J. Endocrinol. Invest. 34: e235-e239, 2011 DOI: 10.3275/7484

Two novel DAX1 gene mutations in Chinese patients with X-linked adrenal hypoplasia congenita: Clinical, hormonal and genetic analysis C.M. Wu1, H.B. Zhang2, Q. Zhou3, L. Wan1, J. Jin1, L. Ni4, Y.J. Pan1, X.Y. Wu1, and L.Y. Ruan1 1Department

of Endocrinology, The Second Affiliated Hospital of Wenzhou Medical College, Wenzhou; of Endocrinology, Key Laboratory of Endocrinology, Ministry of Health, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing; 3Department of Endocrinology, The First Affiliated Hospital of Wenzhou Medical College; 4Department of Clinical Laboratory, The Second Affiliated Hospital of Wenzhou Medical College, Wenzhou, China

2Department

ABSTRACT. Mutations in the DAX1 gene result in X-linked congenital adrenal hypoplasia (AHC). Affected boys usually present with primary adrenal failure in early infancy or childhood and hypogonadotropic hypogonadism (HH) at puberty. This paper describes the clinical, hormonal, radiological, and genetic characteristics of 2 Chinese patients with X-linked AHC. Primary adrenal insufficiency occurred in the 2 patients during their childhood and HH was recognized at puberty. Genomic DNA was extracted from their peripheral blood leukocytes and coding sequence abnormalities of the DAX1

gene were assessed by PCR and direct sequencing analysis. Genetic analysis of the DAX1 gene revealed 2 novel mutations c.572-575 dupGGGC, p.Thr193Gly,fs,205X and c.773774 dupCC, p.Ser259Pro,fs,264X in exon 1, causing frameshifts and yeilding premature stop codons at 205 and 264, respectively. This study identifies 2 novel mutations in the DAX1 gene which can further expand the mutation database and benefit patients in the diagnosis and treatment of AHC. (J. Endocrinol. Invest. 34: e235-e239, 2011) ©2011, Editrice Kurtis

INTRODUCTION

This paper describes 2 unrelated Chinese patients with classic AHC. Mutational analysis of the DAX1 gene identified 2 previously unreported mutations in the first exon.

X-linked adrenal hypoplasia congenita (AHC) (OMIM 300200) is a rare, potentially life-threatening, inherited disorder characterized by the absence of definitive zone of the adrenal cortex and by structural abnormality of the cortex filled with large vacuolated cells. Affected boys, usually develop severe adrenal insufficiency in early infancy or childhood. Hypogonadotropic hypogonadism (HH), another common feature of this disorder, can occur at the expected time of puberty (1). In addition, AHC may also occur as a part of a contiguous gene deletion syndrome including glycerol kinase deficiency and Duchenne’s muscular dystrophy (2). Mutations in the DAX1 gene (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1; also called as NROB1) is responsible for AHC (3). The gene is mapped to the Xp21.3-p21.2 region, composed of 2 exons separated by a 3.4-kb intron. It encodes a member of the nuclear receptor super family and is unusual because it lacks a characteristic zinc-finger DNA-binding domain. Since the first paper on mutational analysis in AHC reported by Muscatelli in 1994 (3), more than 200 DAX1 mutations have been identified worldwide, most of which are deletions or point mutations (4, 5).

MATERIALS AND METHODS Patients We observed 2 Chinese individuals who suffered from primary adrenocortical insufficiency and HH. In both cases, there was no reported history of consanguineous marriage for 3 generations. The clinical diagnosis of AHC was based on their clinical features, medical history, radiological findings, biochemical and hormonal data. Informed written consents were obtained from each patient and the family members who were involved in our study. The study was also approved by the Ethics Committee of the hospital.

hCG stimulation test A single im dose of 5000 IU of hCG (Zhuhai Pharmaceuticals, China) was given, with blood samples taken for serum testosterone measurements both basally and 72 h later.

