Persistent unexplained congenital clitoromegaly in females born extremely prematurely.

OBJECTIVE: Unexplained clitoromegaly is a rare but well recognised feature in girls born premature. Although detected at birth, girls may re-present during childhood to paediatric urologists and gynaecologists who should be aware of this condition. The aim of the study was to describe the clinical findings and management of a series of girls presenting with persistent congenital clitoromegaly associated with prematurity. MATERIALS AND METHODS: This was a retrospective notes review set in a tertiary referral centre for Paediatric and Adolescent Gynaecology (PAG). RESULTS: Eight girls with a mean age of 6 years were seen over an eight year period. In all cases a Disorder of Sex Development (DSD) had been previously excluded. The main symptoms were discomfort or concern about appearance. On examination five girls had excess skin over the clitoris and three had enlarged corporal tissue. Management included reassurance and simple measures to ease discomfort. In two cases the parents requested referral to a paediatric urologist to consider clitoral surgery. CONCLUSION: As survival rates for extreme prematurity improve, paediatric urologists and gynaecologists are likely to see more of these cases. Clinicians must be familiar with this condition to ensure children are managed appropriately.

Clinical and gonadal features and early surgical management of 45,X/46,XY and 45,X/47,XYY chromosomal mosaicism presenting with genital anomalies.

OBJECTIVE: The 45,X/46,XY and 45,X/47,XYY group of patients includes some of those previously diagnosed with 'mixed gonadal dysgenesis'. Our aim was to establish the clinical and gonadal spectrum, and early surgical management, of patients with chromosomal mosaicism presenting with genital anomalies. PATIENTS AND METHODS: We performed a retrospective review of patients with 45,X/46,XY or 45,X/47,XYY mosaicism presenting with genital ambiguity between 1988 and 2009. At least one gonadal biopsy or gonadectomy specimen was available for each patient. Gonadal histology was re-evaluated by a paediatric pathologist. RESULTS: Of 31 patients with 45,X/46,XY (n = 28) or 45,X/47,XYY (n = 3) mosaicism and genital anomalies, 19 (61%) were raised male. Histology of 46 gonads was available from patients who had undergone a gonadectomy or gonadal biopsy, at a median age of 9.5 months. 18 gonads were palpable at presentation, including 5 (28%) histologically unremarkable testes, 2 streak gonads, and 1 dysgenetic gonad with distinct areas of testicular and ovarian stroma but no oocytes. All intra-abdominal gonads were found to be dysgenetic testes (of which 2 were noted to have pre-malignant changes) or streaks, apart from 1 histologically unremarkable testis. 15 (48%) patients had other anomalies, most commonly cardiac and renal; 4 (13%) had a Turner phenotype. CONCLUSION: The anatomy and gonadal histology of 45,X/46,XY and 45,X/47,XYY individuals with genital ambiguity do not conform to a set pattern, and hence management of each patient should be individualized according to detailed anatomical and histological assessment.

Birth after TESE-ICSI in a man with hypogonadotropic hypogonadism and congenital adrenal hypoplasia linked to a DAX-1 (NR0B1) mutation.

DAX1/NR0B1 mutations are responsible for X-linked congenital adrenal hypoplasia (AHC) associated with hypogonadotropic hypogonadism (HH). Few data are available concerning testicular function and fertility in men with DAX1 mutations. Azoospermia as well as failure of gonadotrophin treatment have been reported. We induced spermatogenesis in a patient who has a DAX1 mutation (c.1210C>T), leading to a stop codon in position 404 (p.Gln404X). His endocrine testing revealed a low testosterone level at 1.2 nmol/l (N: 12-40) with low FSH and LH levels at 2.1 IU/l (N: 1-5 IU/l) and 0.1 IU/l (N: 1-4 IU/l), respectively. Baseline semen analysis revealed azoospermia. Menotropin (Menopur(®):150 IU, three times weekly) and human chorionic gonadotrophin (1500 IU, twice weekly) were used. After 20 months of treatment, as azoospermia persisted, bilateral multiple site testicular biopsies were performed. Histology revealed severe hypospermatogenesis. Rare spermatozoa were extracted from the right posterior fragment and ICSI was performed. Four embryos were obtained and, after a frozen-thawed single-embryo transfer, the patient's wife became pregnant and gave birth to a healthy boy. We report the first case of paternity after TESE-ICSI in a patient with DAX1 mutation, giving potential hope to these patients to father non-affected children. Furthermore, this case illustrates the fact that patients with X-linked AHC have a primary testicular defect in addition to HH.

