Dataset Information

Directional next-generation RNA sequencing and examination of premature termination codon mutations in endoglin/hereditary haemorrhagic telangiectasia.

ABSTRACT: 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-? 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.

SUBMITTER: Govani FS

PROVIDER: S-EPMC3666459 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Directional next-generation RNA sequencing and examination of premature termination codon mutations in endoglin/hereditary haemorrhagic telangiectasia.

Govani F S FS Giess A A Mollet I G IG Begbie M E ME Jones M D MD Game L L Shovlin C L CL

Molecular syndromology 20130411 4

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. ...[more]

PMID: 23801935

Similar Datasets

Project description:Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant genetic condition affecting the vascular system and is characterised by epistaxis, arteriovenous malformations and mucocutaneous and gastrointestinal telangiectases. This disorder affects approximately 1 in 8,000 people worldwide. Significant morbidity is associated with this condition in affected individuals, and anaemia can be a consequence of repeated haemorrhages from telangiectasia in the gut and nose. In the majority of the cases reported, the condition is caused by mutations in either ACVRL1 or endoglin genes, which encode components of the TGF-beta signalling pathway. Numerous missense mutations in endoglin have been reported as causative defects for HHT but the exact underlying cellular mechanisms caused by these mutations have not been fully established despite data supporting a role for the endoplasmic reticulum (ER) quality control machinery. For this reason, we examined the subcellular trafficking of twenty-five endoglin disease-causing missense mutations. The mutant proteins were expressed in HeLa and HEK293 cell lines, and their subcellular localizations were established by confocal fluorescence microscopy alongside the analysis of their N-glycosylation profiles. ER quality control was found to be responsible in eight (L32R, V49F, C53R, V125D, A160D, P165L, I271N and A308D) out of eleven mutants located on the orphan extracellular domain in addition to two (C363Y and C382W) out of thirteen mutants in the Zona Pellucida (ZP) domain. In addition, a single intracellular domain missense mutant was examined and found to traffic predominantly to the plasma membrane. These findings support the notion of the involvement of the ER's quality control in the mechanism of a significant number, but not all, missense endoglin mutants found in HHT type 1 patients. Other mechanisms including loss of interactions with signalling partners as well as adverse effects on functional residues are likely to be the cause of the mutant proteins' loss of function.

Project description:Hereditary Haemorrhagic Telangiectasia (HHT) is an autosomal dominantly inherited vascular disease characterized by the presence of mucocutaneous telangiectasia and arteriovenous malformations in visceral organs. HHT is predominantly caused by mutations in ENG and ACVRL1, Which both belong to the TGF-M-NM-2 signalling pathway. Further knowledge on how a disturbance of the TGF-M-NM-2 signalling pathway leads to HHT manifestations is needed in order to identify potential therapeutic targets. As long non-coding RNAs (lncRNAs) are increasingly recognized as key regulators of gene expression and constitute a sizable fraction of the human transcriptome, we wanted to assess whether lncRNAs play a role in the molecular pathogenesis of HHT. Here we used microarray analysis to profile lncRNAs to compare the expression of HHT telangiectasial and HHT non-telangiectasial nasal tissue from the same patient in a paired design. The microarray probes were re-annotated using the GENCODE v.16 dataset, identifying 4,810 probes mapping to 2,811 lncRNAs. By comparing HHT telangiectasial tissue versus HHT non-telangiectasial tissue, we identified 42 lncRNAs that are differentially expressed (q<0.001). Using GREAT, a tool that assumes cis-regulation, we showed that differently expressed lncRNAs are enriched for genomic loci involved in key pathways concerning HHT. Our study identified lncRNAs that are aberrantly expressed in HHT telangiectasia and indicates that lncRNAs may contribute to regulate protein-coding loci in HHT. Which suggest that the lncRNA component of the transcriptome deserves more attention in HHT. A deeper understanding of lncRNAs and their role in telangiectasia formation possesses potential for discovering therapeutic targets in HHT. Gene expression profiling of 80 paired nasal samples from patients with hereditary Haemorrhagic Telangiectasia (HHT), representing telangiectasial and non-telangiectasial (normal) tissue respectively. The patients were divided into HHT1 or HHT2 in regard to the germline mutation in ENG (HHT1) or ACVRL1 (HHT2). Additional controls were healthy siblings (not carrying the germline mutation) and external controls. The study was conducted using Agilent-028004 SurePrint G3 Human GE 8x60K Microarray platform.

Dataset Information

Directional next-generation RNA sequencing and examination of premature termination codon mutations in endoglin/hereditary haemorrhagic telangiectasia.

Publications

Directional next-generation RNA sequencing and examination of premature termination codon mutations in endoglin/hereditary haemorrhagic telangiectasia.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets