- Email: [email protected]
Identification of a novel mutation in CYP17A1 gene
Identification of a novel mutation in CYP17A1 gene LI-QIONG XUE, BING HAN, LI-BO CHEN, CHUN-MING PAN, HUI ZHU, BING-LI LIU, WEI LIU, WAN-LING WU, MING-DAO CHEN, YING-LI LU, JIE QIAO, and HUAI-DONG SONG SHANGHAI AND SHENZHEN, CHINA
17a-hydroxylase/17,20-lyase deficiency (17OHD) is a rare autosomal recessive genetic disease that is characterized by low-renin hypertension, hypokalemia, and abnormal development of the genitalia. Mutations in the CYP17A1 gene account for this disease. We aim to investigate the CYP17A1 mutation and analyze its possible influence on phenotype in a Chinese patient with 17OHD. Steroid hormones were assayed. The 8 exons of the CYP17A1 gene were amplified and directly sequenced. Wild-type and mutant CYP17A1 cDNA were cloned into pcDNA3.1 expression vectors and transfected into 293T cells. Finally, 17-hydroxylase and 17,20-lyase activity were detected by using progesterone and 17-hydroxypregnenolone as the substrates. A novel missense mutation c.716 G.A located in exon 4 that changed the amino acid from arginine to glutamine (R239Q) was discovered in the patient. Steric model analysis of CYP17A1 showed that R239Q changed the local structure and the electrostatic potential. Functional study indicated that the R239Q mutant caused the complete loss of both 17a-hydroxylase and 17,20-lyase activities. Our study expanded the CYP17A1 mutation spectrum. With a functional study, we confirmed that the novel mutation caused the complete loss of both 17a-hydroxylase and 17,20-lyase activities. (Translational Research 2013;161:44–49) Abbreviations: ACTH ¼ adrenocorticotropic hormone; CAH ¼ congenital adrenal hyperplasia; DHEA ¼ dehydroepiandrosterone; P ¼ progesterone; PCR ¼ polymerase chain reaction; RIA ¼ radioimmunoassay; 17OHD ¼ 17a-hydroxylase/17,20-lyase deficiency; 17OHP ¼ 17hydroxyprogesterone; 17OHPreg ¼ 17-hydroxypregnenolone
17a-hydroxylase/17,20-lyase deficiency (17OHD) is a rare type of congenital adrenal hyperplasia (CAH) that accounts for only 1% of all CAH cases.1 The human microsomal enzyme P450c17 (17a-hydroxylase/17,20-
lyase) is one of the key enzymes in adrenal steroid hormone synthesis. This enzyme possesses the activity of both hydroxylase and lyase, with the former catalyzing pregnenolone or progesterone (P) into the cortisol
From the State Key Laboratory of Medical Genomics, Molecular Medical Centre, Shanghai Institute of Endocrinology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Department of Endocrinology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Department of Endocrinology, Shenzhen Sixth People’s Hospital, Shenzhen, China.
Submitted for publication July 6, 2012; revision submitted August 10, 2012; accepted for publication August 30, 2012.
This work was supported by a grant from the National Natural Science Foundation Program (81070666), People’s Republic of China. All authors disclosed no potential conflicts of interest. The authors have read the Journal’s policy on conflicts of interest and have none to declare. L.-Q. X. and B. H. contributed equally to this work.
44
Reprint requests: Jie Qiao, Department of Endocrinology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Rd, Shanghai, China; e-mail: qiaoj2001@ 126.com and Huai-Dong Song, State Key Laboratory of Medical Genomics, Molecular Medical Centre, Shanghai Institute of Endocrinology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2013 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2012.08.007
Translational Research Volume 161, Number 1
Xue et al
45
PATIENTS AND METHODS
AT A GLANCE COMMENTARY Xue L-Q, et al. Background
17OHD is a rare type of CAH that accounts for only 1% of all CAH cases. Mutations in the CYP17A1 gene account for this disease. We investigated the CYP17A1 mutation and analyzed its possible influence on phenotype in a Chinese patient with 17OHD. Translational Significance
Our work expanded the CYP17A1 mutation spectrum. With the functional study, we ascertained the correlation between genotype and clinical phenotype of a patient with 17OHD.
precursors 17-hydroxypregnenolone (17OHPreg) or 17hydroxyprogesterone (17OHP) and the latter cleaving the 17 and 20 carbon chains to produce estrogen and adrenal androgen precursors, mainly dehydroepiandrosterone (DHEA). The P450c17 enzyme is encoded by the CYP17A1 gene, which consists of 8 exons2 and maps to chromosome 10q24.3.3 This gene is primarily expressed in the adrenal glands and gonads.4 When the hydroxylation and cleavage functions are impaired, steroid hormone synthesis in the zona fasciculata and adrenal reticularis can be affected. In addition, the marked decrease of cortisol reactivity increased the secretion of adrenocorticotropic hormone (ACTH) and caused bilateral adrenal hyperplasia. However, the substrate of the enzyme accumulates, resulting in low-renin hypertension and hypokalemia. The deficiency of gonadal hormones, such as estrogen and testosterone, induces primary amenorrhea and hypogonadism in female subjects and pseudohermaphroditism in male subjects. Moreover, sexual infantilism occurs in both genetic sexes. At present, more than 80 different mutations have been reported since Biglieri et al5 reported the first patient in 1966. These mutations, including missense, deletion, insertion, and splice site mutations, in the CYP17A1 gene cause combined or isolated 17OHD.6,7 In this study, we discovered a compound heterozygous mutation including a previously reported c.985_987 del/ins AA in exon 6 and a novel missense mutation R239Q in exon 4 of the CYP17A1 gene in a patient with 17OHD. A functional experiment also was performed to investigate the mechanism of the disease-causing effect of this novel mutation.
