Hereditary haemorrhagic telangiectasia (HHT) affects one in 5−8000, and no longer can be viewed as solely causing anaemia (due to nasal and gastrointestinal bleeding) and characteristic mucocutaneous telangiectasia. Arteriovenous malformations commonly occur, and in the pulmonary and cerebral circulations demand knowledge of risks and benefits of asymptomatic screening and treatment. HHT is inherited as an autosomal dominant trait and there is no age cut off when apparently unaffected offspring of an individual with HHT can be told they are unaffected. This review focuses on the evolving evidence base for HHT management, issues regarding pregnancy and prothrombotic treatments, and discusses the molecular and cellular changes that underlie this disease.

No abstract

Introduction:Hereditary haemorrhagic telangiectasia (HHT) causes chronic nasal and gastrointestinal haemorrhage. Prothrombotic agents are commonly used for severe haemorrhage. Thrombotic risks have not been defined. MethodsTo identify prothrombotic variables in HHT patients, and assess their potential functional significance, a pilot ELISA-based study comparing plasma proteins in healthy individuals with HHT to age/sex-matched non-HHT controls was validated in a full study of 309 consecutive HHT-affected individuals. Results

Coagulation factor VIII is an essential cofactor required for normal hemostatic function. A deficiency in factor VIII results in the bleeding disorder hemophilia A. Despite the fact that the factor VIII gene was cloned a decade ago, the mechanisms which control its transcription remain unresolved. In our studies, we have characterized 12 protein binding sites within the factor VIII promoter by DNase I protection assays performed with rat liver nuclear extracts. Three of these elements (sites 1 to 3) are situated within the 5 untranslated region of the gene, while three other sites (sites 4 to 6) lie within the first 100 bp upstream of the transcriptional start site. We have identified an additional site (site 7) ϳ300 bp upstream from site 6, as well as a cluster of five sites in a 250-bp region which terminates ϳ1 kb from the transcriptional start site. Seven of these binding sites (sites 2, 3, 4, 6, 7, 9, and 10) bind members of the C/EBP family of transcription factors. DBP also binds to five of these sites (sites 3, 4, 6, 7, and 9). Utilizing transient transfection studies in HepG2 cells, we have shown that deletion of the factor VIII promoter sequences distal to nucleotide ؊44 results in a significant but small increase in promoter activity. The activity of each of the various 5 deletion constructs is significantly enhanced by cotransfection of C/EBP␣ and D-site-binding protein expression plasmids, while cotransfection of both C/EBP␣ and C/EBP␤ plasmids resulted in a further enhancement of transactivation. These studies also provide evidence of a repressor element located between nucleotides ؊740 and ؊1002. Since the minimal promoter sequence (؊44 to ؉148) maintains the transcriptional activity of the full-length promoter sequence, we proceeded to identify additional factors binding to sites 1 to 4. Competition studies revealed that a ubiquitous transcription factor, NF-Y, binds to site 4, while the liver-enriched transcription factor hepatocyte nuclear factor I (HNF-1) binds to site 1. Mutation analysis of the minimal promoter demonstrated that HNF-1 is critical for activating transcription of the factor VIII gene in vitro. Our results also suggest that the multiple upstream elements that we have identified may act as a backup regulatory region in the event of disruption of the HNF-1 element in the 5 untranslated region.

SummaryIndividuals with haemophilia B require replacement therapy with recombinant or plasma-derived coagulation factor IX (fIX). More benefit per injected dose might be obtained if fIX clearance could be slowed. The contribution of overall size to fIX clearance was explored, using genetic fusion to albumin. Recombinant murine fIX (MIX), and three proteins with C-terminal epitope tags were expressed in HEK 293 cells: tagged MIX (MIXT), tagged mouse serum albumin (MSAT) and MFUST, in which MIX and MSAT were fused in a single polypeptide chain. Proteins MFUST and MIXT were two-to threefold less active in clotting assays than MIX. In mice, the area under the clearance curve (AUC) was reduced for MFUST compared with MSAT or plasma-derived MSA (pd-MSA); the terminal catabolic halflife (t 0AE5 ) did not differ amongst the three proteins. Two minutes after injection, >40% of the injected MFUST was found in the liver, compared with <10% of either MSAT or pd-MSA. In rabbits, the AUC for MFUST was reduced compared to MIXT, MSAT, or pd-MSA, while the t 0AE5 of the fusion protein fell between that of MIXT and MSAT or pd-MSA. Similar results were obtained with non-radioactive fused or non-fused recombinant human fIX in fIX knockout mice. The clearance behaviour of the fusion protein thus more closely resembled that of fIX than that of albumin despite a modest increase in terminal half-life, suggesting that fIX-specific interactions that are important in determining clearance were maintained in spite of the increased size of the fusion protein.

