Mutation of histidine 373 to leucine in cytochrome P450c17 causes 17 alpha-hydroxylase deficiency.

We identified a new homozygous missense mutation His373-->Leu in the CYP17 gene of two sisters with 17 alpha-hydroxylase deficiency with an elevated plasma aldosterone concentration by sequencing their genomic DNAs amplified by polymerase chain reaction. Using polymerase chain reaction-based site-directed mutagenesis, we prepared a DNA that encoded the Leu373 mutant protein. COS-1 cells transfected with the mutant DNA, despite having an RNA hybridizable to the P450c17 cDNA, did not show 17 alpha-hydroxylase and 17,20-lyase activities. Also, the cells were devoid of 11 beta-hydroxylase and aldosterone synthase activities. To examine the mechanism by which the single amino acid change His373-->Leu eliminates activity, we expressed N-terminally modified P450c17 proteins with and without the Leu373 mutation in Escherichia coli and performed spectral studies. Membrane preparations from E. coli cells expressing the wild-type form of the modified enzyme showed an absorption peak at 449 nm upon addition of carbon monoxide in the reduced state and produced characteristic substrate-induced difference spectra, whereas those from the cells expressing the mutant form did not show these spectral changes. The 17 alpha-hydroxylase and 17,20-lyase activities were observed only in E. coli cells expressing the wild-type enzyme. These results show that the His373-->Leu mutant does not incorporate the heme prosthetic group properly and suggest a critical role of His373 in heme binding.

We identified a new homozygous missense mutation HisS7' + Leu in the CYP17 gene of two sisters with 17a-hydroxylase deficiency with an elevated plasma aldosterone concentration by sequencing their genomic DNAs amplified by polymerase chain reaction. Using polymerase chain reaction-based site-directed mutagenesis, we prepared a DNA that encoded the LeuS7' mutant protein. COS-1 cells transfected with the mutant DNA, despite having an RNA hybridizable to the P450c17 cDNA, did not show 17a-hydroxylase and 17,ZO-lyase activities. Also, the cells were devoid of 1 la-hydroxylase and aldosterone synthase activities. To examine the mechanism by which the single amino acid change His"' + Leu eliminates activity, we expressed N-terminally modified P450c17 proteins with and without the Leu37' mutation in Escherichia coli and performed spectral studies. Membrane preparations from E. coli cells expressing the wild-type form of the modified enzyme showed an absorption peak at 449 nm upon addition of carbon monoxide in the reduced state and produced characteristic substrate-induced difference spectra, whereas those from the cells expressing the mutant form did not show these spectral changes. The 17a-hydroxylase and 17,ZO-lyase activities were observed only in E. coli cells expressing the wild-type enzyme. These results show that the His'73 4 Leu mutant does not incorporate the heme prosthetic group properly and suggest a critical role of HisS7' in heme binding.
Steroid 17a-hydroxylase (P450c17) has a dual function of catalyzing the l7a-hydroxylation of pregnenolone and progesterone and the 17,20-cleavage of the corresponding hydroxylated steroids (1,2). The enzyme is encoded by a single gene CYPl7 that is located on chromosome 10q24-q25 (3,4). The gene consists of eight exons that have been completely sequenced (5) and is expressed in the adrenal and gonadal glands (6). Deficiency of l7a-hydroxylase leads to decreased production of glucocorticoids and sex steroids, and this in * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S turn increases ACTH' secretion. This hormonal imbalance causes hypertension, pseudohermaphroditism in the male, and primary amenorrhea and hypogonadism in the female (7). Since the first description by Biglieri et al. (8) in 1966, well over 120 cases with l7a-hydroxylase deficiency (17-OHD) have been reported (7,9). Patients with this disease are usually characterized by a very low or undetectable plasma level of aldosterone. The depletion of aldosterone is thought to result from sodium retention; expansion of the extracellular fluid volume suppresses the renin-angiotensin system, thereby decreasing aldosterone secretion from the adrenal. However, several cases with this disease in Japan and the United States have been shown to have elevated aldosterone levels with suppressed renin activities (9). Whereas the molecular defects have been elucidated recently in several 17-OHD cases, no structural analysis has been done for the CYPl7 gene of 17-OHD patients with elevated serum aldosterone concentrations. We report here two Japanese sisters with 17-OHD associated with hyperaldosteronism caused by a homozygous missense mutation of His373 to leucine in the CYPl7 gene.

