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Two Novel Mutations Found in a Patient with 17-Hydroxylase Enzyme Deficiency

Department of Pediatrics (B.E.-L., S.T., R.C.W., M.I.N.), Mount Sinai School of Medicine, New York, New York 10029; and Department of Internal Medicine (R.A., M.P.-Z.), Division of Endocrinology and Metabolism, and Donald W. Reynolds Cardiovascular Clinical Research Center, University of Texas Southwestern Medical School, Dallas, Texas 75390

Address all correspondence and requests for reprints to: Maria I. New, M.D., Mount Sinai School of Medicine, Department of Pediatrics, 1 Gustave L. Levy Place, Box 1198, New York, NY 10029. E-mail: maria.new{at}mssm.edu.

	Abstract

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Context: Congenital adrenal hyperplasia resulting from 17-hydroxylase deficiency (17OHD) is a rare disorder associated with hypertension.

Subject and Methods: We describe a phenotypically and hormonally affected female patient with 17OHD. DNA sequencing of her CYP17 gene revealed a maternal heterozygous mutation in exon 2 (R125Q) and a paternal heterozygous mutation in exon 8 (R416H). These are novel mutations in the CYP17 gene that completely eliminate enzyme activity.

Conclusion: Identification of novel mutations in the CYP17 gene is vital in understanding the molecular mechanisms of its deficiency and in providing additional information about the structure and enzymatic functions of P450c17.

	Introduction

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CONGENITAL ADRENAL HYPERPLASIA (CAH) resulting from 17-hydroxylase deficiency (17OHD) is a rare autosomal recessive disorder with an estimated incidence of about 1:50,000 to 1:100,00 in newborns (1). The cytochrome P450c17 enzyme catalyzes both the 17-hydroxylase and the 17,20-lyase activities. There is only one human gene termed CYP17 for the P450c17 enzyme; it is mapped to chromosome 10q24.3 (2). It consists of eight exons and is expressed in several steroidogenic tissues, including the adrenal cortex, ovary, and testis (3). Since the cloning of the CYP17 gene, 50 different mutations have been described, (4, 5, 6, 7) resulting in either combined or isolated 17-hydroxylase/17,20-lyase enzyme deficiencies.

The lack of 17-hydroxylase activity results in decreased cortisol synthesis and compensatory hypersecretion of ACTH. In turn, ACTH stimulates overproduction of 11-deoxycorticosterone (DOC), a mineralocorticoid that causes hypertension and hypokalemia in 17OHD (8). The typical features of 17OHD were first described 40 yr ago as hypertension and hypokalemia, with suppressed plasma renin activity and aldosterone (9). Furthermore, 17OHD is characterized by sexual infantilism in phenotypic females (10), although a spectrum of phenotypes has been reported in genetic males (11). In the gonads, the lack of 17,20-lyase activity prevents gonadal sex steroid production and leads to undervirilization in the 46,XY males and failure of pubertal development (11, 12, 13). Females present with primary amenorrhea and lack of development of secondary sex characteristics (14).

We present the clinical and molecular data of a female patient with CAH resulting from 17OHD. Molecular analysis of the CYP17 gene revealed two novel mutations encoding inactive enzymes.

	Subject and Methods

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Subject

The proband presented at age 31.5 yr to our clinic. She was the only child of nonconsanguineous parents and was of British/Italian origin. She underwent left oopherectomy for ovarian torsion at 12 yr of age. She was initially diagnosed elsewhere at age 15 yr, when she presented with hypertension (148/99 mm Hg), hypokalemia, and delayed puberty (infantile genitalia; Tanner stage 2 for breast and pubic hair). Her plasma cortisol was undetectable before and 2.4 µg/dl after cosyntropin stimulation, suggesting a cortisol synthesis defect. Both her DOC and corticosterone (B) levels were elevated at baseline (145 ng/dl for DOC and 7300 ng/dl for B) and after cosyntropin stimulation (245 ng/dl for DOC and 19,600 ng/dl for B), suggesting 17OHD (reference values for DOC are 18 ± 11 ng/dl and for B 350 ± 390 ng/dl). There was also a defect in gonadal steroid synthesis (estradiol < 10 pg/ml) and elevated gonadotropins (FSH > 145 IU/liter; LH > 168 IU/liter). A pelvic sonogram revealed a bicornuate uterus and a polycystic right ovary. She began treatment with prednisone 5 plus 2.5 mg daily and 50-µg estradiol patch with cyclic 10 mg medroxyprogesterone acetate at age 15, although she did not begin menstruating until she started to take her steroids regularly at age 19. On presentation to our clinic, physical examination showed incomplete female secondary sex characteristics: Tanner stage 3 for breast, Tanner stage 2 for pubic hair, and sparse axillary hair. Her blood pressure was 145/95 mm Hg. The patient admitted that her compliance with her medications was poor.

