Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders caused by deficiency in one or more of the enzymes required for adrenal gland synthesis of cortisol, aldosterone, and the sex steroids. The biosynthetic pathways are shown in Figure 1. In most instances, an enzyme deficiency leads to accumulation of upstream steroids (precursors) and depletion of downstream steroids (products). Depending on the degree and the position of the enzymatic deficiency in the biochemical pathway, shunting of precursor steroids may lead to either increased or decreased production of sex hormones and mineralocorticoids.

The clinical manifestations of CAH vary with the enzyme defect and the degree of deficiency  (Table 1). Nonclassic disease (ie, partial deficiency) is usually minimally symptomatic. Classical CAH, on the other hand, is more often severe and may be characterized by adrenal insufficiency that requires cortisol replacement; salt-wasting; genital ambiguity in males (XY); and virilization ranging from mild to complete masculinization of the external genitalia in females (XX).¹ Additional descriptions of the various CAH enzyme deficiencies follow.

21-Hydroxylase Deficiency

21-Hydroxylase deficiency, caused by mutation of the CYP21A2 gene, accounts for approximately 95% of CAH cases.² Classic 21-hydroxylase deficiency has an incidence of 1:10,000 to 1:20,000 live births in Western populations, though higher frequencies have been identified in certain ethnic groups.² The disorder is characterized by markedly diminished or absent 21-hydroxylase activity, which results in increased levels of 17-hydroxyprogesterone (17-OHP) and androstenedione, and decreased levels of deoxycorticosterone, 11-deoxycortisol, corticosterone, cortisol, and aldosterone (Table 1). Affected individuals typically present at birth or in the neonatal period with either a virilizing or salt-wasting form. Female (XX) infants with virilizing 21-hydroxylase deficiency have varying degrees of masculinization, ranging from clitoral enlargement to complete development of male external genitalia, which may lead to inaccurate sex assignment at birth. Male infants (XY) have normal male genitalia. Approximately three-quarters of affected infants also have mineralocorticoid deficiency that leads to salt-wasting. Symptoms of hyponatremia, hyperkalemia, volume depletion, and decreased blood pressure generally appear within the first 2 weeks of life.

Nonclassic 21-hydroxylase deficiency is thought to have an incidence as high as 1:500 in the general Caucasian population and is characterized by marginally decreased 21-hydroxylase activity.² Affected individuals typically do not have developmental abnormalities or salt-wasting, but present with signs of androgen excess. In boys, increased androgens typically manifest as sexual precocity, whereas in girls increased pubic hair and/or clitoral enlargement is seen. In women, nonclassic 21-hydroxylase deficiency is frequently confused with polycystic ovary syndrome (PCOS) because symptoms such as oligo/amenorrhea and hirsutism are common to both.

11β-Hydroxylase Deficiency

11β-Hydroxlase deficiency, caused by mutation of the CYP11B1 gene, accounts for 5% to 8% of CAH cases.¹ Deficiency of 11β-hydroxlase leads to decreased levels of cortisol, corticosterone, and aldosterone and increased levels of deoxycorticosterone and 11-deoxycortisol. Although it does not cause salt-wasting, virilization of female (XX) fetuses can be as severe as with 21-hydroxylase deficiency. The combination of elevated blood pressure, observed early in life in approximately two-thirds of patients, and elevated deoxycorticosterone and 11-deoxycortisol levels distinguishes 11β- from 21-hydroxylase deficiency (Table 1). A late-onset form that typically presents with signs of androgen excess is analogous to nonclassic 21-hydroxylase deficiency.

