Genetics of vascular malformations

Seminars in Pediatric Surgery 23 (2014) 221–226

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Genetics of vascular malformations Ha-Long Nguyen, PhDa,n, Laurence M. Boon, MD, PhDb, Miikka Vikkula, MD, PhDa,c a b c

Laboratory of Human Molecular Genetics, de Duve Institute, Université catholique de Louvain, Brussels, Belgium Center for Vascular Anomalies, Division of Plastic Surgery, Cliniques universitaires Saint-Luc, Université catholique de Louvain, Brussels, Belgium Walloon Excellence in Lifesciences and Biotechnology (WELBIO), de Duve Institute, Université catholique de Louvain, Brussels, Belgium

a r t i c l e in fo

Keywords: Genetics of vascular malformations Sporadic Familial Somatic mosaicism

a b s t r a c t Vascular anomalies are developmental defects of the vasculature and encompass a variety of disorders. The majority of these occur sporadically, yet a few are reported to be familial. The identification of genes mutated in the different malformations provides insight into their etiopathogenic mechanisms and the specific roles the associated proteins play in vascular development and maintenance. It is becoming evident that somatic mosaicism plays a major role in the formation of vascular lesions. The importance of utilizing Next-Generating Sequencing (NGS) for high-throughput and “deep” screening of both blood and lesional DNA and RNA is thus emphasized, as the somatic changes are present in low quantities. There are several examples where NGS has already accomplished discovering these changes. The identification of all the causative genes and unraveling of a holistic overview of the pathogenic mechanisms should enable generation of in vitro and in vivo models and lead to development of more effective treatments, not only targeted on symptoms. & 2014 Published by Elsevier Inc.

Introduction Vascular anomalies refer to a wide variety of disorders that result from disruptions in the development and maintenance of the vasculature. The majority of vascular anomalies occur sporadically; however, several can be inherited, predisposing patients to developing vascular lesions. Although the familial cases of vascular malformations occur at a much lower rate, they have been a fundamental starting point to provide insight into the molecules and signaling pathways that play major roles in vascular development and maintenance (Table). The inherited malformations seemingly follow a general pattern. The majority of these are transmitted in an autosomal dominant manner, and mutations tend to result in a loss-of-function of the gene. The phenotypic penetrance, age at onset, and severity of lesions vary greatly among patients, even between individuals of the same family. Inherited malformations are typically multifocal, small, and localized. Cutaneous lesions are the most visible, yet visceral and

The studies of the authors are partially funded by the Belgian Science Policy Office Interuniversity Attraction Poles (BELSPO-IAP) programme through the Project IAP P7/43-BeMGI; the National Institutes of Health, USA, Program Project P01 AR048564; and the Fonds de la Recherche Scientifique [F.R.S.-FNRS (Grant nos. FRSM 3.4578.10 and PDR T.0036.14)], Belgium. H.L.N. is supported by a Pierre M. Fellowship. n Corresponding author. E-mail address: [email protected] (H.-L. Nguyen). http://dx.doi.org/10.1053/j.sempedsurg.2014.06.014 1055-8586/& 2014 Published by Elsevier Inc.

deeper lesions can also occur. The variability in penetrance and localized nature of these malformations seem to be explained largely by Knudson's two-hit hypothesis: the combination of a germline mutation with a post-zygotic change resulting in localized complete loss of the gene within the lesion, as demonstrated by glomuvenous malformations.1 Identification of the important role that mutations within the tissue have as second-hits in the familial forms urged studies focusing on somatic changes as the “sole” cause of sporadic lesions.2,3 Next-Generation Sequencing (NGS) has since proven to be a valuable tool to discover these changes, which often have a low frequency in any given tissue.

