Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer.

The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these is NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain, and we demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable transcriptional regulator that supports NRF2-dependent lung cancers.

Cooperation between HDAC3 and DAX1 mediates lineage restriction of embryonic stem cells.

Mouse embryonic stem cells (mESCs) are biased toward producing embryonic rather than extraembryonic endoderm fates. Here, we identify the mechanism of this barrier and report that the histone deacetylase Hdac3 and the transcriptional corepressor Dax1 cooperatively limit the lineage repertoire of mESCs by silencing an enhancer of the extraembryonic endoderm-specifying transcription factor Gata6. This restriction is opposed by the pluripotency transcription factors Nr5a2 and Esrrb, which promote cell type conversion. Perturbation of the barrier extends mESC potency and allows formation of 3D spheroids that mimic the spatial segregation of embryonic epiblast and extraembryonic endoderm in early embryos. Overall, this study shows that transcriptional repressors stabilize pluripotency by biasing the equilibrium between embryonic and extraembryonic lineages that is hardwired into the mESC transcriptional network.

NR0B1 augments sorafenib resistance in hepatocellular carcinoma through promoting autophagy and inhibiting apoptosis.

NR0B1 is frequently activated in hepatocellular carcinoma (HCC). However, the role of NR0B1 is controversial in HCC. In this study, we observed that NR0B1 was an independent poor prognostic factor, negatively correlated with the overall survival of HCC and the relapse-free survival of patients treated with sorafenib. Meanwhile, NR0B1 promoted the proliferation, migration, and invasion of HCC cells, inhibited sorafenib-induced apoptosis, and elevated the IC50 of sorafenib in HCC cells. NR0B1 was further displayed to increase sorafenib-induced autophagic vesicles and activate Beclin1/LC3-II-dependent autophagy pathway. Finally, NR0B1 was revealed to transcriptionally suppress GSK3ß that restrains AMPK/mTOR-driven autophagy and increases BAX-mediated apoptosis. Collectively, our study uncovered that the ectopic expression of NR0B1 augmented sorafenib-resistance in HCC cells by activating autophagy and inhibiting apoptosis. Our findings supported that NR0B1 was a detrimental factor for HCC prognosis.

X-linked congenital adrenal hypoplasia: Report of long clinical follow-up and description of a new complex variant in the NR0B1 gene.

Adrenal hypoplasia congenita, attributed to NR0B1 pathogenic variants, accounts for more than 50% of the incidence of primary adrenal insufficiency in children. Although more than 250 different deleterious variations have been described, no genotype-phenotype correlation has been defined to date. We report a case of an adopted boy who reported the onset of an adrenal crisis at 2 weeks of age, requiring replacement therapy with mineralocorticoids and glucocorticoids for 4 months. For 3 years, he did well without treatment. At almost 4 years of age, the disorder was restarted. A long follow-up showed the evolution of hypogonadotropic hypogonadism. Molecular studies on NR0B1 revealed a novel and deleterious deletion-insertion-inversion-deletion complex rearrangement sorted in the 5'-3' direction, which is described as follows: (1) deletion of the intergenic region (between TASL and NR0B1 genes) and 5' region, (2) insertion of a sequence containing 37 bp at the junction of the intergenic region of the TASL gene and a part of exon 1 of the NR0B1 gene, (3) inversion of a part of exon 1, (4) deletion of the final portion of exon 1 and exon 2 and beginning of the 3'UTR region, (5) maintenance of part of the intergenic sequence (between genes MAGEB1 and NR0B1, telomeric sense), (6) large posterior deletion, in the same sense. The path to molecular diagnosis was challenging and involved several molecular biology techniques. Evaluating the breakpoints in our patient, we assumed that it was a nonrecurrent rearrangement that had not yet been described. It may involve a repair mechanism known as nonhomologous end-joining (NHEJ), which joins two ends of DNA in an imprecise manner, generating an "information scar," represented herein by the 37 bp insertion. In addition, the local Xp21 chromosome architecture with sequences capable of modifying the DNA structure could impact the formation of complex rearrangements.

Hamster DAX1: Molecular insights, specific expression, and its role in the Harderian gland.

