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International Journal of Molecular Sciences logoLink to International Journal of Molecular Sciences
. 2020 Jun 17;21(12):4321. doi: 10.3390/ijms21124321

Germline Mutation Enrichment in Pathways Controlling Endothelial Cell Homeostasis in Patients with Brain Arteriovenous Malformation: Implication for Molecular Diagnosis

Concetta Scimone 1,2, Francesca Granata 3, Marcello Longo 3, Enricomaria Mormina 3,4, Cristina Turiaco 3, Antonio A Caragliano 3, Luigi Donato 1,2,*, Antonina Sidoti 1,2,*, Rosalia D’Angelo 1,2
PMCID: PMC7352422  PMID: 32560555

Abstract

Brain arteriovenous malformation (bAVM) is a congenital defect affecting brain microvasculature, characterized by a direct shunt from arterioles to venules. Germline mutations in several genes related to transforming growth factor beta (TGF-β)/BMP signaling are linked to both sporadic and hereditary phenotypes. However, the low incidence of inherited cases makes the genetic bases of the disease unclear. To increase this knowledge, we performed a whole exome sequencing on five patients, on DNA purified by peripheral blood. Variants were filtered based on frequency and functional class. Those selected were validated by Sanger sequencing. Genes carrying selected variants were prioritized to relate these genes with those already known to be linked to bAVM development. Most of the prioritized genes showed a correlation with the TGF-βNotch signaling and vessel morphogenesis. However, two novel pathways related to cilia morphogenesis and ion homeostasis were enriched in mutated genes. These results suggest novel insights on sporadic bAVM onset and confirm its genetic heterogeneity. The high frequency of germline variants in genes related to TGF-β signaling allows us to hypothesize bAVM as a complex trait resulting from the co-existence of low-penetrance loci. Deeper knowledge on bAVM genetics can improve personalized diagnosis and can be helpful with genotype–phenotype correlations.

Keywords: brain arteriovenous malformations, exome sequencing, endothelial properties, molecular diagnosis

1. Introduction

Brain arteriovenous malformations (bAVM, OMIM #108010) are vascular malformations affecting brain vasculature. Lack of a capillary bed and a direct shunt from arterioles to venules, as well as pericyte reduction, are characteristics of the lesions [1]. A vessel tangle is formed, usually called nidus. During transition from the feeding arteries to the nidus and then, to the draining veins, vessels show dysregulated differentiation patterns and severe enlargement. From arteries, blood perfuses to the nidus with high pressure, increasing risk of lesion rupture. Moreover, the mix of arterial and venous circulations within these lesions leads to a deficit in cerebral tissue oxygenation. This complex condition usually results in major clinical manifestations such as intracerebral hemorrhage and epileptic seizures, appearing in almost 50% of patients. Disease incidence is about 0.01% worldwide and usually arises at an early age [2]. Nevertheless, it most often occurs as a sporadic condition and only a few dozen cases are reported as inherited with an autosomal dominant pattern. Hereditary bAVM usually coexists with other vascular syndromes, such as Osler-Weber-Rendu syndrome, also known as hereditary hemorrhagic telangiectasia (HHT). HHT includes different phenotypes caused by mutations in genes related to the transforming growth factor beta 2 (TGF-βII) transduction pathway, such as ENG, ACVRL1, SMAD4, and GDF2 [3,4,5]. Due to the severe remodeling rate and recidivism risk after total surgical resection, bAVMs are considered highly dynamic lesions. Therefore, lesion growth is now thought of as the result of continuous endogenous stimuli due to genetic factors. The low frequency of inherited bAVM makes molecular characterization of the disease difficult. Impaired expression of the ephrin family genes and of other vascular differentiation markers was reported by several authors [6,7]. At the same time, the sporadic nature of the disease can also be considered a result of numerous single nucleotide polymorphisms (SNPs) in genes involved in vasculogenesis and early angiogenesis pathways, as, for instance, in vascular endothelial growth factor (VEGF) and Notch signaling [8]. To improve knowledge on bAVM pathogenesis, we purified DNA from peripheral blood and performed a whole exome sequencing (WES) analysis on a group of five patients affected by sporadic bAVM. Then, we clustered genes carrying the detected mutations, highlighting pathways and prioritizing genes mainly linked to bAVM development.

2. Results

2.1. WES, Bioinformatic Analysis, and Filtering

An average of 81,170,229 million reads were output by the runs. Of these, 92.29% showed a Phred quality score > 30. After duplicate discard, an average of 75,411,435 reads were filtered and mapped to the GRCh38 human reference genome. The percentage of on-target reads was on average 71.57%, calculated on the deduplicated mapped read numbers. The quality report summary of each run is provided in Table S1. Regarding variant calling, a mean of 40,090 variants was annotated for each exome. Following the application of the above-mentioned filtering criteria, about 230 variants were selected for each sample. These included non-synonymous, nonsense, and frameshift mutations, whose reported minor allele frequency (MAF) was estimated to be < 0.01. Full lists are available in Table S2.

2.2. Gene Clustering and Prioritization

The ClueGO (Gene Ontology) enrichment analysis allowed to cluster the mutated genes for each exome in order to highlight the pathways they are involved in. In Table 1 only annotated terms showing the Bonferroni-adjusted p-value ≤ 0.05 were reported, both for each single term and for the entire cluster. The full lists are available in Table S3. Annotations related to cell adhesion (GO:1903392, GO:0051895, R-HSA:382054, GO:0038089, GO:0007157, GO:0120193, GO:1904886), cardiovascular development (WP:3668, GO:0003279, GO:0035904, GO:0060840, GO:0060976, GO:0060411), Notch signaling (GO:0007221, R-HSA:9021450, R-HSA:9013508, GO:0045747, WP:268, R-HSA:9012852), and TGFβR signaling (GO:1903846, GO:0030511) were highly represented in all the AVM samples. Furthermore, ontologies related to microtubule assembly and cell motility (GO:0003341, GO:0001578, GO:0001539, GO:0031122, GO:0060285) were also widely frequent. Interestingly, pathways related to ion conductance (GO:1904063, GO:0034766, GO:2001258, GO:0043267, GO:0016248, GO:0008200) were highly enriched in the AVM5 sample.

Table 1.

ClueGO (Gene Ontology) enrichment analysis results.

