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CLINICAL CASE SEMINAR |
Department of Paediatric Endocrinology (A.A.V.S., M.A.d.V.), Department of Medical Genetics (J.C.G., R.J.S.), Universitair Medisch Centrum, Lundlaan 6, 3508 AB Utrecht, The Netherlands; Division of Endocrinology, Metabolism, and Molecular Medicine (G.O., T.J.P., R.M.H., J.W., J.L.J.), Northwestern University, The Feinberg School of Medicine, Chicago, Illinois 60611-3008
Address all correspondence and requests for reprints to: J. Larry Jameson, M.D., Ph.D., Department of Medicine, Northwestern Memorial Hospital, Galter Building 3-150, 251 East Huron Street, Chicago, Illinois 60611. E-mail: ljameson{at}northwestern.edu.
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Abstract |
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Objective: The objective was to identify novel clinical phenotypes associated with DAX1 missense mutations.
Patients and Design: We investigated the genetic basis of isolated mineralocorticoid deficiency in a patient who carries a unique missense mutation (W105C) in the amino-terminal region of DAX1.
Results: The W105C DAX1 mutation in the proband was present in three asymptomatic hemizygous males, but it was not detected in the general population. Using in vitro studies of DAX1 expression and function in transfected cells, we demonstrate that the mutant DAX1 protein exhibits mild loss of function, whether studied for genes it represses or for genes it activates. Structure-function studies suggest that the W105C and other mutations in the aminoterminus are compensated by the presence of repeated LXXLL motifs that mediate DAX1 interactions with other proteins.
Conclusions: We describe the first missense mutation in the aminoterminus of DAX1 and conclude that mutations in this region may be partially compensated by redundant functional domains. Mild DAX1 mutations may be a cause of isolated mineralocorticoid deficiency.
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Introduction |
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Mutations or deletions of DAX1 cause X-linked adrenal hypoplasia congenita (AHC) (OMIM, 300200) (1). AHC is an inherited disorder of adrenal gland development, characterized by lack of the permanent zone of the adrenal cortex. Boys with this condition usually present with severe primary adrenal failure in infancy or early childhood. Hypogonadotropic hypogonadism becomes apparent at puberty, and infertility results from gonadotropin deficiency in combination with a primary defect in spermatogenesis (6, 7). Almost 100 different mutations in DAX1 have been described (8, 9), most of which are nonsense or frameshift mutations that cause premature truncation of the protein. Remarkably, missense mutations have been restricted to the carboxy-terminal domain, and have been shown to either impair protein folding and nuclear localization or impair transcriptional repression (10). No missense mutations have been reported in the amino-terminal domain, which contains the repeated LXXLL protein interaction motifs.
In this study, we report a boy who presented with a variant form of AHC (isolated mineralocorticoid deficiency) associated with a W105C missense mutation in the aminoterminus of DAX1. In vitro studies were performed to elucidate the functional effects of this mutation and its interactions with the LXXLL repeats.
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Subject and Methods |
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After obtaining institutional approval and written informed consent, genomic DNA was extracted from peripheral blood leukocytes using standard procedures. The proximal promoter and both exons of DAX1, SF1, and CYP11B2 were amplified by PCR using specific oligonucleotide primer pairs and conditions described previously (11). Direct sequencing of PCR products was performed using a Taq big dye terminator sequencing kit and ABI 310 automated sequencer (PE Applied Biosystems, Foster City, CA).
Construction of human DAX1 expression vectors
DAX1 expression vectors (pCMX) containing W105C and a series of artificial N-terminal missense mutations (W105A, W105P) were created by overlapping PCR using methods described previously (12, 13, 14). Expression vectors containing cDNA for wild-type (WT) DAX1 and the naturally occurring L381H missense mutant (13) were used as positive and negative controls for DAX1 repressor activity. Similarly, triple mutant of the third consensus LXXLL motif were created by introducing alanine substitutions at codons 149 and 150 into the L146A background (AXXAA).
The internally deleted expression vector, which lacks 39 codons flanking codon 105 (
92–132), was constructed by ligation of two PCR-generated fragments (after digestion with SacII and XbaI/NheI, and EcoRI and SplI, respectively) and a spacer double-stranded oligo with sticky ends (i.e. SplI and ScaII recognition sites) into an empty pCMX vector. The expression vector pBKCMV carrying the WT DAX1 cDNA was used as a template for generating these two fragments.
