Compound Heterozygous Mutations in the Cholesterol Side-Chain ...

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The Journal of Clinical Endocrinology & Metabolism 87(8):3808 –3813 Copyright © 2002 by The Endocrine Society

Compound Heterozygous Mutations in the Cholesterol Side-Chain Cleavage Enzyme Gene (CYP11A) Cause Congenital Adrenal Insufficiency in Humans NORIYUKI KATSUMATA, MASATOSHI OHTAKE, TORU HOJO, EISHIN OGAWA, TAKAYUKI HARA, NAOKO SATO, AND TOSHIAKI TANAKA Department of Endocrinology and Metabolism, National Research Institute for Child Health and Development (N.K., N.S., T.T.), Tokyo 154-8567, Japan; Department of Pediatrics, Sendai City Hospital (M.O.), Miyagi 984-8501, Japan; Department of Pediatrics, Watari Hospital (T.Ho.), Fukushima 960-8141, Japan; Department of Pediatrics, Tohoku University School of Medicine (E.O.), Miyagi 980-8574, Japan; and Department of Food and Nutrition, Nakamura Gakuen University (T.Ha.), Fukuoka 814-0198, Japan Cholesterol side-chain cleavage enzyme (P450scc) catalyzes the conversion of cholesterol to pregnenolone in mitochondria, which is the first step in the biosynthesis of all steroid hormones. Until now, no homozygous or compound heterozygous mutations in CYP11A have been described in humans. Here we describe novel compound heterozygous mutations in CYP11A in a patient with congenital adrenal insufficiency born to healthy parents. One mutation, a maternally inherited R353W mutation, resulted in markedly reduced P450scc activity by the single amino acid substitution, indicating that Arg353 is a crucial amino acid residue for P450scc activity. The other mutation, a de novo A189V mutation in the paternal

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HOLESTEROL SIDE-CHAIN cleavage enzyme (P450scc) catalyzes the conversion of cholesterol to pregnenolone in mitochondria in steroidogenic cells, which is the first step in the biosynthesis of all steroid hormones (1). Congenital lipoid adrenal hyperplasia (CLAH) is the most severe form of congenital adrenal hyperplasia, inherited as an autosomal recessive trait, leading to impaired production of all steroids, including glucocorticoids, mineralocorticoids, and sex steroids (2, 3). Because mitochondria from affected adrenal glands and gonads fail to convert cholesterol to pregnenolone, it was once postulated that CLAH could be caused by a defect in P450scc (2, 3), but the CYP11A gene, encoding P450scc, in the patients appeared to be normal (4, 5). Now it is known that in most patients CLAH is caused by mutations in the steroidogenic acute regulatory protein (StAR) gene (6 – 8). In rabbits, a naturally occurring deletion of the P450scc gene was reported; homozygotes of the deletion are affected by CLAH, and heterozygotes of the deletion are phenotypically normal, indicating an autosomal recessive trait of inheritance (9). In humans, a heterozygous mutation, an inframe insertion of Gly and Asp between Asp271 and Val272, in the CYP11A gene has been recently reported in a 46,XY patient affected by adrenal failure and male pseudohermaphroditism (10), and this is the only mutation in the Abbreviations: CLAH, Congenital lipoid adrenal hyperplasia; P450scc, cholesterol side-chain cleavage enzyme; StAR, steroidogenic acute regulatory protein.

allele, did not affect the P450scc activity by the single amino acid substitution and turned out to be a splicing mutation, which created a novel alternative splice-donor site. It resulted in a deletion of 61 nucleotides in the open reading frame and thus partially inactivated CYP11A. These experimental data are consistent with the clinical findings indicating that the patient had partially preserved ability to synthesize adrenal steroid hormones. This is the first report of the compound heterozygote for the CYP11A mutations with congenital adrenal insufficiency and the phenotypically normal heterozygote in humans. (J Clin Endocrinol Metab 87: 3808 –3813, 2002)

CYP11A gene identified to date in humans. The mutant P450scc protein was demonstrated to lose the enzymatic activity completely, but not to exert a dominant negative effect on the wild-type protein. Therefore, the haploinsufficiency of CYP11A was considered to cause adrenal failure and male pseudohermaphroditism. In the present report we describe a patient with congenital adrenal insufficiency caused by compound heterozygous mutations in CYP11A and the phenotypically normal mother who is a heterozygote for one of the inactivating CYP11A mutations. Case Report

