A novel NR2F2 loss-of-function mutation predisposes to congenital heart defect

Abstract

Congenital heart defect (CHD) is the most common type of birth defect in humans and a leading cause of infant morbidity and mortality. Previous studies have demonstrated that genetic defects play a pivotal role in the pathogenesis of CHD. However, the genetic basis of CHD remains poorly understood due to substantial genetic heterogeneity. In this study, the coding exons and splicing boundaries of the NR2F2 gene, which encodes a pleiotropic transcription factor required for normal cardiovascular development, were sequenced in 168 unrelated patients with CHD, and a novel mutation (c.247G > T, equivalent to p.G83X) was detected in a patient with double outlet right ventricle as well as ventricular septal defect. Genetic scanning of the mutation carrier's relatives available showed that the mutation was present in all affected family members but absent in unaffected family members. Analysis of the index patient's pedigree displayed that the mutation co-segregated with CHD, which was transmitted as an autosomal dominant trait with complete penetrance. The nonsense mutation was absent in 230 unrelated, ethnically-matched healthy individuals used as controls. Functional deciphers by using a dual-luciferase reporter assay system revealed that the mutant NR2F2 protein had no transcriptional activity as compared with its wild-type counterpart. Furthermore, the mutation abrogated the synergistic transcriptional activation between NR2F2 and GATA4, another core cardiac transcription factor associated with CHD. This study firstly associates NR2F2 loss-of-function mutation with an increased susceptibility to double outlet right ventricle in humans, which provides further significant insight into the molecular mechanisms underpinning CHD, suggesting potential implications for genetic counseling of CHD families and personalized treatment of CHD patients.

Introduction

Congenital heart defect (CHD) is the most common form of birth malformation in humans, with an estimated prevalence of 1% in live births and as high as 10% in stillbirths (Benjamin et al., 2017, Fahed et al., 2013). Clinically, CHD is usually categorized into 25 different types, encompassing ventricular septal defect (VSD), double outlet right ventricle (DORV) and tetralogy of Fallot (Benjamin et al., 2017, McDermott et al., 2017). Although mild CHD can resolve spontaneously (Benjamin et al., 2017), severe CHD can lead to diminished quality of life (Neiman et al., 2017), decreased exercise tolerance (Chaix et al., 2016), brain development delay or brain injury (Marelli et al., 2016, Morton et al., 2017, Peyvandi et al., 2016), thromboembolism (Jensen et al., 2015, Masuda et al., 2017), infective endocarditis (Diller and Baumgartner, 2017, Kuijpers et al., 2017), pulmonary arterial hypertension (Müller et al., 2017, van der Feen et al., 2017), congestive heart failure (Budts et al., 2016, Hinton and Ware, 2017, Stout et al., 2016), arrhythmias (Holst et al., 2017, Khairy, 2016, Lüscher, 2016, McLeod and Warnes, 2016) and sudden cardiac death (Diller and Baumgartner, 2016, Engelings et al., 2016, Jortveit et al., 2016, Koyak et al., 2017, Williams, 2016). Presently, CHD is still the most common cause of birth defect–related demises in infants, with nearly 24% of infants who died of birth defects having cardiac malformations (Benjamin et al., 2017). Although vast advancement in treatment of CHD during past decades has allowed over 90% of newborns with CHD to survive into adulthood, it results in an increasing number of adults living with CHD, and moreover, the morbidity and mortality in adult CHD cases are much higher than the general population (Bouma and Mulder, 2017, Mandalenakis et al., 2017). Despite significant clinical importance, the etiologies underpinning CHD remain largely elusive.

Previous studies have demonstrated substantial genetic basis for CHD, and in addition to chromosomal anomalies such as trisomy of chromosome 21 and chromosome 22q11 deletion, mutations in over 60 genes have been causally linked to CHD in humans (Andersen et al., 2014, Asadollahi et al., 2017, Blue et al., 2017, Boyle et al., 2016, Cao et al., 2016, Chen et al., 2016, Chen et al., 2017, Edwards and Gelb, 2016, Ellesøe et al., 2016, Fahed et al., 2013, Huang et al., 2016, Huang et al., 2017, LaHaye et al., 2016, Li et al., 2016, Li et al., 2017, Li and Yang, 2017, Liu et al., 2016, Lu et al., 2016, Priest et al., 2016, Ramond et al., 2017, Reijnders et al., 2016, Rocha et al., 2016, Sifrim et al., 2016, Sun et al., 2016a, Sun et al., 2016b, Tong, 2016, Wang et al., 2017a, Wang et al., 2017b, Wells et al., 2016, Werner et al., 2016, Xu et al., 2017, Yoshida et al., 2016, Zaidi and Brueckner, 2017, Zhao et al., 2016, Zhou et al., 2016a, Zhou et al., 2016b). Among these CHD-associated genes, most encode cardiac transcription factors, including NKX2–5, GATA4, HAND1 and TBX20 (Li and Yang, 2017). The expression profiles and functional roles in the heart of these transcription factors partially overlap during embryogenesis, indicating that they constitute a core regulatory network crucial for cardiovascular morphogenesis (Li and Yang, 2017). Nevertheless, CHD is of pronounced genetic heterogeneity, and the genetic determinants for CHD in most patients remain unclear.

Recently, terminal deletions of chromosome 15q, where several genes including the NR2F2 gene are located, have been associated with syndromic CHD in humans, including VSD, atrial septal defect, and coarctation of the aorta (Nakamura et al., 2011). Furthermore, the expression of NR2F2 in the developing human fetal heart including the atria, coronary vessels, and aorta has been substantiated, and NR2F2 mutations have been causally linked to isolated CHD, including atrioventricular septal defect, tetralogy of Fallot, aortic stenosis, VSD, coarctation of the aorta and hypoplastic left heart syndrome (Al Turki et al., 2014). These observational results make it reasonable to screen NR2F2 as a preferable candidate gene for CHD in another cohort of patients.