The factors responsible for the removal of injected factor IX (fIX) from the blood of individuals with haemophilia B are only partly understood, and may include binding to endothelial or subendothelial sites, passive extravasation related to size or charge, or interactions requiring fIX activation. To investigate these issues, we have produced and characterised recombinant fIX proteins with amino acid changes: delta155-177, an internal deletion which removes most of the activation peptide while retaining the activation cleavage sites; S365A, which inactivates the serine protease activity of fIXa; and K5A, previously shown to eliminate fIX binding of endothelial/subendothelial collagen IV. All proteins were expressed in stably transfected HEK 293 cells, purified by immunoaffinity chromatography, and compared to the wild type HEK 293-derived protein (fIX (WT)). Mutant fIX proteins K5A and delta155-177 exhibited 72 and 202% of the specific activity of fIX (WT), respectively; S365A was without activity. Following intravenous injection in haemophilia B (fIX knockout) mice, recoveries did not differ for fIX (WT) and delta155-177, but were higher for K5A and S365A. The terminal catabolic half-life of delta155-177, alone among the mutants, was increased, by 45% versus fIX (WT). Nine hours post-injection, the observed areas under the clearance curve (AUCs) of delta155-177 and K5, but not S365A, were elevated 2-fold. delta155-177 was equally effective as fIX (WT) in reducing blood loss following tail vein transection in haemophilia B mice. Our results suggest that deletion of the multiple sites of fIX post-translational modification found within the activation peptide eliminated important fIX clearance motifs.

The regulatory regions of the genes for coagulation Factors VIII and IX contain binding sites for both liver-enriched and ubiquitous transcriptional regulators. We investigated the role of the liver-enriched protein, hepatic leukemia factor (HLF), in mediating transcriptional regulation of the Factor VIII and IX genes. Using transient transfection assays in HepG2 hepatoma cells, we demonstrated the ability of HLF alone and in synergistic combination with the D-box binding protein (DBP), another proline and acidic-rich (PAR) protein family member, to transactivate these promoters. HLF is capable of binding to multiple sites in both the Factor VIII and Factor IX promoters. At least some of the synergistic activation of the Factor VIII promoter seen with HLF and DBP cotransfection can be attributed to increased binding of HLF-DBP heterodimers to two Factor VIII promoter sites. We have also demonstrated that an E2A-HLF chimera, derived from a t(17;19) translocation in pre-B acute lymphoblastic leukemia (ALL) cells, is capable of mediating expression from the Factor VIII and Factor IX promoters in both hepatoma cells and pre-B ALL cells. These observations indicate that the PAR family of transcription factors plays an important and complex role in regulating expression of the Factor VIII and Factor IX genes, involving the binding of both homodimeric and heterodimeric complexes of HLF and DBP to several sites in the promoters. Finally, these studies reaffirm the potential role of dimeric transcription factor complexes in mediating interactions with specific promoter elements, which, in the case of the Factor VIII promoter, results in dramatically enhanced binding of HLF-DBP heterodimers to two cis-acting sequences. These observations further our understanding of the role played by members of the PAR family of transcription factors in regulating expression of the Factor VIII and Factor IX genes.

Hereditary haemorrhagic telangiectasia (HHT) is a disease characterised by abnormal vascular structures, and most commonly caused by mutations in ENG, ACVRL1 or SMAD4 encoding endothelial cell-expressed proteins involved in TGF-β superfamily signalling. The majority of mutations reported on the HHT mutation database are predicted to lead to stop codons, either due to frameshifts or direct nonsense substitutions. The proportion is higher for ENG (67%) and SMAD4 (65%) than for ACVRL1 (42%), p < 0.0001. Here, by focussing on ENG, we report why conventional views of these mutations may need to be revised. Of the 111 stop codon-generating ENG mutations, on ExPASy translation, all except one were premature termination codons (PTCs), sited at least 50-55 bp upstream of the final exon-exon boundary of the main endoglin isoform, L-endoglin. This strongly suggests that the mutated RNA species will undergo nonsense-mediated decay. We provide new in vitro expression data to support dominant negative activity of stable truncated endoglin proteins but suggest these will not generate HHT: the single natural stop codon mutation in L-endoglin (sited within 50-55 nucleotides of the final exon-exon boundary) is unlikely to generate functional protein since it replaces the entire transmembrane domain, as would 8 further natural stop codon mutations, if the minor S-endoglin isoform were implicated in HHT pathogenesis. Finally, next-generation RNA sequencing data of 7 different RNA libraries from primary human endothelial cells demonstrate that multiple intronic regions of ENG are transcribed. The potential consequences of heterozygous deletions or duplications of such regions are discussed. These data support the haploinsufficiency model for HHT pathogenesis, explain why final exon mutations have not been detected to date in HHT, emphasise the potential need for functional examination of non-PTC-generating mutations, and lead to proposals for an alternate stratification system of mutational types for HHT genotype-phenotype correlations.