`
`THE GENETICS,
`PATHOPHYSIOLOGY, AND
`MANAGEMENT OF HUMAN
`
`DEFICIENCIES OF P450c17
`
`Richard]. Auchus, MD, PhD
`
`P450c17 commands a central role in human steroidogenesis as the
`ualitative regulator of steroid hormone flux (Fig. 1). Analysis of P450c17
`eficiencies 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.
`
`P450017 AND CYP17
`
`Patients who carry the diagnosis of 17-hydroxylase deficiency har-
`bor alterations in the CYPI7 gene that encodes the P450c17 enzyme.
`P450c17 actually performs multiple chemical transformations. Human
`P450c17 170:-hydroxylates A5-pregnenolone and A‘-progesterone with
`roughly equal catalytic efficiency} 35 whereas all other reactions show
`prominent differences between A5 and A‘ substrates. The 17,20-lyase
`activity is roughly 50 times more efficient for the 17oL-hydroxypregneno-
`
`From the Division of Endocrinology and Metabolism,
`University of Texas Southwestern Medical School, Da
`
`artment of Internal Medicine,
`, Texas
`
`ENDOCRINOLOGY AND METABOLISM CLINICS OF NORTH AMERICA
`
`VOLUME 30 - NUMBER 1 0 MARCH 2001
`
`101
`
`ARGENTUM EX1026
`ARGENTUM EX1026
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`MANAGEMENT OF HUMAN DEFICIENCIES OF P450c17
`
`103
`
`lone—to-dehydroepiandrosterone (DHEA) reaction than for the 17oz-hy-
`droxyprogesterone-to-androstenedione reaction?’ 35 Although the rate of
`the lyase reaction can be increased more than 10-fold by the addition of
`cytochrome b5,3' 31/ 35 the A5 preference persists, and the lyase rate never
`quite achieves the rate of the hydroxylase reactions. In addition, human
`P450c17 16a-hydroxylates progesterone but not pregnenolonefi 37'
`‘*2 In
`the presence of cytochrome b5, P450c17 converts approximately 10% of
`pregnenolone substrate to a A15 andiene product,-”5 which is also formed
`by porcine P450c17 and acts as a pheromone precursor in pigs.“ 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 C0li,31' 35 the native P450c17 expressed in yeast
`microsomes3 or intact COS—1 cel1s,37/ 38 and that obtained from human
`tissues and cells3' 52 strengthens these conclusions.
`One consequence of this A5 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,” 5° whereas cortisol production remains
`relatively constant. Adrenarche is an exemplary manifestation of the
`biochemistry of P450c17, in which the 170:-hydroxylase and 17,20-lyase
`activities are differentially regulated. In fact, this dichotomy between
`adrenal 17ot-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 17a—hydroxylase and 17,20-lyase activities of neonatal pig
`testes suggests otherwise.” This controversy was settled when the cDNA
`for bovine P450c17 was expressed in COS—1 cells, conferring 17a—hydrox-
`ylase and 17,20-lyase activities to these nonsteroidogenic cells" and
`proving genetically that the 17oa—hydroxy1ase and 17,20-lyase enzymes
`
`Figure 1. Major steroidogenesis pathways in humans and feedback loops controlling
`glucocorticoid and mineralocorlicoid 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 (P450c11AS) 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
`P450c11[3 in the zona fasciculata also 18-hydroxylates (18OH§ DOC and corticosterone as
`minor products.
`
`Page 3
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`Page 3
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`
`
`104
`
`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-oxidoreductase” and the addition3‘r35' 5“ or coex-
`
`pression3 of cytochrome b5 preferentially augments the 17,20-lyase activ-
`ity, and phosphory1ation7'76 also selectively enhances 17,20-lyase activity.
`Recent data showing high expression of b5 in the zona reticularis of
`monkeys“ and humans“ suggest that the developmentally regulated
`expression of b5 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/‘I 57 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?’ 2° 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 C19 steroids. Curiously, the adrenal glands of patients
`with 17-hydroxylase deficiency are similar to those of rodents, which do
`not express P450c17,"3 such that rodents rely on corticosterone as their
`principal glucocorticoid, and their adrenal glands cannot make C19 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/*5 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 11-deoxycorticosterone
`
`Page 4
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`MANAGEMENT OF HUMAN DEFICIENCIES OF P450cl7
`
`105
`
`(DOC), must accumulate, as well as unusual metabolites, such as 18-
`hydroxycorticosterone33 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
`menstruation in 46,XX subjects; the severity of the hypertension and
`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-
`ciency53 or maternal androgen excess.” This heterogeneity has not been
`completely explained, but many factors, including the severity of the
`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 170:-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 17a—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-
`quently,
`the diagnosis is often not entertained until hypertension,
`hypokalemia, or pubertal delay is evaluated during adolescence or early
`Page 5
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`Page 6
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`MANAGEMENT OF HUMAN DEFICIENCIES OF P450cl7
`
`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.” In addition, 17—hydroxylase deficiency, in distinct con-
`trast to 11-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 11-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 11-hydroxylase deficiency, whereas plasma 18—hydroxy-
`DOC concentrations are not elevated.” The reason for this apparent
`discrepancy is that P450c11[3 (the product of the CYPHB1 gene) is
`not exclusively an 11B-hydroxylase but exhibits weak 18-hydroxylase
`activity“? 64 (Fig. 2) and trace amounts of aldosterone synthase activity.”
`The low 18-hydroxy—DOC production in 11-hydroxylase deficiency, de-
`spite enormous DOC concentrations, is compelling genetic evidence that
`P450c11B 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 CYP11B2/
`11B1 gene“ lead to excessive 18—oxygenated steroid production.“ Pa-
`tients with 17—hydroxylase deficiency with paradoxically measurable, if
`
`Figure 2. Physiologic disturbances in glucocoiticoid, mineralocorticoid, and sex steroid
`homeostasis in complete 17-hydroxylase deficiency. The inability to 17a-hydroxylate C2,
`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 (18OH)-DOC and 18-OH-corticosterone. Because 17-hydroxy
`(17OH)-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.
`
`Page 7
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`Page 7
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`
`
`Table 1. COMPARISON OF COMBINED 1704-HYDROXYLASE/17,20-LYASE DEFICIENCY AND ISOLATED 17,20-LYASE DEFICIENCY
`
`Deficiency
`
`Plasma Steroids
`
`Urinary Steroids
`
`Clinical Presentation
`
`Combined 170:-hydroxylase and
`17,20-lyase
`
`Isolated 17,20—lyase
`
`I 17-OH—steroids, DHEA,
`androgens, estrogens
`T Progesterone, DOC, DOC
`metabolites, corticosterone
`Normal 17—OH—steroids
`I DHEA, androgens
`T 17-OH1’/AD (>10 after hCG)
`
`I 17-OHCS, 17-KS pregnanetriolone
`T Tetrahydro-DOC
`
`Hypertension /hypokalemia,
`sexual infantilism
`
`I 17-KS
`
`Ambiguous genitalia in 46,XY
`
`17-OHP = 17—hydroxyprogesterone; AD : androstenedione; hCG : human chorionic gonadotropin; DHEA : dehydroepiandrosterone; 17—OHCS : 17-hydroxycorti-
`costeroids;17—KS : 17-ketosteroids; DOC : 11-deoxycorticosterone.