Genetic analysis The complete DAX1 gene coding region, containing 2 exons, was amplified from genomic DNA obtained from peripheral blood leukocytes using a commercial DNA extraction kit (QIAamp DNA Micro; Qiagen, Germany). The primers used for PCR were provided by Dr Min Nie, the Department of Endocrinology, Peking Union Medical College Hospital. Exon 1 of the DAX1 gene was amplified by 3 sets of the sense and antisense primers, 1-1s (5’-GCGCCCTTGCCCAGACCGA-3’) and 1-1as (5’-CCCTGGCCTCTGCGCGAAGTAG-3’), 1-2s (5’-GCGACGCTGGGTCCGTGC-TG-3’) and 1-2as (5’-GCATGCTGGGCTCCGAGACTTC-

C.M. Wu and H.B. Zhang contributed equally to this work. Key-words: Adrenal hypoplasia congenita, DAX1, hypogonadotropic hypogonadism. Correspondence: C.M. Wu, Department of Endocrinology, The Second Affiliated Hospital of Wenzhou Medical College, Wenzhou 325027, Zhejiang, China. E-mail: [email protected] Accepted November 17, 2010. First published online January 26, 2011.

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Kindred 2 Kindred 1 I I

II

Fig. 1 - The pedigrees of two kindreds with X-linked congenital adrenal hyperplasia. Partially filled circles denote carrier (heterozygous) females, partially filled squares denote putatively affected males, and filled squares denote affected (hemizygous) males. Arrow indicates patient.

II

3’), 1-3s (5’-CCGGTGGC- GCTCAAGAGTCC-3’) and 1-3as (5’TTCTTCACCTTTGCCCCGACAC-3’), respectively. The sense and antisense primers for exon 2 were 2s (5’-GCTAGCAAAGGACTCTGTGGTG-3’) and 2as (5’-CCCTCATGGTGAACTGCACTAC-3’), respectively. The PCR amplifications were performed for 35 cycles. Each amplification cycle consisted of denaturation for 1 min at 94 C, annealing and extension at 68 Cfor 1 min (primers 1-1s and 1-1as, 1-2s and 1-2as, 1-3s and 1-3as), or annealing at 65 C for 30 sec and extension at 72 C for 1 min (primers 2s and 2as). Then, direct sequencing of PCR products was performed by automated DNA sequencing in an ABI DNA sequencer (Model 3730).

RESULTS Case reports Kindred 1 The index patient (II:5) (Fig. 1 ) was a 36-yr-old male who was born after a 40-week gestation with a birth weight of 3.2 kg and was well during the infant period. At the age of 8 yr he developed progressive skin hyperpigmentation. He subsequently presented to the local hospital at age of 9 yr with vomiting, fever, lethargy, and dehydration, following an episode of coryza, and was diagnosed with adrenal insufficiency of unknown etiology (the specific hormonal data not obtained), and treated with 5-10 mg/day prednisone. Since then he has experienced several adrenal crises during upper respiratory infections or discontinued replacements. At the age of 36 yr, he presented to the emergency department of our hospital with acute adrenal insufficiency. Presenting signs and symptoms were extreme fatigue, vomiting, and hyponatremia after drug withdrawal for 1 week. He also complained of impaired libido and infrequent erections, but he had not sought treatment for these symptoms. His 1 brother (II:2) had died at the age of 3 yr due to severe dehydration and hyponatremia. On admission, his height and weight were 152 cm (below 2.0 SD) and 33 kg (below 2.0 SD), respectively, and blood pressure was 118/62 mmHg. Physical examination revealed no axillary and pubic hair, small penis and testicular volume of 2 ml bilaterally. A few hyperpigmented macules were noted on the oral mucosa and palmar creases. The blood biochemical and the hormonal find-

ings are summarized in Table 1. A computed tomography (CT) scan of the abdomen revealed small non-calcified adrenal glands. Magnetic resonance imaging (MRI) of the hypothalamo-pituitary region was normal. Bone maturation measured at the left hand was 16 yr of age, without closure of the epiphyses. Dual-energy X-ray absorptiometry (DEXA) revealed a markedly low bone mineral density (BMD) (L1-L4: T-score –8.0, Z-score –9.5, and Femoral neck: T-score –5.5, Z-score –4.3). MRI of the spine revealed multiple thoracic compression fractures. The administration of intravenous fluids and hydrocortisone sodium succinate resulted in rapid improvement in his condition, which subsequently was maintained with hydrocortisone acetate: 20-30 mg/day. In addition, to treat hypogonadism and osteoporosis, supplementation with testosterone undecanoate, calcium, calcitriol and alendronate sodium (Merck Sharp & Dohme) was started. Kindred 2 The index case (II:2) (Fig. 1), a 26-yr-old male, was born after a 39-week gestation with a birth weight of 3.7 kg and was well during the infant period. He began to develop

Table 1 - Summarizes the blood biochemical and the hormonal findings in the 2 patients with X-linked congenital adrenal hyperplasia. Na+

