Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia

BACKGROUND: Most patients with familial primary pulmonary hypertension have defects in the gene for bone morphogenetic protein receptor II (BMPR2), a member of the transforming growth factor beta (TGF-beta) superfamily of receptors. Because patients with hereditary hemorrhagic telangiectasia may have lung disease that is indistinguishable from primary pulmonary hypertension, we investigated the genetic basis of lung disease in these patients. METHODS: We evaluated members of five kindreds plus one individual patient with hereditary hemorrhagic telangiectasia and identified 10 cases of pulmonary hypertension. In the two largest families, we used microsatellite markers to test for linkage to genes encoding TGF-beta-receptor proteins, including endoglin and activin-receptor-like kinase 1 (ALK1), and BMPR2. In subjects with hereditary hemorrhagic telangiectasia and pulmonary hypertension, we also scanned ALK1 and BMPR2 for mutations. RESULTS: We identified suggestive linkage of pulmonary hypertension with hereditary hemorrhagic telangiectasia on chromosome 12q13, a region that includes ALK1. We identified amino acid changes in activin-receptor-like kinase 1 that were inherited in subjects who had a disorder with clinical and histologic features indistinguishable from those of primary pulmonary hypertension. Immunohistochemical analysis in four subjects and one control showed pulmonary vascular endothelial expression of activin-receptor-like kinase 1 in normal and diseased pulmonary arteries. CONCLUSIONS: Pulmonary hypertension in association with hereditary hemorrhagic telangiectasia can involve mutations in ALK1. These mutations are associated with diverse effects, including the vascular dilatation characteristic of hereditary hemorrhagic telangiectasia and the occlusion of small pulmonary arteries that is typical of primary pulmonary hypertension

N-linked glycosylation at position ASN98 of the ALK1 receptor protein: relevance for ALK1 function and HHT pathogenesis

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant genetic disorder that results from a mutation of one of two key signaling receptors for the transforming growth factor beta (TGFβ) superfamily: endoglin and activin receptor-like kinase 1 (ALK1). These mutations result in development of HHT Type 1 and HHT Type 2, respectively. Patients suffering from HHT experience spontaneous blood vessel growth that can lead to telangiectasia, arteriovenous malformation (AVM) development, and other related health problems. ALK1 is a serine/threonine kinase receptor found on the cell membrane of endothelial cells. ALK1 and its co-receptor endoglin, are activated by binding to the circulating BMP9 ligand. The ALK1-endoglin-BMP9 complex will then regulate endothelial proliferation by activating the SMAD pathway by phosphorylation. Mutations in the ACVRL1 gene can form a modified ALK1 protein that has a high potential to inhibit this function, causing the hyperproliferation of endothelial cells and the development of AVMs, and ultimately HHT Type 2. It is believed, however unproven, that ALK1 is heavily glycosylated in the extracellular domain. My thesis research was aimed at studying the glycosylation of ALK1 and at exploring the relevance of this glycosylation to the development of HHT. The glycosylation of ALK1 was investigated by using: (i) a computational prediction approach (NetNGlyc 1.0 bioinformatics server), (ii) a glycosylation inhibiting drug (tunicamycin), (iii) an in vitro enzymatic approach of glycosylation breakdown, and (iv) site-directed mutagenesis to identify the ASP residue glycosylated on ALK1. The bioinformatics software NetNGlyc predicted a N-linked glycosylation site on an asparagine (ASN) residue located at position 98 in the extracellular domain of ALK1. I further found that, based on western blot analysis, ALK1 proteins shifted to a lighter molecular weight (5-8 kDa) when treated with tunicamycin, as well as endo H and PNGase F enzymes, which represent two glycosidases able to remove N-linked oligosaccharides on proteins. Western blot analysis also revealed an identical shift in protein size (5-8 kDa) when comparing wild type ALK1 to an asparagine98-to-alanine (N98A) mutant ALK1 construct. The 5-8 kDa shift observed in the drug and enzymatic experiments indicate the removal of a bulky oligosaccharide from the wild type ALK1 protein. This 5-8 kDa shift observed in the mutagenesis experiment indicated that the same oligosaccharide addition could not occur on ALK1 when ASP98 was missing. Thus proving that the asparagine at the 98th position of ALK1 is involved in N-linked glycosylation. These important findings on ALK1 modification offer a greater understanding of the mechanisms behind ALK1 regulation and function, especially its role in controlling angiogenesis. Furthermore, this data provides grounds for further research into the importance of ALK1 glycosylation in the pathogenesis of HHT, as well as the investigation into new treatment regiments

