Mutations In Steroidogenic Factor-1 Research Articles

Abstract 24: NR5A1 mutation: A single centre experience from South India

Background: Inactivating mutations involving Nuclear receptor subfamily 5 group A member (NR5A1) or steroidogenic factor 1 (SF1), is increasingly being recognized as an important cause of 46XY DSD and rarely 46XX DSD. Myriad of presentations include undervirilized male with or without peripubertal virilization, ovotesticular DSD and premature ovarian failure. With the widespread availability of genetic testing it has been found that it constitutes atleast 15% of cases of 46XY DSD.Aims and Objective: To describe the clinical presentation of a series of patients with genetically confirmed SF1 mutations from a south Indian tertiary care centre.Result: Extreme undervirlization was noted in one subject whereas prepubertal virilization was noted in two subjects reared as females. Hormonal investigations revealed various varying degrees of Sertoli cell dysfunction (elevated FSH and low inhibin B), poor Leydig cell function in prepubertal subjects (HCG stimulated testosterone <0.087 nmol/l) and apparent recovery of Leydig cell function in peripubertal period (2.04 & 2.94 ng/ml). Exon Variants-Exon 2 c.58G>T(p.Val20Leu), Exon 5 c.940delinsACGGCAAGG (p.Gin314ThrfsTer23) and Exon 3 c.223A>C (p.Thr75Pro) were noted .Conclusion: 46XY DSD due to inactivating NR5A1 variants can present in unusual ways characterized by extreme undervirlization at birth with apparent recovery of Leydig cell function later on.

The first clinical presentation of disorders of sex development 46 XY due to mutation in Steroidogenic factor 1 (SF1) in Russian Literature

Steroidogenic factor 1 (SF1/AdBP4/FTZF1, NR5A1) is a nuclear receptor transcription factor that plays a critical role in different processes of sex development. Homozygous mutations in SF1 result in adrenal failure and complete testicular disgenesis in 46,XY individuals. According to recent studies heterozygous mutations in SF1 are associated with milder phenotype: they are found in children with 46,XY disorders of sex development (DSD) but with apparently normal adrenal structure and function. Here we present for the first time in Russian literature a case of SF1 deficiency. Molecular genetic analysis of NR5A1 gene revealed a novel heterozygous mutation c.951delC p.H317QfsX17. This clinical case demonstrates the importance of molecular genetic studies in DSD 46,XY, especially severe forms.

Mutation analysis of the SRD5A2, AR and SF-1 genes in 52 Chinese boys with hypospadias

To investigate the prevalence of genetic mutations in steroid 5α-reductase-2 (SRD5A2), androgen receptor (AR) and steroidogenic factor-1 (SF-1) in Chinese children with hypospadias, and to also explore the possible underlying molecular mechanisms of this disease. A total of 52 boys with hypospadias were enrolled. Mutational analyses of the SRD5A2, AR and SF-1 genes were performed by direct sequencing. SRD5A2 gene mutations were found in 13.5% (7/52 cases), including five compound heterozygotic and two homozygotic mutations. One novel heterozygotic SF-1 gene mutation was identified in a patient with perineal hypospadias and cryptorchidism, the patient's mother also had the same mutation. No mutation was found in the AR gene. The clinical manifestations of patients with mutations in SRD5A2 or SF-1 varied. In Chinese patients, SRD5A2 gene mutations were, relatively, frequently associated with hypospadias. The SF-1 gene may be another candidate gene for hypospadias. In contrast, AR gene mutations are not commonly associated with this condition.

Decoding the Functional Role of Fast, a Long Non-Coding RNA Transcribed Antisense to Nr5a1.