Sample GO ID GO Term Ontology Source Term p-Value Corrected with Bonferroni Step Down Group p-Value Corrected with Bonferroni Step Down Associated Genes Found
AVM1 GO:0003341 cilium movement GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.00 0.00 (ASPM, CFAP206, DNAH1, DNAH5, HYDIN, TEKT5)
GO:0035082 axoneme assembly GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.01 0.00 (CFAP206, DNAH1, DNAH5, HYDIN, RP1L1)
R-HSA:2129379 molecules associated with elastic fibers REACTOME_Pathways_27.02.2019 0.01 0.05 (FBN2, FBN3, LTBP1, LTBP4)
GO:2000105 positive regulation of DNA-dependent DNA replication GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.01 0.14 (CDC7, FLG, SYTL2)
GO:0097722 sperm motility GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.02 0.00 (ASPM, CACNA1I, DNAH1, DNAH5, SLC22A16, TEKT5)
R-HSA:1566948 elastic fiber formation REACTOME_Pathways_27.02.2019 0.02 0.05 (FBN2, FBN3, LTBP1, LTBP4)
GO:0030317 flagellated sperm motility GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.02 0.00 (ASPM, CACNA1I, DNAH1, DNAH5, SLC22A16, TEKT5)
GO:0018410 C-terminal protein amino acid modification GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.03 0.02 (ASPM, LCMT2, SH2B1)
GO:0005044 scavenger receptor activity GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.03 0.03 (LRP1, MEGF10, STAB1)
GO:1903078 positive regulation of protein localization to plasma membrane GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.03 0.03 (CARD14, LRP1, NKD2)
WP:3668 hypothesized pathways in pathogenesis of cardiovascular disease WikiPathways_27.02.2019 0.04 0.05 (FBN2, FBN3, LTBP1)
GO:0001578 microtubule bundle formation GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.05 0.00 (CFAP206, DNAH1, DNAH5, HYDIN, RP1L1)
GO:1903392 negative regulation of adherens junction organization GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.05 0.07 (DLEC1, LRP1, TEP1)
GO:0051895 negative regulation of focal adhesion assembly GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.05 0.07 (DLEC1, LRP1, TEP1)
AVM2 GO:0031122 cytoplasmic microtubule organization GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.03 0.02 (GOLGA4, KIF19, MBD1, PCM1, TUBGCP6)
GO:0042267 natural killer cell mediated cytotoxicity GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.03 0.08 (LGALS9, LILRB1, PIK3R6)
GO:0070228 regulation of lymphocyte apoptotic process GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.03 0.09 (IRS2, LGALS9, SLC39A10)
R-HSA:450346 activated human TAK1 phosphorylates MKK3/MKK6 REACTOME_Reactions_27.02.2019 0.04 0.00 (MAP2K3, NOD1, TAB1)
GO:0061098 positive regulation of protein tyrosine kinase activity GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.05 0.02 (AGXT, DGKQ, DOK7, ERCC6, RELN)
R-HSA:450302 activated TAK1 mediates p38 MAPK activation REACTOME_Pathways_27.02.2019 0.06 0.00 (MAP2K3, NOD1, TAB1)
R-HSA:448424 interleukin-17 signaling REACTOME_Pathways_27.02.2019 0.06 0.00 (MAP2K3, NOD1, TAB1)
R-HSA:382054 PDGF binds to extracellular matrix proteins REACTOME_Reactions_27.02.2019 0.07 0.04 (COL6A3, COL6A6, SPP1)
WP:231 TNF alpha signaling pathway WikiPathways_27.02.2019 0.08 0.00 (KSR1, MAP2K3, NFKBIE, NSMAF, TAB1)
R-HSA:186797 signaling by PDGF REACTOME_Pathways_27.02.2019 0.08 0.04 (COL6A3, COL6A6, GRB7, SPP1)
GO:0036498 IRE1-mediated unfolded protein response GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.08 0.04 (ARFGAP3, EXTL1, NR1H4, VAPB, WFS1)
GO:0035176 social behavior GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.09 0.02 (HTT, MBD1, PCM1)
R-HSA:373739 ankyrins link voltage-gated sodium and potassium channels to spectrin and L1 REACTOME_Reactions_27.02.2019 0.10 0.05 (ANK2, SCN7A, SPTA1)
R-HSA:445095 interaction between L1 and ankyrins REACTOME_Pathways_27.02.2019 0.11 0.05 (ANK2, SCN7A, SPTA1)
GO:1901618 organic hydroxy compound transmembrane transporter activity GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.11 0.03 (AQP7, HTT, SLC10A6)
GO:0006890 retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.13 0.05 (ARFGAP3, CENPE, ERGIC1, HTT, TAPBP)
GO:0035036 sperm-egg recognition GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.13 0.05 (CATSPER2, ZAN, ZP1)
GO:0021846 cell proliferation in forebrain GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.13 0.02 (KIF14, MBD1, PCM1)
GO:0018195 peptidyl-arginine modification GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.13 0.05 (NR1H4, PADI2, PRMT7)
GO:0002753 cytoplasmic pattern recognition receptor signaling pathway GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.15 0.00 (ALPK1, DHX58, NOD1, TAB1)
R-HSA:6807875 ARFGAP, cargo, v-SNAREs, and p24 proteins bind nascent COPI complex REACTOME_Reactions_27.02.2019 0.19 0.05 (ANK2, ARFGAP3, SPTA1)
R-HSA:6807877 ARFGAPs stimulate ARF GTPase activity REACTOME_Reactions_27.02.2019 0.19 0.05 (ANK2, ARFGAP3, SPTA1)
R-HSA:450294 MAP kinase activation REACTOME_Pathways_27.02.2019 0.20 0.00 (MAP2K3, NOD1, TAB1)
R-HSA:375165 NCAM signaling for neurite out-growth REACTOME_Pathways_27.02.2019 0.22 0.04 (COL6A3, COL6A6, SPTA1)
R-HSA:2022090 assembly of collagen fibrils and other multimeric structures REACTOME_Pathways_27.02.2019 0.22 0.04 (COL6A3, COL6A6, LAMA3)
R-HSA:168643 nucleotide-binding domain, leucine rich repeat containing receptor (NLR) signaling pathways REACTOME_Pathways_27.02.2019 0.23 0.00 (NOD1, PSTPIP1, TAB1)
GO:1903573 negative regulation of response to endoplasmic reticulum stress GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.23 0.04 (NR1H4, PRKN, WFS1)
AVM3 GO:0038089 positive regulation of cell migration by vascular endothelial growth factor signaling pathway GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.01 0.17 (KDR, MYO1C, PKD1)
GO:0007157 heterophilic cell–cell adhesion via plasma membrane cell adhesion molecules GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.04 0.09 (HMCN1, RP9, SPG7)
GO:0120193 tight junction organization GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.04 0.09 (EPHA2, MYO1C, PDZD4)
GO:0032688 negative regulation of interferon-beta production GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.04 0.08 (LRRFIP1, MYO1C, NLRC3)
GO:0048739 cardiac muscle fiber development GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.08 0.05 (MYOM2, OBSL1, SPG7)
AVM4 GO:0001539 cilium or flagellum-dependent cell motility GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.00 0.00 (DNAH11, DNAH14, DNAH2, DNAH3, DNAH8, GAS8)
GO:0060285 cilium-dependent cell motility GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.00 0.00 (DNAH11, DNAH14, DNAH2, DNAH3, DNAH8, GAS8)
GO:0007221 positive regulation of transcription of Notch receptor target GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.00 0.00 (MAML1, NOTCH3, OPN1LW, PLXND1, SPHKAP)
WP:334 GPCRs, class B secretin-like WikiPathways_27.02.2019 0.01 0.10 (ADCYAP1R1, ADGRG2, GLP2R, SCTR)
GO:0003279 cardiac septum development GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.01 0.00 (CRELD1, DNAH11, LRP2, MAML1, PLXND1, SLIT2, SMO, TAB1)
GO:0071503 response to heparin GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.02 0.01 (AOC1, GPIHBP1, SLIT2)
R-HSA:9021450 PLXND1 gene expression is stimulated by NOTCH1/NOTCH3 coactivator complexes REACTOME_Reactions_27.02.2019 0.02 0.00 (MAML1, NOTCH3, PLXND1)
GO:0061476 response to anticoagulant GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.03 0.01 (AOC1, GPIHBP1, SLIT2)
R-HSA:2162123 synthesis of prostaglandin (PG) and thromboxane (TX) REACTOME_Pathways_27.02.2019 0.03 0.19 (AKR1C3, HPGD, TBXAS1)
GO:0003205 cardiac chamber development GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.04 0.00 (CRELD1, DNAH11, LRP2, MAML1, PKD1, PLXND1, SLIT2, SMO, TAB1)
GO:0048278 vesicle docking GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.05 0.15 (CTBP2, SCFD2, STX11)
GO:0007157 heterophilic cell–cell adhesion via plasma membrane cell adhesion molecules GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.05 0.15 (FAT4, HMCN1, NUFIP2)
GO:0021795 cerebral cortex cell migration GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.05 0.15 (SLIT2, SRGAP2, SUN1)
GO:0002753 cytoplasmic pattern recognition receptor signaling pathway GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.05 0.25 (ALPK1, IRAK2, TAB1)
GO:0007616 long-term memory GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.09 0.04 (CTNS, GRIA1, PJA2, PRNP)
GO:0007613 memory GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.11 0.04 (ADGRF1, CHRNB2, CIC, CTNS, GRIA1, PJA2, PRNP)
R-HSA:9013508 NOTCH3 intracellular domain regulates transcription REACTOME_Pathways_27.02.2019 0.14 0.00 (MAML1, NOTCH3, PLXND1)
GO:0035904 aorta development GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.25 0.00 (LRP2, PLXND1, TAB1)
GO:0060840 artery development GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.25 0.00 (HPGD, LRP2, NOTCH3, PLXND1, TAB1)
GO:0045747 positive regulation of Notch signaling pathway GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.27 0.00 (MAML1, NEPRO, OPN1LW, SPHKAP)
GO:0035082 axoneme assembly GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.33 0.00 (DNAH8, GAS8, RSPH1)
WP:268 Notch signaling WikiPathways_27.02.2019 0.39 0.00 (CTBP2, MAML1, NOTCH3)
GO:1903846 positive regulation of cellular response to transforming growth factor beta stimulus GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.40 0.00 (OPN1LW, RNF111, SPHKAP)
GO:0030511 positive regulation of transforming growth factor beta receptor signaling pathway GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.40 0.00 (OPN1LW, RNF111, SPHKAP)
R-HSA:9012852 signalling by NOTCH3 REACTOME_Pathways_27.02.2019 0.41 0.00 (MAML1, NOTCH3, PLXND1)
GO:0060976 coronary vasculature development GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.41 0.00 (LRP2, PLXND1, TAB1)
GO:0060411 cardiac septum morphogenesis GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.42 0.00 (DNAH11, LRP2, SLIT2, SMO)
AVM5 WP:2572 primary focal segmental glomerulosclerosis (FSGS) WikiPathways_27.02.2019 0.03 0.05 (AGRN, CAMK2B, LAMB2, LMX1B, MKI67)
GO:0006027 glycosaminoglycan catabolic process GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.04 0.08 (AGRN, CD44, GPX4)
GO:0031952 regulation of protein autophosphorylation GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.08 0.04 (CA5A, CAMK2B, ENG)
GO:1904886 beta-catenin destruction complex disassembly GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.08 0.05 (AXIN1, CA5A, DVL1)
GO:0097150 neuronal stem cell population maintenance GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.09 0.05 (BRCA2, FANCC, MBD1)
R-HSA:2142753 arachidonic acid metabolism REACTOME_Pathways_27.02.2019 0.09 0.05 (CYP4B1, CYP4F3, GPX4, PRXL2B)
GO:1904063 negative regulation of cation transmembrane transport GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.10 0.04 (ARHGEF40, CA5A, CAMK2B, EPHB2, KEL)
GO:0007528 neuromuscular junction development GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.15 0.05 (AGRN, DVL1, LAMB2)
GO:0034766 negative regulation of ion transmembrane transport GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.18 0.04 (ARHGEF40, CA5A, CAMK2B, EPHB2, KEL)
GO:0043267 negative regulation of potassium ion transport GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.22 0.04 (CA5A, CPAMD8, KEL)
GO:0044091 membrane biogenesis GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.25 0.04 (CA5A, EPHB2, PTPRH)
GO:2001258 negative regulation of cation channel activity GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.26 0.04 (CA5A, CAMK2B, EPHB2)
GO:0071709 membrane assembly GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.28 0.04 (CA5A, EPHB2, PTPRH)
GO:0016248 channel inhibitor activity GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.28 0.04 (CA5A, CAMK2B, PHPT1)
GO:0008200 ion channel inhibitor activity GO_BiologicalProcess-EBI-UniProt-GOA_27.02.2019_00h00 0.28 0.04 (CA5A, CAMK2B, PHPT1)
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The table reports annotations from ClueGO enrichment analysis. For each sample, the enriched pathways are mentioned as GO Term (3rd column) and the ontology sources (GeneOntology, WikiPathways, Reactome) are reported (4th column), as well as the clustered genes (7th column). Only annotations showing Bonferroni-adjusted p-value ≤ 0.05 for term or group (5th and 6th columns, respectively) are reported. Full results are available in Table S3. ARFGAP: Adenosine diphosphate Ribosylation Factor-GTPase; AVM: Arteriovenous malformation. The number (1–5) indicates the sample; COPI: coating protein 1; GPCR: G protein-coupled receptor; MAP: Mitogen-Activated Protein; MAPK: Mitogen-Activated Protein Kinase; MKK: MAP Kinase Kinase; NCAM: Neural Cell Adhesion Molecule; PDGF: Platelet Derived Growth Factor; PLXND1: Plexin D1; TAK1: TGF-Beta-Activated Kinase 1; TNF: Tumor Necrosis Factor; IRE1: Inositol-Requiring Protein 1; v-SNARE: Vesicle-Soluble NSF (N-Ethylmaleimide-Sensitive Factor) Attachment Protein Receptor.

