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Division of Pediatric Endocrinology (P.C.W.), University of Texas Southwestern Medical Center, Dallas, Texas 75390-9063; and Division of Pediatric Endocrinology (P.W.S.), North Shore University Hospital and New York University School of Medicine, Manhasset, New York 11030
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Abstract |
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 Top Abstract I. IntroductionII. Biochemistry of CAHIII. Pathophysiology of CAHIV. Diagnosis of 21-Hydroxylase...V. TreatmentVI. Molecular Genetic AnalysisVII. SummaryReferences |
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I. Introduction |
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TopAbstractI. IntroductionII. Biochemistry of CAHIII. Pathophysiology of CAHIV. Diagnosis of 21-Hydroxylase...V. TreatmentVI. Molecular Genetic AnalysisVII. SummaryReferences |
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The fundamental defect among patients with CAH due to 21-hydroxylase deficiency is that they cannot adequately synthesize cortisol. Inefficient cortisol synthesis signals the hypothalamus and pituitary to increase CRH and ACTH, respectively. Consequently, the adrenal glands become hyperplastic. But rather than cortisol, the adrenals produce excess sex hormone precursors that do not require 21-hydroxylation for their synthesis. Once secreted, these hormones are further metabolized to active androgens—testosterone and dihydrotestosterone—and to a lesser extent estrogens—estrone and estradiol. The net effect is prenatal virilization of girls and rapid somatic growth with early epiphyseal fusion in both sexes. About three-quarters of patients cannot synthesize sufficient aldosterone to maintain sodium balance and are termed "salt wasters." This predisposes them to episodically develop potentially life-threatening hyponatremic dehydration.
Patients with sufficient aldosterone production and no salt
wasting who have signs of prenatal virilization and/or markedly
increased production of hormonal precursors of 21-hydroxylase
(e.g., 17-hydroxyprogesterone), are termed "simple
virilizers." In addition, a mild nonclassic form of the disorder is
recognized in which affected females have little or no virilization at
birth (Table 1
).
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II. Biochemistry of CAH |
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TopAbstractI. IntroductionII. Biochemistry of CAHIII. Pathophysiology of CAHIV. Diagnosis of 21-Hydroxylase...V. TreatmentVI. Molecular Genetic AnalysisVII. SummaryReferences |
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It is clear that StAR is not the only protein that mediates cholesterol transfer across the mitochondrial membrane. Another protein that appears necessary (but not sufficient, at least in the adrenals and gonads) for this process is the so-called peripheral benzodiazepine receptor, an 18-kDa protein in the mitochondrial outer membrane that is complexed with the mitochondrial voltage-dependent anion carrier in contact sites between the outer and inner mitochondrial membranes (8). This protein does not appear to be directly regulated by typical trophic stimuli, but it is stimulated by endozepines, peptide hormones also called diazepam-binding inhibitors. Endozepines may be regulated by ACTH to some extent, but not with a rapid time course. Thus far, it is not yet clear whether there is a direct physical interaction between StAR and the peripheral benzodiazepine receptor.
The first enzymatic step in steroid synthesis (Fig. 1) is the conversion of cholesterol, a
C27 compound, to the C21
steroid pregnenolone (reviewed in Ref. 9). This is catalyzed by the
mitochondrial cytochrome P450 enzyme CYP11A (P450 scc, cholesterol
desmolase, side-chain cleavage enzyme; see Ref. 10 for further
description of the CYP and P450 enzyme terminology). Pregnenolone is
the common precursor for all other steroids and, as such, may undergo
metabolism by several other enzymes.
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To produce cortisol, the major glucocorticoid in man, CYP17
(P450c17, 17
-hydroxylase/17, 20 lyase) in the endoplasmic reticulum
of the zona fasciculata and zona reticularis converts pregnenolone to
17-hydroxypregnenolone (14). 3ß-Hydroxysteroid dehydrogenase in
the zona fasciculata utilizes 17-hydroxypregnenolone as a substrate,
producing 17-hydroxyprogesterone. The latter is 21-hydroxylated by
CYP21 to form 11-deoxycortisol, which is converted to cortisol by
CYP11B1 (P450c11, 11ß-hydroxylase) in mitochondria.
In the zona reticularis of the adrenal cortex and in the gonads,
the 17,20-lyase activity of CYP17 converts 17-hydroxypregnenolone to
dehydroepiandrosterone (DHEA, a C19 steroid and
sex hormone precursor). DHEA is further converted by 3ß-HSD to
androstenedione. In the gonads, this is reduced by 17ß-hydroxysteroid
dehydrogenase to testosterone [there are several isozymes of
17ß-hydroxysteroid dehydrogenase, some of which possess both
oxidative and reductive activity (15)]. In pubertal ovaries, aromatase
(CYP19, P450c19) can convert androstenedione and testosterone to
estrone and estradiol, respectively (16). Testosterone may be further
metabolized to dihydrotestosterone by steroid 5-reductase in
androgen target tissues (17).
B. Regulation of adrenal steroid secretion
1. Cortisol secretion. Cortisol secretion is regulated mainly
by ACTH. ACTH is a 39-amino acid peptide that is produced in the
anterior pituitary. It is synthesized as part of a larger mol wt
precursor peptide, POMC. This peptide is also the source of
ß-lipotropin (ß-LPH). In addition, ACTH and ß-LPH are cleaved
further to yield -MSH and ß-MSH, 
-LPH, ß- and -endorphin,
and enkephalin. The POMC precursor peptide is found in a variety of
extrahypothalamic tissues, including the gastrointestinal tract,
numerous tumors, and the testis. It is secreted in small amounts from
the anterior pituitary gland and does not bind significantly to the
ACTH receptor. Another pro-ACTH fragment, corticotropin-like
intermediate lobe peptide (CLIP), is made in the rodent anterior
pituitary, but not in the normal human pituitary (18).
ACTH acts through a specific G protein-coupled receptor to increase levels of cAMP (19). cAMP has short-term (minutes to hours) effects on cholesterol transport into mitochondria (6) but longer term (hours to days) effects on transcription of genes encoding the enzymes required to synthesize cortisol (20). The transcriptional effects occur, at least in part, through increased activity of protein kinase A, but it is not known whether the targets of this kinase act directly or indirectly on CYP21 (see Section VI.D.3). ACTH also influences the remaining steps in steroidogenesis as well as the uptake of cholesterol from plasma lipoproteins. It also maintains the size of the adrenal glands. In addition to these effects on the adrenal gland, it stimulates melanocytes and results in hyperpigmentation when secreted in excess, as occurs in Addison’s disease.
CRH is the principal hypothalamic factor that stimulates the pituitary production of ACTH (21, 22). Vasopressin, a peptide product of the posterior pituitary gland, also stimulates ACTH release by acting synergistically with CRH and is an important physiological regulator of ACTH (23). CRH is produced in the paraventricular nuclei of the hypothalamus and is also found in other parts of the central nervous system and in other locations such as peripheral leukocytes. Paracrine action of hypothalamic peptides, e.g., vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), plays a role in CRH release (24). Hypothalamic CRH is transported to the anterior pituitary cells by the hypophysial portal vessels. CRH activates ACTH secretion via a specific receptor coupled to cAMP-dependent signaling. CRH is secreted in a pulsatile fashion that results in the episodic secretion of ACTH and in the diurnal variation of cortisol secretion. The magnitude of the cortisol response to each ACTH burst remains relatively constant. Therefore, it is the number of secretory periods, rather than the magnitude of each pulse of CRH and ACTH, that determines the total daily cortisol secretion.
Numerous factors, such as metabolic, physical, or emotional stress, influence levels of glucocorticoid secretion, mediated by ACTH secreted in response to hypothalamic secretion of CRH and vasopressin. As noted above, paracrine action of various peptides may contribute to modulation of hormone production in the hypothalamus, pituitary, and adrenal. Cortisol is the primary negative regulator of resting activity of the hypothalamic-pituitary-adrenal (HPA) axis through negative feedback on ACTH and CRH secretion. Furthermore, it may inhibit some of the higher cortical activities that lead to CRH stimulation. The negative feedback effects of cortisol are exerted at the level of both the hypothalamus and the pituitary and are mediated by Type II corticosteroid receptors (i.e., classic glucocorticoid receptors) (25).
Whether and to what extent direct glucocorticoid feedback on the adrenal cortex itself regulates cortisol synthesis is unclear. In vitro studies using rat adrenocortical cells suggest that corticosterone may act to inhibit steroidogenesis (26). Northern blotting demonstrates that glucocorticoid receptors are expressed in human adrenals (27), but a physiological role in direct negative regulation of cortisol secretion has not been demonstrated.
2. Aldosterone secretion. The rate of aldosterone synthesis, which is normally 100- to 1,000-fold less than that of cortisol synthesis, is regulated mainly by angiotensin II and potassium levels, with ACTH having only a short-term effect (28). Angiotensin II occupies a G protein-coupled receptor-activating phospholipase C (29). The latter protein hydrolyzes phosphatidylinositol bisphosphate to produce inositol triphosphate and diacylglycerol, which raise intracellular calcium levels and activate protein kinase C and calmodulin dependent protein (CaM) kinases. Similarly, increased levels of extracellular potassium depolarize the cell membrane and increase calcium influx through voltage-gated L-type calcium channels (30). Phosphorylation of as yet unidentified factors by CaM kinases increases transcription of the aldosterone synthase (CYP11B2) enzyme required for aldosterone synthesis (28); as yet, the pathways influencing 21-hydroxylase (CYP21) expression in the zona glomerulosa have not been elucidated.
C. Abnormal steroids in 21-hydroxylase deficiency
1. Elevated 17-hydroxyprogesterone. The most
characteristic biochemical abnormality in 21-hydroxylase deficiency is
elevation of 17-hydroxyprogesterone (17-OHP), the main substrate for
the enzyme. Basal serum 17-OHP values usually exceed 10,000 ng/dl,
although about 10% of severely affected infants have low initial
levels in the newborn period (31), especially if levels are obtained on
the first day of life. Differentiation of 21-hydroxylase deficiency
from other forms of CAH may be accomplished by both clinical features
of the disease (Table 2) and by the complete
adrenocortical hormone profile comparing precursor to product ratios
after ACTH stimulation. It is important to realize that without a
complete adrenocortical profile, other steroidogenic defects—both
3ß-HSD (32) and 11ß-hydroxylase deficiency (34)—may be
misdiagnosed as 21-hydroxylase deficiency. This has significant bearing
on medical therapy since 11ß-hydroxylase patients are often
hypertensive and require specific therapy for this problem. Moreover,
these assays should be performed in laboratories with high standards
for quality control, including preliminary chromatography, to avoid
problems of cross-reactivity when some hormone levels are extremely
high (33). This can be a serious concern when the choice of laboratory
is limited in a managed care environment.
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2. Other abnormal steroids. Other hormones that are elevated in untreated CAH include progesterone, androstenedione, and, to a lesser extent, testosterone. An abnormal steroid, 21-deoxycortisol, is characteristically elevated (39, 40, 41). DHEA, the main adrenal 19-carbon steroid product, is not a good marker of 21-hydroxylase activity. DHEA-sulfate (DHEAS) binds with high affinity to albumin, has a long plasma half-life, and as such is not very responsive to acute ACTH stimulation. Diagnostic assays are discussed in Section IV.C.
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III. Pathophysiology of CAH |
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TopAbstractI. IntroductionII. Biochemistry of CAHIII. Pathophysiology of CAHIV. Diagnosis of 21-Hydroxylase...V. TreatmentVI. Molecular Genetic AnalysisVII. SummaryReferences |
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). During the 7th
week, the male gonads begin to differentiate under the influence of a
cascade of testis-determining genes (reviewed in Refs. 42, 43). In
contrast, the recently characterized signaling molecule WNT-4 plays an
active role in ovarian development (44). Ovaries are recognizable at
about 10 weeks. If there is no secretion of anti-Müllerian
hormone (AMH), a glycoprotein factor synthesized by the Sertoli cells
of the testis (45), development of the Müllerian ducts proceeds
and female internal structures—the Fallopian tubes, uterus, cervix and
upper vagina—are formed (Figs. 3 and 4). In contrast, development of male genital
structures derived from the Wolffian ducts, including the epididymis,
ductus deferens, ejaculatory ducts, and seminiferous tubules, requires
high local concentrations of testosterone secreted from Leydig cells of
the testis beginning at about 7 weeks; in the absence of testosterone,
Wolffian ducts regress. External genital structures are also
bipotential in early gestation and differentiate as male under the
influence of 5-dihydrotestosterone (reviewed in Ref. 17), which must
interact with an intact androgen receptor (Figs. 3 and 4)(46).
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-hydroxylation; the
placenta utilizes 16-DHEAS to produce estriol (48), a traditional
marker of fetal viability. The permanent, or adult, adrenal cortex is
formed in the 9th to 10th week by a second migration of cells that
surround the fetal cortex. At term, the fetal cortex is approximately
10 times the size of the adult cortex, weighing about the same as adult
adrenals, or up to 10 g, but it involutes rapidly in the neonatal
period (49). Thereafter, the permanent cortex assumes the steroidogenic
functions and develops the three-zoned organization of the adult gland.
Several transcription factors are known to be critical for adrenal
development. Steroidogenic factor-1 (SF-1, also called Ad4BP, reviewed
in Ref. 50), induces genes involved in steroid synthesis, and is in
turn negatively regulated by DAX-1, the gene affected in congenital
adrenal hypoplasia (51). Human fetal adrenal development is
regulated primarily by fetal pituitary ACTH. ACTH is not a mitogen
per se; rather its actions on the fetal adrenal cortex are
mediated in autocrine/paracrine fashion by several growth factors. In
cultured human fetal adrenal cortical cells, epidermal growth factor
(EGF), basic fibroblast growth factor (bFGF), human CG (hCG), and
insulin-like growth factors I and II (IGF-I and -II) are mitogenic,
whereas activin and transforming growth factor-ß (TGFß) inhibit
proliferation. IGF-II, activin, and TGFß also modulate
ACTH-stimulated steroidogenesis (reviewed in Ref. 49). In the absence
of ACTH, as in anencephaly, the fetal adrenal involutes in the second
trimester (52). In addition, catecholamines and neuropeptides secreted by the adrenal medulla, as well as direct innervation of the adrenal cortex, may influence development of the cortex (reviewed in Ref. 53).
Anatomic hyperplasia of the adrenal is not seen invariably in steroid 21-hydroxylase deficiency (54). The diagnostic utility of ultrasound diagnosis of CAH may be improved, at least in neonates, by examining not only size but also shape, surface contours, and echogenicity (55). Steroid treatment can reverse the structural abnormalities seen with sonography (55).
Anatomic and physiological data indicate that the hypothalamic-pituitary-adrenal axis does not function until about the eighth week of gestation. Experience with prenatal treatment of CAH (see below, Section V.G), however, suggests that dexamethasone must be administered to the pregnant woman at risk for an affected child as early as possible in the first trimester if virilization of an affected girl is to be prevented. What, then, is the mechanism for dexamethasone’s early action? Is there another ACTH-independent glucocorticoid feedback pathway responsible for fetal adrenal steroid production? Could dexamethasone exert direct suppressive effects on the fetal adrenal? These questions remain unanswered.
C. Adrenarche
Beginning at 5–8 yr of age, there is an increase in the size of
the zona reticularis, correlating with a rise in serum DHEAS and a
modest increase in linear growth rate (56). This process, termed
adrenarche, occurs independently of changes in ACTH, cortisol, or
aldosterone production. Although there has been speculation about a
separate adrenal androgen-stimulating hormone (57), no such factor has
been identified. Premature adrenarche with mildly elevated DHEAS and
more marked elevation of 17-OHP is a known manifestation of untreated
nonclassic CAH (58). On the other hand, DHEAS is suppressed in treated
children with classic CAH (59), probably due to exogenous
glucocorticoid suppression of the adrenal (60).