MATERIALS AND METHODS
Patients-The patients described in this report are two Japanese sisters who live in Nagano Prefecture in central Japan. Their clinical features were described in detail previously (10). These two sisters, currently 24 and 20 years old, sought medical attention for hypertension and sexual infantilism at ages 17 and 13, respectively. Both had a 46 XX karyotype, and neither showed pubertal development. Increased responses of luteinizing and follicle-stimulating hormones to luteinizing hormone-releasing hormone were observed in both cases. They had high plasma levels of 17-deoxysteroids including aldosterone, low to normal levels of 17a-hydroxysteroids, and low levels of sex steroids. The concentrations of 17-deoxysteroids returned to normal levels by administration of dexamethasone. Their plasma concentrations of aldosterone were high, but the plasma renin activity was not increased. Their father and grandmother had hypertension but not hypogonadism, and their mother and other sister were normotensive and showed normal sexual development.

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ase K. After standing overnight at 37 "C, DNA was extracted with phenol/chloroform and precipitated with ethanol. The DNA was dissolved in 10 mM Tris-HC1 (pH 8.0), 1 mM EDTA.
Southern Blotting-Samples of genomic DNA were digested with EcoRI, BamHI, and HindIII, and fractionated by electrophoresis on a 1% agarose gel. The DNA fragments were transferred to nylon membrane (Hybond N, Amersham Corp.) and probes with a 3zPlabeled full-length human P450c17 cDNA (6). Radiolabeling of the cDNA probe was carried out by the random primer labeling method (11) using [a-"P]CTP (111 TBq/mmol, ICN Biomedicals, Inc.). The filter that had been prehybridized in 6 X SSC, 5 X Denhardt's reagent, 1% SDS, 0.1 mg/ml heat-denatured salmon testis DNA at 67 'C for 6 h was incubated with hybridization solution containing 6 X SSC, 1% SDS, and the 32P-labeled probe (lo7 cpm/lO ml) at 67 "C for 16 h. The filter was washed twice with 2 X SSC, 1% SDS, and twice with 0.1 X SSC, 0.1% SDS at 65 "C for 20 min.
DNA Amplification-Exons 1-8 of the CYPl7 gene were individually amplified by PCR (12). Oligonucleotides used as primers were synthesized on an Applied Biosystems 381A DNA synthesizer and are shown in Table I. Each PCR reaction mixture (100 pl) contained 1 pg of genomic DNA, 50 pg of each primer, 200 p~ each of dCTP, dTTP, dGTP, and dATP, 6 mM MgCl,, and 5 units of Taq DNA polymerase in 10 mM Tris-HC1 (pH 8.0). All amplifications were done in a thermal cycler (Perkin-Elmer Cetus Instruments) with the following program: 1) denaturation at 96 'C for 2 min; 2) followed by 40 cycles of denaturation (96 "C, 15 s), annealing (55 "C, 30 s), and extension (72 "C, 90 8 ) ; and 3) final extension (72 "C, 2 min). The PCR products were electrophoresed on a 1% agarose gel, and the DNAs with expected sizes were recovered from the gel using Suprec-01 (Takara Shuzo) and used as templates for asymmetric PCR amplification. Asymmetric PCR was done with a molar ratio of primers of 201 (13).
primers were synthesized primer W, 5'-GGAGTCAACGTI'GGCCT To achieve allele-specific amplification of exon 6, the following TGT-3', and primer M, 5'-GGAGTCAACGTTGGCCTTGA"', where the underlined A was the substituted nucleotide found in the genes of the patients. Using these primers and primer 5-5' (Table I), amplification was carried out under the same conditions described above except that the number of cycles of denaturation, annealing, and extension was reduced to 20.
