21-Hydroxylase Deficiency Congenital Adrenal Hyperplasia - 21-OHD CAH

Medical Experts

Contributor

Doyle

Kelly Doyle, PhD, DABCC, FAACC

Associate Professor of Pathology (Clinical), University of Utah

Medical Director, Special Chemistry and Endocrinology, ARUP Laboratories

Contributor

Krautscheid

Patti Krautscheid, MS, LCGC

Genetic Counselor, ARUP Laboratories

Contributor

Straseski

Joely A. Straseski, PhD, MS, MT(ASCP), DABCC, FAACC

Professor of Pathology (Clinical), University of Utah

Head of Clinical Operations for Clinical Chemistry, Toxicology, and Biochemical Genetics; Medical Director, Endocrinology; Co-Director, Automated Core Laboratory, ARUP Laboratories

Contributor

Wiltse

Heidi Wiltse, MS, LCGC

Genetic Counselor, ARUP Laboratories

Congenital adrenal hyperplasia (CAH) refers to a group of autosomal recessive disorders caused by certain distinct enzymatic defects that result in abnormalities in the balance of adrenal hormones in the body.^, Most cases of CAH are associated with CYP21A2 gene variants and result in 21-hydroxylase deficiency (21-OHD).^,^, Other rare forms of CAH are associated with different genetic and enzymatic defects (eg, 11 beta-hydroxylase deficiency, cytochrome P450 oxidoreductase deficiency, and others).^,

21-OHD CAH is subdivided into classic and nonclassic forms. Classic 21-OHD CAH is further distinguished as either the simple virilizing form or the salt-wasting form, both of which are characterized by aldosterone deficiency and the accumulation of 17-hydroxyprogesterone (17-OHP).^,^, Clinical features of classic 21-OHD CAH include prenatal-onset genital and gonadal developmental abnormalities, and, in the salt-wasting form, electrolyte abnormalities that can be life threatening.^,^, Nonclassic 21-OHD CAH is milder and characterized by postnatal-onset hyperandrogenism.^,^,

In the United States, classic 21-OHD CAH is generally diagnosed early in life, as universal newborn screening includes laboratory testing for classic 21-OHD CAH.^, Nonclassic 21-OHD CAH is typically diagnosed after infancy. Laboratory testing, including serum hormone and electrolyte measurements, with or without molecular genetic testing, is key to the diagnosis and monitoring of both classic and nonclassic 21-OHD CAH.

Quick Answers for Clinicians

What is the preferred sample type for hormone testing in 21-hydroxylase deficiency congenital adrenal hyperplasia?

Serum is generally the preferred sample type for hormonal testing for diagnosis and monitoring of congenital adrenal hyperplasia (CAH).^,^, Testing using a highly sensitive and specific method such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended. Urine may also be tested for adrenal hormones and metabolites if blood sampling is not feasible.^, However, urine collection, particularly 24-hour urine sampling, may not be as easy to perform as serum sampling, especially in newborns.^,

What laboratory testing is available for the prenatal diagnosis of 21-hydroxylase deficiency congenital adrenal hyperplasia?

When familial CYP21A2 pathogenic variants are known, prenatal or preimplantation molecular genetic tests are options for high-risk pregnancies. To minimize unnecessary treatment of unaffected fetuses and associated side effects, an early prenatal sampling method for diagnostic molecular testing is desirable; therefore, chorionic villus sampling, which can be performed at 9-11 weeks, is preferable to amniocentesis, which is generally not performed until later in pregnancy. In low-risk pregnancies with ambiguous genitalia on an ultrasound, fetal karyotyping and fluorescence in situ hybridization (FISH) for SRY are indicated, and results may guide consideration of CYP21A2 molecular testing.

What is the role of laboratory testing in prenatal therapy for 21-hydroxylase deficiency congenital adrenal hyperplasia?

Prenatal treatment is currently considered experimental and should only be pursued under formalized institutional review board (IRB) protocols. Because prenatal treatment is not recommended for male fetuses,^, noninvasive genetic screening for Y chromosomal material in maternal blood is advised if prenatal treatment is being considered for fetuses potentially affected by 21-hydroxylase deficiency congenital adrenal hyperplasia (21-OHD CAH).

