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Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency¹

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

	Abstract

Top Abstract I. Introduction II. Biochemistry of CAH III. Pathophysiology of CAH IV. Diagnosis of 21-Hydroxylase... V. Treatment VI. Molecular Genetic Analysis VII. Summary References

 
More than 90% of cases of congenital adrenal hyperplasia (CAH, the inherited inability to synthesize cortisol) are caused by 21-hydroxylase deficiency. Females with severe, classic 21-hydroxylase deficiency are exposed to excess androgens prenatally and are born with virilized external genitalia. Most patients cannot synthesize sufficient aldosterone to maintain sodium balance and may develop potentially fatal "salt wasting" crises if not treated. The disease is caused by mutations in the CYP21 gene encoding the steroid 21-hydroxylase enzyme. More than 90% of these mutations result from intergenic recombinations between CYP21 and the closely linked CYP21P pseudogene. Approximately 20% are gene deletions due to unequal crossing over during meiosis, whereas the remainder are gene conversions—transfers to CYP21 of deleterious mutations normally present in CYP21P. The degree to which each mutation compromises enzymatic activity is strongly correlated with the clinical severity of the disease in patients carrying it. Prenatal diagnosis by direct mutation detection permits prenatal treatment of affected females to minimize genital virilization. Neonatal screening by hormonal methods identifies affected children before salt wasting crises develop, reducing mortality from this condition. Glucocorticoid and mineralocorticoid replacement are the mainstays of treatment, but more rational dosing and additional therapies are being developed.

I. Introduction

II. Biochemistry of CAH

A. Biochemistry of normal steroid synthesis

B. Regulation of adrenal steroid secretion

C. Abnormal steroids in 21-hydroxylase deficiency

III. Pathophysiology of CAH

A. Normal sexual differentiation

B. Normal prenatal development of adrenal glands

C. Adrenarche

D. Prenatal virilization

E. Salt wasting

F. Postnatal signs of androgen excess

G. Reproductive function in classic CAH

H. Neuropsychology of CAH

I. Tumors

J. Nonclassic CAH phenotypes

K. Heterozygotes

IV. Diagnosis of 21-Hydroxylase Deficiency

A. Evaluation of ambiguous genitalia

B. Newborn screening

C. Further biochemical evaluation

V. Treatment

A. Glucocorticoid replacement

B. Mineralocorticoid replacement

C. Other therapeutic approaches

D. Corrective surgery

E. Psychological counseling

F. Treatment of precocious puberty

G. Prenatal therapy

VI. Molecular Genetic Analysis

A. Biochemistry of CYP21

B. Structure-function relationships

C. CYP21 gene structure

D. Transcription

E. HLA linkage

F. Mutations causing 21-hydroxylase deficiency

G. De novo recombinations

H. Mutation detection and approaches to prenatal diagnosis

I. Correlations between genotype and phenotype

J. Why is CAH so common?

VII. Summary

	I. Introduction

Top Abstract I. Introduction II. Biochemistry of CAH III. Pathophysiology of CAH IV. Diagnosis of 21-Hydroxylase... V. Treatment VI. Molecular Genetic Analysis VII. Summary References

 
VIRILIZING congenital adrenal hyperplasia (CAH) is the most common cause of genital ambiguity, and 90–95% of CAH cases are caused by 21-hydroxylase deficiency. Females affected with severe, classic 21-hydroxylase deficiency are exposed to excess androgens prenatally and are born with virilized external genitalia. First described in the mid-19th century, a more thorough understanding of this disease was not forthcoming until the mid-20th century, when the recessive nature of the genetic trait and identification of hormonal abnormalities were recognized (1).

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).

	II. Biochemistry of CAH

Top Abstract I. Introduction II. Biochemistry of CAH III. Pathophysiology of CAH IV. Diagnosis of 21-Hydroxylase... V. Treatment VI. Molecular Genetic Analysis VII. Summary References

 
A. Biochemistry of normal steroid synthesis
The rate-limiting step in steroid biosynthesis is importation of cholesterol from cellular stores to the matrix side of the mitochondria-inner membrane where the cholesterol side chain cleavage system (CYP11A, adrenodoxin, adrenodoxin reductase) is located. This is controlled by the steroidogenic acute regulatory (StAR) protein (6), the synthesis of which is increased within minutes by trophic stimuli such as ACTH or, in the zona glomerulosa, increased intracellular calcium. StAR is a synthesized as a 37-kDa phosphoprotein that contains a mitochondrial importation signal peptide. However, importation into mitochondria is not necessary for StAR to stimulate steroidogenesis, and it now seems likely that, to the contrary, mitochondrial importation rapidly inactivates StAR (7). The mechanism by which StAR mediates cholesterol transport across the mitochondrial membrane is not yet known.