Over the past decade, research on long non-coding RNAs implicates them in regulatory roles associated with gene expression and chromatin architecture. While searching for conserved regulatory elements, an expressed sequence tag lacking an identifiable open reading frame was identified associated with Ftz-f1 (Nr5a1), the gene encoding steroidogenic factor 1 (SF-1). The nuclear receptor SF-1 is a key determinant and regulator of the adrenal and reproductive axes. Mammals with SF-1 mutations display a range of phenotypes, including absence of adrenal glands and gonads, diminished pituitary gonadotropins, disruption of the ventromedial hypothalamus, ovarian failure, and XY-sex reversal. While there are numerous studies on the function and regulation of SF-1, none involved mechanisms that require a noncoding RNA (ncRNA). Identification of the Ftz-F1 associated transcript, or Fast, and its association with SF-1 suggested a role for the ncRNA in SF-1 expression and/or activity and prompted further investigation of Fast. Directional RT-PCR was used to determine transcriptional orientation of Fast and demonstrated it is transcribed in the opposite orientation of Ftz-F1. Fast full-length structure was delineated using 5' and 3' RACE and sequence analysis of amplified Fast cDNA. This showed Fast has three exons and is polyadenylated and derived from multiple transcriptional start sites that border and extend into exon 1g of Ftz-F1. In addition to identification of a third exon, sequence analysis revealed two amplified, possible spliced variants that lacked part of exon 3. RT-PCR analysis of RNA from multiple mouse tissues and endocrine cell lines showed Fast and SF-1 share the same expression profile. To help define the sub-cellular location of Fast, nuclear and cytoplasmic RNA was isolated from MA-10 cells, a Leydig cell line, and analyzed for the presence of Fast by RT-PCR. This revealed that Fast is predominantly located in the cytoplasm. The similar tissue expression pattern for SF-1 and Fast suggested they share regulatory mechanisms. Further investigation showed that P-19 embryonal-carcinoma cells treated with retinoic acid had decreased SF-1 and Fast transcript levels and that granulosa cells isolated from mice treated with pregnant mare's serum gonadotropin had elevated levels of both Fast and SF-1 mRNA. Together, these findings indicate that Fast is a long non-coding RNA that shares the same tissue-restricted expression profile and hormone regulation as SF-1, suggesting it acts within a similar biological pathway. We hypothesize that Fast acts either in conjunction with SF-1 and thus is required for proper development and function of adrenal glands and gonads.

Isolated ‘idiopathic’ micropenis: hidden genetic defects?

Micropenis is defined as a stretched penile length of less than 2-2.5SD for age. Aetiologies include hypogonadotropic hypogonadism, testicular dysgenesis, defects in testosterone synthesis, androgen resistance [5α-reductase (5αR) deficiency or partial androgen insensitivity] and other rare causes like growth hormone GH deficiency. Often, the cause remains unknown. The aim of this study was to determine whether isolated micropenis with normal plasma testosterone could hide a molecular defect in the androgen pathway. Twenty-six boys with isolated micropenis were included in this study. All of them had 46,XY karyotype, normal luteinizing hormone and follicle-stimulating hormone and a normal plasma testosterone response to human chorionic gonadotropin testing. Androgen receptor (AR), 5αR and steroidogenic factor 1 (SF1) genes were sequenced. A mutation in the AR gene was found in two patients, and a new mutation in the SF1 gene was found in one patient who was the only one to have a low level of inhibin B (InhB). This is the first report of isolated micropenis as a revealing symptom of AR and SF1 mutations. Anti-Mullerian hormone and InhB should thus be evaluated in patients with isolated micropenis, even when plasma testosterone is in the normal range. Detection of gene mutations is helpful for diagnosis, treatment and genetic counselling for probands.

