Congenital adrenal hyperplasias

Best Practice & Research Clinical Endocrinology and Metabolism Vol. 15, No. 1, pp. 17±41, 2001

doi:10.1053/beem.2000.0117, available online at http://www.idealibrary.com on

2 Congenital adrenal hyperplasias Perrin C. White*

MD

Professor of Pediatrics, and Director Division of Pediatric Endocrinology, UT Southwestern Medical Center, Dallas, TX 75235-9063, USA

Congenital adrenal hyperplasia syndromes result from de®ciencies of enzymes involved in corticosteroid biosynthesis. Most commonly, they are due to mutations in 21-hydroxylase. This chapter describes the clinical diagnosis and management of congenital adrenal hyperplasias throughout life, including in the fetus, child and adult. These clinical recommendations are explained in the context of the molecular and biochemical characteristics of the diseases. Key words: steroid 21-monooxygenase; adrenal hyperplasia, congenital; cytochrome P-450; metabolism, inborn errors; sex di€erentiation disorders; virilism; hydrocortisone; aldosterone; androgens; HLA antigens.

Cortisol is normally synthesized from cholesterol in the zona fasciculata of the adrenal cortex in ®ve successive enzymatic conversions (Figure 1). Congenital adrenal hyperplasia (CAH), the inherited inability to synthesize cortisol, may be caused by defective importation of cholesterol into mitochondria (lipoid hyperplasia)1±3 or, more commonly, by mutations in steroidogenic enzymes. More than 90% of cases are caused by a de®ciency of the 21-hydroxylase activity required to convert 17-hydroxyprogesterone to 11-deoxycortisol.4 This chapter focuses on our current knowledge of this disorder, although other de®ciencies causing CAH are brie¯y considered (Table 1). Detailed reviews of these other conditions, including 11b-hydroxylase5, 17a-hydroxylase6,7 and 3b-hydroxysteroid hydrogenase8 de®ciencies, have been published. In patients with 21-hydroxylase de®ciency, poor synthesis of cortisol results in chronic stimulation of the adrenal cortex by corticotropin with consequent overproduction of cortisol precursors. Some of these precursors are shunted into the androgen biosynthetic pathway, causing signs and symptoms of androgen excess including ambiguous genitalia in females and rapid somatic growth with accelerated skeletal maturation in both sexes. The disease occurs in a wide spectrum of clinical variants, including a severe form with a concurrent defect in aldosterone biosynthesis (`salt wasting' type), a form with apparently normal aldosterone biosynthesis (`simple virilizing' type) and a mild `non-classic' form that may be asymptomatic or may be associated with signs of androgen excess developing during childhood or at puberty. *Address for correspondence: UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 7390-9063, USA. 1521±690X/01/010017‡25 $35.00/00

c 2001 Harcourt Publishers Ltd. *

HO

O

3

CH2

CH CH3

17-OH PROGESTERONE

17-OH PREGNENOLONE

CH3

HO

11

18

2

HC

CH

O

21 CH

ALDOSTERONE O

OH

O

18-OH CORTICOSTERONE

CORTICOSTERONE

3

CH3

HO

11

18

HC

17

CH

O

2

21 CH O

O

OH

CORTISOL (CYP11B1)

O

(CYP19)

3

CH3

17

CH3

OH

HO

17

OH CH3

DEHYDROGENASE OESTRADIOL

DIHYDROTESTOSTERONE

5α-REDUCTASE

TESTOSTERONE

ANDROSTENEDIONE

OESTRONE 17β-HYDROXYSTEROID

AROMATASE

ANDROSTERONE

DEHYDROEPI-

11β-HYDROXYLASE

DEOXYCORTICOSTERONE 11- DEOXYCORTISOL

PROGESTERONE

PREGNENOLONE

17α-HYDROXYLASE (CYP17) 17,20-LYASE

CHOLESTEROL

CH2

CH3

Figure 1. Pathways of steroid biosynthesis. The pathways for synthesis of progesterone and mineralocorticoids (aldosterone), glucocorticoids (cortisol), androgens (testosterone and dihydrotestosterone) and oestrogens (oestradiol) are arranged from left to right. The enzymatic activities catalysing each bioconversion are written in boxes. For those activities mediated by speci®c 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 oestradiol are placed near the corresponding labels. Adapted from White and Speiser (2000, Endocrine Reviews 21: 245±291) with permission.

(CYP11B2)

18-OXIDASE

(CYP11B2)

18-HYDROXYLASE

(CYP11B2)

11β-HYDROXYLASE

(CYP21)

21-HYDROXYLASE

DEHYDROGENASE

3β-HYDROXYSTEROID

DESMOLASE (CYP11A)

CHOLESTEROL

CH3

CH3

HC

CH3

18 P. C. White

# # in SW " "

‡ ‡ 17-OHP

Hormones Glucocorticoids Mineralocorticoids Androgens

Oestrogens " Metabolites

nl nl

‡17-OHP

nl nl "

‡ " #

|

DOC, S

# "

‡

B, 18-OHB

#

Rare

1:100 000 # "

CYP11B2 P450aldo

Aldosterone synthase

CYP11B1 P450c11

11b-hydroxylase

‡ " #

{

# DOC, B

# " #

Rare

CYP17 P450c17

17a-hydroxylase

# None

# # #

Rare

STAR

Lipoid adrenal hyperplasia

# "

# "

Severe in {, mild in | { ‡ ‡

# # often # in {; # weak androgens in | # DHEA, 17D5Preg

Rare

HSD3B2 3b-HSD

3b-hydroxysteroid dehydrogenase

‡ ˆ present; # ˆ diminished quantity; " ˆ increased quantity; { ˆ male; | ˆ female; nl ˆ normal; SW ˆ salt-waster; P450 ˆ cytochrome P450; 17-OHP ˆ 17hydroxyprogesterone; DOC ˆ deoxycorticosterone; S ˆ 11-deoxycortisol; B ˆ corticosterone; 18-OHB ˆ 18-hydroxycorticosterone; DHEA ˆ dehydroepiandrosterone; 17D5Preg ˆ 17-D5-pregnenolone.

# in SW " in SW

| ‡ in SW

Classical 1:14 000

Clinical signs Ambiguous genitalia Salt wasting crisis Hypertension Na balance K balance

CYP21 P450c21

Enzyme/gene Alias Subtype Incidence Non-classical 1:500

21-hydroxylase

De®ciency

Table 1. Clinical, biochemical and genetic characteristics of congenital adrenal hyperplasia.

Congenital adrenal hyperplasias 19

20 P. C. White

The salt wasting and simple virilizing forms, collectively referred to as `classic' 21hydroxylase de®ciency, occur in 1/10 000±1/15 000 births in most populations based on newborn screening studies. The frequency of the non-classic form is dicult to determine owing to problems of ascertainment. It may be quite frequent in certain populations such as Jews of Eastern European origin.9 All other forms of CAH occur at frequencies 51/100 000 in most populations.

