CONGENITAL ADRENAL HYPERPLASIA
`
`0889-8529/01 $15.00 + .00
`
`THE GENETICS,
`PATHOPHYSIOLOGY, AND
`MANAGEMENT OF HUMAN
`DEFICIENCIES OF P450c17
`
`Richard J. Auchus, MD, PhD
`
`P450c17 commands a central role in human steroidogenesis as the
`qualitative regulator of steroid hormone flux (Fig. 1). Analysis of P450c17
`deficiencies in humans illustrates many aspects of the physiology of
`steroid biosynthesis and demonstrates poignant features of the genetics
`and biochemistry of P450c17. 17-Hydroxylase deficiency was first de-
`scribed in patients with sexual infantilism and hypertension.'? It is now
`recognized to occur in partial and selective forms with variable pheno-
`types. This article reviews the genetics and biochemistry of P450c17 as
`a prelude for understanding the pathophysiology of such deficiencies
`and approaches to their diagnosis and management.
`
`P450c17 AND CYP17
`
`Patients who carry the diagnosis of 17-hydroxylase deficiency har-
`bor alterations in the CYP17 gene that encodes the P450c17 enzyme.
`P450c17 actually performs multiple chemical transformations. Human
`P450c17 17a-hydroxylates A5-pregnenolone and A4-progesterone with
`roughly equal catalytic efficiency.v " whereas all other reactions show
`prominent differences between A5 and A4 substrates. The 17,20-lyase
`activity is roughly 50 times more efficient for the 17a-hydroxypregneno-
`
`From the Division of Endocrinology and Metabolism, Department of Internal Medicine,
`University of Texas Southwestern Medical School, Dallas, Texas
`
`ENDOCRINOLOGY AND METABOLISM CLINICS OF NORTH AMERICA
`
`VOLUME 30 • NUMBER 1 • MARCH 2001
`
`101
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`1,16<END1>1<END2>14<END3>(414,-14)<E4>22</E4>0<E5>1<E6>18<E7>11<E8>12/1/2015 12:00:00 AM15:01:17.1728490<E9></T>
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`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 1
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`

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`17~HSDJ1i17pHSDn
`
`•Estrone
`
`P450arom
`
`•Estradiol
`
`P450arom
`
`•Cortisol
`
`P450c11~
`
`ll-Deoxycortisol
`
`•
`
`P450c21
`
`,················...180H-Corticosterone&180H-DOC
`~P450cll~
`
`Corticosterone
`
`Figure1.Seelegendonoppositepage.
`
`~Dihydrotestosterone
`
`•Testosterone
`
`Androstenediol
`
`5a-ReductaseI&III
`313HSD
`
`•Aldosterone
`
`P450c21~--...,j.~P450cllAS
`
`•DOC
`
`~
`
`...-.-----.,;;===r-=r-_
`
`_
`
`Progesterone
`
`•
`
`IP450c17!!
`
`3@HSD(l/II)
`
`Pregnenolone
`
`!P450SCC
`
`Cholesterol
`
`!r~~!~:'
`IPituitaryIII
`
`....o
`
`N
`
`T7PHSDTO1il7pHson
`
`•Androstenedione
`
`1
`
`3~HSD
`
`DHEA
`
`p170H-Progesterone
`
`3QHSD..
`
`regnenoone
`
`I
`
`170HP
`
`-
`
`17~HSOT1i17pHSDn
`,~,1
`IP450c171!
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 2
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`MANAGEMENT OF HUMAN DEFICIENCIES OF P450c17
`
`103
`
`lone-to-dehydroepiandrosterone (DHEA) reaction than for the 17u-hy-
`droxyprogesterone-to-androstenedione reaction.v " Although the rate of
`the lyase reaction can be increased more than 10-fold by the addition of
`cytochrome bs/' 31, 35 the as preference persists, and the lyase rate never
`quite achieves the rate of the hydroxylase reactions. In addition, human
`P450c17 16u-hydroxylates progesterone but not pregnenolone.v 37, 62 In
`the presence of cytochrome b., P450c17 converts approximately 10% of
`pregnenolone substrate to a a16 andiene product," which is also formed
`by porcine P450c17 and acts as a pheromone precursor in pigs. 48 Al-
`though experiments to study the chemistry of P450c17 often require
`certain conditions, such as detergent solubilization that could be consid-
`ered nonphysiologic, the remarkable consistency of substrate preferences
`and kinetic constants observed for the modified solubilized P450c17
`expressed in Escherichia coli,31,3s the native P450c17 expressed in yeast
`microsomes" or intact COS-1 cells/7, 38 and that obtained from human
`tissues and cells3,62 strengthens these conclusions.