Arteriovenous malformations Arteriovenous malformations (AVMs) and arteriovenous fistulas (AVFs) occur when arteries connect directly to veins, bypassing the capillary beds. Consequently, vessels become dilated and the veins become arterialized. AVMs are characterized by a nidus. These fast-flow lesions can be found anywhere in the body, including the skin and visceral organs. Hereditary hemorrhagic telangiectasia (HHT, OMIM 187300) is an autosomal dominant inherited disorder where patients are predisposed to developing AVFs/AVMs. The incidence of HHT is 1:5000–1:8000, making it one of the most common inherited vascular malformations. Though it is believed that the vascular defects are present congenitally, most symptoms are seen in older

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Table Genes mutated in vascular anomalies Vascular anomaly

Locus

Gene

Inheritance

Mutation affect and type Normal protein function or involvement

–

–

Sp

Somatic

–

9q34.11 12q13.13 5q31.3–q32 7p14 18q21.2

ENG ALK1 – – MADH4/SMAD4

AD AD AD AD AD

LOF and germline LOF and germline – – LOF and germline

10q11.22

GDF2/BMP9

–

LOF and germline

TGF-β/BMP co-receptor TGF-β/BMP type I receptor – – Common TGF-β signaling co-mediator BMP signaling ligand

9q21

GNAQ

Sp

5q14.3

RASA1

AD/sp

α-subunit in Gq class of proteins, regulation of GPCR RasGTPase-RAS signaling

5q14.3 9q21

RASA1 GNAQ

AD/sp Sp

GOF and somatic mosaicism LOF and germline þ somatic 2nd hit? LOF and germline GOF and somatic mosaicism

7q21.2

KRIT1

AD/sp

Suppress RhoA-GTPase signaling

CCM2

7p13

CCM2/Malcavernin

AD/sp

CCM3

3q26.1

PDCD10

AD/sp

CCM4

3q26.3–27.2

–

–

LOF and germline þ somatic 2nd hit/ somatic LOF and germline þ somatic 2nd hit/ somatic LOF and germline þ somatic 2nd hit/ somatic –

9p21.2

TEK/TIE2

Sp

Cutaneomucosal VM (VMCM)

9p21.2

TEK/TIE2

AD

EC-specific tyrosine kinase receptor for angiopoietins Same as above

Blue rubber bleb nevus syndrome (BRBN)

9p21.2

TEK/TIE2

Sp

Glomuvenous malformation (GVM)

1p22.1

Glomulin

AD

GOF and somatic mosaicism GOF and germline þ somatic 2nd hit GOF and somatic mosaicism LOF and germline þ somatic 2nd hit

–

–

–

–

5q35.3

FLT4/VEGFR3

AD/AR/Sp

4q34 1q41 16q24.1 20q13.33

VEGFC PTPN14 FOXC2 SOX18

AD AR AD AD/AR/Sp

LOF and germline/ somatic LOF and germline LOF and germline LOF and germline LOF?/DN and germline

EC-specific tyrosine kinase receptor Ligand for VEGFR3 Protein tyrosine phosphatase Transcription factor Transcription factor

18q21.32

CCBE1

AR

LOF

10q23.33

KIF11

AD/Sp

LOF and germline/ somatic

Regulates ADAMTS3-mediated activation of VEGFC Spindle motor protein

Xq28

IKBKG/NEMO

X-linked

Hypomorphic

NFκβ transcription regulation

1q41–42 6q22.31 3q21.3

GJC2/CX47 GJA1/CX43 GATA2

AD AD AD

Missense and germline Missense and germline LOF and germline

Gap junction protein Gap junction protein Transcription factor

PIK3CA

Sp

GOF and somatic mosaicism

110-kD catalytic alpha subunit of PI3K

AGGF1/VG5Q PIK3CA AKT1 PTEN

Sp/AD?

Somatic/GOF Somatic/GOF Somatic mosaicism/GOF LOF

Pro-angiogenic factor PI3K/AKT signaling PI3K/AKT signaling Dual-specificity protein phosphatase for PI3K/AKT signaling

Arteriovenous malformations Arteriovenous malformation (AVM) Hereditary hemorrhagic telangiectasia (HHT) HHT1 HHT2 HHT3 HHT4 Juvenile polyposis-HHT (JP-HHT) HHT-like Capillary malformations Capillary malformation (CM) (“port-wine stain”) Capillary malformation–arteriovenous malformation (CM-AVM) Parkes Weber syndrome Sturge–Weber syndrome (SWS) Cerebral cavernous malformation (CCM) CCM1

Venous malformations Venous malformation (VM)