New technologies for the identification of novel genetic markers of disorders of sex development (DSD).

Although the genetic basis of human sexual determination and differentiation has advanced considerably in recent years, the fact remains that in most subjects with disorders of sex development (DSD) the underlying genetic cause is unknown. Where pathogenic mutations have been identified, the phenotype can be highly variable, even within families, suggesting that other genetic variants are influencing the expression of the phenotype. This situation is likely to change, as more powerful and affordable tools become widely available for detailed genetic analyses. Here, we describe recent advances in comparative genomic hybridisation, sequencing by hybridisation and next generation sequencing, and we describe how these technologies will have an impact on our understanding of the genetic causes of DSD.

Update--steroidogenic factor 1 (SF-1, NR5A1).

Steroidogenic factor 1 (SF1, NR5A1, Ad4BP) is a nuclear receptor and regulator of multiple genes involved in adrenal and gonadal development, steroidogenesis, and the reproductive axis. Complete deletion of Nr5a1 in XY mice results in adrenal and gonadal agenesis, female external genitalia and presence of Müllerian structures. These findings were first reported in the early 1990s. Subsequently, NR5A1 mutations were found in two 46,XY phenotypic females with Müllerian structures and adrenal failure and in one 46,XX female with adrenal failure. More recently, heterozygous NR5A1 mutations have been identified in a substantial proportion of patients with 46,XY disorders of sex development (46,XY DSD) without adrenal insufficiency. Most of these individuals display severe underandrogenization with ambiguous genitalia at birth, partial gonadal dysgenesis, and absence of Müllerian structures or remnants. Some of the patients have a milder phenotype such as hypospadias and cryptorchidism, due to less severe defects in androgen synthesis. Testosterone, inhibin B and AMH are usually low indicating a partial (or sometimes progressive) form of gonadal dysgenesis in most cases. However, normal testosterone production at birth might also be present. The frequency of NR5A1 mutations in otherwise unexplained 46,XY DSD with underandrogenization and partial testicular dysgenesis has been estimated to be about 15%. Furthermore, NR5A1 mutations have now been found in women with familial and sporadic 46,XX primary ovarian insufficiency without adrenal failure. These human phenotypes associated with NR5A1 mutations show that SF-1 is a key factor involved in both human testis and ovarian development, but that human adrenal development seems to be more resistant to the effects of SF-1 haploinsufficiency than gonadal development. Patients with 46,XY DSD and mild underandrogenization due to partial testicular dysgenesis should possibly be assigned to the male sex, as small testes with Leydig, Sertoli and germ cells are present in almost all cases. Additionally, spontaneous virilization in puberty might be possible in patients with NR5A1 mutations. However, fertility options and the risk of testicular malignancy and adrenal insufficiency in adulthood are unknown and need to be investigated in long-term outcome studies.

Steroidogenic factor-1 (SF-1, Ad4BP, NR5A1) and disorders of testis development.

Steroidogenic factor-1 (SF-1) (Ad4BP, NR5A1) is a nuclear receptor that regulates many aspects of adrenal and reproductive development and function. Consequently, deletion of the gene (Nr5a1) encoding Sf-1 in XY mice results in impaired adrenal development, complete testicular dysgenesis with Mullerian structures, and female external genitalia. Initial efforts to identify NR5A1 changes in humans focused on 46,XY individuals with combined adrenogonadal failure and Mullerian structures. Although this combination of clinical features is rare, 2 such patients harboring NR5A1 mutations have been described within the past decade. More recently, however, it has emerged that heterozygous loss of function mutations in NR5A1 can be found relatively frequently in children and adults with 46,XY disorders of sex development (DSD) but with apparently normal adrenal function. The phenotypic spectrum associated with these changes ranges from complete testicular dysgenesis with Mullerian structures, through individuals with mild clitoromegaly or genital ambiguity, to severe penoscrotal hypospadias or even anorchia. Furthermore, a non-synonymous polymorphism in NR5A1 may be associated with micropenis or undescended testes within the population. Taken together, these reports suggest that variable loss of SF-1 function can be associated with a wide range of reproductive phenotypes in humans.

