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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 1 199-202
Copyright © 1998 by The Endocrine Society

Original Studies

A New Compound Heterozygous Mutation (W17X, 436+5G->T) in the Cytochrome P450c17 Gene Causes 17{alpha}-Hydroxylase/17,20-Lyase Deficiency

Yukie Suzuki1, Tomohisa Nagashima1, Yoko Nomura, Kazumichi Onigata, Kanji Nagashima and Akihiro Morikawa

Department of Pediatrics, Gunma University School of Medicine, Maebashi, Gunma 371, Japan

Address all correspondence and requests for reprints to: Kanji Nagashima, M.D., Ph.D., Department of Pediatrics, Gunma University School of Medicine, 3–39-22 Showa-machi, Maebashi, Gunma 371, Japan.


   Abstract
Top
 Abstract
Introduction
 Case Report
 Materials and Methods
 Results
 Discussion
 References
 
A genetic disorder in cytochrome P450c17 results in 17{alpha}-hydroxylase/17,20-lyase deficiency. In the present study, a Japanese patient with 17{alpha}-hydroxylase/17,20-lyase deficiency underwent molecular analysis. The patient presented with complete female genitalia with a 46,XY karyotype, absent pubertal development, and hypertension. The exons and exon-intron boundaries of P450c17 genetic region were amplified and sequenced. DNA sequencing revealed a compound heterozygous mutation. One allele showed a G to A transition corresponding to a premature termination codon at tryptophane in codon 17 (W17X). The other allele showed a G to T substitution at the fifth nucleotide from the splice donor site in intron 2 (436+5G->T). W17X was found in one allele of the father, and 436+5G->T was found in one allele of the mother. A previous report presented a patient with 17{alpha}-hydroxylase/17,20-lyase deficiency who was homozygous for W17X. However, the present case is a novel 436+5G->T mutation.

Reverse transcription-PCR analysis using total ribonucleic acid isolated from the testes of the patient revealed that an intron 2 donor site mutation caused abnormal splicing, such that exon 2 was spliced with intron 2. Skipping the exon alters the translational reading frame of exon 3 and introduces a premature termination codon. In semiquantitative analysis, the majority of the transcript for 436+5G->T skips exon 2.

The present findings indicate that in this patient, 17{alpha}-hydroxylase/17,20-lyase deficiency was caused by the compound heterozygous mutation of exon and splice site mutation in cytochrome P450c17 gene.


   Introduction
Top
 Abstract
 Introduction
Case Report
 Materials and Methods
 Results
 Discussion
 References
 
GENETIC disorders in the gene encoding cytochrome P450c17 have been demonstrated to cause 17{alpha}-hydroxylase/17,20-lyase deficiency (1). The consequent defects in the synthesis of cortisol and compensatory hypersecretion of ACTH, which stimulates the synthesis of a large quantity of 11-deoxycorticosterone and corticosterone, lead to hypertension, hypokalemia, and a suppressed renin-angiotensin system. In gonads, the absence of 17,20-lyase activity prohibits the synthesis of androgens. This results in sexual infantilism and a female phenotype in both genetic sexes (1). To date, 21 different genetic lesions have been described in patients suffering from this disorder, indicating that mutations in this gene are due to random events (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). However, the molecular basis for variability in the defect is not clearly understood. Previously reported mutations have primarily consisted of intraexonal mutations. The first case of a splice site mutation in intron 7 was recently reported (12). In the present study, the first case of a compound heterozygous mutation of codon 17 in exon 1 from TTG (Trp) to TAG (stop) (W17X) and a G to T substitution at the fifth nucleotide in the splice donor site of intron 2 in the cytochrome P450cl7 gene (436+5G->T) is described. The splice site mutation, 436+5G->T, is a novel mutation.


   Case Report
Top
 Abstract
 Introduction
 Case Report
Materials and Methods
 Results
 Discussion
 References
 