Lymphatic malformations Lymphatic malformation (LM) Primary lymphedema (LE) Primary congenital LE (Nonne–Milroy disease) Nonne–Milroy-like disease Choanal atresia-LE LE–distichiasis–yellow nail syndrome Hypotrichosis–LE–telangiectasia (HLT) syndrome Hennekam syndrome Microcephaly with or without chorioretinopathy, LE, or mental retardation (MCLMR) X-linked syndrome anhydrotic ectodermal dysplasia with immunodeficiency, osteopetrosis, and LE (OLEDAID) Hereditary LE II (Meige disease) Oculodentodigital dysplasia-LE Primary LE-myelodysplasia (Emberger syndrome)

Combined/complex syndromes Congenital, lipomatous overgrowth, vascular 3q26.32 malformations, epidermal nevi, and scoliosis/skeletal/spinal anomalies (CLOVES) Klippel–Trenaunay syndrome 5q13.3 3q26.32 Proteus syndrome 14q32.33 PTEN hamartoma tumor syndrome (PHTS) 10q23.31

AD

Same as above α-subunit in Gq class of proteins, regulation of GPCR

Suppress RhoA-GTPase signaling

Apoptosis

–

Same as above Intracellular signaling, cell cycle regulation

AD, autosomal dominant; AR, autosomal recessive; DN, dominant negative; Sp, sporadic; LOF, loss-of-function; GOF, gain-of-function; EC, endothelial cell; ECM, extracellular matrix.

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youths and adults. There are five loci linked to HHT, but only three genes have been identified. They code for members of the transforming growth factor-β (TGFβ) signaling pathway. Endoglin (ENG) and activin kinase-like receptor-1 (ALK1/ACVRL1) are a co-receptor and type I receptor, respectively, which are largely expressed on endothelial cells (ECs).4,5 Mutations in these molecules account for 80% of HHT cases.6 Heterozygous Eng and Alk1 mice develop HHT-like phenotypes but with inconsistent penetrance.7,8 EC-specific Alk1 knockout mice develop AVMs/AVFs.9 A subset of patients with mutations in the common TGFβ signaling mediator SMAD4 are inflicted by a combined syndrome of HHT and another autosomal dominant disease, juvenile polyposis (JP-HHT).10 Moreover, exome sequencing was used to find mutations in bone morphogenetic protein (BMP)-9 in three patients with HHT-like symptoms. BMP9 is a likely ligand for ACVRL1.11 This supports the notion that BMP signaling pathway is involved in the development of HHT. The other two genes are on chromosomes 5 (HHT3) and 7 (HHT4), but these genes are unknown.12,13 Despite knowing that the TGFβ signaling pathway is involved, the underlying pathogenetic mechanism remains controversial.

Capillary malformations Capillary malformations (CM; 163000 OMIM) are commonly known as “port-wine” stains. They are usually sporadic, unifocal red, flat, localized, or spread lesions most often localized on the head and neck. CM-AVMs (OMIM 608354) are inherited, small, multifocal lesions commonly surrounded by a pale halo. Parkes Weber syndrome (PWS; OMIM 608355), consisting of a large cutaneous capillary blush associated with underlying multiple micro-AVFs and overgrowth of the affected body part, can be part of CM-AVM.14 CM-AVM is caused by mutations in RASA1, mapped to 5q14.3.15 The various mutations cause loss-of-function of the encoded GTPase-activating protein p120-RasGAP. This loss is thought to lead to over-stimulation of Ras/MAPK signaling, causing aberrant cell growth, differentiation, and proliferation.16–18 Sturge–Weber syndrome (SWS; OMIM 185300) is a sporadic disorder characterized by facial CMs, glaucoma, and possible seizures. Whole-exome sequencing revealed a recurrent somatic single nucleotide variant in the guanine nucleotide-binding protein (G protein), Q polypeptide (GNAQ) in resected tissues. The arginine183-to-glucine (R183Q) substitution was found in cutaneous and cerebral lesions but not in unaffected skin. The change appears to be an activating mutation, as HEK 293T cells transfected with the R183Q mutant showed mildly increased ERK activation compared to control cells.19 Cerebral cavernous malformations (CCM; OMIM 116860) mainly affect the central nervous system but can also be found in the retina and skin. CCMs are composed of dilated vessels or “caverns” that are filled with blood. They are prone to rupture because of the lack of supporting structures, such as the smooth muscle cells.20 Approximately 20% of CCM cases are inherited in an autosomal dominant manner. Patients with sporadic CCMs typically have a single lesion while those with the inherited form have multiple lesions. The loss of three genes has been linked to CCMs: Krev Interaction Trapped-1 (KRIT1), malcalvernin (CCM2), and Programed cell death 10 (PDCD10).20 A second-hit causing complete localized deletion of one of the CCM genes is needed for the formation of CCMs.21,22 The three proteins interact with each other and yet have differential functions.23,24 KRIT1 is involved in the vasculature in regulating endothelial cell–cell junctions via the Delta–Notch pathway.25 CCM2 is a scaffolding protein for mitogenactivated protein kinase kinase kinase (MEKK3).26 In CCM3, which possess PDCD10 mutations, apoptosis and VEGF signaling may be