Hormone Evaluation for Classic 21-OHD Congenital Adrenal Hyperplasia

Indications for Testing

Hormone testing for classic 21-OHD CAH is used to^,^,:

Screen newborns for 21-OHD CAH
Diagnose 21-OHD CAH in individuals with positive newborn screening tests or suggestive clinical features (salt-wasting crisis, ambiguous genitalia or virilization in females, childhood masculinization in males)
Monitor treatment and inform treatment decisions
Clarify and supplement genetic testing results

Criteria for Diagnosis

The diagnosis of classic 21-OHD CAH is established by the detection of elevated concentrations of serum 17-OHP and adrenal androgens (21-deoxycortisol, androstenedione, dehydroepiandrosterone [DHEA], progesterone, testosterone) and the detection of increased plasma renin activity in individuals with salt-wasting classic CAH.

The diagnosis is confirmed by identification of biallelic pathogenic CYP21A2 variants. Refer to the CYP21A2 section for more information.

Laboratory Testing

Newborn Screening and Diagnosis

17-Hydroxyprogesterone

Screening using standardized 17-OHP assays on filter paper blood spot specimens with results stratified by gestational age is recommended for all newborns and is included in universal newborn screening panels in the United States.^,^, In the first 24 hours of life, 17-OHP is elevated; it may also be elevated in infants with low birth weight or premature birth, potentially yielding false-positive results.^, False-negative results may occur in infants being treated with dexamethasone.

Serum 17-Hydroxyprogesterone

If initial screening results are positive, a follow-up test for 17-OHP using liquid chromatography-tandem mass spectrometry (LC-MS/MS) should be performed. (1- ES 2018) However, this is not always performed in practice, and in some cases, other hormones (eg, 21-deoxycortisol) are assessed using a follow-up test.^, Serum 17-OHP testing should also be performed in newborns with siblings diagnosed with CAH.

Adrenocorticotropic Hormone Stimulation Test

The adrenocorticotropic hormone (ACTH) stimulation test (also referred to as a cortrosyn or cosyntropin test) may be performed if LC-MS/MS testing is not available, or as needed to follow up on borderline LC-MS/MS results. Concentrations of 17-OHP are generally elevated in the first 24 hours after birth in all neonates; therefore, the ACTH stimulation test may yield false-positive results.^, Treatment should not be withheld pending ACTH stimulation test results in neonates with suspected adrenal crisis.

Other Hormones and Metabolites

A complete adrenocortical hormone profile is recommended after a diagnosis of CAH to distinguish other enzyme defects and assess for mineralocorticoid deficiency. Androstenedione, DHEA, and testosterone are usually elevated in classic 21-OHD CAH, whereas aldosterone and cortisol are usually low.^, Epinephrine and metanephrine are also low in classic 21-OHD CAH.

Evaluation for Salt Wasting

An electrolyte panel and plasma renin activity or direct renin test are recommended after a diagnosis of 21-OHD CAH. These tests are useful to distinguish between the salt-wasting and simple virilizing forms of CAH.^, Salt wasting is characterized by decreased serum chloride, sodium, and total carbon dioxide and increased serum potassium and urine sodium; these analytes are normal in simple virilizing CAH.^, Plasma renin activity is generally disproportionately elevated relative to serum aldosterone in individuals with salt-wasting CAH.

Hormone Evaluation for Nonclassic 21-OHD Congenital Adrenal Hyperplasia

Indications for Testing

Hormone testing for nonclassic 21-OHD CAH is used to^,^,:

Diagnose CAH in individuals with suggestive symptoms (hirsutism, virilization, precocious puberty; amenorrhea, irregular menses, or polycystic ovary syndrome in females)
Monitor treatment and inform treatment decisions
Clarify and supplement genetic testing results

Criteria for Diagnosis

The diagnosis of nonclassic 21-OHD CAH is suggested by an elevated early morning serum 17-OHP and established by 17-OHP concentrations above cutoff values for the ACTH stimulation test.

The diagnosis is confirmed by the identification of pathogenic CYP21A2 variants. Refer to the CYP21A2 section for more information.

Laboratory Testing

Diagnosis

Serum 17-Hydroxyprogesterone

Serum 17-OHP testing by LC-MS/MS is the recommended first test in individuals with symptoms of CAH.^,^, Serum should be collected in the early morning to assess baseline 17-OHP concentration.

