Steroid 21 hydroxylase deficiency is the most common form of congenital adrenal hyperplasia (CAH). The severity of this disorder depends on the extent of impaired enzymatic activity, which is caused by various mutations of the 21 hydroxylase gene. This article reviews adrenal steroidogenesis and the pathophysiology of 21 hydroxylase deficiency. The three forms of CAH are then discussed in terms of clinical presentation, diagnosis and treatment, and genetic basis. Prenatal diagnosis and treatment are also reviewed. The goal of therapy is to correct the deficiency in cortisol secretion and suppress androgen overproduction. Glucocorticoid replacement has been the mainstay of treatment for CAH, but new treatment strategies continue to be developed and studied.
Congenital adrenal hyperplasia (CAH) refers to a group of genetic enzyme deficiencies that impair normal steroid synthesis by the adrenal cortex. The most common form is 21 hydroxylase deficiency (21 OHD), accounting for more than 90% of the cases. Less frequent causes of CAH include deficiencies of 11β-hydroxylase, 3β-hydroxysteroid dehydrogenase, 17α-hydroxylase/17,20-lyase, steroidogenic acute regulatory (StAR) protein (lipoid hyperplasia) and the newly described form of CAH owing to P450 oxidoreductase defect. In almost all cases, each of these deficiencies results from recessive mutations in the respective genes that encode the enzymes and has distinctive clinical phenotypes. This article focuses on CAH owing to 21 OHD.
Steroid 21 hydroxylase deficiency
CAH owing to 21 OHD is divided into classical and nonclassical (NC) CAH. Patients affected with the classical form have more severe symptoms of hyperandrogenism. Beginning in utero, prenatal exposure to potent androgens such as testosterone and Δ 4 -androstenedione at critical stages of sexual differentiation virilizes the external genitalia of genetic females, often resulting in genital ambiguity at birth in female patients. The classical form is subdivided into the simple virilizing form (SV) and the salt-wasting form (SW), based on the patient’s ability to produce adequate salt-retaining hormone, aldosterone. Salt-wasters account for most of classical 21 OHD CAH patients (≥75%) whereas approximately 25% of individuals are simple virilizers. Newborns who are salt wasters are at risk for life-threatening salt-wasting crises.
Individuals with the NC form of 21 OHD CAH have only mild enzyme deficiency and present postnatally with signs of hyperandrogenism; therefore, females with the NC form are not virilized at birth and do not present with genital ambiguity. Clinical features distinguishing classical and nonclassical forms are shown in Table 1 . In males, the clinical distinction between SV and NC-CAH may not be readily apparent, but genetic analysis and adrenocorticotropic hormone (ACTH) stimulation results may clarify the diagnosis.
| Feature | Classical 21 OHD CAH | Nonclassical 21 OHD CAH |
|---|---|---|
| Prenatal virilization | Present in females | Absent |
| Postnatal virilization | Males and females | Variable |
| Salt wasting | ∼75% of all individuals | Absent |
| Cortisol deficiency | ∼100% | Rare |
Screening studies indicate that the worldwide incidence of classical 21 OHD is 1:15,000 to 1:16,000 live births, of which approximately 75% are salt wasters. The worldwide incidence of NC-CAH is much higher at 1:1000, with a frequency as high as 1:27 among Ashkenazi Jews.
Pathophysiology
Adrenal steroid production occurs in three major pathways: glucocorticoids, mineralocorticoids, and sex steroids, as shown in Fig. 1 . ACTH regulates adrenal steroid production via a rate-limiting step that results in pregnanolone, the principal substrate for the steroidogenic pathway (see Fig. 1 ). It promotes StAR protein function in transporting free cholesterol to the inner mitochondrial membrane, the site where a side chain cleavage occurs and the first step in steroidogenesis takes place. The central nervous system controls the secretion of ACTH, its diurnal variation, and its increase during periods of physiologic stress via the hypothalamus-produced corticotropin-releasing factor (CRF). The hypothalamic-pituitary-adrenal feedback system is mediated through the circulating level of plasma cortisol by its negative feedback on CRF and ACTH secretion. Hence, impeded cortisol secretion in 21 OHD CAH leads to increased ACTH production, which in turn, stimulates (1) excessive synthesis of adrenal products in those pathways unimpaired by the enzyme deficiency and (2) a build-up of precursor molecules, mainly 17 hydroxyprogesterone, in pathways blocked by the enzyme deficiency. The excess 17-OHP is shunted into the intact androgen pathway, where the 17,20-lyase enzyme converts the 17-OHP to Δ 4 -androstenedione, which is converted into androgens. Because the mineralocorticoid pathway requires minimal 21 hydroxylase activity, mineralocorticoid deficiency (salt wasting) is a feature of the most severe form of the disease.