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altered.27 Additionally, delta-like ligand 4 (DLL4) may be a downstream target for CCM3.28

Venous malformations Venous malformations can be divided into four groups. Sporadic venous malformations (VM) are the most common, accounting for 94% of the lesions. Cutaneomucosal venous malformations (VMCM; OMIM 600195) and glomuvenous malformations (GVM; OMIM 138000) are inherited in an autosomal dominant manner. They account for 1% and 5% of patients, respectively.29 The fourth entity is sporadic blue rubber bleb nevus syndrome (BRBN; OMIM 112200). VMCM and GVM are transmitted in a paradominant pattern, i.e., a second-hit is needed for lesion formation.30 The most common second-hit in GVMs is acquired uniparental isodisomy, which renders the inherited mutation homozygous in the affected tissues.29 Histopathologically, VMs appear as dilated venous channels surrounded by thin, irregular layers of smooth muscle cells. GVMs have characteristic abnormal mural cells called glomus cells. Both appear as small blue multifocal (or unifocal, as in most sporadic VMs), flat or raised, hemispherical lesions on the skin at birth and grow with the child. VMs can also appear on other organs, such as mucosa, muscles, the gastrointestinal tract, lungs, and the brain. Complications from VMs and GVMs include functional impairment, organ dysfunction, and esthetic deformations.30 The causative gene for VMs and VMCMs is TIE2/TEK, an ECspecific tyrosine kinase receptor, which binds angiopoietins.2,31,32 It plays a significant role in regulating angiogenesis, proliferation, migration, adhesion, in addition to maintenance of vessel stability and vascular quiescence.31 Though the same gene is mutated, the most frequent mutation found differs between the two; 85% of sporadic VMs have a somatic TIE2 mutation, in which a leucine is changed to a phenylalanine (L914F).3 This change has never been observed in VMCMs, suggesting that it is lethal during embryogenesis. In contrast, an Arg849 to Tryptophan (R849W) substitution is detected in more than half of inherited VMs.31,32 The L914F mutation has a stronger TIE2-hyperphosphorylating effect than the R849W change.2,33 TIE2 functions via the PI3K/AKT signaling pathway. Mutated TIE2 receptors aberrantly activate AKT in a ligand-independent manner, resulting in reduced platelet-derived growth factor-B (PDGFB) production and secretion.34 PDGFB is an important recruiter of mural cells, thus it may contribute to the irregular and attenuated coverage of smooth muscle cells surrounding the endothelium of VMs. BRBN syndrome is characterized by appearance of several cutaneous, visceral, and gastrointestinal VMs, which may hemorrhage. This can lead to severe anemia. Somatic TIE2 mutations have also been identified in the majority of patients. Yet, they differ from VM and VMCM causative mutations, by occurring in double, on the same allele (cis-mutations) (Soblet, submitted for publication). GVMs resemble VMs, yet are distinct. The lesions are pink to purple-blue, superficial, raised, and with a cobblestone appearance. The severity of GVMs varies among patients, from punctate harmless blue spots to large, painful debilitating lesions. New lesions may form over time.35 Most likely all GVMs are caused by mutations leading to loss-of-function in the gene glomulin (GLMN).36–38 Not much is known about GLMN function; however, the specificity of the lesions indicates that this molecule plays an important role in angiogenesis, particularly in vascular smooth muscle cell (vSMC) development. In situ hybridization studies on mouse tissues demonstrate expression in vSMCs.39 A relationship with TGFβ and hepatocyte growth factor (HGF) signaling pathways,