Subjects. A 30-year-old woman presented to the hospital with pain in the abdomen. Approximately 6 years previously, she underwent breast augmentation surgery for breast dysplasia. She presented with primary amenorrhea, and when she was aged 18 years, she underwent estrogen and progestin sequential therapy. However, after 2 years on the treatment, she experienced menstruation and withdrew herself from the drug. Her karyotype was 46,XX. A physical examination showed a height of 168 cm, blood pressure of 190/125 mm Hg, and infantile female external genitalia with the absence of both pubic and axillary hair. She exhibited a decreased level of serum potassium (2.4 mmol/L, 3.5– 5.1 mmol/L). The patient also exhibited an increased level of P and decreased levels of E2 and DHEA. Plasma hormone analysis revealed that the levels of follicle-stimulating hormone, T, luteinizing hormone, and prolactin were within the normal range (Table I). Her supine aldosterone and renin were 406.4 pg/mL (29.4–161.5 pg/mL) and 0.46 ng/mL/h (0.5–1.9 ng/mL/ h), respectively, whereas her orthostatic aldosterone and renin were 407.36 pg/mL (38.1–313.3 pg/mL) and 0.74 ng/mL/h (1.9–6 ng/mL/h), respectively. Her 24-hour urinary 17-hydroxycorticosteroid excretion was 3.4 mg (2–8 mg), and her 24-hour urinary 17-ketocorticosteroid level was 11.8 mg (6–14 mg). The measurement of the basal and ACTH-stimulated steroids revealed slightly elevated levels of cortisol and 17OHP with a poor response to ACTH stimulation (Table I). The adrenal computed tomography scan indicated multibilateral adrenal nodules, and the ultrasonograph revealed a small uterus. The patient was diagnosed with CAH and 17OHD. After the administration of dexamethasone (0.4 mg every night) for 10 days, her P and cortisol (3.9 nmol/L) levels decreased significantly. Her blood pressure (130/80 mm Hg) was within the normal range. Hormone assays. Cortisol, T, DHEA, 17OHP, P, and ACTH concentrations were measured as previously described.8 A commercial radioimmunoassay (RIA) kit (DiaSorin Ltd, Wokingham, UK) was used to measure the cortisol concentration. T, DHEA, 17OHP, and ACTH concentrations were measured using an antibody-coated tube RIA kit (DSL-8600; Diagnostic Systems Laboratories Inc, Webster, TX). The serum P level was measured with the automated Abbott Architect I2000SR System (Abbott Laboratories, Abbott Park, Ill). DNA extraction and mutation analysis. Genomic DNA of the patient and her parents was extracted from peripheral blood leukocytes using a FUJIFILM QuickGene-610L system (Fujifilm Life Science,
46
Translational Research January 2013
Xue et al
Table I. Hormone test results of patient ACTH stimulation
Cortisol (64–536 nmol/L) 17OHP (,3.2 ng/mL)
P (0.08–0.78 ng/mL) E2 (2.59–88.1 ng/L) DHEA (2.66–8.55 mmol/L) FSH (16.74–113.59 mIU/L) LH (10.87–58.64 mIU/L) PRL (3.34–26.72 ng/mL) T (0–0.75 ng/mL)
Basal
30’
60’
71.9 2.77
79.7 1.23
110.9 1.0
Basal
10 d after Dx treatment
31.99 2 0.3 19.3 14.7 10.61 0.09
9.21 12 – 27.87 15.7 4.37 0
Abbreviations: ACTH, adrenocorticotropic hormone; DHEA, dehydroepiandrosterone; Dx, dexamethasone; E2, Estradiol; FSH, follicle-stimulating hormone; LH, luteinizing hormone; P, progesterone; PRL, prolactin; 17OHP, 17a-hydroxylase/17,20-lyase deficiency; T, testosterone.
Tokyo, Japan). All 8 exons and exon-intron boundaries of the CYP17A1 gene were amplified by polymerase chain reaction (PCR) using primers and conditions as previously described.9 The PCR products were purified using a gel extraction kit (QIAGEN, Mississauga, Ontario, Canada) and sequenced in both directions on an ABI 3700 DNA sequencer (Applied Biosystems PerkinElmer, Foster City, Calif). Once a mutation was identified, the PCR fragments amplified from the genomic DNA of 100 normal subjects also were analyzed to exclude polymorphisms. In addition, the exons of the CYP17A1 genes of her parents were amplified and sequenced to clarify the mutation origin. This study was approved by the ethics committee of the Shanghai Ninth People’s Hospital, and informed consent was obtained from each individuals. Molecular modeling of CYP17A1. To further investigate the influence of the R239Q mutation on the enzyme structure, a 3-dimensional computer model was constructed with the NOC program (version 3.0; http://noch.sourceforge.net). CYP17A1 (residues 224–256) was modeled with the SWISS-MODEL software (http://swissmodel.expasy.org/) using the crystal structure of human cytochrome P450 1A2 (PDB accession code 2HI4, chain A) as a template.10 Functional study. The CYP17A1 cDNA was cloned and inserted into a pCDNA3.1 vector (Invitrogen, San Diego, Calif) between the EcoR1 and BamH1 sites. pCDNA3.1-CYP17A1-Y329FS was prepared as previously described. Site-directed mutagenesis was used to create the mutant expression vector of R239Q with the following primers: F, 50 - TTTCTTGAATCC
AGCGGGGACCCAGC-30 and R, 50 -CGCTGGATTCA AGAAACGCTCTGCAG-30 . Both the wild-type and mutant vectors were confirmed by direct sequencing and purified with an anion exchange column. (QIAGEN, Hilden, Germany). HEK-293T cells were seeded in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum at 37 C in a humidified 5% CO2 incubator. Cells were seeded onto 24-well plates to 60% confluence and transfected with 0.8 mg plasmid DNA per well using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. Forty hours after transfection, cells were incubated with incremental concentrations (0.1, 0.2, 1.0, 2.0, 10, and 20 mmol/L) of P and 17OHPreg (Sigma, St Louis, Mo) for another 6 hours. The medium was collected and kept frozen at 220 C for the detection of 17OHP (17ahydroxylase activity) and DHEA (17,20-lyase activity) by RIA kits (Diagnostics Systems Laboratories, Webster, TX) and enzyme-linked immunosorbent assay (IBL, Hamburg, Germany), respectively. All values are expressed as mean 6 standard deviation and represent the results of 3 independent experiments in duplicate. Statistical analysis. The statistical significance of differences between groups was determined by the 2tailed t test. P , 0.05 was considered significant.
