Hereditary Hemorrhagic Telangiectasia Clinical and Molecular Genetics Tom G.W. Letteboer ISBN: 978-90-5335-333-2 Author: Tom G.W. Letteboer Cover design: Wilmy Swaerdens, using painting from Nelleke Docter (Pimpelnell), called “The Family” Lay-out and printing: Simone Vinke, Ridderprint B.V., Ridderkerk, the Netherlands Hereditary Hemorrhagic Telangiectasia Clinical and Molecular Genetics Klinische en moleculair genetische aspecten van hereditaire hemorrhagische teleangiëctasieën (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. J.C. Stoof, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op woensdag 10 november 2010 des middags 12.45 uur door Thomas Gerrit Wim Letteboer geboren op 9 februari 1967 te Zwolle Promotor: Prof. dr. D. Lindhout Co-promotoren: Dr. C.J.J. Westermann Dr. J.K. Ploos van Amstel The author gratefully acknowledges financial support from the Ter Meulen Fund, Royal Netherlands Academy of Arts and Sciences the Netherlands, for supporting collaboration in HHT research with the University of California, San Francisco, the Helen Diller Family Comprehensive Cancer Center Abbreviations ACVRL1 = actvin A receptor type II-like 1 gene (ALK1) AVF = arteriovenous fistula AT = ataxia telangiectasia AVM = arteriovenous malformation BMP = bone-morphogenic-protein CAVM = cerebral arteriovenous malformation CM = capillary malformation CM-AVM = capillary malformation-arteriovenous malformation EC = endothelial cell ENG = endoglin GI = gastrointestinal HAVM = hepatic arteriovenous malformation HBT = hereditary benign telangiectasia HHT = hereditary hemorrhagic telangiectasia HHT1 = hereditary hemorrhagic telangiectasia type 1 (ENG) HHT2 = hereditary hemorrhagic telangiectasia type 2 (ACVRL1) HHT? = hereditary hemorrhagic telangiectasia, unknown genetic cause JP-HHT = juvenile polyposis and hereditary hemorrhagic telangiectasia MLPA = multiplex ligation-dependent probe amplification MNC = mononuclear cells MRI = magnetic resonance imaging PAH = pulmonary arterial hypertension PFO = patent foramen ovale PAVM = pulmonary arteriovenous malformation PH = pulmonary hypertension RLS = right-left shunt ROW = Rendu-Osler-Weber disease SMAD4 = mothers against decapentaplegic, drosophila, homolog of, 4 (MADH4) SMC = smooth muscle cells TGFβ = transforming growth factor beta TIA = transient ischemic attack Contents Chapter 1 General introduction 9 Chapter 2 Molecular genetic basis of hereditary hemorrhagic telangiectasia 25 Chapter 2.1 Hereditary hemorrhagic telangiectasia: ENG and ACVRL1 mutations in Dutch patients. 27 Hum Genet. 2005; 116: 8-16. Chapter 2.2 Multiplex Ligation-dependent Probe Amplification analysis identifies ENG and ACVRL1 deletions/duplications in hereditary hemorrhagic telangiectasia 45 submitted Chapter 2.3 SMAD4 mutations found in unselected HHT patients 55 J Med Genet. 2006; 43: 793-797. Chapter 3 Genotype – phenotype relationship in hereditary hemorrhagic telangiectasia 69 Chapter 3.1 Genotype-phenotype relationship in hereditary hemorrhagic telangiectasia. 71 J Med Genet. 2006; 43: 371-377. Chapter 3.2 Genotype-phenotype relationship for localization and age distribution of telangiectases in hereditary hemorrhagic telangiectasia. 87 Am J Med Genet A. 2008; 146:2733-2739. Chapter 3.3 The onset and severity of epistaxis in patients with ENG or ACVRL1 mutations 101 submitted Chapter 3.4 Assessment of intestinal vascular malformations in patients with hereditary hemorrhagic telangiectasia and anemia. 113 Eur J Gastroenterol Hepatol. 2007; 19: 153-158. Chapter 4 Discussion and perspective 127 Chapter 5 Summary 147 Nederlandse samenvatting 153 Dankwoord 159 Curriculum Vitae 163 List of publications 167 Chapter 1 General introducti on Genotype-phenotype relationship for localization and age distribution of telangiectases in HHT Genotype-phenotype relati onship for localizati on and age distributi on ofGeneral telangiectases introducti in on HHT 11 History Hereditary hemorrhagic telangiectasia (HHT) is also known by its eponym Rendu-Osler- 1 Weber disease (ROW) aft er the names of the fi rst physicians that described the disease. In 1896 Henry Jules Louis Marie Rendu published an arti cle on recurrent epistaxis in a pati ent with “peti ts angiomes cutanés et muqueux”. This was the fi rst complete descripti on of this disease. He was aware of its familial nature, its associati on with bleeding secondary to telangiectases, and its being an enti ty separate from hemophilia [1]. His paper was followed in 1901 by a report from William Osler and later in 1907 by one from Frederick Parkes Weber establishing the disease as an inherited disorder [2,3]. The term hereditary hemorrhagic telangiectasia was in 1909 proposed by Frank Hanes [4]. He advocated the name to be more in conformity with the medical nomenclature by referring to the three major clinical features. Despite the appropriateness and general usage of this descripti ve name, the eponym Rendu-Osler-Weber is sti ll widely used with the order of the names someti mes adapted, depending on the country