Nine novel mutations in NR0B1 (DAX1) causing adrenal hypoplasia congenita

X‐linked adrenal hypoplasia congenita (AHC) is caused by mutations in the NR0B1 gene. This gene encodes an orphan member of the nuclear receptor superfamily, DAX1. Ongoing efforts in our laboratory have identified nine novel NR0B1 mutations in X‐linked AHC patients (Y81X, 343delG, 457delT, 629delG, L295P, 926‐927delTG, 1130delA, 1141‐1155del15, and E428X). Two additional families segregate previously identified NR0B1 mutations (501delA and R425T). Sequence analysis of the mitochondrial D‐loop indicates that the 501delA family is unrelated through matrilineal descent to our previously analyzed 501delA family. © 2001 Wiley‐Liss, Inc.     

Thirteen novel mutations in the NR0B1 (DAX1) gene as cause of adrenal hypoplasia congenita

X-linked adrenal hypoplasia congenita (AHC) is a rare developmental disorder associated with primary adrenal insufficiency and combined primary and secondary male hypogonadism. It is caused by deletions or mutations of the NR0B1 (DAX1) gene encoding DAX1, an atypical orphan member of the nuclear receptor superfamily. The continuous molecular genetic analysis of male patients with primary adrenal insufficiency revealed 13 novel mutations within the coding region of the NR0B1 gene which are predicted to inactivate the DAX1 function. These were three nonsense mutations (c.312C>A, p.Cys104X, c.670C>T, p.Gln224X; and c.873G>A, p.Trp291X), five duplications (c.269_270dup, c.421_422dup, c.895_896dup, c.989dup, c.999_1000dup), and five deletions (c.483del, c.745_746del, c.734_740del, c.1092del, and c.1346del). All of the mutations resulted in a premature stop codon destroying the ligand binding domain of the predictive DAX1 protein.     

Identical NR5A1 Missense Mutations in Two Unrelated 46,XX Individuals with Testicular Tissues

The role of monogenic mutations in the development of 46,XX testicular/ovotesticular disorders of sex development (DSD) remains speculative. Although mutations in NR5A1 are known to cause 46,XY gonadal dysgenesis and 46,XX ovarian insufficiency, such mutations have not been implicated in testicular development of 46,XX gonads. Here, we identified identical NR5A1 mutations in two unrelated Japanese patients with 46,XX testicular/ovotesticular DSD. The p.Arg92Trp mutation was absent from the clinically normal mothers and from 200 unaffected Japanese individuals. In silico analyses scored p.Arg92Trp as probably pathogenic. In vitro assays demonstrated that compared with wild‐type NR5A1, the mutant protein was less sensitive to NR0B1‐induced suppression on the SOX9 enhancer element. Other sequence variants found in the patients were unlikely to be associated with the phenotype. The results raise the possibility that specific mutations in NR5A1 underlie testicular development in genetic females.     

Comprehensive sequence analysis of the NR5A1 gene encoding steroidogenic factor 1 in a large group of infertile males

The steroidogenic factor 1 (SF1) protein, encoded by the NR5A1 gene, plays a central role in gonadal development and steroidogenesis. Mutations in NR5A1 were first described in patients with primary adrenal insufficiency and 46,XY disorders of sexual development and later also in men with hypospadias, bilateral anorchia and micropenis and women with primary ovarian insufficiency. Recently, heterozygous missense mutations were found in 4% of infertile men with unexplained reduced sperm counts living in France, but all mutation carriers were of non-Caucasian ancestry. Therefore, we performed a comprehensive NR5A1 sequence analysis in 488 well-characterised predominantly Caucasian patients with azoo- or severe oligozoospermia. Two-hundred-thirty-seven men with normal semen parameters were sequenced as controls. In addition to several synonymous variants of unclear pathogenicity, three heterozygous missense mutations predicted to be damaging to SF1 protein function were identified. The andrological phenotype in infertile but otherwise healthy mutation carriers seems variable. In conclusion, mutations altering SF1 protein function and causing spermatogenic failure are also found in men of German origin, but the prevalence seems markedly lower than in other populations.     