DAX1 plays an essential role in the differentiation and physiology of the Hypothalamic-Pituitary-Adrenal-Gonadal (HPAG) axis during embryogenesis. However, in adult tissues, in addition to the HPAG axis, evidence has not been found for its differential expression and function. We isolated the DAX1 cDNA to analyze its tissue localization and gene expression profiles in male and female hamsters' Harderian glands (HGs), Mesocricetus auratus. The isolated cDNA clone contains 1848 base pairs (bp), and a 1428-bp open reading frame (ORF) encodes a 476 amino acid protein. Sequence alignments and the phylogenetic tree display a relevant percentage of similarity with human (66%), rat (81%), and mouse (84%) sequences. In adult tissues, the mRNA distribution demonstrated that DAX1 is present in testis, ovaries, and male and female HGs. The highest expression profiles were identified in the adrenal glands, where females exhibit higher mRNA levels than males. The sexually dimorphic expression of DAX1 in adrenals suggests that its presence could be associated with regulating, functioning, and maintaining this endocrine tissue. These findings indicate that the DAX1 gene is limitedly expressed in adult tissues. In the HGs, we demonstrate the absence of sexually dimorphic gene expression. Our results suggest that DAX1 might have an additional physiological function outside of the HPAG axis, specifically in the HG, which may be required for the regulation of intracrine steroidogenesis, secretion, and maintenance of exocrine tissue.

A Novel Look at Dosage-Sensitive Sex Locus Xp21.2 in a Case of 46,XY Partial Gonadal Dysgenesis without NR0B1 Duplication.

A region of 160 kb at Xp21.2 has been defined as dosage-sensitive sex reversal (DSS) and includes the NR0B1 gene, considered to be the candidate gene involved in XY gonadal dysgenesis if overexpressed. We describe a girl with 46,XY partial gonadal dysgenesis carrying a 297 kb duplication at Xp21.2 upstream of NR0B1 initially detected by chromosomal microarray analysis. Fine mapping of the breakpoints by whole-genome sequencing showed a tandem duplication of TASL (CXorf21), GK and partially TAB3, upstream of NR0B1. This is the first description of an Xp21.2 duplication upstream of NR0B1 associated with 46,XY partial gonadal dysgenesis.

Hepatocyte-Specific Deficiency of DAX-1 Protects Mice from Acetaminophen-Induced Hepatotoxicity by Activating NRF2 Signaling.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, but its overdose can cause acute liver failure. The dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1, NR0B1), is an orphan nuclear receptor that acts as a transcriptional co-repressor of various genes. In this study, we identified the role of DAX-1 in APAP-induced liver injury using hepatocyte-specific Dax-1 knockout (Dax-1 LKO) mice. Mouse primary hepatocytes were used as a comparative in vitro study. APAP overdose led to decreased plasma alanine aminotransferase and aspartate aminotransferase levels in Dax-1 LKO mice compared to C57BL/6J (WT) controls, accompanied by reduced liver necrosis. The expression of the genes encoding the enzymes catalyzing glutathione (GSH) synthesis and metabolism and antioxidant enzymes was increased in the livers of APAP-treated Dax-1 LKO mice. The rapid recovery of GSH levels in the mitochondrial fraction of APAP-treated Dax-1 LKO mice led to reduced reactive oxygen species levels, resulting in the inhibition of the prolonged JNK activation. The hepatocyte-specific DAX-1 deficiency increased the protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2) compared with WT controls after APAP administration. These results indicate that DAX-1 deficiency in hepatocytes protects against APAP-induced liver injury by Nrf2-regulated antioxidant defense.

X-linked congenital adrenal hypoplasia: a case presentation.

BACKGROUND: Most patients with congenital adrenal hypoplasia (AHC) develop symptoms during infantile and juvenile periods, with varying clinical manifestations. AHC is a disease that is easily misdiagnosed as Addison's disease or congenital adrenal hyperplasia (CAH). There was also a significant time difference between the age at which patients developed symptoms and the age at which they were diagnosed with AHC. Most patients showed early symptoms during infantile and juvenile periods, but were diagnosed with AHC many years later. CASE PRESENTATION: We are currently reporting a male patient who developed systemic pigmentation at age 2 and was initially diagnosed with Addison's disease. At 22 years of age, he experienced a slipped capital femoral epiphysis (SCFE), a disease mostly seen in adolescents aged 8-15 years, an important cause of which is endocrine disorder. Testes evaluated using color Doppler Ultrasonography suggested microcalcifications. Further genetic testing and auxiliary examinations revealed that the patient had hypogonadotropic hypogonadism (HH) and DAX-1 gene disorders, at which time he was diagnosed with AHC complicated by HH. He was given hormone replacement therapy, followed by regular outpatient review to adjust the medication. CONCLUSIONS: The typical early symptoms of AHC are hyperpigmentation and ion disturbance during infantile and juvenile periods, while few patients with AHC develop puberty disorders as early symptoms. AHC is prone to being misdiagnosed as Addison's disease, and then gradually develops the symptoms of HH in adolescence. The definitive diagnosis of AHC ultimately is based on the patient's clinical presentation, laboratory results and genetic testing results.