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.

View larger version (25K): [in this window] [in a new window]   Figure 1. Pathways of steroid biosynthesis. The pathways for synthesis of progesterone and mineralocorticoids (aldosterone), glucocorticoids (cortisol), androgens (testosterone and dihydrotestosterone), and estrogens (estradiol) are arranged from left to right. The enzymatic activities catalyzing each bioconversion are written in boxes. For those activities mediated by specific cytochromes P450, the systematic name of the enzyme ("CYP" followed by a number) is listed in parentheses. CYP11B2 and CYP17 have multiple activities. The planar structures of cholesterol, aldosterone, cortisol, dihydrotestosterone, and estradiol are placed near the corresponding labels.

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.

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.

	III. Pathophysiology of CAH

Top Abstract I. Introduction II. Biochemistry of CAH III. Pathophysiology of CAH IV. Diagnosis of 21-Hydroxylase... V. Treatment VI. Molecular Genetic Analysis VII. Summary References

 
A. Normal sexual differentiation
Early in gestation, the gonads are indifferent and bipotential (Fig. 2). 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).

View larger version (31K): [in this window] [in a new window]   Figure 2. Time course of prenatal sexual differentiation in male and female fetuses. Top, Amniotic fluid levels of 17-OHP at various ages of gestation. The scale is logarithmic. Open squares denote mean values in fetuses affected with 21-hydroxylase deficiency, and closed circles denote mean values in normal infants. Vertical lines denote 95% confidence limits [adapted from Ref. 500]. Bottom, Timelines for five aspects of sexual differentiation [adapted from Ref. 541]. Note that 17-OHP levels are already markedly elevated in affected fetuses during development of the external genitalia.

View larger version (14K): [in this window] [in a new window]   Figure 3. Normal and abnormal differentiation of the urogenital sinus and external genitalia (cross-sectional view). Diagrams of normal female and male anatomy flank a series of schematic representations of different degrees of virilization of females, graded using the scale developed by Prader (64 ). [Adapted from Refs. 64 and 213]. Note that the uterus persists in virilized females even when the external genitalia have a completely masculine appearance (Prader grade 5).

View larger version (14K): [in this window] [in a new window]   Figure 4. Normal and abnormal differentiation of the external genitalia (external view). Diagrams of normal female and male anatomy flank a series of schematic representations of different degrees of virilization, graded using the scale developed by Prader (64 ). [Adapted from Refs. 64 and 213].

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).

	IV. Diagnosis of 21-Hydroxylase Deficiency

Top Abstract I. Introduction II. Biochemistry of CAH III. Pathophysiology of CAH IV. Diagnosis of 21-Hydroxylase... V. Treatment VI. Molecular Genetic Analysis VII. Summary References

 
A. Evaluation of ambiguous genitalia
Management of the child born with ambiguous genitalia presents a difficult challenge to medical personnel. Insensitive and poorly informed statements made in the delivery room or subsequently may cause long-term psychological problems for the families of such children. It is therefore important to refrain from assigning the sex until diagnostic information can be gathered. Usually test results can be obtained within 24–48 h and parents can be advised as to the child’s chromosomal and gonadal sex and on the anatomy of internal sexual structures.

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.

View larger version (28K): [in this window] [in a new window]   Figure 5. Simplified flowchart for initial evaluation of ambiguous genitalia. Decision points are denoted by diamonds, and endpoints by rectangles. Note that a karyotype is almost invariably performed, although palpation of gonads and a pelvic sonogram permit a tentative sex assignment in many cases.

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).

View larger version (41K): [in this window] [in a new window]   Figure 6. Flowchart for decisions pertaining to newborn screening for 21-hydroxylase deficiency. ACTH stim 17-OHP, 17-Hydroxyprogesterone level after cosyntropin stimulation; 'lytes, electrolytes.

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).

View larger version (24K): [in this window] [in a new window]   Figure 7. Levels of 17-OHP in dried blood samples from the Wisconsin neonatal screening program, plotted against birth weight. The heavy line represents mean values and the dotted lines represent 95% confidence limits. The heavy dashed line denotes threshold notification values in the Wisconsin program for infants of various birth weights. [Adapted from Ref. 217].

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).

View larger version (29K): [in this window] [in a new window]   Figure 8. Nomogram for comparing 17-OHP levels before and 60 min after a 0.25 mg iv bolus of cosyntropin in subjects with or without 21-hydroxylase deficiency. Note that the values for normals and heterozygotes (carriers) overlap. [Adapted from Ref. 35.]

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</