`One consequence of this as preference of human P450c17 for the
`17,20-lyase reaction is that the vast majority of sex steroids in humans
`derive from DHEA as an intermediate. This a5 preference also allows
`the phenomenon of adrenarche to occur in humans, an event that is
`characterized by a dramatic rise in adrenal DHEA production that occurs
`at about age 8 to 10 years,12,60 whereas cortisol production remains
`relatively constant. Adrenarche is an exemplary manifestation of the
`biochemistry of P450c17, in which the 17u-hydroxylase and 17,20-lyase
`activities are differentially regulated. In fact,
`this dichotomy between
`adrenal 17u-hydroxylase activity, reflected by relatively constant cortisol
`production, and 17,20-lyase activity, reflected by drastically age-depen-
`dent changes in DHEA production, previously suggested that distinct
`enzymes performed the two transformations; however, later copurifica-
`tion of the 17u-hydroxylase and 17,20-lyase activities of neonatal pig
`testes suggests otherwise." This controversy was settled when the eDNA
`for bovine P450c17 was expressed in COS-1 cells, conferring 17u-hydrox-
`ylase and 17,20-lyase activities to these nonsteroidogenic cells? and
`proving genetically that the 17u-hydroxylase and 17,20-lyase enzymes
`
`Figure 1. Major steroidogenesis pathways in humans and feedback loops controlling
`glucocorticoid and mineralocorticoid production, Ordinarily, cortisol is the major glucocorti-
`coid produced by the adrenal zona fasciculata/reticularis, and cortisol exerts negative
`feedback inhibition (double vertical bars) to regulate pituitary adrenocorticotropic hormone
`(ACTH) production, Aldosterone is the principal mineralocorticoid of
`the adrenal zona
`glomerulosa, and aldosterone synthase (P450c11 AS) expression is stimulated by volume
`depletion, which activates the renin-angiotensin (All) system, and to a lesser extent, by
`ACTH. Aldosterone acts to stimulate kaluresis and salt and water retention, which feeds
`back on the kidney to suppress renin production. The production of corticosterone, a weak
`glucocorticoid, and of 11-deoxycorticosterone (DOC), a potent mineralocorticoid, is relatively
`low and unimportant
`in healthy individuals with intact
`feedback systems. Note that
`P450c1113 in the zona fasciculata also 18-hydroxylates (180H) DOC and corticosterone as
`minor products.
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 3
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`104
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`AUCHUS
`
`were, in fact, both embodied in a single enzyme, P450c17. Differential
`regulation of the two principal activities of P450c17 is possible because
`the abundance of P450-oxidoreductase37 and the addition"- 3S, so or coex-
`pression" of cytochrome b, preferentially augments the 17,20-lyase activ-
`ity, and phosphorylation7,76 also selectively enhances 17,20-lyase activity.
`Recent data showing high expression of b, in the zona reticularis of
`monkeys'? and humans" suggest that the developmentally regulated
`expression of b, might be a key event in the genesis of adrenarche in
`higher primates.
`
`PATHOPHYSIOLOGY
`
`P450c17 deficiencies are a form of congenital adrenal hyperplasia in
`which not only adrenal but also gonadal steroidogenesis is impaired. In
`humans, one gene for P450c17 is expressed in the adrenals and gonads"
`instead of two tissue-specific isozymes. A single 2.1-kb mRNA species
`yields a 57-kd protein in these tissues, and mutations in this gene
`produce a spectrum of deficiencies in 17-hydroxysteroids and C19 ste-
`roids. Loss of P450c17 in the adrenal gland impairs cortisol and DHEA
`production, whereas gonadal deficiency of P450c17 abrogates sex steroid
`production. The initial description of 17-hydroxylase deficiency was a
`case in which both 17a-hydroxylase and 17,20-lyase products were ab-
`sent.'? When the gene for human P450c17 was cloned/" patients with
`17-hydroxylase deficiency were found to harbor mutations in the CYP17
`gene.v 67 but molecular techniques and subsequent clinical evaluations
`failed to implicate CYP17 mutations as the cause of isolated 17,20-lyase
`deficiency." Recently, three cases of isolated 17,20-lyase deficiency have
`been confirmed by molecular genetics," 20 demonstrating that amino acid
`substitution mutations in P450c17 can cause an isolated loss of 17,20-
`lyase activity.