RESULTS
The patient was found to be a compound heterozygote carrying 2 different mutant alleles of the CYP17A1 gene. The first mutation was a novel missense mutation, c.716 G.A, located in exon 4, which resulted in an amino acid substitution from arginine to glutamine, that is, R239Q (Fig 1). The second mutation was a previously identified, widely reported deletioninsertion mutation (c.985_987 delinsAA) in exon 6 in Chinese patients.11-13 Analysis of the genotype of her parents revealed that the novel R239Q mutation was inherited from her father, and that the other mutation was inherited from her mother; they were both heterozygous carriers. The protein sequence alignment showed that the Arg 239 residue was conserved in CYP21A2 and CYP17A1 (Fig 2). We also investigated the computerized CYP17A1 model based on the crystallized structure of cytochrome P450 1A2 (3rukD) to explain the putative effects of the mutation in CYP17A1. A charged basic amino acid (arginine) was replaced by an amino acid with an uncharged polar side chain (glutamine) at codon 239 (Fig S1), which caused an abnormal electrostatic charge distribution in the G helix (Fig 3).
Translational Research Volume 161, Number 1
Xue et al
47
Fig 1. Sequencing result of the patient. The patient harbors a novel missense mutation, c.716 G.A, which resulted in an amino acid substitution from arginine to glutamine, (R239Q) (A) and a previously reported del/ins mutation (c.985_987del/insAA) (B).
To determine the enzyme activities of the wild-type and mutant CYP17A1, we used site-directed mutagenesis to generate expression vectors containing the wild-type and mutant CYP17A1, which were then transfected into 293T cells. CYP17A1-WT had full enzyme activity, which efficiently catalyzed P to 17OHP and 17OHPreg to DHEA. However, even at a substrate concentration of 20 mmol/L, metabolites were barely detected in the 293T cells transfected with the empty vector or Y329FS. Furthermore, CYP17A1-R239Q showed an activity level similar to those of the empty vector and Y329FS (Fig 4A and B). DISCUSSION
Cytochrome P450c17 (steroid 17a-hydroxylase/17, 20-lyase) plays a key role in the adrenal steroid hormone synthesis process. 17OHD is a rare autosomal recessive disorder that accounts for approximately 1% of all cases of CAH. The estimated incidence is approximately 1 in 50,000 newborns.14 This disease is characterized by the impaired production of cortisol and the secondary hypersecretion of ACTH, which in turn stimulates the adrenal glands to synthesize large amounts of 11-deoxycorticosterone and corticosterone. High concentrations of 11-deoxycorticosterone result in hypertension, hypokalemia, and a suppressed reninangiotensin system.15 Gonadal 17, 20-lyase deficiency prohibits the synthesis of DHEA, T, and estrogen, resulting in a 46,XY disorder of sex development in male subjects. In the present study, we have identified a novel missense mutation in the CYP17A1 gene and elucidated its molecular mechanism. The patient presented significant hypertension, hypokalemia, and lower gonadal hormone levels caused by severe 17a-hydroxylase deficiency, which was significantly relieved after
Fig 2. Sequence alignment of Arg 239 in different P450 enzymes. Amino acid sequences around Arg 239 of human CYP17A1 are aligned with other related human P450s. The amino acid in the red rectangle represents the corresponding amino acid.
dexamethasone treatment (0.4 mg/d). These symptoms and the response to medication are consistent with the absent CYP17A1 enzyme activity in vitro. The novel mutation (R239Q) substituted a basic arginine residue with a neutral glutamine residue. Combined with the clinical and laboratory findings for our patient, we presumed that this locus was essential for enzyme activity. Furthermore, the structural analysis showed that this mutation lies within the G helix of the CYP17A1 protein.16 Molecular modeling demonstrated that this mutation influenced the local structure and the charge distribution. Finally, functional experiments in vitro provided strong evidence for the pathologic effect of this novel mutation, which resulted in the complete loss of the activities of both 17a-hydroxylase and 17,20-lyase. Until now, only 5 different mutations in exon 4 of the CYP17A1 gene have been reported worldwide.17-21 Among them, Rumsby et al20 and Ahlgren et al21 reported a missense mutation at the 239 position that resulted in the truncation of the CYP17A1 protein, which caused the ambiguous external genitalia of the patients. However, no other mutation in the R239 site was reported in patients of Asian origin. Therefore, we have also reported a novel mutation site in Asian countries,
48
Translational Research January 2013
Xue et al
Fig 3. Local steric model of wild-type and mutant CYP17A1. Spacefilling model of CYP17A1-WT and CYP17A1-R239Q is colored by electrostatic potential. Blue represents positive charge, and red represents negative charge. In the CYP17A1-R239Q, local solid surface and electrostatic potential were greatly changed. A and C, Wildtype. B and D, Mutant.
and the investigation of the function of this mutation was important.
