Endocrine Journal 2009, 56 (4), 619-624

NOTE A Novel Heterozygous Mutation of Steroidogenic Factor-1 (SF-1/Ad4BP) (NR5A1) in a 46, XY Disorders of Sex Development (DSD) Patient without Adrenal Failure

Toshihiro Tajima*, Fumie Fujiwara* and Kenji Fujieda**

* Department of Pediatrics, Hokkaido University School of Medicine, Sapporo, Japan ** Department of Pediatrics, Asahikawa Medical College, Asahikawa, Japan

Abstract. Steroidogenic factor-1 [(SF-1/Ad4BP) (MIM184757)] is a nuclear that regulates multiple involved in adrenal and gonadal development, steroidogenesis, , and other metabolic functions. Initially, mutations of SF-1/Ad4BP gene (NR5A1) in humans were identified in two 46, XY female patients with adrenal insufficien- cy and . However, recent studies have revealed that heterozygous mutations are more frequently found in 46, XY disorders of sex development (DSD) patients without adrenal failure than in 46, XY DSD patients with adrenal failure. We encountered a Japanese female patient of 46, XY DSD without adrenal failure and identified a novel mutation (V41G) of NR5A1. Functional analysis revealed that this mutant could not activate CYP19 promoter, indicating loss of function. In conclusion, we add a novel mutation of NR5A1 in 46, XY DSD patient without adrenal failure.

Key words: SF-1/Ad4BP, NR5A1, 46 XY DSD, Adrenal failure (Endocrine Journal 56: 619-624, 2009)

STEROIDOGENIC factor-1 [(SF-1/Ad4BP) (NR5A1) were identified in two 46, XY female pa- (MIM184757)] is a that was first tients with and gonadal dysgen- identified following the search for a common regulator esis [6, 7]. The first mutation (G35E) was a heterozy- of the hydroxylase family of gous mutation, which occurred in the P-box of the first enzymes [1-3]. SF-1/Ad4BP regulates multiple genes domain required for the DNA-binding [6, involved in adrenal and gonadal development, ste- 8]. The second mutation was a homozygous missense roidogenesis, reproduction, and other metabolic func- mutation (R92Q) that disrupts the A-box secondary tions [1-3]. In mice, SF-1/Ad4BP is expressed early DNA binding domain [7]. However, recent several in the urogenital ridge and continues to be expressed studies have demonstrated that heterozygous muta- in the developing adrenal, , ventromedial hy- tions are more frequently identified in 46, XY disor- pothalamus and pituitary [4]. Homozygous knockout ders of sex development (DSD) patients without ad- mice result in gonadal and adrenal agenesis, present of renal failure rather than in 46, XY DSD patients with Mullerian structure and abnormalities of the hypothal- adrenal failure [3, 9-17]. Therefore, the testis is like- amus and pituitary gonadotrope. Heterozygous mice ly to be more sensitive to partial loss of SF-1/Ad4BP have a milder including an impaired adre- function than the in human. In regard nal stress response and reduced testicular size [5]. to females with heterozygous NR5A1 mutations, so far In human, initially, mutations of SF-1/Ad4BP gene seven individuals have been reported [13-18]. Among them, only one patient developed adrenal failure, but she had apparently normal ovarian function [18]. The Received Dec. 22, 2008; Accepted Feb. 27, 2009 as K08E-380 Released online in J-STAGE as advance publication Mar. 24, 2009 remaining individuals were mothers of 46, XY DSD Correspondence to: Toshihiro Tajima, M.D., Ph.D., Department patients and they did not show any symptoms of ad- of Pediatrics, Hokkaido University School of Medicine, N15, W7, renal and ovarian failure [13-17]. Indeed, ovarian de- Sapporo 060-8638, Japan. E-mail: [email protected] velopment during development and at birth is relatively 620 TAJIMA et al. preserved in -specific Sf-1 knockout mice com- Table 1. Endocrinological findings pared to testes in testes-specific Sf-1 knockout mice Blood hormone value Basal Peak [19]. These findings indicate that only one intact SF-1/ LH (mIU/mL)a 14.35 59.81 Ad4BP is sufficient for normal ovarian development. FSH (mIU/mL)a 41.1 59.85 Here, we experienced a Japanese 46, XY DSD pa- Estradiol (pg/ml)b <10 <10 tient and found a novel mutation (V41G) of NR5A1. In (ng/ml)b 0.78 2.57 vitro study demonstrated this mutation lost its function. ACTH (pg/ml) 58.5 Cortisol (mg/dl)c 14.8 20.12