`
`Combined 17a-Hydroxylase/17, 20-Lyase Deficiency
`
`Loss of P450c17 in the human adrenal gland prohibits the biosynthe-
`sis of cortisol and C 19 steroids. Curiously, the adrenal glands of patients
`with 17-hydroxylase deficiency are similar to those of rodents, which do
`not express P450c17,63 such that rodents rely on corticosterone as their
`principal glucocorticoid, and their adrenal glands cannot make C 19 ste-
`roids. Patients with 17-hydroxylase deficiency rarely" manifest symp-
`toms of adrenal insufficiency owing to sustained corticosterone produc-
`tion. Because corticosterone is a weaker glucocorticoid than cortisol,
`abnormally high corticosterone production is necessary before feedback
`inhibition on pituitary corticotropin (ACTH) secretion occurs," establish-
`ing a new steady state (Fig. 2). To produce sufficient corticosterone to
`make up for the absence of cortisol, dramatically elevated quantities of
`intermediate steroids, such as progesterone and U-deoxycorticosterone
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 4
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`MANAGEMENT OF HUMAN DEFICIENCIES OF P450c17
`
`105
`
`(DOC), must accumulate, as well as unusual metabolites, such as 18-
`hydroxycorticosterone'< and 19-nor-deoxycorticosterone.23 This ACTH-
`driven overproduction of mineralocorticoids leads to hypertension, a
`characteristic presenting feature of this disease. The hypertension usually
`develops in early adulthood? but can present in infancy> and can be
`severe." As is true in other hypertensive disorders caused by mineralo-
`corticoid excess," the hypertension can become fixed if the disease is
`not treated for many years."
`Although the general description given herein is true for most
`patients with this disorder, considerable variation in phenotype and
`laboratory findings has been described. These variables include the
`degree of genital virilization in 46,XY subjects and the capacity for
`the severity of the hypertension and
`menstruation in 46,XX subjects;
`hypokalemia; the aldosterone secretion rate;
`the type and amount of
`adrenocortical hyperplasia; the gonadal morphology and histology; and
`the coexistence of additional disorders, such as 21-hydroxylase defi-
`ciency'" or maternal androgen excess." This heterogeneity has not been
`including the severity of the
`completely explained, but many factors,
`P450c17 deficiency, variations in genes regulating hormone respon-
`siveness, diet (sodium consumption), and environment, undoubtedly
`contribute. The reader is referred to a detailed discussion of case re-
`ports," which is beyond the scope of this article.
`
`Isolated 17, 20-Lyase Deficiency
`
`This disoder is extremely rare because mutations that cause this
`phenotype must not only destroy most 17,20-lyase activity but preserve
`most 17a-hydroxylase activity. Patients who are 46,XY present with
`ambiguous genitalia at birth or with inguinal hernias with or without
`pubertal delay as adolescents'? (Table 1). Patients do not show the
`consequences of mineralocorticoid excess because preserved cortisol pro-
`duction prevents excessive DOC and corticosterone accumulation. Clini-
`cal laboratory findings vary considerably owing to the age of diagnosis,
`the severity of the disease, and the discrepancy between the l.Zo-hydrox-
`ylase and 17,20-lyase activities in a given individual. Nonetheless, C19
`steroid production is severely, although not completely,
`impaired,
`whereas 17-hydroxylated steroid production is nearly or completely nor-
`mal.
`
`DIAGNOSIS
`
`Unlike forms of congenital adrenal hyperplasia, such as the lipoid
`type and 21-hydroxylase deficiency, in which glucocorticoid and miner-
`alocorticoid production are impaired, patients with 17-hydroxylase defi-
`ciency do not have an adrenal crisis in the postnatal period. Conse-
`the diagnosis is often not entertained until hypertension,
`quently,
`hypokalemia, or pubertal delay is evaluated during adolescence or early
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 5
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`

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`106
`106
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`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 6
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`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 6
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`MANAGEMENT OF HUMAN DEFICIENCIES OF P450c17
`
`107
`
`adulthood. Patients with a 46,XY karyotype and incomplete deficiency
`may be misdiagnosed with androgen insensitivity or defects in later
`steps of dihydrotestosterone biosynthesis. As is true for all steroidogenic
`enzyme deficiencies, the diagnosis is most convincingly established by
`measuring precursor-to-product ratios during ACTH stimulation testing.