To allow antibody-mediated detection of recombinant DAX1 proteins containing the V5 epitope and a polyhistidine tag, DAX1 cDNAs for WT, W105C, and Q37X (a naturally occurring severe truncation mutation rescued by in-frame alternate translation) (14) were cloned into pcDNA 6/V5-HisA expression vector (Invitrogen, Carlsbad, CA).
The presence of desired mutations/deletion and the integrity of the constructs were confirmed by direct sequencing before studies of protein expression and function.
Western blotting
Human embryonic kidney tsa201 cells were transfected with 10 µg of pcDNA6/V5-HisA DAX1 WT or W105C mutant. Equivalent amounts of protein lysates from transfections were resolved with SDS-PAGE and transferred to polyvinyl difluoride membranes using standard methods. Recombinant DAX1 was probed with a 1:5,000 dilution of the primary antibody toward the V5 epitope and a 1:10,000 dilution of the secondary antimouse antibody (14).
Immunocytochemistry
H295R cells were grown on poly-L-lysine coated coverslips, washed three times with PBS, and fixed with 4% neutral-buffered formalin on ice for 30 min. Cells were permeabilized with 0.5% Triton X-100 for 20 min on ice and washed with PBS three times. Blocking solution consisting of either 10% normal goat serum or 10% normal horse serum was added to the cells for 1 h at room temperature. For immunodetection of endogenous DAX1, cells were incubated with anti-DAX1 rabbit polyclonal antibody (sc-841; Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:50 in blocking solution. For detection of exogenously transfected pcDNA6/V5HisA-WT hDAX1 or pcDNA6/V5HisA-hDAX1 W105C, anti-V5 mouse monoclonal antibody (46–0705; Invitrogen, Carlsbad, CA) was diluted 1:100 in blocking solution. V5 and Dax1 were detected using fluorescein-conjugated antimouse IgG (Vector Laboratories, Burlingame, CA) and Texas Red-conjugated antirabbit IgG (Vector Laboratories), respectively (1:100 in blocking solution).
Functional analysis of WT and mutant DAX1
Transient gene expression studies using human embryonic kidney tsa201 were performed in DMEM supplemented with 10% fetal bovine serum and 1% streptomycin/penicillin in a 5% CO2 atmosphere at 37 C. DAX1 repression of SF1-mediated transactivation was examined using several different assays. A cDNA encoding the DNA-binding domain of the yeast GAL4 protein fused in frame with the ligand-binding domain of SF1 (codons 133–461) was cloned into the pBIND expression vector. Reporter assays were conducted using 20 ng GAL4-SF1, 50 ng DAX1 (cloned into pCMX), and 500 ng UAS-TK109luc (15). In a second set of assays, a luciferase reporter construct (500 ng) containing the native rat LHß promoter (–154 to +5) was cotransfected with expression vectors containing full-length human SF1 (NR5A1) (20 ng), full-length rat early growth response-1 (Egr1) (20 ng), and full-length human WT or mutant DAX1 (50 ng), as described elsewhere (14, 15, 16). Luciferase assays were performed 48 h later.
Transient gene expression studies using H295R cells were performed in a 1:1 mixture of DMEM and Ham’s F12 medium supplemented with 2.5 mM L-glutamine, 15 mM HEPES, 2.5% 
-Serum (BD Biosciences, San Jose, CA), and ITS+Premix (BD Biosciences). DAX1 enhancement of SF1-mediated transactivation was examined in reporter assays using 200-ng full-length human SF1 (NR5A1), 300-ng full-length human WT or mutant DAX1 and 500-ng reporter constructs (hCYP11B1 or hCYP11B2; kindly provided by Dr. William Rainey, University of Texas Southwestern, Dallas, TX).
Statistical analysis
Data are presented as a percentage of maximal stimulation (100%). Bars represent the mean ± SEM for three to four independent experiments. Statistical analysis was performed using ANOVA followed by the Fisher post hoc test. For clarity, only relevant statistical comparisons are indicated in the figures.
