Genetic testing of vascular anomalies of children

I have no conflict of interest Victor Martinez-Glez Vascular Malformations Section, Institute of Medical and Molecular Genetics (INGEMM-CIBERER) La Paz Hospital - Madrid 2018 2018

Capillary malformations (CM) Nevus simplex / salmon patch, “angel kiss”, “stork bite” Unknown Cutaneous and/or mucosal CM (also known as “port-wine” stain) Nonsyndromic CM GNAQ CM with CNS and/or ocular anomalies (Sturge-Weber syndrome) GNAQ CM with bone and/or soft tissues overgrowth GNA11 Diffuse CM with overgrowth (DCMO) GNA11 Reticulate CM CM of MIC-CAP (microcephaly-capillary malformation) STAMBP CM of MCAP (megalencephaly-capillary malformation-polymicrogyria) PIK3CA CM of CM-AVM RASA1 / EPHB4 Cutis marmorata telangiectatica congenita (CMTC) Unknown Others Telangiectasia* Hereditary hemorrhagic telangiectasia (HHT) HHT1: ENG HHT2: ACVRL1 HHT3: Unknown JPHT: SMAD4 Others 2018

Lymphatic malformations (LM) Common (cystic) LM * PIK3CA Macrocystic LM Microcystic LM Mixed cystic LM Generalized lymphatic anomaly (GLA) PIK3CA Kaposiform lymphangiomatosis (KLA) Unknown LM in Gorham-Stout disease Unknown Channel type LM Unknown “Acquired” progressive lymphatic anomaly (so called acquired progressive "lymphangioma") Unknown Primary lymphedema Nonne-Milroy syndrome FLT4 / VEGFR3 Primary hereditary lymphedema VEGFC Primary hereditary lymphedema GJC2 / Connexin 47 Lymphedema-distichiasis FOXC2 Hypotrichosis-lymphedema-telangiectasia SOX18 Primary lymphedema with myelodysplasia GATA2 Primary generalized lymphatic anomaly CCBE1 / FAT4 (Hennekam lymphangiectasia-lymphedema syndrome) Microcephaly with or without chorioretinopathy, lymphedema, or mental KIF11 retardation syndrome Lymphedema-choanal atresia PTPN14 Others 2018

Venous malformations (VM) Common VM TEK (TIE2) / PIK3CA Familial VM cutaneo-mucosal (VMCM) TEK (TIE2) Blue rubber bleb nevus (Bean) syndrome VM TEK (TIE2) Glomuvenous malformation (GVM) Glomulin Cerebral cavernous malformation (CCM) CCM1: KRIT1 CCM2: Malcavernin CCM3: PDCD10 Familial intraosseous vascular malformation (VMOS) ELMO2 Verrucous venous malformation (formerly verrucous hemangioma) MAP3K3 Others

Arteriovenous malformations/Fistula (AVM/F) Sporadic MAP2K1 In HHT HHT1: ENG HHT2: ACVRL1 HHT3: Unknown JPHT: SMAD4 In CM-AVM RASA1 / EPHB4 Others 2018

Vascular malformations associated with other anomalies Klippel-Trenaunay syndrome*: CM + VM +/- LM + limb overgrowth PIK3CA* Parkes Weber syndrome: CM + AVF + limb overgrowth RASA1 Servelle-Martorell syndrome: Limb VM + bone hypothrophy Unknown Sturge-Weber syndrome: facial + leptomeningeal CM + eye anomalies +/- bone and/or soft GNAQ tissue overgrowth Limb CM + congenital non-progressive limb overgrowth GNA11 Maffucci syndrome: VM +/- spindle-cell hemangioma + enchondroma IDH1 / IDH2 Macrocephaly - CM (M-CM or MCAP)*: CM + overgrowth + LM + VM PIK3CA Microcephaly - CM (MICCAP) STAMBP CLOVES syndrome*: LM + VM + CM +/- AVM + lipomatous overgrowth PIK3CA Proteus syndrome: CM, VM and/or LM + asymmetrical somatic overgrowth AKT1 Bannayan-Riley-Ruvalcaba syndrome: AVM + VM +macrocephaly, lipomatous overgrowth PTEN CLAPO Syndrome*: CM + LM + overgrowth PIK3CA

