Fine mapping of the hereditary haemorrhagic telangiectasia (HHT)3 locus on chromosome 5 excludes VE-Cadherin-2, Sprouty4 and other interval genes

BACKGROUND: There is significant interest in new loci for the inherited condition hereditary haemorrhagic telangiectasia (HHT) because the known disease genes encode proteins involved in vascular transforming growth factor (TGF)-beta signalling pathways, and the disease phenotype appears to be unmasked or provoked by angiogenesis in man and animal models. In a previous study, we mapped a new locus for HHT (HHT3) to a 5.7 Mb region of chromosome 5. Some of the polymorphic markers used had been uninformative in key recombinant individuals, leaving two potentially excludable regions, one of which contained loci for attractive candidate genes encoding VE Cadherin-2, Sprouty4 and FGF1, proteins involved in angiogenesis. METHODS: Extended analyses in the interval-defining pedigree were performed using informative genomic sequence variants identified during candidate gene sequencing. These variants were amplified by polymerase chain reaction; sequenced on an ABI 3730xl, and analysed using FinchTV V1.4.0 software. RESULTS: Informative genomic sequence variants were used to construct haplotypes permitting more precise citing of recombination breakpoints. These reduced the uninformative centromeric region from 141.2-144 Mb to between 141.9-142.6 Mb, and the uninformative telomeric region from 145.2-146.9 Mb to between 146.1-146.4 Mb. CONCLUSIONS: The HHT3 interval on chromosome 5 was reduced to 4.5 Mb excluding 30% of the coding genes in the original HHT3 interval. Strong candidates VE-cadherin-2 and Sprouty4 cannot be HHT3

Endothelial Cell Processing and Alternatively Spliced Transcripts of Factor VIII: Potential Implications for Coagulation Cascades and Pulmonary Hypertension

Background: Coagulation factor VIII (FVIII) deficiency leads to haemophilia A. Conversely, elevated plasma levels are a strong predictor of recurrent venous thromboemboli and pulmonary hypertension phenotypes in which in situ thromboses are implicated. Extrahepatic sources of plasma FVIII are implicated, but have remained elusive. Methodology/Principal Findings: Immunohistochemistry of normal human lung tissue, and confocal microscopy, flow cytometry, and ELISA quantification of conditioned media from normal primary endothelial cells were used to examine endothelial expression of FVIII and coexpression with von Willebrand Factor (vWF), which protects secreted FVIII heavy chain from rapid proteloysis. FVIII transcripts predicted from database mining were identified by rt-PCR and sequencing. FVIII mAb-reactive material was demonstrated in CD31+ endothelial cells in normal human lung tissue, and in primary pulmonary artery, pulmonary microvascular, and dermal microvascular endothelial cells. In pulmonary endothelial cells, this protein occasionally colocalized with vWF, centered on Weibel Palade bodies. Pulmonary artery and pulmonary microvascular endothelial cells secreted low levels of FVIII and vWF to conditioned media, and demonstrated cell surface expression of FVIII and vWF Ab–reacting proteins compared to an isotype control. Four endothelial splice isoforms were identified. Two utilize transcription start sites in alternate 59 exons within the int22h-1 repeat responsible for intron 2

Foxn1 regulates key target genes essential for T cell development in postnatal thymic epithelial cells

Thymic epithelial cell differentiation, growth and function depend on the expression of the transcription factor Foxn1; however, its target genes have never been physically identified. Using static and inducible genetic model systems and chromatin studies, we developed a genome-wide map of direct Foxn1 target genes for postnatal thymic epithelia and defined the Foxn1 binding motif. We determined the function of Foxn1 in these cells and found that, in addition to the transcriptional control of genes involved in the attraction and lineage commitment of T cell precursors, Foxn1 regulates the expression of genes involved in antigen processing and thymocyte selection. Thus, critical events in thymic lympho-stromal cross-talk and T cell selection are indispensably choreographed by Foxn1

Hereditary haemorrhagic telangiectasia: a clinical and scientific review

The autosomal-dominant trait hereditary haemorrhagic telangiectasia (HHT) affects 1 in 5–8000 people. Genes mutated in HHT (most commonly for endoglin or activin receptor-like kinase (ALK1)) encode proteins that modulate transforming growth factor (TGF)-β superfamily signalling in vascular endothelial cells; mutations lead to the development of fragile telangiectatic vessels and arteriovenous malformations. In this article, we review the underlying molecular, cellular and circulatory pathobiology; explore HHT clinical and genetic diagnostic strategies; present detailed considerations regarding screening for asymptomatic visceral involvement; and provide overviews of management strategies

Lung expression of FVIII.