Failure of SOX9 Regulation in 46XY Disorders of Sex Development with SRY, SOX9 and SF1 Mutations

BackgroundIn human embryogenesis, loss of SRY (sex determining region on Y), SOX9 (SRY-related HMG box 9) or SF1 (steroidogenic factor 1) function causes disorders of sex development (DSD). A defining event of vertebrate sex determination is male-specific upregulation and maintenance of SOX9 expression in gonadal pre-Sertoli cells, which is preceded by transient SRY expression in mammals. In mice, Sox9 regulation is under the transcriptional control of SRY, SF1 and SOX9 via a conserved testis-specific enhancer of Sox9 (TES). Regulation of SOX9 in human sex determination is however poorly understood.Methodology/Principal FindingsWe show that a human embryonal carcinoma cell line (NT2/D1) can model events in presumptive Sertoli cells that initiate human sex determination. SRY associates with transcriptionally active chromatin in NT2/D1 cells and over-expression increases endogenous SOX9 expression. SRY and SF1 co-operate to activate the human SOX9 homologous TES (hTES), a process dependent on phosphorylated SF1. SOX9 also activates hTES, augmented by SF1, suggesting a mechanism for maintenance of SOX9 expression by auto-regulation. Analysis of mutant SRY, SF1 and SOX9 proteins encoded by thirteen separate 46,XY DSD gonadal dysgenesis individuals reveals a reduced ability to activate hTES.Conclusions/SignificanceWe demonstrate how three human sex-determining factors are likely to function during gonadal development around SOX9 as a hub gene, with different genetic causes of 46,XY DSD due a common failure to upregulate SOX9 transcription.

SF-1, un acteur majeur de la différenciation surrénalienne et sexuelle : implications dans les dysgénésies gonadiques et l’insuffisance ovarienne prématurée

Steroidogenic factor 1 (SF-1) gene, identified by Keith Parker in 1992, encodes for an orphan nuclear receptor, NR5A1, whose expression is detected during fetal life in adrenal and gonadal steroidogenic tissues, but also in the developping hypothalamus and in pituitary gonadotropic cells. SF-1 knock-out mouse models exhibit complete adrenal and gonadal agenesis. Human mutations of this transcription factor, were initially associated with primary adrenal failure and male gonadal dysgenesis with various degrees of underandrogenization. More recently, identification of novel SF-1 mutations responsible for isolated 46, XY gonadal dysgenesis or 46, XX primary ovarian insufficiency, underscores its central role in the control and maintenance of adrenal and reproductive functions. A better understanding in the regulatory mechanisms of SF-1 signaling pathway, will open new avenues for diagnostic and therapeutic managements of sex differentiation disorders and infertilities.

A novel mutation in the accessory DNA-binding domain of human steroidogenic factor 1 causes XY gonadal dysgenesis without adrenal insufficiency

Steroidogenic factor 1 (SF1), officially designated NR5A1, is a nuclear receptor that plays key roles in endocrine development and function. Previous reports of human SF1 mutations revealed a spectrum of phenotypes affecting adrenal function and/or gonadal development and sex differentiation. We present the clinical phenotype and functional effects of a novel SF1 mutation. The patient is a 22-year-old 46, XY Japanese patient who presented with dysgenetic testes, atrophic vasa deferentia and epididymides, lack of Müllerian structures, and clitoromegaly. Endocrine studies revealed normal adrenal function. Analysis of the SF1 gene revealed compound heterozygosity for a previously described p.G146A polymorphism and a novel missense mutation (p.R84C) in the accessory DNA-binding domain. The father carried the p.G146A polymorphism and the mother had the p.R84C mutation; both were clinically and reproductively normal. Functional studies demonstrated that the p.R84C SF1 had normal nuclear localization but decreased DNA-binding affinity and transcriptional activity compared with wild-type SF1; it did not exhibit any dominant negative activity. These results describe the human phenotype that results from compound heterozygosity of the p.G146A polymorphism and a novel p.R84C mutation of SF1, thereby extending the spectrum of human SF1 mutations that impair testis development and sex differentiation in a sex-limited manner while preserving normal adrenal function.

