x - linked adrenal hypoplasia congenita ( ahc , omim#300200 ) is a rare disorder that is characterized by primary adrenocortical failure , occurring due to a lack of permanent adult cortical zone of the adrenal glands . ahc manifests itself mainly in early infancy or in childhood and is caused by deletions or point mutations in the nr0b1 ( dax1 ) ( dosage - sensitive sex reversal , adrenal hypoplasia critical region , on chromosome x , gene 1 ) gene ( omim300473 ) ( figures 1(a)1(c ) ) . nr0b1 ( dax1 ) deletion may occur in isolation or as part of a contiguous gene deletion syndrome at xp21 including the dystrophin gene ( dmd ) and the glycerol kinase gene ( gk ) . such mutations result in ahc , duchenne muscular dystrophy ( dmd ) , and glycerol kinase deficiency ( gkd ) [ 25 ] . in some patients loss of the telomeric locus for the interleukin 1 receptor accessory protein - like gene ( il1rapl1 ) may occur and is associated with developmental delay and intellectual disability [ 4 , 5 ] . nr0b1 ( dax1 ) is located on the short arm of the x chromosome ( xp21.3-p21.2 ) and encodes for an orphan nuclear hormone receptor that functions as a transcriptional regulator of other genes . it is expressed in the adrenal glands , gonads , hypothalamus , and pituitary gland and is crucial for their proper development and function . nr0b1 ( dax1 ) is a transcriptional repressor of other proteins involved in the adrenal steroidogenesis pathway such as steroidogenic factor 1 ( sf-1 ) , steroidogenic acute regulatory protein ( star ) , p450scc , and 3-hydroxysteroid dehydrogenase [ 7 , 8 ] . nr0b1 ( dax1 ) is also involved in sex determination ; it antagonizes the action of the testis - determining factor sry ( sex - determining region y ) during gonadal development . nr0b1 ( dax1 ) is expressed in sertoli cells ; thus it is directly involved in the spermatogenesis process . nr0b1 ( dax1 ) also acts as a shuttling protein between nucleus and cytoplasm . nr0b1 ( dax1 ) protein binds mrnas associated with polyribosomes and regulates other gene expressions on the posttranscriptional level . almost two hundred mutations in the nr0b1 ( dax1 ) gene have been identified to date , and the relationship between mutations in the nr0b1 ( dax1 ) gene and ahc is well documented . frameshift and nonsense mutations may occur within the entire open reading frame of the gene . missense mutations are not so common and are found mainly in the region encoding for the ligand - binding domain ( lbd ) at the c - terminus of the nr0b1 ( dax1 ) protein . other pathogenic alterations that affect the nr0b1 ( dax1 ) gene comprise copy - number variations ( cnvs ) of different size . these changes may involve either the entire coding sequence of nr0b1 ( dax1 ) with or without other adjacent genes or are confined to its intragenic fragments ( e.g. , single exon deletions ) . to date , such mutations have been described in 22 nonconsanguineous cases , including 17 deletions ( hgmd professional 2012.3 ) . ahc is associated with the deletions , which in hemizygous male patients result in a complete loss of the function of nr0b1 ( dax1 ) . nr0b1 ( dax1 ) is crucial for the development of the adrenal glands and the hypothalamic - pituitary - gonadal axis ; hence its disruption can result in various clinical phenotypes leading to adrenal hypoplasia congenita ( ahc ) . the symptoms often occur early in life , in early infancy or childhood , and include salt loss , hypoglycemia , weak gain of weight , vomiting , prolonged period of postnatal jaundice , skin hyperpigmentation , hyponatremia , hyperkalemia , low level of cortisol , low level of aldosterone , high level of acth , and higher plasma renin activity . ahc can be fatal , unless appropriate steroid replacement therapy is provided to patients immediately [ 48 ] . hypogonadotropic hypogonadism ( hh ) is the most frequently observed puberty disorder that is caused by mutations in the nr0b1 ( dax1 ) gene and is due to impaired gonadotropin synthesis and release . hh is frequently followed by absence or delayed puberty and testosterone replacement therapy is usually introduced