Provisionally unclassified vascular anomalies Intramuscular hemangioma * Unknown Angiokeratoma Unknown Sinusoidal hemangioma Unknown Acral arteriovenous "tumour" Unknown Multifocal lymphangioendotheliomatosis with thrombocytopenia / cutaneovisceral Unknown PTEN (type) hamartoma of soft tissue / "angiomatosis" of soft tissue (PHOST) PTEN Fibro adipose vascular anomaly (FAVA) PIK3CA 2018

Benign vascular tumors Infantile hemangioma / Hemangioma of infancy Unknown Congenital hemangioma GNAQ / GNA11 Rapidly involuting (RICH) * Non-involuting (NICH) Partially involuting (PICH) Tufted angioma * ° GNA14 Spindle-cell hemangioma IDH1 / IDH2 Epithelioid hemangioma FOS Pyogenic granuloma (also known as lobular capillary hemangioma) BRAF / RAS / GNA14 Others

Locally aggressive or borderline vascular tumors Kaposiform hemangioendothelioma * ° GNA14 Retiform hemangioendothelioma Unknown Papillary intralymphatic angioendothelioma (PILA) Unknown Composite hemangioendothelioma Unknown Pseudomyogenic hemangioendothelioma FOS Polymorphous hemangioendothelioma Unknown Hemangioendothelioma not otherwise specified Unknown

Malignant vascular tumors Angiosarcoma (Post radiation) MYC Epithelioid hemangioendothelioma CAMTA1 / TFE3 Others Unknown Pathways / Location and timing / Distribution / Expression / Lethal genes / 2º Hit / Function Pathways / Location and timing / Distribution / Expression / Lethal genes / 2º Hit / Function

SOMATIC MOSAICISM

GERMLINE Pathways / Location and timing / Distribution / Expression / Lethal genes / 2º Hit / Function Pathways / Location and timing / Distribution / Expression / Lethal genes / 2º Hit / Function Pathways / Location and timing / Distribution / Expression / Lethal genes / 2º Hit / Function GNAQ • 87% of sporadic or syndromic (SWS) CM (MAF 1-22%).

• Endothelial cells (3-43%), pericytes (0%), stromal cells (0,4-11%), blood (0,3%)

Tissue/organ cell type Pathways / Location and timing / Distribution / Expression / Lethal genes / 2º Hit / Function Pathways / Location and timing / Distribution / Expression / Lethal genes / 2º Hit / Function

• Cells bearing the mutation can survive only in a mosaic state • Schimmelpenning-Feuerstein-Mims syndrome • McCune-Albright syndrome • Klippel-Trenaunay syndrome • Sturge-Weber syndrome • Neurocutaneous melanosis • Proteus syndrome • Delleman-Oorthuys syndrome Pathways / Location and timing / Distribution / Expression / Lethal genes / 2º Hit / Function

• Cells bearing the mutation can survive only in a mosaic state • Schimmelpenning-Feuerstein-Mims syndrome • McCune-Albright syndrome • Klippel-Trenaunay syndrome • Sturge-Weber syndrome • Neurocutaneous melanosis • Proteus syndrome • Delleman-Oorthuys syndrome Pathways / Location and timing / Distribution / Expression / Lethal genes / 2º Hit / Function TIE2 - BRBN Pathways / Location and timing / Distribution / Expression / Lethal genes / 2º Hit / Function

VEGF-C CCBE1 HGF

G-protein C-MET ENG coupled GJA1 GJC2 ITG9 ACVRL1 TIE2 receptor GDF2 KIF11 GNAQ SHP2 GNA11 PTPN14 VEGFR-2 VEGFR-3 PIK3CA STAMBP SOS1 SMAD4 PTEN RAS AKT RASA1 HRAS AKT1 IKBKG KRAS AKT2 CCM NRAS AKT3 KRIT1 CCM2 RAF PDCD10 BRAF mTOR MAP3K3 GOF and somatic GLMN

MEK MAP2K1 NF-KB LOF and germline + IDH1 somatic 2nd hit ERK IDH2 FOXC2 GATA2

SOX18

MOSAICISM & THE PATIENT SAMPLES GLA-Lymphatic Malformation derived Lymphatic Endotelial Cells

PIK3CA pathogenic variant in • 66% of the cells (LECs) NGS • 3% of the LM tissue sample NGS NGS visualization

RASA1: c.2125C>T:p.R709X NGS and genetic variant interpretation and validation Validation (confirmation) of genetic variants Bioinformatic algorithms Real example of analysis in a patient with diffuse phenotype of overgrowth and vascular anomalies. Special casuistry that is divided into two parts:

➢The first step is to calculate the mosaic or frequency of the allele / alternative variant of all variants annotated (without previous filtering).