17α-Hydroxylase Deficiency

17α-Hydroxylase deficiency accounts for approximately 1% of all CAH cases.³ The CYP17 gene encodes an enzyme that catalyzes both 17α-hydroxylation and 17,20-lyase reactions. Isolated deficiency of either activity has been reported; however, the most common form is a combined deficiency with failure of catalysis of both reactions. Affected individuals have decreased levels of cortisol, androgens, and estrogens and increased levels of deoxycorticosterone and corticosterone (Table 1). Presentation is typically at puberty; females (XX) have primary amenorrhea and lack secondary sexual characteristics, and males (XY) are found to have complete pseudohermaphroditism (ie, female external genitalia, absence of uterus and fallopian tubes, and intra-abdominal testes). At the time of diagnosis, individuals are usually found to be hypertensive and hypokalemic.

3β-Hydroxysteroid Dehydrogenase Deficiency

3β-Hydroxysteroid dehydrogenase (3β-HSD) deficiency, due to HSD3B2 gene mutation, is a rare form of CAH characterized by increased levels of pregnenolone, 17-hydroxypregnenolone, and DHEA and decreased levels of all other adrenal steroids (Table 1). Affected individuals usually present in infancy with signs of adrenal insufficiency. Female (XX) infants will typically have mild virilization, and a nonclassic form may appear at adrenarche or at puberty. Phenotypic variation in male (XY) infants may range from hypospadias to complete male pseudohermaphroditism.

Cytochrome P450 Oxidoreductase Deficiency

Cytochrome P450 oxidoreductase (POR) deficiency, caused by mutation of the POR gene, is characterized by elevated serum levels of pregnenolone, progesterone, and 17-OHP. POR deficiency resembles a combination of 21- and 17α-hydroxylase deficiencies. Urine analysis by gas chromatography-mass spectrometry (GC/MS) may show an elevated 5α-tetrahydro-11-dehydrocorticosterone level (suggestive of 17α-hydroxylase deficiency) as well as elevated pregnanetriol, pregnanetriolone, and 17α-hydroxypregnenolone levels (suggestive of 21-hydroxylase deficiency). Affected males are typically undervirilized, whereas females may show virilization due to an alternative steroid synthesis pathway, the so-called “backdoor” dihydrotestosterone (DHT) synthesis pathway. Although not included in Table 1 because of its rarity, POR deficiency should be considered in infants with a moderately elevated serum 17-OHP level that cannot be explained by stress or by 21-hydroxylase deficiency. Evaluation for POR deficiency is especially important in infants with craniofacial or skeletal anomalies. See The Quest Diagnostics Manual: Endocrinology - Chapter 1, for additional details.

• Newborns with a positive CAH screening test (ie, 17-OHP filter paper test)

• Newborns with ambiguous genitalia

• Newborns and infants with adrenal insufficiency and/or unexplained sodium and potassium abnormalities

• Children with evidence of precocious or delayed puberty or unexplained hypertension

• Women with polycystic ovary syndrome, hirsutism, and/or evidence of estrogen deficit

• Individuals with a family history of 21-hydroxylase deficiency, and their partners, who desire carrier screening

• Pregnant women at risk for a fetus affected with CYP21A2 mutations who desire prenatal diagnosis

Test Availability [return to contents]

Steroid Assays

The diagnosis of enzyme deficiencies responsible for CAH relies on accurate measurement of basal steroid and steroid metabolite levels. Quest Diagnostics Nichols Institute utilizes mass spectrometry technology for steroid measurement to address limitations associated with immunoassay measurement.^4,5 Tests may be ordered individually or in panels (Table 2). Table 2 is provided for informational purposes only and is not intended as medical advice. A physician’s test selection and interpretation, diagnosis, and patient management decisions should be based on his/her education, clinical expertise, and assessment of the patient.

Baseline steroid levels are frequently adequate for screening; however, steroid measurement after ACTH stimulation is usually necessary for diagnosis, especially when mild or partial deficiency is suspected. The 60-minute cosyntropin stimulation test is commonly used for this purpose. Cosyntropin stimulates adrenal hormone synthesis, which accentuates steroid levels upstream from the blockage.