The table reports annotations from ClueGO enrichment analysis. For each sample, the enriched pathways are mentioned as GO Term (3rd column) and the ontology sources (GeneOntology, WikiPathways, Reactome) are reported (4th column), as well as the clustered genes (7th column). Only annotations showing Bonferroni-adjusted p-value ≤ 0.05 for term or group (5th and 6th columns, respectively) are reported. Full results are available in Table S3. ARFGAP: Adenosine diphosphate Ribosylation Factor-GTPase; AVM: Arteriovenous malformation. The number (1–5) indicates the sample; COPI: coating protein 1; GPCR: G protein-coupled receptor; MAP: Mitogen-Activated Protein; MAPK: Mitogen-Activated Protein Kinase; MKK: MAP Kinase Kinase; NCAM: Neural Cell Adhesion Molecule; PDGF: Platelet Derived Growth Factor; PLXND1: Plexin D1; TAK1: TGF-Beta-Activated Kinase 1; TNF: Tumor Necrosis Factor; IRE1: Inositol-Requiring Protein 1; v-SNARE: Vesicle-Soluble NSF (N-Ethylmaleimide-Sensitive Factor) Attachment Protein Receptor.Gene prioritization aimed to relate selected loci with others, already linked to bAVM development. ToppGene output a full list of the Test Gene Set, ordered according to their overall p-value. For each gene, the overall p-value is calculated on the basis of the single p-value for each training parameter considered. Therefore, also in this case, only genes showing an overall p-value ≤ 0.05 were selected for each sample. Together with this criterion, “Gene Ontology (GO) biological process” annotations were considered as prioritized for the five AVM exomes. These are LTBP4, LTBP1, LRP1, and FBN2 for AVM1; TAB1, RELN, and MAP2K3 for AVM2; KDR and EPHA2 for AVM3; NOTCH3, PLXND1, TAB1, CTBP2, SLIT2, RNF111, MAML1, and CHRNB2 for AVM4; and AXIN1, EPHB2, DVL1, and CAMK2B for AVM5 (Table 2). The complete lists with detailed statistical parameters as well as the annotations linking the prioritized genes to the training genes are available in Table S4.