Each single-stranded DNA amplified by PCR was sequenced by the dideoxy method (14), using the Sequenase version 2.0 kit (U. S. Biochemical Corp.). A sequencingprimer of 17-20 bp was synthesized for hybridization to each strand, labeled with [y3'P]ATP (DuPont NEN) and T4 polynucleotide kinase (Takara Shuzo), and annealed to the DNA template at 65 "C followed by slow cooling to below 35 "C.
Construction of Eukaryotic Expression Vectors-The vector expressing the wild-type P450c17 protein in COS-1 cell was constructed by ligating the P450c17 cDNA to the EcoRI site of the eukaryotic expression vector pcDL-SRa 296 (15). The P450c17 cDNA encoding the His"3 + Leu mutant protein was constructed by the PCR-based site-directed mutagenesis of P450c17 cDNA. For this purpose, the 5' of the cDNA was ligated to the EcoRI site of pUC19, and the recombinant DNA was used as the PCR template. The M13 reverse the synthetic oligonucleotide 5"TGGAGTCAACGTTGGCCTT sequencing primer was used as the upstream PCR primer, and GAGGGGGA-3' was used as the downstream primer. The downstream primer corresponds to amino acids 371-380 of P450c17 with a single amino acid change HisS73 + Leu (the changed nucleotide is underlined) and contains a HincII site (indicated by boldface letters). PCR amplification was done with the following program: 1) 2 cycles of denaturation (95 "C, 1 rnin), annealing (45 "C, 1 min), and extension (72 "C, 1 min); 2) followed by 18 cycles of denaturation (94 'C, 1 min), annealing (52 "C, 1 min), and extension (72 "C, 1 min); and 3) final extension (72 "C, 2 min). The PCR product (1.3 kb) was digested with AccI and then with HincII, and the resulting 1.2-kb AccI-HincII fragment containing the changed nucleotide was used to replace the corresponding fragment of the normal cDNA. The construct was verified by sequencing. The mutated p450c17 cDNAs were removed from the pUC vector and ligated to pcDL-SRa 296. The recombinant vectors were purified by CsCl gradient centrifugation.
Transient Transfection Assay for P450c17 Actiuity-Transfection was accomplished by incubating COS-1 cells (1 X lo6 cells in a 10cm culture dish) with diethylaminoethyldextran (500 mg/ml, Pharmacia LKB Biotechnology Inc.) and 20 pg of the pcDL-SRa 296 vector with or without P450c17 cDNA inserts in 10 ml of serum-free Dulbecco's modified Eagle's medium (DMEM) containing 50 mM Tris-HC1 (pH 7.3) for 4 h at 37 "C. The medium was changed to the one containing chloroquine (52 mg/ml) and 2% fetal bovine serum, and culture was continued for 3 h. The cells were then washed with DMEM containing 50 mM Tris-HC1 and incubated in DMEM containing 10% fetal bovine serum. At 48 h after transfection, the medium was removed, and 6 ml of DMEM containing 10% fetal bovine serum and 1.0 p~ radiolabeled substrate was added. [ NEN) were used as substrates. After incubation at 37 "C, 5 ml of the reaction mixture were transferred to a borosilicate glass tube, and steroids were extracted with 20 ml of ethyl acetate/isooctane (1:l). The extract was concentrated by evaporation under N, and applied to a Silica Gel 60 F254 TLC plate (Merck). Two kinds of solvents were used chloroform/ethyl acetate (3:l) to separate pregnenolone, progesterone, and their derivatives (17) and methylene chloride/methanol/HzO (300:201) to separate deoxycorticosterone derivatives (18). Radioactive steroids were visualized by autoradiography.
Northern Blotting-Total RNA was prepared from the transformed COS-1 cells by the method of Chomczynski and Sacchi (16). Fifteen pg of RNA were electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde and probed with a 32P-labeled P450c17 cDNA probe. The washing conditions were the same as those described above for Southern blotting.
