Hereditary Haemorrhagic Telangiectasia: mutation analysis, new variant characterization and study of circulating microRNAs in a rare disease

The topic of my PhD project is the Hereditary Haemorrhagic Telangiectasia (HHT), also known as Rendu-Osler-Weber syndrome, a rare genetic disease with an incidence of about 1:5000, inherited as an autosomal dominant trait. Up to date, mutations in four genes (ENG, ACVRL1, MADH4 and GDF2) have been described in HHT, however, ENG and ACVRL1 mutations account for the 85% of patients. All these genes encode for proteins belonging to the TGF-β/BMPs signalling pathway and are involved in the regulation of angiogenesis. HHT is clinically diagnosed if a patient presents at least three out of four criteria, known as “Curaҫao criteria”: (i) spontaneous and recurrent epistaxis; (ii) telangiectases at characteristic sites, as lips, oral cavity, nose, fingertips and gastrointestinal mucosa; (iii) arteriovenous malformations at characteristic sites, as liver, lungs and central nervous system, and (iv) family history, i.e. a first degree relative with a diagnosis of HHT according to the same criteria. The aim of this thesis is to further investigate the genetic background of HHT in order to better understand the underlying molecular mechanisms and the genotype-phenotype correlations. In particular, my work involved the mutation analyses of ENG and ACVRL1 coding exons and the study of circulating miRNAs in a group of HHT patients. Mutation analyses were performed in new index cases with a HHT clinical diagnosis and their relatives, to identify the disease-causing mutation. Then, the results collected were added to data previously obtained by the Medical Genetics Laboratory (headed by Prof. C. Danesino) and used to perform a descriptive study of the HHT Italian Population, carried out in collaboration with Prof. C. Sabbà, from the University of Bari. Moreover, during my PhD I attended a three-months period in the laboratory of Prof. C. Bernabeu, in Madrid, in order to study the putative pathogenic effect of a particular ENG variant (c.1852+42 C>T) found in a HHT family. The study of circulating miRNAs was performed in collaboration with Prof. M. Denti (University of Trento). We collected plasma samples from 15 people (5 controls; 5 patients with ENG mutation and 5 patients with ACVRL1 mutation) and evaluated the expression level of 752 human miRNAs to identify those misregulated in patients compared to controls. Firstly, we analysed results grouping the patients according to the mutated gene and we identified about 20 misregulated miRNAs. Then, we evaluated if some miRNAs were differentially expressed in patients according to their clinical symptoms. In conclusion, the obtained results increased the knowledge on the molecular pathogenesis, and shed a light on the role exploited by miRNAs in this disorder, thus suggesting new genotype-phenotype correlations, although a validation of the results in a bigger population is mandatory

Functional analysis of a novel ENG variant in a patient with Hereditary Hemorrhagic Telangiectasia (HHT) identifies a new Sp1 binding-site

19 p.-6 fig. Plumitallo. Sara et al.Hereditary Hemorrhagic Telangiectasia (HHT) is a rare disease, with an autosomal dominant inheritance and a worldwide incidence of about 1: 5000 individuals. In >80% of patients, HHT is caused by mutations in either ENG or ACVRL1, which code for ENDOGLIN and Activin A Receptor Type II-Like Kinase 1 (ALK1), belonging to the TGF-β/BMP signalling pathway. Typical HHT clinical features are mucocutaneous telangiectases, arteriovenous malformations, spontaneous and recurrent epistaxis, as well as gastrointestinal bleedings. An additional, but less frequent, clinical manifestation in some HHT patients is the presence of Pulmonary Arterial Hypertension (PAH). The aim of this work is to describe the functional role of a novel ENG intronic variant found in a patient affected by both HHT and PAH, in order to assess whether it has a pathogenic role. We proved that the variant lies in a novel binding-site for the transcription factor Sp1, known to be involved in the regulation of ENG and ACVRL1 transcription. We confirmed a pathogenic role for this intronic variant, as it significantly reduces ENG transcription by affecting this novel Sp1 binding-site.This work was supported by grants from the Ministerio de Industria, Economia y Competitividad (SAF2013-43421-R to CB) and the Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 and ER16PIAC707 to CB) in Spain, both supported by European Regional Development Funds (FEDER). CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) in Spain. This work is part of the results of research activity by SP, who holds a research fellowship (2/R.C./2016, n.20160028774) from the IRCCS Fondazione Policlinico San Matteo, Pavia, Italy.Peer reviewe

