The diagnosis and management of the 46,XY infant with severe underandrogenization or genital ambiguity is one of the greatest challenges faced by pediatric endocrinologists, urologists and geneticists. Making a correct diagnosis can have implications for gender assignment, the likely response to hormone treatment, sexual function, fertility options, gonadal malignancy risk and for providing informed and appropriate counseling to the family. Thus, improving our understanding of the genetic basis of DSD in humans has important translational consequences (15
SF1 is a nuclear receptor that plays a central role in adrenal and reproductive function as it influences gene transcription at multiple levels and at different stages of development. Complete loss of Sf1
function in mice results in apoptosis of the developing adrenal gland and gonad during early embryogenesis (3
). The first human SF1 mutation, a heterozygous G35E change, was reported in a 46,XY female patient with primary adrenal failure, relatively severe gonadal dysgenesis and Müllerian structures (8
). Subsequently, the description of a homozygous R92Q mutation in SF1 in an infant with a similar phenotype led to the proposal that functional gene dosage effects of SF1 are important and that loss of SF1 function between haploinsufficiency and null can be associated with a gonadal and adrenal phenotype in humans (9
Recent reports of three heterozygous frameshift or nonsense mutations in NR5A1
/SF1 (1058-1065del8bp, 18delC, C16X) in patients with 46,XY DSD and apparently normal adrenal function support the concept that haploinsufficiency of SF1 (or partial loss of function) can present with a predominantly gonadal phenotype in humans (12
). In this current report, we show that heterozygous missense mutations in critical regions of NR5A1
/SF1 can be associated with 46,XY DSD and normal adrenal function, and that such NR5A1
/SF1 mutations account for 13% of cases of previously undiagnosed 46,XY gonadal dysgenesis/impaired androgenization.
The SF1 mutations identified in our cohort were found predominantly in individuals with impaired Leydig cell function and androgen biosynthesis rather than significant gonadal dysgenesis or classic Swyer syndrome (complete gonadal dysgenesis and Müllerian structures). Testicular architecture was relatively intact post-natally in the three individuals with amino-terminal mutations (V15M, M78I, C91S) who were raised female and underwent early gonadectomy. Müllerian regression had occurred in all cases, and the androgen response to hCG stimulation was surprisingly impaired for testicular size and location. Although AMH levels were low in the two cases studied, this finding may reflect impaired SF1 transcription of the AMH promoter rather than a direct consequence of Sertoli cell dysfunction. Well-developed Wolffian structures (vasa deferentia, epididymes) were present, which are an unusual feature in 46,XY patients with such significant underandrogenization.
The identification of naturally-occurring missense mutations in SF1 is also helping to reveal important functional domains for nuclear receptor action. The three mutations that lie within the very highly conserved amino-terminal region of the protein (V15M, M78I, G91S) affect DNA binding and target gene transactivation (, , ). The valine to methionine mutation at codon 15 (V15M) is a relatively mild disruption, but lies in a critical part of the first zinc-finger of the DBD () (10
). The glycine to serine mutation at position 91 (G91S) affects a crucial amino-acid in the A-box region of the FTZ-F1 domain, which is involved in stabilizing DNA binding by monomeric receptors through an interaction with the PyCA flanking sequencing of the half-site (PyCA
AGGPyCPu) in the minor groove of DNA. A partial loss-of-function mutation in an adjacent codon (R92Q) was reported previously, with phenotypic expression only in the homozygous state (9
). The methionine to isoleucine mutation at position 78 (M78I) affects a highly conserved codon between the zinc-fingers and A-box, in the amino-terminal region of the FTZ-F1 domain () (23
). Although relatively little is known about this region, our studies of GFP-SF1 fusion proteins show that this M78I mutation exhibits marked clustering in sub-nuclear PML (or ND10) bodies in many cells (), as well as impaired DNA binding (). Similar aggregation has been reported for mutations in several other nuclear receptors (e.g. estrogen receptor, androgen receptor, glucocorticoid receptors), or following stimulation with signaling pathway activators or when associated with co-factor (e.g. GRIP1, RIP140) as part of a ubiquitin-proteosome complex or within the pre-assembly transcriptional complex machinery (24
). Recent data have also demonstrated focal clustering for SF1 following protein kinase A pathway stimulation (29
), when associated with co-activators and co-repressors (e.g. GCN5, DAX1) (30
), and following recruitment of SF1 by p300/CBP to a p300-RNA Pol II locus prior to acetylation, DNA binding and transcriptional activation (31
). Thus, it is possible that the M78I mutant (and V15M) becomes aggregated in these complexes and prevents subsequent transcriptional activation, or cannot dissociate from the complexes causing sequestration of co-factors and transcriptional machinery. Such mechanisms may be an important additional means by which mutations in nuclear receptor transcription factors can cause human disease. Finally, disparate effects between the G35E and other point mutants were not seen in the different cell lines studied here. Thus, the G35E mutant may have partial dominant negative effects in more complex or adrenal-specific gene transcription systems, or modifier loci may be important in dictating the predominantly gonadal versus combined gonadal and adrenal phenotype.