Construction of Prokaryotic Expression Vectors-To produce the wild-type and mutant P450c17 proteins in E. coli, we constructed expression vectors using the plasmid pCWori+ (19,20). Barnes et al. (21) succeeded in producing large amounts of bovine P450c17 in E. coli by modifying the 5' sequence of the bovine P450c17 cDNA; the second codon was changed from TGG (Trp) to GCT (Ala), and silent mutations were introduced to codons 4-7 so as to make the 5' sequence AT-rich. Thus, we prepared a human P450c17 cDNA in which the nucleotide sequence of the first seven codons was identical to that of the modified bovine cDNA. A 38-base 5'-primer, 5'encoding the first 13 aminoxlds of modified human P450c17 and a 3'-primer, 5'-AATGATCTTCTCCAGCTTCTGA-3', corresponding to amino acids 139-146 were synthesized and used for PCR amplification (underlined letters are changed nucleotides). The conditions for PCR were the same as those described above under "Construction of Eukaryotic Expression Vectors." The PCR products were digested with XbaI, and the resulting 306-bp blunt end XbaI fragment was inserted into the blunt end NdeI-XbaI site of pCWori+. Finally, the expression plasmids were constructed by ligating the 1.44-kb XbaI-XbaI fragment of P450c17 cDNA (encoding amino acids 102-508 with or without the mutation) into the XbaI site of pcWori+ carrying the PCR product described above. The cDNAs encoding the modified wild-type and the modified Leu373 mutant P450c17s are designated modl7-W and modl7-M, respectively.
Bacterial Expression of Recombinant Plasmids, Preparation of Membranes, and Spectral Studies-An overnight culture of E. coli DH5a transformed with modl7-W or -M was grown at 37 'C in LB medium containing 50 pg/ml ampicillin. A 4-ml aliquot thereof was used to inoculate 200 ml of Terrific broth (22) containing ampicillin. When the cell turbidity measured at 600 nm reached an absorbance of 0.6, IPTG was added to a final concentration of 1 mM, and culture was continued for an additional 24 h at 30 "C. The cells were harvested by centrifugation, washed with 50 mM Mops buffer (pH 7.5) containing 100 mM KC1, 1 mM EDTA, and 1 mM dithiothreitol, and resuspended in the same buffer. For preparation of membranes, the cells were treated with 0.2 mg/ml lysozyme, and the resulting spheroplasts were lysed by sonication. After brief centrifugation at 7,000 X g to remove unbroken cells and debris, the supernatant was made to 6 mM MgC1, and again centrifuged at 60,000 X g for 50 min at 4 "c.
The pellet was suspended in the Mops buffer and homogenized. An aliquot corresponding to 20 mg of protein was made up to 6 ml with the same buffer containing 10 mM glucose, and the mixture was divided equally between two cuvettes. Several grains of sodium dithionite were added to each cuvette, and the base-line spectrum was recorded. CO was then bubbled through the sample cuvette to obtain the reduced CO spectrum (23). Spectral changes induced by substrate binding (24) were measured similarly by adding a steroid substrate dissolved in ethanol to the sample cuvette and an equal volume of ethanol to the reference cuvette. The difference spectrum was re-
Western Blot Anulvsis-Proteins were treated with 1% SDS. 5% 2-mercaptuethanol a t 100 "C for 2 min before electrophoresis. SDSpolyacrylamide gel electrophoresis was carried out according to Laemmli (25) using an 8% polyacrylamide gel. The separated proteins were electrotransferred to nitrocellulose membrane (Schleicher and Schuell). The membrane was incubated for 1 h at room temperature in 20 mM Tris-HC1 (pH 7.5), 137 mM NaCl, 0.1% Tween 20 (TBS-T) containing 5% dry milk to block excess binding sites, followed by washing with TBS-T three times. The membrane was then incubated for 1 h with anti-porcine P450c17 antibody (Oxygene, Dallas) that had been diluted with TBS-T and treated with membrane preparations from untransformed E. coli DH5a. After washing three times with TBS-T, the antigen-antibody complex was reacted with goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (Amersham Corp.) and was washed three times with TBS-T. The antigen-antibody complex was detected by chemiluminescence after addition of luminol and hydrogen peroxide (ECL, Amersham Corp.).
