Novel Missense Mutation (Leu466Arg) of the DAX1Gene in a Patient With X-Linked CongenitalAdrenal Hypoplasia

Shuji Abe,1 Jun Nakae,1 Kouichi Yasoshima,2 Toshihiro Tajima,1 Nozomi Shinohara,1Mari Murashita,1 Kouhei Satoh,1 Akemi Koike,2 Yutaka Takahashi,2 and Kenji Fujieda1*1Department of Pediatrics, Hokkaido University School of Medicine, Sapporo, Japan2Department of Pediatrics, Tonan Hospital, Sapporo, Japan

We identified a DAX1 missense mutation, asubstitution of arginine for leucine at codon466 (Leu466Arg), in an infant with X-linkedcongenital adrenal hypoplasia (AHC). A het-erozygous substitution, Leu466Arg, was alsoidentified in his mother and sister. Sinceleucine at position 466 is well conservedamong other orphan nuclear hormone re-ceptor superfamilies and Leu466Arg wasnot detected among 50 normal Japanesecontrol individuals, the mutation is mostlikely responsible for X-linked AHC. It is in-teresting to note that Leu466Arg among allmutations ever reported is located at themost C-terminal region of the DAX-1 pro-tein. Most mutations identified previouslywere located in the C-terminal presumptiveligand binding domain. Hence, the C-terminal end of the DAX-1 protein may playan important role in the biological function,such as in normal adrenal embryogenesis.Am. J. Med. Genet. 84:87–89, 1999.© 1999 Wiley-Liss, Inc.

KEY WORDS: X-linked AHC; DAX1; mis-sense mutation; C-terminalend

INTRODUCTION

X-linked congenital adrenal hypoplasia (X-linkedAHC) (MIM 300200) is a relatively rare inherited dis-order which manifests adrenal insufficiency duringearly infancy. Hypogonadotropic hypogonadism (HH)is frequently associated with X-linked AHC and is gen-erally observed at pubertal maturation [Migeon and

Donohue, 1994]. Recently, the DAX1 (dosage-sensitivesex-AHC critical region on the X chromosome gene)was identified to be responsible for X-linked AHC andHH [Zanaria et al., 1994; Muscatelli et al., 1994]. DAX1encodes a presumptive transcription factor and is clas-sified as an orphan nuclear receptor. Several muta-tions, such as nonsense, frameshift, and missense mu-tations, have been reported. Identification of these mu-tations gave insight into the relationship between thestructure and function of the DAX-1 protein. However,it is difficult to evaluate precisely a role of deleted do-main of the DAX-1 protein, because nonsense or frame-shift mutations may cause the instability of the DAX1mRNA. Thus, identification of a missense mutationmay give direct insight into an important domain forunderstanding a biological function of the DAX-1 pro-tein. In the present study, we identified a novel mis-sense mutation located at the most C-terminal regionof the DAX-1 protein among the mutations ever re-ported and discuss the importance of the C-terminalend of this protein.

CLINICAL REPORT

The propositus, a 1 7/12-year-old infant, was born at38 weeks of gestation with a birth weigh 3,272 g. Cor-tisol level in the filter paper at 5 days obtained from anewborn screening for congenital adrenal hyperplasiawas 7.3 mg/dl. His mother and sister are healthy andhave no symptoms of adrenal insufficiency. However,his maternal uncle died at age 10 days of an unknowncause. At age 24 days, the patient manifested failure tothrive and dehydration when he caught a cold. Hisblood chemistries showed hyponatremia and hyperka-lemia, suggesting a salt-losing crisis. Intravenous ad-ministration of hydrocortisone improved the symp-toms. After recovering from this episode, he has beenfollowed up without hydrocortisone administration.However, he gradually manifested generalized pigmen-tation. At age 1 7/12 years, he was admitted to ourhospital for further evaluation. On admission, his bodylength and weight were 82.0 cm (+0.06 SD) and 12.6 kg(+1.3 SD), respectively, and serum electrolyte levelswere normal. However, levels of plasma adrenocortico-

*Correspondence to: Kenji Fujieda, M.D., Ph.D., Departmentof Pediatrics, Hokkaido University School of Medicine, N-15,W-7, Kitaku, Sapporo 060-8638, Japan. E-mail: ken-fuji@med.hokudai.ac.jp

Received 11 August 1997; Accepted 8 February 1999

American Journal of Medical Genetics 84:87–89 (1999)

© 1999 Wiley-Liss, Inc.

trophic hormone (ACTH) (1359.9 pg/ml) and plasmarenin activity (23.8 ng/ml/hr) were extremely elevated.Serum cortisol (1.4 mg/dl) was low and serum aldoste-rone (<10 ng/dl) was undetectable. The ACTH admin-istration did not increase serum cortisol level (1.4 mg/dlto 1.5 mg/dl). The administration of luteinizing hor-mone releasing hormone (LHRH) revealed a prepuber-tal response of serum luteinizing hormone (LH) (0.109mIU/ml to 1.170 mIU/ml) and follicle stimulating hor-mone (FSH) (0.488 mIU/ml to 1.547 mIU/ml). However,the administration of human chorionic gonadotropin(hCG) increased serum testosterone level significantly(less than 10 ng/dl to 65.8 ng/dl). The treatment of hy-drocortisone and 9a-fludrocortisone was initiated andhe has been well controlled.