The patient was the second daughter of healthy unrelated Japanese parents. The elder sister is the only sibling of the patient and is healthy. The patient was born by vaginal aspiration delivery after an uneventful 40-wk, 6-d gestation. Her birth weight was 3150 g. The patient was noticed to have dark skin at birth, but otherwise did well. At the age of 7 months, skin pigmentation became prominent, and her plasma ACTH level was markedly elevated (3550 pmol/liter; normal range, ⬍13 pmol/liter). At the age of 9 months the patient was hospitalized for further investigation. The patient had normal electrolyte and plasma glucose levels. Endocrinological investigations revealed increased levels of ACTH (1090 pmol/liter) and plasma renin activity (6.4 ng/ liter䡠sec; normal range, 0.05– 0.75 ng/liter䡠sec) and inappropriately normal levels of cortisol (168 nmol/liter; normal range, 154 –583 nmol/liter), aldosterone (527 pmol/liter; normal range, 83–582 pmol/liter), 17␣-hydroxyprogesterone

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(0.3 nmol/liter; normal range, ⬍1.8 nmol/liter), dehydroepiandrosterone (1.0 nmol/liter; normal range, ⬍3.4 nmol/ liter), androstenedione (0.3 nmol/liter; normal range, ⬍1.7 nmol/liter), and testosterone (1.0 ng/ml; normal range, ⬍1.0 nmol/liter). Serum levels of LH (2.0 IU/liter; normal range, ⬍2.15 IU/liter), FSH (16.1 IU/liter; normal range, 0.8 –18.5 IU/liter), and estradiol (⬍36 pmol/liter; normal range, ⬍36 pmol/liter) were also normal. The karyotype was 46,XX. The patient was diagnosed as having congenital adrenal insufficiency, and replacement therapy with hydrocortisone and fludrocortisone was started. She has been doing well since then. Direct sequencing analyses of the candidate genes of the patient including STAR (6), NR0B1 (formerly known as DAX1) (11), and NR5A1 (formerly known as FTZ1) (12) revealed normal DNA sequences (data not shown). The mother of the patient has been healthy. She has no skin pigmentation or other symptoms or signs of adrenal insufficiency. She has no history of miscarriage and has uneventfully borne two daughters. Materials and Methods PCR amplification and direct sequencing of the CYP11A gene DNA was extracted from peripheral white blood cells of the patient and her family members after informed consent for genetic analyses was obtained. Exons of the CYP11A gene were amplified as reported by Lee et al. (4), except for one modification. We used a sense primer S6 (5⬘GGTCAAGGACCTCAAGGGG-3⬘) for amplification of exon 6 to obtain the splice-acceptor site sequence of exon 6. The amplified PCR products were fractionated and isolated on a 1% agarose gel (Bio-Rad Laboratories, Inc., Richmond, CA), then directly sequenced using a Thermo Sequenase kit (Amersham Japan Ltd., Tokyo, Japan).

Restriction enzyme analysis We designed mismatched primers to introduce restriction enzyme recognition sites for mutant alleles. The sequences of the mismatched primers were as follows: a sense primer S3m, 5⬘-GTCAGTGTCCTGCACAGGCGCAC-3⬘ (the mismatched nucleotide is underlined) for introduction of a BstXI site in exon 3 of the A189V mutant allele; and an antisense primer AS6m, 5⬘-GTGCCGCGCAGCCAAGACCTCTGG-3⬘ (the mismatched nucleotide is underlined) for introduction of an MluNI site in exon 6 of the R353W mutant allele. The other PCR primers were the same as those for amplification of each of exons for the sequencing analysis. The PCR products were digested with BstXI (New England Biolabs, Inc., Beverly, MA) or MluNI (Roche, Mannheim, Germany), followed by electrophoresis on a 3% NuSieve GTG agarose gel (FMC Bioproducts, Rockland, ME).

Haplotype determination The 5.5-kb DNA fragment spanning exons 3– 6 of CYP11A was amplified from the patient using an LA PCR kit (Takara Shuzo Co., Ltd., Otsu, Japan). The primer sequences were as follows: a sense primer S3L, 5⬘-CCAGGCCTGGGGTCTGTGACCATGAGGGCT-3⬘; and an antisense primer AS6L, 5⬘-ACTTCCCTGGCCCTGCCCAGGGATTGGAGT3⬘. The PCR product was cloned into a pCRII plasmid using a TA cloning kit (Invitrogen, San Diego, CA). Each clone was subjected to restriction fragment length polymorphism analysis as described above.