Heterozygous Mutation of Steroidogenic Factor-1 in 46,XY Subjects May Mimic Partial Androgen Insensitivity Syndrome

The clinical and biological features of Sertoli cell and Leydig cell dysfunction are usually investigated when characterizing disorders of sex development in 46,XY individuals: This allows gonadal dysgenesis, a defective development of the gonad, to be distinguished from defects restricted to androgen synthesis or sensitivity. In humans, mutations in steroidogenic factor-1 (SF-1), one of the critical factors involved in testis development, have been reported to cause gonadal dysgenesis with or without adrenal failure in 46,XY individuals. We report a SF-1 mutation that caused ambiguous genitalia associated with strikingly different hormonal phenotypes in two affected 46,XY children from the same family. Hormonal evaluation included testosterone (T), anti-Mullerian hormone (AMH), inhibin B, FSH, and LH measurements during the first weeks of life, a period when physiological activation of the gonadotropin-gonadal system occurs. Direct DNA sequencing of the coding sequence of the SF-1 and the androgen receptor (AR) genes was performed. Both 46,XY children had ambiguous genitalia with no Mullerian structures and no adrenal insufficiency. The older child showed normal elevation of T (up to 7.6 nmol/liter, 2.2 ng/ml), AMH (504 pmol/liter, 70.6 ng/ml), inhibin B (245 pg/ml), FSH, and LH during the first weeks, which led to a presumptive diagnosis of partial androgen insensitivity syndrome. The AR sequence was, however, normal. In the second child, T, AMH, and inhibin B were low, suggesting gonadal dysgenesis. In both children and their mother, a c.536delC frameshift mutation in the SF-1 gene was found. This mutation terminates translation at position 295, removing the ligand-binding domain and the activation function 2 (AF-2) domain, a critical domain for SF-1 transactivating activity. The usual markers of testis dysgenesis may be normal in 46,XY individuals with SF-1 mutation. Screening for SF-1 mutation should be performed in subjects with apparent partial androgen insensitivity syndrome and no mutation in the AR gene.

A Microdeletion in the Ligand Binding Domain of Human Steroidogenic Factor 1 Causes XY Sex Reversal without Adrenal Insufficiency

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that plays key roles in endocrine development and function. Knockout mice lacking SF-1 have adrenal and gonadal agenesis, impaired gonadotropin expression, and structural abnormalities of the ventromedial hypothalamic nucleus. Previous studies have identified three human subjects with mutations in SF-1 causing adrenocortical insufficiency with varying degrees of gonadal dysfunction. We now describe a novel 8-bp microdeletion of SF-1, isolated from a 46, XY patient who presented with gonadal agenesis but normal adrenal function, which causes premature termination upstream of sequences encoding the activation function 2 domain. In cell transfection experiments, the mutated protein possessed no intrinsic transcriptional activity but rather inhibited the function of the wild-type protein in most cell types. To our knowledge, this is the first example of an apparent dominant-negative effect of a SF-1 mutation in humans. These findings, which define a SF-1 mutation that apparently differentially affects its transcriptional activity in vivo in the adrenal cortex and the gonads, may be relevant to the cohort of patients who present with 46, XY sex reversal but normal adrenal function.

Inherited adrenal hypoplasia: not just for kids!

Inherited adrenal hypoplasia: not just for kids!

Convergence of Wnt Signaling and Steroidogenic Factor-1 (SF-1) on Transcription of the Rat Inhibin α Gene

The action of a variety of peptide hormones is critical for proper growth and differentiation of the urogenital ridge, which ultimately gives rise to the kidney, adrenal cortex, and gonad. One such class of peptides is the Wnt family of secreted glycoproteins that is classically involved in development of cell polarity and cell fate determination. Notably, alterations in Wnt-4 expression in mice and humans result in profound defects in urogenital ridge development, including dysregulation of kidney, gonadal, and adrenal growth. The nuclear receptor steroidogenic factor-1 (SF-1) has been implicated as a downstream effector of peptide hormone signaling during urogenital ridge development as evidenced by both the activation of SF-1-dependent transcription in the adrenal cortex by signaling molecules such as protein kinase A and by the adrenal and gonadal agenesis in mice with null mutations in SF-1. We hypothesized that Wnt-dependent signaling cascades regulate SF-1-dependent transcription of genes required for adreno-gonadal development. Specifically, the data demonstrate that beta-catenin synergizes with SF-1 to activate the alpha-inhibin promoter through formation of a transcriptional complex. The activation requires an intact SF-1 RE and is independent of TCF/Lef. These data support the recent observation that beta-catenin can participate in nuclear receptor-mediated transcriptional activation and extend the findings to the monomer binding class of orphan nuclear receptors.

