Multiple lesion-specific somatic mutations and bi-allelic loss of ACVRL1 in a single patient with hereditary haemorrhagic telangiectasia.

Screening for children from families with Rendu-Osler-Weber disease: from geneticist to clinician.

Use of Bevacizumab in Hereditary Hemorrhagic Telangiectasia

Introduction: Hereditary hemorrhagic telangiectasia (HHT) is caused by mutations in TGFβ/BMP9 pathway genes, most commonly ENG or ALK1. HHT patients have arteriovenous malformations (AVM) in brain, lung and liver, leading to severe complications including intracranial hemorrhage (ICH) from brain AVM. ICH presentation of brain AVM in HHT is a marker of high ICH risk. The clinical heterogeneity of HHT suggests a potential role for genetic modifier effects. Common genetic variants in loci that modify phenotype severity in Tgfb knockout mice have been reported to be associated with pulmonary AVM in HHT. We sought to replicate these associations and investigate whether these variants are also associated with brain AVM and ICH presentation of brain AVM in HHT patients. Methods: We genotyped 3 variants (PTPN14 rs2936018, USH2A rs700024 and ADAM17 rs10495565) in 665 Caucasian HHT patients enrolled by the Brian Vascular Malformation Consortium (BVMC). Association of genotype with pulmonary AVM, brain AVM and ICH presentation was evaluated by multivariate logistic regression adjusted for age, gender and family clustering, and also stratified by HHT mutation (ALK1 or ENG). Results: Of 665 Caucasian HHT patients analyzed, 51% had pulmonary AVM and 20% had brain AVM, of whom 17% presented with ICH. None of the 3 SNPs was significantly associated with pulmonary or brain AVM. Among 130 brain AVM patients, USH2A rs700024 was associated with ICH presentation (OR=3.34, 95% CI=1.22-9.15, p=0.019). The effect size was similar in HHT patients with ALK1 and ENG mutations, but only reached statistical significance among the latter (OR=6.77, p=0.014). Conclusions: A common variant in USH2A, rs700024, previously reported to be associated with pulmonary AVM in HHT, was associated with ICH presentation of brain AVM, but not with brain or pulmonary AVM, in the BVMC HHT cohort. Association of the same USH2A variant with different HHT severity phenotypes in different cohorts suggests that it may act as a genetic modifier of HHT phenotype severity in concert with other genetic and environmental factors. Once validated, such genetic modifiers may improve our understanding of the phenotypic heterogeneity of HHT and aid in ICH risk prediction in HHT brain AVM.

Introduction: Brain arteriovenous malformations (AVM) are an important cause of intracranial hemorrhage (ICH) in young adults. Most are sporadic, but also occur in inherited diseases such as hereditary hemorrhagic telangiectasia (HHT). ICH presentation of brain AVM in both sporadic and HHT cases is a marker of high ICH risk. In order to investigate whether the same genetic modifiers influence sporadic and HHT brain AVM, we evaluated candidate genetic polymorphisms reported as associated with sporadic brain AVM, with ICH presentation or ICH during clinical course, in HHT patients. Methods: We genotyped 8 polymorphisms ( APOE E2/3/4 [rs7412, rs429358], ANGPTL rs116724, EPHB4 rs314308, IL6 -174G>C [rs1800795], IL1B -31T>C [rs1143627], ITGB8 rs10486391, TNF -238G>A[rs361525]) in 753 Caucasian HHT patients enrolled by the Brian Vascular Malformation Consortium (BVMC). Genotypes were collapsed into risk allele carriers vs. other for analysis, as published for sporadic AVM. APOE E2/3/4 haplotypes were assigned based on genotypes of the 2 APOE polymorphisms. Association of genotype with phenotype was evaluated by multivariable logistic regression adjusted for age, gender and accounting for family clustering. We used a nominal significance threshold of p=0.05, requiring the same direction of effect as in sporadic brain AVM (odds ratio for risk genotype [OR]>1). Results: Among 753 HHT patients, 155 (21%) had brain AVM, of whom 26 (17%) presented with ICH. Two additional brain AVM patients had ICH during follow-up. None of the 7 variants (6 single nucleotide polymorphisms and APOE haplotype) were significantly associated with brain AVM (OR=0.6-1.3), with ICH presentation of brain AVM (OR=0.4-1.9), or with any brain AVM ICH in HHT patients (OR=0.5-2.1). Conclusions: Common genetic variants previously reported to be associated with sporadic brain AVM were not associated with brain AVM nor with ICH in the BVMC HHT cohort, suggesting different genetic modifiers may influence sporadic and HHT brain AVM. However, the number of ICH cases in the cohort is small, so the confidence intervals are wide and we cannot rule out clinically important associations. The BVMC is enrolling additional HHT patients to expand the cohort and increase power for association analyses.

