Homozygous inactivating mutation in NANOS3 in two sisters with primary ovarian insufficiency

Abstract

Despite the increasing understanding of female reproduction, the molecular diagnosis of primary ovarian insufficiency (POI) is seldom obtained. The RNA-binding protein NANOS3 poses as an interesting candidate gene for POI since members of the Nanos family have an evolutionarily conserved function in germ cell development and maintenance by repressing apoptosis. We performed mutational analysis of NANOS3 in a cohort of 85 Brazilian women with familial or isolated POI, presenting with primary or secondary amenorrhea, and in ethnically-matched control women. A homozygous p.Glu120Lys mutation in NANOS3 was identified in two sisters with primary amenorrhea. The substituted amino acid is located within the second C2HC motif in the conserved zinc finger domain of NANOS3 and in silico molecular modelling suggests destabilization of protein-RNA interaction. In vitro analyses of apoptosis through flow cytometry and confocal microscopy show that NANOS3 capacity to prevent apoptosis was impaired by this mutation. The identification of an inactivating missense mutation in NANOS3 suggests a mechanism for POI involving increased primordial germ cells (PGCs) apoptosis during embryonic cell migration and highlights the importance of NANOS proteins in human ovarian biology.

Figure 1

A Mutational analysis of NANOS3 revealed a homozygous c.358G>A substitution in exon 1 in two sisters with primary amenorrhea. The GAG to AAG change in codon 120 results in a substitution from glutamic acid to lysine in the codified protein (p.Glu120Lys). B, Cartoon representation of the NANOS3 protein (192 amino acids), with the zinc finger domain (amino acid 76 to 130) depicted in yellow, showing the location of the p.Glu120Lys mutation. Glutamic acid 120 (shown as E120) is localized within one of two highly conserved Cys-Cys-His-Cys (C2HC) motifs (shaded red and yellow boxes). Conservation among species of the second C2HC motif sequence (human residues 112 to 128) is shown in detail; glutamic acid 120 (E) is highly conserved. Alignment was performed in Clustal Omega using protein sequences obtained from UniProt.

Figure 2

Detection of apoptosis using flow cytometry and Annexin-V staining in COS-1 cells transfected with wild type (WT) or p.Glu120Lys mutant (MUT) NANOS3-GFP, before and after induction of apoptosis with sodium butyrate (NaBu). Expression of mutant NANOS3 results in significantly higher apoptosis in comparison to wild type (P < 0.05). Error bars represent SEM for nine replicates; NT: nontransfected control.

Figure 3

Confocal microscopy analysis of apoptosis in COS-1 cells following 48 h induction of apoptosis with sodium butyrate. In cells transfected with wild type (WT, upper row) or p.Glu120Lys mutant (Mut, lower row) GFP-tagged NANOS3, expression of green NANOS3-GFP is seen predominantly in the nucleus. Red Annexin-V staining in the cytoplasm and plasma membrane, indicating apoptosis, is seen in cells expressing p.Glu120Lys mutant NANOS3 but not in those expressing WT NANOS3.

Figure 4

In silico analysis of amino acid interaction modification shows the different conformation conferred by the p.Glu120Lys substitution. Glutamic acid 120 (shown as E120) makes a hydrophobic interaction with lysine 96 (K96) and hydrogen bonds with both glycine 116 (G116) and threonine 124 (T124). The substitution to lysine 120 (K120) modifies structural points of contact establishing a new interaction with histidine 123 (His123) through aromatic stacking and abolishes the hydrophobic interaction with K96.

Figure 5

Bulk representation of wild-type NANOS3, with p.Glu120Lys mutant shown in details box. The dashed circle highlights a protein surface region rich in basic residues, shown in blue. At the center of this region, lies the acidic residue glutamic acid 120 (E120), shown in red. In the details, substitution by lysine 120 (K120) disturbs the electrostatic interactions among adjacent residues.