Microsatellite analysis DNA samples from the patient and the parents were analyzed for polymorphic microsatellite markers within CYP11A, CYP19, and NR0B1 as reported previously (13, 14).

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Functional expression of the replacement mutant P450scc The wild-type P450scc cDNA was amplified from a QUICK-Clone human adrenal gland cDNA (CLONTECH Laboratories, Inc., Palo Alto, CA). The amino acid substitutions were introduced in the wild-type P450scc cDNA by the recombinant PCR method (15). The primer sequences were as follows: an antisense primer A189V, 5⬘-TCCGGAGCCCACCTTCTTGAT-3⬘ (the mutated nucleotide is underlined) for introduction of the A189V substitution; and a sense primer R353W, 5⬘-GTGGGCAGAGGATCTTGGCTGCG-3⬘ (the mutated nucleotide is underlined) for introduction of the R353W substitution. The wild-type or the mutant P450scc cDNAs were ligated to a mammalian expression plasmid pRK5. The bovine adrenodoxin and adrenodoxin reductase expression plasmids (16) were provided by Dr. Yoshiyasu Yabusaki (Sumitomo Chemical Co., Ltd., Takarazuka, Japan) and Dr. Shigeaki Kato (Tokyo University, Tokyo, Japan). The human StAR expression plasmid was prepared as described previously (17). COS-1 cells were transfected with 1 ␮g each of bovine adrenodoxin, bovine adrenodoxin reductase, and human StAR expression plasmids and 1 ␮g of either the wild-type or the mutant P450scc expression plasmid by the electroporation method using Gene Pulsor II (Bio-Rad Laboratories, Inc., Richmond, CA). P450scc activity was determined by measuring the amount of pregnenolone, which was synthesized from cholesterol in the medium during 48 h of incubation, as reported previously (17). In addition, pRK-GH1, a hGH expression plasmid, was included in the transfection mixture and used as an internal control for transfection efficiency. Western blot analyses were performed as reported previously (5, 17). Results were presented as the mean ⫾ sd, and statistical analysis was performed using unpaired t test.

In vitro expression of the CYP11A minigene The genomic E1-1 clone, encompassing exons 2– 4 of CYP11A (18), was a gift from Dr. Ken-ichiro Morohashi (National Institute for Basic Biology, Okazaki, Japan). The A189V mutation was introduced in a 159-bp ApaI (Roche)-MroI (TOYOBO, Osaka, Japan) fragment of the E1-1 clone by the recombinant PCR method (15) using the antisense A189V primer, and the recombinant E1-1 bearing the mutation was reconstructed and designated A189V E1-1. The wild-type or the A189V E1-1 was ligated to the mammalian expression plasmid pRK5 to create chimeric minigenes. Four micrograms of wild-type or mutant chimeric plasmid were transfected into COS-1 cells using a lipofectin agent (Invitrogen). After 48 h, total RNA was extracted from the cells with an ISOGEN kit (Nippon Gene Co., Ltd., Tokyo, Japan). The first strand cDNA was synthesized in a 12.5-␮l reaction mixture containing 1 ␮g total RNA, 0.5 ␮g oligo(deoxythymidine)12–18, 0.8 mm of each deoxyNTP, 20 U ribonuclease inhibitor, 50 U TrueScript Reverse Transcriptase, and 1⫻ reaction buffer, which was supplied by the manufacturer (Sawady Technology Co., Ltd., Tokyo, Japan). The resulting cDNA was subjected to PCR amplification with a sense primer, RTS2 (5⬘-TATGTCCATCGACCCTGAAGA-3⬘), located in exon 2 and an antisense primer, RTAS4 (5⬘-GGAACAGGTCTGGGGGAAG-3), located in exon 4. The RT-PCR products were subjected to electrophoresis on a 2% agarose gel. The direct sequencing analyses of the RT-PCR products were performed as described above.