D. Prenatal virilization
Adrenal secretion of excess androgen precursors does not
significantly affect male sexual differentiation. In females affected
with CAH, however, the urogenital sinus is in the process of septation
when the fetal adrenal begins to produce excess androgens; levels of
circulating adrenal androgens are apparently sufficiently high to
prevent formation of separate vaginal and urethral canals. Further
interference with normal female genital anatomy occurs as
adrenal-derived androgens interact with genital skin androgen receptors
and induce clitoral enlargement, promote fusion of the labial folds,
and cause rostral migration of the urethral/vaginal perineal orifice.
However, internal Wolffian structures, such as the prostate gland and
spermatic ducts, are usually not virilized, presumably because
development of the Wolffian ducts requires markedly higher focal
concentrations of testosterone than the external genitalia. This is
supported by animal studies showing that unilateral castration causes
ipsilateral mesonephric duct involution (61). Nevertheless, severely
affected females may occasionally have some development of typically
male internal genital structures; carcinoma of prostate tissue has been
reported in an affected female (62).
Thus, the typical result in severely affected girls is ambiguous or
male-appearing external genitalia with perineal hypospadias, chordee,
and undescended testes (63). The severity of virilization is often
quantitated using a five-point scale developed by Prader (Figs. 3
and 4) (64). Not all classic CAH females develop the same degree of genital
ambiguity. One might speculate that the physical signs of androgen
excess are dependent not only on direct adrenal secretion of androgen
precursors, but also on the efficiency with which such hormones are
converted to more potent products, such as dihydrotestosterone by
peripheral enzymes such as 5-reductase (17). Additionally, the
concentration (65) and transcriptional activity (66) of androgen
receptors (both of which are influenced by a highly polymorphic CAG
repeat sequence within the coding region) may play a further role in
determining genital phenotype.
As another presumed effect of prenatal exposure to excess androgens, both male and female affected infants are longer than average at birth (67). Moreover, infant girls with CAH have higher than typical LH levels—into the range expected for healthy infant boys—presumably due to exposure to higher than normal levels of prenatal androgens and/or other sex hormones (68). Although there have been no studies of LH pulsatility or responsiveness to GnRH in infants with CAH, women with well controlled classic CAH, but not those with nonclassic CAH, have exaggerated LH responses to GnRH and increased production of ovarian androgens. This is consistent with the idea that early exposure to androgens or progestins causes permanent abnormalities in the hypothalamic-pituitary-gonadal axis in CAH women (69) (see Section III.G).
E. Salt wasting
Among classic CAH patients, about three-fourths cannot synthesize
adequate amounts of aldosterone due to severely impaired
21-hydroxylation of progesterone. Aldosterone is essential for normal
sodium homeostasis; deficiency of this hormone results in sodium loss
via the kidney, colon, and sweat glands (70).
Severely affected patients invariably have concomitant cortisol deficiency that exacerbates the effects of aldosterone deficiency. Glucocorticoids normally increase cardiac contractility, cardiac output, sensitivity of both the heart and the vasculature to the pressor effects of catecholamines and other pressor hormones, and work capacity of skeletal muscles (71). In the absence of glucocorticoids, cardiac output decreases. This decreases glomerular filtration, leading to an inability to excrete free water and consequent hyponatremia. Thus, shock and severe hyponatremia are much more likely in 21-hydroxylase deficiency, in which both cortisol and aldosterone biosynthesis are affected, than in (for example) aldosterone synthase deficiency, in which only one biosynthetic pathway is impaired (72).
Although catecholamine secretion has not, to our knowledge, been studied in patients with CAH, high levels of glucocorticoids are required for normal development of the adrenal medulla and for expression of the enzymes required to synthesize catecholamines (73). Indeed, mice with 21-hydroxylase deficiency exhibit abnormal development of the adrenal medulla and secrete reduced levels of catecholamines (74). Catecholamine deficiency could further exacerbate the shock engendered by glucocorticoid and mineralocorticoid deficiency.
In addition, accumulated steroid precursors may directly antagonize the mineralocorticoid receptor and exacerbate mineralocorticoid deficiency, particularly in untreated patients (75). Progesterone is well known to have antimineralocorticoid effects (76, 77, 78, 79), and it and/or a metabolite are likely culprits in this phenomenon. However, there is as yet no evidence that 17-OHP has direct or indirect antimineralocorticoid effects.
Salt wasting may include such nonspecific symptoms as poor appetite, vomiting, lethargy, and failure to gain weight. Severely affected patients with CAH usually present at 1–4 weeks of age with hyponatremia, hyperkalemia, hyperreninemia (see Section IV.C.2) and hypovolemic shock. These "adrenal crises" may prove fatal if proper medical care is not delivered. This problem is particularly critical in infant boys who have no genital ambiguity to alert physicians to the diagnosis of CAH before the onset of dehydration and shock (80). The mortality rate for CAH remains high in such patients, as suggested by the relative paucity of male patients identified in case reports (81). It is for this reason that many states in the United States and a number of countries have adopted newborn screening for CAH (see Section IV.B).
The rapidity of onset and severity of a salt wasting crisis may reflect the individual’s ancillary homeostatic mechanisms for sodium and fluid conservation. Such factors might include the concentration and transcriptional activity of mineralocorticoid receptors in the kidney and elsewhere, and the ability to increase vasopressin or decrease atrial natriuretic factor (82) in response to volume contraction.
Siblings may be discordant for salt wasting (83). Furthermore, CAH patients known to have severe salt wasting episodes in infancy and early childhood may show improved sodium balance and relatively more efficient aldosterone synthesis with age. Unrelated individuals carrying identical mutations may manifest different degrees of salt wasting (84). Although explanations for these observations are not immediately apparent, both genetic and nongenetic factors may contribute to the presence or absence of the salt wasting trait. Extraadrenal 21-hydroxylase has been detected by in vivo metabolic studies (85), but molecular genetic investigation has yielded contradictory results as to whether CYP21 could be a source for this activity (86, 87, 88, 89). Other enzymes with 21-hydroxylase activity have not been identified in humans, although such enzymes have been identified in rabbit liver (90).
F. Postnatal signs of androgen excess
Ongoing adrenal sex steroid production in the untreated or
incompletely treated patient causes several problems. Boys have
inappropriately rapid somatic growth with advancement of epiphyseal
maturation, although this may not be apparent in the first 18 months of
life (91). Pubic hair and apocrine body odor develop, and penile size
increases without testicular enlargement.
Girls may show similar signs of sex steroid excess as well as progressive clitoral enlargement. In adolescence, poorly controlled girls manifest acne, hirsutism, and ovarian dysfunction (see below).
There is considerable interindividual variation in pre- and postnatal signs of androgen excess. This may be attributed directly to differences in the absolute levels of androgen precursors secreted by the affected adrenals, or to the efficiency of conversion of precursors to more potent androgens. Alternatively, variations in androgen receptor expression or activity may contribute to phenotype. For example, expansion of the CAG repeat sequence in exon 1 results in decreased androgen receptor transactivation of target DNA sequences (66). In a correlative study, higher hirsutism scores correlated with fewer CAG repeats in women with idiopathic hirsutism (92).
Although childhood somatic growth is excessive in CAH patients (67), adult height is often suboptimal compared with the surrounding healthy population and with parentally determined target height (93, 94, 95, 96, 97, 98, 99). Whereas untreated patients grow rapidly, patients treated with excessive doses of glucocorticoids may suffer growth retardation. This is discussed below in Section V.A.
Although androstenedione is elevated, DHEAS is suppressed in CAH children (59). This is likely due to exogenous glucocorticoid suppression of the adrenal (60). An adrenal androgen-stimulating hormone (AASH) separate from ACTH has been postulated (100), but this hypothesis cannot be tested in any definitive manner based on data from CAH patients.
G. Reproductive function in classic CAH
1. Females. Reproductive problems for women with CAH become
apparent in adolescence. The average age at which menarche occurs in
inadequately treated girls is late compared with healthy peers (101).
Such girls and women with CAH often have a clinical picture similar to
polycystic ovarian syndrome with sonographic evidence of multiple
cysts, anovulation, irregular bleeding, and hyperandrogenic symptoms
(102). Moreover, a significant reduction in insulin sensitivity,
although not clinical diabetes, is found among young women with
nonclassic CAH as compared with controls of similar age and weight
(103).
The basis for these problems is not precisely known. Several hypotheses have been advanced: 1) Hypothalamic aromatization of excess adrenal androstenedione might interfere with LH-releasing hormone secretion (104). 2) Excess adrenal progesterone might act as a "mini-pill" to inhibit normal cyclicity (101), or it might antagonize estrogen effects (101, 105). 3) Elevated progestins or sex steroids could induce abnormal ovarian function by programming the hypothalamus early in development (69). 4) Androgen excess might directly damage the ovaries. 5) Adrenal rest tissue might displace normal gonadal parenchyma.
The majority of women with CAH eventually undergo menarche. In general, the regularity of menses depends on the adequacy of treatment. A small proportion of women do not undergo menarche and are unable to suppress progesterone levels even when 17-OHP is adequately suppressed (105).
Furthermore, breast development is suppressed in females with CAH. Evidence from animal studies suggests that testosterone exposure in utero may also suppress the breast anlage, resulting in poor breast development at adolescence (106). However, this problem is apparently due mainly to the combined effects of androgen excess and cortisol deficiency, because it is reversible with treatment (107, 108).
Pregnancy outcome in classic CAH has been recently reviewed. During pregnancy, women are optimally managed with hydrocortisone or prednisone (109, 110). Due to pregnancy-induced alterations in steroid metabolism and clearance, doses need be increased compared with doses used in nonpregnant women with CAH. It should be recognized that in this situation, treatment is directed at the mother and not at the fetus, for hydrocortisone and prednisone do not effectively cross the placenta. Interestingly, despite elevated maternal testosterone of 400–600 ng/dl, unaffected female offspring appear to have no genital virilization (109). Apparently, placental aromatase effectively prevents maternal androgens from reaching the fetus. Elevated maternal sex hormone-binding globulin (111) and androgen antagonism by progesterone (112) may also restrict transplacental passage of androgens.
There is no evidence of an excess of congenital malformations in offspring of women with CAH.
2. Males. Men with CAH less frequently have impaired gonadal function compared with affected women. Most affected males are able to father children or at least have normal sperm counts (113). Low sperm counts, when they occur, do not always preclude fertility (114). Among simple virilizing patients, testicular integrity may be normal even in the absence of treatment (115).
A prominent complication in CAH males is the development of testicular adrenal rests (116). This is discussed in Section III.I.2.
H. Neuropsychology of CAH
1. Cognitive effects. Although there have been occasional
reports of elevated IQ among CAH patients (117), this has not been
generally observed. To the contrary, salt wasting patients who suffer
hyponatremic dehydration and shock may sustain permanent brain injury
with resultant lower cognitive test scores (118, 119, 120). Certain sexually
dimorphic cognitive abilities, such as spatial abilities, may be
enhanced among CAH girls (121, 122, 123). Females with CAH are more likely
to be left-handed (as are males) (124) but do not differ from
unaffected women in degree of cerebral lateralization (125). Magnetic
resonance imaging showed white matter abnormalities in the brains of
CAH patients more often (117, 126) than in controls in two of three
recent studies (127). Thus, neurodevelopmental evaluation is warranted
in children with CAH. Patients who have experienced severe hyponatremia
should be considered for enrollment in early intervention programs if
neurodevelopmental milestones are delayed.
2. Effects on gender role and identity. The influence of prenatal sex steroid exposure on personality is controversial (reviewed in Refs. 128, 129, 130, 131, 132); also see Sections V.D and V.E). In considering this question, it is important to distinguish between gender role, sexual orientation, and gender identity.
Gender role refers to gender-stereotyped behaviors such as choice of play toys by young children. Parents of young girls with CAH often report that their daughters prefer to play with trucks as compared with dolls. Indeed, low interest in maternal behavior, beginning with infrequent doll play in early childhood and extending to lack of interest in child rearing for older girls and women, is a recurring theme in CAH research (133, 134, 135, 136). Some investigators have noted tomboyish (137) or aggressive (138) behavior among girls with CAH or a male pattern of distance in social relations (139). Others have found that young patients do not differ significantly from controls for nine parameters of psychopathology including aggression and hyperactivity (140); older girls or women with CAH have tested similarly (133, 141). The amygdala, an androgen-sensitive brain center controlling fear and aggression, is smaller by MRI among children with CAH (142); these MRI-based structural differences have not yet been directly correlated with psychological testing.
Sexual orientation refers to homosexual vs. heterosexual preferences. In most studies, a small but significant percentage of adult women with CAH have been actively homosexual or bisexual or have an increased tendency to homoerotic fantasies (143, 144, 145). These characteristics occur more frequently in women with the salt wasting form of 21-hydroxylase deficiency. A review of German patients found no increase in homosexuality among affected women but did find a decreased frequency of marriage and childbearing, suggesting more general psychosocial dysfunction among patients (146).
Gender identity refers to self-identification as male or female.
Spontaneous gender reassignment back to male has been reported in cases
of males with penile trauma who were raised as females (147, 148) or
male pseudohermaphrodites raised as females, especially in cases of
5-reductase or 17-ketosteroid reductase deficiencies, in which the
brain may be exposed to high circulating levels of androgens (reviewed
in Ref. 132). Conversely, female-to-male transsexuals may have
relatively high levels of androgens and a high incidence of polycystic
ovary syndrome (149). However, self-reassignment to the male sex is
unusual in women with CAH (145, 150). When it occurs, it may be related
to delays in gender assignment or genital surgery or to inadequate
suppression of adrenal androgens with glucocorticoid therapy (151).
Severely virilized females are more likely to be raised as males in
cultures that value boys more highly and/or in third world countries in
which the diagnosis is likely to be delayed (152, 153, 154). There have been
few studies directly comparing psychosexual functioning in severely
virilized genetic females with CAH raised as women or men, but it does
not appear that those raised as men are psychologically better adjusted
than those raised as women (155).
The uncertainty concerning the effects of prenatal and postnatal effects of androgen on gender identity and gender role extends not only to the female CAH population, but also to male pseudohermaphrodites of other etiologies. The role of external genital anatomy before and after genital surgery in fueling problems relating to gender is unclear compared with the roles of prenatal hormone exposure, rearing by the family, and community attitudes. Unfortunately, much of the data in this area are anecdotal (reviewed in Refs. 130, 131). These issues are discussed further in Section V.D.
In summary, most CAH children manifest normal neuropsychological development. Moreover, despite a tendency toward male gender role behavior and homoerotic fantasy, most girls with CAH identify as females and exhibit heterosexual preference.
I. Tumors
1. Adrenal. Almost 60% of patients with incidentally
discovered adrenal masses (incidentalomas) have exaggerated 17-OHP
responses to ACTH stimulation; the frequency of abnormal responses is
even higher in patients with bilateral adrenal masses (156). The
frequency of germline mutations in CYP21 in such patients is
low (157). However, the incidence of adrenal masses appears to be
higher in CAH patients and in heterozygotes than in the general
population (158). Histological types of adrenal tumor include adenoma,
myelolipoma (159, 160), and hemangioma (161). Steroid-responsive
hyperplastic adrenal nodules can present in previously undiagnosed
patients late in life and can potentially be confused with virilizing
adrenal adenomas (159, 162, 163). Because these tumors may regress with
glucocorticoid therapy, it may be unnecessary to resect them if they
are carefully followed. Rarely, virilizing adrenal carcinoma has been
found in CAH patients (164, 165), but most adrenal masses in children
with CAH are benign (166).
Partially autonomous cortisol secretion is rare in adrenal adenomas arising in patients with hormonal evidence of 21-hydroxylase deficiency (167). Acute adrenal insufficiency may develop after resection of such a nodule if steroids secreted by the nodule have suppressed ACTH secretion, leading to atrophy of the remaining adrenal cortex (168).