`
`Page 8
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`MANAGEMENT OF HUMAN DEFICIENCIES OF l’450cl7
`
`109
`
`Table 2. COMPARISON OF STEROID PROFILES IN ADULTS WITH 17-, 11-, AND 21-
`HYDROXYLASE DEFICIENCES
`
`Type of
`Deficiency
`17—OH
`11-OH
`21—OH
`Normal
`
`DOC
`ng/dL
`25-500
`50 to >1000
`10-100
`2-20
`
`18—OH-DOC Corticosterone
`ng/dL
`ng/dL
`100-600
`4000-40,000
`<10
`<200
`3-20
`100-500
`1-20
`100-500
`
`18-OH-corticosterone Aldosterone
`ng/dL
`ngIdL
`60-1000
`< 10
`<10
`<3
`10-200
`10-60
`10-40
`10-30
`
`DOC = 11—deoxyco1-ticosterone;18—OH-DOC = 18-hydroxy-DOC; OH = hydroxylase.
`Data adapted from Kater CE, Biglieri EG: Disorders of steroid 17 alpha-hydroxylase deficiency. Endocrinol Metab
`Clin 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 P450c11B. The latter hypothesis is consistent
`with the finding that most 17-hyclroxylase deficiency cases with measur-
`able aldosterone production are from Japan.” 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 C19
`steroids to those of C21 steroids is low (reflecting low 17,20—lyase activ-
`ity).‘3 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 170:-hydroxylase
`
`Page 9
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`Page 9
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`
`
`Table 3. COMPARISON OF MINERALOCORTICOID EXCESS STATES WITH SUPPRESSED PLASMA FIENIN ACTIVITY
`
`Disease
`
`Laboratory Findings
`
`Key Features
`
`*17-Hydroxylase deficiency
`
`Primary aldosteronism
`
`DOC-oma
`
`"Syndrome of apparent Inineralocorticoid
`excess
`*G1ucocorticoid—remediable aldosteronism
`
`*Cushing syndrome
`
`*Glucocorticoid resistance
`
`T DOC, corticosterone, and 18-OH
`derivatives
`I 17-OH-steroids, C19 steroids
`J, Aldosterone
`TA1dosterone, often T 18-OH-
`corticosterone
`T DOC, normal aldosterone,
`variable other steroids
`,L Cortisone metabolites
`I Aldosterone
`T 18-OH- and 18—oxocortiso1,
`variable aldosterone
`T Cortisol production, Variable
`other steroids
`T Cortisol production
`T C19 steroids
`
`Sexual infantilism/ambiguity
`
`Normal cortisol axis
`
`Normal cortisol axis
`
`Dexamethasone suppression of
`hypertension, kaluresis
`Dexamethasone suppression of
`hypertension, kaluresis
`Symptoms of cortisol excess,
`variable mineralocorficoid excess
`Symptoms of cortisol insufficiency,
`androgen excess
`
`Molecular Basis
`
`CYP1 7 mutations
`
`Unknown
`
`Unknown
`
`HSD1 1 B2 mutations
`
`C YP11B2/11 B1 chimeric gene
`
`Unknown
`
`GR mutations, other
`
`"ACTH—dependent mjneralocorticoid excess.
`DOC = 11-deoxycorticosterone; OH = hydroxy; GR = glucocorticoid receptor.
`
`Page 10
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`MANAGEMENT OF HUMAN DEFICIENCIES OF P450c17
`
`111
`
`deficiency? A 4—bp duplication of the sequence CATC following Ile4793°
`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 B—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 Ile479,3° the deletion of residues 487 to 489,” and the
`mutant Gin461——>stop,73 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.7° The premature truncation Trp
`17—>stop has been found in a homozygous“ and a compound heterozy-
`gous“ patient, and mutations Glu194—>stop and Arg239—>stop each com-
`prise separate alleles in a single patient with complete 17a-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 2“ and an analogous G to A
`substitution at position + 5 of intron 7.56 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-(_3y§-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 17oL—hydroxy—
`lase deficiency and hypokalernic myopathy.58
`
`Amino Acid Substitutions—Combined 17a-
`
`Hydroxylase/17,20-Lyase Deficiency
`
`Careful biochemical and computational analyses of mutant enzymes
`from patients with unusual phenotypes can provide insight into the
`
`Page 11
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`Page 11
`
`
`
`112
`
`AUCHUS
`
`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 studies5 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 Arg440His13 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 Ser106Pro, 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 insI1e112 is devoid of
`measurable activity.” Nearby, mutations Gly9OAsp67 and Arg96Trp34 are
`predicted to reposition the second strand of B—sheet 1, containing the
`key residue Gly95. Computer simulations predict that 3B—hydroxyl and
`3-keto groups of A5 and A4 substrates, respectively, form hydrogen bonds
`to the carbonyl group or the amide hydrogen of Gly95.5' 41 The four
`mutants insIle112, Ser106Pro, Arg96Trp, and Gly9OAsp may all primarily
`impair substrate binding.
