Genetic Aspects of Development*:

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1 Genetic Aspects of Development*: Vascular Anomalies & Overgrowth Syndromes Pascal BROUILLARD, PhD Human Molecular Gene:cs de Duve Ins:tute, Université catholique de Louvain Brussels, BELGIUM * Thompson & Thompson: Genetics in Medicine, 7th ed., chapter 14 1 Vasculogenesis and Angiogenesis mesoderm primary capillary plexus maturation artery bfgf, Vegf A, Nrp 1,2 blood islands Vegf A- Vegfr1,Vegfr2 remodeling capillaries vein Hematopoietic Precursors: Blood Cells Vegf A-Vegfr2, Tgfb1, TgfbR2 Hemangioblasts: Vascular Endothelial Cells Vascular smooth muscle cells Tie2-Angpt 1,2, Vegfr3- Vegfc, Ephrins-Ephs, CoupTFII, Rasa1, Shp2, VE- Cadherin 2 1

2 Vascular Anomalies Diagnosis: clinical history, examination & tests 3 Vascular Anomalies: characteristics Large clinical variability Commonly sporadic; rare familial forms Localized lesions: Single (sporadic) or multiple (familial) Pure forms versus associations & syndromes 4 2

3 Classification of Vascular Anomalies Tumors Malformations Hemangioma Infantile hemangioma (IH) Non-involuting congenital hemangioma (NICH) Rapidly-involuting congenital hemangioma (RICH) Hemangioendothelioma Angiosarcoma Lymphangiosarcoma Mulliken JB and Glowacki, Plast Reconstr Surg 1982; Wassef et al, Pediatrics Classification of Vascular Anomalies Tumors Malformations Capillary CCM CM-AVM Lymphatic LE LM Venous GVM VMCM VM Arterial CM-AVM HHT Mulliken JB and Glowacki, Plast Reconstr Surg 1982; Wassef et al, Pediatrics 2015 Combined: AVM, CVM, CLVM, LVM, CLAVM (Overgrowth) syndromes: Maffucci, KTS, PWS, MLCRD. 6 3

4 Genetic bases of vascular anomalies Predisposing susceptibility genes (vs. causative) Multigenic (vs. monogenic) Polymorphism (vs. mutation) Locus heterogeneity One phenotype, many causes Inherited with reduced penetrance vs. somatic changes Mosaicism/ tissue heterogeneity Clinical phenotypic variability One gene, several clinical presentations Spatio-temporal distribution of mutations/stochastic effect 7 Genetic bases of vascular anomalies Predisposing susceptibility genes (vs. causative): IH Multigenic (vs. monogenic) Polymorphism (vs. mutation) Locus heterogeneity One phenotype, many causes Inherited with reduced penetrance vs. somatic changes Mosaicism/ tissue heterogeneity Clinical/phenotypic variability One gene, several clinical presentations Spatio-temporal distribution of mutations/stochastic effect 8 4

5 Vascular Tumors: Infantile Hemangioma PROLIFERATING Clonal ECs years INVOLUTING INVOLUTED Fibro-fatty matrix 9 Infantile Hemangioma Cells VEGF-dependent increased VEGFR2 phosphorylation Chronic activation of VEGF signaling > Phosphorylation AKT, ERK1/2, RASA1, STAT4. > Transcription VEGF, GLUT1 Hemangioma cells > Hem EC behavior Proliferation Jinnin et al, Nat Med

6 Hemangioma Etiopathogenesis Screening of 24 candidate genes: regulate EC migration/proliferation/adhesion/hif1α Control One TEM8 mutation Control Two VEGFR2 mutations 0/ 110 hemangioma patients, 0/295 controls 10/ 105 hemangioma patients, 12/295 controls Jinnin et al, Nat Med Hemangioma Etiopathogenesis VEGFR1 VEGFR1 R2=VEGFR2 β1= β1 integrin Jinnin et al, Nat Med 2008! All Hem ECs showed pathway dysregulation: Changes not necessary! Associated SNP also present in controls: Changes not sufficient! Particular combinations of predisposing germline changes cause disease 12 6