Pathophysiology
Adrenal steroid production occurs in three major pathways: glucocorticoids, mineralocorticoids, and sex steroids, as shown in Fig. 1 . ACTH regulates adrenal steroid production via a rate-limiting step that results in pregnanolone, the principal substrate for the steroidogenic pathway (see Fig. 1 ). It promotes StAR protein function in transporting free cholesterol to the inner mitochondrial membrane, the site where a side chain cleavage occurs and the first step in steroidogenesis takes place. The central nervous system controls the secretion of ACTH, its diurnal variation, and its increase during periods of physiologic stress via the hypothalamus-produced corticotropin-releasing factor (CRF). The hypothalamic-pituitary-adrenal feedback system is mediated through the circulating level of plasma cortisol by its negative feedback on CRF and ACTH secretion. Hence, impeded cortisol secretion in 21 OHD CAH leads to increased ACTH production, which in turn, stimulates (1) excessive synthesis of adrenal products in those pathways unimpaired by the enzyme deficiency and (2) a build-up of precursor molecules, mainly 17 hydroxyprogesterone, in pathways blocked by the enzyme deficiency. The excess 17-OHP is shunted into the intact androgen pathway, where the 17,20-lyase enzyme converts the 17-OHP to Δ 4 -androstenedione, which is converted into androgens. Because the mineralocorticoid pathway requires minimal 21 hydroxylase activity, mineralocorticoid deficiency (salt wasting) is a feature of the most severe form of the disease.
Clinical presentation and features
External Genitalia
Adrenocortical function begins around the seventh week of gestation ; thus, a female fetus with classical CAH is exposed to adrenal androgens at the critical time of sexual differentiation (approximately 9 to 15 weeks gestational age). In classical CAH patients, the degree of genital virilization may range from mild clitoral enlargement alone to, in rare cases, a penile urethra. Degrees of genital virilization are classified into five Prader stages ( Fig. 2 ).
Stage I: clitoromegaly without labial fusion
Stage II: clitoromegaly and posterior labial fusion
Stage III: greater degree of clitoromegaly, single perineal urogenital orifice, and almost complete labial fusion
Stage IV: increasingly phallic clitoris, urethra-like urogenital sinus at base of clitoris, and complete labial fusion
Stage V: penile clitoris, urethral meatus at tip of phallus, and scrotum-like labia (appear like males without palpable gonads).
Internal Genitalia
As the androgens interact with the receptors on genital skin, they induce changes in the developing external female genitalia such as clitoral enlargement, fusion of the labial folds, and rostral migration of the urethral/vaginal perineal orifice. However, internal female genitalia (uterus, fallopian tubes, and ovaries) are normal, as females cannot produce Müllerian-inhibiting hormone because they do not have testicular Sertoli cells. Therefore the Müllerian ducts do not regress, and the internal female internal genitalia develop normally.
Postnatal Effects and Growth
Lack of appropriate postnatal treatment in boys and girls results in continued exposure to excessive androgens, causing progressive penile or clitoral enlargement, the development of premature pubic (pubarche) and axillary hair and acne. Hyperandrogenism during childhood often leads to rapid linear growth accompanied by premature epiphyseal maturation and closure, resulting in a final adult height that is typically below that expected from parental heights (on average –1.1 to –1.5 SD below the midparental target height). On the other hand, poor growth can occur in patients with 21 OHD as a result of glucocorticoid treatment when replacement therapy exceeds physiologic requirements.
Final height in classical 21 OHD (as well as NC 21 OHD) is one of the features least amenable to glucocorticoid therapy even among patients with excellent adrenal control. A study of growth hormone therapy alone or in combination with a GnRH analog in CAH patients with compromised height prediction showed improvement in short- and long-term growth to reduce the height deficit.