Fig 4. In vitro functional analysis of wild-type and mutant CYP17A1. A, The production of 17-OHP in the presence of various concentrations of P (0.1, 0.2, 1.0, 2.0, 10, and 20 mmol/L) is used to measure 17-hydroxylase activity in 293T cells transfected with CYP17A1-WT, CYP17A1-R239Q, CYP17A1-Y329FS, and empty vector. B, The production of DHEA in the presence of various concentrations of 17-OHPreg (0.1, 0.2, 1.0, 2.0, 10, and 20 mmol/L) is shown as a measure of 17,20-lyase activity in 293T cells transfected with CYP17A1-WT, CYP17A1-R239Q, CYP17A1Y329FS, and empty vector. The experiments are performed at least 3 times, and data are shown as the means 6 standard deviation. DHEA, dehydroepiandrosterone; 17OHP, 17a-hydroxylase/17,20lyase deficiency; WT, wild-type.
CONCLUSIONS
We have identified a novel missense mutation (R239Q) in the CYP17A1 gene of a patient with a 46,XX disorder of sex development. Our study expanded the CYP17A1 mutation spectrum. With a functional study, we confirmed that the novel mutation caused the complete loss of both 17a-hydroxylase and 17,20-lyase activities.
Supplementary Data
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10. 1016/j.trsl.2012.08.007.
REFERENCES
1. Krone N, Dhir V, Ivison HE, Arlt W. Congenital adrenal hyperplasia and P450 oxidoreductase deficiency. Clin Endocrinol (Oxf) 2007;66:162–72. 2. Picado-Leonard J, Miller WL. Cloning and sequence of the human gene encoding P450c17 (steroid 17_-hydroxylase/17,20 lyase): similarity to the gene for P450c21. DNA 1987;6: 439–48. 3. Sparkes RS, Klisak I, Miller WL. Regional mapping of genes encoding human steroidogenic enzymes: P450scc to 15q23q24, adrenodoxin to 11q22; adrenodoxin reductase to 17q24q25; and P450c17 to 10q24-q25. DNA Cell Biol 1991;10: 359–65. 4. Chung BC, Picado-Leonard J, Haniu M, et al. Cytochrome P450c17 (steroid 17_-hydroxylase/17,20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is
Translational Research Volume 161, Number 1
5.
6.
7.
8.
9.
10.
11.
12.
13.
expressed in both tissues. Proc Natl Acad Sci U S A 1987;84: 407–11. Bao X, Ding H, Xu Y, et al. Prevalence of common mutations in the CYP17A1 gene in Chinese Han population. Clin Chim Acta 2011;412:1240–3. Auchus RJ. The genetics, pathophysiology, and management of human deficiencies of P450c17. Endocrinol Metab Clin North Am 2001;30:101–19. Miller WL, Auchus RJ. The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocr Rev 2011;32:81–151. Qiao J, Han B, Liu BL, et al. A unique exonic splicing mutation in the CYP17A1 gene as the cause for steroid 17{alpha}-hydroxylase deficiency. Eur J Endocrinol 2011;164:627–33. Qiao J, Hu RM, Peng YD, et al. A complex heterozygous mutation of His373Leu and Asp487-Ser488-Phe489 deletion in human cytochrome P450c17 causes 17alpha-hydroxylase/17,20-lyase deficiency in three Chinese sisters. Mol Cell Endocrinol 2003;201: 189–95. Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL Workspace: a web-based environment for protein structure homology modelling. Bioinformatics 2006;22:195–201. Wei JQ, Wei JL, Li WC, Bi YS, Wei FC. Genotyping of five Chinese patients with 17alpha-hydroxylase deficiency diagnosed through high-performance liquid chromatography serum adrenal profile: identification of two novel CYP17 mutations. J Clin Endocrinol Metab 2006;91:3647–53. Qiao J, Chen X, Zuo CL, et al. Identification of steroid biosynthetic defects in genotype-proven heterozygous individuals for 17alpha-hydroxylase /17,20-lyase deficiency. Clin Endocrinol (Oxf) 2010;72:312–9. Liu BL, Qiao J, Chen X, et al. Clinical and molecular genetic analysis for 7 patients from 5 pedigrees with 17a-hydroxylase/17, 20
Xue et al
14.
15. 16.
17.
18.
19.
20.
21.
49
lyase deficiency. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2009;26: 282–7. Grumbach MM, Hughes IA, Conte FA. Disorders of sex differentiation. In: Larsen PK, Kronenberg HM, Melmed S, Polonsky KS, eds. Williams Textbook of Endocrinology. 10th ed. Philadelphia, PA: Saunders, 2003:842–1002. Biglieri EG, Herron MA, Brust N. 17-Hydroxylation deficiency in man. J Clin Invest 1966;45:1946–54. Auchus RJ, Miller WL. Molecular modeling of human P450c17 (17alpha-hydroxylase/17,20-lyase): insights into reaction mechanisms and effects of mutations. Mol Endocrinol 1999;13: 1169–82. Takeda Y, Yoneda T, Demura M, et al. Genetic analysis of the cytochrome P-450c17alpha (CYP17) and aldosterone synthase (CYP11B2) in Japanese patients with 17alpha-hydroxylase deficiency. Clin Endocrinol (Oxf) 2001;54:751–8. Katsumata N, Satoh M, Mikami A, et al. New compound heterozygous mutation in the CYP17 gene in a 46, XY girl with 17 alphahydroxylase/17,20-lyase deficiency. Horm Res 2001;55:141–6. Tao H, Zhang B, Lu ZL, Pei Y, Mi SH. New compound heterozygous mutation causes partial combined 17 alpha-hydroxylase/ 17,20-lyase deficiency. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2007;24:19–22. Rumsby G, Skinner C, Lee HA, Honour JW. Combined 17 alphahydroxylase/17,20-lyase deficiency caused by heterozygous stop codons in the cytochrome P450 17 alpha-hydroxylase gene. Clin Endocrinol (Oxf) 1993;39:483–5. Ahlgren R, Yanase T, Simpson ER, Winter JS, Waterman MR. Compound heterozygous mutations (Arg 239——stop, Pro 342——Thr) in the CYP17 (P45017 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 1992;74:667–72.
