0021-972X/06/$15.00/0
`Printed in U.S.A.
`
`The Journal of Clinical Endocrinology & Metabolism 91(6):2428 –2431
`Copyright © 2006 by The Endocrine Society
`doi: 10.1210/jc.2005-2653
`
`BRIEF REPORT
`
`A Novel Point Mutation in P450c17 (CYP17) Causing
`Combined 17␣-Hydroxylase/17,20-Lyase Deficiency
`
`A. M. Brooke, N. F. Taylor, J. H. Shepherd, M. E. Gore, T. Ahmad, L. Lin, G. Rumsby,
`M. Papari-Zareei, R. J. Auchus, J. C. Achermann, and J. P. Monson
`
`Center for Endocrinology (A.M.B., J.P.M.), William Harvey Research Institute, St. Bartholomew’s Hospital, Queen Mary,
`University of London, London EC1A 7BE, United Kingdom; Department of Clinical Biochemistry (N.F.T.), King’s College
`Hospital, London SE5 9RS, United Kingdom; Department of Medical Oncology (J.H.S., M.E.G., T.A.), Royal Marsden
`Hospital, London SW3 6JJ, United Kingdom; Institute of Child Health and Department of Medicine (L.L., J.C.A.), and
`Department of Clinical Biochemistry (G.R.), University College Hospital, London NW1 2BU, United Kingdom; and Division
`of Endocrinology (M.P.-Z., R.J.A.), Department of Internal Medicine, University of Texas Southwestern Medical Center,
`Dallas, Texas 75390-8857
`
`Context: Combined 17␣-hydroxylase/17,20-lyase deficiency is a rare
`cause of congenital adrenal hyperplasia and hypogonadism. Novel
`single amino acid changes in P450c17 provide potentially important
`insights into key structural domains for enzyme function.
`
`Objective, Design, and Setting: We report a novel missense mu-
`tation in P450c17 in a 17-yr-old female presenting with a malignant
`mixed germ cell tumor with yolk sac elements who demonstrated
`clinical and biochemical features of combined 17␣-hydroxylase/17,20-
`lyase deficiency.
`
`Methods: Quantitative urinary steroid analysis was performed by
`high resolution gas chromatography. All eight coding exons of CYP17
`were PCR amplified and sequenced. The position of arginine at codon
`96 was modeled using the CYP17 structure 2c17 (www.rcsb.org). The
`CYP17 genes were subcloned into pcDNA3, expressed in HEK-293
`cells, and chromatographed.
`
`Patient and Results: 17␣-Hydroxylase deficiency was confirmed by
`marked reductions in urinary and serum cortisol, androgens, and
`estradiol. Mutational analysis revealed a novel homozygous R96Q
`missense mutation in P450c17, affecting an amino acid in a key
`substrate-binding region of
`the enzyme,
`leading to complete
`inactivity.
`
`Conclusion: The description of a second missense mutation at codon
`96 (R96W and R96Q) in the substrate-binding region of P450c17
`provides strong evidence for the key role of this amino acid in 17␣-
`hydroxylase/17,20-lyase function. An association between a malig-
`nant germ cell tumor and 17␣-hydroxylase deficiency has not been
`reported previously, although the presence of gonadoblastoma in the
`ovary of a patient with this condition has recently been described.
`(J Clin Endocrinol Metab 91: 2428 –2431, 2006)
`
`COMBINED 17␣-HYDROXYLASE/17,20-LYASE deficiency
`
`is a relatively rare cause of congenital adrenal hyper-
`plasia first described nearly 40 yr ago (1). Genotypic females
`(46,XX) with this condition typically present with hypertension,
`hypokalemia, absent secondary sexual characteristics, and pri-
`mary amenorrhea, whereas genotypic males (46,XY) demon-
`strate impaired virilization (complete or partial pseudoher-
`maphroditism) and absence of pubertal development.
`The gene encoding the dual function P450c17 enzyme,
`CYP17 (10q24.3), was cloned in 1987 and encodes eight exons
`over 6.4 kb DNA (2–4). Mutations in this gene were first re-
`ported in 1988 (5). Since then, approximately 45 different mu-
`tations have been described in the coding regions, splice donor
`sites (resulting in exon skipping), and splice acceptor sites of this
`gene in patients with combined 17␣-hydroxylase/17,20-lyase
`
`deficiency (6). Single amino acid changes in P450c17 are rela-
`tively rare, but provide potentially important insight into key
`structural domains for enzyme function, and a limited number
`of patients with isolated 17,20-lyase deficiency due to P450c17
`point mutations have been reported (7–9).
`In this study, we describe a novel point mutation in a key
`substrate-binding region of P450c17 in a prepubertal 17-yr-
`old girl who presented with a malignant germ cell tumor.
