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DEAR EDITOR, The feasibility of testing multiple genes at once using Next Generation Sequencing, particularly Whole Exome Sequencing (WES), has expanded the phenotypic spectrum associated with many disease genes. Distinguishing atypical presentation from combined gene effects and incidental from causal findings can however be challenging, particularly when composite phenotypes are themselves extremely rare. [...]

Background: Multigene panels are routinely used to assess for predisposing germline mutations in families at high breast cancer risk. The number of variants of unknown significance thereby identified increases with the number of sequenced genes. We aimed to determine whether tumor sequencing can help refine the analysis of germline variants based on second somatic genetic events in the same gene. Methods: Whole-exome sequencing (WES) was performed on whole blood DNA from 70 unrelated breast cancer patients referred for genetic testing and without a BRCA1, BRCA2, TP53, or CHEK2 mutation. Rare variants were retained in a list of 735 genes. WES was performed on matched tumor DNA to identify somatic second hits (copy number alterations (CNAs) or mutations) in the same genes. Distinct methods (among which immunohistochemistry, mutational signatures, homologous recombination deficiency, and tumor mutation burden analyses) were used to further study the role of the variants in tumor development, as appropriate. Results: Sixty-eight patients (97%) carried at least one germline variant (4.7 ± 2.0 variants per patient). Of the 329 variants, 55 (17%) presented a second hit in paired tumor tissue. Of these, 53 were CNAs, resulting in tumor enrichment (28 variants) or depletion (25 variants) of the germline variant. Eleven patients received variant disclosure, with clinical measures for five of them. Seven variants in breast cancer-predisposing genes were considered not implicated in oncogenesis. One patient presented significant tumor enrichment of a germline variant in the oncogene ERBB2, in vitro expression of which caused downstream signaling pathway activation. Conclusion: Tumor sequencing is a powerful approach to refine variant interpretation in cancer-predisposing genes in high-risk breast cancer patients. In this series, the strategy provided clinically relevant information for 11 out of 70 patients (16%), adapted to the considered gene and the familial clinical phenotype., SCOPUS: ar.j, info:eu-repo/semantics/published

Infantile myofibromatosis is one of the most prevalent soft tissue tumors of infancy and childhood. Multifocal nodules with visceral lesions are associated with a poor prognosis. A few familial cases have been linked to mutations in various genes including PDGFRB. In this study, we sequenced PDGFRB, which encodes a receptor tyrosine kinase, in 16 cases of myofibromatosis or solitary myofibroma. Mutations in the coding sequence of PDGFRB were identified in 6 out of 8 patients with the sporadic multicentric form of the disease and in 1 out of 8 patients with isolated myofibroma. Two patients had the same mutation in multiple separated lesions. By contrast, a third patient had three different PDGFRB mutations in the three nodules analyzed. Mutations were located in the transmembrane, juxtamembrane and kinase domains of the receptor. We showed that these mutations activated receptor signaling in the absence of ligand and transformed fibroblasts. In one case, a weakly-activating germline variant was associated with a stronger somatic mutation, suggesting a two-hit model for familial myofibromatosis. Furthermore, the mutant receptors were sensitive to the tyrosine kinase inhibitor imatinib, except D850V, which was inhibited by dasatinib and ponatinib, suggesting a targeted therapy for severe myofibromatosis. In conclusion, we identified gain-of-function PDGFRB mutations in the majority of multifocal infantile myofibromatosis cases, shedding light on the mechanism of disease development, which is reminiscent of multifocal venous malformations induced by TIE2 mutations. Our results provide a genetic test to facilitate diagnosis, and preclinical data for development of molecular therapies. ispartof: Human Molecular Genetics vol:26 issue:10 pages:1801-1810 ispartof: location:England status: published

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.

Somatic mutations in TEK, the gene encoding endothelial cell tyrosine kinase receptor TIE2, cause more than half of sporadically occurring unifocal venous malformations (VMs). Here, we report that somatic mutations in PIK3CA, the gene encoding the catalytic p110α subunit of PI3K, cause 54% (27 out of 50) of VMs with no detected TEK mutation. The hotspot mutations c.1624G>A, c.1633G>A, and c.3140A>G (p.Glu542Lys, p.Glu545Lys, and p.His1047Arg), frequent in PIK3CA-associated cancers, overgrowth syndromes, and lymphatic malformation (LM), account for >92% of individuals who carry mutations. Like VM-causative mutations in TEK, the PIK3CA mutations cause chronic activation of AKT, dysregulation of certain important angiogenic factors, and abnormal endothelial cell morphology when expressed in human umbilical vein endothelial cells (HUVECs). The p110α-specific inhibitor BYL719 restores all abnormal phenotypes tested, in PIK3CA- as well as TEK-mutant HUVECs, demonstrating that they operate via the same pathogenic pathways. Nevertheless, significant genotype-phenotype correlations in lesion localization and histology are observed between individuals with mutations in PIK3CA versus TEK, pointing to gene-specific effects.

