NOBOX homeobox mutation causes premature ovarian failure

Abstract

NOBOX (newborn ovary homeobox gene) is an oocyte-specific homeobox gene that plays a critical role in early folliculogenesis and represents a candidate gene for nonsyndromic ovarian failure. We investigated whether mutations in the NOBOX gene cause premature ovarian failure (POF). We sequenced the NOBOX gene in 96 white women with POF and discovered seven known single-nucleotide polymorphisms and four novel variations, two of which, p.Arg355His and p.Arg360Gln, cause missense mutations in the homeobox domain. Electrophoretic mobility shift assay (EMSA) confirmed that the missense mutation, p.Arg355His, disrupted NOBOX homeodomain binding to NOBOX DNA-binding element (NBE) and had a dominant negative effect on the binding of wild-type NOBOX to DNA. Our findings demonstrate that NOBOX mutations can cause POF.

Figure  1.

Identification of novel variants in the NOBOX gene. A, Schematic diagram of the NOBOX gene, showing three novel nonsynonymous variations (exon 6 and exon 8) and one synonymous variant (exon 1) found in the present study. Exons are a composite of human sequence NM_001080413 (GenBank) and ENST00000389325 (Ensembl). The homeodomain region is coded by exons 4–6. B, Electropherogram showing wild-type (a and c) and mutant (c.1064G→A [Arg355His] and c.1079G→A [Ar360Gln]) sequences. Arrows indicate the G→A change in POF subject 23 (b, Arg355His) and POF subject 34 (d, Ar360Gln). C, NOBOX homeodomain alignment among different species. Arrow heads indicate highly conserved amino acids highly conserved among homeodomains. The two missense mutations (p.Arg355His and p.Arg360Gln) found in this study are indicated by arrows. Asterisks (*) indicate amino acid residues that are conserved in all the species.

Figure  2.

GST-R186H binds to the TAATTG sequences with low affinity. ³²P-labeled TAATTG was incubated with purified recombinant GST-NXHD, GST-R191Q, or GST-R186H. ³²P-labeled TAATTG formed a strong DNA-protein complex with the recombinant GST-NXHD (lane 2) and GST-R191Q protein (lane 4), but not with GST-R186H (lane 6). The DNA-protein complex was supershifted when incubated with polyclonal antibodies against GST (lanes 3, 5 and 7). The presence or absence of recombinant protein and/or antibody is indicated above each lane with a plus (+) or minus (−) sign, respectively. Arrows indicate DNA-protein complex (BP), supershifted complex (SS) and free probe (FP). Oligonucleotide sequence containing TAATTG is 5′-ACG AGC TAC CTT ACT TAA TTG GAC GTT GAA CGC GTA CTG T-3′.

Figure  3.

Dominant negative effect of GST-R186H. Radiolabeled NBE (³²P-TAATTG) was incubated individually with purified recombinant GST-NXHD, GST-R186H, or a 50/50 mixture of both homeodomains. GST-R186H reduced GST-NXHD binding affinity to the ³²P-labeled TAATTG (lane 4). The specificity of the DNA-protein complexes was confirmed by polyclonal antibodies against GST. The presence or absence of recombinant protein and/or antibody is indicated above each lane with a plus (+) or minus (−) sign, respectively. Arrows indicate DNA-protein complex (BP), supershifted complex (SS) and free probe (FP).