A 13-yr-old, phenotypically female, Japanese patient sought medical attention for hypertension (160/92 mm Hg) and absent pubertal development. The hypertension was first noticed at the age of 6 yr. The parents had no history of consanguinity. The patient presented with female genitalia and remarkable pigmentation on her gingivae. Her karyotype was 46,XY. Both serum LH and FSH levels and those after administration of LH-releasing hormone (2 µg/kg, iv) were remarkably high (basal LH and FSH, 47.5 and 571.5 mIU/mL, respectively; peak LH and FSH, 154.7 and 316.3 mIU/mL, respectively). The blood levels of ACTH and potassium were 410 pg/mL and 3.0 mEq/L, respectively. The serum and urinary concentrations of steroids are listed in Table 1Go. High serum levels of 17-deoxysteroids (progesterone and pregnenolone) and low levels of 17-hydroxysteroids (17-hydroxyprogesterone, 17-hydroxypregnenolone, cortisol, and testosterone) were noted. Gas chromatography mass-spectometry demonstrated that the metabolites of C21,17-hydroxysteroids were low, and those of C21,17-deoxysteroids were high. The plasma concentration of aldosterone was slightly elevated, and renin activity was below 0.1 ng/mL·h. The ACTH test revealed the increased response of progesterone and low responses of 17-hydroxyprogesterone and cortisol. The elevated levels of 17-deoxysteroids decreased to normal levels as a result of dexamethasone administration. These findings confirmed the diagnosis of 17{alpha}-hydroxylase/17,20-lyase deficiency.


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  		Table 1. Patient steroid levels

 
At the age of 15 yr, the patient underwent bilateral orchidectomy. The testes with atrophic changes in histological examination were detected lying ip.


   Materials and Methods
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
Results
 Discussion
 References
 
Genetic analysis of P450c17

A genomic DNA of the patient was prepared from peripheral blood using the standard method. The DNA samples of the entire coding region of the P450c17 gene were amplified by PCR using the primers reported by Monno et al. (3). After initial denaturation (96 C for 5 min), PCR was performed for 35 cycles of denaturation (96 C for 60 s), annealing (55 C for 60 s), and extension (72 C for 2 min) in a thermal cycler (Perkin-Elmer/Cetus Instruments, Ciba, Japan). The final extension was added at 72 C for 5 min after the last cycle. Direct sequencing of the PCR products was performed using the automated fluorescence-based dideoxynucleotide termination method (model 373A ABI, Applied Biosystems, Foster City, CA).

Reverse transcription-PCR (RT-PCR) and semiquantitation of cytochrome P450c17 messenger ribonucleic acid (RNA)

Total RNA was extracted from the patient’s testes using the TRIzol reagent (Life Technology, Grand Island, NY). As a control sample, testes removed during castration for treatment of prostate carcinoma were obtained. Informed consents were obtained from the parents of the patient and the control subject. The complementary DNA synthesis reaction was performed using the Superscript preamplification system (Life Technology).

Cytochrome P450c17 complementary DNA was amplified quantitatively using the following primers located in exons 1 and 3: 5'-ACCAAGACTACAGTGATTGTCGGC-3' and 5'-AAAAAATATGGCCCCATCTAT-TCG-3'. The 5'-end of the forward primer was labeled with fluorescein (6'-FAM). PCR was performed under the above-mentioned conditions. To check whether the amplification had stopped in the exponential phase of PCR, duplicate PCR reactions of 38 and 40 cycles were performed. The latter control was used to determine whether the different fragments were amplified equivalently (13). Fluorescein-labeled PCR products were electrophoresed onto a ABI 310 Autosequencer (Applied Biosystems, Foster City, CA). Upon amplification, PCR fragments derived from P450c17 transcripts that contained exon 2 sequences were 294 bp long, whereas those that lacked exon 2 were 155 bp. Quantification of the different fragments was performed using GeneScan (Applied Biosystems) software. The degree of skipping of exon 2 was defined by calculating the ratio between the two peaks.


   Results
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
 Results
Discussion
 References
 
Genomic DNA sequencing analysis of the patient’s cytochrome P450c17 gene revealed a compound heterozygous mutation that was associated with two different mutant alleles (Fig. 1Go). In one allele, a guanine to adenine (G to A) mutation at position 51 resulted in tryptophane (TGG) being substituted by a premature stop codon (TGA) at peptide position 17 (W17X). In the other allele, a guanine to thymine (G to T) substitution at the fifth nucleotide from the splice donor site in intron 2 (436+5G->T) was discovered. DNA analysis of the family confirmed that the father and brother each had one mutant W17X, and the mother and sister each had 436+5G->T. The parents and siblings did not present with any symptoms or abnormal levels of serum steroids (data not shown).



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  		Figure 1. Nucleotide sequences of double strands of PCR products from the patient’s genomic DNA. The upper panel presents a G to A substitution corresponding to a premature termination codon at tryptophan in codon 17 within exon 1 (W17X). The lower panel presents a G to T substitution at the fifth nucleotide from the splice donor site in intron 2 (436+5G->T).