c Methods 17-OHP (ng/dl) 2.3 5.4 a, GnRH test(100mg). b, Three-day hCG stimulation (5000IU). c, Cortisol and 17-hydroxyprogesterone after acute stimulation with DNA amplification and sequence analysis 250 mg ACTH intravenous injection.

Informed consent to participate in the study was ob- tained from the patient and parents. The ethical com- fluency and transiently transfected by lipofectamine mittee of Hokkaido University School of Medicine with either (1) empty expression vector (pCDNA3, approved this study. Genomic DNA was extracted 0.2 µg); (2) WT-SF-1/Ad4BP (0.2 µg); (3) MT- SF-1/ from peripheral leukocytes and each exon of NR5A1 Ad4BP (0.2 µg); (4) WT-SF-1/Ad4BP (0.2 µg) plus was amplified by polymerase-chain-reaction (PCR) MT-SF-1/Ad4BP (0.2 µg); (5) WT-SF-1/Ad4BP (0.2 according to a previous report [10]. After amplifica- µg) plus MT-SF-1/Ad4BP (0.4 µg); or (6) WT-SF-1/ tion, the PCR products were purified and sequenced Ad4BP (0.2 µg) plus MT-SF-1/Ad4BP (1.0 µg) to- directly using an ABI PRISM Dye Terminator Cycle gether with pGL3-CYP19 (0.4 µg). Cell extracts were Sequencing Kit and an ABI 373A automated fluores- prepared 48 hours after transfection and luciferase as- cent sequencer. says were performed. Luciferase measurements were divided by the respective b-galactosidase activity to Mutant SF-1 cDNA construction and plasmid con- control for transfection efficiency. The mean of each struction triplicate reaction was expressed as a percentage of the empty vector control to allow comparison of data Human SF-1/Ad4BP cDNA was inserted into pcD- from different experiments. Data are presented as NA 3.1 (WT-SF-1/Ad4BP). The mutant cDNA was cre- means±S.D. ated by site-directed mutagenesis using an overlapping PCR strategy and was designated MT-SF-1/Ad4BP. A report of case The mutation was verified by direct DNA sequenc- ing. The human cytochrome P450arom gene (CYP19) A Japanese patient was born after 40 weeks of ges- promoter luciferase plasmid was used for analysis of tation by normal vaginal delivery and was the first SF-1/Ad4BP function as described previously [20, child of nonconsanguineous parents. She had no sib- 21]. This construct was designated pGL3-CYP19. lings, and her parents were healthy. Her birth weight was 3490 g and length was 49.5 cm. At birth, clitoro- Cell culture megaly was noticed; however, further medical ex- amination was not performed. Thus, the patient was COS cells were obtained from American Type Cell reared as a female. At 12 years of age, she was re- Culture and grown in Dulbecco’s modified Eagle’s ferred to our hospital because of no development of medium (DMEM) supplemented with 10% fetal bo- secondary sexual characteristics and clitoromegaly. vine serum. Her height was143 cm and body weight was 35 kg. Breast development was at Tanner stage I, and pubic Transient hair development at Tanner stage I. She had clitoro- megaly (~2.2 cm), but no posterior labial fusion and In order to assay CYP19 promoter activity, COS the vaginal and the urethral orifices were separated. cells were plated in 6-well plates, grown to 70% con- Presumed were palpable bilaterally in the in- 46, XY DSD WITHOUT ADRENAL FAILURE 621