17a-hydroxylase/17,20-lyase deficiency (17OHD) is a rare autosomal recessive genetic disease that is characterized by low-renin hypertension, hypokalemia, and abnormal development of the genitalia. Mutations in the CYP17A1 gene account for this disease. We aim to investigate the CYP17A1 mutation and analyze its possible influence on phenotype in a Chinese patient with 17OHD. Steroid hormones were assayed. The 8 exons of the CYP17A1 gene were amplified and directly sequenced. Wild-type and mutant CYP17A1 cDNA were cloned into pcDNA3.1 expression vectors and transfected into 293T cells. Finally, 17-hydroxylase and 17,20-lyase activity were detected by using progesterone and 17-hydroxypregnenolone as the substrates. A novel missense mutation c.716 G.A located in exon 4 that changed the amino acid from arginine to glutamine (R239Q) was discovered in the patient. Steric model analysis of CYP17A1 showed that R239Q changed the local structure and the electrostatic potential. Functional study indicated that the R239Q mutant caused the complete loss of both 17a-hydroxylase and 17,20-lyase activities. Our study expanded the CYP17A1 mutation spectrum. With a functional study, we confirmed that the novel mutation caused the complete loss of both 17a-hydroxylase and 17,20-lyase activities. (Translational Research 2013;161:44–49) Abbreviations: ACTH ¼ adrenocorticotropic hormone; CAH ¼ congenital adrenal hyperplasia; DHEA ¼ dehydroepiandrosterone; P ¼ progesterone; PCR ¼ polymerase chain reaction; RIA ¼ radioimmunoassay; 17OHD ¼ 17a-hydroxylase/17,20-lyase deficiency; 17OHP ¼ 17hydroxyprogesterone; 17OHPreg ¼ 17-hydroxypregnenolone
17a-hydroxylase/17,20-lyase deficiency (17OHD) is a rare type of congenital adrenal hyperplasia (CAH) that accounts for only 1% of all CAH cases.1 The human microsomal enzyme P450c17 (17a-hydroxylase/17,20-
lyase) is one of the key enzymes in adrenal steroid hormone synthesis. This enzyme possesses the activity of both hydroxylase and lyase, with the former catalyzing pregnenolone or progesterone (P) into the cortisol
From the State Key Laboratory of Medical Genomics, Molecular Medical Centre, Shanghai Institute of Endocrinology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Department of Endocrinology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Department of Endocrinology, Shenzhen Sixth People’s Hospital, Shenzhen, China.
Submitted for publication July 6, 2012; revision submitted August 10, 2012; accepted for publication August 30, 2012.
This work was supported by a grant from the National Natural Science Foundation Program (81070666), People’s Republic of China. All authors disclosed no potential conflicts of interest. The authors have read the Journal’s policy on conflicts of interest and have none to declare. L.-Q. X. and B. H. contributed equally to this work.
44
Reprint requests: Jie Qiao, Department of Endocrinology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Rd, Shanghai, China; e-mail: qiaoj2001@ 126.com and Huai-Dong Song, State Key Laboratory of Medical Genomics, Molecular Medical Centre, Shanghai Institute of Endocrinology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2013 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2012.08.007
Translational Research Volume 161, Number 1
Xue et al
45
PATIENTS AND METHODS
AT A GLANCE COMMENTARY Xue L-Q, et al. Background
17OHD is a rare type of CAH that accounts for only 1% of all CAH cases. Mutations in the CYP17A1 gene account for this disease. We investigated the CYP17A1 mutation and analyzed its possible influence on phenotype in a Chinese patient with 17OHD. Translational Significance
Our work expanded the CYP17A1 mutation spectrum. With the functional study, we ascertained the correlation between genotype and clinical phenotype of a patient with 17OHD.
precursors 17-hydroxypregnenolone (17OHPreg) or 17hydroxyprogesterone (17OHP) and the latter cleaving the 17 and 20 carbon chains to produce estrogen and adrenal androgen precursors, mainly dehydroepiandrosterone (DHEA). The P450c17 enzyme is encoded by the CYP17A1 gene, which consists of 8 exons2 and maps to chromosome 10q24.3.3 This gene is primarily expressed in the adrenal glands and gonads.4 When the hydroxylation and cleavage functions are impaired, steroid hormone synthesis in the zona fasciculata and adrenal reticularis can be affected. In addition, the marked decrease of cortisol reactivity increased the secretion of adrenocorticotropic hormone (ACTH) and caused bilateral adrenal hyperplasia. However, the substrate of the enzyme accumulates, resulting in low-renin hypertension and hypokalemia. The deficiency of gonadal hormones, such as estrogen and testosterone, induces primary amenorrhea and hypogonadism in female subjects and pseudohermaphroditism in male subjects. Moreover, sexual infantilism occurs in both genetic sexes. At present, more than 80 different mutations have been reported since Biglieri et al5 reported the first patient in 1966. These mutations, including missense, deletion, insertion, and splice site mutations, in the CYP17A1 gene cause combined or isolated 17OHD.6,7 In this study, we discovered a compound heterozygous mutation including a previously reported c.985_987 del/ins AA in exon 6 and a novel missense mutation R239Q in exon 4 of the CYP17A1 gene in a patient with 17OHD. A functional experiment also was performed to investigate the mechanism of the disease-causing effect of this novel mutation.
