Loss of Function of the Nuclear Receptor NR2F2, Encoding COUP-TF2, Causes Testis Development and Cardiac Defects in 46,XX Children

Abstract

Emerging evidence from murine studies suggests that mammalian sex determination is the outcome of an imbalance between mutually antagonistic male and female regulatory networks that canalize development down one pathway while actively repressing the other. However, in contrast to testis formation, the gene regulatory pathways governing mammalian ovary development have remained elusive. We performed exome or Sanger sequencing on 79 46,XX SRY-negative individuals with either unexplained virilization or with testicular/ovotesticular disorders/differences of sex development (TDSD/OTDSD). We identified heterozygous frameshift mutations in NR2F2, encoding COUP-TF2, in three children. One carried a c.103_109delGGCGCCC (p.Gly35Argfs^∗75) mutation, while two others carried a c.97_103delCCGCCCG (p.Pro33Alafs^∗77) mutation. In two of three children the mutation was de novo. All three children presented with congenital heart disease (CHD), one child with congenital diaphragmatic hernia (CDH), and two children with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). The three children had androgen production, virilization of external genitalia, and biochemical or histological evidence of testicular tissue. We demonstrate a highly significant association between the NR2F2 loss-of-function mutations and this syndromic form of DSD (p = 2.44 × 10^-8). We show that COUP-TF2 is highly abundant in a FOXL2-negative stromal cell population of the fetal human ovary. In contrast to the mouse, these data establish COUP-TF2 as a human "pro-ovary" and "anti-testis" sex-determining factor in female gonads. Furthermore, the data presented here provide additional evidence of the emerging importance of nuclear receptors in establishing human ovarian identity and indicate that nuclear receptors may have divergent functions in mouse and human biology.

Keywords: COUP-TF2; NR2F2; new syndrome; nuclear receptor; sex determination; testicular DSD.

Figure 1

Identification of Three Individuals with 46,XX DSD and Heterozygous Frameshift Mutations in NR2F2 (A) Representative Sanger sequence chromatograms of individuals 1–3 showing the positions of the frameshift mutations. (B) Histology of the right gonad of individual 3 showing testicular tubule-like structures surrounded by stromal-like tissue (scale bar corresponds to 100 μm). (C) Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) of individual 3. (D) Uterus (arrow) of individual 2 observed by MRI. (E) Pelvic radiography of individual 3 showing the vagina (V) and a short urogenital sinus (UrS). (F) Schematic representation of COUP-TF2 showing the main functional domains and the position and downstream consequences of the three frameshift mutations. The first zinc finger motif is highlighted in green in the sequence alignment. The transcript ID is GenBank: NM_021005.3.

Figure 2

Protein Localization of COUP-TF2 and FOXL2 during Early Human Ovarian Development (A–C) Immunofluorescence showing protein localization of COUP-TF2 (green) and FOXL2 (red) at gestational week (GW) 9+1. (D–F) Immunohistochemistry at GW 9+5. Extensive staining of COUP-FT2 is observed in the stromal cell population of the developing fetal ovary. At both stages there appears to be a mutually exclusive presence of FOXL2 and NR2F2, suggesting they mark different somatic cell populations. Dashed box in (A) and (C) indicates the position of the expanded views. Nuclei are counterstained with DAPI. Fetal age was determined by scanning crown-rump length and by evaluation of foot length. Sex of fetal samples was determined by PCR for SRY as previously reported. Immunofluorescence was performed as previously described. Primary antibodies used were COUP-TFII/NR2F2 (Perseus Proteomics, PP-H7147-60, diluted 1:100) and FOXL2 (a kind gift from Dagmar Wilhelm, diluted 1:100). Negative controls were included and processed with the primary antibody replaced by the dilution buffer alone. None of the negative control slides showed staining. Fluorescent images were captured using an Olympus BX61 microscope (Olympus) with the Cell Sens Dimension software v.1.16.