DIAGNOSIS OF CONGENITAL ADRENAL HYPERPLASIA Evaluation of ambiguous genitalia Adrenal androgen secretion is increased prenatally in patients with 21- or 11bhydroxylase de®ciency. This does not signi®cantly a€ect male sexual di€erentiation. In a€ected females, however, the urogenital sinus is in the process of septation when the fetal adrenal begins to produce excess androgens. Apparently, blood levels of adrenal androgens are suciently high to prevent formation of separate vaginal and urethral canals but usually not high enough to sustain Wolan structures. 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 ori®ce. The genitalia in severely a€ected girls are male in appearance with perineal hypospadias, chordee, and undescended testes, but Mullerian structures, including the uterus and Fallopian tubes, are intact. In contrast, males with lipoid adrenal hyperplasia, 17a-hydroxylase/17,20-lyase de®ciency or 3b-hydroxylase de®ciency do not adequately synthesize androgens and are born with ambiguous or female-appearing external genitalia, but they lack female internal genital structures due to secretion of anti-Mullerian hormone. This phenotype is essentially identical to that of androgen insensitivity syndrome. In managing a child born with ambiguous genitalia (Figure 2), it is important to refrain from assigning the sex until diagnostic information can be gathered. Usually test

Gonads

No

Uterus on

Yes

Uterus on

Yes

High 17-

pelvic sono?

palpable?

OHP? Yes

No

No

Yes Karyotype

pelvic sono?

XX

CAH

No X/XY or

XX,XY

X/XXY

XX/XY

XX

XY

Mixed

True

Non-CAH

pseudoherm-

gonadal

hermaphrodite

pseudoherm-

aphrodite

dysgenesis

aphrodite

Figure 2. Simpli®ed ¯owchart 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. Adapted from White and Speiser (2000, Endocrine Reviews 21: 245±291) with permission.

Congenital adrenal hyperplasias 21

results can be obtained within 24±48 hours, and parents can be advised as to the child's chromosomal and gonadal sex and the anatomy of internal sexual structures (see refs 10 and 11). The physical examination should identify the urethral meatus and should include careful palpation for gonads in the inguinal canals and labia or scrotum. Diagnostic tests should include at least a measurement of basal serum 17hydroxyprogesterone (17-OHP), but preferably a complete pro®le of adrenocortical hormones (17-OHP, cortisol, deoxycorticosterone, 11-deoxycortisol, 17-OH-pregnenolone, dehydroepiandrosterone, and androstenedione) before and 1 hour after ACTH1±24 stimulation. These assays should be deferred past the ®rst 24 hours of life. After testing is completed, the child's vital signs should be monitored for any indication of adrenal crisis. Salt-wasting crises may occur in 21-hydroxylase or 3b-HSD de®ciencies or in lipoid hyperplasia, but it is rare for these to occur before 7 days of life. Nevertheless, many clinicians will obtain electrolyte measurements to assess hyponatraemia and hyperkalaemia in a€ected newborns during the ®rst week. Plasma renin activity (PRA) and aldosterone are elevated in many normal infants and do not usually add much useful information within the ®rst days of life. Additional tests that aid in understanding the aetiology of ambiguous genitalia include a rapid karyotype and a pelvic and abdominal sonogram. Further testing will be dictated by the outcome of these initial tests. For instance, a radiological dye study may be done in 46 XX infants to examine the internal genitourinary anatomy; in 46 XY infants, hCG stimulation may help to de®ne androgen synthetic defects such as 5areductase de®ciency. A team consisting of neonatologist, paediatric 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 and the feasibility of surgical correction. Although all available options should be reviewed with the family, these recommendations should be as unequivocal as possible. 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 de®ciency who have normal internal genital structures and the 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 male for more than a few months. Such children will need to be castrated at puberty to avoid feminization. Newborn screening CAH due to 21-hydroxylase de®ciency is a disease well suited to newborn screening because it is a common and potentially fatal childhood disease and early recognition and treatment can, in principle, prevent serious morbidity and mortality. The diagnosis is suspected when one ®nds a markedly elevated ®lter-paper blood 17-hydroxyprogesterone (17-OHP) level by radioimmunoassay.12,13 These assays use the same `Guthrie' cards as are used for screening for phenylketonuria and hypothyroidism. To obtain adequate sensitivity, the cut-o€ levels for 17-OHP are typically set low enough that 0.3±0.5% of all tests are reported as positive. Therefore, speci®city is only 2%, i.e. 98% of all positive tests are false. Moderately elevated 17-OHP levels (40±100 ng/ml for term infants) are followed up with a repeat ®lter-paper specimen. Premature, sick or stressed infants tend to have higher levels of 17-OHP than term

22 P. C. White

infants and generate many false positives unless higher normal cut-o€s are used. High values are evaluated with electrolytes and a serum 17-OHP level; if these are not unequivocally normal, the infant is then referred to a paediatric endocrinologist. An ACTH1±24 stimulation test is then usually performed. 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. This could be accomplished on DNA extracted from the same dried blood spots as are used for hormonal screening (see below). As determined by screening, the highest incidence of classic 21-hydroxylase de®ciency occurs in two geographically isolated populations, the Yupik Eskimos of Western Alaska (1:280) and the French island of La Reunion in the Indian Ocean (1:2100).14 The incidence in most other populations ranges from approximately 1:10 000 to 1:18 000.12,13,15 Screening markedly reduces the time to diagnosis of infants with 21-hydroxylase de®ciency.13,16,17 The main putative bene®ts of this are reduced morbidity and mortality because infants with salt-wasting disease are diagnosed more quickly. Moreover, infants ascertained through screening have less severe hyponatraemia16 and tend to be hospitalized for shorter periods of time.17 CLINICAL PRESENTATION OF CLASSIC CAH Reproductive problems in females As discussed above, females with 21-hydroxylase de®ciency typically present in the neonatal period with ambiguous genitalia. Other reproductive problems become apparent in adolescence. The average age at which menarche occurs in inadequately treated girls is late compared with healthy peers. Such girls and women often appear similar to patients with polycystic ovarian syndrome and have sonographic evidence of multiple cysts, anovulation, irregular bleeding and hyperandrogenic symptoms. The majority of women 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 17hydroxyprogesterone is adequately suppressed.18 During pregnancy, women with classic 21-hydroxylase de®ciency are optimally managed with hydrocortisone or prednisone.19,20 Due to pregnancy-induced alterations in steroid metabolism and clearance, doses need to be increased compared to doses used in non-pregnant women with this condition. In this situation, treatment is directed at the mother and not at the fetus, for these steroids do not e€ectively cross the placenta. Despite elevated maternal testosterone of 400±600 ng/dl, una€ected female o€spring appear to have no genital virilization.19 Apparently, placental aromatase e€ectively prevents maternal androgens from reaching the fetus. Elevated maternal sex hormone binding globulin and androgen antagonism by progesterone may also restrict transplacental passage of androgens. There is no evidence of an excess of congenital malformations in o€spring of women with 21-hydroxylase de®ciency. Women with 11b-hydroxylase de®ciency may have problems similar to those with 21-hydroxylase de®ciency, whereas those with 17-hydroxylase de®ciency cannot synthesize sex hormones and remain sexually infantile unless supplemented with oestrogen. Women with 3b-hydroxysteroid dehydrogenase de®ciency may be slightly virilized due to high levels of dehydroepiandrosterone.

