A novel mutation in DAX1 causes delayed-onset adrenal insufficiency and incomplete hypogonadotropic hypogonadism Antoine Tabarin, … , J. Larry Jameson, Philippe Bouchard J Clin Invest. 2000;105(3):321-328. https://doi.org/10.1172/JCI7212. Article Mutations in the DAX1 gene cause X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HHG). In affected boys, primary adrenal insufficiency occurs soon after birth or during early childhood; HHG is recognized at the expected time of puberty. In this report, we describe the novel phenotype of a man who presented with apparently isolated adrenal insufficiency at 28 years of age. Examination revealed partial pubertal development and undiagnosed incomplete HHG. Gonadotropin therapy did not improve his marked oligospermia, suggesting a concomitant primary testicular abnormality. Genomic analysis revealed a novel missense mutation, I439S, in DAX1. The mutant DAX-1 protein was studied for its ability to function as a transcriptional repressor of target genes. Consistent with the patient’s mild clinical phenotype, the I439S mutation conferred intermediate levels of repressor activity of DAX-1 when compared with mutations associated with classic AHC. This unique case extends the clinical spectrum of AHC to include delayed- onset primary adrenal insufficiency in adulthood and milder forms of HHG. Furthermore, in accordance with findings in Ahch (Dax1) knockout mice, the clinical features in this patient suggest that DAX-1 function is required for spermatogenesis in humans, independent of its known effects on gonadotropin production. Find the latest version: https://jci.me/7212/pdf A novel mutation in DAX1 causes delayed-onset adrenal insufficiency and incomplete hypogonadotropic hypogonadism Antoine Tabarin,1 John C. Achermann,2 Dominique Recan,3 Véronique Bex,1 Xavier Bertagna,4 Sophie Christin-Maitre,5 Masafumi Ito,2 J. Larry Jameson,2 and Philippe Bouchard5 1Département d’Endocrinologie, Diabétologie du CHU de Bordeaux, USN Haut Levêque, 33604 Pessac, France 2Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611, USA 3Service de Biochimie et Génétique Moléculaire, and 4Service d’Endocrinologie, Hôpital Cochin, 75014 Paris, France 5Service d’Endocrinologie et Maladies de la Reproduction, Hôpital Saint-Antoine, 75012 Paris, France Address correspondence to: A. Tabarin, Département d’Endocrinologie, Diabétologie du CHU de Bordeaux, USN Haut Levêque, Ave Magellan, 33604 Pessac, France. Phone: 33-5-56-55-64-24; Fax: 33-5-56-55-68-06; E-mail: [email protected]. Received for publication April 29, 1999, and accepted for publication December 14, 1999. Mutations in the DAX1 gene cause X-linked adrenal hypoplasia congenita (AHC) and hypogo- nadotropic hypogonadism (HHG). In affected boys, primary adrenal insufficiency occurs soon after birth or during early childhood; HHG is recognized at the expected time of puberty. In this report, we describe the novel phenotype of a man who presented with apparently isolated adrenal insuffi- ciency at 28 years of age. Examination revealed partial pubertal development and undiagnosed incom- plete HHG. Gonadotropin therapy did not improve his marked oligospermia, suggesting a con- comitant primary testicular abnormality. Genomic analysis revealed a novel missense mutation, I439S, in DAX1. The mutant DAX-1 protein was studied for its ability to function as a transcription- al repressor of target genes. Consistent with the patient’s mild clinical phenotype, the I439S muta- tion conferred intermediate levels of repressor activity of DAX-1 when compared with mutations asso- ciated with classic AHC. This unique case extends the clinical spectrum of AHC to include delayed-onset primary adrenal insufficiency in adulthood and milder forms of HHG. Furthermore, in accordance with findings in Ahch (Dax1) knockout mice, the clinical features in this patient sug- gest that DAX-1 function is required for spermatogenesis in humans, independent of its known effects on gonadotropin production. J. Clin. Invest. 105:321–328 (2000). Introduction tex and hypogonadotropic hypogonadism (HHG) (1, The DAX1 gene encodes an orphan member of the 11). Affected boys develop adrenal failure shortly after nuclear receptor superfamily that lacks the typical zinc birth or during early childhood, whereas HHG, a uni- finger DNA-binding motif, but retains the ligand-bind- versal feature of the syndrome, is usually recognized at ing domain characteristic of other family members (1). the expected time of puberty (9, 12, 13). Whether or not DAX-1 is expressed in the adrenal cortex, gonads, hypo- DAX1 mutations affect spermatogenesis in humans, thalamus and anterior pituitary (2). It interacts with independent of the effects of gonadotropin deficiency, another orphan nuclear receptor, steroidogenic factor-1 remains unknown (9). (SF-1) (3), which plays a pivotal role in the development In this report, we describe the clinical features of a and function of these tissues (4–9). In