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both of which are important in angiogenesis, is suggested via in vitro experiments.40,41 Glomulin also plays a role in Fbw7mediated protein ubiquitination and degradation. Glomulin inhibits the E3 ubiquitin ligase activity of the Cul1–RING ligase (CRL1) complex by binding to the RING domain of Rbx1. Glomulin loss results in an increased turnover of the CRL1 substrate receptor Fbw7, which binds to proteins such as Cyclin-E and c-Myc. Consequently, GVM lesions and Glmn-null mice express reduced Fbw7 and increased levels of Cyclin-E and c-Myc.42

Lymphatic malformations Lymphatic malformations (LMs) consist of enlarged lymphatic vessels with fibrotic walls of variable thickness. They are commonly classified into macro- and micro-cystic lesions. They are found in the skin and deeper in soft tissue, typically on the head and neck. LMs are commonly localized, but more widespread lesions can be encountered on the thoracic and abdominal areas. There is no evidence of familial forms of LMs. Like sporadic VMs, LMs are likely caused by somatic mutations in a gene important for lymphangiogenesis.43 Lymphedema is another lymphatic anomaly in which the initial and/or collecting lymphatic vessels are affected. Lymphedema is not localized but rather affects entire limbs. In general, more females than males are affected. Lymphedema is typically seen in older patients, and the affected regions of the body gradually become enlarged as lymph fluid is not efficiently cleared. In lymphoscintigraphy, lymphedema is characterized by hypoplasia, aplasia, or hyperplasia of lymphatic channels.43 There are two main categories of lymphedema. “Secondary” refers to lymphedema that develops in response to a trauma, such as surgery. When the cause is not known, lymphedema is considered “primary.” Most cases of primary lymphedema are sporadic; however, 35% of patients have a positive family history.43,44 At least 21 genes have been linked to lymphedema, though a clear pathogenetic mechanism connecting these is unknown. The VEGFC/VEGFR3 and phosphatidylinositol-4,5bisphophate 3-kinase (PI3K)–AKT signaling pathway as well as the development and maintenance of lymphatic valves seem to be major players.43 Familial congenital lymphedema, or Nonne–Milroy disease, is linked to mutations in the vascular endothelial growth factor receptor 3 (VEGFR3; OMIM 153100).45,46 Patients with VEGFR3 mutations tend to have bilateral lower limb lymphedema. VEGFR3 is a tyrosine kinase receptor in ECs, essential for angiogenesis and later on for lymphangiogenesis in embryonic development. Mutations lead to lack of receptor phosphorylation, thus decreasing downstream signaling. Although mutations are classically dominant, a recessive mutation in the ATP-binding site has also been identified.47 Studies in mice established the importance of VEGFR3 in lymphangiogenesis, and Vegfr3 KO mice are embryonic lethal at midgestation due to cardiovascular defects.48 One family has been reported to have a loss-of-function mutation in the ligand that binds VEGFR3 specifically in lymphatic vessels, VEGFC.49 The Chy-3 mouse has Vegfc ablated and characteristically develops chylous ascites, in addition to lymphedema in the hindlimbs.50 Vegfc heterozygous mice are born with hypoplasic lymphatic vessels and develop lymphedema in adulthood.50 A homozygous deletion of exon 7 of the protein tyrosine phosphatase-14 (PTPN14) was discovered in one consanguineous family. The mutation leads to a premature stop codon that ultimately results in nonsense-mediated mRNA decay. Ptpn14 knockout mice develop hyperplasia of vessels and lymphedema, recapitulating the human syndrome. As PTPN14 is recruited to VEGFR3 via VEGFC stimulation in order to dephosphorylate the