➢The second part consists in detecting variants of the somatic mosaic using the paired blood / tissue samples and "control" samples.

Kristina Ibañez → https://github.com/kibanez/mOsAiC Filtering variants in NGS 303750 variants Filters: Real example of analysis in a patient with ➢Population frequency : <1% diffuse phenotype of overgrowth and vascular ➢Samples run anomalies. ➢Impact: Modifiers ➢DP: <70 ➢Alt. depth: <7 ➢Predictors of pathogenicity ➢MAVAF Filtering variants in NGS 62564 variants Filters: Real example of analysis in a patient with ➢Population frequency : <1% diffuse phenotype of overgrowth and vascular ➢Samples run anomalies. ➢Impact: Modifiers ➢DP: <70 ➢Alt. depth: <7 ➢Predictors of pathogenicity ➢MAVAF Filtering variants in NGS 6256449957 variantes variants Filters: Real example of analysis in a patient with ➢Population frequency : <1% diffuse phenotype of overgrowth and vascular ➢ Samples run anomalies. ➢Impact: Modifiers ➢DP: <70 ➢Alt. depth: <7 ➢Predictors of pathogenicity ➢MAVAF Filtering variants in NGS 34133 variants Filters: Real example of analysis in a patient with ➢Population frequency : <1% diffuse phenotype of overgrowth and vascular ➢Samples run anomalies. ➢Impact: Modifiers ➢DP: <70 ➢Alt. depth: <7 ➢Predictors of pathogenicity ➢MAVAF Filtering variants in NGS 6202 variants Filters: Real example of analysis in a patient with ➢Population frequency : <1% diffuse phenotype of overgrowth and vascular ➢Samples run anomalies. ➢Impact: Modifiers ➢DP: <70 ➢Alt. depth: <7 ➢Predictors of pathogenicity ➢MAVAF Filtering variants in NGS 2395 variants Filters: Real example of analysis in a patient with ➢Population frequency : <1% diffuse phenotype of overgrowth and vascular ➢Samples run anomalies. ➢Impact: Modifiers ➢DP: <70 ➢Alt. depth: <7 ➢Predictors of pathogenicity ➢MAVAF Filtering variants in NGS 303 variants Filters: Real example of analysis in a patient with ➢Population frequency : <1% diffuse phenotype of overgrowth and vascular ➢Samples run anomalies. ➢Impact: Modifiers ➢DP: <70 ➢Alt. depth: <7 ➢Predictors of pathogenicity ➢MAVAF Filtering variants in NGS 303 variants Filters: Real example of analysis in a patient with ➢Population frequency : <1% diffuse phenotype of overgrowth and vascular ➢Samples run anomalies. ➢Impact: Modifiers ➢DP: <70 ➢Alt. depth: <7 ➢Predictors of pathogenicity ➢MAVAF: Maximum alternative variant allele frequency Filtering variants in NGS

IGV visualization

1 candidate gene

EVALUATION OF VASCULAR ANOMALIES • Classification • Mosaicism • Embryology / Timing • Gene / Pathway / Expression / Lethality • Location / distribution • Second Hit • Samples, testing, analysis

Implications for sampling, testing, variant identification, interpretation and recurrence risks