Table 2.

Genes prioritized by ToppGene tool.

Ontology Feature ID Name Genes
GO: Biological Process Vessel development GO:0001525 Angiogenesis ACVRL1 ENG EPHA2 EPHB2 GDF2 KDR NOTCH3 PLXND1 SLIT2 TGFBR2
GO:0001568 Blood vessel development ACVRL1 ENG EPHA2 EPHB2 GDF2 KDR PKD1 LRP1 LTBP1 TAB1 HPGD LRP2 NOTCH3 PLXND1 SLIT2 SMO TGFBR2
GO:0001569 Branching involved in blood vessel morphogenesis ENG GDF2 PLXND1 TGFBR2
GO:0001570 Vasculogenesis ENG EPHA2 GDF2 SMO KDR TGFBR2
GO:0001944 Vasculature development ACVRL1 ENG EPHA2 GDF2 KDR PKD1 LRP1 LTBP1 HPGD LRP2 NOTCH3 PLXND1 SLIT2 SMO TAB1 TGFBR2
GO:0048514 Blood vessel morphogenesis ACVRL1 ENG EPHA2 EPHB2 GDF2 LRP1 KDR HPGD LRP2 NOTCH3 PLXND1 SLIT2 SMO TGFBR2
GO:1901342 Regulation of vasculature development ACVRL1 ENG EPHA2 GDF2 KDR PLXND1 TGFBR2
TGFBR signaling GO:0007179 Transforming growth factor beta receptor signaling pathway ACVRL1 AXIN1 ENG FBN2 GDF2 LTBP1 LTBP4 HPGD RNF111 SMAD4 TAB1 TGFBR2
GO:0017015 Regulation of transforming growth factor beta receptor signaling pathway AXIN1 ENG FBN2 GDF2 LTBP1 LTBP4 RNF111 SMAD4 TGFBR2
GO:0071559 Response to transforming growth factor beta ACVRL1 AXIN1 ENG FBN2 GDF2 HPGD RNF111 LTBP1 LTBP4 SMAD4 TAB1 TGFBR2
GO:0071560 Cellular response to transforming growth factor beta stimulus ACVRL1 AXIN1 ENG FBN2 GDF2 LTBP1 LTBP4 HPGD RNF111 SMAD4 TAB1 TGFBR2
GO:1903844 Regulation of cellular response to transforming growth factor beta stimulus AXIN1 ENG FBN2 GDF2 LTBP1 LTBP4 SMAD4 TGFBR2
Heart development GO:0003007 Heart morphogenesis ACVRL1 DNAH11 ENG SMAD4 FAT4 LRP2 PLXND1 SLIT2 SMAD4 SMO TAB1 TGFBR2
GO:0003205 Cardiac chamber development DNAH11 ENG LRP2 MAML1 PLXND1 SLIT2 LRP1 LTBP1 SMAD4 SMO TAB1 TGFBR2
GO:0003206 Cardiac chamber morphogenesis DNAH11 ENG LRP2 SLIT2 SMAD4 SMO TGFBR2
GO:0003208 Cardiac ventricle morphogenesis ENG LRP2 SMAD4 TGFBR2
GO:0003231 Cardiac ventricle development ENG LTBP1 LRP2 SLIT2 SMAD4 TGFBR2
GO:0003279 Cardiac septum development DNAH11 ENG LRP1 LTBP1 LRP2 MAML1 PLXND1 SLIT2 SMAD4 SMO TAB1 TGFBR2
GO:0007507 Heart development ACVRL1 DNAH5 DNAH11 DVL1 ENG KDR PKD1 LRP1 LTBP1 FAT4 LRP2 MAML1 PLXND1 SLIT2 SMAD4 SMO TAB1 TGFBR2
GO:0060411 Cardiac septum morphogenesis DNAH11 ENG LRP2 SLIT2 SMAD4 SMO TGFBR2
GO:0072358 Cardiovascular system development ACVRL1 ENG EPHA2 EPHB2 GDF2 KDR PKD1 LRP1 LTBP1 HPGD LRP2 NOTCH3 PLXND1 SLIT2 SMO TAB1 TGFBR2
GO:0072359 Circulatory system development ACVRL1 DNAH5 DNAH11 DVL1 ENG EPHA2 EPHB2 FAT4 GDF2 LRP1 LTBP1 KDR PKD1 HPGD LRP2 MAML1 NOTCH3 PLXND1 SLIT2 SMAD4 SMO TAB1 TGFBR2
GO:2000826 Regulation of heart morphogenesis ENG SMAD4 SMO TGFBR2
BMP signaling GO:0030509 BMP signaling pathway ACVRL1 ENG GDF2 KDR LRP2 SMAD4
GO:0030510 Regulation of BMP signaling pathway ACVRL1 ENG GDF2 KDR LRP2 SMAD4
GO:0030513 Positive regulation of BMP signaling pathway ACVRL1 ENG GDF2 KDR SMAD4
GO:0071772 Response to BMP ACVRL1 ENG GDF2 KDR LRP2 SMAD4
GO:0071773 Cellular response to BMP stimulus ACVRL1 ENG GDF2 KDR LRP2 SMAD4
Endothelial/mesenchymal differentiation GO:0001935 Endothelial cell proliferation ACVRL1 ENG EPHA2 GDF2 KDR
GO:0001936 Regulation of endothelial cell proliferation ACVRL1 ENG GDF2 KDR
GO:0003158 Endothelium development ACVRL1 ENG GDF2 KDR SMAD4
GO:0045446 Endothelial cell differentiation ACVRL1 ENG GDF2 KDR SMAD4
GO:0048762 Mesenchymal cell differentiation ACVRL1 ENG SMAD4 SMO TGFBR2
GO:0060485 Mesenchyme development ACVRL1 ENG SMAD4 SMO TGFBR2
Hypoxia response GO:0001666 Response to hypoxia ACVRL1 CHRNB2 ENG KDR SMAD4 TGFBR2
GO:0036293 Response to decreased oxygen levels ACVRL1 CHRNB2 ENG KDR SMAD4 TGFBR2
GO:0070482 Response to oxygen levels ACVRL1 CHRNB2 ENG KDR SMAD4 TGFBR2
Human Phenotype Cerebrovascular malformations HP:0100026 Arteriovenous malformation ACVRL1 BRCA2 DVL1 ENG FANCC GDF2 SMAD4
HP:0001009 Telangiectasia ACVRL1 BRCA2 ENG FANCC GDF2 SMAD4
HP:0001048 Cavernous hemangioma ACVRL1 AXIN1 ENG GDF2 SMAD4
Benign/malignant neoplasm HP:0005306 Capillary hemangioma ACVRL1 DVL1 ENG GDF2 KDR SMAD4
HP:0001028 Hemangioma ACVRL1 AXIN1 DVL1 ENG GDF2 KDR PRKN SMAD4
HP:0100742 Vascular neoplasm ACVRL1 AXIN1 DVL1 ENG GDF2 KDR PRKN SMAD4
Vessel dilatation HP:0002624 Abnormal venous morphology ACVRL1 AXIN1 BRCA2 ENG EPHB2 GDF2 NOTCH3 SMAD4
HP:0002619 Varicose veins ACVRL1 AXIN1 ENG GDF2 NOTCH3 SMAD4
HP:0004414 Abnormality of the pulmonary artery ACVRL1 ENG FBN2 GDF2 LTBP4 SMAD4 TGFBR2
HP:0004930 Abnormality of the pulmonary vasculature ACVRL1 AXIN1 ENG FBN2 GDF2 LTBP4 SMAD4 TGFBR2
HP:0100659 Abnormality of the cerebral vasculature ACVRL1 BRCA2 ENG GDF2 NOTCH3 PKD1 PRNP SMAD4 SMO TGFBR2 WFS1
HP:0009145 Abnormal cerebral artery morphology ACVRL1 BRCA2 ENG FANCC GDF2 NOTCH3 PKD1 SMAD4 TGFBR2
HP:0011004 Abnormal systemic arterial morphology ACVRL1 DNAH11 ENG FBN2 GDF2 NOTCH3 PKD1 SMAD4 TGFBR2
Mouse Phenotype Cerebrovascular malformations MP:0006093 Arteriovenous malformation ACVRL1 ENG GDF2 NOTCH3
Vessel dilatation MP:0000259 Abnormal vascular development ACVRL1 CD44 CTBP2 ENG EPHA2 KDR HPGD MAML1 NOTCH3 PLXND1 LTBP1 SMAD4 SMO SPP1 TGFBR2
MP:0001614 Abnormal blood vessel morphology ACVRL1 CD44 CTBP2 DNAH11 DNAH5 ENG EPHA2 FBN2 GDF2 KDR PKD1 LMX1B LRP1 LTBP1 LTBP4 HPGD LRP2 MAML1 NOTCH3 PLXND1 SLIT2 NR1H4 SMAD4 SPP1 TAB1 TGFBR2
MP:0003410 Abnormal artery development ACVRL1 ENG NOTCH3 PLXND1 SMO KDR LTBP1 TGFBR2
MP:0004787 Abnormal dorsal aorta morphology ACVRL1 ENG KDR TGFBR2
MP:0000267 Abnormal heart development ACVRL1 AXIN1 CTBP2 DNAH5 ENG KDR MAML1 PKD1 SMAD4 SMO TGFBR2
Defects in embryo vasculogenesis MP:0001719 Absent vitelline blood vessels ACVRL1 CTBP2 ENG KDR SMO TGFBR2
MP:0003229 Abnormal vitelline vasculature morphology ACVRL1 CTBP2 ENG KDR SMO SMAD4 TGFBR2
Disease Cerebrovascular malformations C0003857 Congenital arteriovenous malformation ACVRL1 ENG NOTCH3 SMAD4
Vessel disorders C0334533 Arteriovenous hemangioma ACVRL1 ENG NOTCH3 SMAD4
C0007820 Cerebrovascular Disorders ACVRL1 ENG GDF2 NOTCH3 PKD1 RELN SMAD4 SPP1 TGFBR2
C0042373 Vascular Diseases ACVRL1 ENG KDR NOTCH3 PRKN SMAD4 SPP1 TGFBR2
C0002940 Aneurysm ACVRL1 ENG LRP1 LTBP4 PKD1 TGFBR2
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The table reports results obtained by ToppGene prioritization analysis giving as Training Gene Set ACVRL1, ENG, GDF2, SMAD4, and TGFBR2. According to the specific ontology, prioritized genes are shown in bold. Results are here summarized and grouped in relation to annotations. Data obtained for the single patient are available in Table S4. BMP: Bone Morphogenetic Protein; TGFBR: Transforming Growth Factor Beta Receptor.