The olfactory and gustatory functions of hereditary hemorrhagic telangiectasia (HHT) patients have not been documented by validated tests. Disorders of the nasal/oral cavity may interfere with the olfactory and gustatory functions. Fifty-four HHT patients were investigated by smell/taste tests. HHT patients provided subjective ratings in areas such as ability to perceive smell/taste. "Sniffin' Sticks" were used for smell tests, and taste strips were used for taste tests. HHT patients rated their subjective olfactory and gustatory function on a visual analog scale from 0 (none) to 100 (high) as 65.3 ± 27.7 and 68.1 ± 25.1, respectively. Comparison of smell test results of HHT patients with normative data of sex- and age-matched controls from Hummel et al. revealed that HHT patients had lower threshold values, whereas there was no difference in identification and discrimination values. HHT patients were hyposmic. In the case of taste qualities, all values (sweet, sour, salty, and bitter) in HHT patients were lower than those in normative data of Mueller et al. However, HHT patients were not hypogeusic. The duration of disease, extranasal manifestation, and treatments did not significantly correlate with smell/taste test values. Compared with healthy people, HHT patients exhibit reduced olfactory and gustatory function; however, HHT patients are hyposmic and not hypogeusic. This chemosensory deficit may highlight an early sign of disease and has no correlation with disease severity. HHT patients should be informed about these potential disease manifestations, thus enabling them to improve their quality of life.

Introduction: Hereditary Hemorrhagic Telangiectasia (HHT) is a genetic disorder with vascular anomalies of various organ systems including the skin, brain, heart, lungs, and liver. While the most commonly recognized disease burden of HHT are epistaxis and mucocutaneous telangiectasia, the development of vascular malformations can result in more severe symptoms such as cerebral hemorrhage, liver cirrhosis, and stroke. Various case reports/series have reported high output cardiac failure, secondary to presence of a single large AVM or multi organ AVMs, as a major cause of morbidity and mortality in HHT population. To the best of our knowledge, this is the first study analyzing the cardiovascular disease burden in HHT patients from a large national database. Methodology: All HHT patients were identified using ICD-9 code 448.0 using the 2014 National Inpatient Sample (NIS) database. Comorbidities - intracerebral hemorrhage (431), cerebrovascular anomaly (747.81), congestive heart failure (428.0), anemia (285.9) were also identified using the ICD-9 coding system. Descriptive and inferential statistical analysis were performed using SAS 2015. Results: A total of 842 HHT patients were identified with relatively older patients having increased prevalence of anemia (29% vs 15%), congestive heart failure (16% vs 8%), intra-cerebral hemorrhage (0.5% vs 0.3%), hypertension (47% vs 42%) compared to general population, respectively. We report HHT to be linked with having higher odds (p < 0.05) of congestive heart failure (1.6 times), chronic pulmonary lung disease (1.7 times), liver disease (3 times), anemia (1.6 times), cerebrovascular anomaly including cerebral AVMs (26 times), and other neurological disorders than patients without HHT, after adjusting for confounders. Conclusion: This is the first database study confirming higher prevalence of CHF in HHT. We infer that chronic anemia and AVM/AVF can cumulatively cause high output congestive heart failure causing increased morbidity and mortality in HHT population especially in advanced ages.