to induce sexual maturation in patients with ahc and hh [ 912 ] . several rare cases with ahc and normal puberty , peripheral precocious puberty , acth - dependent precocious puberty , or central precocious puberty have also been described hitherto [ 7 , 1316 ] . abnormalities in spermatogenesis ( oligo- , azoospermia ) may also occur in patients with ahc and nr0b1 ( dax1 ) mutations [ 6 , 9 , 12 , 17 , 18 ] . nr0b1 ( dax1 ) gene duplication leads to dosage - sensitive sex reversal ( dss ) ( 46,xy male individuals develop as phenotypic females ) and has been found in several patients . duplications of the x chromosome region containing nr0b1 ( dax1 ) also lead to 46,xy - gonadal dysgenesis or ambiguous genitalia . nr0b1 ( dax1 ) is known for its  antitestis  function ; it acts antagonistically to sry , wt1 , and sf1 and hence inhibits amh ( anti - mllerian hormone ) production . an extra copy of nr0b1 ( dax1 ) can therefore block the development of the male reproductive system . schematic representation of this gene is shown in figures 1(a)1(c ) [ 9 , 1930 ] . the aim of this study was to investigate the clinical and molecular background of congenital adrenal hypoplasia in a boy , in whom a nr0b1 ( dax1 ) mutation was suspected . the phenotypic boy was born to nonconsanguineous parents in the 41st week of gestation with a birth weight of 3880  g and apgar score 7/8/10 . he was referred and admitted to a regional pediatric hospital in the neonatal period , on the 25th day of life with severe vomiting . he was breast fed and also received extra portions of humanized milk . immediately after birth he was treated with combined antibiotic therapy since a congenital infection physical examination at admission revealed normal skin pigmentation and a skin rash on his trunk . his weight ( 4000  g ) was almost the same as at the time of delivery . > 400  nmol / l ( 150  ng / ml ) ] however coexisting with an elevated acth level [ 319.0  pg / ml ( ref . 1060 ) ] , thereby showing its insufficient release from the adrenal glands in a severe clinical state . the urinary steroid profile ( gc / ms ) suggested adrenal insufficiency ( congenital hypoplasia or bilateral massive adrenal hemorrhage ) ( table 1 ) . this test also suggested that there is no adrenal reserve in terms of cortisol secretion . by the clinical and biochemical findings , adrenal insufficiency due to congenital adrenal hypoplasia the remaining adrenal hormones [ 17-hydroxyprogesterone 1.8 and 0.8  ng / ml ( ref . 0.21.6 ) , androstenedione 0.6  ng / ml ( ref . 0.10.9 ) and dheas < 0.40 and 1.27  mol / l ( ref . 1.027.16 ) ] as well as testosterone level [ 1.30  nmol / l ( ref . 0.514.0 ) ] , were all in a wide spectrum of normal range for age and sex . combined treatment with iv / oral hydrocortisone , oral fludrocortisone , supplementation with saline , and rehydration was used . written informed consent was obtained from the patient and his mother and the study was approved by the local ethics committee ( number 621/11 ) . genetic analysis of the nr0b1 ( dax1 ) gene was performed in the molecular endocrinology laboratory of the department of pediatric endocrinology and rheumatology , poznan university of medical sciences , poland , as well as in the chair and department of medical genetics university of medical sciences in poznan . biochemical measurements were performed in the central laboratory of k. jonscher 's clinical hospital of the university using commercial kits . molecular studies of the nr0b1 ( dax1 ) gene were performed using the standard pcr method . genomic dna was isolated from peripheral blood leukocytes using qiaamp dna blood mini kit ( qiagen ) . two sets of primers came from genomed and were used to amplify the coding sequence , 5utr and 3utr , and flanking intronic sequences of the nr0b1 ( dax1 ) gene . polymerase chain reactions ( pcr ) were performed in a 20  l mixture using hotstartaq dna polymerase ( qiagen ) under the following parameters : denaturation at 95c for 15  min , followed by 4045 cycles of 95c for 60  s , 53c ( exon 1 ) or 50c ( exon 2 ) for 30  s , and elongation in 72c for 90  s ( exon 