`In particular, circulating concentrations of the 17-deoxysteroids proges-
`terone, corticosterone, and DOC rise to 5 to 10 times normal after ACTH
`administration. IS In addition, 17-hydroxylase deficiency, in distinct con-
`trast to Il-hydroxylase and 21-hydroxylase deficiencies, is characterized
`by elevated production of 18-hydroxycorticosterone and 18-hydroxy-
`DOC (Table 2).33 The ratio of corticosterone to DOC (or of their 18-
`hydroxy-derivatives) distinguishes 17- from ll-hydroxylase deficiency.
`Table 3 compares the clinical, laboratory, and genetic characteristics of
`the various mineralocorticoid excess states that may arise in children
`and young adults.
`Although production of the precursors corticosterone and DOC is
`markedly elevated in 17-hydroxylase deficiency, DOC production can be
`much greater in ll-hydroxylase deficiency, whereas plasma 18-hydroxy-
`DOC concentrations are not elevated." The reason for this apparent
`is
`discrepancy is that P450cll13 (the product of the CYP11B1 gene)
`not exclusively an 1113-hydroxylase but exhibits weak 18-hydroxylase
`activity49,64 (Fig. 2) and trace amounts of aldosterone synthase activity,"
`The low 18-hydroxy-DOC production in ll-hydroxylase deficiency, de-
`spite enormous DOC concentrations, is compelling genetic evidence that
`P450cll13 is responsible for elevated 18-hydroxy-DOC and 18-hydroxy-
`corticosterone production in 17-hydroxylase deficiency. Analogously, in
`glucocorticoid-remediable aldosteronism, abundant 18-oxygenase activi-
`ties in the zona fasciculata owing to the presence of a chimeric CYP11B2j
`11B1 gene36 lead to excessive 18-oxygenated steroid production." Pa-
`tients with 17-hydroxylase deficiency with paradoxically measurable, if
`
`Figure 2. Physiologic disturbances in glucocorticoid, mineralocorticoid, and sex steroid
`homeostasis in complete 17-hydroxylase deficiency. The inability to 17a-hydroxylate C2 1
`steroids in the adrenal gland eliminates all steroids within shaded region and shunts
`pregnenolone flux to progesterone, 11-deoxycorticosterone (DOC), corticosterone, and
`possibly aldosterone (large open arrows). Absence of negative feedback by cortisol (dashed
`line) causes overproduction of adrenocorticotropic hormone (ACTH) (top-most large open
`arrow), and the resultant abundance of the weak glucocorticoid corticosterone provides
`adequate systemic glucocorticoid action and feedback on ACTH secretion (solid line). The
`hypothalamic-pituitary-adrenal axis then reaches a steady state at a higher set-point;
`however, the drive to overproduce corticosterone allows the accumulation of intermediates
`such as the potent mineralocorticoid DOC, and high DOC production stimulates salt and
`water retention, which suppresses renin secretion (dashed arrow). Thus, aldosterone pro-
`duction is low (dashed arrow), but hypertension and hypokalemia develop because of DOC
`excess. In addition, the unusually high concentrations of DOC and corticosterone in the
`presence of robust P450c1113 expression leads to excessive production of ordinarily minor
`metabolites 18-hydroxy (180H)-DOC and 18-0H-corticosterone. Because 17-hydroxy
`(170H)-pregnenolone and dehydroepiandrosterone (DHEA) synthesis is nil (shaded region)
`in the fetus and at puberty, no androgen or estrogen synthesis is possible, and sexual
`infantilism results.