Venous malformations (VMs) are localized defects in vascular morphogenesis frequently caused by mutations in the gene for the endothelial tyrosine kinase receptor TIE2. Here, we report the analysis of a comprehensive collection of 22 TIE2 mutations identified in patients with VM, either as single amino acid substitutions or as double-mutations on the same allele. Using endothelial cell (EC) cultures, mouse models and ultrastructural analysis of tissue biopsies from patients, we demonstrate common as well as mutation-specific cellular and molecular features, on the basis of which mutations cluster into categories that correlate with data from genetic studies. Comparisons of double-mutants with their constituent single-mutant forms identified the pathogenic contributions of individual changes, and their compound effects. We find that defective receptor trafficking and subcellular localization of different TIE2 mutant forms occur via a variety of mechanisms, resulting in attenuated response to ligand. We also demonstrate, for the first time, that TIE2 mutations cause chronic activation of the MAPK pathway resulting in loss of normal EC monolayer due to extracellular matrix (ECM) fibronectin deficiency and leading to upregulation of plasminogen/plasmin proteolytic pathway. Corresponding EC and ECM irregularities are observed in affected tissues from mouse models and patients. Importantly, an imbalance between plasminogen activators versus inhibitors would also account for high d-dimer levels, a major feature of unknown cause that distinguishes VMs from other vascular anomalies.

The localized structural abnormalities that arise during vasculogenesis, angiogenesis and lymphangiogenesis, the developmental processes which give rise to the adult vasculature, are collectively termed vascular anomalies. The last 2 years have seen an explosion of studies that underscore paradominant inheritance, the combination of inherited changes with somatic second-hits to the same genes, as underlying rare familial forms. Moreover, local, somatic genetic defects that cause some of the common sporadic forms of these malformations have been unraveled. This highlights the importance of assessing for tissue-based genetic changes, especially acquired genetic changes, as possible pathophysiological causes, which have been largely overlooked except in the area of cancer research. Large-scale somatic screens will therefore be essential in uncovering the nature and prevalence of such changes, and their downstream effects. The identification of disease genes combined with exhaustive, precise clinical delineations of the entire spectra of associated phenotypes guides better management and genetic counseling. Such a synthesis of information on functional and phenotypic effects will enable us to make and use animal models to test less invasive, targeted, perhaps locally administered, biological therapies.

Germline substitutions in the endothelial cell tyrosine kinase receptor TIE2 (encoded by TEK) cause a rare, inherited form of venous anomaly known as a mucocutaneous venous malformation (VMCM; refs. 1, 2, 3 and V.W., N.L., M.U., A. Irrthum, L.M.B. et al., unpublished data). We identified a somatic 'second hit' causing loss of function of TIE2 in a resected VMCM and assessed whether such localized, tissue-specific events have a role in the etiology of sporadic venous malformations, which are far more common. We identified eight somatic TEK mutations in lesions from 28 of 57 individuals (49.1%) with sporadic venous malformations; the mutations were absent from the individuals' blood and control tissues. The somatic mutations included one causing a frequent L914F substitution and several double mutations in cis, all of which resulted in ligand-independent TIE2 hyperphosphorylation in vitro. When overexpressed in human umbilical vein endothelial cells, the L914F mutant was abnormally localized and responded to ligand, in contrast to wild-type TIE2 and the common, inherited R849W mutant, suggesting that the mutations have distinct effects. The presence of the same mutations in multifocal sporadic venous malformations in two individuals suggests a common origin for the abnormal endothelial cells at the distant sites. These data show 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 venous malformations.

Infantile hemangiomas are localized and rapidly growing regions of disorganized angiogenesis. We show that expression of vascular endothelial growth factor receptor-1 (VEGFR1) in hemangioma endothelial cells (hemECs) and hemangioma tissue is markedly reduced compared to controls. Low VEGFR1 expression in hemECs results in VEGF-dependent activation of VEGFR2 and downstream signaling pathways. In hemECs, transcription of the gene encoding VEGFR1 (FLT1) is dependent on nuclear factor of activated T cells (NFAT). Low VEGFR1 expression in hemECs is caused by reduced activity of a pathway involving beta1 integrin, the integrin-like receptor tumor endothelial marker-8 (TEM8), VEGFR2 and NFAT. In a subset of individuals with hemangioma, we found missense mutations in the genes encoding VEGFR2 (KDR) and TEM8 (ANTXR1). These mutations result in increased interactions among VEGFR2, TEM8 and beta1 integrin proteins and in inhibition of integrin activity. Normalization of the constitutive VEGFR2 signaling in hemECs with soluble VEGFR1 or antibodies that neutralize VEGF or stimulate beta1 integrin suggests that local administration of these or similar agents may be effective in hemangioma treatment.