Introduction: Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disease caused by mutations in transforming growth factor-beta signaling genes (ENG, ALK1, or SMAD4), and is characterized by mucocutaneous telangiectasia and arteriovenous malformations (AVM) primarily in brain, lung and liver. In contrast to sporadic brain AVM, HHT brain AVMs are often multiple though small series to date have suggested that they may have a lower risk of intracranial hemorrhage (ICH). We sought to describe ICH rates and characteristics in HHT patients with brain arteriovenous malformations (HHT-BAVM). Methods: We collected data from the first 134 HHT-BAVM patients with follow-up enrolled in the BVMC HHT Project, including age, sex, race, HHT gene mutation, and ICH at diagnosis. We performed Kaplan-Meier survival analysis to estimate ICH rates after diagnosis censoring at date of first treatment, death or last follow-up, out to 15 years. Results: The majority of patients were female (57%) and Caucasian (93%). HHT gene mutation status was known in 57%, of which 74% had ENG mutations, 23% ALK1, and 3% SMAD4. The mean age at BAVM diagnosis was 30±19 years (range: 0-70), with over half of cases diagnosed based on asymptomatic screening (54%). Among symptomatic cases, 32% presented initially with ICH. During 482 person-years of follow-up, there were 5 ICH events corresponding to a rate of 1.04% per year (95% CI: 0.43 - 2.49%). ICH-free survival differed significantly by ICH presentation (P=0.003); ruptured cases had a higher rate (5.23%, 95% CI: 1.31 - 20.89%) than unruptured cases (0.51%, 95% CI: 0.13 - 2.04%). ICH-free survival did not differ by gender (P=0.285). Conclusions: HHT-BAVM patients who present with hemorrhage are at a higher risk for rehemorrhage compared to BAVMs detected presymptomatically in HHT patients. Correlation with unruptured sporadic BAVM cases will however need a larger sample and number of events in the HHT BAVM cohort. Ongoing recruitment of HHT-BAVM patients into the BVMC study will increase precision of ICH estimates and ability to examine additional risk factors such as angiographic features that may differ from sporadic BAVM.

Hereditary hemorrhagic telangiectasia is a genetic disorder characterized by visceral and mucocutaneous arteriovenous malformations (AVMs). Clinically indistinguishable hereditary hemorrhagic telangiectasia 1 and hereditary hemorrhagic telangiectasia 2 are caused by mutations in ENG and ALK1, respectively. In this study, we have compared the development of visceral and mucocutaneous AVMs in adult stages between Eng- and Alk1-inducible knockout (iKO) models. Eng or Alk1 were deleted from either vascular endothelial cells (ECs) or smooth muscle cells in adult stages using Scl-CreER and Myh11-CreER lines, respectively. Latex perfusion and intravital spectral imaging in a dorsal skinfold window chamber system were used to visualize remodeling vasculature during AVM formation. Global Eng deletion resulted in lethality with visceral AVMs and wound-induced skin AVMs. Deletion of Alk1 or Eng in ECs, but not in smooth muscle cells, resulted in wound-induced skin AVMs. Visceral AVMs were observed in EC-specific Alk1-iKO but not in Eng-iKO. Intravital spectral imaging revealed that Eng-iKO model exhibited more dynamic processes for AVM development when compared with Alk1-iKO model. Both Alk1- and Eng-deficient models require a secondary insult, such as wounding, and ECs are the primary cell type responsible for the pathogenesis. However, Alk1 but not Eng deletion in ECs results in visceral AVMs.

BackgroundThe severity of Hereditary Hemorrhagic Telangiectasia (HHT) disease is generally related to vascular visceral involvement represented by arteriovenous malformations (AVMs). Pulmonary function tests (PFTs) remain normal in HHT patients without Pulmonary AVMs (PAVMs) and respiratory comorbidity. The aim of our study was to compare the diffusing capacity of the lung for carbon monoxide (DLCO) and nitric oxide (DLNO) and its 2 components: the pulmonary capillary blood volume (Vc) and the alveolar-capillary membrane conductance (Dm), in HHT patients with PAVMs, PAVMs and liver AVMs (LAVMs), LAVMs without PAVM, no PAVM and LAVM, and controls.MethodsSixty one consecutive adult patients (HHT without PAVM and LAVM: n = 7; HHT with PAVMs: n = 8; HHT with PAVMs and LAVMs: n = 25; HHT with LAVMs: n = 21) and controls matched for age and sex ratio without respiratory, heart and liver pathology (n = 15) were non-invasively evaluated using PFTs, combined DLCO/DLNO, arterial blood gas at rest, contrast echocardiography and enhanced computed tomography scan of the liver and chest the day of pulmonary function testing.ResultsWe found that patients with LAVMs but without PAVMs exhibited increased Vc/Dm ratio. Interestingly, HHT patients with hepatic artery enlargement showed higher Vc/Dm ratio than HHT patients with normal hepatic artery diameter.ConclusionVc/Dm ratio may have practical impact in HHT patients’ management to detect precociously the occurrence of LVAMs. However, further studies are needed to assess the accuracy and potential prognostic value of pulmonary gas exchange measurements in HHT patients with LVAMs.

