-specific pairwise SNP-chip method. Overall, we identified 16 somatic mutations, most of which were not intragenic but were cases of acquired uniparental isodisomy (aUPID) involving chromosome 1p. The breakpoint of each aUPID is located in an A- and T-rich, high-DNA-flexibility region (1p13.1-1p12). This region corresponds to a possible new fragile site. Occurrences of these mutations render the inherited glomulin variant in 1p22.1 homozygous in the affected tissues without loss of genetic material. This finding demonstrates that a double hit is needed to trigger formation of a GVM. It also suggests that somatic UPID, only detectable by sensitive pairwise analysis in heterogeneous tissues, might be a common phenomenon in human cells. Thus, aUPID might play a role in the pathogenesis of various nonmalignant disorders and might explain local impaired function and/or clinical variability. Furthermore, these data suggest that pairwise analysis of blood and tissue, even on heterogeneous tissue, can be used for localizing double-hit mutations in disease-causing genes., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

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 fourfold 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 new, 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 new 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, to the best of our knowledge 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.

Glomuvenous malformations (GVMs) are cutaneous venous lesions characterized by the presence of smooth-muscle--like glomus cells in the media surrounding distended vascular lumens. We have shown that heritable GVMs link to a 4--6-cM region in chromosome 1p21-22. We also identified linkage disequilibrium that allowed a narrowing of this VMGLOM locus to 1.48 Mb. Herein, we report the identification of the mutated gene, glomulin, localized on the basis of the YAC and PAC maps. An incomplete cDNA sequence for glomulin had previously been designated "FAP48," for "FKBP-associated protein of 48 kD." The complete cDNA for glomulin contains an open reading frame of 1,785 nt encoding a predicted protein of 68 kD. The gene consists of 19 exons in which we identified 14 different germline mutations in patients with GVM. In addition, we found a somatic "second hit" mutation in affected tissue of a patient with an inherited genomic deletion. Since all but one of the mutations result in premature stop codons, and since the localized nature of the lesions could be explained by Knudson's two-hit model, GVMs are likely caused by complete loss of function of glomulin. The abnormal phenotype of vascular smooth-muscle cells (VSMCs) in GVMs suggests that glomulin plays an important role in differentiation of these cells--and, thereby, in vascular morphogenesis--especially in cutaneous veins.

Venous malformations with glomus cells are localised cutaneous lesions of vascular dysmorphogenesis. They are usually sporadic, but sometimes familial. Using five families, we mapped the locus, VMGLOM, to chromosome 1p21-p22. In order to refine this locus, spanning 4-6 Mbp, we then studied seven additional families. They exhibited linkage to VMGLOM and the combined lod score for all 12 families was 18.41 at theta = 0.0 for marker D1S188. We found a distinct haplotype shared by seven families, comprising seven alleles which are rare in the general population (P < 0.01). This indicates that the haplotype is identical by descent in all seven families, and hence the locus can be refined by inferring ancestral crossovers. Using this approach, we position the causative gene between two markers on the same non-chimeric YAC of 1.48 Mbp, a feasible size for positional cloning. As there is no known gene involved in vasculogenesis and/or angiogenesis in this YAC, the identification of the causative gene is likely to reveal a novel regulator or vascular development.

Vascular malformations are frequent in newborns, and they persist throughout life, which differentiates them from vascular tumors (eg, hemangiomas). Arteriovenous malformations are high-flow vascular malformations. They are considered nonmalignant but can expand and become a significant clinical risk when extensive. To characterize endothelial cells from arteriovenous malformations (AMEC), we cultured cells obtained from surgical specimens and studied their properties. After selection, the cells that grew out from explants had phenotypic and antigenic features (platelet endothelial cell adhesion molecule, von Willebrand factor) of human endothelial cells. Their spontaneous proliferation rate was higher (1.8 to 6.4 times) than that of human umbilical vein, arterial, or microvascular endothelial cells. The proliferation rate of AMEC was not sensitive to the inhibitory activity of various cytokines (interleukin-1beta, tumor necrosis factor-alpha, transforming growth factor-beta, Interferon-gamma). In basal conditions, intercellular adhesion molecule (ICAM-1) was detected at a higher level of expression (6- to 10-fold) on AMEC, but these cells failed to express E-selectin or the vascular cell adhesion molecule (VCAM-1) after cytokine stimulation. Expression of c-ets-1 proto-oncogene was shown by in situ hybridization. The low response to cytokines, the higher propensity to proliferate, and the ets-1 expression suggest that AMEC have a defective regulation of proliferation that may be due to a reduced apoptotic process.

Cystic lymphatic malformations are haemodynamically inactive vascular malformations that never regress. During a nine-year period, 52 patients with cervico-facial superficial cystic lymphatic malformations underwent percutaneous sclerotherapy with Ethibloc (a corn-based alcohol derivative). In 57 percent (30/52) of cases, the outcome of sclerotherapy alone was excellent or good: in 19 percent (10/52), the procedure had to be combined with surgery for aesthetic reasons; and in the remaining 24 percent (12/52), the treatment failed. The results suggest percutaneous sclerotherapy with Ethibloc to be safe and effective, and that it is to be recommended as the treatment of choice in cases of superficial cystic lymphatic malformations.

Neonatal hemangioma is a common benign proliferation of unorganized structures containing stromal and capillary endothelial cells. We tested the hypothesis that such cell proliferation might result from the release by stromal cells of endothelial cell mitogens. Stromal cells cultured from biopsies of surgically removed life-threatening hemangiomas released an endothelial cell mitogen in vitro that was indistinguishable from vascular endothelial growth factor (VEGF) based on independent criteria such as affinity chromatography for heparin or anti-VEGF IgG and radioreceptor assay. A functional product of the KDR gene encoding a cognate VEGF receptor was also expressed by these stromal cells. Transient transfection with antisense oligonucleotides targeted on the translation initiation codon of KDR abolished its tyrosine phosphorylation and mitogenic response of neonatal hemangioma cells to VEGF, confirming the existence of an autocrine loop of proliferation. When grafted in nude mice, these stromal cells elicited an angiogenic response that was blocked by neutralizing anti-VEGF IgG. These results might provide a clue to the importance of stromal cells in the pathogeny of neonatal hemangiomas.

Purpose: To study cerebral developmental venous anomalies in patients with extensive venous malformations of the head and neck., Methods: All patients had undergone carotid angiography 10 to 15 years previously. Four-vessel cerebral angiography was carried out in 40 patients with venous malformations. All patients had a physical examination, 16 had CT, and 22 were examined with MR imaging. One patient had MR angiography., Results: Eighteen developmental venous anomalies were noted in 8 (20%) of 40 patients. Four patients had multiple anomalies, and these were bilateral in 1 patient. Developmental venous anomalies seen in association with cervicofacial, cutaneous, and mucosal venous malformations were remarkable in their absence of neurologic events and associated cavernoma; significance of ectatic venous convergence, extension, and preponderance of deep drainage routes; and frequency with which they multiple in occurrence., Conclusion: Developmental venous anomalies have a remarkable prevalence of 20% in patients with extensive superficial venous malformations. Therefore, it is important to search for a cerebral developmental venous anomaly when confronted with a cervicofacial venous malformation.