Early reprogramming regulators identified by prospective isolation and mass cytometry.

In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of non-productive and staggered productive intermediates arise at different reprogramming time points. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells, prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that, during reprogramming, cells progressively lose donor cell identity and gradually acquire iPS cell properties. Here we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen, we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200, that are absent in both fibroblasts and iPS cells. Single-cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic, reprogramming phase. Expression profiling reveals early upregulation of the transcriptional regulators Nr0b1 and Etv5 in this reprogramming state, preceding activation of key pluripotency regulators such as Rex1 (also known as Zfp42), Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus represent some of the earliest known regulators of iPS cell induction. Our study deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition.

The Genetic Backdrop of Hypogonadotropic Hypogonadism.

The pituitary is an organ of dual provenance: the anterior lobe is epithelial in origin, whereas the posterior lobe derives from the neural ectoderm. The pituitary gland is a pivotal element of the axis regulating reproductive function in mammals. It collects signals from the hypothalamus, and by secreting gonadotropins (FSH and LH) it stimulates the ovary into cyclic activity resulting in a menstrual cycle and in ovulation. Pituitary organogenesis is comprised of three main stages controlled by different signaling molecules: first, the initiation of pituitary organogenesis and subsequent formation of Rathke's pouch; second, the migration of Rathke's pouch cells and their proliferation; and third, lineage determination and cellular differentiation. Any disruption of this sequence, e.g., gene mutation, can lead to numerous developmental disorders. Gene mutations contributing to disordered pituitary development can themselves be classified: mutations affecting transcriptional determinants of pituitary development, mutations related to gonadotropin deficiency, mutations concerning the beta subunit of FSH and LH, and mutations in the DAX-1 gene as a cause of adrenal hypoplasia and disturbed responsiveness of the pituitary to GnRH. All these mutations lead to disruption in the hypothalamic-pituitary-ovarian axis and contribute to the development of primary amenorrhea.

Timing of adrenal regression controlled by synergistic interaction between Sf1 SUMOylation and Dax1.

The nuclear receptor steroidogenic factor 1 (Sf1, Nr5a1, Ad4bp) is crucial for formation, development and function of steroidogenic tissues. A fetal adrenal enhancer (FAdE) in the Sf1 gene was previously identified to direct Sf1 expression exclusively in the fetal adrenal cortex and is bound by both Sf1 and Dax1. Here, we have examined the function of Sf1 SUMOylation and its interaction with Dax1 on FAdE function. A diffused prolonged pattern of FAdE expression and delayed regression of the postnatal fetal cortex (X-zone) were detected in both the SUMOylation-deficient-Sf12KR/2KR and Dax1 knockout mouse lines, with FAdE expression/activity retained in the postnatal 20αHSD-positive postnatal X-zone cells. In vitro studies indicated that Sf1 SUMOylation, although not directly influencing DNA binding, actually increased binding of Dax1 to Sf1 to further enhance transcriptional repression of FAdE. Taken together, these studies define a crucial repressor function of Sf1 SUMOylation and Dax1 in the physiological cessation of FAdE-mediated Sf1 expression and the resultant regression of the postnatal fetal cortex (X-zone).

Roles of two cyp11 genes in sex hormone biosynthesis in Japanese flounder (Paralichthys olivaceus).