Adrenocorticotropic Hormone Stimulation Test

The ACTH stimulation test is used to establish the diagnosis of nonclassic CAH.^, Briefly, the test is performed by taking a baseline serum measurement of 17-OHP, followed by the administration of a dose of synthetic ACTH; after 60 minutes, serum 17-OHP is measured again.^, Concentrations of 17-OHP may vary based on the menstrual phase in individuals who are postmenarche, which will potentially impact results.^,

Other Hormones and Metabolites

A complete adrenocortical hormone profile is recommended after a diagnosis of CAH to distinguish other enzyme defects and assess for mineralocorticoid deficiency. Androstenedione, DHEA, and testosterone are usually elevated in nonclassic 21-OHD CAH, particularly with ACTH stimulation, whereas aldosterone is usually moderate and cortisol is normal or slightly low.^,

Molecular Testing for 21-OHD CAH

Indications for Testing

Molecular genetic testing may be appropriate in individuals^,^,:

Diagnosed biochemically with either classic or nonclassic 21-OHD CAH
Suspected of having either classic or nonclassic 21-OHD CAH based on clinical features

Molecular testing is also used to:

Determine carrier status for 21-OHD CAH
Assess for familial CYP21A2 variant(s) in at-risk family members or fetuses

Laboratory Testing

CYP21A2

Most cases of CAH are caused by biallelic pathogenic CYP21A2 gene variants.^,^, Confirmatory molecular genetic testing of the CYP21A2 locus may be considered when an ACTH stimulation test cannot be performed in an individual suspected of being affected, if adrenocortical profile results are equivocal, or in the context of genetic counseling. Of note, the CYP21A2 locus is very complex; hundreds of variants have been characterized that may lead to different phenotypes and disease severity.^,^, Single gene testing, panel testing, or genomic testing may all be considered, depending on the clinical scenario.

Single gene testing may involve sequencing and deletion/duplication analysis. A potential gene conversion event can occur, resulting in two pathogenic variants on one chromosome; therefore, both parents should undergo genotyping to clarify whether the variants are on the same or opposite chromosomes to avoid errors in interpretation (such as false-positive results).

Multigene panels require careful clinical consideration, as different panels may include different genes (eg, genes not associated with CAH) and may differ in sensitivity. Several techniques may be used, including sequencing, deletion/duplication analysis, focused exome analysis, and others, depending on the panel.

Genomic testing methods such as exome sequencing, genome sequencing, and mitochondrial sequencing may be useful if other techniques fail to yield a diagnostic result in an individual with clinical features of CAH.

Genetic testing in at-risk family members, particularly infants, is recommended if familial variants are known. Genetic testing may also be used for carrier screening, ideally, before pregnancy. Ideally, carrier testing in individuals with a family history of 21-OHP CAH is performed with knowledge of the CYP21A2 variants in the family. CYP21A2 duplications may occur that involve a functional gene coexisting on the same chromosome with a CYP21A2 pathogenic variant, leading to situations in which an individual who is not an obligate carrier is falsely identified as a carrier during carrier screening. Deletion/duplication or haplotype analysis can distinguish this situation from true carrier status.

Other Genes Associated With CAH

Pathogenic variants in several other genes have been associated with rare forms of CAH, which are usually characterized by endocrine abnormalities different from those associated with 21-OHD CAH. These include CYP11A1, CYP11B1, CYP17A1, HSD3B2, POR, and STAR. Molecular genetic testing may be appropriate if familial variants are known.

Monitoring

Therapeutic Monitoring

Laboratory results should not be used as the sole basis for treatment decision-making, which should rely on the complete clinical context.

Monitoring recommendations vary with patient age. In pediatric patients being treated for classic CAH, close monitoring with clinical evaluation is recommended until 3 months of age, and then every 3 months in individuals 3-18 months of age, and every 4-6 months thereafter.^, In adult patients being treated for classic CAH, annual clinical evaluation is recommended and should include a lipid profile.^, Close monitoring is recommended in pregnant individuals.

Monitoring of Glucocorticoid Therapy

Laboratory testing in individuals on glucocorticoid therapy should include serum androstenedione, 17-OHP, and testosterone.^,^, Testosterone does not need to be measured in male neonates. In adult males, follicle-stimulating hormone may also be measured. In adult females, progesterone may be measured in the follicular phase. Samples should all be collected at the same time of day or at the same time relative to corticosteroid dosing (preferably, early morning) because circadian variations and dosage timing affect steroid concentrations.^,^, Reference intervals should be age- and sex-specific and may vary by laboratory.

ACTH measurement is not recommended for monitoring in classic CAH.

Monitoring of Mineralocorticoid and Salt Therapy

Monitoring of mineralocorticoid and salt therapy should include either a direct renin assay or early morning plasma renin activity.^,^, Serum potassium and sodium measurements should also be performed and may be used to adjust the dosing of some treatments.