Experience of an Italian reference laboratory for a rare disease: Hereditary Haemorragic Telangiectasia

Introduction: Hereditary Haemorragic Teleangiectasia (HHT) is an autosomal dominant disorder affecting 1:5000-8000 individuals worldwide. Mucocutaneous telangiectases and Arteriovenous malformations in internal organs (mostly lungs, liver and central nervous system) are the disease hallmarks. HHT is caused by pathogenetic variants in ENG, ACVRL1, SMAD4 and GDF2, belonging to the TGFβ/BMPs pathway. We report the experience of our research laboratory in the last five years (2015-2020), focusing on mutation analysis. Materials and Methods: Patients’ samples were collected by HHT reference centres in Pavia and Crema (CR). Index cases’ samples were analysed by NGS sequencing panel of the four HHT causative genes and MLPA; the molecular investigation in patients’ relatives was performed by Sanger sequencing. Results: We collected 334 patients’ samples; 99/334 were index cases. Results are summarized in the table below. Subjects ACVRL1 ENG SMAD4 Not Found In progress Unaffected Index case 99 39 (39.4%) 26 (26.3%) 1 (1%) 27 (27.3%) 6 (6%) - Patient’s relatives 234 86 60 - - 8 80 Total patients 333 125 86 1 28 13 80 Conclusions: Our data confirm that HHT is mostly underdiagnosed; however, the presence of a reference center enhances the quality of genetic and clinical results. Moreover, we also corroborate the previous observation that in our country ACVRL1 is the major HHT gene. Not found subjects can harbor variants in intronic or regulatory regions rather than in novel genes. However, we are collecting WES data to re-analyse these cases. Grant: CO: Italian Ministry of Education, University and Research to the DMM-University of Pavia “Dipartimenti di Eccellenza (2018-2022)

Characterization of a mutation in the zona pellucida module of Endoglin that causes Hereditary Hemorrhagic Telangiectasia

7 p.-4 fig.Hereditary hemorrhagic telangiectasia (HHT) is a vascular rare disease characterized by nose and gastrointestinal bleeding, skin and mucosa telangiectasias, and arteriovenous malformations in internal organs. HHT shows an autosomal dominant inheritance and a worldwide prevalence of approximately 1:5000 individuals. In >80% of patients, HHT is caused by mutations in either ENG (HHT1) or ACVRL1 (HHT2) genes, which code for the membrane proteins Endoglin and Activin A Receptor Type II-Like Kinase 1 (ALK1), respectively, both belonging to the TGF-β/BMP signaling pathway. In this work, we describe a novel mutation in exon 9 of ENG (c.1145 G > A) found in five affected members of a family, all of them with characteristic symptoms of HHT. This mutation involves Cys382 residue of the Endoglin protein (p.Cys382 > Tyr) in the zona pellucida (ZP) module of its extracellular region. This is a critical residue involved in a conserved intrachain disulphide bond and in the correct folding of the protein. In fact, transfection studies in human cells using Endoglin expression vectors demonstrated that the p.Cys382 > Tyr mutation results in a marked reduction in the levels of the Endoglin protein. These results demonstrate the pathogenic role for this variant in HHT1 and confirm the key function of Cys382 in Endoglin expression.This work was supported by the Ministerio de Economía, Industria y Competitividad (Grant SAF2013-43421-R to CB); the Consejo Superior de Investigaciones Científicas (Grant 201420E039 and 201920E022 to CB); and the Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER; Grant ISCIII-CB06/07/0038 to CB and contract to LR-L) of Spain. CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds.Peer reviewe

Functional analysis of a novel ENG variant in a patient with hereditary hemorrhagic telangiectasia (HHT) identifies a new Sp1 binding-site

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare disease, with an autosomal dominant inheritance and a worldwide incidence of about 1: 5000 individuals. In >80% of patients, HHT is caused by mutations in either ENG or ACVRL1, which code for ENDOGLIN and Activin A Receptor Type II-Like Kinase 1 (ALK1), belonging to the TGF-β/BMP signalling pathway. Typical HHT clinical features are mucocutaneous telangiectases, arteriovenous malformations, spontaneous and recurrent epistaxis, as well as gastrointestinal bleedings. An additional, but less frequent, clinical manifestation in some HHT patients is the presence of Pulmonary Arterial Hypertension (PAH). The aim of this work is to describe the functional role of a novel ENG intronic variant found in a patient affected by both HHT and PAH, in order to assess whether it has a pathogenic role. We proved that the variant lies in a novel binding-site for the transcription factor Sp1, known to be involved in the regulation of ENG and ACVRL1 transcription. We confirmed a pathogenic role for this intronic variant, as it significantly reduces ENG transcription by affecting this novel Sp1 binding-site