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 7
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`

`
`....o
`
`oe
`
`Table 1. COMPARISON OF COMBINED 17a-HYDROXYLASE/17,20-LYASE DEFICIENCY AND ISOLATED 17,20-LYASE DEFICIENCY
`
`Deficiency
`
`Combined 17a-hydroxylase and
`17,20-lyase
`
`Isolated 17,20-lyase
`
`Plasma Steroids
`t 17-0H-steroids, DHEA,
`androgens, estrogens
`t Progesterone, DOC, DOC
`metabolites, corticosterone
`Normal 17-0H-steroids
`t DHEA, androgens
`t 17-0HP/AD (>10 after hCC)
`
`Urinary Steroids
`t 17-0HCS, 17-KS pregnanetriolone
`t Tetrahydro-DOC
`
`Clinical Presentation
`
`Hypertension/hypokalemia,
`sexual infantilism
`
`t 17-KS
`
`Ambiguous genitalia in 46,XY
`
`17-0HP = 17-hydroxyprogesterone; AD = androstenedione; hCG = human chorionic gonadotropin; DHEA
`costeroids; 17-KS = 17-ketosteroids; DOC = ll-deoxycorticosterone.
`
`dehydroepiandrosterone; 17-0HCS ~ 17-hydroxycorti-
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 8
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`

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`MANAGEMENT OF HUMAN DEFICIENCIES OF P450c17
`
`109
`
`Table 2. COMPARISON OF STEROID PROFILES IN ADULTS WITH 17-,11-, AND 21-
`HYDROXYLASE DEFICIENCES
`
`Type of
`Deficiency
`
`DOC
`ng/dL
`
`18-0H-DOC
`ng/dL
`
`Corticosterone
`ng/dL
`
`18-0H-Corticosterone
`ng/dL
`
`Aldosterone
`ng/dL
`
`17-0H
`11-0H
`21-0H
`Normal
`
`25-500
`50 to >1000
`ID-l00
`2-20
`
`100-600
`<10
`3-20
`1-20
`
`4000-40,000
`<200
`100-500
`100-500
`
`6D-1000
`<10
`10-200
`10-40
`
`<10
`<3
`10-60
`10-30
`
`DOC = ll-deoxycorlicosterone; 18-0H-DOC = 18-hydroxy-DOC; OH = hydroxylase.
`Data adapted from Kater CE, Biglieri EG: Disorders of steroid 17 alpha-hydroxylase deficiency. Endocrinol Metab
`Clio North Am 23:341-357, 1994.
`
`not elevated, aldosterone production have been described. It is possible
`that, in these instances, artifacts owing to laboratory methods or intercur-
`rent glucocorticoid therapy confound the data." It is equally likely that
`other genetic and environmental modifiers contribute to these variations
`in disease manifestations, such as polymorphisms that alter the aldoste-
`rone synthase activity of P450c1113. The latter hypothesis is consistent
`with the finding that most 17-hydroxylase deficiency cases with measur-
`able aldosterone production are from [apan.v- 72 Until a large series
`of patients with 17-hydroxylase deficiency is compiled with uniform
`evaluation, these conundrums will persist.
`Although heterozygous family members of patients with 17-hydrox-
`ylase deficiency without other endocrine abnormalities usually have
`clinically normal adrenal and gonadal physiology, it is sometimes possi-
`ble to detect heterozygosity using biochemical testing. Elevated cortico-
`sterone and 18-hydroxycorticosterone concentrations, as well as the 18-
`hydroxycorticosterone-to-aldosterone ratio, after ACTH stimulation are
`perhaps the most readily available means to detect heterozygotes if an
`index case has been identified." More precisely, the ratio of total urinary
`metabolites of corticosterone to those of cortisol is elevated (reflecting
`low 17a-hydroxylation), and the ratio of total urinary metabolites of CI9
`steroids to those of C21 steroids is low (reflecting low 17,20-lyase activ-
`ity)." If a compelling reason for ascertainment of an individual's zygos-
`ity exists, molecular genetics provides a highly sensitive, although te-
`dious, method that must be performed in a research laboratory."