Puberty and Secondary Sexual Characteristics
In most patients treated adequately from early life, the onset of puberty in both girls and boys with classical 21 OHD occurs at the expected chronologic age. However, a recent careful study showed that the mean ages at onset of puberty in both males and females were somewhat younger than the general population, but did not differ significantly among the three forms of 21 OHD. Following the onset of puberty, in most successfully treated patients, the milestones of further development of secondary sex characteristics in general appear to be normal.
Inadequate treatment leads to an advanced epiphyseal development. This can result in precocious puberty. Also in cases where a significant body maturation at the initial presentation, such as simple virilizing males who were not detected by newborn screening, the exposure to elevated androgens followed by the sudden decreased androgen levels after initiation of glucocorticoid treatment may cause an early activation of the hypothalamic-pituitary-gonadal axis. Studies suggest that excess adrenal androgens (aromatized to estrogens) inhibit the pubertal pattern of gonadotropin secretion by the hypothalamic-pituitary axis. This inhibition, which probably takes place via a negative feedback effect can be reversed by glucocorticoid treatment.
In adolescents and adults, signs of hyperandrogenism in females may include male-pattern alopecia (temporal balding), acne, or infertility, and in females, irregular or absent menses and hirsutism. There is also an association of CAH and polycystic ovarian syndrome (PCOS).
Gender Role Behavior
Prenatal androgen exposure in females with classical forms of 21 OHD CAH has a virilizing effect on the external genitalia and childhood behavior. Prenatal androgen exposure correlates with a decrease in self-reported femininity by adult females, but not an increase in self-reported masculinity by adult females. The rates of bisexual and homosexual orientation were increased in women with all forms of 21 OHD CAH. They were found to correlate with the degree of prenatal androgenization. Of interest, bisexual/homosexual orientation was correlated with global measures of masculinization of nonsexual behavior and predicted independently by the degree of both prenatal androgenization and masculinization of childhood behavior.
Changes in childhood play behavior correlated with reduced female gender satisfaction and reduced heterosexual interest in adulthood. Affected adult females are more likely to have gender dysphoria, and experience less heterosexual interest and reduced satisfaction with the assignment to the female sex. In contrast, males with 21 OHD CAH do not show a general alteration in childhood play behavior, core gender identity, or sexual orientation.
Fertility
Difficulty with fertility in females with CAH may arise for various reasons, including anovulation, secondary polycystic ovarian syndrome, irregular menses, nonsuppressible serum progesterone levels, or an inadequate introitus. Fertility is reduced in SW 21 OHD with rare reports of pregnancy. In a retrospective survey of fertility rates in a large group of females with classical CAH, simple virilizers were shown to be more likely to become pregnant and carry the pregnancy to term. However, more recent data suggest that fertility rates have significantly improved from 60% in SW and 80% in SV patients. Adequate glucocorticoid therapy is probably an important variable with respect to fertility outcome. The development of PCOS in CAH patients is not uncommon and may be related to both prenatal and postnatal excess androgen exposure, which can affect the hypothalamic-pituitary-gonadal axis.
In male patients with classical CAH, several long-term studies indicate that those who have been adequately treated undergo normal pubertal development, have normal testicular function, and normal spermatogenesis and fertility. However, small testes and reduced sperm count can occur in patients as a result of inadequately controlled disease. A complication in postpubertal boys with inadequate control of CAH is hyperplastic nodular testes. Almost all these patients were found to have adenomatous adrenal rests within the testicular tissue, so-called testicular adrenal rest tumor (TART), as indicated by the presence of specific 11β-hydroxylated steroids in the blood from gonadal veins. TART can lead to end-stage damage of testicular perenchyma, most probably as a result of long-standing obstruction of the seminiferous tubules. These tumors have been reported to be ACTH dependent and to regress following adequate steroid therapy.
Ironically, some investigators have reported normal testicular maturation as well as normal spermatogenesis and fertility in patients who had never received glucocorticoid treatment.