 
Based on previous report that the homozygotic W17X is a pathogenic mutation of 17{alpha}-hydroxylase/17,20-lyase (7), we investigated whether 436+5G->T in the P450c17 gene results in the skipping of exon 2 in the patient’s resected testes by RT-PCR. RT-PCR analysis revealed two different sized samples (155 and 294 bp; Fig. 2Go). The 155-bp sample was presumed to represent products resulting from skipping of exon 2, and the 294-bp sample represented normal transcription of the 436+5G->T allele and the mutant W17X. Direct sequencing of a 155-bp product confirmed the skipping of exon 2 (Fig. 3Go). These data indicate that alternative splicing by the mutation, 436+5G->T, had occurred in the patient’s testes. Skipping exon 2 results in a frame shift and a premature stop codon at codon 175.



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  		Figure 2. RT-PCR. Left panel, Electrophoresis of RT-PCR products amplified using primers located in exons 1 and 3. RT-PCR products were separated by 4% Nuseive-agarose (3:1; FMC Co., Rockland, ME) gel electrophoresis and stained by ethidium bromide. The patient (left lane) had both 155- and 294-bp fragments, whereas the normal control (right lane) had only the 294-bp fragment. The 155-bp sample was presumed to be the transcript that skipped exon 2, and the 294-bp sample was the normal transcription. Right panel, Schematic representation of transcription of mutant (436+5G->T) and wild cytochrome P450c17 genes. Numbered boxes represent exons; lines between these boxes represent introns. The arrows indicate the primers used in the RT-PCR.

 


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  		Figure 3. Nucleotide sequences of the 155-bp RT-PCR product. Direct sequencing of smaller fragments of RT-PCR products revealed a skipping of exon 2.

 
Semiquantitative P450cl7 transcript analysis demonstrated that the ratio of transcript skipping exon 2 to normal transcript is 0.925:1 (Fig. 4Go). Because the transcription of W17X resulted in a 294-bp sample, a considerable proportion of transcripts of the mutant 436+5G->T must skip exon 2.



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  		Figure 4. Semiquantitation of cytochrome P450c17 messenger RNA. Fluorescein-labeled RT-PCR products were electrophoresed with ROX mol wt markers for loading onto a ABI 310 Autosequencer. Peak heights were analyzed using GeneScan software. Small peaks represent molecular size markers.

 

   Discussion
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
 Results
 Discussion
References
 
The clinical and laboratory characteristics of the patient agree with the diagnosis of 17{alpha}-hydroxylase/17,20-lyase deficiency (1). This disorder is generally associated with a very low serum aldosterone level. However, in the present case, the serum concentration of aldosterone was slightly elevated. Several other researchers have reported similar findings. Although the reason for high aldosterone levels in these patients has not yet been clarified, all cases expect one were Japanese (1). Thus, this disorder may be linked ethnically to steroid genesis and regulation.

The patient was found to be a compound heterozygote, carrying two different inherited mutant alleles in the cytochrome P450c17 gene. One mutation, W17X, was inherited from the father, and the other mutation, 436+5G->T, was inherited from the mother. Because the W17X mutation occurs at the N-terminal side of the heme binding sequence, the putative resultant truncated protein may be nonfunctional. A previous study reported the presence of a homozygotic W17X in an unrelated Japanese patient with 17{alpha}-hydroxylase deficiency (7). On the other hand, the 436+5G->T mutation is a novel mutation located at the exon 2/intron 2 junction. The fifth nucleotide in the splice donor site of the intron is absolutely conservative and plays an important role in normal splicing (14). The most common effect of an alternation at this position is the complete skipping of the preceding exon. In the present case, the results of RT-PCR and semiquantitative analysis indicate that most transcriptions of the 436+5G->T mutation spliced out of exon 2. Skipping exon 2 results in a frame shift and a premature stop codon at codon 175. These findings indicate that the compound heterozygous mutations (W17X and 436+5G->T) are responsible for the pathogenesis of complete 17{alpha}-hydroxylase/17,20-lyase deficiency.


   Acknowledgments
 
The authors thank Dr. T Yanase (Third Department of Internal Medicine, Kyushu University) for his helpful advice, as well as Dr. K. Honma (Department of Laboratory Medicine, Keio University School of Medicine) for the examination of urinary steroids.


   Footnotes
 
1 These authors contributed equally to this work. Back

Received July 9, 1997.

Revised September 15, 1997.