`
`MOLECULAR GENETICS
`
`Deletions, Premature Truncations, Frameshifts, and
`Splicing Errors
`
`Among the genetic abnormalities described in the CYP17 gene, the
`largest deletion reported involves the substitution of 518 bp (most of
`exon 2 and part of exon 3) with 469 bp of unknown DNA, disrupting
`the protein near its beginning and causing complete 17a-hydroxylase
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 9
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`........o
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`Table 3. COMPARISON OF MINERALOCORTICOID EXCESS STATES WITH SUPPRESSED PLASMA RENIN ACTIVITY
`
`Disease
`
`*17-Hydroxylase deficiency
`
`Primary aldosteronism
`
`DOC-oma
`
`"Syndrome of apparent mineralocorticoid
`excess
`"Clucocorticoid-remediable aldosteronism
`
`"Cushing syndrome
`
`*Glucocorticoid resistance
`
`Laboratory Findings
`t DOC, corticosterone, and 18-0H
`derivatives
`! 17-0H-steroids, C19 steroids
`! Aldosterone
`t Aldosterone, often t 18-0H-
`corticosterone
`t DOC, normal aldosterone,
`variable other steroids
`! Cortisone metabolites
`! Aldosterone
`t 18-0H- and 18-oxocortisol,
`variable aldosterone
`i Cortisol production, variable
`other steroids
`i Cortisol production
`i C19 steroids
`
`*ACTH-dependent mineralocorticoid excess.
`DOC = ll-deoxycorticosterone; OH = hydroxy; GR = glucocorticoid receptor.
`
`Key Features
`
`Molecular Basis
`
`Sexual infantilism/ambiguity
`
`CYP17 mutations
`
`Normal cortisol axis
`
`Normal cortisol axis
`
`Unknown
`
`Unknown
`
`Dexamethasone suppression of
`hypertension, kaluresis
`Dexamethasone suppression of
`hypertension, kaluresis
`Symptoms of cortisol excess,
`variable mineralocorticoid excess
`Symptoms of cortisol insufficiency,
`androgen excess
`
`HSDllB2 mutations
`
`CYPllB2/11Bl chimeric gene
`
`Unknown
`
`GR mutations, other
`
`MYLAN PHARMS. INC. EXHIBIT 1026 PAGE 10
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`MANAGEMENT OF HUMAN DEFICIENCIES OF P450cl7
`
`111
`
`deficiency." A 4-bp duplication of the sequence CATC following Ile47930
`was originally observed in Canadian Mennonites" and has been subse-
`quently found in at least six Dutch Frieslander families." This duplica-
`tion leaves 95% of the protein unaffected and creates a mutant P450c17
`that has an altered sequence in its last 25 residues, that is truncated
`three residues prematurely, and that
`is wholly devoid of enzymatic
`activity. The crucial nature of the carboxy terminus of P450c17 is also
`shown by the complete absence of activity in the 9-bp, in-frame deletion
`of residues Asp487, Ser488, and Phe48919 and in a Gln461~stop muta-
`tion." Although these mutants retain the heme-binding region, these
`ostensibly minor alterations in the extreme carboxy terminus are cata-
`strophic for enzymatic activity.
`A computer model of human P450c17 suggests why the enzyme is
`so sensitive to alterations in its carboxy terminus," The last 48 residues
`of P450c17 are involved in an extended f3-sheet structure that folds down
`from the protein surface to form the "roof" of the active site, which is
`critical for proper substrate binding and subsequent catalysis. The CATC
`duplication after Ile479,30 the deletion of residues 487 to 489/9 and the
`mutant Gin461~stop/3 which all retain the heme-binding site, disrupt
`or lack this critical stretch of residues required for activity.
`The mutation delTG300,301 shifts the reading frame and alters the
`codon use beginning within exon 5.43 Mutation 7bp dup 120 changes the
`reading frame from exon 2 onward." The premature truncation Trp
`17~stop has been found in a homozygous" and a compound heterozy-
`gous" patient, and mutations Glu194~stopand Arg239~stopeach com-
`prise separate alleles in a single patient with complete 17u-hydroxylase
`deficiency." These three early truncations are not
`informative for
`structure/function studies because they delete the heme-binding region
`as well as residues important for substrate and redox partner binding.
`Two deleterious intronic mutations have been described, a G to T
`substitution at nucleotide +5 in intron 261 and an analogous G to A
`substitution at position + 5 of intron 7.66 These splice junction mutations
`delete exons 2 or 7, respectively, during RNA processing ("exon skip-
`ping"). The excision of these exons introduces early premature stop
`codons well before the heme-binding region. The deletion of a G within
`codon 438 has been found in a homozygous patient." This mutant gene
`encodes a protein in which the Gly-Pro-Arg-Ser-Cys-Ile motif at residues
`438--443 (the underlined Cys ordinarily donates the axial sulfhydryl to
`the heme iron) is converted to Asp-Leu-Ala-Pro-Val-Stop, which destroys
`all enzymatic activity. An ATG~ATC substitution in the intiating methi-
`onine codon has been described in a patient with complete 17u-hydroxy-
`lase deficiency and hypokalemic myopathy."