Congenital adrenal hyperplasias 23

Reproductive problems in males Men with 21-hydroxylase de®ciency usually have normal sperm counts and are able to father children.21 Low sperm counts, when they occur, do not always preclude fertility. Among simple virilizing patients, testicular function may be normal even in the absence of treatment. A€ected males are prone to develop testicular adrenal rests.22 These are seen most often, but not exclusively, in inadequately treated patients, particularly those with the salt-wasting form of 21-hydroxylase de®ciency.23 These tumours, although most often benign, have prompted biopsies and sometimes even orchiectomy. The preferred mode of treatment consists of e€ective adrenal suppression with dexamethasone because many of these tumours are ACTH-responsive. When they do not respond to dexamethasone, testis-sparing surgery may be performed after imaging the tumour by sonography and/or magnetic resonance imaging (MRI). Testicular masses have been detected in children as young as 3 years with 21-hydroxylase de®ciency22, suggesting that boys should undergo a baseline testicular sonogram by adolescence. The testes of a€ected males should be carefully examined throughout childhood, adolescence and adulthood. Men with 11b-hydroxylase de®ciency may have problems similar to those with 21hydroxylase de®ciency. Genetic males with 3b-HSD or 17-hydroxylase de®ciencies or lipoid hyperplasia are usually raised as females. As is the case with patients with complete androgen insensitivity syndrome, they are usually castrated during or before adolescence to prevent malignant transformation of abdominal testes.

E€ects on gender role and identity The in¯uence of prenatal sex steroid exposure on personality is controversial24±27; in considering this question, it is important to distinguish between gender role, sexual orientation and gender identity. Gender role refers to gender-sterotyped behaviours such as choice of play toys by young children. Girls with 21-hydroxylase de®ciency often have low interest in maternal behaviour, extending from lack of doll play in early childhood to lack of interest in childrearing in women.28 Sexual orientation refers to homosexual versus heterosexual preferences. Although most adult women with 21-hydroxylase de®ciency are heterosexual, a small but signi®cant proportion are actively homosexual or bisexual or have an increased tendency to homo-erotic fantasies.29,30 These characteristics occur more frequently in women with the salt-wasting form of 21-hydroxylase de®ciency. Gender identity refers to self-identi®cation as male or female. Spontaneous gender re-assignment back to male has been reported in cases of males with penile trauma who were raised as females31 or male pseudohermaphrodites raised as females, especially in cases of 5a-reductase or 17-ketosteroid reductase de®ciencies, in which the brain may be exposed to high circulating levels of androgens.27 However, selfreassignment to the male sex is unusual in women with 21-hydroxylase de®ciency.30 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. 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.

24 P. C. White

The uncertainty concerning the e€ects of prenatal and postnatal e€ects of androgen on gender identity and gender role extends to male pseudohermaphrodites of other aetiologies. Unfortunately, many of the data in this area are anecdotal.25,26 Growth disturbances Children with 21-hydroxylase (or 11b-hydroxylase) de®ciency should have an annual bone age X-ray and careful monitoring of linear growth. Whereas untreated patents grow rapidly and have accelerated skeletal maturation due to high levels of androgen, patients treated with excessive doses of glucocorticoids (hydrocortisone dose 420 mg/m2/day) may su€er growth retardation.32 Despite careful monitoring and good patient compliance, ®nal height usually averages one to two standard deviations below the population mean or the target height based on parental heights.33,34 Nevertheless, bone mineral density does not appear to be compromised in CAH patients receiving typical glucocorticoid doses.35±37 Salt wasting Among patients with classic 21-hydroxylase de®ciency, approximately three-fourths cannot synthesize adequate amounts of aldosterone due to severely impaired 21hydroxylation of progesterone. Aldosterone is essential for normal sodium homeostasis; de®ciency of this hormone results in sodium loss via the kidney, colon and sweat glands.38 Severely a€ected patients invariably have concomitant cortisol de®ciency which exacerbates the e€ects of aldosterone de®ciency. Glucocorticoids normally increase cardiac contractility, cardiac output, sensitivity of both the heart and the vasculature to the pressor e€ects of catecholamines and other pressor hormones, and work capacity of skeletal muscles.39 In the absence of glucocorticoids, cardiac output decreases. This decreases glomerular ®ltration, leading to an inability to excrete free water and consequently to hyponatraemia. Thus, shock and severe hyponatraemia are much more likely in 21-hydroxylase de®ciency, in which both cortisol and aldosterone biosynthesis are a€ected, than in (for example) aldosterone synthase de®ciency, in which only aldosterone biosynthesis is impaired and cortisol and sex hormone biosynthesis are entirely normal.40 Although catecholamine secretion has not 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.41 Indeed, mice with 21-hydroxylase de®ciency exhibit abnormal development of the adrenal medulla and secrete reduced levels of catecholamines.42 Catecholamine de®ciency could further exacerbate the shock engendered by glucocorticoid and mineralocorticoid de®ciency. In addition, accumulated steroid precursors may directly antagonize the mineralocorticoid receptor and exacerbate mineralocorticoid de®ciency, particularly in untreated patients.43 Progesterone is well known to have anti-mineralocorticoid e€ects44,45 and it and/or a metabolite are probable culprits in this phenomenon. However, there is as yet no evidence that 17-hydroxyprogesterone has such e€ects. Salt wasting may include non-speci®c symptoms such as poor appetite, vomiting, lethargy, and failure to gain weight. Severely a€ected patients with CAH usually present at 1±4 weeks of age with hyponatraemia, hyperkalaemia, hyperreninaemia and hypovolaemic shock. These `adrenal crises' may prove fatal if proper medical care is