Functional characterization of five NR5A1 gene mutations found in patients with 46,XY disorders of sex development

Steroidogenic factor‐1 (SF1), encoded by the NR5A1 gene, is a key regulator of steroidogenesis and reproductive development. NR5A1 mutations described in 46,XY patients with disorders of sex development (DSD) can be associated with a range of conditions of phenotypes; however, the genotype–phenotype correlation remains elusive in many cases. In the present study, we describe the impact of five NR5A1 variants (three novel: p.Arg39Cys, p.Ser32Asn, and p.Lys396Argfs*34; and two previously described: p.Cys65Tyr and p.Cys247*) on protein function, identified in seven patients with 46,XY DSD. In vitro functional analyses demonstrate that NR5A1 mutations impair protein functions and result in the DSD phenotype observed in our patients. Missense mutations in the DNA binding domain and the frameshift mutation p.Lys396Argfs*34 lead to both, markedly affected transactivation assays, and loss of DNA binding, whereas the mutation p.Cys247* retained partial transactivation capacity and the ability to bind a consensus SF1 responsive element. SF1 acts in a dose‐dependent manner and regulates a cascade of genes involved in the sex determination and steroidogenesis, but in most cases reported so far, still lead to a sufficient adrenal steroidogenesis and function, just like in our cases, in which heterozygous mutations are associated to 46,XY DSD with intact adrenal steroid biosynthesis.     

LXXLL motifs and AF-2 domain mediate SHP (NR0B2) homodimerization and DAX1 (NR0B1)-DAX1A heterodimerization

Small heterodimer partner (SHP; NR0B2) is an unusual orphan member of the nuclear receptor superfamily that functions as a corepressor of other nuclear receptors through heterodimeric interactions. Mutations in SHP are associated with mild obesity and insulin resistance. The protein domain structure of SHP is similar to Dosage-sensitive sex reversal adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1 (DAX1; NR0B1). Mutations in DAX1 cause AHC with associated hypogonadotropic hypogonadism. DAX1A is an alternatively spliced isoform of DAX1 that lacks the last 80 amino acids of the DAX1 C-terminal repressor domain and is replaced by a novel 10-amino acid motif. We have previously shown homodimerization of SHP and DAX1 individually, heterodimerization of DAX1 with SHP, and heterodimerization of DAX1 with DAX1A. In these studies, we investigated the domains and residues of SHP involved in SHP homodimerization and DAX1-SHP heterodimerization and also further characterized DAX1-DAX1 homodimerization and DAX1-DAX1A heterodimerization. We showed involvement of the SHP LXXLL motifs and AF-2 domain in SHP homodimerization and DAX1-SHP heterodimerization. We demonstrated redundancy of the LXXLL motifs in DAX1 homodimerization. While DAX1A subcellular localization is mostly cytoplasmic, DAX1-DAX1A heterodimers existed in the nucleus, suggesting differential functions for DAX1A in each compartment. We showed that the AF-2 domain of DAX1 is involved in DAX1-DAX1A heterodimerization. These results indicate that NR0B family members use similar mechanisms for homodimerization as well as heterodimerization. These resemble coactivator-receptor interactions that may have potential functional consequences for molecular mechanisms of the NR0B family.     

Novel mutation of the DAX1 gene in a patient with X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism

X-linked adrenal hypoplasia congenita (AHC) is characterized by primary adrenal insufficiency and is frequently associated with hypogonadotropic hypogonadism (HHG). Mutations of the DAX1 gene have been reported in patients with AHC and HHG. We found a novel DAX1 mutation in our patient. Sequence analysis of the patient's DAX1 demonstrated a 1-bp (G) deletion at codon 49 in exon 1. The mutation shifts the reading frame, resulting in completely different amino acid sequences from codon 49 to the premature stop codon at 84. The G was present at this position in the sequences of the father and 2 younger brothers. Direct sequence and single-strand conformation polymorphism analyses of polymerase chain reaction fragments revealed that the mutation at codon 49 was heterozygously present in the mother's DAX1 gene. The codon 84 is located in the first half of the DNA binding domain, and the mutation site is closer to the N-terminus than those in previously reported cases. The onset of adrenal insufficiency in the neonatal period as seen in our patient has also been reported in other patients with different DAX1 mutations, especially in a patient with DAX1 protein lacking 11 amino acids at the C-terminus. Thereore, it is less likely that position of termination codons correlate to clinical manifestations. Am. J. Med. Genet. 76:62–66, 1998. © 1998 Wiley-Liss, Inc.     