Accepted October 3, 1997.


   References
Top
 Abstract
 Introduction
 Case Report
 Materials and Methods
 Results
 Discussion
 References
 

  1. Yanase T, Simpson ER, Waterman MR. 1991 17{alpha}-Hydroxylase/17,20-lyase deficiency from clinical investigation to molecular definition. Endocr Rev. 12:91–107.[Abstract/Free Full Text]
  2. Yanase T. 1995 17{alpha}-Hydroxylase/17,20-lyase defects. J Steroid Biochem Mol Biol. 53:153–157.[CrossRef][Medline]
  3. Monno S, Ogawa H, Date T, Fujioka M, Miller WL, Kobayashi M. 1993 Mutation of histidine 373 to leucine in cytochrome P450c17 causes 17{alpha}-hydroxylase deficiency. J Biol Chem. 268:25811–25817.[Abstract/Free Full Text]
  4. Fardella CE, Zhang LH, Mahachoklertwattana P, Lin D, Miller WL. 1993 Deletion of amino acids Asp487-Ser488-Phe489 in human cytochrome P450c17 causes severe 17{alpha}-hydroxylase deficiency. J Clin Endocrinol Metab. 77:489–493.[Abstract]
  5. Ahlgren R, Yanase T, Simpson ER, Winter JSD, Waterman MR. 1992 Compound heterozygous mutations (Arg239->Stop, Pro342->Thr) in the CYP17(P450 17{alpha}) gene lead to ambiguous external genitalia in a male patient with partial combined 17 {alpha}-hydroxylase/17,20-lyase deficiency. J Clin Endocrinol Metab. 74:667–672.[Abstract]
  6. Fardella CE, Hum DW, Homoki J, Miller WL. 1994 Point mutation of Arg440 to His in cytochrome P450c17 causes severe 17{alpha}-hydroxylase deficiency. J Clin Endocrinol Metab. 79:160–164.[Abstract]
  7. Yanase T, Kagimoto M, Matsui N, Simpson ER, Waterman MR. 1988 Combined 17{alpha}-hydroxylase/17,20-lyase deficiency due to a stop codon in the N-terminal region of 17{alpha}-hydroxylase cytochrome P-450. Mol Cell Endocrinol. 59:249–253.[CrossRef][Medline]
  8. Laflamme N, Leblanc JF, Mailloux J, Faure N, Labrie F, Simard J. 1996 Mutation R96W in cytochrome P450c17 gene causes combined 17{alpha}-hydroxylase/17,20-lyase deficiency in two french canadian patients. J Clin Endocrinol Metab. 81:264–268.[Abstract]
  9. Lin D, Harikrishna JA, Moore CCD, Jones KL, Miller WL. 1991 Missense mutation serine106->proline causes 17{alpha}-hydroxylase deficiency. J Biol Chem. 266:15992–15998.[Abstract/Free Full Text]
  10. Miura K, Yasuda K, Yanase T, et al. 1996 Mutation of cytochrome P450c17 gene (CYP17) in a Japanese patient previously reported as having glucocorticoid-responsive hyperaldosteronism: with a review of Japanese patients with mutations of CYP17. J Clin Endocrinol Metab. 81:3797–3801.[Abstract]
  11. Oshiro C, Takasu N, Wakugami T, et al. 1995 Seventeen alpha-hydroxlase deficiency with one base pair deletion of the cytochrome P450c17 (CYP17) gene. J Clin Endocrinol Metab. 80:2526–2529.[Abstract]
  12. Yamaguchi H, Nakazato M, Miyazato M, Kangawa K, Matsukura S. 1997 A 5'-splice site mutation in the cytochrome P450 steroid 17{alpha}-hydroxylase gene in 17{alpha}-hydroxylase deficiency. J Clin Endocrinol Metab. 82:1934–1938.[Abstract/Free Full Text]
  13. Teng H, Jorissen M, Van Poppel H, et al. 1997 Increased proportion of exon 9 alternatively spliced CFTR transcripts in vas defens compared with nasal epithelial cells. Hum Mol Genet. 6:85–90.[Abstract/Free Full Text]
  14. Zielenski J, Markiewicz D, Lin SP, Huang FY, Yang-Feng TL, Tsui LC. 1995 Skipping of exon 12 as a consequence of a point mutation (1898+5G->T) in the cystic fibrosis transmembrane conductance regulator gene found in a consanguineous Chinese family. Clin Genet. 47:125–132.[Medline]



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