Salt Wasting in 21 OHD
When the loss of 21 hydroxylase function is severe, adrenal aldosterone secretion is not sufficient for sodium reabsorption by the distal renal tubules, and individuals suffer from salt wasting, in addition to cortisol deficiency and androgen excess. Classical CAH patients who make adequate aldosterone are considered to have SV-CAH. Infants with renal salt wasting have poor feeding, weight loss, failure to thrive, vomiting, dehydration, hypotension, hyponatremia, and hyperkalemic metabolic acidosis progressing to adrenal crisis (azotemia, vascular collapse, shock, and death). Adrenal crisis can occur as early as age 1 to 4 weeks. The salt wasting is presumed to result from inadequate secretion of salt-retaining steroids, primarily aldosterone. In addition, hormonal precursors of the 21 OH enzyme may act as antagonists to mineralocorticoid action in the sodium-conserving mechanism of the immature newborn renal tubule. The adrenal medulla may also be incompletely formed in some CAH patients. Merke and colleagues noted that the epinephrine and metanephrine concentrations in CAH patients who manifested salt-wasting crisis was approximately 50% of that of controls, in line with adrenal gland histopathologic studies that showed depleted secretory vesicles.
Affected males who are not detected in a newborn screening program are at high risk for a salt-wasting adrenal crisis because their normal male genitalia do not alert medical professionals to their condition; they are often discharged from the hospital after birth without diagnosis and experience a salt-wasting crisis at home. On the other hand, salt-wasting females are born with ambiguous genitalia that trigger the diagnostic process and appropriate treatment. It is important to recognize that the extent of virilism may not differ among the two classical CAH SV and SW forms. Thus, even a mildly virilized newborn with 21 OHD should be observed carefully for signs of a potentially life-threatening crisis within the first few weeks of life.
It has been observed that an aldosterone biosynthetic defect apparent in infancy may be ameliorated with age. Speiser and colleagues reported the spontaneous partial recovery in adulthood of a patient with documented severe salt wasting in infancy. Therefore, it is desirable to follow the sodium and mineralocorticoid requirements carefully by measuring plasma renin activity (PRA) in patients who have been diagnosed in the neonatal period as salt wasters.
Although insufficient aldosterone biosynthesis is clinically apparent only in the SW form of the disease, impaired adrenal capacity to produce aldosterone in response to renin stimulation appeared to be a spectrum in all forms of 21 OHD CAH.
Nonclassical Congenital Adrenal Hyperplasia
Individuals with the NC form of 21 OHD, also known as late-onset 21 OHD, have only mild to moderate enzyme deficiency and present postnatally with signs of hyperandrogenism. NC-CAH may present at any age after birth with a variety of hyperandrogenic symptoms, excluding ambiguous genitalia. This form of CAH results from a mild deficiency of the 21 hydroxylase enzyme. Similar to classical CAH, NC-CAH may cause premature development of pubic hair, advanced bone age, and accelerated linear growth velocity in both males and females. Severe cystic acne has also been attributed to NC-CAH.
Women may present with symptoms of androgen excess, including hirsutism, temporal baldness, and infertility. Menarche in females may be normal or delayed, and secondary amenorrhea is a frequent occurrence. Further virilization may include hirsutism, male habitus, deepening of the voice, or male-pattern alopecia (temporal recession). Clinical course of NC form varies among individuals and it is difficult to predict the appearance of any virilization symptoms.
Polycystic ovarian syndrome may also be seen as a secondary complication in these patients. Possible reasons for the development of PCOS include reprogramming of the hypothalamic-pituitary-gonadal axis from prenatal exposure to androgens, or chronic levels of excess adrenal androgens that disrupt gonadotropin release and have direct effects on the ovary, ultimately leading to the formation of cysts. Because of the overlap of hyperandrogenic symptoms, it is important to consider NC 21 OHD in a patient diagnosed with PCOS. Of note, NC 21 OHD CAH was identified in 2.2% to 10.0% among women who presented with hyperandrogenic symptoms.
Symptoms in adult males with NC-CAH may include short stature, acne, or oligozoospermia and diminished fertility. Infertility is often caused by TART, although some untreated men have been fertile, as discussed before.
A subset of individuals with NC 21 OHD are completely asymptomatic when detected (usually as part of a family study). Based on longitudinal follow-up of such patients, we observed that symptoms of hyperandrogenism wax and wane with time. The presence of 21 OHD can also be discovered during the evaluation of an incidental adrenal mass. An increased incidence of adrenal incidentalomas has been found, reported as high as 82% in patients with 21 OHD and up to 45% in subjects heterozygous for 21 OHD mutations. This probably arises from hyperplastic tissue areas and does not require surgical intervention.