`
`Amino Acid Substitutions-Combined 17«-
`Hydroxylase/17,20-Lyase Deficiency
`
`Careful biochemical and computational analyses of mutant enzymes
`from patients with unusual phenotypes can provide insight into the
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`112
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`AUCHUS
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`functional roles of specific amino acids in P450c17. For example, the
`mutation His373Leu, when expressed in E. coli, lacks the classical P450
`difference spectrum." strong evidence that this protein does not bind
`heme properly. Modeling studies" predict that His373 lies distant from
`the heme moiety, suggesting that structural changes elsewhere in the
`His373Leu mutant secondarily abolish heme binding. In contrast, the
`mutation Arg440His18 lies two residues away from the heme-liganding
`Cys442, and the reason for
`loss of activity in this mutant is more
`straightforward. In most P450 enzymes, an analogous arginine residue
`in this position is critical for neutralization of a negative charge on a
`heme proprionate and stabilization of heme incorporation": hence, this
`mutation also interferes with heme binding.
`The mutation Serl06Pro, found in two apparently unrelated Gua-
`manian patients," introduces a helix-breaking proline into what is pre-
`dicted to be the B'-helix, near residues that form a lateral boundary of
`the substrate-binding pocket. P450c17 is sensitive to perturbations in
`this region, such that even the conservative replacement of Ser106 with
`Thr (the corresponding residue found in rainbow trout P450c1757) abol-
`ishes most enzymatic activity." Specifically, Ile112 is predicted to interact
`directly with substrate, suggesting why mutation insIle112 is devoid of
`measurable activity." Nearby, mutations Gly90Asp 67 and Arg96Trp 34 are
`predicted to reposition the second strand of f3-sheet I, containing the
`key residue Gly95. Computer simulations predict that 3f3-hydroxyl and
`3-keto groups of 115 and 114 substrates, respectively, form hydrogen bonds
`to the carbonyl group or the amide hydrogen of Gly95. 5, 41 The four
`mutants insIle112, Serl06Pro, Arg96Trp, and Gly90Asp may all primarily
`impair substrate binding.
`Three mutations that retain partial enzymatic activity have also
`been described. Mutations Tyr64Ser 27 and Pro342Thr1 retain approxi-
`mately 15% and 20% of wild-type activity, respectively. The loss of one
`of two contiguous Phe residues in the IlPhe53/54 mutations" destroys
`all but a trace of enzymatic activity,'? and this mutation has been found
`in other cases of 17-hydroxylase deficiency in [apan," suggesting a
`founder effect. The structural alterations responsible for
`the loss of
`activity in these mutants are not entirely clear, but these regions of the
`protein must be somewhat more tolerant of such structural changes
`than, for example, the active site and the heme-binding region.
`
`Mutations Causing Isolated 17,20-Lyase Deficiency
`
`The first patient with isolated 17,20-lyase deficiency in whom the
`CYP17 gene was sequenced proved to be a compound heterozygote for
`the Gln461~stop and Arg496Cys mutations." When studied in trans-
`fected cells, the Gln461~stop mutant was inactive, but the Arg496Cys
`mutant retained a small amount of 17a-hydroxylase and 17,20-lyase
`activities." When restudied as an adult," the patient's steroid hormone
`profile reflected nearly complete deficiencies of 17a-hydroxylase and
`17,20-lyase activities, consistent with the molecular genetics and bio-
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`MANAGEMENT OF HUMAN DEFICIENCIES OF P450cl7
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`113
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`chemistry of the mutant proteins. This case illustrates many of the
`pitfalls in the diagnosis of isolated 17,20-lyase deficiency and emphasizes
`that the clinical features, the molecular genetics, and the biochemistry
`of the mutant P450c17 protein(s) must all be congruent to ensure an
`accurate diagnosis.
