We identified a series of missense, in-frame, and frameshift mutations in the NR5A1
gene in association with anomalies of ovarian development and function. In four of the probands, whose condition was diagnosed as 46,XY disorders of sex development, the mutation was familial. In each of these families, another member of the family with primary ovarian insufficiency also harbored the mutation. We identified two additional heterozygous mutations by screening 25 female subjects with primary ovarian insufficiency. In all subjects, there was no evidence of adrenal dysfunction, a finding that is consistent with reported mutations in NR5A1
in patients with 46,XY disorders of sex development with apparently normal adrenal function.27-34
Taken together, these data indicate that mutations in NR5A1
can cause ovarian insufficiency.
The subjects we describe show variations in expressivity of the phenotype, penetrance, and modes of inheritance. Families 1, 3, and 4 show a dominant mode of inheritance, whereas Family 2 shows a recessive mode. Families 1 and 4 carry heterozygous frameshift mutations that generate a premature termination codon predicted to result in truncated proteins. Nonsense-mediated decay usually results in the degradation of mRNAs containing a premature termination codon at least 50 nucleotides upstream of the last exon–exon boundary.36
The two mutations, c.666delC and c.390delG, result in a premature termination codon at amino acid position 295, predicted to be recognized by the nonsense-mediated decay surveillance complexes and degraded. Our functional analyses indicated that even if a truncated protein were produced, it would have severely impaired transcriptional activity (data not shown).
In Family 3, both XY and XX affected family members harbored the same heterozygous c.3G→A transition as their apparently unaffected mother. This mutation abolishes the Kozak consensus sequence for translation initiation, which may either abolish translation or result in a defect in translation. An alternative initiation codon is located downstream at codon position 78. Translation initiation at this codon is predicted to truncate the N-terminal of the protein, eliminating the DNA-binding domain. The absence of a phenotype in the mother carrying the same mutation as the affected family members suggests incomplete penetrance of the mutant allele, owing to a variable translation defect, the existence of modifier genes, or environmental effects. This is consistent with incomplete penetrance of NR5A1
mutations present in 46,XY dizygotic twins with disorders of sex development: one boy presented with anorchia, and the other had normal testicular development.33
In six reported patients with 46,XY disorders of sex development, the NR5A1
mutation was inherited from the mother, who had apparently normal ovarian function.30-34
These cases may be explained by incomplete penetrance of the mutant allele or by a progressive failure of ovarian function. In one published familial case, the mother of a 46,XY boy with anorchia who also harbored a p.Val355Met mutation in NR5A1
underwent left ovariectomy and homolateral fallopian tube ablation for ovarian cysts at the age of 22 years and subsequently had two spontaneous miscarriages, an outcome that suggests impaired ovarian function.33
In Family 2, it is simplest to interpret the mode of inheritance as recessive. The two affected family members had a homozygous p.Asp293Asn mutation in a region of the gene that encodes the ligand-binding domain of NR5A1. Unaffected family members who were genotyped were either heterozygous or lacked the mutation. A recessive mode of inheritance for this family can be explained by the residual activity of the NR5A1 p.Asp293Asn protein, which was about half as active as wild-type NR5A1.
During further screening of isolated cases of ovarian insufficiency, we detected heterozygous NR5A1
mutations in two subjects. The mutation in Subject 1 was predicted to result in the deletion of three amino acids (p.Leu231_Leu233) in the ligand-binding domain of NR5A1. These three amino acids are highly conserved in subclass V of nuclear receptors and overlap with the first helix (Helix 1) of the ligand-binding domain. Helix 1 contains an activation function domain (AFH1) that is essential for maximal receptor function through interaction with coactivators.37
Disruption of this structure is predicted to dramatically alter NR5A1 stability and activity, as demonstrated by the failure of mutant NR5A1 (p.Leu231_Leu233del) to transactivate the CYP11A1
promoters. We observed two changes on a single chromosome in the second subject with isolated primary ovarian insufficiency (data not shown). One of these changes, p.Pro129Leu, is probably pathogenic, since it severely impairs transcriptional activity.
We have shown that mutations in NR5A1
are associated with human ovarian insufficiency, an observation consistent with the hypoplastic ovaries and infertility of mice lacking Nr5a1
in their granulosa cells.23
plays an important role in human ovarian development and function. During early human embryonic development, NR5A1
is expressed in the bipotential gonad in both sexes and is not sexually dimorphic after sex determination. A broad range of expression persists in the developing human fetal ovary, as well as in the testis.38,39
In cycling human ovaries, NR5A1
is expressed in the undeveloped follicles during the preantral phase.38
It is also expressed in theca interna cells, in luteinized and nonluteinized granulosa cells, and in corpus luteum during the luteal phase, as well as in both atretic follicles and degenerating corpora lutea.38
In both theca and granulosa cells, NR5A1
regulates genes required for ovarian steroidogenesis and follicle growth and maturation, including, STAR, CYP11A1, CYP17A1, CYP19A1, LHCGR
(encoding luteinizing hormone receptor), and INHA
Dysregulation of any of the proteins encoded by these genes could lead to ovarian dysfunction. Our data show that mutated forms of NR5A1
, detected in subjects with anomalies of ovarian development and function, show quantitative impairment in the transactivation of two of these factors (CYP11A1
Our data suggest that mutated NR5A1 is associated with a progressive loss of ovarian reproductive capacity. A diagnostic genetic test could aid in counseling for the possibility of familial recurrence and in evaluation of prospects for treatment of primary ovarian insufficiency.