of origin of the authors or on the appreciati on of the contributi on of the fi rst physicians. Hemorrhage of telangiectati c origin HHT was established as a disorder that primarily results in bleeding (e.g. nosebleeds or epistaxis) due to “inadequate vessels”, in which visceral involvement could occur and present in multi ple subjects in a family, in men as well as in women [2]. HHT is an autosomal dominant vascular disorder characterized by the presence of multi ple arteriovenous malformati ons (AVM). These AVMs are direct connecti ons between arteries and veins, thereby lacking the (normally) intervening capillary bed. Telangiectases are in fact small AVMs, which can present on the face, lips, tongue, fi ngers and in the nasal, oral and gastrointesti nal mucosa. AVMs in HHT usually refer to the larger arteriovenous connecti ons, commonly occurring in the lung (pulmonary arteriovenous malformati ons or PAVM), the brain (cerebral arteriovenous malformati ons or CAVM) and/or the liver (hepati c arteriovenous malformati ons or HAVM). These AVMs cause direct shunti ng of blood, resulti ng in the absence of normal capillary exchange and the absence of a normal capillary fi lter functi on [5]. Prevalence The prevalence of HHT varies considerably between countries and even between regions within a country. In part, this may be due to diff erences in ascertainment and diagnosis, but diff erences between populati ons truly exist, thought to be due to founder eff ects. As an example, in the Netherlands the frequency is esti mated to be 1-2:10,000, whereas in the Dutch Anti lles the prevalence is esti mated to be as high as 1:1,330 [6]. Also within-country 12 Chapter 1 General introduction regional variability has been documented: in some regions in France prevalence nears 1:3,500 [7]. Genetics of HHT Linkage studies on HHT families identified two major gene loci associated with HHT. In 1994, the ENG gene encoding endoglin was identified on chromosome 9q34 as the gene associated with HHT1 (MIM 187300) [8]. However, locus heterogeneity was obvious, since a number of families were not linked to chromosome 9 [9]. Two years later, in 1996, mutations in ACVRL1 (activin-like receptor kinase: ACVRL1 or ALK1) located on chromosome 12q13 were found to cause HHT2 (MIM 600376) [10]. Both genes encode proteins involved in the TGFβ pathway. The spectrum of mutations for both the ENG gene and the ACVRL1 gene consists of all kinds of mutations: missense, nonsense, frame shift, splice site etc. These mutations are typical for a loss of function. Mutations in HHT are collected in two major mutation databases (http://www.hhtmutation.org/ ; http:// www.hgmd.cf.ac.uk/ac/index.php). In January 2010, the Human Gene Mutation Database reported 316 mutations for the ENG gene; 100 missense/nonsense mutations, 134 insertions/deletions, 51 splice site mutations, 30 large deletions or duplications and 1 regulatory region mutation. For the ACVRL1 gene 273 different mutations are reported; 160 missense/nonsense mutations, 84 insertions/deletions, 20 splice site mutations and 9 large deletions/duplications. Protein expression studies in human umbilical vein endothelial cells and peripheral blood monocytes have confirmed haploinsufficiency, mutations that cause loss of function of one allele, as the main model in HHT1 and HHT2 [11-13]. SMAD4 has been implicated as a third gene involved in HHT. In 2004 Gallione et al.[14] reported mutations in the SMAD4 gene in a rare group of patients that show clinical features of two diseases: juvenile polyposis and HHT or JP-HHT (MIM 175050). The SMAD4 gene is located on chromosome 18q21.1, codes for the protein SMAD4 and is expressed in a variety of cell types. The protein, like ENG and ACVRL1, has a role in the TGFβ pathway as well as in the bone-morphogenic-protein (BMP) pathway. HHT as part of the JP-HHT syndrome can thus also be explained by SMAD4 mutations. Other candidate loci for HHT are linked to chromosome 5 and chromosome 7 [15,16]. These loci are now described as being responsible for HHT3 and HHT4 respectively. Cole et al.[16] described linkage to chromosome 5q31 in a family, in which linkage with ENG, ACVRL1 and SMAD4 was excluded and without mutations in any of these genes. They narrowed the linkage region on the chromosome down to 6 Mb with a maximum two point LOD score of 3.45. Bayrak-Toydemir et al.[15] excluded linkage to the HHT causing genes and detected a maximum two point LOD score of 3.6 with an STR marker on chromosome 7p14, in one family. They narrowed the region to 7 Mb. To date, no causative genes have been found in these two regions. General introducti on 13 The HHT1 gene: ENG The ENG gene (depicted in fi gure 1) is located on 9q33-q34.1 and encompasses approximately 1 40 kb of genomic DNA. The gene consists of 14 exons or 15 exons if the alternati ve splicing within the most distal exon is taken into considerati on. They respecti vely encode a longest mRNA of 3196 nt (NM_000118) and a shorter one with a length of 3072 nt (NM- 01114753).