(mmol/l)

Patient 1

Patient 2

Normal adult values

124.4

137.5

135.0-145.0

K+ (mmol/l)

5.05

4.72

3.50-5.30

Glucose (mmol/l)

4.75

5.56

3.90-6.10

Cortisol at 08:00 h (nmol/l)

11

27

240-618

ACTH at 08:00 h (pmol/l)

117

159

2-10

Aldo supine (pmol/l)

42

68

83-250

PRA supine (ng/l/s)

3.20

2.80

0.06-0.64

T (nmol/l)

1.10

2.40

6.07-27.10

LH (IU/l)

0.26

0.99

1.24-8.62

FSH (IU/l)

4.15

2.10

1.27-19.46

17OHP (nmol/l)

<0.3

<0.3

<6.0

hCG stimulation test

4.86

10.24

T at 72 h (nmol/l) Aldo: aldosterone; PRA: plasma renin activity; T: testosterone; 17OHP: 17α-hydroxyprogesterone.

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Novel mutations in the DAX1 gene

Mutational analysis of the DAX1 gene After the entire DAX1 gene had been sequenced, we found two novel mutations in our patients (Fig. 2). In patient 1, sequence analysis revealed a 4-nucleotide (c.572575 dup GGGC) duplication in exon 1. This duplication converts codon 193 from threonine to glycine followed by a 12-amino frameshift that results in premature termination after codon 205 (p.Thr193Gly,fs,205X). His mother was found to be heterozygous for this mutation. His father, brother (II:4) and sisters (II:1 and II:3), however, did not carry any genomic mutation in this gene. Patient 2 has a 2-base pair duplication (c.773-774 dupCC) in exon 1, which is predicted to result in a frameshift and premature stop codon after 5 amino acids (p.Ser259Pro, fs,264X). His mother was a heterozygous carrier for this mutation, whereas his father and brother (II:3) had a normal sequence of the DAX1 gene. The above results were confirmed in 2 different PCR reactions on both strands using a forward and a reverse primers.

progressive skin and buccal mucosa pigmentation from the age of 3 yr. At the age of 6 yr he presented to the local hospital following a 2-day history of drowsiness, nausea, vomiting, and fever. A provisional diagnosis of acute adrenal crisis was made based on an abnormal elevated serum ACTH, low cortisol level, and hyponatremia (the specific data was not obtained). He was commenced on prednisone replacement with saline supplementation. At the age of 10 yr he again presented with a history of a collapse following a vomiting illness. He was resuscitated with intravenous fluids and hydrocortisone sodium succinate. On this admission, he complained of frequent fatigue and hypogonadism was noted. Family history revealed the his older brother (II:1) had died at the age of 3 yr from a salt-wasting crisis, but unfortunately few other details are known. His height was 173 cm (approximately 0 SD) and weight was 51 kg (approximately –1.0 SD). Blood pressure was 90/58 mmHg. The moderate hyperpigmentation of buccal mucosa and gums was noted. Physical examination revealed no axillary hair, sparse pubic hair (Tanner stage 2), small penis (Tanner stage 2), and low testicular volume (2 ml bilaterally). The blood biochemical and the hormonal findings are summarized in Table 1. Bone age was markedly delayed (15.5 yr). A CT scan of the abdomen revealed small non-calcified adrenal glands. MRI scan of the sella showed “partially empty sella”. DEXA revealed a low BMD (L1-L4: T-score –3.1, Z-score –3.3, and Femoral neck: T-score –1.7, Z-score –1.6). The patient was maintained with hydrocortisone acetate: 20-40 mg/day and was advised to take a rich-salt diet. In addition, he was commenced on testosterone replacement therapy and the supplementation with calcium and calcitriol.

DISCUSSION In this study, 2 Chinese patients with X-linked AHC and HH in which the sequence analysis of the DAX1 gene identified 2 novel mutations. Based on the medical histories of several salt-wasting crises and HH, results of imaging studies, and measurements of hormones and electrolytes, the clinical diagnosis of AHC in our patients was relatively easy to make. In both cases, the family history of unexpected deaths in males was the potential indicator of X-linked AHC. Sometimes it may be misdiagnosed in clinic with the more frequent salt wasting form

Kindred 1

WT/WT

c.572-575 dupGGGC

c.572-575 dupGGGGC/WT

Kindred 2

WT

c.773-774 dupCC

c.773-774 dupCC/WT

Fig. 2 - The sequencing results for two kindreds: 1) Kindred 1: the hemizygous mutation (c.572-575 dupGGGC, p.Thr193Gly,fs,205X) for patient 1 (middle), the normal sequence for his sister (left) and the heterozygous carrier for his mother (right); 2) Kindred 2: the hemizygous mutation (c.773-774 dupCC, p.Ser259Pro,fs,264X) for patient 2 (middle), the normal sequence for his father (left) and the heterozygous state for his mother (right).