Subjects. A 30-year-old woman presented to the hospital with pain in the abdomen. Approximately 6 years previously, she underwent breast augmentation surgery for breast dysplasia. She presented with primary amenorrhea, and when she was aged 18 years, she underwent estrogen and progestin sequential therapy. However, after 2 years on the treatment, she experienced menstruation and withdrew herself from the drug. Her karyotype was 46,XX. A physical examination showed a height of 168 cm, blood pressure of 190/125 mm Hg, and infantile female external genitalia with the absence of both pubic and axillary hair. She exhibited a decreased level of serum potassium (2.4 mmol/L, 3.5– 5.1 mmol/L). The patient also exhibited an increased level of P and decreased levels of E2 and DHEA. Plasma hormone analysis revealed that the levels of follicle-stimulating hormone, T, luteinizing hormone, and prolactin were within the normal range (Table I). Her supine aldosterone and renin were 406.4 pg/mL (29.4–161.5 pg/mL) and 0.46 ng/mL/h (0.5–1.9 ng/mL/ h), respectively, whereas her orthostatic aldosterone and renin were 407.36 pg/mL (38.1–313.3 pg/mL) and 0.74 ng/mL/h (1.9–6 ng/mL/h), respectively. Her 24-hour urinary 17-hydroxycorticosteroid excretion was 3.4 mg (2–8 mg), and her 24-hour urinary 17-ketocorticosteroid level was 11.8 mg (6–14 mg). The measurement of the basal and ACTH-stimulated steroids revealed slightly elevated levels of cortisol and 17OHP with a poor response to ACTH stimulation (Table I). The adrenal computed tomography scan indicated multibilateral adrenal nodules, and the ultrasonograph revealed a small uterus. The patient was diagnosed with CAH and 17OHD. After the administration of dexamethasone (0.4 mg every night) for 10 days, her P and cortisol (3.9 nmol/L) levels decreased significantly. Her blood pressure (130/80 mm Hg) was within the normal range. Hormone assays. Cortisol, T, DHEA, 17OHP, P, and ACTH concentrations were measured as previously described.8 A commercial radioimmunoassay (RIA) kit (DiaSorin Ltd, Wokingham, UK) was used to measure the cortisol concentration. T, DHEA, 17OHP, and ACTH concentrations were measured using an antibody-coated tube RIA kit (DSL-8600; Diagnostic Systems Laboratories Inc, Webster, TX). The serum P level was measured with the automated Abbott Architect I2000SR System (Abbott Laboratories, Abbott Park, Ill). DNA extraction and mutation analysis. Genomic DNA of the patient and her parents was extracted from peripheral blood leukocytes using a FUJIFILM QuickGene-610L system (Fujifilm Life Science,
46
Translational Research January 2013
Xue et al
Table I. Hormone test results of patient ACTH stimulation
Cortisol (64–536 nmol/L) 17OHP (,3.2 ng/mL)
P (0.08–0.78 ng/mL) E2 (2.59–88.1 ng/L) DHEA (2.66–8.55 mmol/L) FSH (16.74–113.59 mIU/L) LH (10.87–58.64 mIU/L) PRL (3.34–26.72 ng/mL) T (0–0.75 ng/mL)
Basal
30’
60’
71.9 2.77
79.7 1.23
110.9 1.0
Basal
10 d after Dx treatment
31.99 2 0.3 19.3 14.7 10.61 0.09
9.21 12 – 27.87 15.7 4.37 0
Abbreviations: ACTH, adrenocorticotropic hormone; DHEA, dehydroepiandrosterone; Dx, dexamethasone; E2, Estradiol; FSH, follicle-stimulating hormone; LH, luteinizing hormone; P, progesterone; PRL, prolactin; 17OHP, 17a-hydroxylase/17,20-lyase deficiency; T, testosterone.
Tokyo, Japan). All 8 exons and exon-intron boundaries of the CYP17A1 gene were amplified by polymerase chain reaction (PCR) using primers and conditions as previously described.9 The PCR products were purified using a gel extraction kit (QIAGEN, Mississauga, Ontario, Canada) and sequenced in both directions on an ABI 3700 DNA sequencer (Applied Biosystems PerkinElmer, Foster City, Calif). Once a mutation was identified, the PCR fragments amplified from the genomic DNA of 100 normal subjects also were analyzed to exclude polymorphisms. In addition, the exons of the CYP17A1 genes of her parents were amplified and sequenced to clarify the mutation origin. This study was approved by the ethics committee of the Shanghai Ninth People’s Hospital, and informed consent was obtained from each individuals. Molecular modeling of CYP17A1. To further investigate the influence of the R239Q mutation on the enzyme structure, a 3-dimensional computer model was constructed with the NOC program (version 3.0; http://noch.sourceforge.net). CYP17A1 (residues 224–256) was modeled with the SWISS-MODEL software (http://swissmodel.expasy.org/) using the crystal structure of human cytochrome P450 1A2 (PDB accession code 2HI4, chain A) as a template.10 Functional study. The CYP17A1 cDNA was cloned and inserted into a pCDNA3.1 vector (Invitrogen, San Diego, Calif) between the EcoR1 and BamH1 sites. pCDNA3.1-CYP17A1-Y329FS was prepared as previously described. Site-directed mutagenesis was used to create the mutant expression vector of R239Q with the following primers: F, 50 - TTTCTTGAATCC
AGCGGGGACCCAGC-30 and R, 50 -CGCTGGATTCA AGAAACGCTCTGCAG-30 . Both the wild-type and mutant vectors were confirmed by direct sequencing and purified with an anion exchange column. (QIAGEN, Hilden, Germany). HEK-293T cells were seeded in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum at 37 C in a humidified 5% CO2 incubator. Cells were seeded onto 24-well plates to 60% confluence and transfected with 0.8 mg plasmid DNA per well using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. Forty hours after transfection, cells were incubated with incremental concentrations (0.1, 0.2, 1.0, 2.0, 10, and 20 mmol/L) of P and 17OHPreg (Sigma, St Louis, Mo) for another 6 hours. The medium was collected and kept frozen at 220 C for the detection of 17OHP (17ahydroxylase activity) and DHEA (17,20-lyase activity) by RIA kits (Diagnostics Systems Laboratories, Webster, TX) and enzyme-linked immunosorbent assay (IBL, Hamburg, Germany), respectively. All values are expressed as mean 6 standard deviation and represent the results of 3 independent experiments in duplicate. Statistical analysis. The statistical significance of differences between groups was determined by the 2tailed t test. P , 0.05 was considered significant.