Results Sequencing analysis of CYP11A and haplotype determination

We directly sequenced the CYP11A gene from the patient and found two heterozygous mutations (Fig. 1A). One mutation was a C to T transition in the second nucleotide of codon 189 in exon 3, apparently resulting in a substitution of Val for Ala and was designated A189V. The other mutation was a C to T transition in the first nucleotide of codon 353 in exon 6, which resulted in a substitution of Trp for Arg and was designated R353W. No other mutations were found in the remainder of the coding region or the splice sites of the CYP11A gene.

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FIG. 1. Mutations in the CYP11A gene. A, Nucleotide sequence of the patient’s CYP11A. The C to T transition in the second nucleotide of codon 189 in exon 3 apparently results in a substitution of Val for Ala (A189V). The C to T transition in the first nucleotide of codon 353 in exon 6 results in a substitution of Trp for Arg (R353W). B, Pedigree of the family and restriction enzyme analyses. The arrow indicates the patient. BstXI digestion of a 120-bp fragment from the A189V mutant allele yields 90and 30-bp fragments, whereas the normal fragment remains undigested. The MluNI digestion of a 163-bp fragment from the R353W mutant allele yields 139- and 24-bp fragments, whereas the normal fragment remains undigested. Short fragments (⬍30 bp) are undetectable on the gels. The patient and the mother are heterozygous for the R353W mutation, whereas only the patient is heterozygous for the A189V mutation. ␸X indicates HaeIII-digested ␸X174 RF DNA, which was used as a size marker. C, Restriction enzyme analyses of the cloned alleles of the patient’s CYP11A. The clones 1, 2, 4, 6, and 8 bear only the A189V mutation, whereas clones 3, 5, 7, and 9 bear only the R353W.

To confirm the mutations and perform a family study, we designed mismatched primers to introduce BstXI and MluNI sites for the A189V and R353W mutant alleles, respectively. The BstXI digestion of a 120-bp PCR fragment from the A189V mutant allele should yield 90- and 30-bp fragments, whereas the normal fragment remains uncut. The MluNI digestion of a 163-bp PCR fragment from the R353W mutant allele should yield 139- and 24-bp fragments, whereas the normal fragment remains undigested. As shown in Fig. 2B, the patient and the mother were heterozygous for the R353W mutation, whereas only the patient was heterozygous for the A189V mutation, indicating that the A189V mutation in the patient was a de novo event. To confirm the compound heterozygosity of the

mutations, a 5.5-kb DNA fragment containing exons 3– 6 of CYP11A was amplified from the patient, and cloned into the plasmid. Each clone was subjected to the restriction fragment length polymorphism analyses and was demonstrated to have either of the mutations (Fig. 1C). Thus, the patient was considered to be a compound heterozygote with the de novo A189V mutation in the paternal allele and the inherited R353W mutation in the maternal allele. Microsatellite analysis

As de novo mutations represent a rare event, we confirmed the paternity by amplification of the microsatellites within CYP11A (13), CYP19 (13), and NR0B1 (14) (data not shown).



FIG. 2. CYP11A minigene expression study. A, Comparison of the A189V allele sequence and consensus splicedonor sequence. B, Schematic presentation of the CYP11A minigene. The arrows below exons 2 and 4 denote the positions of the primers used in RTPCR. C, Schematic presentation of mRNA splicing of the CYP11A minigene. Should splicing occur normally, an RT-PCR product of 469 bp is expected. Should aberrant splicing occur between codons 188 and 189, an RTPCR product of 408 bp is expected. D, Electrophoretic patterns of the RT-PCR products. The wild-type minigene yielded a 469-bp product, whereas the A189V minigene yielded 408- and 469-bp products. ␸X indicates HaeIIIdigested ␸X174 RF DNA, which was used as a size marker. E, Sequencing analyses of the RT-PCR products. The 469-bp products showed the normal splicing pattern. In the 408-bp product, codon 188 was followed by exon 4 as the result of skipping the last 61 nucleotides of exon 3.

P450scc activity of replacement mutants

To examine the functional consequences of the amino acid substitutions, we introduced substitutions in the wild-type P450scc cDNA by the recombinant PCR method and transiently expressed the replacement mutants along with adrenodoxin, adrenodoxin reductase, and StAR in COS-1 cells. Western blot analyses detected similar amounts of wild-type P450scc and the replacement mutants (data not shown). COS-1 cells expressing wild-type P450scc successfully converted cholesterol to pregnenolone, whereas those transfected with the empty pRK5 plasmid produced virtually no pregnenolone (Table 1). The R353W replacement resulted in marked reduction in pregnenolone production, but retained some residual activity (Table 1). A189V replacement did not affect the ability to convert cholesterol to pregnenolone (Table 1).