Steroid Metabolism by E. coli Expressing P450cl7-E. coli DH5a cells were cultured and induced as described above. At 24 h after addition of IPTG, a radiolabeled steroid (["C]pregnenolone, ["CC] progesterone, 17-OH [3H]pregnenolone, or 17-OH [3H]progesterone) was added to 2 ml of the culture, and the mixture was incubated for 24 h at 30 'C with gentle shaking. The products were extracted and analyzed by TLC as described above.

RESULTS
Southern Blot Analysis-The patients described here are two sisters who were diagnosed as having 17-OHD from steroid profiles and other symptoms (10). Genomic DNAs obtained from leukocytes of the two patients and an unaffected person were each digested with restriction endonucleases EcoRI, BamHI, and HindIII. Fig. 1 shows Southern blot analysis of the DNA digests. In each case, EcoRI yielded fragments of 5.7 1 was found to contain a homozygous missense mutation changing codon 373 from histidine (CAC) to leucine (CTC) (Fig.  2A). The sequence ladders for other members of the family examined all showed a heterozygous pattern CAC/CTC for codon 373, indicating that they have the same mutation in one allele and the normal sequence in the other (Fig. 2B). We also found, in all subjects examined, silent mutations in exons 1 and 5: His46, CAT + CAC; Ser65, TCT + TCG; Asp2=, GAT + GAC. The same silent mutations were reported by Kagi-mot0 et al. (26) in a patient with 17-OHD and a normal control.

FIG.
Allele-specific Amplification-To confirm the results of the sequence analysis, we performed allele-specific PCR amplification of genomic DNA (Fig. 3). The 5'-primer used was 5-5', and the 3'-primer was either W, containing the normal sequence, or M, containing the mutated nucleotide (Table I) (Fig. 5A). With A4 substrates, the wild-type cells demonstrated the 16a-and 17a-hydroxylase (conversion of ['4C]progesterone to 16-OH and 17-OH ['4C]progesterone) and 17,20-lyase activity. Again, the mutant cells showed neither enzyme activity (Fig. 5B). To   one as substrate. No conversion of the substrate to corticosterone, 18-OH corticosterone, or aldosterone was observed, as with the wild-type cells (data not shown).
Expression of Modified P450c17 cDNA in E. coli and Spectral Study-To obtain sufficient amounts of the wild-type and mutant P450c17 for spectral studies, we attempted to produce these proteins in E. coli. To this end, we constructed a pCWori+ vector containing the coding sequence of the wildtype or mutant human P450c17 cDNA. The vector, however, failed to produce detectable amounts of P450c17 protein in E. coli DH5a. Barnes et al. (21) also observed that the pCWori+ vector constructed by introducing the coding sequence of bovine P450c17 cDNA did not produce the P450 protein. They found that modification of the nucleotide sequence corresponding to N-terminal seven amino acids led to production of large amounts of the recombinant protein.
Thus, we modified the 5' sequences of the wild-type and mutant human P450c17 cDNAs as described by Barnes et al. (21); the second codon was changed from TGG (Trp) to GCT (Ala), the third codon from GAG (Glu) to CTG (Leu), the fifth codon from GTG (Val) to TTA (Leu), and the seventh codon from CTC (Leu) to GTT (Val). As shown in Fig. 6, Western blot analysis revealed that E. coli transformed with the vectors containing the modified cDNAs produced proteins immunoreactive to anti-porcine P450c17 antibody in the presence of IPTG. Membrane fractions prepared from E. coli transformed with the pCWori+ carrying the modified wildtype cDNA showed, in the reduced state, a peak at 449 nm upon addition of CO and a characteristic substrate-induced difference spectrum when 17-OH pregnenolone, progesterone, or 17-OH progesterone was added. (Only the data for pregnenolone are shown in the figure.) On the other hand, membrane preparations from E. coli transformed with the mutant vector failed to produce these spectral changes (Fig. 7). These results suggest that the single amino acid substitution + Leu causes a defect in heme binding.
Barnes et al. (21) showed that E. coli contained an electron transport system that could support the activity of recombi-

36.