Immunocytochemical study of NR1, NR2A and NR2B subunits of NMDA receptor in frog retina (Rana ridibunda)

The NMDA receptors are ionotropic glutamate receptors that are involved in a variety of functions in the nervous system and in particular in the retina. They are composed of NR1 and NR2 subunits. The NMDA receptors have been fairly well studied in the retina of mammals, however, there is only limited information concerning these receptors in the retinas of lower vertebrates. The aim of the present study was to investigate immunocytochemically the NR1, NR2A and NR2B subunits of the NMDA receptors in the frog retina. Six primary antibodies were used. Three of them were directed to different splice variants of the NR1 subunit and the remaining three variants directed to NR2 subunits. All antibodies showed well expressed labeling in the frog retina. The labels had a punctate character and were located mainly in the inner and the outer plexiform layers. The results obtained indicate that the NR1, NR2A and NR2B subunits of NMDA receptor may participate in the glutamatergic neurotransmission from photoreceptors to second order retinal neurons, as well as from bipolar cells to third order retinal neurons. It has been proposed that in the frog retina, several subtypes of NMDA receptors exist each involved with different functions.     

Immunolocalization of NR1, NR2A, and PSD-95 in rat hippocampal subregions during postnatal development

Although the expression of NMDARs and synaptic-associated proteins has been widely studied, the temporospatial distribution of NMDAR subunits and synaptic proteins in different hippocampal subregions during postnatal development still lacks detailed information, and the relationship between NR1 or NR2 subunits and PSD-95 family proteins is controversial. In this study, we used immunofluorescent staining to assess NR1 or NR2A and PSD-95 expressions and the relationship between them in CA1, CA3, and DG of rat hippocampus on postnatal (P) days: P0, P4, P7, P10, P14, P21, P28, P56. The results showed that from P0 to P56, NR1, NR2A, and PSD-95 expressions increased gradually, and the time points of their expression peak differed in CA1, CA3, and DG during postnatal development. Interestingly, although the expression of PSD-95 was positively correlated to both NR1 and NR2A, the NR1 and PSD-95 coexpressed puncta were greatest in CA3, while NR2A and PSD-95 coexpressed puncta were greatest in CA1, compared to other subregions. Surprisingly, at P21, among different strata of CA1, the area of highest expression of NR2A was dramatically changed from stratum pyramidale to stratum polymorphum and stratum moleculare, and returned to stratum pyramidale gradually on the later observed days again, indicating that P21 may be one critical timepoint during postnatal development in CA1. The specific temporospatial distribution pattern of NR1, NR2A, and PSD-95 might be related to the different physiological functions during postnatal development. Discovering the alteration of the relationship between PSD-95 and NMDAR subunits expression may be helpful for understanding mechanisms and therapy of neurodegenerative diseases.     

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.     

NR5A1 gene variants repress the ovarian‐specific WNT signaling pathway in 46,XX disorders of sex development patients

Several recent reports have described a missense variant in the gene NR5A1 (c.274C>T; p.Arg92Trp) in a significant number of 46,XX ovotesticular or testicular disorders of sex development (DSDs) cases. The affected residue falls within the DNA‐binding domain of the NR5A1 protein, however the exact mechanism by which it causes testicular development in 46,XX individuals remains unclear. We have screened a cohort of 26 patients with 46,XX (ovo)testicular DSD and identified three unrelated individuals with this NR5A1 variant (p.Arg92Trp), as well as one patient with a novel NR5A1 variant (c.779C>T; p.Ala260Val). We examined the functional effect of these changes, finding that while protein levels and localization were unaffected, variant NR5A1 proteins repress the WNT signaling pathway and have less ability to upregulate the anti‐testis gene NR0B1. These findings highlight how NR5A1 variants impact ovarian differentiation across multiple pathways, resulting in a switch from ovarian to testis development in genetic females.     