Although prioritization analysis was quite exhaustive, further loci were considered. These added loci were selected as they are involved in vasculogenesis and carry missense and nonsense variants. In particular, they were FLT4, NCoR2, CCN1, and GIMAP1 in the AVM2 sample; NOTCH4 in AVM3; and ENG and TGFBR2 in AVM5. In particular, ENG and TGFRB2 are known to be highly linked to brain AVM and HHT development. NCoR2 was affected by the novel nonsense variant c.2078G>T (p.Glu693Ter) (Ensembl Transcript ID: ENST00000405201.5). As a consequence, the mutated protein counts 692 amino acids rather than the 2514 amind acids of the wild-type one. The mutation was detected in a heterozygous condition and resulted as “Damaging” and “Disease causing” in SIFT (https://sift.bii.a-star.edu.sg/) [9] and MutationTaster (http://www.mutationtaster.org/) [10] prediction tools, respectively (not shown).

2.3. Sanger Validation

Table 3 lists the variants detected in the genes previously prioritized. All these variants were confirmed by Sanger sequencing and were not detected in our internal 10 control exomes obtained from healthy subjects. Figure 1 reports only the electropherogram of the novel nonsense variant, c.2078G>T (p.Glu693Ter) affecting the NCoR2 locus.

Table 3.

Variants affecting prioritized genes.

Locus Gene HGNC ID SNP ID Ensembl Transcript ID Coding Sequence Change Ensembl Protein ID Protein Sequence Change
1p22.3 CCN1 * 2654 rs765069158 ENST00000451137 c.G463C ENSP00000398736 p.Gly155Arg
1p36.12 EPHB2 3393 rs142113032 ENST00000400191 c.847G>C ENSP00000383053 p.Asp283His
1p36.13 EPHA2 3386 rs35484156 ENST00000358432 c.830C>T ENSP00000351209 p.Ser277Leu
1p36.33 DVL1 3084 rs61735963 ENST00000378891 c.469G>A ENSP00000368169 p.Ala157Thr
1q21.3 CHRNB2 1962 rs55685423 ENST00000368476 c.1191G>C ENSP00000357461 p.Gln397His
2p22.3 LTBP1 6714 rs80163321 rs149319598 ENST00000404816 c. 2248 G>A
c. 2410C>T		 ENSP00000386043 p.Val750Ile
p.Pro804Ser
3p24.1 TGFBR2 * 11773 rs35766612 ENST00000295754 c.1159G>T ENSP00000295754 p.Val387Leu
3q22.1 PLXND1 9107 rs137955512 ENST00000324093 c.2275C>T ENSP00000317128 p.Pro759Ser
4p15.31 SLIT2 11086 rs115629108 ENST00000504154 c.4049G>A ENSP00000422591 p.Ser1350Asn
4q12 KDR 6307 rs755067067 ENST00000263923 c.1990C>T ENSP00000263923 p.Arg664Cys
5q23.3 FBN2 3604 rs28763954 ENST00000262464 c.976C>T ENSP00000262464 p.Pro326Ser
5q35.3 MAML1 13632 rs113636707 ENST00000292599 c.569G>A ENSP00000292599 p.Arg190His
5q35.3 FLT4 * 3767 rs200763913 ENST00000261937 c.1133G>A ENSP00000261937 p.Arg378His
6p21.32 NOTCH4 * 7884 rs8192573 ENST00000375023 c.4037G>A ENSP00000364163 p.Arg1346Gln
7p13 CAMK2B 1461 rs528355050 ENST00000395749 c.1577C>T ENSP00000379098 p.Pro526Leu
7q22.1 RELN 9957 rs114684479 rs79499902 ENST00000428762 c.3477C>A
c.5284G>A		 ENSP00000392423 p.Asn1159Lys p.Val1762Ile
7q36.1 GIMAP1 * 23237 rs1326399257 ENST00000307194 c.699G>A ENSP00000302833 p.W233X
9q34.11 ENG * 3349 rs139398993 ENST00000373203 c.392C>T ENSP00000362299 p.Pro131Leu
10q26.13 CTBP2 2595 rs1058301 ENST00000334808 c.387C>G ENSP00000357816 p.Asp129Glu
12q13.3 LRP1 6692 rs113379328 ENST00000243077 c.7636G>A ENSP00000243077 p.Gly2546Ser
12q24.31 NCoR2 * 7673 Novel ENST00000405201 c..2078G>T ENSP00000384018 p.Glu693Ter
15q22.1-q22.2 RNF111 17384 rs142916216 ENST00000557998 c.888T>G ENSP00000452732 p.Ile296Met
16p13.3 AXIN1 903 rs200741961 ENST00000262320 c.644C>T ENSP00000262320 p.Ser215Leu
17p11.2 MAP2K3 6843 rs33911218
rs36047035		 ENST00000342679 c.118C>G
c.164G>C		 ENSP00000345083 p.Pro40Ala p.Arg55Thr
19p13.12 NOTCH3 7883 rs141320511 ENST00000263388 c.4552C>A ENSP00000263388 p.Leu1518Met
19q13.2 LTBP4 6717 rs35809725 ENST00000308370 c.4499A>T ENSP00000311905 p.Tyr1500Phe
22q13.1 TAB1 18157 rs536084162 rs140879164 ENST00000216160 c.19A>C
c.560G>A		 ENSP00000216160 p.Ser7Arg p.Arg187His
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For each variant, chromosomal band, gene name, HUGO Gene Nomenclature Committee gene name, variant ID, transcript and protein reference IDs of the Ensembl Genome Browser, coding sequence, and amino acid position changes are reported. * Loci not output by the ToppGene tool.