2. Testicular. Although seen most often in inadequately treated patients, testicular adrenal rests accompanied by deficient spermatogenesis may occur despite treatment, particularly in males with the salt wasting form of 21-hydroxylase deficiency (104, 169, 170). These tumors, although most often benign, have prompted biopsies and sometimes even orchiectomy (171). The preferred mode of treatment consists of effective adrenal suppression with dexamethasone, since many of these tumors are ACTH responsive. When they do not respond to dexamethasone, testis-sparing surgery may be performed after imaging of the tumor by sonography and/or MRI (171). Adrenalectomy (Section V.C.2) would not be expected to alleviate problems caused by gonadal adrenal rests (172). Testicular masses have been detected in children as young as 3 yr with CAH (116, 173), prompting the recommendation that boys undergo a baseline testicular sonogram by adolescence (174). The testes of affected males should be carefully examined throughout childhood, adolescence, and adulthood.
The main differential in the diagnosis of a virilizing testicular mass is a Leydig cell tumor. Such tumors can occasionally secrete high levels of 17-OHP suggesting CAH, but the secretion of 17-OHP from such a tumor will not be suppressed by dexamethasone or stimulated by ACTH (175). In general, bilateral tumors or those that decrease in size with dexamethasone are very likely to be adrenal rests. An adrenal rest may also be diagnosed if selective spermatic vein catheterization to assay steroids produced by the testis reveals high levels of 11ß-hydroxylated steroids (e.g., 21-deoxycortisol) (176), because the 11ß-hydroxylase enzyme is not active in testicular tissue.
3. Pituitary. Although glucocorticoid replacement doses exceed physiological cortisol production, CRH and ACTH often are not fully suppressed by treatment as evidenced by basal levels and by stimulation testing with ovine CRH. In one study, four of seven CAH patients undergoing MRI of the head showed pituitary abnormalities (three apparent microadenomata and one empty sella) (177). However, to the best of our knowledge symptomatic pituitary tumors have not been reported.
J. Nonclassic CAH phenotypes
1. Signs. Patients with the mild, nonclassic form of
21-hydroxylase deficiency may have any of the signs of postnatal
androgen excess listed above, but affected females are born with
nonambiguous (normal or with mild clitoromegaly) external genitalia.
Adrenal steroid precursors of 21-hydroxylase are only mildly elevated
in nonclassic CAH and are intermediate between those of heterozygote
carriers of the enzyme deficiency and those who are severely affected
(35). Depending on the laboratory, affected individuals have serum
17-OHP levels of greater than 1,000 or 1,500 ng/dl 60 min after an
intravenous bolus of cosyntropin (ACTH 1–24). Due to circadian
variability of adrenal cortical hormones (178), the diagnosis may be
missed by measuring only baseline serum 17-OHP late in the day. The
severity of signs and symptoms of mild androgen excess varies widely,
and probably many affected individuals are asymptomatic. The most
common presenting symptoms are premature pubarche in children (179, 180), or severe cystic acne (181), hirsutism, and oligomenorrhea in
young women (58, 182).
Nonclassic male patients diagnosed after puberty have presented with acne or infertility, but are most often diagnosed in the course of family studies and are entirely asymptomatic (183, 184). Rarely, a nonclassic male has presented with unilateral testicular enlargement (183). Precise clinical distinction between classic simple virilizing disease and the nonclassic disorder is often difficult among boys, since 17-OHP levels form a continuum between the mild and severe cases, and signs of androgen excess are much less apparent than in females.
Aldosterone synthesis and sodium balance are not compromised to any clinically significant extent in patients with nonclassic 21-hydroxylase deficiency (185), although under stress conditions subtle abnormalities may be elicited (186). Likewise, cortisol synthesis during stress is not impaired to any clinically significant degree (185), and there have been no deaths from adrenal insufficiency reported with this condition.
There are conflicting reports as to whether adult height is compromised in nonclassic CAH. Height SD scores were lower in one study compared with the population (-0.99 ± 0.98) but not when compared with midparent heights (0.43 ± 0.77) (94). Similarly, other investigators found no differences between nonclassic patients and their unaffected siblings (187).
The pathophysiology of the less frequent and milder reproductive problems associated with nonclassic CAH is presumably similar to that suggested for classic CAH. Data regarding reproductive function in nonclassic CAH come mainly from studies of populations referred for symptoms and signs of hyperandrogenism and/or infertility; ascertainment bias obviously affects such studies. In one report 39% of women presented with hirsutism, 39% with oligomenorrhea or other signs of polycystic ovaries, and 22% with no obvious signs of androgen excess (188). Based on data derived from family studies, it is clear that many individuals with mild 21-hydroxylase deficiency have minimal symptoms and are not brought to medical attention. French investigators found that half of the patients in their clinic became pregnant before the diagnosis of nonclassic CAH was made. All the others who desired pregnancy successfully conceived during hydrocortisone treatment; only 1 of 20 women required clomiphene citrate to conceive (189). Clomiphene without hydrocortisone has also successfully induced ovulation (190). For women who have conceived without hydrocortisone treatment, it is not necessary to initiate therapy during pregnancy; testosterone levels in nonpregnant women with nonclassic CAH are generally lower than typical testosterone levels in normal women during the second trimester of pregnancy (i.e., less than about 150 ng/dl).
2. Incidence. Because the signs of androgen excess in nonclassic 21-hydroxylase deficiency can be difficult to discern, particularly in males, the most reliable estimates of allele and disease frequencies come from ascertainment of affected individuals in the course of studies of kindreds in which classic and nonclassic 21-hydroxylase deficiency are segregating (191, 192, 193). The disease frequency is estimated at 0.1% of the general population but it occurs in 1–2% of Hispanics and Yugoslavs and 3–4% of Ashkenazi (Eastern European) Jews. Similar frequencies have been estimated from a small screening study using morning salivary 17-OHP levels (194). In New Zealand, molecular screening of normal newborns showed that 5% are carriers for mutations in the 21-hydroxylase gene (CYP21) associated with either classic or nonclassic 21-hydroxylase deficiency (see Section VI.F). This implies a disease frequency for nonclassic 21-hydroxylase deficiency of 0.06%, in good agreement with estimates in the general American population (195).
Although it has been suggested that nonclassic 21-hydroxylase deficiency represents the most frequent autosomal recessive genetic disorder in man (191, 194), the proportion of affected individuals who have problems with androgen excess is not known. To the best of our knowledge there has been no prospective study of symptomatology in any nonclassic 21-hydroxylase deficiency patient population. Because of the stigma and anxiety that may be associated with the diagnosis of a genetic disease, we suggest that nonclassic 21-hydroxylase deficiency be initially considered a "genetic polymorphism" and discussed as a disease only if signs of androgen excess develop.
Conversely, only a small percentage of individuals presenting with signs of androgen excess prove to be affected with nonclassic 21-hydroxylase deficiency. Among children referred for precocious pubarche, 4–7% have nonclassic 21-hydroxylase deficiency (180, 196, 197, 198). Among 31 women referred for acne, none had 21-hydroxylase deficiency, although a majority of patients had exaggerated adrenal responsiveness to ACTH stimulation (199). In the largest study of hyperandrogenic women, only 6% of 400 hirsute French women had hormonal profiles compatible with the diagnosis of late-onset CAH (200). These statistics have been borne out in other large clinic population samples (201, 202, 203). The lowest incidence of nonclassic CAH was 1.2% in 83 hyperandrogenic Californian women (204), whereas the highest incidence of nearly 14% was detected in New York women (205). Variations in the frequency of nonclassic alleles among different ethnic groups may account for some of the discrepancies noted. New York has a high proportion of Ashkenazi Jews, and this group has the highest frequency of the typical nonclassic CYP21 allele, valine-to-leucine 281 (V281L), associated with HLA-B14,DR1 (see Section VI.E) (191, 206).
There does not appear to be a high prevalence of nonclassic CAH in men with infertility (207), and, conversely, most men with nonclassic CAH ascertained through family studies have proved fertile. However, oligospermia and infertility have occasionally been described (104, 183, 208). In some cases, these problems may be reversed by glucocorticoid treatment (104, 208).
The incidence of classic CAH is discussed in Section IV.B.
K. Heterozygotes
Heterozygotes carrying a single mutant allele have slightly
elevated 17-OHP levels after ACTH stimulation, but there is substantial
overlap with unaffected individuals (35). The range of most
heterozygotes’ 17-OHP response at 60 min after cosyntropin
stimulation is approximately 200–1,000 ng/dl (209). Of 53 women with
signs of hyperandrogenism who were suspected by hormonal testing of
being carriers for CYP21 mutations, such mutations could be
detected in only 37; in contrast, mutations could be detected on both
alleles in 15/16 women who had 17-OHP or 21-deoxycortisol levels in the
range expected for nonclassic CAH (210).
In view of these problems with hormonal detection of heterozygotes, genotyping would appear to be a superior heterozygote detection method. This is discussed further in Sections IV.B and VI.H.
Mothers of children with classic CAH are no more likely to show signs of androgen excess than age, sex, and BMI-matched controls (211). However, children referred to an endocrine clinic for premature pubarche or hirsutism showed a higher prevalence of heterozygous CYP21 mutations compared with 80 adult controls who were not screened for hyperandrogenic signs or symptoms (212). Since potential heterozygotes in the latter study were culled from a symptomatic referral population, they may not represent the population-at-large carrying CYP21 mutations. With estimated nonclassic and classic heterozygote frequencies of 10% (191) and 1.5% in the general population, respectively, it is unlikely that heterozygosity confers a clinically significant reproductive disadvantage. Screening of men referred for evaluation of infertility has not revealed a high prevalence of nonclassic 21-hydroxylase deficiency patients or heterozygotes (207).
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IV. Diagnosis of 21-Hydroxylase Deficiency |
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TopAbstractI. IntroductionII. Biochemistry of CAHIII. Pathophysiology of CAHIV. Diagnosis of 21-Hydroxylase...V. TreatmentVI. Molecular Genetic AnalysisVII. SummaryReferences |
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A detailed review of the evaluation of ambiguous genitalia is beyond
the scope of this paper (see Refs. 213, 214), but some general
principles may be stated (Fig. 5). The
physical examination should identify the urethral meatus and should
include careful palpation for gonads in the inguinal canals and labia
or scrotum. Standard diagnostic tests should include at least a
measurement of basal serum 17-OHP, but preferably a complete profile of
adrenocortical hormones before and 1 h after cosyntropin
stimulation. These assays should be deferred past the first 24 h
of life (also see Section IV.C.1). They will identify
potential defects in adrenal steroidogenesis; salt wasting
21-hydroxylase deficiency is the most commonly encountered cause of
female pseudohermaphroditism. After testing is completed, the child’s
vital signs should be monitored for any indication of adrenal crisis.
It is rare for salt wasting crisis to occur before 7 days of life, but
many clinicians will obtain electrolyte measurements to assess
hyponatremia and hyperkalemia in CAH newborns during the first week.
PRA and aldosterone are elevated in many normal infants and do not
usually add much useful information within the first days of life.
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-reductase deficiency. A team consisting of neonatologist,
pediatric endocrinologist, urologist, and preferably an experienced
social worker and/or child psychiatrist should promptly review the
essential early diagnostic data and make a recommendation to the family
as to the sex of rearing and any medical and/or surgical treatments.
These recommendations should be based on both the current state of
knowledge of psychosexual development in intersex individuals
(discussed in Section III.H) and the feasibility of surgical
correction (Section V.D). Although all available options
should be reviewed with the family, these recommendations should be as
unequivocal as possible.
B. Newborn screening
CAH is a disease well suited to newborn screening since it is a
common and potentially fatal childhood disease, it is easily diagnosed
by a simple hormonal measurement in blood, and early recognition and
treatment can, in principle, prevent serious morbidity and mortality
(Fig. 6).
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Premature, sick, or stressed infants tend to have higher levels of
17-OHP than term infants and generate many false positives unless
higher normal cut-offs are used (Fig. 7).
Suggested weight-adjusted cut-offs range from 165 ng/ml for infants
under 1,300 g to 40 ng/ml for infants over 2,200 g in Wisconsin (217);
in Texas, cut-offs of 40 and 65 ng/ml are used for infants greater or
less than 2,500 g, respectively (218). Elevated 17-OHP levels in
preterm infants have been confirmed by HPLC and are thus not due to
cross-reaction with other steroids [however, some 17-OHP RIAs do
cross-react with other steroids; these include 15ß-hydroxylated
compounds, which are apparently generated by gut bacteria and resorbed
through the enterohepatic circulation (219)]. The steroid profiles in
preterm infants suggest a functional deficiency of several adrenal
steroidogenic enzymes with a nadir in function at 29 weeks of gestation
(220).
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, and summarized in Ref.
81) the highest incidence of classic CAH occurs in two geographically
isolated populations, the Yupik Eskimos of Western Alaska (1:280) (221)
and the French island of La Reunion in the Indian Ocean (1:2,100). The
incidence in most other populations ranges from approximately 1:10,000
to 1:18,000 (81, 195, 217, 218, 222, 223, 224).
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As regards morbidity, infants ascertained through screening have less severe hyponatremia (225) and tend to be hospitalized for shorter periods of time (although the difference falls short of statistical significance) (226).
Although salt wasting males would seem to derive the greatest benefit from screening programs, the delay before correct sex assignment of severely virilized females is also markedly reduced (79, 81, 225). Moreover, males with simple virilizing disease may otherwise not be diagnosed until rapid growth and accelerated skeletal maturation are detected later in childhood, at which time final height may already be adversely affected. However, it is debatable whether this last benefit itself justifies the costs of a screening program.
The estimated cost of screening each newborn infant was $2.70 in Sweden. With a disease incidence of 1:9,800 in this population, 102 affected newborns are expected per million infants screened; 51 should be males, of whom 75% should be salt wasters. The total cost for screening each million infants is $2,700,000, and thus the cost for each of 38 salt wasting males expected to be detected by screening is $71,000. The cost of newborn CAH screening in Texas was higher at $87,000 per CAH case, as separate hormonal assays were performed on each infant at birth and again at 1–2 weeks of age (218). All infants with salt-wasting CAH were detected on the first screen, so that the second screen may not be cost effective (230).
Nevertheless, these costs are within the general range estimated for other newborn infant disease detection programs. By comparison, targeted newborn screening for hemoglobinopathy in Alaska cost approximately $200,000 per death averted (231).
Patients with nonclassic 21-hydroxylase deficiency are occasionally detected by newborn screening. In Texas, 87% are detected on the second of the two routine screening tests, with an overall frequency of nonclassic disease of 1:35,870 (218). This is much less than the 1:1,000 frequency in the general population estimated from nonclassic allele frequencies in kindreds in which classic 21-hydroxylase deficiency is segregating (191, 192). Thus, neonatal screening using hormonal assays is not an efficient way to detect nonclassic disease. As yet, there are no follow-up studies of patients with nonclassic disease who have been ascertained by neonatal screening to determine how frequently they develop signs of androgen excess. There have also not been any systematic genotyping studies of nonclassic patients ascertained through neonatal screening to determine whether their genotypes differ from nonclassic patients ascertained through family studies or because they had developed signs of androgen excess. In a small study in Japan, all four patients ascertained through neonatal screening were compound heterozygotes for classic mutations (232), consistent with the higher 17-OHP levels seen after cosyntropin stimulation in compound heterozygotes (36)(also see Section VI.I). These data suggest that infants who are homozygous for mild CYP21 mutations are less likely to be detected by basal hormone screening.
