21-Hydroxylase-Deficient Congenital Adrenal Hyperplasia

Clinical characteristics.

21-hydroxylase deficiency (21-OHD) is the most common cause of congenital adrenal hyperplasia (CAH), a family of autosomal recessive disorders involving impaired synthesis of cortisol from cholesterol by the adrenal cortex. In 21-OHD CAH, excessive adrenal androgen biosynthesis results in virilization in all individuals and salt wasting in some individuals. A classic form with severe enzyme deficiency and prenatal onset of virilization is distinguished from a non-classic form with mild enzyme deficiency and postnatal onset. The classic form is further divided into the simple virilizing form (~25% of affected individuals) and the salt-wasting form, in which aldosterone production is inadequate (≥75% of individuals). Newborns with salt-wasting 21-OHD CAH are at risk for life-threatening salt-wasting crises. Individuals with the non-classic form of 21-OHD CAH present postnatally with signs of hyperandrogenism; females with the non-classic form are not virilized at birth.

Diagnosis/testing.

The diagnosis of classic 21-OHD CAH is established in newborns with characteristic clinical features, elevated serum 17-OHP, and elevated adrenal androgens. The diagnosis of non-classic 21-OHD is established by comparison of baseline serum 17-OHP and ACTH-stimulated serum 17-OHP or early morning elevated 17-OHP. Identification of biallelic pathogenic variants in CYP21A2 confirms the clinical diagnosis and allows for family studies.

Management.

Treatment of manifestations: Classic 21-OHD CAH: glucocorticoid replacement therapy, which needs to be increased during periods of stress. Salt-wasting form: mineralocorticoid 9α-fludrohydrocortisone therapy and often sodium chloride. Females who are virilized at birth may require feminizing genitoplasty and/or vaginal dilation. Symptomatic individuals with non-classic 21-OHD CAH may require treatment.

Prevention of primary manifestations: Newborn screening programs aim to identify infants with classic 21-OHD CAH in order to initiate glucocorticoid and mineralocorticoid treatment prior to a potentially life-threatening salt-wasting crisis.

Surveillance: Monitor:

• Efficacy of glucocorticoid and mineralocorticoid replacement therapy every three to four months while children are actively growing, and less often thereafter;
• For testicular adrenal rest tumors in males every three to five years after onset of puberty;
• Weight, bone mineral density, fertility, cardiovascular and metabolic risks in adults.

Evaluation of relatives at risk: It is appropriate to measure 17-hydroxyprogesterone (17-OHP) of at-risk sibs to facilitate early diagnosis and treatment.

Genetic counseling.

21-OHD CAH is inherited in an autosomal recessive manner. Most parents are heterozygous for a pathogenic variant. Approximately 1% of pathogenic variants are de novo; thus, 1% of probands have only one parent who is heterozygous. In some instances during evaluation of a proband, a parent not previously known to be affected may be found to have biallelic pathogenic variants and the non-classic form of 21-OHD CAH. At conception, if the parents of a proband are both known to be heterozygotes, each sib has a 25% chance of being affected, a 50% chance of being an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier. Carrier testing for at-risk relatives and prenatal testing for pregnancies at increased risk are possible if the pathogenic variants in the family are known.

Suggestive Findings

21-hydroxylase-deficient congenital adrenal hyperplasia (21-OHD CAH) should be suspected in the following individuals:

• Females who are virilized at birth, or who become virilized postnatally, or who have precocious puberty or adrenarche. Virilization affects maturation, growth (leading to tall stature), and sex hormone-sensitive areas (external genitalia, skin, and hair) (leading to secondary sexual characteristics).
• Males with masculinization in childhood (i.e., premature adrenarche)
• Any infant with a salt-losing crisis in the first four weeks of life. Individuals with untreated or poorly controlled salt wasting may have a decreased serum concentration of sodium, chloride, and total carbon dioxide (CO₂), an increased serum concentration of potassium, and inappropriately increased urine concentration of sodium.
• An infant with elevated 17-OHP concentration detected as positive newborn screening

Note: Females with 21-OHD CAH have a normal 46,XX karyotype; males with 21-OHD CAH have a normal 46,XY karyotype.