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Results |
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The patient described here is an 11-yr-old prepubertal Caucasian boy of Dutch origin who presented with failure to thrive and vomiting at the age of 4 wk. He was moderately dehydrated but not hyperpigmented. Clinical laboratory investigations revealed hyponatremia (110 mmol/liter) and hyperkalemia (7.2 mmol/liter). He was started on hydrocortisone and fludrocortisone replacement with a preliminary diagnosis of congenital adrenal hyperplasia (Tables 1
and 2). Serum creatine kinase, glycerol kinase, and very long chain fatty acids were normal. Ornithine transcarbamylase deficiency was excluded by measuring a normal serum ammonium level. He was subsequently withdrawn from hydrocortisone replacement therapy and has not experienced adrenal crisis while on mineralocorticoid treatment only. Although ACTH levels were not elevated, the first ACTH stimulation test showed subnormal cortisol results. A second test showed normal cortisol values, suggesting sufficient adrenal function (Table 2). Family history was unremarkable except for a diagnosis of unilateral Wilms tumor in a 17-yr-old male cousin at the age of 2 yr.
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After excluding mutations in the genes encoding aldosterone synthase (CYP11B2) (Kiel, M. P., personal communication) and SF1, direct DNA sequencing revealed a novel tryptophan to cysteine missense mutation (W105C, TGG
TGC) in the amino-terminal region of DAX1 (Fig. 1). This mutation was detected in the proband’s mother as well as five other females and, unexpectedly, in three unaffected males in the family (Fig. 2). All three unaffected hemizygous males (IV.5, V.5, and V.8) had no signs and symptoms of adrenal insufficiency or hypogonadism. These males were evaluated further after a 5-d salt-restricted diet. Renin and aldosterone responses were normal, indicating adequate mineralocorticoid reserve.
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Functional studies of the W105C mutation
Functional studies were performed to assess whether the W105C substitution may represent a hypomorphic allele. First, we confirmed that the W105C mutant was expressed and efficiently translated in mammalian cells using V5 epitope-tagged WT and W105C DAX mutants (data not shown). Although the DAX1 W105C mutation was predominantly nuclear localized, the receptor was partially retained in the cytoplasm.
The DAX1 W105C mutation was tested in several different transfection assays to assess its ability to modulate SF1-mediated transcription. When cotransfected with Gal4-SF1, WT DAX1 exerted 65% repression, whereas a control AHC mutant (L381H) (13) mediated approximately 10% repression (Fig. 3). The W105C mutant induced approximately 50% repression, indicating that it has diminished repressive activity compared with WT.
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). Whereas the control AHC mutant (L381H) (13) exhibited minimal SF1 stimulation (2.3-fold), the W105C mutant conferred partial SF1 stimulation (4.3-fold), indicating that it retains substantial enhancing activity but is less effective than WT.
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). WT DAX effectively suppressed SF1-mediated transcription (86% repression), whereas transcriptional repression was largely eliminated in the carboxy-terminal L381H mutant (18% repression). Using this reporter system, the W105C mutant retained transcriptional repression (84% repression), although it was consistently lower than WT DAX1 in multiple experiments. The substitution of a Cys residue at codon 105 creates the possibility of forming nonproductive disulfide bonds. Thus, to assess further the functional importance of the Trp105, this residue was also substituted with alanine and proline. Each of these substitutions caused greater loss of repression (64% repression) than the Cys mutant. Thus, mutations at Trp105 consistently impair transcriptional repression but the Trp to Cys substitution appears to be better tolerated than certain other residues.
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). Because the W105C missense mutation lies between the second and third LXXLL motifs, we considered the possibility that alterations in this region might disrupt interactions between these hydrophobic protein binding sites. However, a deletion (92-132) between these motifs minimally reduced transcriptional repression. Taken together, these findings suggest that the amino terminus of DAX1 is relatively resistant to mutational effects because of redundant LXXLL motifs. Specifically, mutation of W105 or deletion of the region containing this residue minimally impairs DAX1 repression of SF1-mediated transcription. |
Discussion |
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The clinical presentation of AHC in the proband described here is unusual in two respects. First, the patient had prominent hypoaldosteronism without clear evidence of glucocorticoid insufficiency. Although transient neonatal hypoaldosteronism or progressive mineralocorticoid deficiency with elevated plasma renin activity have been described in AHC (W39X and Y380D, respectively) (14, 15), these patients had concomitant glucocorticoid deficiency. Second, several male relatives who carry the W105C mutation are clinically unaffected, without evidence of adrenal or reproductive dysfunction. These features indicate phenotypic heterogeneity, presumably caused by the effects of other genes that modify or compensate for DAX1 function (20, 21). Alternatively, environmental events such as illness or exposure to medications could unmask underlying adrenal dysfunction. The variable expression of the AHC phenotype is reminiscent of other genetic disorders, particularly when the mutation has partial effects on protein function. For example, in the syndrome of resistance to thyroid hormone, the R316H mutation in thyroid hormone receptor ß is associated with variable degrees of hormone resistance among family members with the same mutation (22, 23).