Venous malformations (VMs) are composed of ectatic veins with scarce smooth muscle cell coverage. Activating mutations in the endothelial cell tyrosine kinase receptor TIE2 are a common cause of these lesions. VMs cause deformity, pain, and local intravascular coagulopathy, and they expand with time. Targeted pharmacological therapies are not available for this condition. Here, we generated a model of VMs by injecting HUVECs expressing the most frequent VM-causing TIE2 mutation, TIE2-L914F, into immune-deficient mice. TIE2-L914F–expressing HUVECs formed VMs with ectatic blood-filled channels that enlarged over time. We tested both rapamycin and a TIE2 tyrosine kinase inhibitor (TIE2-TKI) for their effects on murine VM expansion and for their ability to inhibit mutant TIE2 signaling. Rapamycin prevented VM growth, while TIE2-TKI had no effect. In cultured TIE2-L914F–expressing HUVECs, rapamycin effectively reduced mutant TIE2-induced AKT signaling and, though TIE2-TKI did target the WT receptor, it only weakly suppressed mutant-induced AKT signaling. In a prospective clinical pilot study, we analyzed the effects of rapamycin in 6 patients with difficult–to-treat venous anomalies. Rapamycin reduced pain, bleeding, lesion size, functional and esthetic impairment, and intravascular coagulopathy. This study provides a VM model that allows evaluation of potential therapeutic strategies and demonstrates that rapamycin provides clinical improvement in patients with venous malformation.

Signaling lymphocyte activation molecules (SLAMs) play an integral role in immune regulation. Polymorphisms in the SLAM family receptors are implicated in human and mouse model of lupus disease. The lupus-associated, somatically mutated, and class-switched pathogenic autoantibodies are generated in spontaneously developed germinal centers (GCs) in secondary lymphoid organs. The role and mechanism of B cell–intrinsic expression of polymorphic SLAM receptors that affect B cell tolerance at the GC checkpoint are not clear. In this study, we generated several bacterial artificial chromosome–transgenic mice that overexpress C57BL/6 (B6) alleles of different SLAM family genes on an autoimmune-prone B6.Sle1b background. B6.Sle1b mice overexpressing B6-derived Ly108 and CD84 exhibit a significant reduction in the spontaneously developed GC response and autoantibody production compared with B6.Sle1b mice. These data suggest a prominent role for Sle1b-derived Ly108 and CD84 in altering the GC checkpoint. We further confirm that expression of lupus-associated CD84 and Ly108 specifically on GC B cells in B6.Sle1b mice is sufficient to break B cell tolerance, leading to an increase in autoantibody production. In addition, we observe that B6.Sle1b B cells have reduced BCR signaling and a lower frequency of B cell–T cell conjugates; the reverse is seen in B6.Sle1b mice overexpressing B6 alleles of CD84 and Ly108. Finally, we find a significant decrease in apoptotic GC B cells in B6.Sle1b mice compared with B6 controls. Our study establishes a central role for GC B cell–specific CD84 and Ly108 expression in maintaining B cell tolerance in GCs and in preventing autoimmunity.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the appearance of autoantibodies against a variety of nucleosomal and kidney antigens. Targeting of glomeruli, the basic units of filtration in the kidneys, by these autoantibodies is one of the crucial events initiating nephritis, leading to the chronic renal insufficiency seen in lupus. Here we review published reports that directly tested the in vivo pathogenicity of antinuclear/glomerular autoantibodies, which indicate that intermediating nucleosomal bridges might be what empower antibodies to target glomeruli. Further studies that systematically evaluate the contribution of clearly non-DNA binding nephritogenic antibodies, and elucidate the additional factors essential for renal pathology to ensue once the golmeruli are targeted, remain to be done.