3. Strategies for follow-up. To obtain adequate sensitivity, the cut-off levels for 17-OHP are typically set low enough that 0.3–0.5% of all tests are reported as positive. Therefore, specificity is only 2%, i.e., 98% of all positive tests are false. The above estimates for cost of detection do not include costs for follow-up of false positives. In Texas (218), both the infant’s primary physician and a pediatric endocrinologist are notified of all positive screens. Moderately elevated 17-OHP levels (40–100 ng/ml for term infants) are followed up with a repeat filter paper specimen. Higher values are evaluated with electrolytes and a serum 17-OHP level; if these are not unequivocally normal, the infant is then referred to a pediatric endocrinologist. A cosyntropin stimulation test is then usually performed.
4. Molecular genetic screening. Much of the expense of following up positive newborn screening tests could be avoided with a second level of screening based on detection of actual mutations (see Section VI.H). This could be accomplished on DNA extracted from the same dried blood spots as are used for hormonal screening. Because 90–95% of mutant alleles carry one or more of a discrete number of mutations (see Section VI.F), samples that carry none of these mutations may be presumed with more than 99% confidence to be unaffected. Heterozygous carriers of a mutation for classic 21-hydroxylase deficiency would still need to be followed up due to the chance that the other allele might carry a novel mutation, but less than 2% of individuals are carriers of classic 21-hydroxylase deficiency alleles.
Two large-scale studies of the utility of genotyping in screening programs have shown that this is a useful adjunct to hormonal measurements (195, 233). One study examined cost and found it to be approximately $5 per sample analyzed (195). At present, however, there are few laboratories equipped to do rapid, accurate, and large-scale CYP21 genotyping.
C. Further biochemical evaluation
1. The cosyntropin stimulation test. As previously
mentioned, a basal serum or filter paper 17-OHP may not be fully
informative, and it may be necessary to evaluate the patient further.
In cases where there is no newborn screening program, but one suspects
CAH based on ambiguous genitalia, cosyntropin stimulation should be
deferred beyond the first 24 h of life. There is a high incidence
of both false-positive and false-negative results when samples are
obtained immediately after birth. Another justification for performing
stimulation testing is that 17-OHP may be elevated in other enzymatic
defects, e.g., 11ß-hydroxylase or 3ß-hydroxysteroid
dehydrogenase deficiencies. Ideally, to fully differentiate the various
enzymatic defects, the clinician should measure 17-OHP, cortisol, DOC,
11-deoxycortisol, 17-OH-pregnenolone, DHEA, and
androstenedione at 0 min and 60 min (Fig. 8). If blood volume is an issue in small
infants, a sample is collected only at 60 min. Precursor to product
ratios are particularly useful in distinguishing the different
enzymatic defects. If the diagnosis remains unclear, it may be
desirable to treat the child and later retest after partially or
completely tapering glucocorticoids. Our practice is to use a uniform
dose of 0.25 mg cosyntropin, providing a pharmacological stimulus to
the adrenal cortex. This diagnostic test should be distinguished from
the low-dose ACTH stimulation test now becoming increasingly popular
for evaluating the integrity of the hypothalamic-pituitary-adrenal axis
(234, 235).
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3. Other hormones useful in diagnosis and monitoring of
CAH. Several other diagnostic biochemical assays have been
proposed, but few are widely available. Assays of 21-deoxycortisol can
detect more than 90% of CAH heterozygotes (39, 40). Levels of an
androgen metabolite, 3-androstanediol glucuronide, are elevated in
nonclassic 21-hydroxylase deficiency (240) and highly correlated with
levels of androstenedione and testosterone (241). The main urinary
metabolite of 17-OHP, pregnanetriol, can also be used to diagnose
21-hydroxylase deficiency. Moreover, urinary levels of pregnanetriol
glucuronide may be a way to monitor therapeutic efficacy and possible
overtreatment (242). As an alternative to enzyme-linked immunoassays or
RIAs, urinary steroid metabolites can be analyzed by GS/MS, in which
case several relevant markers for CAH and other disorders of steroid
metabolism can be assayed simultaneously (243).
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V. Treatment |
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TopAbstractI. IntroductionII. Biochemistry of CAHIII. Pathophysiology of CAHIV. Diagnosis of 21-Hydroxylase...V. TreatmentVI. Molecular Genetic AnalysisVII. SummaryReferences |
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The short half-life of hydrocortisone minimizes growth suppression and other adverse side effects of longer acting, more potent glucocorticoids such as prednisone and dexamethasone. On the other hand, a single daily dose of a short-acting glucocorticoid is ineffective in controlling adrenocortical hormone secretion (247)
Cortisone acetate is not a drug of first choice for CAH. It has only 80% of the bioavailability of hydrocortisone and approximately two thirds of its potency (248). Moreover, since cortisone must be converted to cortisol to be biologically active, defective 11ß-hydroxysteroid dehydrogenase reductase activity can further reduce the efficacy of this drug (249).
Older adolescents and adults may be treated with modest doses of prednisone (e.g., 5–7.5 mg daily in two divided doses) or dexamethasone (total 0.25–0.5 mg given as one or two daily doses). Patients should be monitored carefully for signs of iatrogenic Cushing’s syndrome such as rapid weight gain, hypertension, pigmented striae, and osteopenia. Men with testicular adrenal rests may require higher dexamethasone doses to suppress ACTH.
Treatment efficacy (i.e., suppression of adrenal hormones) is assessed by monitoring 17-OHP and androstenedione levels. Testosterone can also a useful parameter in females and prepubertal males. Because of the adverse effects of overtreatment (see the next section) it is not desirable to completely suppress endogenous adrenal corticosteroid secretion. A target 17-OHP range might be 100-1000 ng/dl with commensurate age and gender-appropriate androgen levels (247, 250). Hormones should be measured at a consistent time in relation to medication dosing, preferably at 0800 h at the physiological peak of ACTH secretion, or at least at the nadir of hydrocortisone blood levels just before the next dose is to be given. Remote monitoring of hormonal control in CAH patients is possible through the use of either salivary hormone measurements (194, 251, 252) or finger-prick blood samples collected on filter paper and assayed for 17-OHP (247, 250, 253). The latter methodology is routinely used for neonatal screening for CAH (see Section IV.B).
Children should have an annual bone age x-ray and careful monitoring of linear growth. Despite careful monitoring and good patient compliance, most retrospective reviews (94, 95, 96, 97, 98, 99) indicate that final height averages 1–2 SDs below the population mean or the target height based on parental heights.
2. Adverse effects of overtreatment. Early excessive glucocorticoid treatment (hydrocortisone dose > 20 mg/m2/day) is potentially detrimental to growth (67). A randomized prospective crossover trial showed that patients treated with 15 mg/m2/day of hydrocortisone were less likely to show growth suppression compared with those taking doses of 25 mg/m2/day (254). High body mass index in childhood also correlates with poor final height and may be a surrogate marker for overtreatment (67, 255). However, patients with CAH may be more prone to obesity than other children, and they begin gaining weight earlier in childhood (nadir for adiposity of 1.7 yr in British children with CAH as compared with 5.5 yr in the general UK population) even when height is normal (256). Despite linear growth averaging approximately 1 SD below the mean, bone mineral density does not appear to be compromised in CAH patients receiving typical glucocorticoid doses (98, 257, 258, 259). Only one study of Finnish patients showed low bone density in the femoral and L2–4 lumbar regions; the authors attributed their findings to excessive glucocorticoid dosing in some subjects (260). However, decreased bone turnover has also been associated with CAH (261).
If control cannot be achieved with hydrocortisone, it is reasonable to use either prednisone or dexamethasone for a 2- to 4-day course of suppressive therapy before resuming hydrocortisone. After epiphyseal fusion, prednisone or dexamethasone may be used as maintenance therapy, but doses should not exceed the equivalent of 20 mg/m2 hydrocortisone daily, and patients should be carefully monitored for signs of iatrogenic Cushing’s syndrome.
3. Stress dosing. Patients with classic CAH cannot mount a sufficient cortisol response to stress and require pharmacological doses of hydrocortisone in such situations as febrile illness and surgery under general anesthesia. Such treatment should approximate typical endogenous adrenal secretion in critically ill and perioperative patients (71). Dose guidelines include tripling the maintenance dose of oral hydrocortisone (administered in three divided doses) in minor febrile illnesses. If a patient is unable to tolerate oral medication, intramuscular hydrocortisone sodium succinate (Solu-Cortef) may be given, but medical advice concerning the need for intravenous hydration should be promptly sought. Patients and parents should receive instructions for these types of emergency contingencies, and patients should carry or wear identification with information about their medical condition. For major surgery, administration of hydrocortisone (100 mg/m2/day) divided in four intravenous doses is warranted for at least 24 h peri- and postoperatively before tapering over several days to a maintenance dose. Intravenous hydrocortisone is preferred over equivalent glucocorticoid doses of methylprednisolone (Solu-Medrol) or dexamethasone because (when it is administered in high doses) its mineralocorticoid activity is able to substitute for oral fludrocortisone.
Patients with nonclassic CAH do not require stress doses of hydrocortisone for surgery unless they have iatrogenically been rendered hypoadrenal by prior chronic administration of glucocorticoids. In our experience no patient with nonclassic CAH has ever shown evidence of adrenal insufficiency. However, one assumes that all patients treated over long periods of time with glucocorticoids have some degree of endogenous adrenal suppression. It is therefore prudent to treat with supplemental glucocorticoids in times of extreme stress, and patients receiving such therapy should wear medical alert tags. Alternatively, if given adequate advance notice, one could discontinue treatment and test the integrity of the hypothalamic-pituitary-adrenal axis with a low-dose ACTH stimulation test (234, 235).
4. Indications for therapy in patients with nonclassic CAH. Individuals diagnosed with nonclassic CAH should be offered treatment when they manifest signs or symptoms of androgen excess. Low-dose glucocorticoid therapy may be initiated in children with precocious pubarche, i.e., inappropriately early onset of body hair and odor, accompanied by advanced bone age. A small group of Jewish nonclassic CAH patients was able to achieve final heights within the range predicted from parental heights as long as glucocorticoid therapy was started at the first signs of precocious adrenarche or bone age acceleration; delaying initiation of therapy until after central puberty began was associated with decreased final height (262). Other studies have found no adverse effect of nonclassic CAH on height (187).
Other common indications for treatment are hirsutism, oligomenorrhea, and acne in young women. Infertility patients diagnosed with nonclassic CAH should also be treated, as they may more readily become pregnant if the hormonal imbalance is the principal obstacle to conception. Treatment with glucocorticoids suppresses adrenal androgen production, resulting in gradual improvement in clinical signs of androgen excess. Remission of hirsutism is the most difficult objective to achieve with glucocorticoid monotherapy, as established hair follicles are difficult to eradicate. Cosmetic therapy is therefore advised as an adjunct to hormonal therapy in women for whom the hirsutism is unsightly. An exact timetable to regression of each clinical sign has yet to be established.
Men with nonclassic CAH may achieve improved sperm counts and fertility with glucocorticoid treatment (263, 264). Although rare, testicular enlargement in nonclassic males is also an indication for glucocorticoid therapy (183).
Nonclassic patients whose symptoms have resolved (e.g., a boy treated for precocious pubarche, now fully grown), or affected women past child-bearing age, should be given the option of discontinuing therapy.
B. Mineralocorticoid replacement
Infants with the salt wasting form of 21-hydroxylase deficiency
require mineralocorticoid (fludrocortisone, usually 0.1–0.2 mg but
occasionally up to 0.4 mg daily) and sodium chloride supplements (1 to
2 g daily; each gram of sodium chloride contains 17 mEq of sodium)
in addition to glucocorticoid treatment. The sodium content of either
breast milk or the most popular infant formulae is about 8 mEq/liter,
which is only sufficient for maintenance sodium requirements in healthy
infants. Considerably more sodium (
8 mEq/kg/day) must be supplied to
keep up with ongoing losses in aldosterone-deficient infants. Often,
older children acquire a taste for salty food and do not require daily
supplements of sodium chloride tablets. Moreover, fludrocortisone doses
may often be decreased after early infancy.
Although patients with the simple virilizing form of the disease by definition secrete adequate amounts of aldosterone, they are nevertheless often treated with fludrocortisone. This can aid in adrenocortical suppression, reducing the dose of glucocorticoid required to maintain acceptable 17-OHP levels (236).
PRA may be used to monitor mineralocorticoid and sodium replacement. Hypertension, tachycardia, and suppressed PRA are clinical signs of overtreatment with mineralocorticoids (265). Excessive increases in fludrocortisone dosage may also retard growth (266).
C. Other therapeutic approaches
1. Pharmacological. A novel four-drug regimen for CAH,
consisting of flutamide (an androgen receptor-blocking drug),
testolactone (an aromatase inhibitor), low-dose hydrocortisone, and
fludrocortisone, showed promising results after a 6-month trial.
Children in the experimental treatment group showed less bone age
advancement and more appropriate linear growth velocity than those in
the standard treatment group (267). After 2 yr, the 16 children in the
experimental group showed higher levels of 17-OHP, androstenedione,
DHEA and its sulfate, and testosterone, plus a slower rate of growth
and bone maturation with improved predicted height compared with
children on standard therapy. However, central precocious puberty
occurred and required treatment with LHRH analog in 3 of 8 males in the
experimental therapy group and in 0 of 9 control males (268). It
remains to be seen whether longer term, larger scale studies will show
a favorable effect of the experimental regimen on final height. Other
questions include whether the average family could cope with such a
medical regimen in a school-aged child, and what the cost of such
therapy would be over many years.
Another interesting experimental CAH therapy is the addition of carbenoxolone, an inhibitor of 11ß-hydroxysteroid dehydrogenase (11-HSD). The latter is an enzyme important in inactivating cortisol and preventing its access to the mineralocorticoid receptor (269). The rationale for carbenoxolone as an adjunct to therapy of CAH is that inhibition of the oxidative 11-HSD reaction should generate higher endogenous bioactive cortisol levels without administering larger doses of steroids. In a short-term pilot study with an open-label, crossover design involving six CAH patients aged 15 to 39 yr, there were significant reductions in 17-OHP, androstenedione, renin, and urinary pregnanetriol when carbenoxolone was added to the standard therapeutic regimen (270, 271). Hypertension is potentially a complication of such a regimen (269).
Two adult patients with CAH and concurrent malignancies were treated with chlormadinone acetate, an antiandrogen used overseas for prostate cancer. In both cases, secretion of ACTH and adrenal androgens was suppressed (272). At present, antiandrogens are not recommended for treatment of children and young women with CAH outside the research setting, since the risks of adverse side effects, including hepatic toxicity and teratogenicity, are significant.
2. Adrenalectomy. Consequences of inadequate treatment or noncompliance for the female include ongoing virilization in addition to compromise of linear growth. For this reason, it has been suggested that (laparoscopic) adrenalectomy may represent an alternative to suppressive medical therapy with glucocorticoids (273). Severely affected patients, especially females, could perhaps be more easily managed as Addisonians with low-dose gluco- and mineralocorticoids than with adrenal glands that secrete excessive sex steroids. Opponents of surgical treatment feel that this is too radical a step, potentially placing patients at risk from the surgical procedure, and later incurring further risks from iatrogenic adrenal insufficiency. Moreover, the beneficial effects of adrenalectomy may be confounded by the development of gonadal adrenal rests that can secrete androgen precursors (172). Finally, there is a clear benefit in terms of improved lipid profile, libido, and quality of life from physiological adrenal DHEA production (274, 275) that would be lost with adrenalectomy. Although patients have been managed in this manner (172, 276), further data must be collected before deciding whether adrenalectomy is a viable therapeutic alternative. It is likely to be used, if at all, in patients with severe 21-hydroxylase deficiency refractory to standard medical management.