Figure 1.

Figure 1

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The c.2078G>T (p.Glu693Ter) at the NCoR2 locus. Electropherogram shows the novel nonsense mutation affecting the NCor2 gene, detected in heterozygous condition in AVM2 sample. Nucleotide substitution is indicated by the arrow. The brace indicates the DNA sequence corresponding to the triplet carrying the mutated nucleotide (the first one).

3. Discussion

The genetic landscape of bAVM is to date quite elusive probably due to the very low frequency of inherited cases. Most patients are affected by sporadic forms whose molecular causes are waiting to be clarified. In many cases, lesions are congenital, increasing in size over the years until they become symptomatic. Due to the dynamic nature of bAVM and its high recidivism rate, it is well accepted that lesions arise as a consequence of continuous endogenous stimuli, as inherited and de novo genetic variants or epigenetic modifications occurring during embryo development [11,12]. Therefore, we performed WES analysis on a group of five patients affected by sporadic bAVM, highlighting the main pathways enriched by germline mutated genes. Despite mutated genes differing among patients, the pathways in which they converge are the same. Firstly, we checked for variants in ENG, NOTCH4, and TGFβR2 that are known to be involved in arteriovenous malformation development [13,14]. Then, we searched for KRAS c.35G>T (p.Gly12Val) that was recurrently detected in bAVM patients [15] and none of them carried the mutation, as well as other variants within the gene. However, this was expected as our analysis was performed on DNA purified by peripheral blood. Mosaic-activating KRAS mutations, indeed, were found in sporadic AVM-derived specimens [16,17].

The ClueGO enrichment analysis revealed pathways related to microtubule formation, cell adhesion, and vascular remodeling as being highly enriched (Table 1). Moreover, prioritization analysis was performed for each sample to detect the main genes involved in TGFβR transduction pathways and, therefore, more likely associated to bAVM onset. As reported in Table S4a,b, several loci are noteworthy of consideration and the most relevant are listed in Table 2. However, among prioritized genes, here we briefly discuss those more likely related to bAVM development, in relation to the single sample. This selection was made considering significant p-values related to phenotypes and pathway, outputted by the ToppGene tool (Table S4a).

3.1. AVM1

Regarding AVM1, we focused on LTBP1, LTBP4, FBN2, and LRP1 loci. FBN2, encoding for Fibrillin 2, and LTBP1 and LTBP4 belonging to the “latent transforming growth factor beta binding proteins” family, are ligands of TGF-β receptors [18,19]. With regard to LRP1, encoding for the LDL receptor related protein 1, expression data showed it is expressed in brain endothelial cells where it contributes to chemotactic cell migration, inducing sphingosine-1-phosphate proangiogenic signaling [20]. Moreover, depletion of LRP1 determines defects of both large and small vessel morphogenesis leading to a lethal phenotype [21].

3.2. AVM2

Regarding data obtained from AVM2 exome, we considered variants affecting TAB1, FLT4, RELN, MAP2K3, CCN1, GIMAP1, and NCoR2 loci. TAB1 encodes for the TGF-β activated kinase 1 binding protein 1 and increases endothelial permeability, mediated by the non-canonical TGF-β pathway following inflammation stimuli [22]. Moreover, TAB1 activates the TAK1 kinase, an upstream modulator of the p38 MAPK signaling. MAP2K3 is also involved [23] in the same pathway and, in particular, a physical interaction between TAK1 and MAP2K3 has been reported [24]. Involvement of inflammatory response in bAVM is, to date, well accepted [25,26] and in this context, we detected a nonsense mutation, the c.699G>A (p.Trp233Ter) in GIMAP1 gene. Together with the TGFβR2 signaling, Notch transduction pathways were also reported as promoting AVM development [27]. Therefore, we also considered prioritizing the NCor2 locus, encoding for nuclear receptor co-repressor. We found the novel nonsense mutation, c.2078G>T (p.Glu693Ter) (Figure 1). FLT4, instead, encodes for the vascular endothelial growth factor receptor 3 (VEGFR3) [28]. Finally, we considered CCN1 locus, encoding for the cellular communication network factor 1. Its expression is increased in the extracellular matrix surrounding microvessels and is growth factor-inducible. The protein promotes integrin-mediated endothelial cell adhesion in response to mechanotransduction signaling [29]. This evidence is well congruent with bAVM pathogenesis due to frequent insult by the high blood pressure within the lesions.

3.3. AVM3

Together with NOTCH4, variants carried by EPHA2, EPHB4, and KDR were detected in sample AVM3. EPHA2 and EPHB2 encode for two proteins belonging to the ephrin family, a subgroup of protein-tyrosine kinase receptors. Ephrins are vessel differentiation markers and their role is pivotal during early vasculogenesis. In particular, mesenchymal cells express EPHB2, a feature of arterial morphogenesis. A model proposed by Adams et al. hypothesizes interaction among type-B ephrins differentially expressed in arteries and veins as the basis of a remodeling process that leads to sprouting and capillary network development [30]. EPHB2 expression is upregulated in capillaries during inflammation. This results in increased endothelial permeability and loss of vessel differentiation [31]. The absence of EphA2, instead, was shown to impair the blood–brain barrier, resulting in inhibition of endothelial cell migration and in enhancement of tight junction formation in human brain microvascular endothelial cells (HBMECs) [32]. KDR encodes for the vascular VEGFR2, essential for the organization of the embryonic vasculature and angiogenic sprouting [33].