`
`Patient and Methods
`
`Laboratory analysis
`
`Quantitative urinary steroid analysis was performed by high reso-
`lution gas chromatography as previously described (10).
`
`Mutational analysis
`
`First Published Online March 28, 2006
`Abbreviations: DHEA, Dehydroepiandrosterone; 17OHP, 17-hydroxy-
`progesterone.
`JCEM is published monthly by The Endocrine Society (http://www.
`endo-society.org), the foremost professional society serving the endo-
`crine community.
`
`After institutional review board approval and informed consent were
`obtained, genomic DNA was extracted from the patient’s leukocytes. All
`eight coding exons of CYP17 were PCR amplified using variations of the
`primers and conditions described previously (11). PCRs were purified
`using exonuclease and shrimp alkaline phosphatase and were se-
`quenced directly, forward and reverse, using a BigDye Terminator ver-
`sion 1.1 cycle sequencing kit (Applied Biosystems, Warrington, UK).
`
`2428
`
`Amerigen Exhibit 1179
`Amerigen v. Janssen IPR2016-00286
`
`

`
`Brooke et al. • CYP17 Mutation Causing 17␣-Hydroxylase/17,20-Lyase Deficiency
`
`J Clin Endocrinol Metab, June 2006, 91(6):2428 –2431 2429
`
`Sequencing was performed on a MegaBACE1000 capillary DNA se-
`quencer (Amersham Biosciences, Little Chalfont, UK).
`
`Structural modeling and functional studies
`
`The position of arginine at codon 96 was modeled using the CYP17
`structure 2c17 (www.rcsb.org). Images were generated with MidasPlus
`software on a Silicon Graphics (Mountain View, CA) octane workstation.
`The cDNA for CYP17 mutation R96Q was generated by overlapping
`PCR using oligonucleotides T7⫹R96Q-S (5⬘-CTCTGGGCAGCCT-
`CAAATGGCAAC-3⬘) and R96Q-AS (5⬘-TTGAGGCTGCCCAGAGAA-
`GTCCTTG-5⬘)⫹SP6, and pLW01-c17 as a template (12, 13). HEK-293
`cells were seeded in six-well plates and transfected with 1 ␮g pcDNA3-
`c17 plasmids using FuGene 6 as previously described (12). Cells were
`incubated with 2 ml complete medium containing 0.03– 0.1 ␮m preg-
`nenolone with 150,000 cpm [3H]pregnenolone (PerkinElmer NEN, Shel-
`ton, CT). Aliquots (1 ml) were removed after 2– 4 h, extracted, and
`chromatographed as described previously (12, 13).
`
`Case report
`
`Results
`
`from the
`A 17-yr-old, phenotypically female patient
`United Arab Emirates came to medical attention because of
`abdominal pain and was found to have a mixed germ cell
`tumor with yolk sac elements. Remission was achieved with
`six cycles of bleomycin, etoposide, cisplatinum chemother-
`apy (serum ␣-fetoprotein reduced from 12,714 to 5 mU/liter).
`It was noted that the patient was prepubertal, with normal
`female external genitalia, absent axillary and pubic hair, and
`primary amenorrhea. Her height was at the third percentile
`for her age. Her parents were first cousins. Examination
`demonstrated palmar pigmentation, buccal and streak pig-
`mentation, and hypertension. Abdominal computed tomog-
`raphy revealed bilateral adrenal hyperplasia.
`Her karyotype was 46,XX. Serum LH and FSH levels were
`32 and 60 IU/liter, respectively, and serum estradiol was 88
`pmol/liter (24 pg/ml). Serum potassium was 2.5 mmol/liter,
`serum bicarbonate was 24 mmol/liter, recumbent serum al-
`dosterone was 643 pmol/liter (23.2 ng/dl), and plasma renin
`activity was undetectable. Serum dehydroepiandrosterone
`(DHEA) sulfate, 17-hydroxyprogesterone (17-OHP), and 11-
`deoxycortisol were undetectable; androstenedione and tes-
`tosterone were decreased at 1.1 nmol/liter (0.32 ␮g/liter) and
`0.4 nmol/liter (0.12 ng/ml), respectively. Serum cortisol was
`decreased at 15 nmol/liter (0.54 ␮g/dl) with a 0900 h plasma
`ACTH level of 21 ␮g/liter. There was no response of cortisol
`or any androgens to standard tetracosactrin stimulation (250
`␮g, iv). A clinical diagnosis of complete 17␣-hydroxylase
`deficiency was supported by measurement of urinary ste-
`roids by gas liquid chromatography (10), which showed
`grossly elevated corticosterone metabolites (42,925 ␮g/24 h)
`and pregnanediol (1,360 ␮g/24 h), and absent cortisol, an-
`drostenedione, and dehydroandrosterone metabolites.