1 ) or for 45  s ( exon 2 ) . a final amplification at 72c for 10  min completed the pcr program . pcr products were then separated by electrophoresis on a 1.3% agarose gel in the presence of ethidium bromide ( merck ) used for visualization . the samples were then purified from the gel using qiaquick gel extraction kit ( qiagen ) and directly sequenced with the same primer pairs used in the pcr . sequencing reactions were performed with a bigdye terminator v3.1 cycle sequencing kit ( applied biosystems ) on an abi prism 3130xl genetic analyzer ( applied biosystems ) . finally , the sequences were analyzed using vector nti9.0 software ( invitrogen ) and compared to the ncbi reference sequence ng_009814.1 ( 22.07.2010 ) . blastn was used in analysis of the sequence of pcr product obtained in the patient that should correspond to exon 2 of the nr0b1 ( dax1 ) gene . array comparative genomic hybridization ( acgh ) was performed using a cgx format nimblegen 12-plex microarray with a resolution of 135  k ( 134829 oligonucleotide probes ) per haploid genome . cgx chips are designed to target gene - rich chromosomal regions at the highest density . dna of the index case as well as commercially available reference dna ( promega ) were differentially labeled with fluorescent dyes ( cy3 and cy5 , resp . ) and cohybridized onto the array slide according to the manufacturer 's protocols ( nimblegen ) . the array was subsequently scanned with the use of a roche nimblegen platform ( ms 200 microarray scanner ) . data generated on scanning was extracted by nimblescan software ( roche nimblegen ) and then analyzed for clinical relevance by means of genoglyphix software ( signature genomics ) . pcr amplification of the nr0b1 ( dax1 ) gene showed no amplification of exon 1 in the ahc patient ( figure 1(d ) , lanes 1 and 2 ) . sequencing of the pcr product obtained in the presence of primers specific for exon 2 of the nr0b1 ( dax1 ) gene showed a 92% identity of the sequence with human chromosome 7 . this product was obtained due to the deletion of the nr0b1 ( dax1 ) gene and hybridization of the primers for exon 2 to their complementary sequences located in chromosome 7 . using standard pcr method we were not able to define if the patient 's mother is a carrier of the mutation or not ( figure 1(d ) , lanes 3 and 4 ) . array comparative genomic hybridization revealed a loss of 24 consecutive oligonucleotide probes , thus showing hemizygous interstitial deletion on chromosome xp21.2 . minimal size of the detected cnv encompassed 284.01 ( genomic coordinates 3001753230301543 according to hg18 ) , whereas estimated maximum size was 329.80  kb ( figure 1(e ) ) . the deletion involved the five following genes : mageb2 , mageb3 , mageb4 , mageb1 , and nr0b1 ( dax1 ) . furthermore , a younger maternal cousin affected by ahc and his mother ( a healthy sister of the mother of our proband ) were also demonstrated to carry identical deletion ( figure 2 ) . in this study we present a boy with ahc , with normal testes and normal male external genitalia development in whom a novel type of deletion in the nr0b1 ( dax1 ) gene comprising the entire coding sequence of the nr0b1 ( dax1 ) was detected . this deletion resulted in a complete loss of function of the nr0b1 ( dax1 ) protein . the nr0b1 ( dax1 ) gene defect coexisted with the deletion of other neighbouring genes ( mage family genes ) and resulted from a submicroscopic microdeletion of the short arm of chromosome x. the role of the mage genes ( mageb1 , mageb2 , mageb3 , and mageb4 ) is still unknown ; however it is known that they are expressed in embryonic tissues , in some tumors , and after birth only in the testes . the microdeletion of the short arm of chromosome x found in our patient does not include the dmd gene ; therefore the features of muscular dystrophy should not appear in the future . x - linked ahc is caused predominantly by point mutations in the nr0b1 ( dax1 ) gene ; however it can also result from its partial or complete deletion . genomic rearrangements may account for either classical ahc or a phenotype with additional features , depending on the cnv size and number of genes that are affected . in the patient presented here , ahc was caused by a pathogenic deletion encompassing the nr0b1 ( dax1 ) and four mage family genes . therefore , we suggest that quantitative screening , preferably array cgh , should be included in the molecular testing of ahc cases , especially if the sequencing fails to detect causative alterations . unlike other targeted methods , such as qpcr or mlpa , array cgh allows for both confirmation of nr0b1 ( dax1 ) deletion and precise delineation of its size , which is extremely important for proper counseling and clinical management of affected individuals . most patients with x - linked ahc present adrenal insufficiency early in life and have combined glucocorticoid and mineralocorticoid deficiency . ahc is also characterized by puberty failure and hypogonadotropic hypogonadism as well as growth hormone deficiency later in life . individuals with milder forms of ahc have also been described , but the incidence is not common . in such patients adrenal insufficiency is incomplete and their sexual maturation is delayed or arrested due to mild gonadotropin deficiency [ 18 , 23 , 33 , 34 ] . these milder clinical phenotypes result from incomplete nr0b1 ( dax1 ) loss - of - function mutations , mostly including missense variants . for instance , two such mutations in the nr0b1 ( dax1 ) carboxy terminal region were described ( i439s , y380d ) with partial repression activity of sf1-mediated transcription [ 18 , 35 ] . in turn , a q37x nonsense mutation in nr0b1 ( dax1 ) led to an alternative translation of the nr0b1 ( dax1 ) protein generated from an alternate in - frame translation initiation site ( codon 83 for methionine ) . translation of this truncated isoform of the nr0b1 ( dax1 ) protein , possibly exerting some residual activity , might prevent the patient from the classical ahc phenotype and delay the onset of adrenal insufficiency until early adulthood . the clinical signs and symptoms of ahc are similar to that observed in patients with the salt wasting form of congenital adrenal hyperplasia caused by 21-hydroxylase deficiency ; thus it is very important to distinguish between these two disorders , because they have a different clinical course and implications for genetic counseling . here we presented a boy with a novel deletion of the entire nr0b1 ( dax1 ) gene , associated with congenital adrenal insufficiency and normal development of male external genitalia . this report thus highlights the value of nr0b1 ( dax1 ) genetic analysis in confirming an ahc diagnosis and emphasizes the critical role of genetic counseling in families with ahc patients , particularly in those with a significant deletion of x chromosome material including the nr0b1 ( dax1 ) gene .
x - linked adrenal hypoplasia congenita ( ahc ) is caused by deletions or point mutations in the nr0b1 ( dax1 ) gene . 
 we present a boy with ahc who came at the age of 25 days in a severe state due to prolonged vomiting and progressive dehydration . 
 laboratory studies showed prominent hyponatremia and hyperkaliemia but not hypoglycemia . 
 primary adrenal insufficiency was confirmed with low serum cortisol levels and high plasma acth levels . 
 hydrocortisone therapy combined with saline and glucose infusions was started immediately after blood collection . 
 two exons of the nr0b1 ( dax1 ) gene were impossible to amplify using the standard pcr method . 
 array cgh was used to confirm the putative copy - number variation of nr0b1 ( dax1 ) revealing a novel hemizygous deletion encompassing the entire nr0b1 ( dax1 ) gene together with the mageb genes . 
 this genetic defect was also present in heterozygosity in the patient 's mother . 
 we show that nr0b1 ( dax1 ) gene analysis is important for confirmation of ahc diagnosis and highlights the role of genetic counseling in families with ahc patients , particularly those with x chromosome microdeletions , covering more than nr0b1 ( dax1 ) alone . 
 we hope that further clinical follow - up of this patient and his family will shed a new light on the role of mageb genes .