3.4. AVM4

The highest number of prioritized genes was in the AVM4 sample. GO annotations for biological process revealed involvement in vasculature morphogenesis for NOTCH3, PLXND1, SLIT2, and MAML1 loci. At the embryo stage, VEGF and Notch transduction signaling modulates PLXND1 expression to guide organ vasculogenesis by promoting endothelial cell migration and proliferation [34]. In adults, instead, PLXND1 expression is physiologically low and limited to a few cell types, such as endothelial cells [35]. Balancing effects on migration were reported for SLIT2 [36]. MAML1 is described as a NOTCH coactivator, even if its role in angiogenesis needs more elucidation [37]. Regarding the TGF-β/BMP pathway, we focused on the RNF111 gene, encoding for the E3 ubiquitin-protein ligase. One of its targets is the SMAD7 protein that acts by inhibiting TGF-β/BMP signaling. Therefore, RNF111 activity is required to promote SMAD7 degradation and to enhance the TGF-β/BMP pathway [38]. TGF-β signaling is also upregulated by increased levels of CTBP2 (C-terminal binding protein 2), driving endothelial-to-mesenchymal transition (EMT) [39]. This gene was also mutated in the AVM4 patient. In the end, we focused our attention on the GO terms regarding the response to hypoxia (GO:0001666, GO:0036293, GO:0070482) with the patient as a carrier of rs55685423 (c.1191G>C, p.Gln397His) in the CHRNB2 locus. This gene encodes for the cholinergic receptor nicotinic beta 2 subunit [40]. Its expression was demonstrated in HBMECs, where it contributes to capillary network formation and to angiogenic response to inflammation [41]. Notably, the same ontologies were also found in the AVM3 patient, annotated by the KDR locus.

3.5. AVM5

Finally, in the AVM5 sample we identified two variants in ENG and TGFBR2 loci, rs139398993 (c.392C>T, p.Pro131Leu) and rs35766612 (c.1159G>T, p.Val387Leu), respectively. Moreover, EPHB2 was also affected by a missense variant. Based on human and mouse phenotype ontologies, DVL1 and AXIN1 were annotated to the “cerebrovascular disease” term and, in particular, with AVM and telangiectasia phenotypes. DVL1 is known to control postnatal angiogenesis [42]. AXIN1 encodes for a negative regulator of the Wnt pathway, also enhancing TGF-β signaling by promoting the degradation of the inhibitory SMAD7, in a RNF111-dependent manner [43]. Surprisingly, it was recently described as an important regulator of embryo central nervous system (CNS) angiogenesis, and overexpression leads to premature vascular regression, followed by progressive dilation and inhibition of vascular maturation [44].

3.6. Novel Insights

Together with TGF-β/Notch signaling, GO annotations derived from the ClueGO enrichment analysis (Table 1) highlight a relevant presence of ontologies related to microtubule and cilia organization (GO:0003341, GO:0001578, GO:0031122, GO:0001539, GO:0060285). A recent study demonstrated that cilia are widely represented in endothelial cells during early vasculogenesis and in the later stages as vessel bifurcation point anastomosis. Zebrafish knock-down for cilia biogenesis gene models showed cilia disassembly following shear stress, resulting in remodeling of endothelial cell architecture and increased permeability and hemorrhagic events. Moreover, hemorrhages were only observed in head vasculature and were not observed in the trunk or caudal vessels [45]. Based on this evidence, germline defects in genes controlling cilia assembly might also contribute to brain AVM development as the result of mechanical stress induced by high blood flow and pressure. Clearly, this hypothesis needs to be adequately validated.

Another important property of the blood–brain barrier is the highly selective control of solute transport which is maintained by the exact spatial distribution of membrane transporters and ion channels. Polarization is a key factor for morpho-functional homeostasis of endothelial cells and was shown to be driven by VEGF via Ca2+ specific signaling pathways [46]. Moreover, dysregulation of K+ ion influx in non-excitable cells was shown to lead to hyperpolarization of membrane potential with consequent increased intracellular Ca2+. This results in enhancement of cell proliferation and was also demonstrated in brain capillary endothelial cells [47]. However, if physiological Ca2+ concentration is abnormally excessive, endothelial cells undergo apoptosis [48]. Therefore, we focused attention on GO terms from the ClueGO analysis of sample AVM5. As shown in Table 1, GO terms related to transmembrane ion transport (GO:1904063, GO:0034766, GO:2001258, GO:0043267, GO:0016248, GO:0008200) are largely enriched, as well as those related to membrane biogenesis (GO:0044091, GO:0071709, GO:1904886) making the hypothesis of misregulation in bAVM onset conceivable.

Although these are preliminary findings, they are in accordance with what was recently published by Hauer and colleagues. They describe dysregulated expression of genes also involving cytoskeleton network and transmembrane transport in bAVM-derived specimens, when compared to intracranial control arteries [49]. Therefore, these results allow to elicit other mechanisms in pathogenesis of bAVM not only confined to the canonical TGFβR2 pathway.

3.7. Final Considerations

We discussed loci affected by germline variants in five bAVM samples. Despite these loci differing among the samples, they converged in regulation of the same cellular signaling pathways. This interconnection is represented in Figure 2. The image was obtained by STRING tool Version 11.0 (https://string-db.org/) [50]. Details on nodes and edges are supplied in Table S5.

Figure 2.

Figure 2

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Functional network of prioritized genes. The image describes observed and inferred functional interactions linking prioritized genes. Nodes represent input proteins while edges represent protein–protein associations. Each edge color indicates a specific annotation. Green: activation; red: inhibition; blue: binding; light blue: phenotype; black: reaction; violet: catalysis; pink: posttranslational modification; yellow: transcriptional regulation.

According to prioritized genes, our data support findings previously reported [51,52] and, in particular, the genetic heterogeneity of the disease. These results suggest that sporadic bAVM is probably not a monogenic condition, rather it arises during early vasculogenesis at the embryo stage. In particular, following fertilization, the combination of both inherited and eventually de novo genetic variants in numerous loci controlling vessel development could result in early vasculogenesis impairment and lesion onset. However, the evidence that these patients develop lesions only in the CNS underlines the importance of the cross-talk between glial cells and endothelium during neurodevelopment and blood–brain barrier morphogenesis. Most genes considered here show an early peculiar expression in neural progenitor cells that contributes to correct vasculogenesis and angiogenetic processes. In this context, proteins related to axon guidance such as Slits, plexins, and ephrins are exhaustive examples [53,54]. However, a last consideration regards genes involved in DNA repair such as WRN, FANCC, BRCA2, TP53BP1, and others carrying rare missense variants. These variants might cause protein functional alteration and, subsequently, DNA repair impairment. At the embryo stage, this might trigger DNA errors resulting in somatic mutations. Clearly, as our study focused on germline variants, the role of somatic mutations is not evaluated here.

Germline genetic variants are endogenous and permanent factors affecting both early vasculogenesis and late angiogenesis. Endothelial remodeling is a continuous phenomenon, and this can explain the increased recidivism rate of bAVM. Therefore, our hypothesis regards the possibility of considering bAVM as the result of the co-existence of numerous low-penetrance loci controlling different processes during endothelial cell differentiation and maturation. This idea is supported by two observations. We selected only loci affected by rare variants (MAF < 0.01) and then, those most likely related to rare disease onset. Rare variants at the same loci were searched for in our internal 10 control exomes obtained from healthy subjects and none was detected.

Clearly, the main limitation of the study is related to the few samples considered and, certainly, results require further validations on a larger patient cohort.