7 Genetic bases of vascular anomalies Predisposing susceptibility genes (vs. causative): IH Multigenic (vs. monogenic) Polymorphism (vs. mutation) Locus heterogeneity: CCM, PLE One phenotype, many causes Inherited with reduced penetrance vs. somatic changes Mosaicism/ tissue heterogeneity Clinical/phenotypic variability One gene, several clinical presentations Spatio-temporal distribution of mutations/stochastic effect 13 Cerebral Cavernous Malformation (CCM) Incidence: % Single or multiple lesions Epilepsy, headache, haemorrhage; Asymptomatic: 15-20% Autosomal dominant inheritance (>80%) Variable expressivity EPI 14 7

8 Genetic basis of CCM CCM2 20%, penetrance 100% CCM3 40%, penetrance 63% CCM1 40%, penetrance 88% X Y Dubovsky et al., Hum Mol Genet, 1995; Craig et al., Hum Mol Genet, Pathophysiology of CCM fibronectin cell spreading ERK1/2 α v ß1 NOTCH integrin ICAP1α PI3K/AKT CCM1 HEG1 RAP1 CCM3 membrane MEKK3 CCM2 RHOA SMURF1 hyperosmotic shock MKK3 RAC1 p38mapk ROCK proliferation apoptosis actin stress fiber formation microtubule (cell morphology) apoptosis? osmoregulation ICAP1α KRIT1? cell proliferation? nucleus CCM1: KRIT1, CCM2: malcavernin/mgc4607, CCM3: PDCD10 Adapted from Revencu et al, J Med Genet

9 Primary lymphedema Chronic accumulation of lymph within tissues Predisposition to infections Important dysfunction of extremities abnormal development and/or function of lymphatic vessels genetic predisposition 17 Primary Lymphedema: Highly heterogeneous (28 genes explain only ~30% of cases) LEC PIEZO1 HGF c-met c-met SHP2 KIF11 ADAMTS3 CCBE1 PTPN14 VEGF-C VEGFR3 VEGFR3 FAT4 CELSR1 EPHB4 CX43 CX47 (GJA1) (GJA2) ITGA9? RELN RASGAP SOS1 PI3K PTEN RAS AKT SHP2 = PTPN11 RAS = KRAS,RIT1 RASGAP = RASA1 RAF1 TSC1 TSC2 IKBKG MEK RHEB NF-κB MAPK mtor Proliferation Survival GATA2 FOXC2 FOXC2 LEC and valves Nucleus NF-κB GATA2 FOXC2 SOX18 PROX1 PROX1 ITGA9 ETS NF-κB PROX1 FLT4 Adapted from Brouillard et al, J Clin Invest 2014; Brouillard et al, Hum Mol Genet

10 Genetic bases of vascular anomalies Predisposing susceptibility genes (vs. causative): IH Multigenic (vs. monogenic) Polymorphism (vs. mutation) Locus heterogeneity: CCM, LE One phenotype, many causes Inherited with reduced penetrance vs. somatic changes: GVM, VM Mosaicism/ tissue heterogeneity Clinical/phenotypic variability One gene, several clinical presentations Spatio-temporal distribution of mutations/stochastic effect 19 Glomuvenous Malformation (GVM)! 5% of venous anomalies! Caused by loss-of-function mutations in glomulin! Autosomal dominant, with reduced penetrance & phenotypic heterogeneity! Does lesion-formation require an additional somatic event? Boon et al, Hum Mol Genet

11 First somatic 2 nd hit identified in a vascular malformation Inherited: (IVS7-2884)-(IVS13+255) del 8.4 kb+insgg E8- E13 Ad- 1 Ad- 3 Ad- 4 * * Ad- 12 Ad- 11 Somatic: 980delCAGAA (E10) Brouillard et al. Am. J Hum Genet Complete local loss of glomulin wt 2 nd somatic mutation inherited mutation inherited mutation SMC α ac:n 22 11

12 Local loss of wild-type glomulin expression: genomic deletion? Affymetrix SNP arrays on lesion-dna Chromosome 1 Affymetrix 250K (NspI) GVM71_12 1pter 1p qter Copy number (1 SNP) c.107insg GLMN Average copy number (10 SNPs) Cytoband Heterozygous SNPs Cen Amyere et al, Am J Hum Genet Somatic 2 nd hits in GVM Pairwise comparison of allele-specific copy number Blood DNA Allele A Blood Allele B Blood Pairwise analysis Allele A Blood Allele A GVM GVM lesion DNA Allele A GVM Allele B GVM Allele B Blood Allele B GVM Amyere et al, Am J Hum Genet