Simultaneous measurement of multiple steroids, rather than measurement of a single steroid, has improved the differential diagnosis of CAH.^2,6 Quest Diagnostics offers several test panels (Table 2): 1) 3 panels for diagnosing the various CAH enzyme deficiencies, 2) a panel for distinguishing CAH from polycystic ovary syndrome (PCOS), and 3) a panel to differentiate 21-hydroxylase deficiency from stress as the cause of an increased 17-OHP level. Stress is commonly seen in newborns and pediatric patients subsequent to prolonged birth, infection, or blood collection and can cause a positive 17-OHP screen. For example, premature infants have significantly higher serum levels of 17-OHP than do term infants. Determining the trend in 17-OHP levels over time may help differentiate premature infants who are affected by CAH from those who are unaffected (levels steadily decrease over time).

Genetic Testing

Quest Diagnostics offers 2 genetic tests that can be used to confirm a 21-hydroxylase deficiency diagnosis (Table 2).

CAH (21-Hydroxylase Deficiency) Common Mutations

The genetic confirmation of 21-hydroxylase deficiency is complex because of the large number of unique mutations in CYP21A2 that may result in decreased enzyme activity and because of interactions between CYP21A2 and its pseudogene, CYP21AP. This test detects the most common mutations of CYP21A2: P30L, In2G, G110del8, I172N, exon 6 cluster mutation (I236N, V237E, M239K), V281L, F306+1nt, Q318X, R356W, P453S, and a 30-kb deletion. These 12 mutations and the 30-kb deletion are responsible for approximately 90% of the CYP21A2 changes that can result in 21-hydroxylase deficiency.

CAH (21-Hydroxylase Deficiency) Rare Mutations

This test provides complete DNA sequence information for the CYP21A2 gene.

Steroid Assays

Classic CAH

Neonatal screening for 21-hydroxylase deficiency, the most common cause of CAH, is performed routinely in the United States by measuring 17-OHP in filter paper blood samples collected from newborns. A positive screen can be followed up with measurement of baseline and ACTH-stimulated 17-OHP levels (Figure 2, option A); steroid panels (using serum or urine) for differential diagnosis of CAH, especially when virilization or ambiguous genitalia is present (Figure 2, option B); or a steroid panel that can distinguish stress from 21-hydroxylase deficiency as the cause of a 17-OHP elevation (Figure 2, option C).

Once CAH is diagnosed, treatment requires monitoring adrenal steroid levels to ensure correct dosing of glucocorticoid and/or mineralocorticoid replacement (Table 2). Additionally, a plasma renin activity (PRA) assay is used to monitor mineralocorticoid replacement in individuals with salt-wasting.

Nonclassic CAH

Nonclassic CAH, which presents with evidence of androgen excess in childhood or postpuberty, is commonly caused by 21-hydroxylase deficiency and rarely caused by 11β-hydroxylase deficiency. A steroid panel is available to distinguish these 2 forms of nonclassic CAH and also to distinguish PCOS (Figure 3).

Genetic Testing

In cases of ambiguous genitalia, chromosome analysis should be performed to determine the genetic gender of a newborn (Table 2).

Mutation testing is recommended to confirm a 21-hydroxylase deficiency diagnosis in newborns, to follow-up equivocal ACTH stimulation results after infancy, for prenatal diagnosis, and for purposes of genetic counseling.²

Mutation testing begins with an assay for common mutations, which includes the 30-kb deletion. Follow-up testing for rare mutations is indicated for individuals with 21-hydroxylase deficiency in whom only 1 or none of the common mutations have been identified, and for individuals whose family history includes a rare mutation.

Steroid Assays

Classic CAH

Table 3 shows the pattern of results expected for the various types of classic CAH. This pattern can be used to identify the specific enzyme deficiency causing the CAH. In normal children, adolescents, and adults, precursor-product ratios associated with the 3β-hydroxysteroid dehydrogenase, 21-, and 17-hydroxylase enzymes will typically not exceed 10. The normal ratio associated with 11β-hydroxylase will not exceed 15. Precursor-product ratios are similar before and after ACTH stimulation in unaffected individuals. Pre- and post ACTH stimulation test results for infants and children are provided in Table 4.