Skewed X inactivation is associated with phenotype in a female with adrenal hypoplasia congenita.

Adrenal hypoplasia congenita (AHC) can occur due to deletions or mutations in the DAX 1 (NR0B1) gene on the X chromosome (OMIM 300200). This form of AHC is therefore predominantly seen in boys. Deletion of the DAX 1 gene can also be part of a larger contiguous deletion including the centromeric dystrophin and glycerol kinase (GK) genes. We report a girl with a de novo deletion at Xp21.2 on the maternal chromosome, including DAX1, the GK gene and 3' end of the dystrophin gene, who presented with salt losing adrenal insufficiency and moderate developmental delay, but relatively mild features of muscular dystrophy. Investigation using the androgen receptor as a marker gene identified skewed inactivation of the X chromosome. In the patient's leucocytes, the paternal X chromosome was completely inactive, but in muscle 20% of the active chromosomes were of paternal origin. Thus skewed X inactivation (deletion on the active maternal X chromosome with an inactive paternal X chromosome) is associated with AHC in a female. Variability in X inactivation between tissues may account for the pronounced salt loss and adrenal insufficiency but mild muscular dystrophy.

A novel point mutation in P450c17 (CYP17) causing combined 17alpha-hydroxylase/17,20-lyase deficiency.

CONTEXT: Combined 17alpha-hydroxylase/17,20-lyase deficiency is a rare cause of congenital adrenal hyperplasia and hypogonadism. Novel single amino acid changes in P450c17 provide potentially important insights into key structural domains for enzyme function. OBJECTIVE, DESIGN, AND SETTING: We report a novel missense mutation in P450c17 in a 17-yr-old female presenting with a malignant mixed germ cell tumor with yolk sac elements who demonstrated clinical and biochemical features of combined 17alpha-hydroxylase/17,20-lyase deficiency. METHODS: Quantitative urinary steroid analysis was performed by high resolution gas chromatography. All eight coding exons of CYP17 were PCR amplified and sequenced. The position of arginine at codon 96 was modeled using the CYP17 structure 2c17 (www.rcsb.org). The CYP17 genes were subcloned into pcDNA3, expressed in HEK-293 cells, and chromatographed. PATIENT AND RESULTS: 17alpha-Hydroxylase deficiency was confirmed by marked reductions in urinary and serum cortisol, androgens, and estradiol. Mutational analysis revealed a novel homozygous R96Q missense mutation in P450c17, affecting an amino acid in a key substrate-binding region of the enzyme, leading to complete inactivity. CONCLUSION: The description of a second missense mutation at codon 96 (R96W and R96Q) in the substrate-binding region of P450c17 provides strong evidence for the key role of this amino acid in 17alpha-hydroxylase/17,20-lyase function. An association between a malignant germ cell tumor and 17alpha-hydroxylase deficiency has not been reported previously, although the presence of gonadoblastoma in the ovary of a patient with this condition has recently been described.

Two novel missense mutations in g protein-coupled receptor 54 in a patient with hypogonadotropic hypogonadism.