DNA analysis

After informed consent was obtained from the proband and the parents, genomic DNA was extracted from peripheral leukocytes (Pure Gene DNA Isolation Kit D-5000, Gentra Systems, Minneapolis, MN). Molecular genetic analysis was performed by PCR and by DNA sequencing of the exon and intron boundaries of the CYP17 gene. The CYP17 gene was amplified in six fragments using the primers in Table 1 (15). The reactions were performed in 50 µl containing 100–300 ng genomic DNA and 0.3 µmol/liter of each primer pair. The samples were denatured at 94 C for 10 min. This was followed by 35 cycles at 94 C for 30 sec, 55 C for 30 sec, and 72 C for 30 sec. A final cycle was then performed at 72 C for 7 min. Amplified gene fragments were purified using 1% agarose gel electrophoresis and QIA Quick Purification kit (QIAGEN, Valencia, CA). Sequencing of all eight exon and intron boundaries of CYP17 was performed using the primers outlined in Table 1, as well as BIG DYE terminators from PE Applied Biosystems (Foster City, CA), and run on ABI 3700 DNA Sequencers at the Institute for Biotechnology and Life Sciences Technology, Cornell University, Ithaca, NY.

The cDNA for CYP17 mutations were generated by overlapping PCR and subcloned into pcDNA3. HEK-293 cells were seeded in six-well plates and transfected with 1 µg pcDNA3-c17 plasmids using FuGENE6 as described (16, 17). Cells were incubated with 2 ml complete medium containing 0.03 µmol/liter pregnenolone with 200,000 cpm of [³H]pregnenolone (PerkinElmer NEN Life Science Products Life Sciences, Shelton, CT). Aliquots (1 ml) were removed after 2 and 4 h, extracted, and chromatographed on plastic silica gel plates using 3:1 chloroform/ethyl acetate as described (16, 17). The dried plates were coated with EN³HANCE spray (PerkinElmer), and steroids were visualized by autoradiography. The regions of the plates corresponding to substrate and products were excised with scissors and quantitated by liquid scintillation counting (16, 17).

	Results

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At the time of presentation, the patient was treated with 0.5 mg dexamethasone daily, 0.625 mg conjugated estrogen daily, and 10 mg medroxyprogesterone acetate between the 15th and 25th days of her cycle. Hormonal data during treatment showed low serum cortisol (1.1 µg/dl), low estradiol (4.3 ng/dl), and undetectable testosterone, androstenedione, and dehydroepiandrosterone (DHEA). The rest of her adrenal profile revealed concentrations of 17-hydroxyprogesterone of 49 ng/dl, DOC of 40 ng/dl, B of 170 ng/dl, and aldosterone of 8 ng/dl.

Molecular analysis of the patient’s cytochrome CYP17 gene revealed two different mutant alleles, indicating that she is a compound heterozygote. Genomic DNA sequencing analysis revealed a maternal heterozygous mutation in exon 2 in codon 125 (CGA to CAA) resulting in a substitution of arginine with glutamine (R125Q) and a paternal heterozygous mutation in exon 8 in codon 416 (CGT to CAT) resulting in a substitution of arginine with histidine (R416H) (Fig. 1). These represent two novel mutations in the CYP17 gene.

View larger version (23K): [in this window] [in a new window]   FIG. 1. Direct sequencing of CYP17 PCR products showing the two missense mutations found in the patient on her respective exons. The bold lines represent the codons where the substitutions occurred, and they are also represented as expected in the CYP17 gene.

	Discussion

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We report a patient who presented with the clinical and hormonal features typical of 17OHD. The hormonal profile of the patient demonstrated a marked ACTH-driven elevation of all 17-deoxysteroids, including mineralocorticoids produced by the zona fasciculata in the 17-deoxy pathway, as well as a significant reduction of cortisol and androgens typical of the combined form of 17-hydroxylase and 17,20-lyase deficiency (18). Reduced production of sex steroids leads to the hypergonadotropic hypogonadal state observed in both our patient and other 17OHD patients (18).