Capillary malformations (CM) Arteriovenous malformations/fistula (AVM/F) Cutaneous and/or mucosal CM (also known as “port-wine” stain) Sporadic MAP2K1 SOMATIC Nonsyndromic CM GNAQ SOMATIC In HHT HHT1: ENG CM with CNS and/or ocular anomalies (Sturge-Weber syndrome) GNAQ SOMATIC HHT2: ACVRL1 Germline CM with bone and/or soft tissues overgrowth GNA11 SOMATIC HHT3: Unknown Diffuse CM with overgrowth (DCMO) GNA11 SOMATIC JPHT: SMAD4 Reticulate CM In CM-AVM RASA1 / EPHB4 Germline + 2nd Hit CM of MIC-CAP (microcephaly-capillary malformation) STAMBP Germline Vascular malformations associated with other anomalies CM of MCAP (megalencephaly-capillary malformation-polymicrogyria) PIK3CA MOSAIC Klippel-Trenaunay syndrome*: CM + VM +/- LM + limb overgrowth PIK3CA* SOMATIC CM of CM-AVM RASA1 / EPHB4 Germline + 2nd Hit Parkes Weber syndrome: CM + AVF + limb overgrowth RASA1 Germline Telangiectasia* Sturge-Weber syndrome: facial + leptomeningeal CM + eye anomalies +/- bone and/or soft Hereditary hemorrhagic telangiectasia (HHT) HHT1: ENG GNAQ SOMATIC tissue overgrowth HHT2: ACVRL1 Germline Limb CM + congenital non-progressive limb overgrowth GNA11 SOMATIC HHT3: Unknown Maffucci syndrome: VM +/- spindle-cell hemangioma + enchondroma IDH1 / IDH2 SOMATIC JPHT: SMAD4 Lymphatic malformations (LM) Macrocephaly - CM (M-CM or MCAP)*: CM + overgrowth + LM + VM PIK3CA SOMATIC Common (cystic) LM * PIK3CA SOMATIC Microcephaly - CM (MICCAP) STAMBP STAMBP Generalized lymphatic anomaly (GLA) PIK3CA Unknown CLOVES syndrome*: LM + VM + CM +/- AVM + lipomatous overgrowth PIK3CA SOMATIC Primary lymphedema Proteus syndrome: CM, VM and/or LM + asymmetrical somatic overgrowth AKT1 SOMATIC Germline and/or Nonne-Milroy syndrome FLT4 / VEGFR3 Germline Bannayan-Riley-Ruvalcaba syndrome: AVM + VM +macrocephaly, lipomatous overgrowth PTEN Primary hereditary lymphedema VEGFC Germline somatic Primary hereditary lymphedema GJC2 / Connexin 47 Germline CLAPO Syndrome*: CM + LM + overgrowth PIK3CA SOMATIC Lymphedema-distichiasis FOXC2 Germline Provisionally unclassified vascular anomalies Hypotrichosis-lymphedema-telangiectasia SOX18 Germline PTEN (type) hamartoma of soft tissue / "angiomatosis" of soft tissue (PHOST) PTEN Germline Primary lymphedema with myelodysplasia GATA2 Germline Fibro adipose vascular anomaly (FAVA) PIK3CA SOMATIC Primary generalized lymphatic anomaly CCBE1 / FAT4 Germline Benign vascular tumors (Hennekam lymphangiectasia-lymphedema syndrome) Congenital hemangioma GNAQ / GNA11 SOMATIC Microcephaly with or without chorioretinopathy, lymphedema, or mental KIF11 Germline Tufted angioma * ° GNA14 SOMATIC retardation syndrome Spindle-cell hemangioma IDH1 / IDH2 SOMATIC Lymphedema-choanal atresia PTPN14 Germline Epithelioid hemangioma FOS SOMATIC Venous malformations (VM) Pyogenic granuloma (also known as lobular capillary hemangioma) BRAF / RAS / GNA14 SOMATIC Common VM TEK (TIE2) / PIK3CA SOMATIC Locally aggressive or borderline vascular tumors Familial VM cutaneo-mucosal (VMCM) TEK (TIE2) Germline + 2nd Hit Blue rubber bleb nevus (Bean) syndrome VM TEK (TIE2) Two somatic (cis) Kaposiform hemangioendothelioma * ° GNA14 SOMATIC Glomuvenous malformation (GVM) Glomulin Germline + 2nd Hit Pseudomyogenic hemangioendothelioma FOS SOMATIC Cerebral cavernous malformation (CCM) CCM1: KRIT1 Malignant vascular tumors Germline and/or CCM2: Malcavernin Angiosarcoma (Post radiation) MYC SOMATIC somatic CCM3: PDCD10 Epithelioid hemangioendothelioma CAMTA1 / TFE3 SOMATIC Familial intraosseous vascular malformation (VMOS) ELMO2 ELMO2 Verrucous venous malformation (formerly verrucous hemangioma) MAP3K3 SOMATIC Constitutional RASA1 pathogenic variants causing CM-AVM, detected in mosaic in blood samples from two unrelated patients and in the affected tissue sample in one of the patients are described. Future

Target cells LECs, BECs, osteoclasts, macrophages, etc.

Are we ready for multiple single cell NGS experiments? True mosaic detection

Is always the genetic cause in the exome? Genome, methylome, etc… cloud computing.

Digenic causes? Modulation of the phenotype

Pharmacological / Molecular Therapies Rapamycin, CRISPR-Cas9, etc. Thanks