Congenital adrenal hyperplasias 25

not delivered. This problem is particularly critical in infant boys, who have no genital ambiguity to alert physicians to the diagnosis of CAH prior to the onset of dehydration and shock. The mortality rate for CAH remains high in such patients as suggested by the relative paucity of male patients identi®ed in case reports.14 After the ®rst days of life, the ratio of plasma renin activity to 24-hour urinary aldosterone may be used as a marker of impaired aldosterone synthesis12, but this ratio may also be elevated in poorly controlled simple virilizers. Siblings may be discordant for salt wasting. Furthermore, CAH patients known to have severe salt-wasting episodes in infancy and early childhood may show improved sodium balance and more ecient aldosterone synthesis with age. Unrelated individuals carrying identical mutations may manifest di€erent severeties of salt wasting.46 Explanations for these observations are not immediately apparent. Extraadrenal 21-hydroxylase has been detected by in vivo metabolic studies but molecular genetic investigation has yielded contradictory results as to whether CYP21 could be a source for this activity.47,48 Other enzymes with 21-hydroxylase activity have not been identi®ed in humans, although such enzymes have been identi®ed in rabbit liver.49 Patients with 3b-HSD de®ciency, aldosterone synthase de®ciency or lipoid hyperplasia are also unable to synthesize aldosterone and may present with saltwasting crises. In contrast, patients with 11b- or 17-hydroxylase de®ciency have elevated levels of steroids such as deoxycorticosterone with mineralocorticoid activity, and they tend to develop hypertension (see Chapter 3). NON-CLASSICAL CAH PHENOTYPES Signs Patients with the mild, non-classical form of 21-hydroxylase de®ciency may have signs of postnatal androgen excess but a€ected females are born with non-ambiguous (normal or with mild clitoromegaly) external genitalia. Adrenal steroid precursors of 21-hydroxylase are only mildly elevated in non-classical 21-hydroxylase de®ciency, and are intermediate between those of heterozygote carriers of the enzyme de®ciency and those who are severely a€ected.50 Depending on the laboratory, a€ected individuals have serum 17-hydroxyprogesterone levels greater than 1000 or 1500 ng/dl 60 minutes after an intravenous bolus of ACTH1±24. Due to circadian variability of adrenal corticol hormones, the diagnosis may be missed by measuring only baseline serum 17hydroxyprogesterone late in the day. The most common presenting symptoms are premature pubarche in children or severe cystic acne, hirsutism and oligomenorrhea in young women.51 However, the severity of these symptoms varies widely and probably many a€ected individuals are asymptomatic. Conversely, only a small percentage of individuals presenting with signs of androgen excess prove to be a€ected with non-classical 21-hydroxylase de®ciency.52,53 Incidence Because the signs of androgen excess in non-classical 21-hydroxylase de®ciency can be dicult to discern, the most reliable estimates of allele and disease frequencies come from ascertainment of a€ected individuals in the course of studies of kindreds in which classical and non-classical 21-hydroxylase de®ciency are segregating.9 The disease frequency is estimated at 0.1% of the general population but 1±2% of Hispanics and

26 P. C. White

Yugoslavs and 3±4% of Ashkenazi (Eastern European) Jews. In New Zealand, molecular screening of normal newborns showed that 5% are carriers for mutations in the 21-hydroxylase gene (CYP21) associated with either classical or non-classical 21hydroxylase de®ciency. This implies a disease frequency for non-classical 21hydroxylase de®ciency of 0.06%, in good agreement with estimates in the general American population.15 Other forms of non-classical CAH Non-classical 11b-hydroxylase de®ciency presents similarly to non-classical 21hydroxylase de®ciency, but it occurs rarely.54 It should be suspected in individuals with 11-deoxycortisol levels greater than ®ve times the upper limit of normal. Nonclassical 3b-HSD de®ciency has been suspected when DHEA levels are elevated in women with signs of androgen excess, and by this de®nition the disorder may be as common as non-classical 21-hydroxylase de®ciency. However, mutations in the HSD3B2 gene encoding the enzyme (see below) are not found in such patients55, suggesting that they do not have a congenital enzymatic de®ciency.

TREATMENT Glucocorticoid replacement All patients with classic 21-hydroxylase de®ciency, and symptomatic patients with nonclassical disease, are treated with glucocorticoids. This suppresses the excessive secretion of CRH and ACTH by the hypothalamus and pituitary, and reduces the abnormal blood levels of adrenal sex steroids. In children, the preferred cortisol replacement is hydrocortisone (i.e. cortisol itself) in doses of 10 to 20 mg/m2/day in two or three divided doses. These doses exceed physiological levels of cortisol secretion, which are 6±8 mg/m2/day in children and adolescents56, but they seem to be required to suppress adrenal androgens adequately and to minimize the possibility of developing adrenal insuciency. 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. For optimal suppression of adrenal androgens, glucocorticoid replacement can be given in `reverse diurnal rhythm', i.e. with a larger dose of a longer half-life glucocorticoid taken at night-time and a smaller dose in the morning. This approach allows suppression of the early morning rise in ACTH secretion, although it may increase the risk of glucocorticoid excess by preventing the nadir in glucocorticoid levels which occurs overnight. Treatment ecacy (i.e. suppression of adrenal hormones) is assessed by monitoring 17-OHP and androstenedione levels. Early excessive glucocorticoid treatment (hydrocortisone dose 420 mg/m2/day) is potentially detrimental to growth.32 Therefore, it is not desirable to suppress endogenous adrenal corticosteroid secretion completely. A target 17-OHP range might be 100±1000 ng/dl with commensurate age and gender-appropriate androgen levels. Hormones should be measured at a consistent time in relation to medication dosing. Patients with classic 21-hydroxylase de®ciency cannot mount a sucient cortisol response to stress and require increased doses of hydrocortisone (50±100 mg/m2/day) in situations such as febrile illness and surgery under general anaesthesia.39