Diagnosis of congenital adrenal hyperplasia
Hormonal Diagnosis
Biochemical diagnosis of 21 OHD can be confirmed by hormonal evaluation. In a randomly timed blood sample, a very high concentration of 17-hydroxyprogesterone (17-OHP), the precursor of the defective enzyme, is diagnostic of classical 21 OHD. The gold standard to establish hormonal diagnosis for NC 21 OHD and for certain enigmatic cases is the corticotropin stimulation test (250 μg cosyntropin intravenously), measuring levels of 17-OHP and Δ 4 -androstenedione at baseline and 60 minutes. The 17-OHP values can then be plotted in the published nomogram to ascertain disease severity ( Fig. 3 ). The corticotropin stimulation test is crucial in establishing hormonal diagnosis of the NC form of the disease, as even early-morning values of 17-OHP may not be sufficiently elevated to allow accurate diagnosis. For example, an NC patient may have a normal baseline 17-OHP of 100 ng/dL, yet stimulate to greater than 2000 ng/dL, and that would be diagnostic for NC-CAH. Patients with classical CAH typically have stimulated 17-OHP levels of 20,000 to 100,000 ng/dL. The corticotropin stimulation test may also be helpful in males for distinguishing between the NC and SV forms, as males with 21 OHD have normal genitalia.
Molecular Genetics
Hormonally and clinically defined forms of 21 OHD CAH are associated with distinct genotypes characterized by varying enzyme activity demonstrated though in vitro expression studies. The gene encoding 21 hydroxylase is a microsomal cytochrome P450-termed cytochrome P450, family 21, subfamily A, polypeptide 21 ( CYP21A2) (Online Mendelian Inheritance in Man [OMIM] database number 201,910) located on the short arm of chromosome 6, within the human leukocyte antigen (HLA) complex. CYP21A2 and its homolog, the pseudogene CYP21A1P alternate with two genes, C4B and C4A , that encode the two isoforms of the fourth component of the serum complement system.
More than 100 mutations have been described including point mutations, small deletions, small insertions, and complex rearrangements of the gene. Table 2 demonstrates the common mutations in CYP21A2 and their related phenotypes. In recessive disorders, the less severe mutation of the two alleles typically dictates phenotype. Classical 21 OHD is most often caused by two alleles with severe mutations. In contrast to the classical form, patients with NC 21 OHD are predicted to have mild mutations on both alleles or one severe and one mild mutation (compound heterozygosity) of CYP21A2 . It is not always possible, however, to accurately predict the phenotype on the basis of the genotype—such predictions have been shown to be 79% to 88% accurate with some nonconcordance. Studies have demonstrated that there is often a divergence in phenotypes within mutation-identical groups, the reason for which requires further investigation.
| Study | Exon/Intron | Mutation Type | Mutation | Phenotype | Severity of Enzyme Defect (% Enzyme Activity) | References |
|---|---|---|---|---|---|---|
| Nonclassical mutations | ||||||
| Tusie-Luna, 1991 | Exon 1 | Missense mutation | P30L | NC | Mild (30–60) | |
| Speiser, 1988 | Exon 7 | Missense mutation | V281L | NC | Mild (20–50) | |
| Helmberg, 1992 | Exon 8 | Missense mutation | R339H | NC | Mild (20–50) | |
| Helmberg, 1992; Owerbach, 1992 | Exon 10 | Missense mutation | P453S | NC | Mild (20–50) | |
| Classical mutations | ||||||
| White, 1984 | Deletion | 30kb Deletion | — | SW | Severe (0) | |
| Higashi, 1988 | Intron 2 | Aberrant splicing of Intron 2 | 656 A/C-G | SW, SV | Severe (ND) | |
| White, 1994 | Exon 3 | Eight-base deletion | G110 Δ8nt | SW | Severe (0) | |
| Tusie-Luna, 1990; Amor, 1988 | Exon 4 | Missense mutation | I172N | SV | Severe (1) | |
| Tusie-Luna, 1990; Amor, 1988 | Exon 6 | Cluster | I236N, V237E, M239K | SW | Severe (0) | |
| Globerman, 1988 | Exon 8 | Nonsense mutation | Q318X | SW | Severe (0) | |
| Chiou, 1990 | Exon 8 | Missense mutation | R356W | SW, SV | Severe (0) | |
| Wedell, 1993 | Exon 10 | Missense mutation | R483P a | SW | Severe (1–2) | |
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