The Genetics Of Vein Of Galen Malformation And Assessment Of Candidate Genes In Xenopus Tropicalis

The Vein of Galen Malformation (VOGM) is a specific subtype of arteriovenous malformation (AVM) that becomes evident in weeks 6-11 of embryonic development. VOGM comprise less than 1% of all vascular malformations, yet represent 30% of all pediatric intracranial vascular malformations. Depending on their specific characteristics, i.e. its feeding vessels, it can present clinically as devastating congestive heart failure in neonates, hydrocephalus in children, or seizures and headaches in young adults. Advances in treatment provide improved survival, primarily through endovascular surgery. The genetic and molecular etiology of VOGM remains relatively unknown, with the only associated genes being in the context of other syndromes, including seven mutations in p120-RasGAP (RASA1) in Cutaneous Malformations-Arteriovenous Malformations (CM-AVM), as well as one mutation each in activin A receptor type II-like 1 (ACVRL1) mutation and Endoglin (ENG) in Hereditary Hemorraghic Teleangiectasia. Our limited knowledge of the molecular genetics of VOGM has hindered the development of novel therapies. We hypothesized that the apparent sporadic occurrence of VOGM may frequently be attributable to damaging de novo mutation events or incomplete penetrance of rare transmitted variants. Unbiased whole-exome sequencing (WES) can overcome these barriers for gene discovery. We recruited 55 patients, including 52 parent-offspring trios. WES revealed statistically significant rare, damaging de novo mutations in chromatin modifier genes involved in brain and vascular development (p=8.9 x 10-4). VOGM probands also had inherited, missense deleterious and loss of function, in ephrin signaling genes, specifically a whole exome significant mutation burden in EPHB4 (p=7.47 x 10−10). Finally, we observed a whole exome significant inherited mutation in Claudin 14 (p= 6.44 x 10−7). Inherited mutations demonstrated incomplete penetrance and variable expressivity with mutation carriers often exhibiting cutaneous vascular abnormalities suggesting a two-hit mechanism. The identified mutations account for 30% of studied VOGM cases. To functionally validate candidate mutations and establish causality, we developed a screening platform using Xenopus tropicalis by using CRISP/Cas9 gene editing and observing the effects of candidate gene knockdown on vasculogenesis and brain vasculature. CRISPR/Cas9 knockdown of EPHB4 and CLDN14 yielded significantly abnormal vasculogenesis (p=0.0028 and p=0.0001 respectively) observed by in situ hybridization. We implemented and modified established clearing techniques to allow visualization of the full thickness of the Xenopus brain vasculature. The evolutionary precursor to the vein of Galen, the posterior vascular plexus (PVP), and the mesencephalic veins (MSV), were imaged. EPHB4 and CLDN14 CRISPR/Cas9 knockdown revealed significant decreases in MSV length (p Together these findings are the first step in better understanding the mechanism and pathogenesis of VOGM and potential novel therapeutic targets. While Xenopus is not a perfect model system, it does show promise as a tool to assess candidate VOGM that merit further study in mammalian systems harboring a true vein of Galen

Advocating Intraluminal Radiation Therapy in Cerebral Arteriovenous Malformation Treatment