(A) GTG (Val) GTG(Val)/GGG (Gly) V41G

WT Patient

(B) G Y H (C) S Y V G K L D L P-box G T Human CESCKGFFKRT V QNNHY C C Murine CESCKGFFKRT V QNNHY V Zn E P S Rat CESCKGFFKRT V QNNHY C C L K G F F K R V Q N N K H Y Bovine CESCKGFFKRT V QNNHY

35 41

Fig. 1. (A) Sequence analysis demonstrated a T to G transition. This change substitutes glycine for valine at residue 41 in the first zinc finger domain of SF-1/Ad4BP as denoted by an arrow. (B) A part of the amino acid sequence of the first zinc-finger domain of SF-1/Ad4BP protein is shown. A hatched box indicates P-box. (C) Part of the amino acid sequence of the first zinc finger domain of SF-1/Ad4BP is shown. A box indicates the conserved valine residue at codon 41. guinal region. Skin pigmentation was not observed. rare germ cells, and loose interstitium had a few clus- She had no episode of adrenal insufficiency during her ters of Leydig cells. life. Her was 46, XY. Her endocrinologi- She is now 19 years-old and being treated with es- cal evaluation is summarized in Table 1. Her serum trogen supplementation. Her height is 161 cm and estradiol concentration was less than 10 pg/ml. Basal body weight is 42 kg. Her basal cortisol and ACTH serum testosterone concentration was 0.78 ng/ml and at 9:00 A.M. are 7.5 µg/dl and 27.5 pg/ml. Until this after human chorionic-gonadotropin (hCG) stimula- time, she has not developed adrenal failure. tion (5000 IU intramuscularly daily for 3 days), serum testosterone increased up to 2.57 ng/ml. Basal gonad- otropin levels were elevated [follicle stimulating hor- Results mone (FSH) 41.1 mIU/ml, luteinizing hormone (LH) LH 14.35 mIU/ml]. Sequencing analysis of NR5A1 revealed a heterozy- Her basal cortisol and adrenocorticotropin (ACTH) gous point mutation in exon 3 at codon 41 [GTG (Val) levels were within normal range (14.8 µg/dl and 58.5 to GGG (Gly)] (Fig. 1A). Fifty normal Japanese sub- pg/ml, respectively). After ACTH stimulation, se- jects did not show this base change. This mutation is rum cortisol increased up to 20.1 µg/dl without any present in the first zinc finger domain (Fig. 1B) and abnormal accumulation of adrenal steroid precursors. this valine at residue 41 is well conserved in different Pelvic magnetic resonance imaging demonstrated no species (Fig. 1C). Her parents were not subjected to uterus or vaginal pouch. Laparoscopy did not show DNA analysis. any Mullerian derivatives. Genitoplasty and gonad- In vitro transfection study demonstrated that WT- ectomy were performed. Histological examination SF-1/Ad4BP activated the CYP19 promoter ac- revelaed dysgenetic testis. Microscopic examination tivity, whereas MT-SF-1/Ad4BP did not (Fig. 2). showed that seminiferous contained Sertoli cells, but Cotransfection of mutant with WT-SF-1/Ad4BP did 622 TAJIMA et al. not show a dominant negative effect even when 5:1 ra- tios of MT:WT-SF-1/Ad4BP were transfected (Fig. 2). 100

80 (%WT)