13 Somatic 2 nd hits in GVM GVM22 Chromosome 1 Copy number (1 SNP) Average copy number (10X SNPs) Cytoband Heterozygous SNPs Discrepancy calls Pairwise CN c.157delaagaa 1pter 1qter LOH Loss of WT allele expression in GVM tissue Cen Similar observation in 12 GVM lesions Amyere et al, Am J Hum Genet aupid: A novel mechanism for glomulin loss Allelic imbalance (LOH) without copy number change in tissue: acquired Uniparental Isodisomy 1p aupid detected in 9/25 GVM tissues, not present in 54 control pairwise comparisons Amyere et al, Am J Hum Genet

14 Autosomal dominant with incomplete penetrance: recessive at the level of the cell! Glomuvenous malformation Cutaneomucosal Venous Malformation (VMCM) Cerebral Cavernous Malformation (CCM) 27 Venous Malformations: mostly sporadic Enlarged venous channels Single EC layer, with patchy vsmc Typically sporadic (>98%) Familial forms caused by TIE2/TEK mutations 28 14

15 Sporadic VM: Tissue heterogeneity hides mutation Sanger sequencing: cumulative signal 40% mutant <allele 10% mutant allele 5% mutant allele 29 Overcoming tissue heterogeneity: cdna-based screens DNA VM Tissue WT Mutant = cdna = abnormal EC = normal EC = other cells - DNA screens: TIE2 gene present in all cell types - cdna screens: TIE2 expression primarily from ECs (Semi quantitative minisequencing): SNaPshot Limaye et al., Nat Genet

16 Overcoming tissue heterogeneity: Deep (Next Generation) Sequencing 9 mut (T): 170 wt (C) " Should include negatives to distinguish low-freq alleles from background/noise! 31 Somatic TIE2/TEK mutations cause >60% of VMs (1-10% of mutant alleles detected) Ig2 74% EGF Ig1 Ig3 FN III L914F Y897F R915L R849W G1115* Y897H, F, C, S L914F R915C, L S917I TK R849W R918C TK F960I R1099* T1105N T1106P E1109Lfs*5 G1115* Limaye et al, Nat Genet 2009; Soblet et al, Mol Syndromol 2013 T1105N T1106P Y897F R1099* T1105N G1115* 32 16

17 Somatic TIE2/TEK mutations cause >60% of VMs EGF Ig2 Ig3 Ig1 Hyper-phosphorylation of mutants vec WT L914F R849W vec WT Y897F+ R915L Y897F R915L p-tyr FN III TIE2 B Growth factor RTK Cell membrane Y897H, F, C, S L914F R915C, L S917I TK R849W R918C TIE2 P P PIP 2 PIP 3 p85 PIK3CA AKT TK F960I R1099* T1105N T1106P E1109Lfs*5 G1115* Cell proliferation, cell survival, transformation, cell motility, insulin response pathways Apoptosis, cell cycle, cell cycle arrest pathways Limaye et al, Nat Genet 2009; Soblet et al, Mol Syndromol 2013, Uebelhoer et al, 2013, Natynki et al, Hum Mol Genet Somatic PIK3CA mutations cause 20% of VM A B E Skin Involvement No skin Involvement 40 p < C D Number of indivuduals 20 0 p < vs. TEK p = vs. N.D. p = vs. TEK TEK PIK3CA N.D. Mutation detected VM: Venous malformation Limaye et al, Am J Hum Genet

18 Genetic bases of vascular anomalies Predisposing susceptibility genes (vs. causative): IH Multigenic (vs. monogenic) Polymorphism (vs. mutation) Locus heterogeneity: CCM, LE One phenotype, many causes Inherited with reduced penetrance vs. somatic changes: GVM, VM Mosaicism/ tissue heterogeneity Clinical/phenotypic variability: VM-LM-PROS One gene, several clinical presentations Spatio-temporal distribution of mutations/stochastic effect 35 Somatic PIK3CA mutations also cause 72% of LM (Lymphatic malformations)! No-flow malformations! Filled with lymph! Macro- or micro-cystic! Present in utero! Grow with the individual Boscolo et al, 2015; Osborne et al, 2015; Luks et al, 2015; Schlogel et al, In Prep 36 18