Naturally-Occurring Mutations in Steroidogenic Factor-1 (Sf-1) Provide Insight into Dose-Dependent Regulation of Gene Transcription by Monomeric Orphan Nuclear Receptors

Steroidogenic factor-I (SF-I, NR5A1) is an orphan nuclear receptor that regulates the transcription of multiple target genes involved in steroidogenesis, reproduction and male sexual differentiation. Unlike most nuclear receptors, SF-I binds to the response elements of these genes as a monomer and recognizes variations on the extended half site, PyCA AGGTCA. Recently, we described a heterozygous G35E mutation in the P-box of SF-I in a patient with severe primary adrenal failure, complete XY sex-reversal and persistent Miillerian structures (Nat Genet 1999;22: 125-6). The P-box amino-acid sequence is critical for recognizing the core half-site element of target genes (AGGTCA) and for determining DNA-binding specificity. Consequently, the G35E mutation impairs binding to SFI responsive promoters, particularly when “imperfect” binding sites are present, and a heterozygous mutation is sufficient to cause a severe phenotype in this patient. We have now identified a homozygous R92Q mutation in the A-box of SF-I in a baby with a similar phenotype and consanguineous parents. This A-box arginine residue is highly conserved among nuclear receptors that bind to DNA as monomers (e.g., SF-I, LRH, ERR, NGFIB) and is thought to intcract primarily with the 5’ flanking sequence within the minor groove of DNA (PyCA) to stabilize the receptor-DNA complex. Functional studies reveal that the R92Q mutant has partial loss of binding to and transactivation of several SF-I target genes when compared to the G35E P-box change. Further, heterozygous carriers of the R92Q mutation have a normal phenotype and adrenal function. Thus, a homozygous R92Q A-box mutation is necessary for full phenotypic penetrance and autosomal dominant inheritance is seen. Taken together, these two naturally-occurring SF-I mutations reveal the relative importance of the P-box and A-box regions for monomeric binding by nuclear receptors, demonstrating that functional gene dosage effects are important when one factor controls the transcription of many different target genes in humans.

Phenotypic spectrum of mutations in DAX-1 and SF-1

SF-1 (steroidogenic factor-1) (NR5A1) and DAX-1 (dosage-sensitive sex-reversal, adrenal hypoplasia congenital, X chromosome) (NR0B1) are orphan nuclear receptors that are expressed in the adrenal gland, gonads, ventromedial hypothalamus (VMH), and pituitary gonadotrope cells. The function of these genes has been clarified by examining the consequences of naturally occurring mutations in humans, as well as targeted disruption of the genes in mice. Mutations in DAX1 cause adrenal hypoplasia congenita (AHC), an X-linked disorder characterized by adrenal insufficiency and failure to undergo puberty because of hypogonadotropic hypogonadism. Most DAX1 mutations introduce frameshifts and/or cause premature termination of the protein. Relatively few missense mutations have been described and all are located within the carboxy-terminal half of the protein. Transfection assays demonstrate that AHC-associated DAX1 mutations abrogate its ability to act as a transcriptional repressor of SF-1. Most boys affected with AHC present with adrenal insufficiency in early infancy, although a significant fraction present in later childhood or even as young adults. The degree of gonadotropin deficiency is also variable. With the exception of one mild missense DAX1 mutation, genotype–phenotype correlations have been elusive, suggesting an important role for modifier genes. Targeted mutagenesis of Dax1 ( Ahch) in mice reveals an additional role in testis development and spermatogenesis. Similar abnormalities appear to be present in humans. Targeted mutagenesis of Sf1 ( FtzF1) prevents gonadal and adrenal development, and causes male-to-female sex-reversal. A human XY individual with a heterozygous SF1 mutation presented with adrenal insufficiency and complete sex-reversal; this DNA-binding domain mutation prevents SF-1 stimulation of its target genes. In addition to their clinical relevance, studies of SF1 and DAX1 are proving useful for unraveling the genetic pathways that govern adrenal and gonadal development.

