Rare variants in NR2F2 cause congenital heart defects in humans

Abstract

Congenital heart defects (CHDs) are the most common birth defect worldwide and are a leading cause of neonatal mortality. Nonsyndromic atrioventricular septal defects (AVSDs) are an important subtype of CHDs for which the genetic architecture is poorly understood. We performed exome sequencing in 13 parent-offspring trios and 112 unrelated individuals with nonsyndromic AVSDs and identified five rare missense variants (two of which arose de novo) in the highly conserved gene NR2F2, a very significant enrichment (p = 7.7 × 10(-7)) compared to 5,194 control subjects. We identified three additional CHD-affected families with other variants in NR2F2 including a de novo balanced chromosomal translocation, a de novo substitution disrupting a splice donor site, and a 3 bp duplication that cosegregated in a multiplex family. NR2F2 encodes a pleiotropic developmental transcription factor, and decreased dosage of NR2F2 in mice has been shown to result in abnormal development of atrioventricular septa. Via luciferase assays, we showed that all six coding sequence variants observed in individuals significantly alter the activity of NR2F2 on target promoters.

Figure 1

Structure of NR2F2 and the Encoded Protein (A) NR2F2 has three coding exons and four transcripts (see Figure S3C). The transcript that generates the full-length protein (RefSeq NM_021005) is shown here annotated with functional variants in cases (red) and controls (blue). (B) Similar to other nuclear receptors, NR2F2 has three main domains: a ligand-binding (LBD), DNA-binding (DBD), and an activation binding motif (AF2). Three mutations in cases are located in the ligand-binding domain (LBD). Asterisk (^∗) denotes de novo variant. (C) The Grantham score for the missense mutations. (D) Two missense variants mapped onto the partial crystal structure for the NR2F2 ligand-binding domain. (E) c.753G>C (RefSeq NM_021005.3); p.Glu251Asp (RefSeq NP_066285.1) (purple) falls in the ligand-binding groove of the dimer, which in the repressed conformation is occupied by helix AF2 (red), and thus this variant is likely to perturb ligand binding. (F) c.1022C>A (RefSeq NM_021005.3); p.Ser341Tyr (RefSeq NP_066285.1) (blue) is likely to destabilize helix A10 through steric hindrance and thus decrease the stability of NR2F2 homodimerization (see Figure S5).

Figure 2

Pedigree Charts and Capillary Sequencing Results of NR2F2 Variants in Eight CHD-Affected Families Solid lines in pedigree charts indicate both whole-exome sequencing data and capillary sequencing are available; dashed lines indicate samples with NR2F2 capillary sequencing data only. See Table 1 for details.

Figure 3

Nr2f2 Expression in the Developing Mouse Embryo Nr2f2 mRNA expression (red) is detected in the atria of the heart, branchial arches, somites, and olfactory placode at 10.5 dpc by whole-mount in situ hybridization.

Figure 4

NR2F2 Localization in the Developing Human Heart Immunofluorescent analysis of NR2F2 in fixed human fetal heart via anti-NR2F2 (D–F, J–L, P–R, U–W, green) and colabelling (red) with CD34 (E, K, Q), troponin C (F), SMA (L, R), D2-40, and DAPI (W, blue). Haematoxylin and eosin staining (A–C, G–I, M–O, S). An additional autofluorescence artifact was detected (arrowhead F, P–R) from hemaglobin within erythrocytes. Negative control for NR2F2 shown in (T). The fields shown in (C), (I), (O), and (S) are from hematoxylin and eosin-stained serial sections adjacent to the fields shown in (D)–(F), (J)–(L), (P)–(R), and (T)–(W), respectively. The boxed areas in hematoxylin and eosin-stained fields represent the area shown in higher magnification in the adjacent field to the right. Abbreviations are as follows: LA, left atrium; Ao, aorta; Co.Art, coronary artery; CoVn, coronary vein; Lym, lymphatic vessel. Scale bars represent 100 μm.

Figure 5

NR2F2 Variants in AVSD-Affected Probands Affect Transcriptional Activity An NR2F2-responsive luciferase reporter driven by the NGFI-A or APOB upstream region was cotransfected with wild-type NR2F2, or identified coding variants (p.Gln75dup, p.Asp170Val, p.Asn205Ile, p.Glu251Asp, p.Ser341Tyr, and p.Ala412Ser) into HEK293T (NGFI-A and APOB) and HEPG2 (APOB) cells (see Subjects and Methods for details). Bar chart values are activity relative to wild-type NR2F2 (mean percentage ± SD). Repression of the APOB promoter in HEPG2 cells is shown as negative values to illustrate the direction of change from negative control. In HEK293 cells, all variants show significant difference from wild-type on one or both promoters. The p.Asn205Ile variant shows the reverse direction of change depending on which promoter was used. In HEPG2 cells, all variants retain wild-type levels of repressive activity. Asterisk indicates significant change from wild-type activity. Triangle indicates significant difference between promoters.