vitro studies sug- patient with a mild phenotypic presentation of AHC gest that DAX-1 represses SF-1–mediated transcription, and examine the functional properties of the mutant but the roles of SF-1 and DAX-1 in the development and DAX-1 protein. In addition, we describe the results of function of these tissues remain unclear (5–7). Recent exogenous gonadotropin therapy on spermatogenesis. results obtained in Ahch (the mouse Dax1 homologue) Recognition of this unique phenotype is of practical knockout mice suggest that DAX-1 may also play a importance because it extends the clinical spectrum of direct role in spermatogenesis (10). the disease to include mild forms of HHG and delayed Mutations in the DAX1 gene in humans cause the X- onset of adrenal insufficiency. Studies in this patient linked cytomegalic form of adrenal hypoplasia con- also suggest that DAX-1 function is required for sper- genita (AHC), a rare disorder characterized by impaired matogenesis in humans, independent of gonadotropin development of the permanent zone of the adrenal cor- and testosterone production. The Journal of Clinical Investigation | February 2000 | Volume 105 | Number 3 321 Methods (∆448–470) was made by restriction enzyme digestion Hormone assays and pulse analysis. Follicle-stimulating of wild-type cDNA (6). Each mutant DNA fragment hormone (FSH) and luteinizing hormone (LH) were was inserted into wild-type DAX1 cDNA using appro- measured using chemiluminescent immunoassays priate restriction enzyme sites. Mutations were veri- (Chiron Diagnostics, Cergy-Pontoise, France). The FSH fied by direct DNA sequencing. and LH assays had an analytical sensitivity of 0.3 IU/L Full-length wild-type or mutant (R267P, ∆V269, and 0.07 IU/L, respectively. The intra- and interassay I439S, and ∆448–470) DAX1 cDNA was introduced coefficients of variation were, respectively, 2.8% and into a pCMX vector to allow expression of full-length 4.6% at 4 IU/L for FSH, and 4.7% and 6.3% at 5 IU/L for wild-type or mutant DAX-1 protein. In addition, cDNA LH. Free α subunit (FAS) was measured using an corresponding to the putative ligand-binding (LBD) immunoradiometric assay (Immunotech, Marseilles, domain of DAX-1 alone (codons 207–470) was intro- France). The FAS assay had an analytical sensitivity of duced into a pBIND vector (Promega Corp., Madison, 0.02 IU/L. The intra- and interassay coefficients of vari- Wisconsin, USA) and linked to a GAL4 DNA-binding ation were 6.8% and 8.6% at 0.44 IU/L. Serum inhibin domain (DBD) to allow expression of wild-type and B was measured as described previously (14). LH and mutant GAL4–DAX-1 fusion proteins. FAS pulsatility was determined using cluster analysis Construction of SF-1 and Egr-1 expression vectors and (method number 7)(15). reporter genes. Human SF1 (FTZF1) cDNA (kindly pro- DNA sequencing and mutation analysis. Genomic DNA vided by M.S. Ramayya, University of Washington, was extracted from peripheral blood leukocytes using Seattle, Washington, USA) was introduced into a standard procedures. The gonadotropin-releasing hor- pCMX vector to express full-length SF-1 protein. In mone (GnRH) receptor gene was sequenced as addition, cDNA corresponding to the transcription- described previously (16). Exons 1 and 2 of DAX1 were al activation domain of SF-1 (codons 133–461) was PCR-amplified with specific primers as described pre- introduced into a pBIND vector and linked to the viously (11). The following primer pair was used to GAL4 DBD to express a GAL4–SF-1 fusion protein amplify and sequence exon 2: 2F (sense): 5′-GCTAG- (6). Rat early growth response 1 (Egr1) cDNA was CAAAGGACTCTGTGGTG-3′ and 2R (antisense): 5′-CCCT- introduced into a pCMX vector for expression of full- CATGGTGAACTGCACTAC-3′. PCR was performed in 50- length Egr-1 protein (17). µL volumes containing 200 ng of genomic DNA, 50 The UAS-TK109luc reporter was used for GAL4 pM of each dNTP, 10 pmol each of primer (2F and 2R), fusion protein studies, as described previously (6). This 16.6 mM (NH4)2SO4, 6.7 mM MgCl2, 67 mM Tris, 6.7 reporter contains 2 GAL4 binding sites (UAS) upstream µM EDTA, 1% DMSO, and 0.4 units of Taq-I poly- of the –109 TK promoter. A rat LHβ gene reporter con- merase (Roche Diagnostics, Meylan, France). PCR con- taining nucleotides –154 to +5 of the LHβ gene pro- ditions were 30 cycles as follows: 30 seconds at 96°C, moter was inserted into a pA3 luciferase vector. 90 seconds at 60.5°C, and 2 minutes at 72°C. This Transient gene expression studies. Transient gene ex- sequence was followed by a final extension step at 72°C pression studies were performed using a human for 7 minutes. PCR products were run on a 1.5% embryonic kidney tsa201 cell line (18). These cells NuSieve gel. Purified PCR products (50 ng) were were grown in DMEM supplemented with 10% FBS, sequenced directly using the Taq Big Dye Terminator 1% penicillin, and 1% streptomycin in a 5% CO2 sequencing kit (Perkin-Elmer Applied Biosystems, Fos- atmosphere at 37°C.