Efficacy and safety of thalidomide for the treatment of severe recurrent epistaxis in hereditary haemorrhagic telangiectasia: results of a non-randomised, single-centre, phase 2 study

Hereditary haemorrhagic telangiectasia is a genetic disease that leads to multiregional angiodysplasia. Severe recurrent epistaxis is the most common presentation, frequently leading to severe anaemia. Several therapeutic approaches have been investigated, but they are mostly palliative and have had variable results. We aimed to assess the efficacy of thalidomide for the reduction of epistaxis in patients with hereditary haemorrhagic telangiectasia that is refractory to standard therapy. We recruited patients aged 17 years or older with hereditary haemorrhagic telangiectasia who had severe recurrent epistaxis refractory to minimally invasive surgical procedures into an open-label, phase 2, non-randomised, single-centre study at IRCCS Policlinico San Matteo Foundation (Pavia, Italy). We gave patients thalidomide at a starting dose of 50 mg/day orally. If they had no response, we increased the thalidomide dose by 50 mg/day increments every 4 weeks, until a response was seen, up to a maximum dose of 200 mg/day. After patients had achieved a response, they continued treatment for 8-16 additional weeks. The primary endpoint was the efficacy of thalidomide measured as the percentage of patients who had reductions of at least one grade in the frequency, intensity, or duration of epistaxis. We followed up patients each month to assess epistaxis severity score and transfusion need, and any adverse events were reported. We included all patients who received any study drug and who participated in at least one post-baseline assessment in the primary efficacy population. The safety population consisted of all patients who received any dose of study treatment. This trial is registered with ClinicalTrials.gov, number NCT01485224. Between Dec 1, 2011, and May 12, 2014, we enrolled 31 patients. Median follow-up was 15·9 months (IQR 10·1-22·3). Three (10%, 95% CI 2-26) patients had a complete response, with bleeding stopped, 28 (90%, 95% CI 74-98) patients had partial responses. Overall, all 31 (100%, 89-100) patients responded to therapy with a significant decrease in all epistaxis parameters (p<0·0001 for frequency, intensity, and duration). A response was achieved by 25 (81%) patients at 50 mg/day of thalidomide, five (16%) patients at 100 mg/day, and one (3%) patient at 150 mg/day. Patients had only non-serious, grade 1 adverse effects, the most common of which were constipation (21 patients), drowsiness (six patients), and peripheral oedema (eight patients). One patient died a month after the end of treatment, but this was not deemed to be related to treatment. Low-dose thalidomide seems to be safe and effective for the reduction of epistaxis in patients with hereditary haemorrhagic telangiectasia. Our findings should be validated by further studies with larger patient populations, longer follow-up, and that also assess the benefit for quality of life

Different forms of pulmonary hypertension in a family with clinical and genetic evidence for hereditary hemorrhagic teleangectasia type 2

Hereditary hemorrhagic telangiectasia (HTT) is an autosomal dominant disease, most frequently caused by a mutation in either ENG or ACVRL1, which can be associated with pulmonary arterial hypertension (PAH). In this report, we describe a new unpublished ACVRL1 mutation segregating in three members of the same family, showing three different types of pulmonary hypertension (PH) in the absence of BMPR2 mutations. The first patient has a form of heritable PAH (HPAH) in the absence of hepatic arteriovenous malformations (AVMs); the second one has a severe form of portopulmonary hypertension (PoPAH) associated with multiple hepatic AVMs; the third one has hepatopulmonary syndrome (HPS) with numerous hepatic arteriovenous fistulas and a form of post-capillary PH due to high cardiac output. In summary, a single mutation in the ACVRL1 gene can be associated, in the same family, with an extreme phenotypic variability regarding not only the clinical presentation of HHT but also the type of PH in the absence of BMPR2 mutations. More studies are needed to evaluate if this variability can be explained by the presence of additional variants in other genes relevant for the pathogenesis of HHT