It has recently been shown that loss-of-function mutations of the G protein-coupled receptor (GPR)54 lead to isolated hypogonadotropic hypogonadism (IHH) in mice and humans. Such mutations are thought to be rare, even within the clinical IHH population, and only a handful of alleles have been described, making further screening of IHH populations imperative. We examined the genes encoding GPR54 and its putative endogenous ligand, kisspeptin-1, for mutations in a cohort of 30 patients with normosmic HH or delayed puberty. One subject with HH, of mixed Turkish-Cypriot and Afro-Caribbean ancestry, was found to be a compound heterozygote for two previously undescribed missense mutations in GPR54: cysteine 223 to arginine (C223R) in the fifth transmembrane helix and arginine 297 to leucine (R297L) in the third extracellular loop. Assessed in vitro using a previously described sensitive signaling assay in cells stably expressing GPR54, the C223R variant was found to exhibit profoundly impaired signaling, whereas the R297L variant showed a mild reduction in ligand-stimulated activity across the ligand dose range. These novel mutations provide further evidence that human HH may be caused by loss-of-function mutations in GPR54.

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.

Variable presentation of X-linked adrenal hypoplasia congenita.

We present a family with X-linked adrenal hypoplasia congenita (AHC) due to a truncation mutation in the DAX1 gene. The three patient reports demonstrate variable clinical and biochemical features at presentation. They presented with adrenal crises at 3 years, 4 weeks, and 3 weeks. Mineralocorticoid deficiency preceded glucocorticoid deficiency in patient 3 and an early ultrasound indicated normal sized adrenal tissue. Genetic analysis showed that potential female carriers were unaffected.

Molecular and structural analysis of two novel StAR mutations in patients with lipoid congenital adrenal hyperplasia.

Mutations in the gene encoding steroidogenic acute regulatory protein (StAR) cause lipoid congenital adrenal hyperplasia. We report a novel homozygous splice site mutation (IVS1 + 2T --> G) in STAR in two sisters (46XY, 46XX) who presented with primary adrenal insufficiency at birth and a novel homozygous R182H missense mutation in the putative lipid transfer domain of StAR in a phenotypic female (46XY) with adrenal failure and a parotid tumor. These cases highlight the importance of StAR-dependent steroidogenesis during fetal development and early infancy and of the critical functional role of R182 in cholesterol transport.

Missense mutations cluster within the carboxyl-terminal region of DAX-1 and impair transcriptional repression.

DAX-1 is an orphan nuclear receptor that plays a key role in the development and function of the adrenal gland and hypothalamic-pituitary gonadal axis. Mutations in the gene encoding DAX-1 result in X-linked adrenal hypoplasia congenita (AHC). Affected boys typically present with primary adrenal failure in infancy or childhood and hypogonadotropic hypogonadism at the time of puberty. The majority of DAX1 mutations described to date are nonsense or frameshift mutations that result in premature truncation of the DAX-1 protein and loss of DAX-1 repressor function. Relatively few missense mutations in DAX1 have been reported. Here, we describe missense mutations in three additional families with X-linked AHC. When combined with previous reports, the DAX1 missense mutations appear to cluster within restricted regions of the putative ligand-binding domain of DAX-1 and affect amino acids that are evolutionarily conserved, suggesting that these regions correspond to critical functional domains. Transcription assays, using a variety of artificial and native target genes, were performed to assess the effects of these mutations on the function of DAX-1. All DAX-1 missense mutant constructs showed marked loss of repressor function, with the exception of I439S, a mutation previously shown to be associated with delayed-onset adrenal failure and incomplete hypogonadotropic hypogonadism. These data indicate that most DAX1 missense mutations associated with classic AHC exhibit marked loss of function. The locations of these mutations thereby identify important functional domains in the carboxyl-terminus of the protein.

Inherited disorders of the gonadotropin hormones.