FIG. 6. Western blot analysis of bacterially expressed P450c17 proteins.  one was not converted to androstenedione.) E. coli expressing the mutant form of the enzyme showed none of these activities (Fig. 8).

DISCUSSION
This is the first report of molecular analysis of the CYP17 gene in a family with l7a-hydroxylase deficiency accompanied by elevated plasma aldosterone concentrations. We have found a new homozygous missense mutation His373 4 Leu in two Japanese siblings with this subtype of the disease. We also documented heterozygosity of this mutation in the patients' family members. The plasma levels of basal and ACTH-stimulated deoxycorticosterone, corticosterone, 18-OH corticosterone, and 18-OH deoxycorticosterone are re-ported to be high in 17-OHD heterozygotes (27). Although these clinical features are useful for diagnosis, they are obviously not definitive. We established the diagnosis by allelespecific PCR amplification of genomic DNAs.
To date, 11 different genetic lesions have been reported in patients with 17-OHD. Three of these were nonsense mutations causing immature chain termination (29-31); two were small duplications of 4 and 7 bp, respectively, resulting in the change in the reading frame (26,32); two were small deletions (28,33); and one was a 518-bp deletion with a 469-bp insertion (34). Amino acid replacement mutations were described in only three patients; two were compound heterozygotes (Arg4= -+ C y s / G l~~~' -+ Stop (30) and -+ Thr/Arg3' + Stop (31), respectively), and only one was homozygous for a single amino acid change (SerlOG -+ Pro) (17 der "Materials and Methods." The Leu373 mutant expressed in COS-1 cells lacked the 17ahydroxylase and 17,20-lyase activities. To gain insight into the mechanism by which this single amino acid mutation eliminated activity, we attempted to produce large amounts of P450c17 proteins in E. coli and perform spectral studies. Although pCWori+ vectors constructed by insertion of the coding sequences of the wild-type and mutant forms of cDNA failed to produce detectable amounts of P450c17 proteins in E. coli, modification of the 5'-nucleotide sequences as described by Barnes et al. (21) affected production of the recombinant proteins (Fig. 6). Spectral changes characteristic of P450 (CO and substrate-induced difference spectra in the reduced state), however, were observed only with the membrane preparations from E. coli expressing the modified wild type (Fig. 7). It is generally accepted that the fifth ligand of the heme iron of a P450 enzyme is a conserved cysteine residue in the C-terminal half of the enzyme. The failure of the Leu373 mutant to exhibit characteristic spectrophotometric properties of P450 indicates that the mutant does not incorporate the heme moiety properly and suggests that His373, in addition to the iron-binding cysteine, plays an important role in heme binding. A molecular model of human P450c17 based on the known crystallographic structure of bacterial P450cam (35) suggests that this histidine, which is conserved across mammalian and chicken P450c179, interacts with the heme propionate group (36). Thus, the amino acid residue interacting with the heme propionate may be crucial for heme incorporation.
While the mutant expressed in COS-1 or E. coli cells is devoid of the activities of P450c17, the patients' plasma contained considerable amounts of l7a-hydroxysteroids (10). Thus, it is possible that the Leu373 mutant might have retained a small amount of activity, which we could not detect by our in vitro methods. A similar discrepancy between the enzyme activities in vivo and in vitro has been reported by Yanase et al. (31).
The reason our patients have an elevated plasma aldosterone level is not clear. The mutant enzyme is not able to synthesize aldosterone from deoxycorticosterone. Recently, Lifton et al. (37) studied the molecular basis of glucocorticoidsuppressible hyperaldosteronism and found a chimeric llphydroxylase/aldosterone synthase gene. This gene is thought to arise from gene duplication by unequal crossing over, fusing the 5' regulatory region of llp-hydroxylase gene to the coding sequence of the aldosterone synthase gene. Like glucocorticoid-suppressible hyperaldosteronism patients, the plasma aldosterone level in our patients appears to be controlled by ACTH. Analysis of the IlB-hydroxylase and the aldosterone synthase genes should be done in our patients.