RESULTS
The patient was found to be a compound heterozygote carrying 2 different mutant alleles of the CYP17A1 gene. The first mutation was a novel missense mutation, c.716 G.A, located in exon 4, which resulted in an amino acid substitution from arginine to glutamine, that is, R239Q (Fig 1). The second mutation was a previously identified, widely reported deletioninsertion mutation (c.985_987 delinsAA) in exon 6 in Chinese patients.11-13 Analysis of the genotype of her parents revealed that the novel R239Q mutation was inherited from her father, and that the other mutation was inherited from her mother; they were both heterozygous carriers. The protein sequence alignment showed that the Arg 239 residue was conserved in CYP21A2 and CYP17A1 (Fig 2). We also investigated the computerized CYP17A1 model based on the crystallized structure of cytochrome P450 1A2 (3rukD) to explain the putative effects of the mutation in CYP17A1. A charged basic amino acid (arginine) was replaced by an amino acid with an uncharged polar side chain (glutamine) at codon 239 (Fig S1), which caused an abnormal electrostatic charge distribution in the G helix (Fig 3).
Translational Research Volume 161, Number 1
Xue et al
47
Fig 1. Sequencing result of the patient. The patient harbors a novel missense mutation, c.716 G.A, which resulted in an amino acid substitution from arginine to glutamine, (R239Q) (A) and a previously reported del/ins mutation (c.985_987del/insAA) (B).
To determine the enzyme activities of the wild-type and mutant CYP17A1, we used site-directed mutagenesis to generate expression vectors containing the wild-type and mutant CYP17A1, which were then transfected into 293T cells. CYP17A1-WT had full enzyme activity, which efficiently catalyzed P to 17OHP and 17OHPreg to DHEA. However, even at a substrate concentration of 20 mmol/L, metabolites were barely detected in the 293T cells transfected with the empty vector or Y329FS. Furthermore, CYP17A1-R239Q showed an activity level similar to those of the empty vector and Y329FS (Fig 4A and B). DISCUSSION
Cytochrome P450c17 (steroid 17a-hydroxylase/17, 20-lyase) plays a key role in the adrenal steroid hormone synthesis process. 17OHD is a rare autosomal recessive disorder that accounts for approximately 1% of all cases of CAH. The estimated incidence is approximately 1 in 50,000 newborns.14 This disease is characterized by the impaired production of cortisol and the secondary hypersecretion of ACTH, which in turn stimulates the adrenal glands to synthesize large amounts of 11-deoxycorticosterone and corticosterone. High concentrations of 11-deoxycorticosterone result in hypertension, hypokalemia, and a suppressed reninangiotensin system.15 Gonadal 17, 20-lyase deficiency prohibits the synthesis of DHEA, T, and estrogen, resulting in a 46,XY disorder of sex development in male subjects. In the present study, we have identified a novel missense mutation in the CYP17A1 gene and elucidated its molecular mechanism. The patient presented significant hypertension, hypokalemia, and lower gonadal hormone levels caused by severe 17a-hydroxylase deficiency, which was significantly relieved after
Fig 2. Sequence alignment of Arg 239 in different P450 enzymes. Amino acid sequences around Arg 239 of human CYP17A1 are aligned with other related human P450s. The amino acid in the red rectangle represents the corresponding amino acid.
dexamethasone treatment (0.4 mg/d). These symptoms and the response to medication are consistent with the absent CYP17A1 enzyme activity in vitro. The novel mutation (R239Q) substituted a basic arginine residue with a neutral glutamine residue. Combined with the clinical and laboratory findings for our patient, we presumed that this locus was essential for enzyme activity. Furthermore, the structural analysis showed that this mutation lies within the G helix of the CYP17A1 protein.16 Molecular modeling demonstrated that this mutation influenced the local structure and the charge distribution. Finally, functional experiments in vitro provided strong evidence for the pathologic effect of this novel mutation, which resulted in the complete loss of the activities of both 17a-hydroxylase and 17,20-lyase. Until now, only 5 different mutations in exon 4 of the CYP17A1 gene have been reported worldwide.17-21 Among them, Rumsby et al20 and Ahlgren et al21 reported a missense mutation at the 239 position that resulted in the truncation of the CYP17A1 protein, which caused the ambiguous external genitalia of the patients. However, no other mutation in the R239 site was reported in patients of Asian origin. Therefore, we have also reported a novel mutation site in Asian countries,
48
Translational Research January 2013
Xue et al
Fig 3. Local steric model of wild-type and mutant CYP17A1. Spacefilling model of CYP17A1-WT and CYP17A1-R239Q is colored by electrostatic potential. Blue represents positive charge, and red represents negative charge. In the CYP17A1-R239Q, local solid surface and electrostatic potential were greatly changed. A and C, Wildtype. B and D, Mutant.
and the investigation of the function of this mutation was important.