The P450 side-chain cleavage enzymes P450scc (Cyp11a) and 11ß-hydroxylase (Cyp11b) play important roles in sex steroid and cortisol production. Here, two duplicates of cyp11 genes were identified in Japanese flounder (Paralichthys olivaceus): Pocyp11a and Pocyp11b, respectively. Phylogenetic analysis and amino acid sequence alignment revealed that Pocyp11a and Pocyp11b shared significant identity with sequences of other teleost fish species. The quantitative real-time polymerase chain reaction (qRT-PCR) results indicated that among the studied tissues, brain tissue showed the highest expression of Pocyp11a, followed by kidney and testis tissues, whereas Pocyp11b expression was highest in the testis. The expression patterns of these two genes showed sexual dimorphism, with both genes showing higher expression in the testis than in the ovary. In-situ hybridization analysis demonstrated that Pocyp11a and Pocyp11b mRNA were both detected in oocytes, spermatocytes, and Sertoli cells, indicating that they might be involved in hormone synthesis. The expression levels of Pocyp11a and Pocyp11b were significantly downregulated by treatment with 17α-methyltestosterone (17α-MT) in the testis and ovary in both in vivo and studies. In vivo studies showed that Pocyp11a and Pocyp11b transcripts were suppressed by 17ß-estradiol (E2 ) treatment in both the testis and ovary. In addition, in vitro studies showed that the expression level of Pocyp11b was decreased by treatment with E2 , whereas that of Pocyp11a was largely unaffected. Moreover, the expression levels of Pocyp11a and Pocyp11b in the testis cell line were significantly upregulated after NR0b1 and NR5a2 (p < .05) treatment. These results indicate that Pocyp11a and Pocyp11b might play important roles in sex hormone biosynthesis. Our research can assist future studies of the mechanisms of steroid biosynthesis and functional differences between cyp11a and cyp11b in Japanese flounder.

Spontaneous fertility and variable spectrum of reproductive phenotype in a family with adult-onset X-linked adrenal insufficiency harboring a novel DAX-1/NR0B1 mutation.

BACKGROUND: Adrenal hypoplasia congenita (AHC) is an X-linked disorder that affects the adrenal cortex and hypothalamus-pituitary-gonadal axis (HPG), leading to primary adrenocortical insufficiency (PAI) and hypogonadotropic hypogonadism. AHC is caused by a mutation in the DAX-1 gene (NR0B1). More commonly, this disease is characterized by early-onset PAI, with symptoms in the first months of life. However, a less severe phenotype termed late-onset AHC has been described, as PAI signs and symptoms may begin in adolescence and adulthood. Here we describe a family report of a novel mutation within NR0B1 gene and variable reproductive phenotypes, including spontaneous fertility, in a very late-onset X-linked AHC kindred. CASE PRESENTATION: Three affected maternal male relatives had confirmed PAI diagnosis between 30 y and at late 64 y. The X-linked pattern has made the endocrinology team to AHC suspicion. Regarding the HPG axis, all males presented a distinct degree of testosterone deficiency and fertility phenotypes, varying from a variable degree of hypogonadism, oligoasthenoteratozoospermia to spontaneous fertility. Interestingly, the other five maternal male relatives unexpectedly died during early adulthood, most likely due to undiagnosed PAI/adrenal crisis as the probable cause of their premature deaths. Sequencing analysis of the NR0B1 gene has shown a novel NR0B1 mutation (p.Tyr378Cys) that segregated in three AHC family members. CONCLUSIONS: NR0B1 p.Tyr378Cys segregates in an AHC family with a variable degree of adrenal and gonadal phenotypes, and its hemizygous trait explains the disease in affected family members. We recommend that NR0B1 mutation carriers, even those that are allegedly asymptomatic, be carefully monitored while reinforcing education to prevent PAI and consider early sperm banking when spermatogenesis still viable.

Report: central diabetes insipidus and schwannoma in a male with X-linked congenital adrenal hypoplasia.

BACKGROUND: DAX1 mutations are related to the X-linked form of adrenal hypoplasia congenita (AHC) in infancy and to hypogonadotropic hypogonadism (HH) in puberty. We report a male patient affected by X-linked AHC who presented with central diabetes insipidus and schwannoma in adulthood, which has not been described in association with AHC. CASE PRESENTATION: A 36-day-old male infant who presented with severe dehydration was admitted to the intensive care unit. His laboratory findings showed hyponatremia, hyperkalemia, hypoglycemia, and metabolic acidosis. After hormonal evaluation, he was diagnosed with adrenal insufficiency, and he recovered after treatment with hydrocortisone and a mineralocorticoid. He continued to take hydrocortisone and the mineralocorticoid after discharge. At the age of 17, he did not show any signs of puberty. On the basis of a GnRH test, a diagnosis of HH was made. At the age of 24, he was hospitalized with thirst, polydipsia and polyuria. He underwent a water deprivation test for polydipsia and was diagnosed with central diabetes insipidus. By quantitative polymerase chain reaction analysis, we identified a hemizygous frameshift mutation in DAX1 (c.543delA). CONCLUSIONS: We suggest that DAX1 mutations affect a wider variety of endocrine organs than previously known, including the posterior pituitary gland.