3. Gene therapy. Because 21-hydroxylase deficiency is an inherited metabolic defect, the question arises of the feasibility of gene therapy (277). Indeed, mice with 21-hydroxylase deficiency have been rescued by transgenesis with a murine Cyp21 gene (278). However, this disorder does not seem a promising test system for human gene therapy. As discussed above, medical therapy, albeit not perfect, is effective and relatively inexpensive. High level expression would need to be targeted to the adrenal cortex, where adequate levels of steroid precursors are available. As the most difficult therapeutic goal to achieve is adequate suppression of adrenal androgens, expression would need to be sufficiently high to permit nearly normal levels of cortisol biosynthesis under both normal and stress conditions, and such levels of expression would need to be maintained indefinitely to be cost effective in comparison with conventional treatment. These criteria seem unlikely to be met for the foreseeable future.
D. Corrective surgery
The general approach to evaluating the newborn with ambiguous
genitalia has been discussed in Section IV.A. In general,
the recommended sex assignment should be that of the genetic/gonadal
sex, if for no other reason than to retain the possibility of
reproductive function. This is especially true for females with
21-hydroxylase deficiency who have normal internal genital structures
and potential for child-bearing. An exception to this rule might be the
genetically female patient with completely male appearing genitalia,
especially if the child has been raised as a male for more than a few
months. Such children will need to be castrated at puberty to avoid
feminization.
Whether, how, and when to intervene surgically in the correction of genital anomalies is the subject of continuing debate (279, 280). Some adult patients with CAH and other intersex conditions who are unhappy with their gender assignment, as well as some physicians, have advocated postponing genital surgery until the affected individual is able to provide informed consent for cosmetic genital surgery, and select the gender with which he/she will be most comfortable (279, 281, 282, 283). It is not clear, however, whether families would readily accept the idea of raising a child with indeterminate gender and/or ambiguous genitalia, whether children would then be psychologically traumatized due to lack of societal acceptance of such conditions, and whether such children would be able to develop an unambiguous gender identity at all.
It must also be recognized that recommendations for sex assignment are to some extent culture specific. In cultures that value infant boys over girls, parents may strongly resist rearing a female with ambiguous genitalia as a girl, and many girls with severely virilized external genitalia will be raised as males (152, 153).
The most common current approach to surgical correction is for clitoroplasty (284, 285), rather than clitoridectomy, to be done in infancy. In adolescence the patient can be taught to perform vaginal dilation with acrylic molds (286, 287). Vaginal reconstruction is often postponed until the age of expected sexual activity (288, 289), but single-stage corrective surgery has also been successfully performed in children (284, 290, 291). Correction in infancy may be more successful for cases of simple labial fusion than in cases where the distal vagina must be reconstructed (289, 292). Newer modifications in vaginoplasty procedures may improve outcome in patients with urogenital sinus for whom simple dilation is not helpful (286, 293, 294). According to self-assessment surveys among sexually active women with CAH, approximately 60% are able to have satisfactory intercourse (295). Reoperation is frequently required to achieve satisfactory results (292).
As surgical and medical treatment regimens have improved in recent years, more women with CAH have successfully conceived spontaneously, completed pregnancies, and given birth (296). Most often delivery is by cesarean section due to an inadequate introitus, but vaginal delivery is possible in some cases (109).
E. Psychological counseling
Families of CAH patients should be assessed for emotional health.
The initial screening will most likely be done by the pediatrician and
pediatric endocrinologist. The child behavior checklist and the
self-perception profile can be used in school-aged children (140).
Parents should be offered psychological counseling soon after the
diagnosis is made. Intermittent assessment of family functioning, as
has been done in other disease states, may be a useful tool in
predicting future problems (297). Children should, as they mature, be
repeatedly informed about their condition by parents and physicians in
a sensitive and age-appropriate manner. When psychotherapy is
undertaken, medical and psychiatric caregivers should maintain
communication so that both are aware of the patient’s and family’s
status. Unfortunately, many locales lack mental health professionals
with experience in counseling patients and families with intersex
conditions.
Although psychosexual development of females with classic CAH is incompletely understood (Section III.H), we believe anticipatory counseling of patients’ families should initially address the high likelihood that affected girls will exhibit tomboyish behavior, masculine play preferences and perhaps, when older, a preference for a career over domestic activities (133). In the contemporary United States, these preferences usually have a high degree of social acceptance, considering the increased availability of and interest in girls’ competitive sports as well as the many women who work. Parents should also be reassured that the majority of (but not all) girls function heterosexually, although they may require repeated genital surgeries to have satisfactory intercourse. The endocrinologist and/or mental health professional (depending on inclination and experience) caring for the adolescent girl with CAH should address sexual orientation, both fantasized and actual. The patient should be reassured that some degree of attraction to other girls, although it does not always occur, is a typical feature of her condition. A discussion of psychotherapy for homosexuality is beyond the scope of this review, but it should be accepted by health care professionals that a minority of women with CAH may be most comfortable as homosexuals and that such individuals should be helped to come to terms with their situation. Adult patients should also be made aware of relevant patient advocacy groups.
F. Treatment of precocious puberty
Central precocious puberty may occur in the setting of excess
adrenal sex steroids and advanced bone age, especially when
glucocorticoid treatment is initiated in children with markedly
advanced bone age. Under such circumstances, chronic exposure to
adrenal androgens may cause the hypothalamic-pituitary gonadal axis to
mature. A sudden decrease in androgen levels with adequate treatment
may then trigger secretion of gonadotropins by the pituitary.
Clinical suspicion of central precocious puberty in affected boys may be aroused when physical examination in boys reveals increased testicular growth, or when girls show increased breast growth. In boys, serum testosterone may increase in the face of well controlled 17-OHP. However, since testosterone is also a byproduct of excessive adrenal sex hormone production, this hormone alone is not an accurate marker for central precocious puberty. An elevated ratio of testosterone to androstenedione suggests a gonadal, rather than an adrenal, source of hormone production. Serum estrogen levels are not typically part of the hormonal profile for CAH management.
Definitive diagnosis of precocious puberty requires GnRH stimulation testing. LH and FSH levels drawn before and 30 min after a 100-µg bolus of GnRH (Factrel) will show a marked rise in LH > FSH; the absolute levels depend on the type of assay employed.
Although spontaneous resolution of central precocious puberty has been anecdotally reported (298), this condition usually requires separate pituitary suppressive treatment with GnRH analogs (299). The goals of GnRH analog treatment are to suppress pituitary gonadotropins and, consequently, gonadal sex steroid production and to attempt to enhance adult height by preventing premature epiphyseal fusion. Whereas the first goal is readily achieved, the latter objective is more difficult. Preliminary data consisting solely of predicted height in but a few children allows cautious optimism about such treatment, but more long-term data are needed (300). Another goal of therapy is to avoid the adverse psychological consequences of premature puberty; anecdotal experience suggests this is aided by suppressive medical therapy.
GH-deficient children with precocious puberty who do not have CAH appear to benefit from combined treatment with GH and GnRH agonists if such treatment is begun at a relatively young bone age (301). However, short but otherwise healthy children with normally timed puberty do not benefit in terms of final height outcome from GnRH agonist plus GH therapy (302). CAH children are typically not GH deficient. Thus, multidrug regimens such as these remain experimental and very costly, and their effectiveness has not been established in CAH.
G. Prenatal therapy
1. Overview. In pregnancies at risk for a child affected
with virilizing adrenal hyperplasia, suppression of fetal adrenal
androgen production and decreased genital ambiguity in females have
been achieved by administering dexamethasone to the mother (303, 304, 305, 306, 307, 308, 309, 310, 311).
As compared with hydrocortisone, dexamethasone has no salt retaining
activity and it is able to cross the placenta because it is not
metabolized significantly by placental 11ß-hydroxysteroid
dehydrogenase (269). The dose is typically 20 µg/kg/day based on
prepregnancy weight to a maximum of 1.5 mg daily in three divided
doses, beginning before the 7th to 8th week of gestation (307, 311).
Approximately 70% of prenatally treated females are born with normal
or only slightly virilized genitalia. Treatment failures,
i.e., affected females requiring genital reconstruction,
have been attributed to late onset of treatment, cessation of therapy
in midgestation, noncompliance, or suboptimal dosing (312), whereas
others had no ready explanation (313). The fetal adrenal cortex may
not always be adequately suppressed by these doses of maternally
administered dexamethasone. Unaffected newborns treated until birth
usually have suppressed steroid secretion for at least 1 week
after birth, especially of steroids such as
16-hydroxypregnenolone that originate in the fetal zone of
the cortex. However, 17-OHP and 21-deoxycortisol metabolites in
affected infants may not be suppressed at birth even with continuous
treatment beginning early in gestation (314).
Very few data are available regarding natural variability in genital virilization among family members with the same CYP21 genotype, although nearly normal genitalia without treatment in relatives of highly virilized patients have been reported anecdotally (315, 316). Nevertheless, the likelihood of severe genital ambiguity is apparently reduced among those prenatally treated compared with their untreated sisters and to all affected girls with similar genotypes. Anecdotal reports suggest that suppression of adrenal androgen secretion is easier to achieve after prenatal treatment (310). The first prenatally treated female has reached late adolescence with normal cognitive development (310). It is not yet possible to determine whether prenatal treatment will induce marked differences in the psychosexual outcome for women with CAH.
Prenatal therapy is usually coupled with prenatal diagnosis (see
Section VI.H). Since dexamethasone treatment suppresses
amniotic fluid adrenocortical hormones, genetic diagnosis must be
performed. Although the incidence of fetal deaths in treated
pregnancies does not appear to exceed that for the general population
[9% spontaneous abortion rate in treated pregnancies, compared with
14% in untreated pregnancies (317)], a high rate of spontaneous
abortions has been observed after chorionic villus sampling (CVS)
performed to obtain tissue for genetic diagnosis (318). Either
amniocentesis or CVS may be done for diagnostic purposes, but the
latter should be carried out only in experienced centers at 10–12
weeks gestation (319). If the sex is male, or CYP21 genotype
indicates the fetus is unaffected, dexamethasone should promptly be
discontinued to minimize potential risks of glucocorticoid toxicity
(Fig. 9).
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3. Therapeutic risks to the mother. The incidence of maternal complications has varied among investigators; overall, it is about 10% (310). In evaluating such data, it is important to correlate the types of adverse side effects observed and the duration of therapy. Both American (331) and European (311) investigators have found a higher incidence of side effects in women treated from the first through third trimesters. Cushingoid features, excessive weight gain, severe striae, hypertension, and hyperglycemia are seen in this setting. These side effects most often resolve with discontinuation of treatment. Weight, blood pressure, and glucose tolerance should be closely monitored in all treated women treated to term. Serial maternal serum dexamethasone levels, if available, might prevent over- and undertreatment, but they have not been followed routinely. Maternal urinary estriol measurements have also been suggested as a guide to adjusting maternal treatment (304). A gradual decrease in the dose of dexamethasone later in gestation might decrease the incidence of maternal side effects, but there are as yet no data concerning the efficacy of such a regimen. More common maternal side effects in those treated for a shorter duration include edema, gastrointestinal upset, mood fluctuations, acne, and hirsutism; one or more of these symptoms are seen in 10–20% of women treated in early pregnancy.
Because of these concerns, caution should be exercised in recommending prenatal therapy with dexamethasone, and women must be fully informed of potential fetal and maternal risks, some of which may be as yet unrecognized. Additionally, the possibility of lack of therapeutic benefit should be disclosed when obtaining informed consent. However, we emphatically disagree with a recent suggestion (330) that prenatal treatment is so experimental as to require approval by institutional review boards.
Caveats notwithstanding, many parents of affected girls still opt for prenatal medical treatment because of the severe psychological impact of ambiguous genitalia on the child and on the family (332). Similar diagnostic and therapeutic approaches can also be effective in families at risk for 11ß-hydroxylase deficiency, in which affected female fetuses may also suffer severe prenatal virilization (333).
4. Other considerations for genetic counseling. The most common circumstance in which prenatal treatment is offered to a pregnant woman is when she and her partner have already had a child with CAH, in which case the likelihood of her bearing an affected girl with each successive pregnancy is 1/8.
Other scenarios may arise in genetic counseling. What if one partner has classic CAH and the carrier status of the partner is not known (recognizing that an affected mother will remain on glucocorticoid replacement regardless, but her dose may need to be increased to treat the fetus)? If the carrier frequency for classic CAH in the general population is 1.6% (equivalent to a disease frequency of 1/16,000, see Section IV.B), then the a priori likelihood of these parents giving birth to an affected girl is 0.4%, or 1/250 ([1 parent carrying 2 classic alleles] x [1.6% carrier frequency in general population] x [1/2 chance the carrier parent will pass his or her affected allele to the fetus] x [1/2 chance the fetus is female]).
Infants of mothers affected with the nonclassic disorder are also at slightly increased risk of developing classic CAH. Most studies examining genotypes in CAH (Section VI.I) did not specifically target nonclassic patients, and ascertainment biases and ethnic differences make it difficult to draw firm conclusions regarding allele frequencies in the nonclassic CAH patient population. Nevertheless, it appears that at least 50% of women clinically ascertained to have nonclassic CAH are compound heterozygotes for a classic and a nonclassic mutation (209, 210, 334). Accepting this figure, there is a priori approximately a 0.1% (1/1000) chance that a mother with nonclassic disease will give birth to a daughter affected with classic CAH ([50% carrier frequency of classic alleles among women with nonclassic disease] x [1.6% carrier frequency in general population] x [1/4 chance both classic alleles will be passed to the fetus] x [1/2 chance the fetus is female]).
In each of these scenarios, the risk of having an affected daughter is far less than the 1:8 risk with two known classic 21-hydroxylase deficiency carriers. Thus, prenatal therapy is not warranted unless the carrier status of the mate (as well as the genotype of a nonclassic patient) is first ascertained by hormonal testing and/or genotyping as part of preconception genetic counseling. This may be desired by some but not all couples. It may nevertheless be prudent to measure 17-OHP in such at-risk infants after birth, remembering that basal hormone measurements may not be sufficient to diagnose nonclassic CAH in infants and young children.
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VI. Molecular Genetic Analysis |
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TopAbstractI. IntroductionII. Biochemistry of CAHIII. Pathophysiology of CAHIV. Diagnosis of 21-Hydroxylase...V. TreatmentVI. Molecular Genetic AnalysisVII. SummaryReferences |
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Human CYP21 normally contains 494 amino acid residues (343, 344) [a normal variant has an extra leucine within the N-terminal hydrophobic domain and thus contains 495 residues (345)] and has a molecular mass of approximately 52 kDa. When expressed in mammalian cells, human recombinant CYP21 has apparent Km values for 17-OHP and progesterone of 1.2 and 2.8 µM, respectively, and the apparent Vmax for 17-OHP is twice that of progesterone (346).
B. Structure-function relationships
Alignment of the amino acid sequences of many P450s have
identified a small number of strongly conserved residues that are
presumed to be important for catalytic function (347, 348). The basic
three-dimensional structure of P450 enzymes has been deduced from x-ray
crystallographic studies of four bacterial P450s. The first of these,
P450cam (CYP101, camphor 5-exo-hydroxylase from Pseudomonas
putida), is a soluble molecule that bears little similarity in
primary structure (roughly 15%) to eukaryotic P450s (349); P450terp
(350) and P450eryF (351), bacterial P450s structurally related to
P450cam, have also been characterized. In contrast, P450BM-3 (CYP102
from Bacillus megaterium), is a complex protein consisting
of a P450-like N-terminal domain and a C-terminal domain that is 35%
identical to eukaryotic cytochrome P450 reductase. The P450 domain is
25–30% identical to the eukaryotic CYP4 and CYP52 families (352) and
approximately 20% identical to CYP21 (Fig. 10). This domain has been subjected to
crystallographic analysis (353). Its sequence has been aligned with
CYP21 and used as the basic for three-dimensional modeling of CYP21
(354). Based on thermodynamic considerations, this model may not be as
accurate as analogous models of other eukaryotic P450s such as CYP19
(aromatase) (355) or CYP17 (17-hydroxylase) (356). Nevertheless, based
on these analyses and functional studies, several conclusions may be
drawn.
