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BS29 FLT4 pleiotropy in congenital cardiac or lymphovascular disease is mediated through distinct cellular mechanisms
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  1. Richard Monaghan1,
  2. Simon Williams2,
  3. Bernard Keavney2
  1. 1University of Manchester, AV Hill Building, Oxford Road, Manchester, GTM M13 9PT, UK
  2. 2University of Manchester

Abstract

Background The FLT4 gene encodes vascular growth factor receptor 3 (VEGFR3). Studies by several groups have shown that genetic variation in FLT4 is implicated in susceptibility to sporadic, non-syndromic occurrences of the most prevalent cyanotic congenital heart disease, Tetralogy of Fallot (TOF). FLT4 is also implicated in Milroy disease (MD), the most common form of hereditary lymphoedema. MD is caused by dominant negative heterozygous FLT4 variants that ablate the activity of the kinase domain of the receptor in response to its ligands VEGFC and D, which is required for lymphangiogenesis. FLT4 genetic variation giving rise to TOF is distinct to MD, and the conditions do not have overlapping phenotypic features. The mechanism whereby FLT4 contributes to TOF risk has not been elucidated.

Methods FLT4 wild type (WT), a confirmed MD FLT4 variant, or two TOF-associated variants from previously published studies (either a missense de novo variant, DNV; or a protein truncating variant, PTV), with C-terminal V5 epitope tags, were expressed in primary human endothelial cells and their subcellular localisation was compared by immunofluorescence. Escape from nonsense-mediated decay of TOF FLT4 PTVs, and activation of proteostatic signalling of both TOF variants was assessed by immunoblotting techniques. Differentially expressed genes (DEGs) were examined by RNAseq analysis. Rescue of gene expression changes was investigated following treatment with chemical inhibitors of the three main pathways of proteostasis.

Results TOF FLT4 variants of both types display predominantly endoplasmic reticulum (ER)/perinuclear subcellular localisation, and activation of proteostatic signalling responses, compared to WT and MD FLT4, which exhibit mainly plasma membrane staining (Figure 1). TOF FLT4 PTVs are expressed, but at a lower level than WT, however, this can be augmented by treatments simulating low oxygen levels. Transcriptomic analyses revealed a subset of DEGs that are TOF FLT4-specific compared to both WT and MD expressing cells (TFSGs, Table 1). Gene ontology analysis showed that TFSGs were enriched for proteostatic, metabolic and developmental signalling processes. TFSGs were also compared with stably heart expressed developmental genes (SHDGs) and showed significant overlap when examined by permutation testing (Figure 2), directly linking in vitro and in vivo transcriptomic data. Inhibitors of all three proteostatic signalling pathways rescued TFSG expression changes, to differing degrees, confirming the mechanism of pathogenesis for FLT4 variants in TOF.

Abstract BS29 Figure 1

IF of subcellular localisation of FLT4 variants associated with MD and TOF

Abstract BS29 Figure 2

TFSGs overlapping SHDGs compared to an equal number of random genes by permutation analysis

Abstract BS29 Table 1

TFSGs identified by RNAseq analysis of FLT4 variant expressing primary human endothelial cells

Conclusions We demonstrate a gain-of-function mechanism, with varying degrees of penetrance, that is responsible for the TOF phenotype. This contrasts with the already established dominant negative mechanism, that leads to Milroy lymphoedema with other FLT4 variants. Our results succinctly delineate the mechanisms of FLT4 pleiotropy in two unrelated cardiovascular conditions and suggest that targeting proteostatic signalling could identify potential pathways to therapeutic interventions in FLT4-associated TOF.

  • congenital heart disease
  • genetic pleiotropy
  • vascular endothelial growth factor receptor 3

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