Rare variants in KDR, encoding VEGF Receptor 2, are associated with tetralogy of Fallot

Doris Škorić‐Milosavljević ,David Brook,Amy Roberts,Sarah E Sheppard,Fleur V.Y Tjong,Laura Cross,Fernanda M Bosada,Simon G Williams,Marc‐Phillip Hitz ,Tomi Pastinen,S Atmani ,Irene M Kuipers ,Vincent M Christoffels,Anna Wilsdon,Paul Kruszka,Katharina Steindl,Heather C Mefford ,Sally‐Ann B Clur ,Seema Mital,Aho Ilgun,Alanna Strong,Elaine H Zackai ,Elisabeth M Lodder,Leander Beekman,Ilham Ratbi,Anita Rauch,Najlae Adadi,Najim Lahrouchi,Boris Keren,Nico A Blom,Karim Ouldim,Dihong Zhou,Jeroen Breckpot,Gregor Dombrowsky,Bernard Keavney ,Ihssane El Bouchikhi,Solveig Heide,Alex V Postma,Enrique Audain,Pascal Joset,Isabelle Thiffault,Barbara J.m Mulder ,Robert Lesurf,Maximilian Muenke,Mariam Hababa,Roddy Walsh,Alison M Muir,Connie R Bezzina

doi:10.1038/s41436-021-01212-y

Abstract

PurposeRare genetic variants in KDR, encoding the vascular endothelial growth factor receptor 2 (VEGFR2), have been reported in patients with tetralogy of Fallot (TOF). However, their role in disease causality and pathogenesis remains unclear. MethodsWe conducted exome sequencing in a familial case of TOF and large-scale genetic studies, including burden testing, in >1,500 patients with TOF. We studied gene-targeted mice and conducted cell-based assays to explore the role of KDR genetic variation in the etiology of TOF. ResultsExome sequencing in a family with two siblings affected by TOF revealed biallelic missense variants in KDR. Studies in knock-in mice and in HEK 293T cells identified embryonic lethality for one variant when occurring in the homozygous state, and a significantly reduced VEGFR2 phosphorylation for both variants. Rare variant burden analysis conducted in a set of 1,569 patients of European descent with TOF identified a 46-fold enrichment of protein-truncating variants (PTVs) in TOF cases compared to controls (P = 7 × 10-11). ConclusionRare KDR variants, in particular PTVs, strongly associate with TOF, likely in the setting of different inheritance patterns. Supported by genetic and in vivo and in vitro functional analysis, we propose loss-of-function of VEGFR2 as one of the mechanisms involved in the pathogenesis of TOF.

Highlights

Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart defect (CHD).[1]
Genetic analysis and knock-in mouse model of index family with tetralogy of Fallot We performed exome sequencing on DNA from two siblings of Moroccan descent affected by TOF with suspected recessive inheritance
In an effort to shed light on the biological mechanisms of the two KDR variants of the index family, we examined the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) at Tyr1175, one of its major phosphorylation sites[17] and crucial for the recruitment of proteins in the signaling cascade downstream of VEGFR2.17,18 HEK 293T cells heterologously expressing the two identified variants or wild-type KDR were stimulated with VEGF165

Summary

Introduction

Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart defect (CHD).[1] TOF can present in combination with extracardiac defects, in the majority of cases it presents as an isolated defect.[2] An increased risk of CHD among first-degree relatives and offspring of TOF patients[3,4] provides evidence for a genetic contribution to the disease etiology. A microdeletion on chromosome 22q11.2 is the most common genetic abnormality identified in patients with TOF, accounting for ~15% of cases.[5] In addition, several other genes, mainly encoding cardiac transcription factors,[6,7] have been implicated in TOF, these account for a minority of patients, and the majority of cases remain genetically elusive. Over the last few years, dysregulated vascular endothelial growth factor (VEGF) signaling h-

Methods

Results

Conclusion