Establishing the Diagnosis

21-OHD CAH. The diagnosis is established in a newborn with the following laboratory findings:

• Serum 17-OHP is markedly elevated.
• Adrenal androgens are elevated; Δ⁴-androstenedione, 21 deoxycortisol, and progesterone are increased in males and females with 21-OHD CAH; Testosterone and adrenal androgen precursors (Δ⁴-androstenedione, DHEA) are increased in affected females and prepubertal males.
• Plasma renin activity is markedly elevated in individuals with the salt-wasting form of 21-OHD CAH.
  Note: In individuals with the salt-wasting form of 21-OHD CAH, the serum concentration of aldosterone is inappropriately low compared to the level of plasma renin activity (PRA) elevation. A reduced ratio of aldosterone to PRA indicates impaired aldosterone synthesis and can differentiate those individuals with the salt-wasting form of CAH from those with the simple virilizing form of CAH after the newborn period [Nimkarn et al 2007].
• Identification of biallelic pathogenic variants in CYP21A2 (see Table 2)

Non-classic 21-OHD CAH. The diagnosis is established in a proband based on the results of ONE of the two following laboratory tests (see Figure 1 and Table 1):

Figure 1.

17-OHP nomogram for the diagnosis of steroid 21-hydroxylase deficiency (60-minute Cortrosyn™ stimulation test). The data for this nomogram was collected between 1982 and 1991 at the Department of Pediatrics, the New York Hospital-Cornell Medical (more...)

• 60-minute ACTH stimulation test. The serum concentration of 17-OHP measured at baseline and at 60 minutes after intravenous injection of a standard 250-µg bolus of synthetic ACTH (Cortrosyn™) are plotted on the nomogram in Figure 1.
• 17-hydroxyprogesterone (17-OHP). A single early-morning (<8AM) measurement of plasma 17-OHP concentration (baseline values in affected individuals are not always elevated; see Table 1)
  Note: Normal ranges of 17-OHP for gender and pubertal status vary by laboratory, reflecting the methods used. In adult females, normal ranges depend on the phase of the menstrual cycle.

Molecular testing. Identification of biallelic pathogenic variants in CYP21A2 (see Table 2) confirms the diagnosis and allows for family studies. Molecular testing approaches can include single-gene testing, use of a multigene panel, and more comprehensive genomic testing:

• Single-gene testing. Sequence analysis of CYP21A2 is performed first and followed by gene-targeted deletion/duplication analysis if only one or no pathogenic variant is found.
  Note: A large-scale gene conversion (see Molecular Genetics) can replace a large segment of functional CYP21A2 sequence with a segment of the CYP21A1P pseudogene that is nonfunctional as a result of more than one deleterious variant [Mao et al 2002]. Thus, when targeted analysis detects multiple pathogenic variants, it is possible that the pathogenic variants are either in trans configuration (i.e., are on separate chromosomes, one inherited from each parent) or in cis configuration (i.e., are on the same chromosome and thus represent only one mutated allele rather than two; most likely arising from gene conversion). To avoid diagnostic errors, studying both parents as well as the proband is recommended to confirm the pathogenic variants and to determine if they are in cis configuration or trans configuration.
• A multigene panel that includes CYP21A2 and other genes of interest (see Differential Diagnosis) may also be considered. Note: (1) The genes included in the panel and the diagnostic sensitivity of the testing used for each gene vary by laboratory and are likely to change over time. (2) Some multigene panels may include genes not associated with the condition discussed in this GeneReview; thus, clinicians need to determine which multigene panel is most likely to identify the genetic cause of the condition at the most reasonable cost while limiting identification of variants of uncertain significance and pathogenic variants in genes that do not explain the underlying phenotype. (3) In some laboratories, panel options may include a custom laboratory-designed panel and/or custom phenotype-focused exome analysis that includes genes specified by the clinician. (4) Methods used in a panel may include sequence analysis, deletion/duplication analysis, and/or other non-sequencing-based tests.
  For an introduction to multigene panels click here. More detailed information for clinicians ordering genetic tests can be found here.
• More comprehensive genomic testing (when available) including exome sequencing, genome sequencing, and mitochondrial sequencing may be considered if serial single-gene testing (and/or use of a multigene panel) fails to confirm a diagnosis in an individual with features of 21-OHD CAH.
  For an introduction to comprehensive genomic testing click here. More detailed information for clinicians ordering genomic testing can be found here.

Clinical Description

21-hydroxylase-deficient congenital adrenal hyperplasia (21-OHD CAH) occurs in a classic form and a non-classic form (Table 3).

In classic 21-OHD CAH prenatal exposure to potent androgens such as testosterone and Δ⁴-androstenedione at critical stages of sexual development virilizes the external genitalia of genetic females, often resulting in genital ambiguity at birth. The classic form is further divided into the simple virilizing form (~25% of individuals) and the salt-wasting form, in which aldosterone production is inadequate (≥75% of individuals). Newborns with salt-wasting CAH caused by 21-OHD CAH are at risk for life-threatening salt-wasting crises.

Individuals with the non-classic form of 21-OHD CAH have only moderate enzyme deficiency and present postnatally with signs of hyperandrogenism; females with the non-classic form are not virilized at birth.