Despite this being a preliminary investigation, the possibility of detecting novel loci and germline mutations potentially involved in bAVM onset will allow to hypothesize a strategy for molecular diagnosis, preferably based on a panel of selected genes.

4. Materials and Methods

4.1. Patient Recruitment and WES Analysis

The study was performed on a group of five Italian patients (AVM1–5) diagnosed with bAVM following cerebral angiography investigation (Figure 3). A severity lesion score was assigned to each patient based on the Spetzler–Martin grading system [55]. Anamnestic data are presented in Table 4. No familiar history of bAVM was reported for the patients and they were classified as sporadic. Patients were fully informed on their enrolment in the study and informed consent was obtained, and for underaged patients as well. DNA samples were collected from peripheral blood and purified by the QIAamp DNA Blood Mini Kit (Qiagen). Qualitative and quantitative measurements of the samples were performed by NanoDrop spectrophotometer (Thermo Fisher Scientific) and by a Qubit fluorometer (Thermo Fisher Scientific). Paired-end libraries were obtained by the SureSelect Human All Exon V7 (Agilent) kit and sequenced on a HiSeq 2500 Illumina platform.

Figure 3.

Figure 3

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Imaging for neuroradiological diagnosis. The figure reports cerebral angiographies that confirm the presence of bAVM lesions. The five images refer to the five patients as reported in the text: (A) AVM1, (B) AVM2, (C) AVM3, (D) AVM4, (E) AVM5. AVM lesions are framed in the rectangular box.

Table 4.

Anamnesis data of the patients enrolled in the study.

Patient Sex Age (Years) Age of Onset (Years) Symptoms Lesion Number Spetzler–Martin Grading
AVM1 M 14 12 Intracerebral hemorrhage following AVM rupture 1—parietal left area 2
AVM2 M 31 18 Tremor of the left leg, diffuse tremor, seizures 1—front-parietal left area (not bleeding) 2
AVM3 F 32 29 Dizziness, tinnitus, seizures nausea right hemiparesis visus reduction 1— parietal left area 3
AVM4 F 8 At birth Drowsy status, finalistic limb movement 1—proliferative microangiopathy, central left area 3
AVM5 M 7 5 Sudden headache, vertigo, seizures 1—anterior-parietal paramedian right area 2
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The patients are named as mentioned in the text (AVM1–5). Gender, age, and clinical features are reported for each patient. AVM: Arteriovenous malformation. The number (1–5) indicates the sample.

4.2. Bioinformatic Analysis

FastQ data obtained from sequencing runs were quality checked by the FastQC (v.0.11.7) tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Only reads presenting a Phred score ≥ 28 were selected after trimming and aligned to the GRCh38 Human Reference Genome by the Burrows–Wheeler Aligner (BWA) algorithm [56]. Duplicate reads were removed by the MarkDuplicate tool provided by Picard toolkit (v.2.18.23) (“Picard Toolkit.” 2019. Broad Institute, GitHub Repository. http://broadinstitute.github.io/picard/; Broad Institute). Then, the Indel realignment and the base recalibration were performed by the Genome Analysis Toolkit (GATK) (v.4.1.3.0) (https://software.broadinstitute.org/gatk/). Variant calling was executed by FreeBayes [57], while ANNOVAR v.2018Apr16 [58] was used for variant annotation.

4.3. Variant Filtering Criteria

Before proceeding with downstream analysis, annotated genes and variants were filtered on the basis of several criteria. Variants showing quality score < 150 were discarded. This threshold value was established by the observation that several variants with depth lower than 150 were not confirmed by the following Sanger sequencing validation. Filtered variants were classified by functional class and intronic, synonymous, non-coding RNA, and untranslated regions affecting variants were discarded. Missense, nonsense, frameshift, and short indels presenting an MAF < 0.01 were selected. Rare variants were preferred based on the low worldwide incidence of bAVM. For the MAF-based filtering, the values reported in the Genome Aggregation Database (https://gnomad.broadinstitute.org/) [59] and in the 1000 Genomes phase 3 project [60] were considered.

4.4. Gene Clustering and Prioritization

To visualize and functionally group genes carrying filtered variants, the ClueGO plug-in of Cytoscape software was used for each sample [61]. Clustering was performed on the basis of the GO Biological Process, REACTOME and WikiPathways ontologies. Groups showing Bonferroni step down corrected p-value ≤ 0.05 were considered significant and therefore selected for the purpose. Genes within the chosen groups were added to the Test Gene Set in ToppGene (https://toppgene.cchmc.org/) [62], a web-based tool for prioritization of candidate genes based on functional similarity to a training gene list. The Training Gene Set group was made up of ENG, ACVRL1, TGFBR2, SMAD4, and GDF2 genes, already known to be causative of HHT and of a few familiar bAVM cases without HHT. The training parameters selected were “GO:Biological Process”, “Human Phenotype”, “Mouse Phenotype”, “Pathway”, “PubMed”, “Interaction”, and “Disease”. Five different analyses were run, one for each exome data. Statistical parameters were calculated applying the Bonferroni correction, and p-values ≤ 0.05 were considered significant.

4.5. Sanger Validation

Variants carried by prioritized genes were validated by Sanger sequencing, next to polymerase chain reaction (PCR) amplification. Primer sequences and PCR conditions are available upon request. Sanger sequencing was carried out using the BigDyeTerminator© v3.1 Cycle Sequencing Kit chemistry and run on a 3130xl Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific). Moreover, all variants here considered were further searched in an in-house exome-control dataset obtained by WES data, collected on a cohort of 10 Caucasian healthy subjects, heterogeneous for sex and age. The healthy condition was confirmed by computed tomography.

Patients agreed to be enrolled in the study and their informed consents were obtained. The manuscript does not contain information attributable to their identity. The study involves human participants and was approved by the local Ethics Committee “A.O.U. G. Martino”, N.11/2011 date of approval: 14 December.2011.

5. Conclusions

As knowledge on bAVM is still very elusive, we recruited a group of patients affected by sporadic bAVM and performed WES analysis. Clustering of genes which were affected by rare variants highlighted cytoskeleton impairment as well as defective ion conduction in endothelial cells. Therefore, we hypothesize perturbations at these pathways as possible mechanisms involved in bAVM pathogenesis. We prioritized genes more likely linked to lesion development as FBN2, TAB1, NCoR2, SLIT2, RNF111, CAMK2B, EPHA2, and EPHB2. Although to date no correlation has been reported between gene mutations and clinical phenotype, further characterization of pathways involved in bAVM development could provide a valid criterion to relate molecular features to clinical presentation. In particular, lesion site, bleeding risk, and patient outcome could represent valid prognostic factors linked to patient genotype.

Supplementary Materials

Supplementary materials are available at https://www.mdpi.com/1422-0067/21/12/4321/s1. Table S1: AVM Sample Reports of WES Analysis, Table S2: Selected Variants, Table S3: ClueGO Pathways, Table S4: ToppGene Results. Table S5.: STRING Results.

Click here for additional data file. (1.8MB, zip)

Author Contributions

Conceptualization, C.S.; methodology, C.S., L.D. and C.T.; investigation: M.L. and F.G.; resources: E.M. and A.A.C.; data curation: L.D.; writing—original draft preparation, C.S.; writing—review and editing, A.S.; supervision, R.D. All authors have read and agreed the final version of the manuscript.

Funding

This research received no external funding.

Conflicts of Interest

The authors declare no conflict of interest.

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