Screening for mutations in the steroidogenic acute regulatory protein and steroidogenic factor-1 genes, and in CYP11A and dosage-sensitive sex reversal-adrenal hypoplasia gene on the X chromosome, gene-1 (DAX-1), in hyperandrogenic hirsute women.

Abstract Abnormalities in adrenal and/or ovarian steroidogenesis are found in most patients with hirsutism. The rate-limiting step in the synthesis of steroids in the ovary and the adrenal is the conversion of cholesterol into pregnenolone by cholesterol side-chain cleavage enzyme (P450scc), encoded by the gene CYP11A, after cholesterol is introduced into the mitochondria by the steroidogenic acute regulatory protein (StAR). DAX-1 is a repressor of StAR gene expression, and steroidogenic factor-1 (SF-1) is a regulator of CYP11A, DAX-1, and StAR gene. Mutations in any of these factors resulting in gain of function, or loss of repression, of StAR or P450scc might contribute to the steroidogenic abnormalities present in hirsute patients. In the present study we have screened, using heteroduplex analysis, the genes encoding StAR and SF-1 as well as DAX-1 and CYP11A for mutations in genomic DNA from 19 women presenting with hirsutism and increased serum androgen levels. When variants were found, analysis was extended to a larger group of hyperandrogenic patients and nonaffected women. Two variants were identified in the SF-1 gene. A G-->C change in exon 6, resulting in an Arg(365)Pro mutation, was found in 1 of 45 patients, but not in controls. Also, a Gly(146)Ala missense mutation, resulting from a G-->C change in exon 4, was found in 2 of 48 patients and in 2 of 50 nonaffected individuals. We identified a C-->T base pair change at position -33 of the StAR gene. Three of 48 patients and 3 of 43 controls presented this variant. No mutations were found in coding regions of the StAR gene. Analysis of CYP11A-coding regions identified a G-->A change in exon 3, resulting in a Val(179)Ile missense mutation. This mutation was found in 1 of 29 patients studied and was not present in 50 controls. Finally, analysis of DAX-1 showed no variant in any of the women studied. In conclusion, mutations in StAR, SF-1, CYP11A, and DAX-1 are seldom found in hirsute patients and do not explain the steroidogenic abnormalities found in these women.

A Naturally Occurring Steroidogenic Factor-1 Mutation Exhibits Differential Binding and Activation of Target Genes

Steroidogenic factor-1 (SF-1) is an orphan nuclear receptor that binds DNA as a monomer and regulates the transcription of multiple target genes. A mutation in the proximal (P)-box of the first zinc finger of SF-1 (G35E) has been reported to cause complete XY sex reversal and adrenal insufficiency. Because this P-box region dictates DNA binding specificity, we investigated the effect of this mutation on DNA binding and regulation of target genes. Binding of the P-box mutant was markedly impaired for most native SF-1 response elements. However, mutant SF-1 bound to a subset of response elements containing a CCA AGGTCA motif. Mutagenesis studies of response elements revealed that the first nucleotide position in the 5'-flanking sequence triplet and the central part of the half-site dictate DNA binding specificity by the mutant SF-1. Further, introduction of a mutation into the SF-1 A-box, which has been proposed to bind to the 5'-flanking sequence triplet, eliminated binding by mutant SF-1 to all response elements tested. These data support the idea that the A-box stabilizes monomeric binding by nuclear receptors. This action may be particularly important when P-box binding affinity is compromised either by mutations in SF-1 or by sequence alterations in its binding site.