DAX1 and its network partners: exploring complexity in development

DAX1 encoded by NR0B1, when mutated, is responsible for X-linked adrenal hypoplasia congenita (AHC). AHC is due to failure of the adrenal cortex to develop normally and is fatal if untreated. When duplicated, this gene is associated with an XY sex-reversed phenotype. DAX1 expression is present during development of the steroidogenic hypothalamic-pituitary-adrenal-gonadal (HPAG) axis and persists into adult life. Despite recognition of the crucial role for DAX1, its function remains largely undefined. The phenotypes of patients and animal models are complex and not always in agreement. Investigations using cell lines have proved difficult to interpret, possibly reflecting cell line choices and their limited characterization. We will review the efforts of our group and others to identify appropriate cell lines for optimizing ex vivo analysis of NR0B1 function throughout development. We will examine the role of DAX1 and its network partners in development of the hypothalamic-pituitary-adrenal/gonadal axis (HPAG) using a variety of different types of investigations, including those in model organisms. This network analysis will help us to understand normal and abnormal development of the HPAG. In addition, these studies permit identification of candidate genes for human inborn errors of HPAG development.     

Two sisters with IMAGe syndrome: cytomegalic adrenal histopathology, support for autosomal recessive inheritance and literature review

Adrenal hypoplasia congenita (AHC) is a rare condition and causes primary adrenal insufficiency. X-linked (OMIM 300200) and autosomal recessive (OMIM 240200) forms are recognized. Recently, an association between Intrauterine growth restriction, Metaphyseal dysplasia, Adrenal hypoplasia congenita, and Genital abnormalities (IMAGe syndrome; OMIM 300290) has been described. We present the clinical features of two sisters with intrauterine growth restriction, AHC, and dysmorphic features. Interesting histopathologic findings of one sister are also presented. We suggest that IMAGe syndrome is the most plausible diagnosis and that autosomal recessive inheritance is likely. We analyzed genes that were postulated candidates for IMAGe syndrome (SF1, DAX-1, and STAR), and no mutations were found. Other cases of IMAGe syndrome are reviewed.     

Haplotypes of the NR4A2/NURR1 gene and cardiovascular disease: The Rotterdam Study

Nuclear receptor subfamily 4, group A, member 2 (NR4A2, also called Nurr1) has lately become of interest with regard to atherogenesis. We examined the association between common variation in the NR4A2 gene and cardiovascular disease in the Rotterdam Study, a prospective population‐based study among persons aged ≥55 years. Three SNPs that tag common haplotypes across a 36‐kb region surrounding the NR4A2 gene were determined. Four haplotypes with frequencies >1% covered 96% of the genetic variation. In 5,650 participants without history of coronary heart disease, 729 coronary heart disease events occurred during a median follow‐up time of 11.9 years. NR4A2 haplotypes were neither associated with coronary events nor with intima‐media thickness (IMT), carotid plaques, or ankle‐arm index (AAI). NR4A2 haplotypes showed a tendency toward associations with aortic and coronary calcification (haplo.score global simulation P values 0.076 and 0.075, respectively), which seemed to be based on haplotype 2 (individual P values were both P=0.015). Furthermore, NR4A2 haplotype 3 was associated with higher high‐density lipoprotein (HDL) cholesterol and haplotype 4 with lower systolic blood pressure. In conclusion, NR4A2/NURR1 haplotypes were not associated with coronary events, carotid IMT, carotid plaques, or AAI. There was a tendency toward associations with aortic calcification and coronary calcification. Associations for NR4A2 were found with both HDL levels and blood pressure. It remains to be investigated which pathophysiological mechanisms pertain to NR4A2 function in cardiovascular disease. Hum Mutat 0, 1–7, 2009. © 2009 Wiley‐Liss, Inc.