Sequencing of the CYP17 gene identified two novel mutations in exon 2 and exon 8. Expression of mutant alleles in HEK-293 cells revealed no detectable activity of the encoded proteins. Since the first mutations in the CYP17 gene were identified by Kagimoto et al. (4), 50 different mutations have been described including missense mutations, deletions, insertions, and single base changes (19). It is of interest that the majority of mutations identified to date, including R416H, lie in the coding region of exon 8 (20).

Although 17OHD represents approximately 1% of all cases of CAH worldwide (7), the prevalence of 17OHD appears to be more common in certain ethnic groups, notably in Canadian Mennonites (21) and Brazilians (7). The report by Costa-Santos et al. (7) indicates founder effects for the CYP17 mutations because their study on 24 Brazilian patients from 19 families with 17OHD showed that 50% of affected alleles in this population contained mutation W406R and 32% showed mutation R362C. Previous studies in other populations also suggested the presence of founder effects for certain mutations in the CYP17 gene (18).

In general, the biochemical diagnosis of 17OHD, as for all steroidogenic enzyme deficiencies, is established by measuring precursor-to-product ratios in the course of an ACTH stimulation test (12). A 5- to 10-fold increase in the 17-deoxysteroids, B, DOC, and progesterone (P) after ACTH administration leads to diagnosis of 17OHD (12). In addition, 17OHD is characterized by elevated production of 18-hydroxycorticosterone and 18-hydroxy-DOC, in contrast to 11-hydroxylase and 21-hydroxylase deficiencies (12). Elevation of B is also seen in patients with cytochrome P450 oxidoreductase deficiency in conjunction with elevated 17-OHP (22). Analysis of the CYP17 gene in cytochrome P450 oxidoreductase deficiency, however, is normal (22). P is elevated in 17OHD and available in most hospital laboratories, but P is also elevated in other forms of CAH. Martin et al. (18) and others reported elevated basal P levels in patients with combined 17-hydroxylase/17,20-lyase deficiency, confirmed by CYP17 genotyping. Basal P measurement seems a useful and practical screen for diagnosis of 17OHD, particularly if the clinical presentation excludes other forms of CAH (18).

Various phenotypic features have been described despite carrying the same mutations in the CYP17 gene (23). Although phenotypic features and biochemical findings are consistent with 17OHD, some patients may not carry any obvious mutation in the CYP17 gene (23, 24). It is not clear why there is a discrepancy between genotype and phenotype in patients with 17OHD. It was hypothesized that unknown intracellular factors may variably increase the stability of mutant CYP17 proteins within the adrenals and/or gonads of patients with 17OHD, which may allow for some enzyme activity in rare cases when enzyme expression is maximally induced, despite the lack of demonstrable activity in heterogeneous expression systems (7).

In summary, we report two novel mutations in a female Caucasian patient with 17OHD. Our patient is a compound heterozygote for novel mutations of exon 2 (R125Q) and exon 8 (R416H), which are responsible for the pathogenesis of complete 17-hydroxylase/17,20-lyase deficiency. Identification of novel mutations in the CYP17 gene is important in understanding the molecular mechanisms of its deficiency and in providing additional information about the structure and enzymatic functions of P450c17. Furthermore, molecular analysis of patients with 17OHD may help in identifying regulator genes or cofactors responsible for variations in the clinical phenotype when a phenotype-genotype discrepancy is apparent.

	Footnotes

 
Disclosure summary: The authors have nothing to disclose.

This work was supported by National Institutes of Health Grants HD 00072, Rare Disease Grant RR19484 (to M.I.N.), and R21DK059942 (to R.A.).

Results from this work were presented in abstract and poster forms at the Ninth Annual Meeting and Scientific Exposition of the American Society of Hypertension, New York, May 2004.

First Published Online July 18, 2006

Abbreviations: B, Corticosterone; CAH, congenital adrenal hyperplasia; DHEA, dehydroepiandrosterone; DOC, deoxycorticosterone; 17OHD, 17-hydroxylase deficiency; P, progesterone.

Received March 1, 2006.

Accepted July 12, 2006.

	References

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