Vascular anomalies are localized developmental defects of the vasculature. They include vascular tumors, primarily infantile hemangioma, and vascular malformations, which are categorized by the compartment they affect into capillary, lymphatic, venous, arterial, and combined malformations. While typically isolated and sporadic, hemangiomas and vascular malformations can occur as inherited traits or as part of certain syndromes. The identification of the genetic changes that cause disease holds out the promise of accurate (molecular) diagnosis, as well as insights into disease mechanisms and novel targets for improved therapy. The advances initially made were largely confined to rare, familial forms, with much less known about the bases of the common sporadic versions of vascular malformations. The shift toward the analysis of affected tissues in addition to blood samples has now proven to be key in improving our understanding, as it is becoming increasingly evident that somatic changes play an important role in these developmental disorders. In addition, rapidly evolving next-generation sequencing techniques allow for unprecedented throughput and depth of coverage, greatly increasing the ability to detect mosaic changes anywhere in the exome or genome. Expression profiling and functional studies, in vitro and in animal models, have in parallel begun to uncover how the aberrant genes affect the different cellular and molecular components of the vasculature.

The vasculature is the first organ system to develop dur- ing embryogenesis, delivering nutrients, growth factors, and oxygen to tissues and removing waste products. It is therefore essential for development. The vascular sys- tem comprises four major compartments (arterial, capil- lary, and venous blood vessels, and lymphatic vessels), which have in common the presence of endothelial cells (ECs) forming the innermost layer. In blood vessels, the endothelial tubes are supported by a layer of vascular smooth muscle cells (vSMCs) and/or pericytes (together called mural cells) of variable thickness, whereas only a few SMCs are present in certain areas of large lymphatic vessels. The mechanisms that give rise to the mature vascular network are complex. These developmental processes, termed vasculogenesis, angiogenesis, and lymphangio- genesis, involve a plethora of signaling molecules and their receptors at various time-points. The best known are the angiopoietins (ANGPTs), ephrins, fibroblast growth factors, platelet-derived growth factor, trans- forming growth factor beta (TGFβ), and vascular endothelial growth factors. As suggested by numerous murine models showing abnormal vascular develop- ment, many other factors, including those participating in cell–cell and cell–matrix interactions, are probably also involved. Vascular anomalies result from localized defects that occur during vascular development (1,2). They are sub- divided into vascular tumors (mainly hemangiomas) and vascular malformations, which in turn are subcatego- rized according to the type(s) of vessel(s) altered (3,4). Thus, capillary, venous, arteriovenous, lymphatic, and combined malformations can be distinguished.

Mutations in the endothelial cell (EC) tyrosine kinase receptor TIE2 cause inherited and sporadic forms of venous malformation. The recurrent somatic mutation L914F and common germline mutation R849W differ in terms of phosphorylation level, as well as sub-cellular localization and trafficking of the receptor. Previous studies have shed light on certain pathogenic properties of R849W, but the mechanisms of action of L914F are unknown. We used global gene expression profiling to study the effects of L914F on ECs. We found that L914F strongly dysregulates genes involved in vascular development, cell migration and extracellular matrix processing, while R849W has weak effects. We also demonstrate, for the first time, that TIE2-mutant ECs are deficient in the production of PDGFB, both in vitro and ex vivo in patient tissues. This defect is mediated by the chronic, ligand-independent activation of AKT by the mutant receptors. Inadequate secretion of the major mural cell attractant likely plays an important role in the development of abnormal vascular channels, contributing to the characteristic paucity of surrounding vascular smooth muscle cells.

Angiopoietin 1 (Ang1) is an activating ligand for the endothelial receptor tyrosine kinase Tie2, whereas Ang2 acts as a context-dependent agonist or antagonist that has a destabilizing effect on the vasculature. The molecular mechanisms responsible for the versatile functions of Ang2 are poorly understood. We show here that Ang2, but not Ang1, induces Tie2 translocation to the specific cell-matrix contact sites located at the distal end of focal adhesions. The Ang2-specific Tie2 translocation was associated with distinct Tie2 activation and downstream signals which differed from those of Ang1, and lead to impaired cell motility and weak cell-matrix adhesion. We demonstrate that the different oligomeric/multimeric forms of the angiopoietins induce distinct patterns of Tie2 trafficking; the lower oligomerization state of native Ang2 was critical for the Ang2-specific Tie2 redistribution, while multimeric structures of Ang1 and Ang2 induced similar responses. The Ang2-specific Tie2 trafficking to cell-matrix contacts was also dependent on cell substratum, α2β1 integrin-containing cell-matrix adhesion sites and intact microtubules. Our data indicate that the different subcellular trafficking of Tie2/Ang2 and Tie2/Ang1 complexes generates ligand-specific responses in the angiopoietin-Tie signaling pathway, including modulation of cell-matrix interactions.