Role for the EWS domain of EWS/FLI in binding GGAA-microsatellites required for Ewing sarcoma anchorage independent growth.

Ewing sarcoma usually expresses the EWS/FLI fusion transcription factor oncoprotein. EWS/FLI regulates myriad genes required for Ewing sarcoma development. EWS/FLI binds GGAA-microsatellite sequences in vivo and in vitro. These sequences provide EWS/FLI-mediated activation to reporter constructs, suggesting that they function as EWS/FLI-response elements. We now demonstrate the critical role of an EWS/FLI-bound GGAA-microsatellite in regulation of the NR0B1 gene as well as for Ewing sarcoma proliferation and anchorage-independent growth. Clinically, genomic GGAA-microsatellites are highly variable and polymorphic. Current data suggest that there is an optimal "sweet-spot" GGAA-microsatellite length (of 18-26 GGAA repeats) that confers maximal EWS/FLI-responsiveness to target genes, but the mechanistic basis for this remains unknown. Our biochemical studies, using recombinant Δ22 (a version of EWS/FLI containing only the FLI portion), demonstrate a stoichiometry of one Δ22-monomer binding to every two consecutive GGAA-repeats on shorter microsatellite sequences. Surprisingly, the affinity for Δ22 binding to GGAA-microsatellites significantly decreased, and ultimately became unmeasureable, when the size of the microsatellite was increased to the sweet-spot length. In contrast, a fully functional EWS/FLI mutant (Mut9, which retains approximately half of the EWS portion of the fusion) showed low affinity for smaller GGAA-microsatellites but instead significantly increased its affinity at sweet-spot microsatellite lengths. Single-gene ChIP and genome-wide ChIP-sequencing (ChIP-seq) and RNA-seq studies extended these findings to the in vivo setting. Together, these data demonstrate the critical requirement of GGAA-microsatellites as EWS/FLI activating response elements in vivo and reveal an unexpected role for the EWS portion of the EWS/FLI fusion in binding to sweet-spot GGAA-microsatellites.

Isolated hypoaldosteronism as first sign of X-linked adrenal hypoplasia congenita caused by a novel mutation in NR0B1/DAX-1 gene: a case report.

BACKGROUND: X-linked Adrenal Hypoplasia Congenita (AHC) is a rare cause of primary adrenal insufficiency due to mutations in the NR0B1 gene, causing a loss of function of the nuclear receptor protein DAX-1. Adrenal insufficiency usually appears in the first 2 months of life, but can sometimes emerge during childhood. Hypogonadotropic Hypogonadism is often associated later in life and patients may develop azoospermia. We describe an unusual onset of AHC started with isolated hypoaldosteronism as first and only sign of the disease. CASE PRESENTATION: A 18-days-old newborn presented with failure to thrive and feeding difficulties. Blood tests showed severe hyponatremia, hyperkalemia and hypochloremia. Renin was found over the measurable range and aldosterone was low whereas cortisol level was normal with a slightly increased ACTH. In the suspicion of Primary Hypoaldosteronism, correction of plasmatic electrolytes and replacement therapy with Fludrocortisone were promptly started. The subsequent evidence of low plasmatic and urinary cortisol and increased ACTH required the start of Hydrocortisone replacement therapy and it defined a clinical picture of adrenal insufficiency. Genetic analysis demonstrated a novel mutation in the DAX-1 gene leading to the diagnosis of AHC. CONCLUSIONS: AHC onset may involve the aldosterone production itself, miming an isolated defect of aldosterone synthesis. NR0B1/DAX-1 mutations should be considered in male infants presenting with isolated hypoaldosteronism as first sign of adrenal insufficiency.

Novel frameshift mutation of the NR0B1(DAX1) in two tall adult brothers.

NR0B1 (nuclear receptor subfamily 0, group B, member 1) is a transcription factor encoded by DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) responsible for the development and maintenance of the steroidogenic tissues. In humans the DAX1 mutations cause congenital adrenal hypoplasia (AHC) and hypogonadotropic hypogonadism (HHG) in boys. Here we report two brothers who were assessed by endocrinologist at the age of 51 and 43 because of their serious osteoporosis. They had been substituted with prednisolone since the age of 4 and 9 years because of their primary adrenal insufficiency (PAI). Due to their late puberty caused by HHG at the age of 16 and 17 years their heights were - 3.1 and - 3.3 SD, but then they had a significant growth during their adulthood and reached the + 1.85 SD and + 3.78 SD respectively. During this period, they received glucocorticoid supplementation, but the treatment of their HHG was inadequate. At the age of 51 and 43 years insulin tolerance test (ITT) and gonadotropin releasing hormone (GnRH) test confirmed their PAI and HHG. Genetic test performed at this time revealed a novel, four nucleotides deletion (del.586-571c.GGGC or 572-575c.GGGC) of DAX1 gene. The two brothers with AHC and HHG caused by a novel DAX1 mutation, reached tall final heights, despite of the disadvantageous prednisolone treatment during their childhood. We assume that the long-term lack of the sexual hormone substitution was a significant reason of their above average height as well as their serious osteoporosis.