19 Somatic PIK3CA mutations also cause PROS (PIK3CA-Related Overgrowth Syndromes) MCAP: Megalencephaly Capillary Malformation KT: Klippel Trenaunay CLVM with overgrowth CLOVES: Congenital Lipomatous Overgrowth, Vascular malformation, Epidermal nevi, Scoliosis Macrodactyly Keppler-Noreuil et al, Am J Med Genet Somatic PIK3CA mutations cause a spectrum of phenotypes Keppler-Noreuil et al, Am J Med Genet

20 Genetic bases of vascular anomalies Predisposing susceptibility genes (vs. causative): IH Multigenic (vs. monogenic) Polymorphism (vs. mutation) Locus heterogeneity: CCM, LE One phenotype, many causes Inherited with reduced penetrance vs. somatic changes: GVM, VM Mosaicism/ tissue heterogeneity Clinical/phenotypic variability: VM-LM-PROS, CM-AVM One gene, several clinical presentations Spatio-temporal distribution of mutations/stochastic effect 39 RASA1 and EPHB4 Mutations cause CM-AVM (Capillary Malformation-Arteriovenous Malformation) RASA1 c.806deltttac c.951insg c.957g>a c.613delcttat c.1208insc ATG c g>t c.1575insatgt c g>a c.2341g>t c.2365c>t c.2422c>t c.1490t>g c.1636c>t c delg c a>g c.1683deltc c delgtta c.2125c>t c.2532deltttaa c.1277a>g c g>a c.1279c>t c inst c g>t c.3028c>t c.3052delg TAG SH2 SH3 SH2 PH C2 Ras-GAP c.1336c>t c g>a c.1619g>a c insa c.828+3a>t c.1192c>t c.1870c>t c inst c.656c>g c.1350inst c.1579delgtct c.2026c>t c.2514insa c.512delt c.1480inst c.2288a>t c.2579deltcat c.475delct c.1362instcagt c.2336delgc c.2450delct EPHB4 c.41261g>t) c.389g>a) c.123+3g>c) c.814_816delcta) c.730c>t) c g>a) c.632_633deltg) c.2215c>t) c.481_485delgtcainstt) c.447_448insgaag) c g>c) c.1558c>t) c.2037_2038inst) c.1291c>t) c.1177_1177delg) c.1788t>g) c.1123g>t) c t>g) c.1077_1081delccgct) c g>a) c.1054c>t) c g>a) c.2418c>g) c.1615delg) c.2379delc) c.33delg) c.1733_1734insa) c.221g>c) c.1406t>c) c.175g>a) c.1546g>a) c.802t>c) c.560t>c) Revencu et al, Hum Mutat 2008; Amyere et al, Circulation 2017 p.e664k;)c.1990g>a) c.1966c>t) c.2173g>a) C.2233A>G) c.2366c>g) c.2365c>t) c.2458c>a) c.2459c>t) c.2419g>a) c.2621t>c) c.2590c>t;)p.r864w) c.2567g>a) c.2533t>c;)p.c845r) c.2512c>t;)r838w) 40 20

21 Phenotypic variability in CM-AVM B Eerola et al, Am J Hum Genet 2003; Revencu et al, Inborn errors of development 2nd Ed, RASA1 Phenotypes RASA1 mutations in 314 individuals from 132 families 306: multifocal CM (97%) 101: Accompanying fast-flow lesions 26 Parkes Weber syndrome (8.5%) 32 Intra-CNS AVM/AVF (10%) 43 Extra-CNS AVM/AVF (13%) Large inter- and intra-familial variability I. II. III. Revencu et al, Hum Mutat

22 Genetic bases of vascular anomalies Predisposing susceptibility genes (vs. causative): IH Multigenic (vs. monogenic) Polymorphism (vs. mutation) Locus heterogeneity: CCM, LE One phenotype, many causes Inherited with reduced penetrance vs. somatic changes: GVM, VM Mosaicism/ tissue heterogeneity Clinical/phenotypic variability: VM-LM-PROS, CM-AVM Spatio-temporal distribution of mutations/genetic background/environment/ stochastic effect Thanks for your attention! 43 22