Nonclassic CAH

Except in cases of mild enzyme deficiency, an early morning 17-OHP level <200 ng/dL rules out nonclassic CAH due to either 21-hydroxylase or 11β-hydroxylase deficiency.² Levels >200 ng/dL should be followed up with ACTH stimulation; patients with nonclassic 21-hydroxylase deficiency will exhibit post-ACTH levels of 17-OHP >1000 ng/dL.² Exaggerated (higher than normal) ACTH response of 11-deoxycortisol or deoxycorticosterone is characteristic of 11β-hydroxylase deficiency.

Genetic Testing

When both partners are carriers of a CAH mutation, a newborn has a 50% chance of being a carrier and a 25% chance of being affected. The following information will assist in understanding test results. A complete family history and parental and affected sibling genotypes are required for the most accurate interpretation of 21-hydroxylase deficiency DNA testing.

CAH (21-Hydroxylase Deficiency) Common Mutations

A negative result indicates that neither the 30-kb deletion nor any of the 12 common mutations responsible for 21-hydroxylase deficiency was detected. In an individual without clinical symptoms of CAH, this lowers but does not eliminate the risk of a mutation being present; the residual risk of being a carrier depends upon the individual’s family history. In an individual affected with CAH, a negative result suggests the presence of a rare mutation or a cause other than 21-hydroxylase deficiency.

Three different positive results may be reported:

1. Positive: 1 copy of CYP21A2 contains at least 1 common mutation, while the other copy does not contain any of the 12 common mutations or the 30-kb deletion. In most instances the individual is considered a carrier and may exhibit mild symptoms depending upon the mutation present. DNA testing of parents and affected siblings may be necessary to eliminate the possibility of gene duplication, or the individual may be incorrectly classified as a carrier.

2. Positive: both copies of CYP21A2 contain common mutations (ie, 2 distinct copies of CYP21A2 are identified and both contain 1 or more of the 12 common mutations or the 30-kb deletion). Individuals will be affected with 21-hydroxylase deficiency, with the severity determined by the mutations present.

3. Positive for the heterozygous presence of at least 2 common mutations (ie, mutations are present, but it cannot be determined if they are on the same or separate chromosomes). If the mutations all reside on the same chromosome (cis configuration), the individual is a carrier; if they are on separate chromosomes (trans configuration), the individual will be affected with 21-hydroxylase deficiency. DNA testing of parents and affected siblings is frequently necessary to determine if the mutations are in cis or trans configuration.

Mutations will be identified in approximately 90% of carriers; the other 10% may have a rare mutation not tested for in this assay. Furthermore, this test will detect both relevant mutations in approximately 81% of affected individuals, only 1 relevant mutation in another 18%, and no mutations in the final 1% of affected individuals. Thus, rare mutation analysis may be needed to detect additional mutations in affected individuals.

CAH (21-Hydroxylase Deficiency) Rare Mutations

Because the rare mutation analysis does not detect certain sequence alterations (eg, large deletions), negative results reduce but do not eliminate the possibility that an unaffected individual is a carrier. The residual risk is influenced by the individual’s family history.

Positive results include: 1) sequence alterations known or predicted to be pathogenic; 2) sequence alterations known or predicted to be benign; and 3) sequence alterations of unknown clinical significance. In the presence of positive clinical findings, the detection of 2 mutations (either common or rare) known or predicted to be pathogenic is consistent with a diagnosis of 21-hydroxylase deficiency. The detection of 2 mutations known or predicted to be pathogenic in the absence of clinical symptoms of CAH suggests cis configuration of the mutations.

Results must be interpreted in light of the individual’s clinical status and family history including genotypes of parents and siblings. Assistance with the interpretation of results is available from our Genetic Counselors; call 1-866-GENE-INFO (1-866-436-3463).