`The patient commenced reverse circadian prednisolone
`treatment (2 mg in the morning and 4 mg in the evening), and
`her hypokalemia and hypertension resolved. Puberty was
`induced initially with 5 ␮g ethinyl estradiol daily, increased
`gradually to 20 ␮g daily over 18 months, followed by the
`addition of levonorgestrel. She remains in remission after 18
`months of follow-up.
`
`Mutational analysis, modeling, and functional studies
`
`Mutational analysis revealed a novel homozygous R96Q
`missense mutation in P450c17. The parents were consan-
`guineous, but were unavailable for investigation. This R96Q
`change converts a highly conserved, charged, side chain
`amino acid (arginine) to an amino acid with a polar un-
`charged side chain (glutamine) in flanking strand 2 of ␤-sheet
`1 (Fig. 1). This mutation showed complete loss of P450c17
`function (Fig. 2).
`
`Discussion
`
`We describe a novel homozygous R96Q missense muta-
`tion in P450c17 affecting an amino acid in a key substrate-
`binding region of the enzyme, resulting in complete absence
`of 17␣-hydroxylase/17,20-lyase activity. 17␣-Hydroxylase
`deficiency is an autosomal recessive condition that accounts
`for approximately 1% of all cases of congenital adrenal hy-
`perplasia (14). The estimated incidence is 1 in 50,000 new-
`borns (15). This condition was first described by Biglieri et al.
`(1) in an adult female (genotype 46,XX) with hypertension,
`hypokalemia, and sexual infantilism. This patient had in-
`creased urinary excretion of corticosterone with absence of
`17␣-hydroxylated metabolites. As in the present case, the
`lack of sexual development and impaired estrogenization
`suggested that P450c17 has 17,20-lyase activity in addition to
`its 17␣-hydroxylase activity, and most patients reported to
`date have combined 17␣-hydroxylase/17,20-lyase deficiency
`due to deleterious changes in this P450c17 enzyme system.
`The biochemical and structural mechanisms involved in
`P450c17 dual function (17␣-hydroxylase and 17,20-lyase ac-
`tivity) are being elucidated, and the localization of critical
`domains for enzyme function has been supported by the
`
`FIG. 1. Computer model of human CYP17 (RCSB pdbid 2c17). The
`protein backbone is shown in ribbon rendering (yellow), with heme in
`red. R96 (blue) protrudes into the cleft that separates the ␤-sheet
`domain (right) from the ␣-helical domain (left). The guanidinium
`group of R96 appears to form hydrogen bonds with carbonyl groups
`of residues A113 and F114 (purple), but not with the side chains.
`Removal of this positively charged group, which occurs in mutation
`R96Q or R96W, is predicted to destabilize the protein by disrupting
`this interaction between the two domains.
`
`

`
`2430 J Clin Endocrinol Metab, June 2006, 91(6):2428 –2431
`
`Brooke et al. • CYP17 Mutation Causing 17␣-Hydroxylase/17,20-Lyase Deficiency
`
`(11, 18). The W406R mutation was more common in southern
`Brazilians of Spanish origin, and the R362C mutation was
`more common in northern Brazilians of Portuguese origin.
`Interestingly, neither of these mutations has been found in
`Spain or Portugal, suggesting a founder arriving in Brazil
`many years before.
`In contrast, a homozygous R96W mutation has been re-
`ported previously in two siblings of French Canadian origin
`with 46,XY pseudohermaphroditism and combined 17␣-
`hydroxylase/17,20-lyase deficiency (19). Although certain
`genetic sequences are more vulnerable to mutation, the oc-
`currence of two different point mutations in the same amino
`acid suggests that this locus has a particularly important
`functional role and is supported by the complete loss of dual
`enzyme function reported in studies of P450c17 R96W ac-
`tivity. Furthermore, modeling of this amino acid change sug-
`gests that this amino acid lies within the flanking strand 2 of
`the ␤-sheet 1. Mutation of the charged side chain of arginine
`to glutamine would lead to complete inactivity of the
`enzyme.
`The severity of the clinical disease tends to be milder with
`mutations that retain partial catalytic activity, but the age of
`onset of hypertension, the degree of hypokalemia, and the
`aldosterone production rate appear to vary, even in those
`with the same mutation (14). For example, up to 15% of
`patients with a diagnosis of 17␣-hydroxylase deficiency are
`normokalemic and normotensive at diagnosis (14) (although
`not all of these have had the diagnosis proven by genotyp-
`ing). An elevated aldosterone level, as in our patient, is
`unusual, but not unique (14), and the relatively normal value
`of ACTH is probably due to some glucocorticoid potency of
`corticosterone leading to partial suppression of the hypo-
`thalamic-pituitary (HPA) axis.