TABLE 1. Enzyme activities of wild-type and mutant P450scc Pregnenolone (pmol/dish)

pRK5 pRK-SCC pRK-A189V pRK-R353W

1.0 ⫾ 0.13 1030 ⫾ 107a 1140 ⫾ 82a,b 83.5 ⫾ 5.0a,c

pRK5, Empty plasmid; pRK-SCC, wild-type P450scc; pRK-A189V, A189V replacement P450scc; pRK-R353W, R353W replacement P450scc. The values are shown as the mean ⫾ SD from four dishes. a P ⬍ 0.01 vs. pRK5. b P ⬎ 0.05 vs. pRK-SCC. c P ⬍ 0.01 vs. pRK-SCC.

Expression study of the CYP11A minigene

As the mother, bearing the heterozygous R353W mutation, had no symptoms or signs of adrenal insufficiency, we con-

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sidered that the A189V mutation ought to inactivate the CYP11A gene in some way. As we realized that the A189V mutation results in a nucleotide sequence with considerable homology to the consensus splice-donor site sequence (19) (Fig. 2A), we hypothesized that the mutation would create a novel alternative splice-donor site. Because adrenal, gonadal, or placental RNA of the patient was not available, we could not perform in vivo analyses of these to verify the hypothesis. Therefore, we made an in vitro expression study using the chimeric minigene system to analyze transcription of the A189V mutant CYP11A gene. We introduced the A189V mutation in an E1-1 clone, which spans exons 2– 4 of the CYP11A gene (18), and constructed two minigenes consisting of the mammalian expression plasmid and the wild-type or the A189V mutant E1-1 (Fig. 2B). After the respective minigenes were expressed in COS-1 cells, the splice site selection patterns for exons 2– 4 were assessed from the cDNA sizes that corresponded to the expressed mRNAs by means of RT-PCR. If the mRNA splicing of the minigene proceeds normally, the RT-PCR amplification should yield a 469-bp product; a 408-bp product would be amplified should the aberrant splicing occur between codons 188 and 189 (Fig. 2C). As shown in Fig. 2D, the RT-PCR product of the chimeric minigene that included wild-type E1-1 was 469 bp, whereas two species of the transcripts, 469 and 408 bp, were detected in COS-1 cells expressing the chimeric A189V mutant minigene. DNA sequencing analyses confirmed that the 469-bp transcripts had the normal splicing pattern, whereas the 408-bp transcript had a deletion of 61 nucleotides of exon 3 downstream from codon 189 as the result of the aberrant splicing (Fig. 3E). Discussion

In the present study we demonstrated that the patient was a compound heterozygote with the de novo A189V mutation in the paternal allele of CYP11A and the inherited R353W mutation in the maternal allele. Neither the A189V nor the R353W mutation has been described previously, and the functional consequences of the mutations had to be elucidated. Although Ala189 is not conserved, Arg353, located in the adrenodoxin-binding site, is conserved in the human (20), bovine (21), horse (GenBank accession no. AF031664), sheep (22), goat (22), rabbit (9), rat (23), mouse (GenBank accession no. AF195119), hamster (GenBank accession no. AF323965), trout (24), stingray (25), and catfish (GenBank accession no. AF063836) P450scc proteins, suggesting that Arg353 is an important amino acid residue for P450scc function. The functional expression analyses of the mutant P450scc proteins with the amino acid substitutions demonstrated that R353W replacement resulted in marked reduction in enzymatic activity, but A189V replacement did not affect the activity. These experimental findings together with the conservation of Arg353 in all P450scc molecules indicate that Arg353 is a very crucial amino acid residue for P450scc function. As the mother, bearing the heterozygous R353W mutation, had no symptoms or signs of adrenal insufficiency, we hypothesized that the A189V mutation should inactivate the CYP11A gene by creating an alternative splice-donor site. The in vitro expression analyses of the chimeric minigenes