In 2014, ARUBA (a randomized trial on cerebral Arteriovenous Malformation – AVM) found patients treated using prevalent interventional strategies are three times more likely to suffer a stroke/die compared with those treated conservatively (blood pressure reduction). Subsequent controversy led the European societies dealing with AVM to organize a consensus conference. Among the statements made was: “There may be indications for treating patients with higher Spetzler-Martin (SM) grades, based on a case-to-case consensus decision of the experienced team”. Thus, a clear accord emerges. There is a lacuna/weakness of interventional modalities when addressing high SM grade AVMs. This lack of a clear treatment choice originated our review. We attempt to identify the advantages and challenges of each present treatment/evaluation modality and highlight core requirements for future strategies. We conclude that existing modalities provide substantial recent improvements, yet the core challenge persists. Finally, we advocate testing a novel modality – intraluminal radiotherapy (active implants) by exploiting the “candy wrapper” or edge effect. If proven effective, this approach could offer gradual vessel occlusion with minimal abrupt hemodynamic changes known to induce hemorrhage, the lowest recurring session number (reduced costs), minimally invasive attributes and very low radiation (dose/dose rate) kinetics minimizing potential Adverse Radiation Effects (AREs)

Mosaic RAS/MAPK variants cause sporadic vascular malformations which respond to targeted therapy.

BACKGROUND: Sporadic vascular malformations (VMs) are complex congenital anomalies of blood vessels that lead to stroke, life-threatening bleeds, disfigurement, overgrowth, and/or pain. Therapeutic options are severely limited, and multidisciplinary management remains challenging, particularly for high-flow arteriovenous malformations (AVM). METHODS: To investigate the pathogenesis of sporadic intracranial and extracranial VMs in 160 children in which known genetic causes had been excluded, we sequenced DNA from affected tissue and optimized analysis for detection of low mutant allele frequency. RESULTS: We discovered multiple mosaic-activating variants in 4 genes of the RAS/MAPK pathway, KRAS, NRAS, BRAF, and MAP2K1, a pathway commonly activated in cancer and responsible for the germline RAS-opathies. These variants were more frequent in high-flow than low-flow VMs. In vitro characterization and 2 transgenic zebrafish AVM models that recapitulated the human phenotype validated the pathogenesis of the mutant alleles. Importantly, treatment of AVM-BRAF mutant zebrafish with the BRAF inhibitor vemurafinib restored blood flow in AVM. CONCLUSION: Our findings uncover a major cause of sporadic VMs of different clinical types and thereby offer the potential of personalized medical treatment by repurposing existing licensed cancer therapies. FUNDING: This work was funded or supported by grants from the AVM Butterfly Charity, the Wellcome Trust (UK), the Medical Research Council (UK), the UK National Institute for Health Research, the L'Oreal-Melanoma Research Alliance, the European Research Council, and the National Human Genome Research Institute (US)

Master of Science

thesisPort wine stains are capillary malformations that are typically located in the dermis of the head and neck. They affect 0.3% of the population. Current theories suggest that port wine stains are caused by somatic mutations that disrupt vascular development. I hypothesize that port wine stains are multifactorial and understanding their genetic determinants could provide insight into novel treatments. This study used a custom next generation sequencing panel and digital polymerase chain reaction to investigate genetic variants in 12 isolated port wine stain cases (i.e., individuals with port wine stain alone). Novel variants were identified in GNAQ, SOX10, and RASA1. The previously identified GNAQ c.548G>A, p.Arg183Gln mutation was confirmed in 9 of 12 cases with an allele frequency ranging from 1.73 to 7.42%. The custom next generation sequencing panel allowed for detection of low level mosaicism found in 10 of 12 isolated port wine stain cases. A novel GNAQ c.547C>G, p.Arg183Gly mutation was identified in one case. Digital polymerase chain reaction confirmed novel variants detected by next generation sequencing. The results from both methods were highly concordant providing an effective way to detect low level mosaicism in port wine stains. Importantly, the identification of novel genetic mutations in port wine stains may facilitate the development of novel treatments

Genetic testing for vascular anomalies

Abstract Vascular anomalies (VAs) have phenotypic variability within the same entity, overlapping clinical features between different conditions, allelic and locus heterogeneity and the same disorder can be inherited in different ways. Most VAs are sporadic (paradominant inheritance or de novo somatic or germline mutations), but hereditary forms (autosomal dominant or recessive) have been described. This Utility Gene Test was developed on the basis of an analysis of the literature and existing diagnostic protocols. The genetic test is useful for confirming diagnosis, as well as for differential diagnosis, couple risk assessment and access to clinical trials