Congenital adrenal hyperplasias 27

Patients with CAH caused by other enzymatic de®ciencies also require glucocorticoid replacement, and treatment ecacy is assessed by monitoring the appropriate precursor hormones. Mineralocorticoid replacement Infants with the salt-wasting form of 21-hydroxylase de®ciency require mineralocorticoid (¯udrocortisone, 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. Fludrocortisone doses may often be decreased after early infancy, and older children usually acquire a taste for salty food and do not require daily supplements of sodium chloride tablets. Although patients with the simple virilizing form of the disease by de®nition secrete adequate amounts of aldosterone, they are nevertheless often treated with ¯udrocortisone. This can aid in adrenocortical suppression, reducing the dose of glucocorticoid required to maintain acceptable 17-hydroxyprogesterone levels.57 Plasma renin activity may be used to monitor mineralocorticoid and sodium replacement. Hypertension, tachycardia and suppressed plasma renin activity are clinical signs of overtreatment with mineralocorticoids.58 Fludrocortisone is also required to treat 3b-HSD de®ciency, aldosterone synthase de®ciency or lipoid hyperplasia. Other therapeutic approaches Pharmacological A novel four-drug regimen for 21-hydroxylase de®ciency, consisting of ¯utamide (an androgen receptor blocking drug), testolactone (an aromatase inhibitor), low-dose hydrocortisone and ¯udrocortisone, produced less bone age advancement and more appropriate linear growth velocity than standard treatment, but central precocious puberty occurred and required treatment with gonadotrophin releasing hormone analog in three of eight males in the experimental therapy group and in none of nine control males.59 It remains to be seen whether longer-term, larger-scale studies will show a favourable e€ect of the experimental regimen on ®nal height. Another interesting experimental 21-hydroxylase de®ciency therapy is the addition of carbenoxolone, an inhibitor of 11b-hydroxysteroid dehydrogenase (11-HSD). The latter is an enzyme important in inactivating cortisol and preventing its access to the mineralocorticoid receptor (see Chapter 4).60 The rationale for carbenoxolone as an adjunct to therapy for 21-hydroxylase de®ciency is that inhibition of the oxidative 11HSD reaction should generate higher endogenous bioactive cortisol levels without administering larger doses of steroids. In a short-term pilot study with an open-label, cross-over design involving six 21-hydroxylase de®ciency patients aged 15 to 39 years, there were signi®cant reductions in 17-OHP, androstenedione, renin, and urinary pregnanetriol when carbenoxolone was added to the standard therapeutic regimen.61 Hypertension is potentially a complication of such a regimen60, which needs further evaluation before it should be employed routinely. Adrenalectomy The consequences of inadequate treatment or non-compliance for the female include ongoing virilization in addition to compromise of linear growth while subtle

28 P. C. White

over-treatment with glucocorticoid may also retard growth. For this reason, it has been suggested that (laparoscopic) adrenalectomy may represent an alternative to suppressive medical therapy with glucocorticoids.62 Severely a€ected patients, especially females, could perhaps be more easily managed with low-dose gluco- and mineralocorticoids after adrenalectomy, rather than with adrenal glands which 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 insuciency. Moreover, there may be bene®ts in terms of improved lipid pro®le, libido, and quality of life from physiological adrenal DHEA production63 which would be lost with adrenalectomy. 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 de®ciency refractory to standard medical management. Gene therapy Because 21-hydroxylase de®ciency is an inherited metabolic defect, the question arises of the feasibility of gene therapy.64 Indeed, mice with 21-hydroxylase de®ciency have been rescued by transgenesis with a murine Cyp21 gene.65 However, this disorder does not seem a promising test system for human gene therapy. Medical therapy, albeit not perfect, is e€ective 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 dicult therapeutic goal to achieve is adequate suppression of adrenal androgens, expression would need to be suciently 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 inde®nitely to be cost-e€ective in comparison with conventional treatment. These criteria seem unlikely to be met for the foreseeable future. Corrective surgery The general approach to evaluating the newborn with ambiguous genitalia has been previously discussed. Whether, how and when to intervene surgically in the correction of genital anomalies is the subject of continuing debate.66,67 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 a€ected individual is able to provide informed consent for cosmetic genital surgery, and select the gender with which he/she will be most comfortable.31 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 be recognized that recommendations for sex assignment are to some extent culture-speci®c. 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. The most common current approach to surgical correction is for clitoroplasty, rather than clitoridetectomy, to be done in infancy. Vaginal reconstruction is often postponed until the age of expected sexual activity68, but single-stage corrective

Congenital adrenal hyperplasias 29

surgery has also been successfully performed in children.69 According to selfassessment surveys among sexually active women with 21-hydroxylase de®ciency, approximately 60% are able to have satisfactory intercourse.70 Re-operation is frequently required to achieve satisfactory results.71 As surgical and medical treatment regimens have improved in recent years, more women with 21-hydroxylase de®ciency have successfully conceived spontaneously, completed pregnancies and given birth.19 Prenatal therapy Overview In pregnancies at risk for a female child a€ected with virilizing adrenal hyperplasia, fetal adrenal androgen production may be suppressed and genital ambiguity decreased by administering dexamethasone to the mother.72±74 As compared with hydrocortisone, dexamethasone has no salt-retaining activity and it is able to cross the placenta because it is not metabolized signi®cantly by placental 11b-hydroxysteroid dehydrogenase.60 The dose is typically 20 mg/kg/day based on pre-pregnancy weight to a maximum of 1.5 mg daily in three divided doses, beginning before the 7±8th week of gestation. Approximately 70% of pre-natally treated females are born with normal or only slightly virilized genitalia. Prenatal therapy is usually coupled with prenatal genetic diagnosis (see below). If the sex is male, or CYP21 genotype indicates that the fetus is una€ected, dexamethasone should promptly be discontinued to minimize potential risks of glucocorticoid toxicity (Figure 3). Intrauterine growth retardation and unexplained fetal death have been observed in 2% or less of treated pregnancies; these statistics are not signi®cantly di€erent from those in the general population. The risk of overt human fetal defects appears to be

Pregnancy test (<6wks) Positive

Begin

Chorionic

dexamethasone

villus sample

Male Fetal sex?

Stop dexamethasone

Female Affected Continue dexamethasone

Unaffected CYP21 genotype?

Stop dexamethasone

Figure 3. Flow chart for decisions pertaining to prenatal diagnosis of 21-hydroxylase de®ciency. The format is identical to that in Figure 2. Reproduced from White and Speiser (2000, Endocrine Reviews 21: 245±291) with permission.