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of 21-hydroxylase deficiency in the neonatal or infant period due to the absence of early-onset hypogonadism, although the steroid profiles of these two conditions are much different. Genetic testing may be a alternative approach for a definitive diagnosis of AHC. X-linked AHC is a rare cause of adrenal insufficiency in boys. The mutational analysis of AHC on Chinese boys were firstly reported in 2007 (6). The exact population prevalence of this disease is not currently known. Several studies suggest that X-linked AHC might occur worldwide between 1:140,000 and 1:1,200,000 in children (or between 1:70,000 and 1:600,000 in males) (7, 8). Clinical phenotype of patients with AHC is variable. Approximately 60% of boys affected by this disease have an early onset of salt-wasting primary adrenal insufficiency, usually within the first 2 months of life (9). Children who do not present with signs and symptoms early in life may undergo a period of relatively good health and will tend to present with more insidious symptoms throughout childhood or, although rarely, in adulthood (10). In this study, the first clinical presentation in these patients was progressive skin and mucosa hyperpigmentation beginning in childhood. During infancy, however, there were no symptoms of adrenal insufficiency, such as a salt-losing crisis. HH has been identified as another characteristic of Xlinked AHC in affected individuals who survive into adulthood. The HH resulting from hypothalamic or pituitary dysfunction still remains an unsettled issue. In most patients, there is a poor LH response, but variable FSH responses to a single injection of synthetic GnRH are evident (1, 11, 12). Azoospermia has been observed in several patients with classic AHC, and some researchers made unsuccessful attempts to induce fertility in a limited number of patients using gonadotropins (13-16). Thus, it seems likely that an intrinsic defect in spermatogenesis results in a greater infertility for individuals with AHC compared with isolated idiopathic HH (2, 13, 14). This assertion is based on findings in DAX1 knock-out mice showing the DAX1 protein is essential for the integrity of the seminiferous tubule epithelium (17-19). Both patients presented with markly reduced BMD, especially patient 1 who had multiple thoracic compression fractures, which have not been previously described in AHC patients. Low BMD has been previously reported in patients with idiopathic HH (20). Thus, low BMD in patients with AHC may be attributed to hypogonadism rather than a genetic defect and long-time corticosteroid replacement therapy may also have a negative effect on BMD. It is interesting to note that patient 2 presented with a partially empty sella. So far, only two cases of AHC have been reported to have the similar phenotype (21, 22). Whether or not the association between the DAX1 gene mutation and empty sella is significant will require further study. DAX1 is an unusual member of the nuclear receptor super family. It is an orphan receptor. The C-terminal portion has the structure characteristic of a ligand-binding domain (LBD). However, the N-terminal portion is an atypical DNA-binding domain (DBD) with 3.5 amino acid repeats. DAX1 is a negative regulator that interacts with steroidogenic factor 1 (SF1, NR5A1) to inhibit SF1-mediated trans-

activation of numerous genes involved in the development of the hypothalamic-pituitary-adrenal-gonadal axis and in the biosynthesis of steroid hormones (23). Over 200 different mutations in the DAX1 gene have been described (4, 5), most of which are nonsense or frameshift mutations that cause premature truncation of the protein. In this study, we identified 2 novel mutations of the DAX1 gene (c.572-575 dupGGGC, p.Thr193Gly,fs,205X and c.773-774 dupCC, p.Ser259Pro,fs,264X) in 2 unrelated families with AHC. These 2 frameshift mutations in patient 1 and patient 2 lead to premature stops at codon 205 and 264, respectively. If translated, these will result in drastically truncated proteins that lack the most or entire LBD. Thus, it is conceivable that the DAX1 activity was abolished. In conclusion, our study identified 2 novel mutations in the DAX1 gene. The expanded database of mutations should benefit patients in the diagnosis and treatment of AHC. Molecular evaluation is much needed for carriers of the defect, because there is an increased risk for their offspring being affected. ACKNOWLEDGMENTS We are grateful to the patients and their families for their cooperation in this study.

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