Gene dosage of DAX-1, determining in sexual differentiation: duplication of DAX-1 in two sisters with gonadal dysgenesis.

Two sisters phenotypically normal females, presenting with tumor abdominal mass with histopathological findings of teratoma and gonadoblastoma associated to 46,XY male-to-female sex reversal syndrome, secondary to a duplication in DAX-1, possibly inherited of maternal gonadal mosaicism. Copy number variation and functional effects of the duplication were done by MLPA multiplex ligation-dependent probe amplification and real time PCR. DAX-1, also known as dosage sensitive sex reversal gene (DSS), is considered the most likely candidate gene involved in XY gonadal dysgenesis when overexpressed. The excess of DAX-1 gene disturbs testicular development by down regulation of SF-1, WT1, and SOX9. This is the first report of 46,XY sex reversal in two siblings who have a maternally inherited duplication of DAX-1 associated with reduced levels of expression of downstream genes as SOX9-SF1.

Molecular cloning, characterization of dax1 gene and its response to progesterone in Misgurnus anguillicaudatus.

Progesterone (P4) are aquatic contaminants that can impair fish reproduction even in low concentrations. The aim of this study was to investigate the effects of P4 on the sex differentiation, by quantitative determination of transcriptional changes of a candidate target gene (dax1, has a function in the sex determination and gonadal differentiation of several vertebrate species) in Misgurnus anguillicaudatus. We first cloned and characterized the full-length cDNAs for the dax1 in M. anguillicaudatus (designated as Ma-dax1). Sequence analysis reveals that Ma-dax1 shares high homology with dax1 in other species. Quantitative real-time PCR (qRT-PCR) and in situ hybridization showed that Ma-dax1 gene was highly conserved during vertebrate evolution and involved in a wide range of developmental processes including embryogenesis, central nervous system development and gonad development. For the P4 administration assay, groups of mature fish were exposed for 1, 7, 14, 21 and 28 days to nominal concentrations of 10, 100, and 1000 ng/L P4 in a flow-through system. Quantification of Ma-dax1 transcripts revealed the expression of Ma-dax1 mRNA is altered after P4 treatment in mature gonads. Those showed that P4 could influence the sexual development and sex differentiation in M. anguillicaudatus by disturbing sex differentiation-associated gene expression, and dax1 can be used as a sensitive molecular biomarker for early warning to monitor the environmental progestins chemicals in fresh water environment.

Novel mutation in the nuclear receptor subfamily 0, group B, member 1 (NR0B1) gene associated with intrafamilial heterogeneity in three boys with X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism from India.

Background: Nuclear receptor subfamily 0, group B, member 1 (NR0B1) gene previously known as DAX1 is a transcription factor that plays a key role in the development of hypothalamo-pituitary-gonadal and adrenal axis. Primary adrenal failure may result from metabolic, infection, autoimmune or developmental causes resulting in a life-threatening condition needing immediate intervention. This study aimed to analyse NR0B1 (DAX1) gene mutation resulting in adrenal hypoplasia congenita (AHC) in three brothers presenting with hypogonadotropic hypogonadism and primary adrenal failure either in infancy or in early childhood. Methods: We studied three boys with primary adrenal failure and hypogonadotropic hypogonadism presenting at different ages at the Paediatric Endocrinology Clinic. Muta- tional analysis of NR0B1 gene was carried out by bidirectional sequencing. Results: All the three boys had deletion of G in exon 1 at position 189 (c.189_189delG) of the gene resulting in frame shift mutation (Y64Tfs*21). Conclusion: Novel mutation in NR0B1 detected by this study explained the cause ofhypogonadotropic hypogonadism with primary adrenal failure in this Indian family. Intrafamilial variability was seen in this family. Early diagnosis by genetic testing, genetic counselling and family screening can help to manage this life-threatening condition.