<p>Serial sections of frozen normal human lung tissue from two donor blocks (a and b) stained with <b>i)</b> control IgG₁, <b>ii</b>) anti-CD31, or <b>iii</b>) anti-FVIII (C5). The 200x images are representative of data from all five donors.</p

Secreted and cell surface FVIII.

<p><b>a</b>) <b>Quantification of FVIII on the surface of EC.</b> Relative fluorescence intensity (RFI) of confluent HUVEC, HPAEC and HPMEC stained with FVIII mAb C2, compared to EC from the same well in which C2 was omitted. <b>b</b>)<b> Quantification of HPAEC surface FVIII: bi</b> Comparison of EC RFI for HPAEC from the same well treated with IgG₁ control, or C2 mAb; p values calculated by Mann Whitney. <b>bii-iii</b>. Representative raw data plots of log expression (FL1 log) versus forward scatter as a marker of cell size for HPAEC from the same well treated with <b>bii</b>) IgG₁ control, <b>biii</b>) C2 mAb. <b>c</b>)<b> Quantification of HPAEC surface vWF. ci</b>) Comparison of EC RFI for HPAEC from the same well treated with IgG₁ control, or vWF pAb, p values calculated by Mann Whitney. <b>cii-iii</b>. Representative raw data plots of log expression (FL1 log) versus forward scatter as a marker of cell size for HPAEC from the same well treated with <b>cii</b>) IgG₁ control, <b>ciii</b>) vWF pAb. <b>d</b>) <b>Quantification of FVIII:Ag in control and EC-conditioned media by ELISA</b>. M₁ denotes control EC media (2% FCS), M₂ denotes control MEC media (5% FCS). M₁ control data are presented twice for clarity. <i>P</i> values are presented for the 48 hour data sets of EC from passages 4 and 5. Differences at 24 hours did not reach statistical significance. <b>e</b>)<b> Manual FVIII:c assay standard curve</b>. Ln FVIII, logarithm of FVIIIc activity (U/ml). Note clotting time in samples exceeded 240 seconds.</p

FVIII splice isoforms.

<p><b>a: Variants identified by ExonMine</b>: Variants 1 and 2 correspond to major and minor RefSeq isoforms; variants 3–5 to expressed sequence tag (EST) sequences deposited in Genbank. Note none of the alternate variants encode the FVIII mAb epitopes. <b>b: Variants identified in EC</b>. Simultaneous expression of variants 1–4 in HPAEC (PA), HPMEC (PM), and HUVEC (H). Gels: φx, HaeIII-digested φx marker, C^A negative water control for HPAEC/HUVEC, C^M negative water control for HPMEC. The apparent difference in size of variant 4 is an artefact due to gel running (note differential site of 194 marker band in first and last lanes). Cartoons: Thin and thick arrows indicate sites of PCR and sequencing oligonucleotide primers respectively. Sequence chromatograms were obtained using nested reverse internal primers in exon 23 (variants 1–3) or exon 3 (variant 4; low concentration first round product sequenced). Note V5 sequences (exons U1-1-2-3) were not amplified from EC in any reaction.</p

Immunofluorescence images of FVIII expression by EC.

<p><b>a</b>: Sequential confocal fluorescence microscopy images in primary human EC. <b>a</b> and <b>b</b>: Representative HPMEC and HPAEC control images using To-Pro-3 nuclear counterstain (first panel, monochrome), murine control IgG₁ (second panel monochrome) and merged images (third panel: To-Pro-3 nuclear counterstain white, control IgG₁ red) using maximum gain used for imaging. <b>c</b>) Comparison of proportion of <b>i</b>) <b>HPMEC</b> and <b>ii</b>) <b>HPAEC</b> expressing vWF and FVIII protein (expression levels defined in methods). <b>d, e, f, g</b>) Sequential confocal fluorescence microscopy images comparing vWF (monochrome in first panel); FVIII (monochrome in second panel, specific mAb as denoted), and merged images (third panel; vWF green, anti-FVIII reacting protein red) in <b>d</b>): HPMEC, and <b>e, f, g</b>): HPAEC. FVIII mAb images displayed here are representative of all FVIII mAbs examined, and all cell lots. Note yellow merged images suggesting degree of FVIII/vWF colocalisation in <b>e, f, g</b>, with white colouring denoting the nuclei (TO-PRO3 nuclear counterstain). Scale bars indicate 5 µm.</p