revealed that the A189V mutation created a new alternative splice-donor site and gave rise to two species of the transcripts: one of normal size and the other with a 61-nucleotide deletion of exon 3 downstream from codon 189. The normal size transcript is expected to encode a P450scc protein with a substitution of Val for Ala at position 189, which was shown to have normal P450scc activity (Table 1). The 61-nucleotide deletion in the transcript is expected to cause a shift in the open reading frame after codon 189 and a premature termination at codon 205. This truncated product is predicted to have no P450scc activity, because it lacks more than half of the native molecule including the highly conserved and functionally critical heme-binding region of P450s. Thus, we conclude that the A189V mutation partially inactivates the CYP11A gene by introducing the alternative splice-donor site. It is noteworthy that our patient did not develop clinically manifest adrenal insufficiency until 7 months of age and was able to synthesize adrenal steroid hormones to some extent in the presence of elevated ACTH and plasma renin activity levels. This partially preserved steroidogenic ability in the patient is consistent with the results of in vitro studies, which demonstrated that the A189V mutation partially inactivates the CYP11A gene, and the R353W mutation results in the markedly reduced activity. Although it was postulated that no one has P450scc deficiency caused by homozygous mutations in CYP11A (26), this postulation appears to be wrong. Our present study clearly indicated that homozygous or compound heterozygous mutations in the CYP11A gene are not necessarily lethal, if the mutations preserve P450scc activity sufficiently to maintain pregnancy. The patient was prepubertal, and we did not examine the detailed gonadal functions of the patient, but the serum FSH level of the patient approached the upper limit of the normal range, perhaps denoting early ovarian insufficiency. We are planning to evaluate detailed ovarian functions in future when the patient reaches puberty. We revealed that the patient is a compound heterozygote for the inactivating CYP11A mutations, A189V and R353W, and that her mother, who is a heterozygote for the R353W mutation, is healthy and totally asymptomatic. Therefore, congenital adrenal insufficiency in this family is inherited as an autosomal recessive trait. Similarly, congenital adrenal hyperplasia in humans caused by mutations in the genes coding all other adrenal steroidogenic enzymes [3BHSD2 (27), CYP17 (28), CYP21A2 (29), CYP11B1 (30), and CYP11B2 (30)] and CLAH in rabbits caused by deletion of the P450scc gene (9) are inherited as autosomal recessive traits. In contrast, the 46,XY patient reported by Tajima et al. (10) had apparent haploinsufficiency of CYP11A and developed congenital adrenal insufficiency and male pseudohermaphroditism, suggesting an autosomal dominant trait of inheritance. There are at least three ways to explain the discrepancy in the manners of inheritance. The first is to hypothesize that a sex difference might exist in the sensitivity to dosage effect of the CYP11A insufficiency; that is, the haploinsufficiency of CYP11A might develop symptoms and signs in 46,XY subjects, but not in 46,XX subjects. The second is to hypothesize that the patient reported by Tajima et al. might harbor another loss of function mutation somewhere in the apparently


normal allele of the CYP11A gene. The last is to hypothesize that the patient reported by Tajima et al. (10) might lack expression of the normal CYP11A allele in the adrenal glands and testes. Accumulation of other cases with CYP11A mutations would lead to more knowledge of the inheritance of CYP11A insufficiency in humans. In conclusion, we have identified compound heterozygous inactivating mutations in the CYP11A gene in a patient with congenital adrenal insufficiency and demonstrated that there exists some relationship between clinical findings and P450scc activity in the patient. This is the first report of compound heterozygous mutations of CYP11A in humans. Acknowledgments We are grateful to Ms. Shoko Mikami and Ms. Atsuko NagashimaMiyokawa for their excellent technical assistance. We thank Dr. Yoshiyasu Yabusaki (Sumitomo Chemical, Co., Ltd., Takarazuka, Japan) and Dr. Shigeaki Kato (Tokyo University, Tokyo, Japan) for providing the bovine adrenodoxin and adrenodoxin reductase expression plasmids. We also thank Dr. Ken-ichiro Morohashi (National Institute for Basic Biology, Okazaki, Japan) for providing the genomic CYP11A E1-1 clone. Received March 21, 2001. Accepted May 7, 2002. Address all correspondence and requests for reprints to: Noriyuki Katsumata, M.D., Department of Endocrinology and Metabolism, National Research Institute for Child Health and Development, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8567, Japan. E-mail: nkatsumata@ nch.go.jp. This work was supported in part by Grants for Pediatric Research (10C-2 and 10C-3) from the Ministry of Health and Welfare, Japan, and a Grant for Organized Research Combination System from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.


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