Discussion ty vi ti

ac 60

We identified a novel mutation of V41G in a 46, se ra

XY DSD patient without adrenal insufficiency. In fe 40 vitro promoter assay demonstrated that V41G pro- ci ed lu tein lost activating function. So far C33S and G35E iz 20 mutations were identified in the P-box region of the al rm

first zinc finger domain, and these two mutants lost No a DNA-binding activity [6, 17]. As shown in Figure 0 Empty WT MT WT: MT WT: MT WT: MT 1, the V41 residue is located near the P-box region in 1:1 1:2 1:5 the first zinc finger domain, and is highly conserved Fig. 2. Transactivation function of WT-SF-1/Ad4BP and MT- among different species. Therefore, although we did SF-1/Ad4BP. While cotransfection of WT-SF-1/Ad4BP not analyze DNA binding, our mutant protein would with CYP19 promoter stimulated the luciferase reporter affect DNA binding, resulting in loss of function. gene relative to the empty vector, MT-SF-1/Ad4BP did Despite ambiguous genitalia, our patient showed not. Transfection of increasing amounts of MT-SF-1/ low but increased serum testosterone level after hCG Ad4BP did not impair the transactivation capacity of the wild-type protein, suggesting no dominant negative effect stimulation at 12 years of age. These findings might of the mutant protein. Data are presented as means±S.D. be explained by insufficient testosterone production, especially during the development of the external gen- italia. Most patients with SF-1/Ad4BP mutation dem- the familial case. Accordingly, not only the genotype onstrated severe defect in testosterone production. of SF-1/Ad4BP, but also other genetic or environmen- However, low but detectable testosterone levels bas- tal factors seem to affect testosterone production of al or after hCG stimulation were observed in five pa- during the critical period of the develop- tients [9, 12, 14, 15, 17]. Among these patients, one ment of the male external genitalia. This must be fur- had very mild phenotype of penoscrotal hypospadias ther studied. and was raised as a male [15]. Consistent with the So far, 16 of the 46, XY DSD patients without ad- mild phenotype, the mutation (L437Q) of this patient renal failure caused by SF-1/Ad4BP mutations were retained partial function in vitro. The authors sug- reported (9-17). Lin et al. [15] have reported four 46, gest the genotype may partly explain the mild phe- XY DSD patients with SF-1/Ad4BP mutations among notype. However, the other patient with increased 30 patients with 46, XY DSD. Köhler et al. [17] testosterone (2.5 ng/ml) level after hCG stimulation have reported 5 patients with SF-1/Ad4BP mutations demonstrated significant undervirilization [17]. This in a cohort of 27 patients with 46, XY DSD. Four patient had C33S mutation with complete loss of func- Japanese 46, XY DSD patients without adrenal failure tion. Furthermore, Coutant et al. [14] have reported caused by SF-1/Ad4BP mutations have already been two siblings caused by a severe SF-1/Ad4BP mutation reported [10, 12, 13]. Thus, it is plausible that SF-1/ (c.536delC), who lack the ligand-binding domain and Ad4BP mutations are more frequent than previously the activation function 2 domain. The elder 46, XY suspected causes of 46, XY DSD in Japan. To clarify female showed ambiguous genitalia and elevated tes- this hypothesis, a systemic cohort of 46, XY DSD pa- tosterone (2.2 ng/ml) level in neonatal period, which tients throughout Japan is necessary. led to a presumable diagnosis of partial androgen in- In conclusion, we identified a novel mutation of sensitivity syndrome. By contrast, the second child of NR5A1 in a Japanese 46, XY DSD patient without ad- 46, XY female had less virilized genitalia than the old- renal failure. er child and her testosterone production was severely defect. This report indicated that the difference of the and Leydig cell function existed even in 46, XY DSD WITHOUT ADRENAL FAILURE 623

Acknowledgments Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu We thank Dr. Ken-Ichirou Morohashi, Department University, for providing human CYP19 promoter of Molecular Biology Faculty of Medical Sciences, vector and Dr. Tomonobu Hasegawa, Department of Kyushu University, for providing us with the human Pediatrics, Keio University School of Medicine, for Ad4/BP expression vector, Dr. Toshihiko Yanase, PCR primers for NR5A1.

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