`Three mutations that retain partial enzymatic activity have also
`been described. Mutations Tyr64Ser27 and Pro342Thr1 retain approxi-
`mately 15% and 20% of wild-type activity, respectively. The loss of one
`of two contiguous Phe residues in the APhe53/ 54 mutation” destroys
`all but a trace of enzymatic activity,” and this mutation has been found
`in other cases of 17-hydroxylase deficiency in Iapan,“ 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 17<x—hydroxylase and 17,20-lyase
`activities.” When restudied as an adult,” the patient’s steroid hormone
`profile reflected nearly complete deficiencies of 17oz-hydroxylase and
`17,20-lyase activities, consistent with the molecular genetics and bio-
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`MANAGEMENT OF HUMAN DEFICIENCIES OF P450c17
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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—1
`cells, the mutants hydroxylated progesterone and pregnenolone,2° but
`only a trace of 17,20-lyase activity could be reconstituted by coexpressing
`an excess of oxidoreductase and b5.“ Although 17oL—hydroxypregneno-
`lone is a poor substrate for the mutant enzymes, competition experi-
`ments unequivocally show that the affinity of the mutant proteins for
`17oL-hydroxypregnenolone is equivalent
`to that of
`the wild-type
`enzyme,“ 21 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.5' 2° Biochem-
`ical studies confirm that mutations R347H and R358Q impair interactions
`of P450c17 with its electron donor P450-oxidoreductase and with cyto-
`chrome b521; therefore, isolated 17,20-lyase deficiency is not caused by an
`inability of the mutant enzymes to bind the intermediate 17oz-hydr0xy-
`pregnenolone but rather by subtle disturbances in interactions with
`redox partners?’ 20' 21 Another patient subsequently shown to have iso-
`lated 17,20-lyase deficiency was found to harbor mutation F417C,3 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 b5 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 b5, the
`cofactor protein that stimulates 17,20-lyase activity. Neither circulating
`steroid hormone concentrations nor a genetic analysis of the CYP17 gene
`were reported for this subject. If this patient has isolated 17,20-lyase
`deficiency owing to the loss of b5, the physiologic importance of b5 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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`AUCHUS
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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 Tumer’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
`affected individuals. In most cases, estrogen replacement therapy is
`initiated at the time of expected puberty or on diagnosis if that time has
`already passed. Estrogen replacement not only allows the development
`of female secondary sexual characteristics but stimulates the increase in
`bone mass that normally occurs during puberty.“ In a few cases, testos-
`terone supplementation has been given to mildly affected 46,XY patients
`to stimulate penile development”; however, as is true for patients with
`partial androgen insensitivity, the rearing of these individuals as males
`and the choice of appropriate therapy are complex decisions that unfor-
`tunately may yield less than satisfactory results.
`The treatment of 17-hydroxylase deficiency in the adult patient
`strives to achieve four goals: (1) reduction of the production or action of
`mineralocorticoids; (2) avoidance of the untoward effects of glucocorti-
`coid excess; (3) replacement of sex steroids; and (4) prevention of the
`long-term consequences of the abnormal physiology. Al