Fig 4. In vitro functional analysis of wild-type and mutant CYP17A1. A, The production of 17-OHP in the presence of various concentrations of P (0.1, 0.2, 1.0, 2.0, 10, and 20 mmol/L) is used to measure 17-hydroxylase activity in 293T cells transfected with CYP17A1-WT, CYP17A1-R239Q, CYP17A1-Y329FS, and empty vector. B, The production of DHEA in the presence of various concentrations of 17-OHPreg (0.1, 0.2, 1.0, 2.0, 10, and 20 mmol/L) is shown as a measure of 17,20-lyase activity in 293T cells transfected with CYP17A1-WT, CYP17A1-R239Q, CYP17A1Y329FS, and empty vector. The experiments are performed at least 3 times, and data are shown as the means 6 standard deviation. DHEA, dehydroepiandrosterone; 17OHP, 17a-hydroxylase/17,20lyase deficiency; WT, wild-type.
CONCLUSIONS
We have identified a novel missense mutation (R239Q) in the CYP17A1 gene of a patient with a 46,XX disorder of sex development. Our study expanded the CYP17A1 mutation spectrum. With a functional study, we confirmed that the novel mutation caused the complete loss of both 17a-hydroxylase and 17,20-lyase activities.
Supplementary Data
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10. 1016/j.trsl.2012.08.007.
REFERENCES
1. Krone N, Dhir V, Ivison HE, Arlt W. Congenital adrenal hyperplasia and P450 oxidoreductase deficiency. Clin Endocrinol (Oxf) 2007;66:162–72. 2. Picado-Leonard J, Miller WL. Cloning and sequence of the human gene encoding P450c17 (steroid 17_-hydroxylase/17,20 lyase): similarity to the gene for P450c21. DNA 1987;6: 439–48. 3. Sparkes RS, Klisak I, Miller WL. Regional mapping of genes encoding human steroidogenic enzymes: P450scc to 15q23q24, adrenodoxin to 11q22; adrenodoxin reductase to 17q24q25; and P450c17 to 10q24-q25. DNA Cell Biol 1991;10: 359–65. 4. Chung BC, Picado-Leonard J, Haniu M, et al. Cytochrome P450c17 (steroid 17_-hydroxylase/17,20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is
Translational Research Volume 161, Number 1
5.
6.
7.
8.
9.
10.
11.
12.
13.
expressed in both tissues. Proc Natl Acad Sci U S A 1987;84: 407–11. Bao X, Ding H, Xu Y, et al. Prevalence of common mutations in the CYP17A1 gene in Chinese Han population. Clin Chim Acta 2011;412:1240–3. Auchus RJ. The genetics, pathophysiology, and management of human deficiencies of P450c17. Endocrinol Metab Clin North Am 2001;30:101–19. Miller WL, Auchus RJ. The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocr Rev 2011;32:81–151. Qiao J, Han B, Liu BL, et al. A unique exonic splicing mutation in the CYP17A1 gene as the cause for steroid 17{alpha}-hydroxylase deficiency. Eur J Endocrinol 2011;164:627–33. Qiao J, Hu RM, Peng YD, et al. A complex heterozygous mutation of His373Leu and Asp487-Ser488-Phe489 deletion in human cytochrome P450c17 causes 17alpha-hydroxylase/17,20-lyase deficiency in three Chinese sisters. Mol Cell Endocrinol 2003;201: 189–95. Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL Workspace: a web-based environment for protein structure homology modelling. Bioinformatics 2006;22:195–201. Wei JQ, Wei JL, Li WC, Bi YS, Wei FC. Genotyping of five Chinese patients with 17alpha-hydroxylase deficiency diagnosed through high-performance liquid chromatography serum adrenal profile: identification of two novel CYP17 mutations. J Clin Endocrinol Metab 2006;91:3647–53. Qiao J, Chen X, Zuo CL, et al. Identification of steroid biosynthetic defects in genotype-proven heterozygous individuals for 17alpha-hydroxylase /17,20-lyase deficiency. Clin Endocrinol (Oxf) 2010;72:312–9. Liu BL, Qiao J, Chen X, et al. Clinical and molecular genetic analysis for 7 patients from 5 pedigrees with 17a-hydroxylase/17, 20
Xue et al
14.
15. 16.
17.
18.
19.
20.
21.
49
lyase deficiency. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2009;26: 282–7. Grumbach MM, Hughes IA, Conte FA. Disorders of sex differentiation. In: Larsen PK, Kronenberg HM, Melmed S, Polonsky KS, eds. Williams Textbook of Endocrinology. 10th ed. Philadelphia, PA: Saunders, 2003:842–1002. Biglieri EG, Herron MA, Brust N. 17-Hydroxylation deficiency in man. J Clin Invest 1966;45:1946–54. Auchus RJ, Miller WL. Molecular modeling of human P450c17 (17alpha-hydroxylase/17,20-lyase): insights into reaction mechanisms and effects of mutations. Mol Endocrinol 1999;13: 1169–82. Takeda Y, Yoneda T, Demura M, et al. Genetic analysis of the cytochrome P-450c17alpha (CYP17) and aldosterone synthase (CYP11B2) in Japanese patients with 17alpha-hydroxylase deficiency. Clin Endocrinol (Oxf) 2001;54:751–8. Katsumata N, Satoh M, Mikami A, et al. New compound heterozygous mutation in the CYP17 gene in a 46, XY girl with 17 alphahydroxylase/17,20-lyase deficiency. Horm Res 2001;55:141–6. Tao H, Zhang B, Lu ZL, Pei Y, Mi SH. New compound heterozygous mutation causes partial combined 17 alpha-hydroxylase/ 17,20-lyase deficiency. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2007;24:19–22. Rumsby G, Skinner C, Lee HA, Honour JW. Combined 17 alphahydroxylase/17,20-lyase deficiency caused by heterozygous stop codons in the cytochrome P450 17 alpha-hydroxylase gene. Clin Endocrinol (Oxf) 1993;39:483–5. Ahlgren R, Yanase T, Simpson ER, Winter JS, Waterman MR. Compound heterozygous mutations (Arg 239——stop, Pro 342——Thr) in the CYP17 (P45017 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 1992;74:667–72.