Genotype-Phenotype Correlations

A study by New et al [2013] that included the largest cohort of individuals with 21-OHD CAH demonstrated that the predictability of phenotype was less certain than previously thought. A direct genotype-phenotype correlation was found in approximately 50% of genotypes. The most unreliable predictions occurred in the simple virilizing form, where a wide phenotypic variety was observed with the same genotype. However, a strong correlation was noted for some genotypes that were exclusively found in salt-wasting and non-classic forms. For example, the Val281Leu pathogenic variant is exclusively associated with the non-classic form. In individuals with this form, the phenotype reflected the pathogenic variant with the less severe phenotypic effect of the two alleles.

Alleles can be grouped as severe or mild, based on residual enzyme activity (Table 4).

• Salt-wasting 21-OHD CAH usually has the most severe pathogenic variants (e.g., homozygous deletions).
• Non-classic 21-OHD CAH usually has one mild allele or both mild alleles.

In the context of prenatal diagnosis, it is important to distinguish classic and non-classic genotypes in order to determine the need to offer prenatal treatment.

• In families in which the proband is a virilized female, predicting the risk of genital virilization in subsequent affected female fetuses is feasible.
• In families in which the proband is a male, predicting the risk of genital virilization in subsequent affected female fetuses based on genotype is less reliable.

Classic 21-OHD CAH. The genotype for the classic form of 21-OHD CAH is predicted to be a severe pathogenic variant on both CYP21A2 alleles, with completely abolished enzyme activity determined by in vitro expression studies.

Note: The single-nucleotide variants c.293-13A>G or c.293-13C>G, among the most frequent pathogenic variants in classic 21-OHD CAH, cause premature splicing of the intron and a shift in the translational reading frame. Although most individuals (>90%) who are homozygous for one of these pathogenic variants have salt-wasting 21-OHD CAH, variation in severity of salt wasting is observed. This genotype-phenotype non-concordance can be explained by increased alternate splicing that can occur when the normal splicing is abolished by the splice site variant, allowing some protein production but with variable activity [Higashi et al 1988].

Among affected individuals who were compound heterozygotes for the pathogenic single-nucleotide variant p.Ile173Asn and a second severe variant, 76% had the simple virilizing phenotype while 23% had the salt wasting phenotype [New et al 2013]. It is postulated that subtle variations in transcription regulation or downstream protein translation may account for reduced 21-OH enzyme activity.

Non-classic 21-OHD CAH. Individuals with non-classic CAH are predicted to have two mild variants or one mild and one severe variant. Approximately two-thirds of individuals with non-classic 21-OHD CAH are compound heterozygotes. Pathogenic missense variants p.Pro31Leu in exon 1 and p.Val282Leu in exon 7 reduce enzyme activity and are generally associated with this form of the disease. However, variation in the phenotype associated with one mild variant can be observed:

• In a small number (<3%) of affected individuals with the p.Val282Leu or p.Pro31Leu pathogenic variant and a severe variant, the classic phenotype was observed when a non-classic phenotype was expected.
• In a very small percentage of affected individuals with the p.Ile173Asn pathogenic variant and a severe variant, the non-classic phenotype (rather than the expected classic phenotype) was observed [Stikkelbroeck et al 2003].

Contiguous gene deletion. A contiguous gene deletion involving CYP21A2 and TNX led to a combination of Ehler-Danlos syndrome, hypermobility type and 21-OHD CAH [Burch et al 1997, Schalkwijk et al 2001].

Nomenclature

Terms used in the past for 21-OHD CAH include adrenogenital syndrome (AG syndrome) and congenital adrenocortical hyperplasia.

The non-classic form of 21-OHD CAH was previously referred to as the "attenuated" or "late-onset" form.

The salt-wasting form of 21-OHD CAH has also been called "salt-losing CAH."

Prevalence

Classic 21-OHD CAH. Analysis of data from almost 6.5 million newborns screened in different populations worldwide has demonstrated an overall incidence of 1:15,000 live births for the classic form of 21-OHD [van der Kamp & Wit 2004].

Prevalence in specific populations:

• 1:300 in Yup'ik Eskimos of Alaska
• 1:5,000 in Saudi Arabia
• 1:10,000-1:16,000 in Europe and North America
• 1:21,000 in Japan
• 1:23,000 in New Zealand

Non-classic 21-OHD CAH. The prevalence of non-classic 21-OHD CAH in the general heterogeneous population of New York City was estimated at 1:100. The highest ethnic-specific non-classic disease prevalence (1:27) is found among Ashkenazi Jews. Other ethnic groups exhibiting high non-classic disease prevalence are: Hispanics (1:40), Slavs (1:50), and Italians (1:300) [Speiser et al 1985].