In contrast to these DNA-binding region mutations, the L437Q mutation is the first ligand-binding region mutation reported in SF1 and is the first reported case of a mild phenotype (penoscrotal hypospadias) in a patient raised male. Consistent with this phneotype, the L437Q SF1 mutant retained partial function in several SF1 expressing cell lines (). In contrast, his testicular biopsy at 6 years of age showed more marked changes than in Subjects 1-3. Whether these histological changes represent a progressive post-natal deterioration in testicular integrity with time or the consequences of testicular maldescent is unclear. Analysis of further patients with SF1 mutations who are raised male will be necessary to confirm whether progressive testicular changes occur with time.
Recently, the putative LBD of SF1 has been crystalized and shown to interact with phospholipid ligands (19
). These ligands may be important in the interface between PI3-kinase signaling pathways and SF1 activation. The L437Q mutation reported here affects an amino acid thought to interact directly with the phospholipid ligand as part of the ligand-binding pocket. The leucine to glutamine mutation would be predicted to de-stabilize this domain through replacement of the hydrophobic side chain with the polar amide group (19
). Of note, this patient appears to be developing a partial form of hypogonadotropic hypogonadism in addition to a primary testicular defect. PI3-kinase-dependent signaling may be more important in the gonadotrope compared to the adrenal (32
). Further studies through adolescence will be necessary to validate this hypothesis, once puberty has been induced with sex steroid supplementation.
Taken together, these cases show that heterozygous missense mutations in NR5A1
/SF1 are emerging as a relatively frequent association with 46,XY disorders of sex development with intact adrenal steroid biosynthesis. Whether these patients will develop adrenal failure with time remains to be seen, but this study supports the hypothesis that, in humans, the developing testis may be more sensitive to disruption by partial loss of SF1 function than the developing adrenal gland (22
), and that androgen biosynthesis is more severely affected than testicular integrity. Furthermore, the apparent sex-limited dominant transmission of NR5A1
/SF1 mutations in two cases here (M78I, G91S) support reports of preserved ovarian development and function in a 46,XX girl who has adrenal failure due to a heterozygous R255L mutation in SF1 (35
). Indeed, ovarian development is relatively preserved following tissue specific targeted deletion of Sf1
in mice, possibly due to the compensatory role of the related nuclear receptor LRH1 (NR5A2
). The families described here could be at risk of having future affected 46,XY fetuses or carrier daughters, similar to an X-linked pattern of inheritance. Further studies will be necessary to establish the exact prevalence of NR5A1
/SF1 mutations in patients with reproductive disorders, the long-term risk of adrenal dysfunction, whether different disease mechanisms (e.g., ligand-binding defects; nuclear aggregation) have different clinical consequences, and whether polymorphic variants of SF1 (e.g. G146A) are important modifiers of phenotypic expression of reproductive disorders (38