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2. Oxygen and water binding. The ligand at the other axial position of heme is either a water or an oxygen molecule. When an oxygen molecule is bound, it is parallel to the axis of coordination with the iron atom. An H2O molecule is consistently present in a groove in the "I" helix adjacent to strongly conserved acidic (aspartate or glutamate) and threonine residues (E294 and T295 in CYP21). Mutation of the corresponding threonine in several other P450s destroys or drastically decreases enzymatic activity (358, 359).
According to one of the several proposed models of P450 catalysis (360), the first step of the reaction is binding of substrate to oxidized (ferric, Fe+3) enzyme. One electron is donated from P450 reductase to the enzyme so that the iron is in the reduced (ferrous, Fe+2) state. This complex binds molecular oxygen and then accepts a second electron from the accessory protein, leaving the bound oxygen molecule with a negative charge. Two protons are then donated in succession to the water molecule by the carboxyl group of the acidic residue, transferred to the hydroxyl of the conserved threonine, and finally donated to the distal oxygen atom (353). The distal oxygen atom is then released as a water molecule, leaving the iron in the Fe+3 state. The remaining oxygen atom is highly reactive (the iron-oxygen complex is a "ferryl" moiety) and attacks the substrate, resulting in an hydroxylation.
3. Substrate binding. Like most P450 substrates, steroids are relatively hydrophobic molecules. Thus, it is likely that the substrate binding site(s) will consist primarily of hydrophobic amino acid residues.
A priori, it was possible that the substrate binding sites of steroid-metabolizing P450s would more closely resemble each other in sequence than the substrate binding sites of other P450s such as xenobiotic metabolizing enzymes. Comparisons of the sequences of 21 and 17-hydroxylase and cholesterol desmolase (CYP21, CYP17, and CYP11A) identified two highly conserved areas, one near the N terminus (Q53-R60 in CYP21) and the other toward the C terminus (L342-V358 in CYP21) (361, 362).
The crystal structure of CYP102 confirms that part of the first of these indeed corresponds to a portion of a deep pocket constituting the substrate binding site (ß-sheet 1–1, residues E38-A44 in CYP102). However, the second conserved area corresponds to helix K (L311-W325 of CYP102), which does not interact with substrate. Instead, this region forms part of the docking site for the accessory electron transport protein, cytochrome P450 reductase. The remainder of segments that form the substrate binding pocket are widely distributed in the peptide (remainder of ß-sheet 1, B' and F helices) and the sequence conservation among steroid metabolizing P450s is not particularly strong in these regions.
4. Binding to accessory proteins. Microsomal and mitochondrial P450s accept electrons from cytochrome P450 reductase or adrenodoxin, respectively. In either case chemical modification studies suggest that basic amino acids (usually lysine) on the P450 interact with acidic residues on the accessory protein. The crystallographic studies of CYP102 suggest a docking site for reductase formed in part by helices B, C, D, J', and K. Helix K, as mentioned, was previously thought to interact with substrate. Support for the idea that it is instead required for redox interactions (with cytochrome P450 reductase or adrenodoxin, depending on the type of P450) comes from mutagenesis studies of CYP11A, wherein modification of either of two lysine residues in helix K destroys enzymatic activity without affecting substrate binding (363). In similar studies of CYP17, mutagenesis of arginine residues in this region disrupts interactions with cytochrome P450 reductase and cytochrome b5 (364).There are several naturally occurring mutations of arginine residues (R354 and R356) in this region of CYP21 that drastically decrease enzymatic activity (365, 366, 367).
Only two basic residues, one in helix K and the other in the heme binding peptide (R323 and R398 in CYP102, corresponding to R354 and R426 in CYP21), are completely conserved in all eukaryotic P450s, suggesting that other positively charged residues that are not completely conserved may be necessary for binding to the accessory protein (368).
C. CYP21 gene structure
The structural gene encoding human CYP21 (CYP21,
CYP21A2, or CYP21B) and a pseudogene (CYP21P,
CYP21A1P, or CYP21A) are located in the HLA
major histocompatibility complex on chromosome 6p21.3 approximately 30
kb apart, adjacent to and alternating with the C4B and
C4A genes encoding the fourth component of serum complement
(369, 370) (Fig. 11). In addition the
RP1 (G11) gene is located immediately 5' of
C4A and encodes a putative nuclear protein similar to DNA
helicase; a truncated copy of this gene, RP2, is located
between CYP21P and C4B (371, 372). The
CYP21, C4, and RP genes are transcribed in the
same direction. CYP21 overlaps a gene on the opposite DNA
strand (OSG, XB, or TNXB) that encodes
a putative extracellular matrix protein, tenascin-X (373).
CYP21P overlaps a truncated copy of this gene
(TNXA) that does not encode a functional protein.
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D. Transcription
1. Naturally occurring transcripts. For purposes of this
review, the most important gene transcript in the C4-CYP21
region is that of CYP21 itself, which begins 10–11 nt
before the initial AUG codon. Whereas the C4A, C4B, and
TNXB genes are mainly expressed in other tissues, the
truncated TNXA gene is transcribed in an adrenal-specific
manner (374).
CYP21P is also transcribed specifically in the intact adrenal cortex at a level 10–20% that of CYP21(375). However, the first 2 introns are inconsistently spliced out, and an uncertain proportion of transcripts include additional exons in the region between the end of CYP21P and the beginning of C4B. Some of these exons may overlap the truncated TNXA gene (375). In contrast, CYP21P transcripts cannot be detected in primary cultures of human adrenocortical cells, whereas CYP21 is appropriately expressed under the same conditions (376, 377). In any case, CYP21P transcripts do not contain a long open reading frame and are of uncertain functional significance (378). Adrenal transcripts in the same direction as CYP21 have also been detected overlapping TNXB; these are also of uncertain functional significance (375, 378).
2. Hormonally induced expression. The primary factor regulating CYP21 expression in the zona fasciculata of the adrenal cortex is ACTH (reviewed in Ref. 20). ACTH induction is mediated mainly through increased transcription (379, 380), is duplicated by cAMP and related agonists such as forskolin, and requires protein kinase A (381). This is consistent with the known mode of action of ACTH.
Factors inducing expression of CYP21 in H295R human adrenocortical carcinoma cells include cAMP (the second messenger for ACTH) and angiotensin II, which acts primarily through the protein kinase C pathway but also through Ca2+ signaling (382). The cAMP and protein kinase C pathways also induce CYP21 expression in primary cultures of human adrenocortical cells, as do insulin and IGF-I (377).
3. 5'-Flanking sequences controlling transcription. In
cultured mouse Y-1 or human H295 adrenocortical cells, the 5'-flanking
region of human CYP21 drives basal expression of reporter
constructs at levels 2.5–8 times higher than the corresponding region
of CYP21P (376, 383, 384). Sequences responsible for this
difference have been localized to the first 176 nucleotides (376)
although sequences upstream of this region are required for full
expression (in this discussion, we will number nucleotides from the
start of translation, as different numbering systems have been used by
different authors). There are only 4 nucleotide differences between
CYP21P and CYP21 in the proximal 176 nucleotides
(Fig. 12). It appears that the most
important differences are at nucleotide -113, which is a G in
CYP21 and an A in CYP21P, and at -126, which is
a C in CYP21 and a T in CYP21P (384). The latter
polymorphism is in the middle of a binding site for the Sp1
transcription factor from -123 to -129; the CYP21P
sequence binds this factor much less well. Moreover, -126C is at one
end of an overlapping binding site for an additional transcription
factor termed "adrenal specific protein" (ASP). ASP has not yet
been fully characterized but is presumed to bind DNA through zinc
fingers as do nuclear hormone receptors (385, 386, 387). In contrast, -113G
does not lie within a canonical binding site for any known
transcription factor, but it is similar to an Sp1 site, and mutation of
this nucleotide does interfere with binding of Sp1 (384). There is a
site at -110/-103 that could bind the nuclear factor NF-GMb, which,
as far as is known, is specific for granulocytes and macrophages (388).
Mutating nucleotide -110 does not have major effects on expression,
and so the significance of this putative binding site is uncertain.
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Although CYP21 is known to respond to cAMP, the 5'-flanking region does not contain a canonical cAMP response element (a CRE, which is TGACGTCA or a variant thereof). Two regions have been implicated in cAMP responsiveness because they confer such responsiveness on heterologous reporter constructs. The first is a segment from -140/-107, which contains the aforementioned Sp1 and ASP recognition sites (385, 386), and the second extends from -244/-237 and binds the transcription factor NGFI-B (nerve growth factor inducible-B, also called Nur77)(390). Mutation of either of these sites destroys cAMP responsiveness (376, 386); moreover, cotransfection with an NGFI-B expression plasmid transactivates CYP21 reporter constructs (376, 390). NGFI-B is constitutively present in Y-1 mouse adrenocortical cells but is phosphorylated within the DNA-binding domain and does not bind DNA. Treatment with ACTH results in de novo synthesis of unphosphorylated protein that is able to bind DNA and is transcriptionally active (391).
4. More distal elements. Whereas 330 nucleotides of 5'-flanking sequences from the mouse Cyp21 gene are sufficient for expression of reporter constructs in cultured Y-1 cells (381), even 2.2 kb of flanking sequences are unable to direct expression to the adrenal gland in transgenic mice (392). At least 6.4 kb of such sequences are required; the necessary sequences are localized to two short sequences 5–6 kb upstream of Cyp21, located within the adjacent Slp gene (an inactive homolog of complement C4)(392). These sequences may constitute a locus control region. Such regions are required to establish a tissue-specific open chromatin domain in the vicinity of a particular locus and thus permit appropriate tissue-specific expression; the first one identified was in the ß-globin cluster (393). In addition to their effects on chromatin, elements within certain locus control regions can act as transcriptional enhancers, and that is the case for both of the elements in Slp when they are tested in mouse Y-1 cells.
Even these sequences may not be sufficient for full expression of Cyp21, because the levels of expression of reporter constructs in transgenic animals are low compared with the intrinsic Cyp21 gene (394).
It is not yet certain whether a similar region exists in humans, but a cryptic adrenal specific promoter has been located within the C4A gene (395). The finding that the truncated TNXA gene is expressed specifically in the adrenal gland (396), although its promoter has been deleted by the duplication of the entire C4-CYP21-TNX locus, suggests that there is, at least, an adrenal-specific enhancer that is able to influence expression of several adjacent genes.
E. HLA linkage
CAH due to 21-hydroxylase deficiency is inherited as a monogenic
autosomal recessive trait closely linked to the HLA complex,
meaning that siblings who have 21-hydroxylase deficiency are almost
invariably HLA identical (397, 398). Before cloning of
CYP21, HLA typing was the main way to perform
prenatal diagnosis (399)(see Section VI.H). In addition,
particular forms of 21-hydroxylase deficiency are associated with
particular combinations of HLA antigens, or haplotypes; this
phenomenon is referred to as genetic linkage disequilibrium. The most
interesting is an association between the salt wasting form of the
disease and HLA-A3;Bw47;DR7 most characteristically seen in
Northern European populations. In addition to 21-hydroxylase
deficiency, this haplotype usually carries a null allele at one of the
two C4 loci encoding the fourth component of serum
complement (400, 401). Before cloning of CYP21, this was
strongly suspected to represent a contiguous gene syndrome due to a
single deletion of C4 and 21-hydroxylase genes; this was confirmed
shortly after CYP21 was cloned (2, 370). The deletion
apparently occurred after the haplotype was generated, because the
identical haplotype without the deletion has been identified in the Old
Order Amish (402). The nonclassic form of 21-hydroxylase deficiency is
often associated with HLA-B14;DR1, particularly in Eastern
European Jewish populations (58, 403, 404). This haplotype is
associated with the V281L mutation in CYP21 (see below) and
with a duplication of complement C4A and the
CYP21P pseudogene (405, 406). Finally,
HLA-A1;B8;DR3 is negatively associated with 21-hydroxylase
deficiency. This haplotype has a C4A null allele and is associated with
deletion of the C4A and CYP21P genes (370, 407).
Thus, comparison of a very few individuals homozygous for
HLA-A3;Bw47;DR7 or A1;B8;DR3 strongly suggested
that the CYP21 gene (then called the 21-hydroxylase "B"
gene) was an active gene, whereas the CYP21P gene
(21-hydroxylase "A") was a pseudogene (370).
Using pulsed field gel electrophoresis, CYP21 has been mapped approximately 600 kb centromeric of HLA-B and 400 kb telomeric of HLA-DR. It is transcribed in the telomeric to centromeric direction (408, 409).
F. Mutations causing 21-hydroxylase deficiency
Most mutations causing 21-hydroxylase deficiency that have been
described thus far are apparently the result of either of two types of
recombinations between CYP21, the normally active gene, and
the CYP21P pseudogene. These two mechanisms are unequal
crossing over during meiosis, resulting in a complete deletion
of C4B and a net deletion of CYP21 (2, 405, 410),
or apparent gene conversion events that transfer deleterious mutations
normally present in CYP21P to CYP21 (206, 411, 412, 413, 414, 415, 416, 417).
The deleterious mutations in CYP21P include an A
G
substitution 13 nucleotides (nt) before the end of intron 2 that
results in aberrant splicing of pre-mRNA, an 8-nt deletion in exon 3
and a 1-nt insertion in exon 7, each of which shifts the reading frame
of translation, and a nonsense mutation in codon 318 of exon 8 (343, 344). There are also 8 missense mutations in CYP21P, 7 of
which have been observed in patients with 21-hydroxylase deficiency
(Fig. 13 and Table 4).
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, 4, and 5, and
see Section VI.I) (34, 209, 334, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432).
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1. Deletions and large gene conversions. Large deletions
involving C4B and CYP21 comprise approximately
20% of alleles in patients with classic 21-hydroxylase deficiency in
most populations (Table 5) but are rarer in some Latin American
countries (443, 444). Many deleted alleles are associated with the
HLA haplotype A3;Bw47;DR7 (2). Deletions usually
extend approximately 30 kb from somewhere between exons 3 and 8 of
CYP21P through C4B to the corresponding point in
CYP21, yielding a single remaining CYP21 gene in
which the 5'-end corresponds to CYP21P, and the 3'-end
corresponds to CYP21 (Fig. 14)
(396, 410, 445). Deleterious mutations within the CYP21P
portion render such a gene incapable of encoding an active enzyme. All
patients who carry homozygous deletions suffer from the salt wasting
form of the disorder.
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One kindred carries an unusual deletion extending into the TNXB gene; the patients in this kindred have a contiguous gene syndrome including 21-hydroxylase deficiency and a form of Ehlers-Danlos syndrome caused by loss of function of tenascin-X (449). However, an unrelated patient with 21-hydroxylase deficiency and a similar heterozygous deletion apparently had no additional problems (446).
In most studies (2, 34, 370, 405, 406, 410, 419, 420, 421, 423, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461),
these deletions have been detected by genomic blot hybridization as
absence (or diminished intensity in heterozygotes) of gene-specific
fragments produced by digestion with several restriction enzymes (Fig. 14). Large gene conversions, in which multiple mutations are
transferred from CYP21P to CYP21, are also
detected by this approach when gene-specific restriction endonuclease
sites are affected. Large conversions account for approximately 10% of
alleles in classic 21-hydroxylase deficiency (Table 5).