active VEGFR3, the lymphatic defects may arise from hyperactivity of VEGFR3 in the absence of PTPN14.51 Hereditary lymphedema II often develops in combination with distichiasis (and to a lesser degree, ptosis and yellow nails). It is linked to truncating and missense mutations in the forkhead box protein C2 (FOXC2) transcription factor.52,53 FOXC2 has many functions, notably in angiogenesis by regulating the expression of important endothelial target genes, such as Ang-2, integrinβ3, D114, and Hey2.54 Foxc2 heterozygous mice develop lymphatic vessel and lymph node hyperplasia, in which increased pericyte recruitment interferes with proper function of collecting lymphatics.55 FOXC2 is a downstream target of VEGFC/VEGFR3 signaling.43 A rare form of syndromic lymphedema with variable onset is Hypotrichosis–Lymphedema–Telangiectasia (HLT; OMIM 607823). Both recessive and dominant mutations in the transcription factor SRY-containing box 18 (SOX18) have been reported. SOX18 regulates the essential initiator of lymphangiogenesis, prospero homeobox 1 (PROX1), which in turn regulates VEGFR3 expression. The reported dominant nonsense mutation is found within the transactivation domain of SOX18, while recessive substitutions lie within the DNA-binding domain.56 The dominant mutations are associated with an additional renal defect.57 The lymphatic defects may be due to competitive transcription factor binding. Mice with disrupted SOX18 only have a mild coat phenotype.58 Hennekam syndrome is inherited in autosomal recessive manner, with alterations within collagen and calcium-binding EGF domaincontaining protein-1 (CCBE; OMIM 235510). In the lymphatics, CCBE1 is bound to the extracellular matrix and influences ADAMTS3-mediated proteolytic activation of VEGFC.59 Hennekam syndrome is characterized by extensive lymphedema with visceral involvement, intellectual disability, and distinct facial features (flat facies, hypertelorism, and a broad nasal bridge); occasionally hydrops fetalis is seen.60 Knockdown of the zebrafish homolog full of fluid (fof) indicates that CCBE1 is involved in lymphangioblast budding and sprouting from the venous endothelium.61 Microcephaly associated with or without chorioretinopathy, lymphedema, or mental retardation (MCLMR; OMIM 152950) is linked to the homotetrameric kinesin-like protein-11 (KIF11)/ EG5.62 KIF11 is a motor protein involved in chromosome positioning and centrosome separation during mitosis, by forming a bipolar spindle. KIF11/EG5 inhibition activates PI3K/AKT signaling. Kif11 null mice are early embryonic lethal, but Kif11 heterozygotes are phenotypically normal.63 It is unclear how KIF11 mutations lead to lymphedema. Inhibitor of kappa light polypeptide gene enhancer in B-cells (IKBKG), also as known as NEMO, is altered in the X-linked syndrome osteopetrosis, lymphedema, anhidrotic ectodermal dysplasia, and immunodeficiency (OLEDAID; OMIM 308300). In this particular form of lymphedema, mutations are hypomorphic. Thus, IKBKG is greatly reduced, not completely lost.64 IKBKG indirectly regulates VEGFR3 expression by inducing nuclear factor-κβ activation and consequently PROX1.65 Defective lymphatic valve development/maintenance seems to contribute to lymphedema as mutations in two connexins (CX), CX47 and CX43, which are expressed on lymphatic valves, have been identified in some patients.66,67 The reported mutations are amino acid substitutions that alter connexin activity. The specific molecular role on the valves is not clear. Cx47 knockout mice do not have lymphatic problems.68,69 Moreover, loss of CX47 is seen in the autosomal recessive disorder hypomyelinating leukodystrophy, which affects the central nervous system, without lymphedema. Only one patient was reported to have a CX43 mutation along with oculodentodigital dysplasia.70 The zinc-finger transcription factor GATA2 is mutated in Emberger syndrome (OMIM 614038); patients are susceptible to

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developing acute myeloid leukemia in addition to lymphedema.71,72 GATA2 is mainly expressed within ECs and hematopoietic stem cells, as well as progenitors and valves of lymphatic vessels. The lymphatic defects are likely due to loss of GATA2 function. Gata2-null mice are embryonic lethal at midgestation due to severe anemia and lowered myeloid–erythroid progenitor cells. However, no vascular defects are observed. This may be due to redundancy among GATA family members.73,74 The use of NGS has been an asset in finding some of these genes. It enables to study even small families, in which classic linkage analysis is not informative. Thus, it is critical in exploring new mutations and genes, including the sporadic cases.