`Recently, two 46,XY Brazilian patients presented with convincing
`clinical evidence of isolated 17,20-lyase deficiency, that is, genital ambi-
`guity and diminished C19 steroid production yet normal 17-hydroxycorti-
`costeroid production. One patient was homozygous for mutation
`Arg347His and the other for Arg358Gln, whereas each parent was het-
`erozygous for the respective mutant allele." When expressed in COS-l
`cells, the mutants hydroxylated progesterone and pregnenolone,'? but
`only a trace of 17,20-lyase activity could be reconstituted by coexpressing
`an excess of oxidoreductase and bs.2l Although 17a-hydroxypregneno-
`lone is a poor substrate for the mutant enzymes, competition experi-
`ments unequivocally show that the affinity of the mutant proteins for
`the wild-type
`to that of
`17a-hydroxypregnenolone is equivalent
`enzyme.s- " suggesting that arginines 347 and 358 do not lie in or near
`the active site.
`Computer modeling studies demonstrate that R347H and R358Q
`neutralize positive charges in the redox partner binding site.s, 20 Biochem-
`ical studies confirm that mutations R347H and R358Q impair interactions
`of P450c17 with its electron donor P450-oxidoreductase and with cyto-
`chrome bi!; therefore, isolated 17,20-lyase deficiency is not caused by an
`inability of the mutant enzymes to bind the intermediate 17a-hydroxy-
`pregnenolone but rather by subtle disturbances in interactions with
`redox partners.v 20, 21 Another patient subsequently shown to have iso-
`lated 17,20-lyase deficiency was found to harbor mutation F417C,8 which
`is predicted to lie on the edge of this redox partner binding surface."
`The biochemistry of the F417C mutant has not been studied in detail, so
`it is not known if the same mechanisms as for the R347H and R358Q
`mutants apply to F417C.
`A male pseudohermaphrodite with congenital methemoglobinemia
`owing to a mutation in the gene for cytochrome b, has been described."
`It is possible that this patient was incompletely virilized because of low
`(but not absent) testicular 17,20-lyase activity and testosterone deficiency
`in utero owing not to a P450c17 mutation but rather to the loss of b., the
`cofactor protein that stimulates 17,20-lyase activity. Neither circulating
`steroid hormone concentrations nor a genetic analysis of the CYP17gene
`were reported for this subject. If this patient has isolated 17,20-lyase
`deficiency owing to the loss of bs, the physiologic importance of b, in
`P450c17 chemistry would be proved.
`
`MANAGEMENT
`
`The child with 17-hydroxylase deficiency is chronically exposed to
`elevated circulating mineralocorticoid (DOC) concentrations but roughly
`normal amounts of glucocorticoids (as corticosterone). Mineralocorticoid
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`excess in the neonatal period is of no consequence because mineralocorti-
`coid (aldosterone) production is normally high in infants": however, as
`the child ages and begins to consume solid foods, sodium intake rises,
`and mineralocorticoid excess can lead to sodium retention, hypertension,
`and hypokalemia. The hypertension can become fixed if not treated for
`many years": hence, some control of DOC production is desirable.
`Moderation of dietary sodium content is prudent as an adjunct to phar-
`macologic therapy, which consists of glucocorticoid supplementation to
`reduce aberrant DOC production. Special considerations in the child
`with 17-hydroxylase deficiency include the avoidance of highly potent
`fluorinated glucocorticoids, such as dexamethasone, that have dispro-
`portionately large detrimental effects on linear growth and bone mineral
`accrual. Hydrocortisone administered in two or three divided doses will
`generally suffice, although direct comparison of steroid regimens in this
`uncommon disease are lacking. The glucocorticoid dose should be ti-
`trated to normalization of blood pressure and plasma potassium concen-
`trations, as well as restoring plasma renin activity to the measurable
`range as endpoints. The frank normalization of plasma DOC and cortico-
`sterone concentrations may require overtreatment with glucocorticoids."
`It is preferable to err on the side of undertreatment because the dire
`consequences of glucocorticoid excess throughout childhood are less
`desirable than modest mineralocorticoid excess.
`As is true for patients with Turner's syndrome, gonodal dysgenesis,
`androgen insensitivity, or some other steroid biosynthetic defects, pa-
`tients with 17-hydroxylase deficiency fail to exhibit pubertal develop-
`ment, and fetal
`testosterone deficiency causes all but the most mildly
`affected patients to present phenotypically as prepubertal females. In
`addition, the testosterone surge that occurs during the first year of life
`in 46,XY children is absent in 17-hydroxylase deficiency, which could
`theoretically impair responsiveness to testosterone later in life for mildly
`therapy is
`affected individuals. In most cases, estrogen replacement
`initiated at the time of expected puberty or on dia