30 P. C. White

low, but it is conceivable that more subtle e€ects of glucocorticoids on the developing human brain may go unnoticed during early life. The ®rst prenatally treated female has reached late adolescence with normal cognitive development73, but prenatally treated children have not undergone thorough neuropsychological testing. Recent editorials called attention to these issues, citing numerous studies in animal models showing the dangers of prenatal exposure to glucocorticoids with respect to impairment of somatic growth, brain development, fuel metabolism and blood pressure regulation.75,76 An international registry might facilitate long-term studies that could answer many of these questions. Pregnant mothers should be closely monitored for features of Cushing's syndrome ± such as excessive weight gain, severe striae, hypertension and hyperglycaemia. These may occur in approximately 10% of women treated to term, but they usually resolve when treatment is discontinued.73 Side-e€ects in those treated for a shorter time include oedema, gastrointestinal upset, mood ¯uctuations, acne and hirsutism; one or more of these symptoms are seen in 10±20% of women treated in early pregnancy. Therefore, 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. Caveats notwithstanding, many parents of a€ected girls still opt for prenatal medical treatment because of the severe psychological impact of ambiguous genitalia on the child and on the family. Similar diagnostic and therapeutic approaches can also be e€ective in families at risk for 11bhydroxylase de®ciency, in which a€ected female fetuses may also su€er severe prenatal virilization.77 MOLECULAR GENETICS OF 21-HYDROXYLASE DEFICIENCY Mutations causing 21-hydroxylase de®ciency Steroid 21-hydroxylase (P450c21, CYP21) is a microsomal cytochrome P450 enzyme that converts 17-hydroxyprogesterone to 11-deoxycortisol and progesterone to deoxycorticosterone. The CYP21 gene encoding 21-hydroxylase and a homologous pseudogene, CYP21P, are located 30 kb apart in the HLA complex on chromosome 6p21.3, adjacent to and alternating with the C4B and C4A genes encoding the fourth component of serum complement (Figure 4). Although CYP21 and CYP21P are 98% identical in nucleotide sequence, the latter has accumulated at least nine mutations that, together, render the putative gene product completely inactive.78,79 Almost all mutations causing 21-hydroxylase de®ciency arise by either of two types of recombination between CYP21 and CYP21P. About 20% of mutations are deletions of a 30 kb DNA segment80, encompassing the 30 end of the CYP21P pseudogene, all of C4B, and the ®rst three to seven exons of CYP21. This yields a non-functional chimeric pseudogene. Alternatively, deleterious mutations found in the pseudogene may be transferred to CYP21 by a process known as gene conversion. The most common such mutation, found in 25% of a€ected chromosomes, is a point mutation in the second intron (nt 656 A to G) which results in an abnormally spliced gene product.81 Studies of de novo recombinations involving the CYP21 genes in human sperm and peripheral blood lymphocytes suggest that, whereas deletions are the result of unequal crossing over during meiosis, gene conversions take place during mitosis and are thus the result of a distinct molecular process.82

Congenital adrenal hyperplasias 31

A

6p21.3 RP1

CA4

CYP21P

RP2 C4B

CYP21

XA

XB

30,000 base pairs (30kb)

B

normal

21-hydroxylase deficiency

C Non-classical

1

2

3

P30L

6

7

8

9

10

V281L

Simple virilizing

I172N a

Salt wasting

4 5

g I236N

R356W

V237E M239K G110∆8nt F306+1nt

Q318X 1 kb

Figure 4. Mutations causing steroid 21-hydroxylase de®ciency. (A) Map of the genetic region around the 21hydroxylase (CYP21) gene. Arrows denote direction of transcription. CYP21P 21-hydroxylase pseudogene; C4A and C4B ˆ genes encoding the fourth component of serum complement; RP1 ˆ gene encoding a putative nuclear protein of unknown function; RP2 ˆ truncated copy of this gene. XB ˆ tenascin-X gene (not shown full length); XA ˆ a truncated copy of this gene, are on the opposite chromosomal strand. (B) An unequal cross-over generating a CYP21 deletion. For clarity, only the two C4 and two CYP21 genes are shown on each chromosome. When these are misaligned during meiosis, a cross-over can generate two daughter chromosomes, one with three copies of the C4-CYP21 tandem and the other with one copy. The latter is often a 21-hydroxylase de®ciency allele. (C) Diagram of a CYP21P gene. Exons are numbered. CYP21P has nine mutations that may be transferred into CYP21 by gene conversion, causing 21-hydroxylase de®ciency. These are arranged in the diagram so that those causing increasing enzymatic compromise are arrayed from top to bottom. Dotted lines divide mutants into groups with similar activities. The mutations at the bottom are nonsense mutations or frameshifts that are predicted to prevent protein synthesis completely. The mutations are associated with di€erent forms of 21-hydroxylase de®ciency as marked. As an example of mutation terminology, P30L is Proline-30 to Leucine. a ! g, a mutation at nucleotide (nt) 656 in intron 2. The three mutations in the box are invariably inherited together. There are several dozen additional rare mutations that collectively account for 5% of all 21-hydroxylase de®ciency alleles; these are not shown. D ˆ deletion; ‡ ˆ insertion. Other single-letter amino acid codes: A ˆ alanine; C ˆ cysteine; D ˆ aspartic acid; E ˆ glutamic acid; F ˆ phenylalanine; G ˆ glycine; H ˆ histidine; I ˆ isoleucine; K ˆ lysine; L ˆ leucine; M ˆ methionine; N ˆ asparagine; P ˆ proline; Q ˆ glutamine; R ˆ arginine; S ˆ serine; T ˆ threonine; V ˆ valine; W ˆ tryptophan; Y ˆ tyrosine.

32 P. C. White

General considerations for molecular diagnosis If an a€ected child (a proband) and both parents are available, 21-hydroxylase de®ciency can be diagnosed prenatally using highly polymorphic microsatellite markers to determine whether the fetus (the propositus) has inherited the same alleles as the proband.83 However, because a small number of gene conversions account for most cases, it is usually possible to detect these mutations directly after gene-speci®c ampli®cation using the polymerase chain reaction (PCR). A gene conversion may be suciently large that it includes several mutations. If only a DNA sample from the patient is analysed, this is impossible to distinguish from compound heterozygosity for di€erent mutations. Therefore, both parents should also be analysed whenever possible so as to determine most reliably the phase of di€erent 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. A large number of techniques have been used to detect mutations in PCR-ampli®ed material, including allele-speci®c PCR, allele-speci®c oligonucleotide hybridization, and ligase chain reaction. With the possible exception of the last, none of these readily lends itself to automation, and other methods such as `gene chip' technology might be used if large numbers of tests were to be performed. Phenotype-genotype correlations Many studies have correlated speci®c mutations with phenotype.81,84±86 These correlations are most reliably made in patients who are homozygous or hemizygous (i.e. the other chromosome carries a deletion) for a given mutation. Mutations in CYP21 can be grouped into three categories according to the predicted level of enzymatic activity based on in vitro mutagenesis and expression.84 The ®rst group consists of mutations such as deletions, frameshifts or nonsense mutations that totally ablate enzyme activity; these are most often associated with salt-wasting disease. The second group of mutations, consisting mainly of the missense mutation Ile172Asn (I172N), yields enzymes with 1±2% normal activity and is carried predominantly by patients with simple virilizing disease. The ®nal group includes mutations such as Val281Leu (V281L) and Pro30Leu (P30L) that produce enzymes with 20±60% of normal activity and are most often associated with the non-classical disorder. Patients who are compound heterozygotes for two di€erent CYP21 mutations most often have a phenotype compatible with that of the less severe of the gene defects.84 Thus, the various 21-hydroxylase de®ciency phenotypes are primarily the result of allelic variation in CYP21. When quantitative scores for signs of androgen excess or salt wasting are used, approximately 80% of phenotypic variation is accounted for by CYP21 genotype. Obviously, other genes must in¯uence sensitivity to androgens or renal sodium conservation. Another source of genetic variability is `leakiness' of splice mutations. The common point mutation in the second intron (nt 656 A to G) results in an abnormally spliced mRNA transcript.81 If a small amount of mRNA is normally spliced, resulting in some functional enzyme being synthesized, as little as 1±2% of normal enzyme activity might change the phenotype. Indeed, a minority of patients carrying this mutation have a simple virilizing phenotype, although most are salt wasters. Asymptomatic individuals homozygous for the nt 656G mutation have been reported87, but pedigree analysis and