`This patient presented with a mixed germ cell tumor. A
`gonadoblastoma has previously been described in a 46,XX
`Brazilian patient with combined 17␣-hydroxylase/17,20-
`lyase deficiency who presented at 17 yr of age (18). There has
`also been another report of a 46,XX patient with 17,20-lyase
`deficiency presenting with giant ovarian cysts that were re-
`duced in size by GnRH antagonist therapy (20). This asso-
`ciation remains likely to be a chance finding, but an aware-
`ness of the possible development of gonadal tumors in
`patients with disordered gonadal
`steroidogenesis
`is
`necessary.
`Our description of a second missense mutation at codon
`96 (R96Q) in the substrate-binding region of P450c17 pro-
`vides strong evidence for the key role of this amino acid in
`17␣-hydroxylase/17,20-lyase function.
`
`Acknowledgments
`
`Received December 7, 2005. Accepted March 20, 2006.
`Address all correspondence and requests for reprints to: Dr. J. P.
`Monson, Center for Endocrinology, William Harvey Research Institute,
`St. Bartholomew’s Hospital, Queen Mary, University of London, Lon-
`don EC1A 7BE, United Kingdom. E-mail: j.p.monson@qmul.ac.uk.
`A.M.B. was supported by a clinical training fellowship awarded by
`the Joint Research Board of St. Bartholomew’s Hospital. J.C.A. holds a
`Wellcome Trust Clinician Scientist Fellowship (068061). R.J.A. was sup-
`ported by National Institutes of Health Grant R21-DK-56641.
`
`FIG. 2. Activity of the CYP17 mutation R96Q. Autoradiogram of a
`thin layer chromatogram used to separate [3H]pregnenolone metab-
`olites is shown, with the locations of steroids indicated. HEK-293 cells
`transfected with cDNAs for mutation R96Q or wild-type CYP17 were
`incubated with 0.1 ␮M [3H]pregnenolone for 2– 4 h, and medium was
`extracted and chromatographed. Wild-type CYP17 converts preg-
`nenolone (Preg) to 17␣-hydroxypregenolone (17Preg) and then to
`DHEA; mutation R96Q shows no activity.
`
`identification of a limited number of point mutations in the
`protein in patients with disorders of steroidogenesis.
`Under normal physiological conditions, pregnenolone
`and progesterone undergo 17␣-hydroxylation to 17␣-
`hydroxypregnenolone and 17OHP, respectively. Scission of
`the C17,20 carbon bond in 17␣-hydroxypregnenolone yields
`DHEA; however, very little 17OHP is converted to andro-
`stenedione, because the human P450c17 enzyme catalyzes
`this reaction at only 3% the rate of conversion of 17␣-
`hydroxypregnenolone to DHEA (16). If neither 17␣-hydrox-
`ylase nor 17,20-lyase activity is present, pregnenolone is
`converted to mineralocorticoids. If only 17␣-hydroxylase
`activity is present, pregnenolone is converted to cortisol, and
`if both activities are present, pregnenolone is converted to
`DHEA. The P450 enzyme is bound to the smooth endoplas-
`mic reticulum, where it accepts electrons from P450 oxi-
`doreductase. Electron transfer for the lyase reaction is pro-
`moted by the allosteric action of cytochrome b5, but also
`requires phosphorylation of serine residues on P450c17 by a
`cAMP-dependent kinase. The availability of electrons seems
`to determine whether P450c17 performs only 17␣-hydroxy-
`lation or also 17,20-lyase activity. Optimal functioning of the
`redox partner-binding site is particularly important for the
`lyase reaction. Thus, point mutations (e.g. R347H and R358Q)
`that change the distribution of surface charges on the redox
`binding site of P450c17 impair electron transfer and can
`result in isolated 17,20-lyase deficiency with preserved 17␣-
`hydroxylase function (8, 9, 13).
`Point mutations resulting in combined 17␣-hydroxylase/
`17,20-lyase deficiency have also provided insight into critical
`amino acids involved in dual-enzyme function. To date, ap-
`proximately 18 different missense and in-frame point mu-
`tations have been described (7). A significant proportion of
`these mutations may have arisen due to a genetic founder
`effect, rather than through selection advantage of heterozy-
`gote carriers (17). For example, in studies of combined 17␣-
`hydroxylase/17,20-lyase deficiency in Brazil, only seven mu-
`tations from 28 unrelated alleles in 19 families were found
`
`

`
`Brooke et al. • CYP17 Mutation Causing 17␣-Hydroxylase/17,20-Lyase Deficiency
`
`J Clin Endocrinol Metab, June 2006, 91(6):2428 –2431 2431
`
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`JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the
`endocrine community.