Fig. 7: A NEDD4 variant identified in an individual with congenital heart disease has impaired ubiquitination of DKK1. | Nature Communications

Fig. 7: A NEDD4 variant identified in an individual with congenital heart disease has impaired ubiquitination of DKK1.

From: Neural crest cell-derived DKK1 and NEDD4 modulate Wnt signalling in the second heart field to orchestrate outflow tract development

Fig. 7: A NEDD4 variant identified in an individual with congenital heart disease has impaired ubiquitination of DKK1.

A Pedigree of family with a NEDD4 variant. Genomic sequencing of the trio (parents and proband) revealed a homozygous NEDD4 missense variant c.2297 A > G (p.K766R) in the proband with Tetralogy of Fallot. Hole in the heart was the term used by the family to describe distant relatives, with no clinical data available. B Protein domain structure of NEDD4, indicating the position of the K766R amino acid substitution. C Western blot of in vitro ubiquitination assay. Representative image from n = 3 independent experiments. HEK293T cells were co-transfected with DKK1-GFP, Ub-HA, and either NEDD4 WT, FLAG-NEDD4 CS (cysteine mutant), or FLAG-NEDD4 K766R. Immunoprecipitation for GFP demonstrates HA positivity with NEDD4 WT, indicating ubiquitination of DKK1, but minimal ubiquitination with NEDD4 CS or NEDD4 K766R. D HeLa cells transfected with DKK1-GFP and FLAG-NEDD4 WT, CS, or K766R, immunostained for FLAG and DKK1. Representative images from n = 3 independent experiments. Mean fluorescence intensity of DKK1 and NEDD4 was quantified for individual cells and plotted graphically. Simple linear regression analysis reveals a negative correlation between DKK1 and NEDD4 WT levels, but not for NEDD4 CS or K766R. For transfection control for DKK1 construct, see Supplementary Fig. 16. ns not significant. E Representative image of control and Nedd4K766R/K766R E15.5 and E17.5 embryos, from six independent litters. F Frequency of heart defects in E15.5-E17.5 Nedd4K766R/K766R embryos, with defects noted in the right ventricle and interventricular septum. G Histological sections of control and Nedd4K766R/K766R E15.5 embryos showing rugged surface of right ventricular wall (arrowheads), right ventricular non-compaction, and ventricular septal defect (VSD). Representative images from n = 3 Nedd4+/+ and n = 4 Nedd4K766R/K766R embryos. H Histological sections of control and Nedd4K766R/K766R E17.5 embryos showing right ventricular non-compaction. Representative images from n = 3 Nedd4+/+ and n = 5 Nedd4K766R/K766R embryos. Source data are provided as a Source Data file.

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