Combined and complex disorders Vascular anomalies are occasionally found as one of several symptoms in complex syndromes, such as the overgrowth syndromes, in which hyperplasia of connective tissues and bones is also present. As in the isolated vascular malformations, somatic or mosaic mutations are likely the major cause of these phenotypes.3 The associated genetic changes identified thus far are related to the PI3K–AKT signaling pathways.75,76,81 Whole-exome sequencing will likely further clarify the genetic basis of these syndromes enabling more accurate diagnosis. Additionally, as overgrowth syndromes can predispose affected patients to cancer, the identification of the causative genes should help evaluate prognosis and identify more specific therapeutic molecules. Congenital lipomatous asymmetric overgrowth with vascular malformation, epidermal nevi, and skeletal anomalies (CLOVES; OMIM 612918) is a noninherited disorder that is linked to mosaic missense mutations in PIK3CA. PIK3CA is the 110-kDa catalytic subunit of PI3K, activated by ligand binding to tyrosine kinase receptors. The mutations cause gain-of-function with increased AKT phosphorylation in affected tissues.77 Klippel–Trenaunay syndrome (KTS; OMIM 149000), consisting of slow-flow capillo-lymphatico-venous malformations with soft tissue overgrowth, is usually a sporadic disease. Rare chromosomal translocations between 8q22.3 and 14q13, and 5q13.3 and 11p15.1 have been reported. The latter resulted in the identification of a gain-of-function effect of angiogenic factor with G patch and FHA domains 1 (AGGF1).78 Zebrafish overexpressing aggf1 exhibit enhanced venous differentiation and increased AKT signaling.79 However, preliminary data suggests that most KTS, like CLOVES, is due to post-zygotic modifications in PIK3CA.77 Proteus syndrome (PLS, OMIM 176920) is another noninherited overgrowth syndrome characterized by skin, connective tissue, brain, and tissue hyperplasia, which often appear patchy. There is a susceptibility to developing tumors. Whole-exome sequencing of paired affected and unaffected tissues from patients allowed the determination of a recurrent somatic AKT1 mutation. The aberrant genetic variant likely results in gain-of-function of AKT1.80 A transgenic mouse expressing activated Akt1 exhibited skin overgrowth.81 Additionally, cartilage calcification, a major event in Proteus syndrome, is induced when Akt1 is activated in murine chondrocytes in vitro.82 PTEN hamartoma tumor syndrome (PHTS) is inherited in an autosomal dominant manner. PHTS encompasses several disorders: Cowden syndrome (OMIM 158350), Bannayan–Riley–Ruvalcaba syndrome (OMIM 153480), PTEN-related Proteus syndrome, and “Proteus-like” syndrome. In addition to the appearance of slow- and fastflow vascular anomalies, PHTS patients may exhibit tissue overgrowth, gastrointestinal polyps, and macrocephaly. PHTS is characterized by pathogenic phosphatase and tensin homolog (PTEN) mutations. PTEN is a tumor-suppressing phosphatase that counteracts PI3K signaling. In the absence of PTEN, PI3K/AKT signaling is dysregulated.83,84

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Concluding remarks Vasculogenesis, angiogenesis, and lymphangiogenesis are tightly regulated and complex processes that involve several signaling molecules and pathways. Although the sporadic forms of vascular anomalies are more common, the familial forms have been fundamental in discovering the molecules and pathways involved in their pathogenesis. These discoveries have allowed to extrapolate the findings to the more common sporadic forms, even before the era of NGS. This led to the discovery of somatic mutations in VMs laying down the foundation for somatic screens for all localized vascular malformations as well as mosaic screens for more widespread syndromic forms. Tissue heterogeneity is now becoming increasingly appreciated. Standard sequencing methods initially used to search for genetic mutations is not sensitive enough to detect low-level somatic mutations. NGS, and especially targeted NGS of the (likely) pathogenic genes, is a great tool to unravel etiopathogenic variants in resected tissues. This will soon allow efficient molecular genetic diagnostics of vascular anomalies. Identification of causative somatic mutations will lead to a better molecular clarification of vascular anomalies, and address time-point relatedness influencing severity and localization of lesions. Our understanding of the processes relevant to vascular development and maintenance will also become clearer. This will help reproduce the disease in an animal model. Such models can be used to screen for beneficial effects of therapeutic molecules in use for other disorders and to develop precise targeted treatments.

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