An additional unusual feature of the W105C mutation is its location in the amino-terminal region of DAX1, because each of the previously identified missense mutations cluster within the carboxy-terminal region (13). The idea that the W105C mutation is a disease-causing allele, as opposed to a polymorphism, is supported by its absence among healthy individuals. Of note, this tryptophan residue is highly conserved in several other species including mouse, monkey, and pig (24). The functional studies of the W105C mutation indicate that it consistently reduces DAX1 repression of SF1-mediated transcription, although the loss of function is much less than seen with other DAX1 mutations. Of note, we also found loss of DAX1 enhancement of SF1-mediated transcription of the CYP11B1 promoter by the W105C mutant. This is the first report of DAX1 regulation of CYP11B1 and the observed transcriptional stimulation is reminiscent of that seen previously for the CYP11A1 promoter. Although the mechanistic basis remains unknown for how DAX1 can mediate transcriptional activation for some promoters and repression for others, the loss of DAX1 function occurs for both DAX1 mediated repression and activation. These findings are consistent with other studies suggesting a correlation between DAX1 function in vitro with the severity of clinical phenotype as assessed by age of onset and severity of adrenal and reproductive abnormalities (12, 13, 14, 15). It should also be recognized, however, that these transcriptional assays may not necessarily reflect all of the functions of DAX1, including its important developmental roles (25).
The function of the DAX1 amino terminus is incompletely understood, although there is increasing evidence that this region mediates interactions with other proteins such as SF1 or ER (3, 4, 17). This region has been shown to use three distinct LXXLL-like motifs (4, 17) that were initially identified as binding sites of nuclear receptors coregulators (26). All three copies of the LXXLL motifs are conserved among different species (human, mouse, and pig) except in chicken and alligator, which only have one copy (24). Structure–function studies of this region suggest partial functional redundancy since mutations in all three repeats are necessary to abrogate protein–protein interactions and transcriptional repression by DAX1 (4, 17). The location of the W105C mutation between the second and third LXXLL motifs (Fig. 1) led us to consider that it might function by disrupting the actions of these motifs. However, consistent with previous studies (4, 17), we find that mutations in either the second or third LXXLL motif, or deletion of the intervening region, has minimal effect on DAX1-mediated repression, suggesting alternative explanations. For example, Trp105 may play some other structural role, such as altering DAX1 nuclear localization (10) or inducing protein misfolding, particularly because a Cys residue is introduced by the mutation. Because mutations to several other residues (Ala, Pro) have even more pronounced effects than the identified Cys mutation, it is possible that Trp105 is directly involved in DAX1 structure and function. A nonsense mutation (W105X) in a boy with classic AHC (27) also raises the possibility that codon 105 may be a hot spot for mutations.
In summary, we describe the first amino-terminal missense mutation in DAX1 in a subject with atypical and mild AHC, suggesting that missense mutations in the DAX1 amino terminus may impair protein function sufficiently to cause clinical presentation of AHC. These findings expand the phenotypic spectrum of AHC and suggest that DAX1 mutations may cause hypoaldosteronism, with normal glucocorticoid and gonadotropin production.
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Acknowledgments |
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Footnotes |
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Current address for G.O.: Division of Endocrinology and Metabolism, GATA School of Medicine, Haydarpasa Teaching Hospital, Istanbul 34668, Turkey.
A.A.V.S., G.O., M.A.d.V., J.C.G., R.J.S., T.J.P., R.M.H., J.W., and J.L.J. have nothing to declare.
First Published Online December 12, 2006
1 A.A.V.S. and G.O. contributed equally to this work. 
Abbreviations: AF2, Activation function-2 domain; AHC, adrenal hypoplasia congenita; DBD, DNA-binding domain; ER, estrogen receptor; LBD, ligand-binding domain; LXXLL motif, leucine-rich receptor binding motif; SF1, steroidogenic factor-1; WT, wild type.
Received November 7, 2005.
Accepted December 4, 2006.
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, Hemizygous males; 
, heterozygous carrier females. Generations are indicated in Roman numerals. Arrow indicates proband. Genotyping was performed in all individuals labeled with "DNA." IV:5, Asymptomatic, normal semen analysis; V:5, asymptomatic male, unilateral Wilms tumor at age 2 yr.