Congenital adrenal hyperplasias 33

microsatellite typing demonstrate that many such cases in fact represent a PCR artifact termed allele drop-out.83

MOLECULAR GENETICS OF OTHER FORMS OF CAH We brie¯y summarize the genetic aetiologies of forms of congenital adrenal hyperplasia other than 21-hydroxylase de®ciency. The reader is directed to the referenced review articles for citations of original research on these genes. 11b b-Hydroxylase and aldosterone synthase de®ciencies Steroid 11b-hydroxylase isozymes Humans have two 11b-hydroxylase isozymes that are respectively responsible for cortisol and aldosterone biosynthesis, CYP11B1 (P450c11, 11b-hydroxylase) and CYP11B2 (P450aldo, aldosterone synthase). These isozymes are mitochondrial cytochromes P450. CYP11B1 11b-hydroxylates 11-deoxycorticosterone to corticosterone and 11deoxycortisol to cortisol. It can also convert 11-deoxycorticosterone to 18hydroxy,11-deoxycorticosterone, but it 18-hydroxylates corticosterone poorly and cannot convert corticosterone into aldosterone. In contrast, CYP11B2 has strong 11bhydroxylase activity but also 18-hydroxylates and then 18-oxidizes corticosterone to aldosterone. When deoxycorticosterone is converted to aldosterone, the same steroid molecule probably remains bound to the enzyme for all three conversions without release of the intermediate products. In humans, CYP11B1 and CYP11B2 are encoded by two genes approximately 40 kb apart on chromosome 8q24; CYP11B2 is on the left if the genes are pictured as being transcribed left to right (Figure 5). The nucleotide sequences of these genes are 95% identical in coding sequences, and the predicted proteins are 93% identical in amino acid sequence.5 11b-Hydroxylase de®ciency De®ciency of 11b-hydroxylase results from mutations in CYP11B1. In contrast to 21hydroxylase de®ciency, these do not include gene conversions. Although CYP11B1 and CYP11B2, like CYP21 and CYP21P, are closely linked homologs, CYP11B1 and CYP11B2 both encode active enzymes. Thus, gene conversions that transfer polymorphic sequences between CYP11B1 and CYP11B2 should not produce 11b-hydroxylase de®ciency alleles. This may explain why 11b-hydroxylase de®ciency is less frequent than 21-hydroxylase de®ciency. Unequal cross-overs between CYP11B1 and CYP11B2 should yield a chromosome carrying a single CYP11B gene with a 50 end corresponding to CYP11B2 and a 30 end corresponding to CYP11B1. Like CYP11B2, such a gene should be expressed at low levels and only in the zona glomerulosa, and thus might function as an 11b-hydroxylase de®ciency allele. As yet, such chromosomes have not been detected. On the other hand, the reciprocal product of an unequal cross-over, a chromosome carrying three CYP11B genes, has been observed in glucocorticoid-suppressible hyperaldosteronism (see Chapter 3).

34 P. C. White

A

8q22 CYP11B2

CYP11B1

40,000 base pairs (40kb)

B 1

2

3

4

5

6

7 8

9

CYP11B1 Nonclassical P42S

N133H

V129M

Classical

T319M T318M A331V R384Q V441G E371G R384Q R448H R448C

R374Q

D

P32 1nt W116X

K174X W247X Q338X N394+2nt Y423X

Q121+5nt

Q356X

D

L464+3nt

L105 28nt 1 kb

1

2

3

4

5

6

7 8

9

CYP11B2 Partial activity No activity

V386A

R18WE198D

D

V35 2nt

T318M

D

C372 1nt R384P

Figure 5. Mutations involving the 11b-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes. (A) The arrangement of genes is diagrammed. (B) Mutations in CYP11B1 causing non-classical or classical 11bhydroxylase de®ciency, and mutations in CYP11B2 causing aldosterone synthase de®ciency. Terminology and arrangement of mutations is similar to that in Figure 2. Although there are two types of aldosterone synthase de®ciency, they do not correspond exactly to in vitro levels of enzymatic activity. R181W and V386A do not cause disease separately, but double homozygosity causes type II aldosterone synthase de®ciency. Other combinations with this phenotype include T318M ‡ V386A and R181W ‡ C372D1nt. Homozygosity for either V35D2nt and R384P has been associated with type I de®ciency.

Aldosterone synthase de®ciency Aldosterone synthase de®ciency results from mutations in CYP11B2 that reduce enzymatic activity to less than 0.5% of normal. Individuals who are homozygous for mutations that severely compromise but do not destroy enzymatic activity may have entirely normal aldosterone biosynthesis with normal ratios of plasma renin activity to aldosterone. Similarly, patients with 21-hydroxylase de®ciency carrying CYP21 mutants that retain 1±2% of normal activity are able to synthesize aldosterone normally. The ®nding that such reductions in enzymatic activity are not rate-limiting may re¯ect the very low levels at which aldosterone is normally secreted. Thus the apparent rarity of aldosterone synthase de®ciency may re¯ect problems of ascertainment because any but the most severe defects will in fact have no obvious phenotypic e€ects. Patients with type I and type II de®ciencies di€er in having low or high levels of the immediate precursor of aldosterone, 18-hydroxycorticosterone, respectively. It might be supposed that type I de®ciency would result from more severe enzymatic defects, but patients with similar genotypes have been identi®ed with both types of the