Ollier disease and Maffucci syndrome are non-hereditary skeletal disorders characterized by multiple enchondromas (Ollier disease) combined with spindle cell hemangiomas (Maffucci syndrome). We report somatic heterozygous mutations in IDH1 (c.394C>T encoding an R132C substitution and c.395G>A encoding an R132H substitution) or IDH2 (c.516G>C encoding R172S) in 87% of enchondromas (benign cartilage tumors) and in 70% of spindle cell hemangiomas (benign vascular lesions). In total, 35 of 43 (81%) subjects with Ollier disease and 10 of 13 (77%) with Maffucci syndrome carried IDH1 (98%) or IDH2 (2%) mutations in their tumors. Fourteen of 16 subjects had identical mutations in separate lesions. Immunohistochemistry to detect mutant IDH1 R132H protein suggested intraneoplastic and somatic mosaicism. IDH1 mutations in cartilage tumors were associated with hypermethylation and downregulated expression of several genes. Mutations were also found in 40% of solitary central cartilaginous tumors and in four chondrosarcoma cell lines, which will enable functional studies to assess the role of IDH1 and IDH2 mutations in tumor formation.

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. European Journal of Human Genetics (2010) 18, 414-420; doi:10.1038/ejhg.2009.193; published online 4 November 2009

Vascular malformations are comprised of a variety of developmental defects of the vasculature. Typically sporadic in nature, they can sometimes occur as incompletely penetrant, inherited traits. The genetic bases of several of these anomalies have been identified, and are described in this review. This has had a hugely beneficial impact in terms of the precise diagnosis and appropriate, effective treatment of different disease entities; it has also revealed potential therapeutic targets for the future. The advances made thus far, however, are largely confined to the rare, familial forms, and much remains to be uncovered about the genes that mediate common sporadic versions of vascular malformations. Moreover, the pathogenic pathways and molecular mechanisms by which the aberrant genes cause these defined, often heterogeneous lesions, remain to be thoroughly dissected. Previous studies have largely focused on the analysis of blood samples, as these are more accessible. Further progress in identifying the somatic events that cause sporadic lesions or locally exacerbate the pathogenic effects of germline-heterozygous mutant alleles, will require the additional assessment of irregularities of gene expression and function at the level of lesion-derived tissue.

The evolutionary origin of genetic diversity in the SLAM/CD2 gene cluster, implicated in autoimmune lupus susceptibility in mice, was investigated by sequence analysis of exons from six members of the cluster in 48 wild mouse samples derived from the global mouse population. A total of 80 coding region SNPs were identified among the six genes analyzed, indicating that this gene cluster is highly polymorphic in natural mouse populations. Phylogenetic analyses of these allelic sequences revealed clustering of alleles derived from multiple Mus species and subspecies, indicating alleles at several SLAM/CD2 loci were present in ancestral Mus populations prior to speciation and have persisted as polymorphisms for more than 1 million years. Analyses of nonsynonymous/synonymous ratios using likelihood codon substitution models identified several segments in Cd229, Cd48 and Cd84 that were impacted by positive diversifying selective pressures. These findings support the interpretation that selection favoring the generation and retention of functional polymorphisms has played a role in the evolutionary origin of genetic polymorphisms that are predisposing to autoimmunity.

Susceptibility to autoimmunity in B6.Sle1b mice is associated with extensive polymorphisms between two divergent haplotypes of the SLAM/CD2 family of genes. The B6.Sle1b-derived SLAM/CD2 family haplotype is found in many other laboratory mouse strains but only causes autoimmunity in the context of the C57Bl/6 (B6) genome. Phenotypic analyses have revealed variations in the structure and expression of several members of the SLAM/CD2 family in T and B lymphocytes from B6.Sle1b mice. T lymphocytes from B6.Sle1b mice have modified signaling responses to stimulation at 4–6 weeks of age. While autoimmunity may be mediated by a combination of genes in the SLAM/CD2 family cluster, the strongest candidate is Ly108, a specific isoform of which is constitutively upregulated in B6.Sle1b lymphocytes.

Systemic lupus erythematosus (SLE) is the paradigm of a multisystem autoimmune disease in which genetic factors strongly influence susceptibility. Through genome scans and congenic dissection, numerous loci associated with lupus susceptibility have been defined and the complexity of the inheritance of this disease has been revealed. In this review, we provide a brief description of animal models of SLE, both spontaneous models and synthetic models, with an emphasis on the B6 congenic model derived from analyses of the NZM2410 strain. A hypothetical model of disease progression that organizes many of the identified SLE susceptibility loci in three distinct biological pathways that interact to mediate disease pathogenesis is also described. We finally discuss our recent fine mapping analysis, which revealed a cluster of loci that actually comprise the Sle1 locus.