MOLECULAR ANALYSIS

Genomic DNA was isolated from peripheral bloodleukocytes of the patient, mother, and sister. Polymer-ase chain reaction (PCR) of DAX1 was performed for 30cycles using primer pairs 1–2, 3–4, 5–6, 7–8, 7–10, and11–12 (Table I) as described previously [Nakae et al.,1996]. Direct sequencing of PCR products was per-formed from both strands utilizing an automated se-quencer (ABI PRISMTM 310, Applied Biosystems, Edi-son, NJ). Mutations detected were confirmed by partialsequencing and HhaI-restriction analysis of DNA fromthe mother and sister.

RESULTS AND DISCUSSION

We identified a novel missense mutation encoding asubstitution of arginine for leucine at codon 466(Leu466Arg) in the patient’s DAX1 (Fig. 1). No othersubstitutions in his DAX1 could be found. In addition,the mother and sister had a heterozygous substitutionof G for T at nucleotide 1631. The Leu466Arg mutationgenerates a recognition site of HhaI (GCG/C). Thus,digestion of each PCR product with HhaI produced twofragments of 226 bp and 78 bp in the propositus, threebands of 304 bp, 226 bp, and 78 bp in the mother andthe sister, and one band of 304 bp in three normalcontrols, respectively (Fig. 2). These data suggest thatthis substitution is responsible for X-linked AHC andthat the mother and sister are carriers of the disorder.

DAX-1 protein is composed of 470 amino acids, andits N-terminal portion contains four incomplete repeats

of a new structural motif encoding a DNA-bindingfunction. The C-terminal half of the protein has highhomology with the ligand-binding domain (E domain)of the nuclear hormone receptor superfamily, espe-cially with the E domain of the retinoid X receptor andthe orphan receptor subfamily [Zanaria et al., 1994].Most mutations previously reported are located in theC-terminal presumptive ligand-binding domain. In ad-dition, all the missense mutations reported are alsolocated in the C-terminal domain. These mutationsgave insight into the relationship between the struc-ture and function of the DAX-1 protein. From this pointof view, the mutation (Leu466Arg) observed in our fam-ily merits comment. Since leucine at position 466 isconserved among other orphan nuclear hormone recep-tor superfamilies [Bae et al., 1996; Mangelsdorf et al.,1992; Leid et al., 1992; Oro et al., 1990; Mlozik et al.,1990; Wang et al., 1989; Ladias and Karathanatis,1991] and this mutation was not found among 50 nor-mal Japanese, the Leu466Arg may be responsible for

TABLE I. Primers Used for PCR Amplification of DAX1

Primerno. Sequence (58–38)

Nucleotideposition (nt)

1 CAC TGG GCA GAA CTG GGC TAC 199–2202 TGG TCT TCA CCA CAA AAG CAG 650–6303 CGA AGG CGC CCG AGG CGA C 515–5334 CGC TTG ATT TGT GCT CGT GG 903–8845 GGT AAA GAG GCG CTA CCA GGC 781–8016 AGT ACT TGA CGA AGC GCA GC 1048–10297 AGC ACA AAT CAA GCG CAG GC 889–9088 CTG CAG CAT GCT GGG CTC CG 1188–1170

10 GTA CCC TTA CCC GGG TTA AAG 1403–138311 CTC CCT CCA GAC GTG CCG GGC 1404–142412 CAG CTC TTT ATT CTT CCC TCA 1708–1688

Fig. 1. G-to-T substitution (arrow) at nucleotide 1631 of DAX1 in thepropositus. This mutation leads to a substitution of arginine for leucine atcodon 466 (Leu466Arg).

Fig. 2. HhaI-restriction analysis, showing 226-bp and 78-bp fragmentsin the propositus (lane 1), these plus 304-bp fragments in the mother (lane2) and sister (lane 3), and a 304-bp fragment in the normal controls (lanes4–6) (M, size marker FX174 HaeIII).

88 Abe et al.

X-linked AHC in the family. It was previously sug-gested that the C-terminal domain of the DAX-1 pro-tein is important for its function [Nakae et al., 1996;Ito et al., 1997]. It is interesting to note that theLeu466Arg mutation is located in the most C-terminalend among all mutations reported. Thus, this mutationsupports the hypothesis of the functional importance ofthe C-terminal end of the protein.

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