Hereditary Hemorrhagic Telangiectasia

Systemic Bevacizumab for the Treatment of Chronic Bleeding in Hereditary Hemorrhagic Telangiectasia

Editorial on Albinana et al. Thromb Haemost 2012; 108: 41-53. Hereditary haemorrhagic telangiectasia (HHT) is a rare autosomal dominant vascular disorder, which occurs at a frequency of about one in 5,000 people (1–3). Vascular malformations in HHT patients range from regions of capillary dilation (telangiectases) in the nasal septum, oral mucosa, skin, or gastrointestinal tract, to large arterial-venous malformations (AVM) in organs such as lung, brain, or liver. The lesions can give rise to spontaneous recurrent nasal or gastrointestinal bleeding, leading to significant blood loss. Therapeutic options include surgery, or manipulation of coagulation and fibrinolytic pathways (2). However, these treatments are insufficient, and novel therapies are needed. The most frequent cases of HHT are caused by mutations in the Endoglin (ENG) or ALK1 genes. Endoglin and Alk1 are cell surface receptors for members of the TGF-β family of cytokines. The biology of TGF-β is complex and many of the observed activities are dependent on the biological context (4); notably, TGF-β can either stimulate or inhibit angiogenesis. Yet, genetic studies in mice have provided clear evidence for a role of Endoglin and Alk1 in vascular development and malformation. Mice carrying heterozygous mutations in the ENG or ALK1 genes develop HHT-like vascular phenotypes. The current hypothesis suggests haploinsufficiency of these genes as the major cause of HHT in men (1, 3). However, the development of the vascular lesions in HHT has not only been attributed to decreased TGF-β signalling but also to increased activity of the pro-angiogenic VEGF pathway. Treatments that result in elevated TGF-β signalling or in angiogenesis inhibition might reduce pathological endothelial cell activation in HHT lesions and appear promising. Various candidate drugs have been proposed for the treatment of HHT on the basis of these considerations. For example, estrogen therapy using raloxifene has been shown to increase the expression of ENG and ALK1 in cultured endothelial cells, and to improve endothelial cell functions like tubulogenesis and migration. This suggests that raloxifene counteracts the haploinsufficiency of ENG and ALK1 and may be beneficial for epistaxis treatment in HHT menopausal women (5). Inhibitors of VEGF, a key regulator of physiological and pathological angiogenesis, are obvious candidates for angiogenesis inhibition. The VEGF-specific antibody bevacizumab, which is approved for the treatment of several malignancies, can be beneficial for the treatment of epistaxis and anaemia in HHT patients (6). However, VEGF has important functions in the healthy adult organism, and inhibiting its activity can have undesired effects. Cancer patients who received bevacizumab therapy suffered from fever, headache, rash, or chills; notably, also epistaxis occurred (7). Although these events were generally mild to moderate in severity, caution is necessary. This is also true for thalidomide. This drug was originally used to treat nausea during pregnancy, but was removed from the market after severe congenital defects became overt, which were attributed to its anti-angiogenic activity resulting in defective vascular development (8). However, thalidomide enjoyed a renaissance in medicine recently. It was reported to inhibit gastrointestinal bleeding in a HHT patient who received the drug as anti-cancer therapy (9), and to reduce the frequency of nosebleeds in HHT patients (8). Although these effects might be attributed to thalidomide s anti-angiogenic activity, a recent study revealed an alternative potential mechanism of action which may guide the development of novel therapeutics for HHT (8). Thalidomide was shown to promote vessel maturation in ENG+/mice which develop HHT-like lesions, as well as in nasal mucosal biopsies of HHT patients. Vessel maturation involved enhanced pericyte and vascular smooth muscle cell coverage in a PDGF-B dependent manner. Ideally, drugs for anti-angiogenesis or normalisation of the endothelium in HHT lesions should be in clinical use without severe side effects. In this issue of Thrombosis and Haemostasis, Albinana et al. propose the use of the β-blocker propanolol for the treatment of HHT (10). This non-selective β-adrenergic receptor antagonist inhibits β1 and β2 adrenergic receptors. It causes vasoconstriction and has been used for the treatment of cardiovascular disorders like hypertension, angina, or cardiac arrhythmias. Propanolol has in addition antiangiogenic properties: it can reduce the expression of VEGF and inhibits typical angiogenic responses in endothelial cells, such as VEGFR-2 phosphorylation, cell proliferation and migration, as well as tube formation (11). Recently, propanolol has been reported to improve capillary haemangiomas of infancy (12). These vascular tumors are formed by endothelial cells that proliferate under the influence of elevated VEGF levels. The question arises whether propanolol might be useful for the treatment of HHT as well. Albinana and colleagues address this question by studying endothelial responses in vitro. Apart from confirming the antiangiogenic activity of pro-