It is now well appreciated that reliable differentiation of deletions
and large gene conversions requires analysis of several different
restriction digests (typically using the enzymes Taq I and
Bgl II) in which the sites used to distinguish
CYP21P and CYP21 are widely spaced (410, 454, 457). This is required because the remaining CYP21-like gene
on a chromosome with a deletion consists of the 3'-end of
CYP21 "spliced" onto the 5'-end of CYP21P, so
that the missing fragment in each restriction digest does not
necessarily correspond in size to the CYP21-specific
fragment in normal chromosomes. In fact, deletions of CYP21
and CYP21P are indistinguishable in certain restriction
digests (e.g., Bgl II, see Fig. 14). On the other
hand, transfers of polymorphic restriction sites from CYP21P
to CYP21 by gene conversion can also be difficult to
distinguish from actual deletions of CYP21in single
restriction digests (e.g., Taq I).
Moreover, deletion or duplication of C4A and CYP21P (which, as mentioned, occurs frequently) can be very confusing when a C4B-CYP21 deletion or a large CYP21 gene conversion is present on the other chromosome. Thus, DNA from both parents should be examined whenever possible to confirm segregation of putative deletions or gene conversions. Reprobing the same blots with a probe for C4 is a useful measure to confirm deletions (410, 447, 452).
Deletions have also been directly documented by resolving very large restriction fragments using pulsed field gradient electrophoresis (448, 462, 463) and by high resolution fluorescent in situ hybridization (464).
In practice, genomic blot hybridizations are no longer routinely used for molecular diagnosis because they are more laborious and less informative than PCR-based techniques. However, PCR is unable to distinguish deletions from large gene conversions (see Section VI.H).
2. Nonsense and frameshift mutations. Two other mutations normally found in CYP21P completely prevent synthesis of an intact enzyme and cause salt wasting 21-hydroxylase deficiency if they occur in CYP21: the nonsense mutation in codon 318 (Q318X) (416) and the 8-nt deletion in exon 3 (413). The 1-nt insertion in exon 7 of CYP21P has generally not been identified as an independent mutation in patients with 21-hydroxylase deficiency.
3. A or CG mutation in intron 2. The nucleotide 13 bp
before the end of intron 2 (nt 656) is A or C in normal individuals.
Mutation to G constitutes the single most frequent allele causing
classic 21-hydroxylase deficiency.
This mutation causes aberrant splicing of intron 2 with retention of 19 nucleotides normally spliced out of mRNA, resulting in a shift in the translational reading frame (414, 418). Almost all of the mRNA is aberrantly spliced, but in cultured cells a small amount of normally spliced mRNA is detected. If no other mutations were present, a small amount of normal enzyme might thus be synthesized.
Although it is not known what proportion of mRNA is normally spliced in the adrenal glands of patients with this mutation, most (but not all) patients who are homozygous or hemizygous for this mutation have the salt wasting form of the disorder, indicating that they have insufficient enzymatic activity to permit adequate aldosterone synthesis. Occasionally, presentation of salt wasting signs is delayed until several months of age in patients carrying this mutation (465). Putative asymptomatic nt 656g homozygotes have been reported but in fact represent PCR typing artifacts (see Section VI.I.3)(466).
4. Pro-30Leu (P30L). This mutation yields an
enzyme with 30–60% of normal activity when expressed in cultured
cells (438). However, enzymatic activity is rapidly lost when the cells
are lysed, suggesting that the enzyme is relatively unstable. Patients
carrying this mutation tend to have more severe signs of androgen
excess than patients carrying the more common nonclassic mutation V281L
(420, 438). This mutation is found in approximately one-sixth of
alleles in patients with nonclassic disease, but it may comprise a
higher percentage of such alleles in Japan (467).
As is the case with other microsomal P450 enzymes, CYP21 is targeted and anchored to the membrane of the endoplasmic reticulum mainly by a hydrophobic "tail" at the amino terminus; this tail is required for enzymatic activity and stability (468, 469). Most P450 enzymes have one or more proline residues separating this tail from the remainder of the polypeptide. These residues are predicted to create a turn in the polypeptide chain, and P30L may abolish this turn. Based on studies in other P450 enzymes, this leads to improper folding of the polypeptide and may interfere with localization in the endoplasmic reticulum (470). Indeed, the P30L mutant of CYP21 is poorly localized to the endoplasmic reticulum in some (438) but not other (435) studies.
5. Ile-172Asn (I172N). This mutation, the only one
specifically associated with the simple virilizing form of the disease,
results in an enzyme with approximately 1% of normal activity (346, 365) with normal substrate affinity (Km) but
reduced activity (Vmax) (346, 434). The
isoleucine residue normally at this position in the E helix is strongly
conserved in many different P450 enzymes, and this region of the
protein in other P450s interacts with the membrane of the endoplasmic
reticulum (471). Mutation of this hydrophobic residue to a polar
residue might disrupt such an interaction, weakening the association of
the enzyme with the endoplasmic reticulum, and indeed improper
localization to the endoplasmic reticulum has been demonstrated in some
(346) but not other (434) studies. Alternatively, the mutation may
disrupt an intramolecular hydrophobic interaction stabilizing the
secondary structure of the enzyme; the mutant enzyme is abnormally
sensitive to protease digestion and doesn’t incorporate heme properly
(434).
Because aldosterone is normally secreted at a rate 100-1000 times lower than that of cortisol, it is apparent that 21-hydroxylase activity would have to decrease to very low levels before it became rate-limiting for aldosterone biosynthesis. Apparently, as little as 1% of normal activity allows sufficient aldosterone synthesis to prevent significant salt wasting in most cases (see Section VI.I).
6. Ile-Val-Glu-Met-235–238Asn-Glu-Glu-Lys
(I235N/V236E/M238K). This cluster of three missense mutations in
the G helix also abolishes enzymatic activity (346, 418). Interference
with substrate binding has been suggested (based on sequence
conservation with cholesterol side chain cleavage enzyme, another
cytochrome P450) (414) but is not supported by molecular modeling of
CYP21 based on the crystal structure of CYP102.
7. Val-281Leu (V281L). V281L occurs in all or nearly all
patients with nonclassic 21-hydroxylase deficiency who carry the
HLA haplotype B14;DR1, an association that is
presumably due to a founder effect (see Section VI.J) (206).
In certain populations (such as Jews of Eastern European origin) this
is a very common genetic polymorphism with a gene frequency of more
than 10% (191, 192); in contrast, direct molecular screening of normal
newborns in New Zealand yielded a carrier frequency of 2% (195).
Overall, approximately 70% of all nonclassic alleles carry the V281L
mutation (210, 334). However, the HLA-B14,DR1- associated
haplotype (and/or V281L) are less common among nonclassic CAH patients
in certain ethnic groups such as Yugoslavs (193) and Japanese (467).
This mutation results in an enzyme with 50% of normal activity when
17-OHP is the substrate but only 20% of normal activity for
progesterone (346, 357). One study suggested that the mutant enzyme is
not normally localized in the endoplasmic reticulum (346), whereas
another proposed that heme binding was affected (357). As another
possibility, this mutation is located in the relatively well conserved
I helix, which contains residues thought to be involved in proton
transfer (see above).
8. Arg-356Trp (R356W). This mutation
abolishes enzymatic activity when expressed in mammalian cells (365, 418). It is located in a region of the gene encoding the K helix of the
enzyme, which suggests that the mutation affects interactions with the
cytochrome P450 reductase, but this has not been demonstrated
experimentally (366).
9. Other mutations. Mutations that are apparently not gene conversions (i.e., they are not usually found in CYP21P) account for 5–10% of 21-hydroxylase deficiency alleles in most populations. The most frequent of these is P453S, which occurs in a number of different populations. This suggests that CYP21P may carry P453S as an occasional polymorphism and that this mutation is transferred to CYP21 in the same way as the other mutations frequently causing 21-hydroxylase deficiency (439, 472, 473).
Novel mutations are easy to detect using automated sequencing technologies in centers with well developed prenatal or neonatal screening programs and thus have been reported at an increased rate over the past few years. Such mutations include W22X (474), P30Q (469), G90V (367, 475), Y97X (476), P105L (436, 473), G187A (367, 475), deletion of E196 (437), G291S (437, 473), G291C (367, 475), W302X (477), R316X (478), R339H (439), R354H (367, 475), R356P, R356Q (these two are independent of the R356W mutation that can be generated as a gene conversion) (366), E380D (479), W405X (480), G424S (481), and R483P (482) and frameshifts at W22 (483),Y47 (484), C168 (485), and R483 (473). Several codons including W22, P30, G291, R356, and R483 have undergone several independent mutations and thus these areas may be "hotspots" for such events. Larger rearrangements include a deletion of 10 nucleotides in exon 8 and a duplication of 16 nucleotides in exon 9 (478). Additional mutations affecting splicing include a mutation of the splice acceptor of intron 1 (474) and the splice donors of introns 2 (478) and 7 (480).
Most of these have been reported on only one chromosome. P30Q (469), G90V and G178A (475), delE196 and G291S (437), G291C (475), R339H (439), R354H (475), R356P and R356Q (366), and R483P (437) decrease or abolish enzymatic activity, and delE196 and R483P also adversely affect enzyme stability (437); P105L acts synergistically with P453S, with which it is associated in one kindred (436).
Despite an apparently exhaustive search, mutations could be not detected in CYP21 in one patient with apparent simple virilizing 21-hydroxylase deficiency (486). The patient was homozygous for an HLA haplotype shared (on one chromosome) by a second cousin with salt wasting 21-hydroxylase deficiency who carried the 8-bp deletion in exon 3 on his other chromosome. This strongly suggests that the presumed 21-hydroxylase deficiency in the patient is genetically linked to HLA and thus to CYP21. Trivial explanations aside, this suggests that a site outside the gene may be able to significantly influence its expression (see Section VI.D.3).
10. Normal polymorphisms. Several normal polymorphisms have been detected in CYP21 in the course of initial sequencing of cloned genes by several groups (343, 344, 345, 365). An extra leucine near the N terminus (this has confused numbering of other mutations in some reports) and D183E also occur in CYP21P and presumably represent gene conversions that don’t affect activity (418). K102R (345), S268T (345, 357, 487), and N493S (345, 365) do not represent gene conversions.
G. De novo recombinations
CAH is unusual among genetic diseases in the high proportion of
mutant alleles generated by intergenic recombination. Both de
novo deletions (488, 489) and de novo apparent gene
conversions (34, 420, 490) have been documented; the latter usually
involve the intron 2 nt 656g mutation and comprise approximately
1% of 21-hydroxylase deficiency alleles. In such cases, the proband
carries a mutation clearly not inherited from the genetically confirmed
parents. As the frequency of 21-hydroxylase deficiency alleles in the
general population is approximately 2%, the allele frequency of
de novo gene conversions in intron 2 in the general
population should be approximately 1 in 2 x
104.
De novo recombinations involving CYP21 have also
been documented by PCR in sperm and leukocytes (491). Unequal
crossing-over is detected only in sperm (1 in
105-106 genomes),
confirming that this process takes place only during meiosis. Gene
conversions, however, take place at equal frequencies in somatic cells
and gametes, suggesting that gene conversions occur mainly in mitosis
and that meiotic recombination (i.e., double crossing-over)
contributes little, if at all, to this process. The frequency of gene
conversions observed by this strategy (1 in
104) is consistent with the reported rate of
de novo gene conversions in patients with 21-hydroxylase
deficiency.
The high rate of recombinations involving the CYP21 genes may reflect their location in the major histocompatibility complex, in which a high recombination rate between genes encoding transplantation antigens may increase the diversity of the immune response and be evolutionarily favored. The mechanism by which recombination rates might be increased is not known. It is also not known whether deletions or gene conversions within CYP21 and CYP21P, or more generally within the 30-kb tandem duplication containing these genes, take place within certain discrete regions, or hotspots. It has been suggested that sequences resembling bacteriophage lambda chi sites, which are present at relatively high frequencies within CYP21/CYP21P, might promote recombination (415, 492). This hypothesis has not been directly tested, but a recombination between TNXA and TNXB also occurred near a chi site (446).
In addition, both CYP21 (493) and CYP21P (494) have high rates of single nucleotide polymorphisms, particularly in intron 2. The significance of this is uncertain, but it may mean that additional mechanisms other than intergenic recombination generate sequence diversity within the major histocompatibility complex.
H. Mutation detection and approaches to prenatal diagnosis
1. Nonmolecular techniques. Although it is possible to
prenatally diagnose 21-hydroxylase deficiency by measuring 17-OHP
levels in amniotic fluid obtained by amniocentesis, this technique
cannot be used for pregnancies in which the mother takes dexamethasone
to suppress the fetal adrenal (see Section V.G) unless she
stops the medication for 5–7 days before the amniocentesis (309, 495).
Because 21-hydroxylase deficiency was known to be closely linked to HLA, the first alternative strategy was to HLA type the proband (i.e., a living affected child in the same family) and fetal amniocytes using serological techniques (399, 496). If these were identical, the fetus could be diagnosed with 21-hydroxylase deficiency with a high degree of confidence. This technique could not be applied in kindreds in which the proband had died or was otherwise unavailable, and new mutations, HLA homozygosity, and rare complex recombinations could confound the diagnosis (496). Moreover, serological HLA typing is a complex and expensive technique, and amniocytes must be cultured for several weeks to obtain adequate quantities (497). Nevertheless, it was used until recently in some locales (498).
Initial attempts to use the CYP21 gene itself for prenatal
diagnosis relied solely on genomic blot hybridization using cDNA
probes, which were able to detect only deletions and large gene
conversions (i.e., 30% of affected alleles) (305, 499, 500, 501). When it was apparent that other gene conversions accounted
for most of the remaining alleles, it became feasible to carry out
prenatal diagnosis by detection of a limited number of mutations.
Several strategies have been used; these are considered in approximate
chronological order.
2. General considerations for molecular diagnosis. Although it
is possible to detect mutations by hybridization to genomic DNA (see
below), gene amplification using PCR dramatically improves the
sensitivity of these techniques. However, it was initially difficult to
use PCR to detect CYP21 mutations because of the paucity of
primers that would amplify CYP21 without amplifying the
highly homologous CYP21P pseudogene, which already carries
most of the mutations of interest. Eventually PCR conditions were
identified that permitted gene-specific amplification of
CYP21 in two segments (Fig. 13). For each of these segments,
the CYP21- specific primer includes an 8-bp segment in exon
3 that is deleted in CYP21P (502). This strategy fails to
amplify CYP21 if the gene is deleted, but deletions can be
detected by conventional Southern blotting. A mutant CYP21
gene would also not be amplified if it contains a gene conversion
including exon 3, but such rearrangements can be detected by a second
pair of PCRs encompassing exons 1–6 and 6–10. The
CYP21-specific primers for these reactions are located in
exon 6, in which there is a cluster of 4 nucleotides that are mutated
in CYP21P.
Conversely, "back conversion" of CYP21P to include CYP21-specific sequences could lead to spurious amplification of CYP21P and false positives. This problem is also minimized by amplification of overlapping segments.
PCR-based diagnosis may be complicated by cross-contamination of samples if rigorous controls are not implemented. Furthermore, failure to amplify one haplotype may result in misdiagnosis (466) (see Section VI.I.3). Examination of flanking microsatellite markers in all family members can minimize these problems.
Finally, it must be kept in mind that a gene conversion may be sufficiently large that it includes several mutations. If only a DNA sample from the patient is analyzed, this is impossible to distinguish from compound heterozygosity for different mutations. Therefore, both parents should also be analyzed whenever possible so as to most reliably determine the phase of different mutations (i.e., whether they lie on the same or opposite alleles). Analysis of parental alleles also permits homozygotes and hemizygotes (i.e., individuals who have a mutation on one chromosome and a deletion on the other) to be distinguished.
3. Allele-specific oligonucleotide hybridization. Once CYP21 cDNA had been cloned, deletions and gene conversions that affected gene-specific restriction sites could be detected by Southern blotting. As previously discussed, these accounted for approximately 25% of alleles in most populations.