Congenital adrenal hyperplasias 35

disorder. The di€erence between these disorders may be due to associated polymorphisms in the adjacent CYP11B1 gene.88 17a a-Hydroxylase/17,20-lyase de®ciency Biochemistry Steroid 17a-hydroxylase/17,20 lyase (CYP17, P450c17) catalyses conversion of pregnenolone to 17a-hydroxypregnenolone. It also catalyses an oxidative cleavage of the 17,20 carbon±carbon bond, converting 17a-hydroxypregnenolone and 17ahydroxyprogesterone to dehydroepiandrosterone and androstenedione respectively. Activity of 17a-hydroxylase is required for the synthesis of cortisol, whereas both 17a-hydroxylase and 17,20-lyase activities are required for androgen and oestrogen biosynthesis. As signi®cant amounts of 17a-hydroxylated steroids are secreted by the human adrenal cortex but not by the gonads, it is apparent that the 17,20-lyase activity of CYP17 is stronger in the gonads than in the adrenals, relative to 17a-hydroxylase activity. Tissue-speci®c variations in lyase activity may re¯ect the activity of the electron transport protein, cytochrome b5 , or perhaps phosphorylation of the enzyme.6,89 Genetics In humans, a single copy of the CYP17 gene is located on chromosome 10q24.3. Mutations in this gene cause de®ciency of 17a-hydroxylase and 17,20 lyase activities. Complete de®ciency is associated with frameshifts or nonsense mutations. Partial de®ciency is associated with particular missense mutations. Certain mutations a€ect hydroxylase and lyase activities di€erently. The in vitro activities associated with each mutation may be correlated with clinical signs of the disorder in a€ected individuals. It seems that more than 20% of normal activity is required to synthesize sucient androgens to permit normal male sexual development. On the other hand, 50% of normal activity must be sucient for normal development because males who are obligate heterozygotes for severe mutations are asymptomatic. 3b b-Hydroxysteroid dehydrogenase de®ciency Biochemistry Conversion of D5-3b-hydroxysteroids (pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone) to D4-3-ketosteroids (progesterone, 17-hydroxyprogesterone, androstenedione) is mediated by 3b-hydroxysteroid dehydrogenase. These conversions are mediated by several isozymes in the endoplasmic reticulum, all of which are of the short-chain alcohol dehydrogenase type. They utilize NAD‡ as an electron acceptor. In addition to 3b-hydroxysteroid dehydrogenase activity, these enzymes mediate a D5-D4-ene isomerase reaction that transfers a double bond from the B ring to the A ring of the steroid so that it is in conjugation with the 3-keto group. Thus far, two isozymes have been identi®ed in humans. The human type I enzyme is expressed in the placenta, skin and adipose tissue, whereas the type II enzyme is expressed in the adrenal gland and the gonads. There are as many as six HSDB3 genes

36 P. C. White

in humans, all of which are located on chromosome 1p11-13, but only two, HSD3B1 and HSD3B2, encode active enzymes.8 3b-Hydroxysteroid dehydrogenase de®ciency Patients with the classical form of the disease have mutations in the HSD3B2 gene which abolish activity. As this gene is expressed in the adrenals and gonads, these mutations account fully for the pathogenesis of the disease. Because the HSD3B1 gene is intact, steroid biosynthesis in the placenta should be normal during gestation. Moreover, 3b-hydroxysteroid dehydrogenase activity in the skin and adipose tissue should remain intact postnatally, suggesting that complete 3b-hydroxysteroid dehydrogenase de®ciency should occur rarely if at all. Although non-classical 3b-hydroxysteroid dehydrogenase de®ciency has been reported to occur frequently, mutations in HSD3B2 have rarely been identi®ed in such patients, and this disorder is apparently not usually due to a speci®c enzymatic de®ciency. Lipoid adrenal hyperplasia By analogy with other forms of congenital adrenal hyperplasia, it was originally hypothesized that patients with lipoid adrenal hyperplasia had mutations in the cholesterol desmolase (CYP11A, P450scc) enzyme, but no such mutations have been detected. Because the maintenance of human pregnancy is critically dependent on placental progesterone synthesis, such mutations may be embryonically lethal. Instead, mutations have been detected in all a€ected individuals in the gene encoding the steroidogenic acute regulatory (StAR) protein, which controls importation of cholesterol into mitochondria. The a€ected gene is located on chromosome 8p11.2; a processed pseudogene is located on chromosome 13 and is not involved in this disease.3 SUMMARY Over 90% of cases of virilizing congenital adrenal hyperplasia (CAH, the inherited inability to synthesize cortisol) are caused by 21-hydroxylase de®ciency. Females with severe, classical 21-hydroxylase de®ciency are exposed to excess androgens prenatally and are born with virilized external genitalia. Approximately three-quarters of patients cannot synthesize sucient 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. Over 90% of these mutations result from intergenic recombinations between CYP21 and the closely linked CYP21P pseudogene. The degree to which each mutation compromises enzymatic activity is strongly but not completely correlated with the clinical severity of the disease in patients carrying it. Prenatal diagnosis by direct detection of mutation permits prenatal treatment of a€ected females to minimize genital virilization. Neonatal screening by hormonal methods identi®es a€ected children before saltwasting 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. Other forms of CAH are rare. Excessive secretion of androgens and mineralocorticoids occurs in 11b-hydroxylase

Congenital adrenal hyperplasias 37

Practice points . glucocorticoids (usually hydrocortisone) are invariably used to treat congenital adrenal hyperplasia. The dose should be adequate to suppress abnormal production of sex steroids by the adrenal (in 21- and 11b-hydroxylase de®ciencies), but not so high as to suppress growth. Growth, skeletal maturation and levels of precursor steroid should be periodically monitored . mineralocorticoid (¯udrocortisone) replacement is required in most cases of 21hydroxylase and 3b-hydroxysteroid dehydrogenase de®ciencies and in lipoid hyperplasia. Serum electrolytes and plasma renin activity should be periodically monitored . many locales mandate neonatal screening for 21-hydroxylase de®ciency, so that a€ected infants may be detected before salt-wasting develops

Research agenda . psychosexual development in all forms of CAH . natural history of the non-classical form of 21-hydroxylase de®ciency when prospectively ascertained . optimal timing and form of genital surgery . safety of prenatal treatment of 21- or 11b-hydroxylase de®ciency . safety and ecacy of novel forms of pharmacological management of CAH . role of adrenalectomy in managing 21- or 11b-hydroxylase de®ciency de®ciency, the second most frequent form of congenital adrenal hyperplasia. Mineralocorticoid excess is also seen in 17a-hydroxylase de®ciency, but in this disorder sex steroid synthesis is defective. Lipoid hyperplasia (a defect in importation of cholesterol into mitochondria) and 3b-hydroxysteroid dehydrogenase de®ciency a€ect synthesis of all classes of steroids in both the adrenals and gonads. Acknowledgement Supported by grant NIH R37DK37 867.

REFERENCES * 1. Bose HS, Sugawara T, Strauss JF 3rd & Miller WL. The pathophysiology and genetics of congenital lipoid adrenal hyperplasia. International Congenital Lipoid Adrenal Hyperplasia Consortium. New England Journal of Medicine 1996; 335: 1870±1878. 2. Lin D, Sugawara T, Strauss JF et al. Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis. Science 1995; 267: 1828±1831. * 3. Stocco DM & Clark BJ. Regulation of the acute production of steroids in steroidogenic cells. Endocrine Reviews 1996; 17: 221±244. * 4. White PC & Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase de®ciency. Endocrine Reviews 2000; 21: 245±291. * 5. White PC, Curnow KM & Pascoe L. Disorders of steroid 11 beta hydroxylase isozymes. Endocrine Reviews 1994; 15: 421±438.

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