Pulsatile GnRH acts at the GnRH receptor on gonadotropes to stimulate gonadotropin gene expression, hormone synthesis and secretion. The pituitary gonadotropins, LH and FSH, stimulate steroid production and gametogenesis in males and in females. Gonadotropin production thus requires the normal development and function of hypothalamic GnRH-producing neurons and pituitary gonadotrope cells. Genes involved in gonadotrope development and/or gene expression include SF1, DAX1, KAL, GNRHR, PC1, HESX1, LHX3, PROP1, LH beta, and FSH beta. Given the complex control of gonadotropin biosynthesis and secretion, it is not surprising that genetic abnormalities have been identified at several of these steps. Some of the mutations that will be reviewed include: (1) SF1 and DAX1-orphan nuclear receptors that are expressed at multiple levels throughout the reproductive axis; (2) KAL-X-linked Kallmann syndrome, where there is abnormal development of hypothalamic GnRH-producing neurons; (3) PC1-causing abnormal processing of GnRH and GNRHR mutations that impair action at the GnRH receptor; (4) HESX1, LHX3, PROP1-abnormal development/function of the gonadotrope cell lineage; (5) LH beta and FSH beta-mutations in the gonadotropin genes that cause structural abnormalities in the hormones. Although all of these gene defects lead to gonadotropin deficiency, each disorder is associated with unique phenotypic or hormonal features. Characterization of the molecular basis of gonadotropin deficiency is useful for directing therapy and for genetic counseling. Identification of these mutations also provides insight into the pathways that govern reproduction.

Aromatase (Cyp19) expression is up-regulated by targeted disruption of Dax1.

DAX-1 [dosage-sensitive sex reversal, adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1] is an orphan nuclear receptor that represses transcription by steroidogenic factor-1 (SF-1), a factor that regulates expression of multiple steroidogenic enzymes and other genes involved in reproduction. Mutations in the human DAX1 gene (also known as AHC) cause the X-linked syndrome AHC, a disorder that is associated with hypogonadotropic hypogonadism also. Characterization of Dax1-deficient male mice revealed primary testicular defects that included Leydig cell hyperplasia (LCH) and progressive degeneration of the germinal epithelium, leading to infertility. In this study, we investigated the effect of Dax1 disruption on the expression profile of various steroidogenic enzyme genes in Leydig cells isolated from Dax1-deficient male mice. Expression of the aromatase (Cyp19) gene, which encodes the enzyme that converts testosterone to estradiol, was increased significantly in the Leydig cells isolated from mutant mice, whereas the expression of other proteins (e.g., StAR and Cyp11a) was not altered. In in vitro transfection studies, DAX-1 repressed the SF-1-mediated transactivation of the Cyp19 promoter but did not inhibit the StAR or Cyp11a promoters. Elevated Cyp19 expression was accompanied by increased intratesticular levels of estradiol. Administration of tamoxifen, a selective estrogen-receptor modulator, restored fertility to the Dax1-deficient male mice and partially corrected LCH, suggesting that estrogen excess contributes to LCH and infertility. Based on these in vivo and in vitro analyses, aromatase seems to be a physiologic target of Dax-1 in Leydig cells, and increased Cyp19 expression may account, in part, for the infertility and LCH in Dax1-deficient mice.

Advances in the molecular genetics of hypogonadotropic hypogonadism.

Mutations in several genes have been shown to cause hypogonadotropic hypogonadism (HHG) in humans. This condition may result from abnormalities in hypothalamic gonadotropin-releasing hormone (GnRH) secretion, impaired pituitary gonadotropin release, or both. Here, we consider mutations in KAL in X-linked Kallmann syndrome; DAX1 in X-linked adrenal hypoplasia congenita; the related orphan nuclear receptor, steroidogenic factor-1; leptin and prohormone convertase-1, which may influence GnRH release and processing; the GnRH receptor; the pituitary transcription factors, HESX-1, LHX3 and PROP-1; and the gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH). Identifying naturally occurring mutations in these genes provides important information about the role of these factors in the development and function of the hypothalamic-pituitary gonadal axis in humans. Different approaches to treatment and counseling may be needed, depending on the condition. Furthermore, the pathophysiological basis of HHG in the majority of individuals remains unclear, despite recent advances. Other candidate genes may be involved in these patients.

Presymptomatic diagnosis of X-linked adrenal hypoplasia congenita by analysis of DAX1.

A novel DAX1 mutation (L381H) was discovered in the asymptomatic 8-month-old brother of a boy with primary adrenal failure. The infant had impaired adrenal reserve despite normal basal adrenal steroid concentrations. This case highlights the value of genetic testing in children at risk of the development of X-linked adrenal hypoplasia congenita before the onset of a potentially life-threatening adrenal crisis.

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.