Point mutations could be identified by hybridization with
allele-specific oligonucleotide (ASO) probes, which are short
(typically 19–21 nucleotides) single-stranded DNA segments that are
centered on each polymorphic or mutant nucleotide in the gene (Fig. 15). These probes are usually radioactively
labeled. Under appropriate hybridization and washing conditions, a
single nucleotide mismatch is sufficient to destabilize hybridization
between the probe and the gene. Each mutation could thus be tested for
by duplicate hybridization with pairs of probes, each corresponding to
either the normal or mutant sequence.
|
PCR makes this technique much more sensitive (34, 334, 419, 422, 428, 502, 503, 504, 505, 506, 507, 508). Amplified DNA is dotted on filters and hybridized with 18 probes corresponding to the normal and mutant sequences for each of the frequently occurring gene conversions. Because many samples can be dotted on a single filter and this process can be automated, this approach is relatively efficient when large numbers of samples are to be analyzed simultaneously. However, 18 independent hybridizations are laborious for small numbers of samples as are typically encountered by laboratories performing prenatal diagnosis. This procedure is also usually performed with radioactively labeled probes, although other labeling techniques are possible. Therefore, several alternative mutation detection strategies have been used. Most require a second round of PCR after relatively long gene-specific segments have been amplified.
4. Amplification-created restriction sites. Several mutations
causing 21-hydroxylase deficiency (e.g., V281L and Q318X)
create or destroy restriction sites and can thus be detected in
restriction digests of PCR-amplified DNA by staining agarose gels with
ethidium bromide (Fig. 15). Most mutations that do not involve
restriction sites can be detected by locating a PCR primer adjacent to
each mutation and changing its sequence to introduce a polymorphic
restriction site into the amplified segment (426, 467, 509, 510, 511, 512, 513). This
technique thus involves a series of second round PCRs and several
different restriction digests but does not require radioactivity or
specialized equipment. It has been used to screen for mutations in
preimplantation embryos in a couple at risk for CAH who were undergoing
in vitro fertilization (514).
5. Single-stranded conformation polymorphisms. If double-stranded DNA is denatured and then quickly returned to native conditions, it will remain in a single-stranded state with a characteristic conformation stabilized by intramolecular hydrogen bonding. Under these conditions, many mutations change the conformation of the single-stranded segment. This can be detected by a change in the mobility of the segment during PAGE under nondenaturing conditions (478, 515, 516, 517, 518). This technique has the theoretical advantage of being able to detect novel mutations that would be missed by allele-specific approaches. However, it becomes insensitive with segments longer than a few hundred nucleotides and thus requires a series of second- round PCRs to cover the entire gene as well as electrophoresis on sequencing gels to resolve the conformation differences. Moreover, typically two rounds of electrophoresis are carried out under different conditions (e.g., with or without glycerol in the gel) to increase the likelihood that most mutations will be detected. Thus, this technique has little to recommend it over available alternatives.
Some of these disadvantages may be minimized by use of a related technique, denaturing gradient gel electrophoresis (519, 520); this technique may be more sensitive but it usually requires specialized electrophoresis apparatus, and it has not been widely used for diagnosis of CAH.
6. Allele-specific PCR. A single nucleotide mismatch at the
3'-end of a PCR primer dramatically decreases the efficiency of the
reaction. Thus a mutation can be detected by running two alternative
reactions. Both use the same primer on one end, but at the other end
each reaction uses a primer that corresponds to either the normal or
mutant sequence, respectively. Once CYP21-specific sequences
have been amplified, a second round of 18 allele-specific PCRs
(recognizing the normal and mutant sequences at each mutation) will
identify the 9 most common mutations (209, 420, 423, 424, 429, 430, 431, 432, 480, 521, 522). This technique has similar advantages to the
amplification-created restriction site approach; it requires more PCR
reactions but doesn’t involve restriction digests. Most recent large
genotyping studies have used this strategy (Fig. 15).
7. Ligation detection reaction. DNA ligase can discriminate
point mutations by sequential rounds of linear template-dependent
ligation. The ligase will preferentially seal adjacent oligonucleotides
hybridized to target DNA in which there is perfect complementarity at
the nick junction. A single base mismatch at the nick junction inhibits
ligation. Nicks with matches or mismatches can be readily made at a
possible mutation site by hybridizing two adjacent oligonucleotides at
the desired nucleotide position. Suitably labeling one of these
oligonucleotides allows for detection of ligation products, thus
permitting sequence discrimination at the single nucleotide level.
Ligated and unligated products are easily resolved on a sequencing gel
(Fig. 15) (523).
This type of reaction can be readily multiplexed, i.e., all 18 (or more) necessary typing reactions can be run simultaneously in a single tube using PCR-amplified CYP21 gene segments. Multiplexing is achieved by synthesizing oligonucleotides with synthetic nonhybridizing poly-dA tails such that each ligation product has a unique length. If the oligonucleotides are fluorescently labeled, the entire genotyping can be performed on an automated DNA sequencer. Several different fluorescent labels can also be employed.
This technique has been successfully used for a second round of neonatal screening after hormonally based primary screening (195).
8. Oligonucleotide arrays ("DNA chips").Allele-specific oligonucleotides can be covalently linked to a solid support and hybridized with fluorescently labeled PCR-amplified DNA. The pattern of hybridization is electronically scanned to identify each mutation (524). This technique has been used to demonstrate that CYP21 is one of the most frequently polymorphic genes in the human genome (493). To our knowledge, it has not yet been applied to routine diagnosis of 21-hydroxylase deficiency, but the potential high throughput of this technique makes it attractive for mass screening applications.
9. DNA sequencing. The best way to make certain that novel mutations are not missed is to sequence the entire gene. This can readily be accomplished using automated sequencing methods (473, 525), and computer analysis eliminates the tedium and potential for error in manually reading sequencing gels. This technique is most commonly used after other approaches have failed to identify a mutation on at least one known 21-hydroxylase deficiency allele, but it has been used for some population-based studies (427).
10. Linked microsatellites. Despite efforts to increase the ease and accuracy of direct mutation detection, it still requires several PCRs. The interpretation of resulting data is often complex when gene conversions affect the sites recognized by gene-specific primers. Moreover, as discussed below, certain alleles may be preferentially amplified by PCR, leading to errors in typing. Although this strategy is required for research purposes or for confirmation of neonatal screening results, it may not be necessary or even optimal for prenatal diagnosis, especially in countries lacking a centralized laboratory with expertise in this technique. Linkage analysis using highly polymorphic "microsatellite" loci is an alternative or supplementary technique that can be readily performed by most genetics laboratories (431, 466, 526). Microsatellite loci contain several (typically 10–20) dinucleotide (such as CA), trinucleotide (such as CAG), or tetranucleotide tandem repeats. Alleles differ in the number of repeats. PCR using primers flanking the repeated segments yield products of varying lengths that can be measured using manual or automated sequencing gel electrophoresis. For prenatal diagnosis of 21-hydroxylase deficiency, this is conceptually similar to HLA typing in its requirement that a sample from an affected child in the same family be compared with the fetal sample, but it may be carried out on DNA from a chorionic villus sample. Typing of at least two flanking microsatellites will yield the most informative and reliable results (195).
I. Correlations between genotype and phenotype
1. Classification of disease severity. The classification of
21-hydroxylase deficiency into salt wasting, simple virilizing, and
nonclassic types is a useful way to roughly grade the severity of the
disease and to predict the therapeutic interventions that will likely
be required. If molecular diagnosis could predict this classification,
it would increase the utility of prenatal diagnosis and neonatal
screening, and it might serve as a useful diagnostic adjunct to ACTH
stimulation tests.
The simplest way to correlate genotype and phenotype is to determine which mutations are characteristically found in each type of 21-hydroxylase deficiency. This is most informative for frequently occurring mutations. Deletions and large conversions are most often found in salt wasting patients, the intron 2 nt 656g mutation is found in both salt wasting and simple virilizing patients, I172N is characteristically seen in simple virilizing patients, and V281L and P30L are found in nonclassic patients (34, 418, 419). This distribution is consistent with the compromise in enzymatic activity conferred by each mutation (see Section VI.F).
However, patients are usually compound heterozygotes for different mutations, and so this approach has little predictive value in itself. A useful analytic strategy is to consider that 21-hydroxylase deficiency is a recessive disease, and thus the phenotype of each patient is likely to reflect his or her less severely impaired allele. If mutations are provisionally classified by the degree of enzymatic compromise—severe (also termed "type A"), moderate (type B), or mild (type C)—then one might hypothesize that salt wasting patients would have severe/severe genotypes, simple virilizing patients would have severe/moderate or moderate/moderate genotypes, and nonclassic patients would have severe/mild, moderate/mild or mild/mild genotypes. In one study of 88 families (34), these three predictions were correct in 90%, 67%, and 59% of cases, respectively. The overall correct classification rate was 79%. An expanded follow-up study of the same population (209) yielded even better results, with 177 of 197 patients (88%) being correctly classified in this manner. Similar results were obtained in other studies using the same approach (420, 527).
The salt wasting, simple virilizing, and nonclassic categories are qualitative in nature, and the distinction between simple virilizing and nonclassic disease is necessarily difficult in males in whom signs of androgen excess cannot be detected at birth. Therefore, attempts have been made to correlate genotype with quantitative measures of disease severity such as basal and ACTH-stimulated 17-OHP levels, plasma renin/urinary aldosterone ratios, and Prader genital virilization scores. In general, these are no better correlated with genotype than the broader clinical categories are. There is excellent discrimination between severe and mild genotypes, but a high degree of overlap between moderate genotypes and those either more and less affected (34).
2. Explanations for discordance of genotype and phenotype. Several explanations for the less-than-complete correspondence between genotype and phenotype are possible. The most obvious is that the severity of the disease falls on a continuum and patients with disease severity near the borders of the various classifications may easily fall on either side of these borders. Several mutations and genotypes seem to be particularly associated with this problem. First, although the intron 2 nt 656g mutation is classified as severe, it is clearly "leaky" and may yield enough normally spliced mRNA to ameliorate the enyzmatic deficiency in some patients. Second, the I172N mutant has marginal enzymatic function (1% of normal), and this apparently is not always sufficient to prevent salt wasting. These two explanations accounted for 12 of 20 examples of apparent discordance between genotype and phenotype in one study (209), and significant phenotypic variation was noted in a kindred in which all five affected individuals were compound heterozygotes for these two mutations (315). Third, many patients who are discordant for genotype and phenotype are compound heterozygotes for mutations that compromise enzymatic activity to different extents (209, 420); thus it appears that some of these patients actually have in vivo enzymatic activities intermediate between those seen in patients who are homozygous for each mutation. Consistent with this idea, presumed compound heterozygotes for a classic and nonclassic allele as a group have higher stimulated 17-OHP levels than presumed homozygotes for nonclassic alleles (36).
Fourth, in studies relying on detection of known mutations, additional novel mutations within CYP21 might not be detected and might adversely affect activity.
Finally, genetic or environmental factors other than 21-hydroxylase activity may influence phenotype. As discussed in Section III.E, the degree of salt wasting tends to improve with time, even in subjects who are genetically predicted to have no 21-hydroxylase activity (84), and genetically identical siblings are occasionally discordant for severity of salt wasting (34, 83); this might reflect expression of additional 21-hydroxylase activities not encoded by CYP21 (85, 86). Similarly, genetically based variations in androgen biosynthesis or sensitivity to androgens would be expected to influence expression of signs of androgen excess (Section III.D).
3. The problem of allele dropout. Inaccurate genotyping can obviously confound genotype-phenotype correlations. An important cause of inaccurate genotyping of CYP21 is unequal PCR amplification of different alleles, sometimes termed "allele dropout." In particular, nt 656g is sometimes preferentially amplified over the corresponding two normal alleles, 656a and especially 656c, so that heterozygous carriers of nt 656g are typed as homozygotes (466). This led to several reports of high frequencies of asymptomatic homozygotes for this mutation; such individuals were usually obligate heterozygous carriers (such as parents of patients) detected in family studies (514, 528, 529, 530). This seemed physiologically possible because this mutation activates a cryptic splice site but allows some amount of normal mRNA to be synthesized (see above). However, the carrier frequencies of nt 656g predicted by these studies were implausibly high. Moreover, the "extra" nt 656g alleles failed to segregate within kindreds (466).
Preferential amplification or allele dropout, in some instances, can be alleviated by altering the PCR amplification conditions such as lowering the KCl concentration (423, 531). In any case, confusion in critical circumstances such as prenatal diagnosis is minimized by genotyping both parents whenever possible and by concomitant use of microsatellite linkage markers (466).
J. Why is CAH so common?
Classic CAH is a relatively common inherited disease, yet it is
potentially lethal if untreated. As death tends to remove mutant
alleles from the population, the question arises as to mechanisms
maintaining the carrier frequency at 1–2%. Could there be a selective
advantage to heterozygosity, such as exists for sickle cell anemia and
resistance to malaria? If so, one would expect to find a carrier
frequency for classic CAH that exceeds that predicted from the
frequency of individuals with the disease (i.e., a
violation of Hardy-Weinberg equilibrium). In New Zealand, the
carrier frequency estimated from direct genetic testing of 600 normal
newborns—2.8%—indeed exceeds the estimate derived from the frequency
of classic CAH observed in newborn screening, 1.3% (195, 223). This
difference, however, falls short of statistical significance. Moreover,
no mechanism for a heterozygote advantage is immediately apparent.
The observed carrier frequency might be accounted for by a series of founder effects; i.e., a mutation arising in a single ancestor in a particular population is spread through the population via one or more prolific carriers. This is the likely explanation for the high frequency of CYP21 deletions on the HLA haplotype A3;Bw47;DR7 among Northern Europeans or the V281L mutation on the HLA-B14;DR1 haplotype observed at high frequency in Ashkenazi Jews. As matings between carriers are relatively infrequent at the observed carrier frequencies, there may have been insufficient time for these mutations to be selected against, a phenomenon termed "genetic drift" (532).
New mutations may replace mutant alleles lost through death of affected individuals. As discussed in Section VI.G, the incidence of de novo gene conversions is estimated to be approximately 1 in 1 x 10-4 (491), similar to the incidence of CAH itself, suggesting that the rates of new mutation and loss from selection may indeed balance.
A selective advantage to the carrier state might arise indirectly due to the position of CYP21 within the major histocompatibility complex. There seem to be selective advantages to heterozygosity for HLA antigens as regards disease resistance (533, 534), and this might select for heterozygosity for CYP21 mutations when these are in genetic linkage disequilibrium with particular HLA antigens. It has also been speculated (535) that gametes carrying particular HLA haplotypes might be preferentially inherited—a phenomenon termed "transmission ratio distortion"—but although this is well documented in mice (536), it has not been consistently observed in the human HLA complex.
As discussed in Section III.J, nonclassic CAH is very frequent in some populations despite putative deleterious effects on fertility, leading to analogous questions about a heterozygote advantage for this form of 21-hydroxylase deficiency (191). However, the actual prevalence of infertility in this disorder is difficult to determine due to the problem of ascertainment bias. It has been speculated that there could be a selective immunological advantage for individuals carrying the nonclassic CYP21 defect who have slightly higher cortisol levels after cosyntropin stimulation than controls. There is no evidence, however, that the response to pharmacological doses of cosyntropin has any physiological relevance to immunological function (537, 538). Obviously, the alternative explanations of founder effect, genetic drift, and a high frequency of de novo mutations all apply equally to classic and nonclassic CAH.
In summary, several explanations account for the observed frequency of mutant CYP21 alleles in the general population. Although the initial report of an increased carrier frequency of mutant CYP21 alleles (195) needs to be extended and confirmed, it is unlikely that a direct selective advantage for heterozygosity can be demonstrated.
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1 Supported by NIH Grant R37 DK-37867. 
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8nt) or a cluster of three missense
mutations (I236N/V237E/M239K), respectively. A 1-kb scale is displayed.