Hereditary haemorrhagic telangiectasia (HHT) is a complex, multisystemic vascular dysplasia affecting approximately 85,000 European Citizens. In 2016, eight founding centres operating within 6 countries, set up a working group dedicated to HHT within what became the European Reference Network on Rare Multisystemic Vascular Diseases. By launch, combined experience exceeded 10,000 HHT patients, and Chairs representing 7 separate specialties provided a median of 24 years' experience in HHT. Integrated were expert patients who focused discussions on the patient experience. Following a 2016–2017 survey to capture priorities, and underpinned by more than 40 monthly meetings, and new data acquisitions, VASCERN HHT generated position statements that distinguish expert HHT care from non-expert HHT practice. Leadership was by specialists in the relevant sub-discipline(s), and 100% consensus was required amongst all clinicians before statements were published or disseminated. One major set of outputs targeted all healthcare professionals and their HHT patients, and include the new Orphanet definition; Do's and Don'ts for common situations; Outcome Measures suitable for all consultations; COVID-19; and anticoagulation. The second output set span aspects of vascular pathophysiology where greater understanding will assist organ-specific specialist clinicians to provide more informed care to HHT patients. These cover cerebral vascular malformations and screening; mucocutaneous telangiectasia and differential diagnosis; anti-angiogenic therapies; circulatory interplays between anaemia and arteriovenous malformations; and microbiological strategies to counteract loss of normal pulmonary capillary function. Overall, the integrated outputs, and documented current practices, provide frameworks for approaches that augment the health and safety of HHT patients in diverse health-care settings.

Objective: Circulating plasma protein profiling may aid in the identification of cerebrovascular disease signatures. This study aimed to identify circulating angiogenic and inflammatory biomarkers that may serve as biomarkers to differentiate sporadic brain arteriovenous malformation (bAVM) patients from other conditions with brain AVMs, including hereditary hemorrhagic telangiectasia (HHT) patients. Methods: The Quantibody Human Angiogenesis Array 1000 (Raybiotech) is an ELISA multiplex panel that was used to assess the levels of 60 proteins related to angiogenesis and inflammation in heparin plasma samples from 13 sporadic unruptured bAVM patients (69% male, mean age 51 years) and 37 patients with HHT (40% male, mean age 47 years, n=19 (51%) with bAVM). The Quantibody Q-Analyzer tool was used to calculate biomarker concentrations based on the standard curve for each marker and log-transformed marker levels were evaluated for associations between disease states using a multivariable interval regression model adjusted for age, sex, ethnicity and collection site. Statistical significance was based on Bonferroni correction for multiple testing of 60 biomarkers (P< 8.3x10 - 4 ). Results: Circulating levels of two plasma proteins differed significantly between sporadic bAVM and HHT patients: PDGF-BB (P=2.6x10 -4 , PI= 3.37, 95% CI:1.76-6.46) and CCL5 (P=6.0x10 -6 , PI=3.50, 95% CI=2.04-6.03). When considering markers with a nominal p-value of less than 0.01, MMP1 and angiostatin levels also differed between patients with sporadic bAVM and HHT. Markers with nominal p-values less than 0.05 when comparing sporadic brain AVM and HHT patients also included angiostatin, IL2, VEGF, GRO, CXCL16, ITAC, and TGFB3. Among HHT patients, the circulating levels of UPAR and IL6 were elevated in patients with documented bAVMs when considering markers with nominal p-values less than 0.05. Conclusions: This study identified differential expression of two promising plasma biomarkers that differentiate sporadic bAVMs from patients with HHT. Furthermore, this study allowed us to evaluate markers that are associated with the presence of bAVMs in HHT patients, which may offer insight into mechanisms underlying bAVM pathophysiology.

Causes and Severity of Anemia in Hereditary Hemorrhagic Telangiectasia