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Challenges of prenatal diagnosis of 21-hydroxylase deficiency in the era of expanded carrier screening

The current era of expanded carrier screening has been successful in identifying couples at increased risk for having a child with certain genetic conditions, but also poses genetic counseling and testing challenges. One example is 21-hydroxylase deficiency (21-OHD), which is caused by genetic variants in the CYP21A2 gene. Twenty-one OHD has 2 clinical forms, classic and non-classic; the classic form frequently results in ambiguous genitalia (virilization) and/or life-threatening salt-wasting episodes while the non-classic form is much less severe, but more common. Traditionally, prenatal diagnosis of 21-OHD was offered only when there was known family history of an affected child or a prenatal ultrasound finding of ambiguous genitalia, both of which are associated with the severe, classic form of 21-OHD. Some genetic laboratories offering expanded carrier screening include CYP21A2 analysis, which has identified more at-risk couples who are then offered diagnostic fetal testing. However, what is not always clear to the provider is that certain variants in the CYP21A2 gene are often specific to each clinical type and the couple may only be at risk for having a child with the non-classic form of 21-OHD rather than the classic form. It is especially important for providers to be aware of the distinction between these 2 clinical forms and the associated CYP21A2 variants so that patients have the most accurate information and are appropriately counseled about which clinical form their children are at risk for.

What is 21-OHD?
Twenty-one hydroxylase deficiency (21-OHD) is the most common form of congenital adrenal hyperplasia (CAH), a family of genetic conditions associated with an increased production of androgens, is inherited in an autosomal recessive pattern. The gene associated with 21-OHD, CYP21A2, has 7 common pathogenic variants, some of which are associated with a specific clinical type. As mentioned above, there are 2 clinical forms of 21-OHD, classic and non-classic.  The form is determined by the amount of the 21-hydroxylase enzyme present; lower enzyme levels result in classic/severe form, whereas higher enzyme levels are associated with non-classic/mild form. Clinical features of each form are below:

Classic form:

  • newborn onset
  • 75% of cases have salt-wasting, which can be life-threatening in the newborn period if not identified and treated promptly
  • 25% of cases have simple-virilizing effects, which only affects females.

Non-classic form:

  • later age of onset
  • precocious (early) puberty, accelerated growth with advanced bone age resulting in reduced adult height, and acne.
  • females have an increased risk of infertility1

Is 21-OHD common?
Yes, in terms of genetic disorders. The classic form occurs in approximately 1 in 15,000 live births 2. Non-classic CAH has a much higher incidence, approximately 1 in 100, with the frequency being higher in individuals of Ashkenazi Jewish ancestry (1 in 27). 

How is 21-OHD identified?
The clinical diagnosis of classic CAH due to 21-OHD is typically made by a combination of clinical and biochemical findings, which include: ambiguous genitalia in a female fetus/infant, either by prenatal ultrasound finding or in the newborn period; abnormal newborn screening results; and/or abnormal biochemical testing (see below). 21-OHD is included in many state newborn screening programs. If a newborn screen is positive for CAH, biochemical testing is typically used to confirm the diagnosis.  Biochemical findings that confirm a clinical diagnosis in both classic and non-classic forms include elevated 17-hydroxyprogesterone levels, increased adrenal androgens, and/or plasma renin activity. It is important to note that the findings of ambiguous genitalia and salt-wasting crises are seen only in the classic form of 21-OHD due to extremely low enzyme activity.

Non-classic 21-OHD is typically not identified in infancy, but when a child goes through early puberty or when a female has issues with conceiving2.   The diagnosis can be confirmed with biochemical testing. In some cases, adults are found to have 21-OHD after having expanded carrier screening as a part of their routine prenatal care.

What does the specific variant tell us about clinical presentation?
It is well established that some of the common variants in the CYP21A2 gene are typically associated with the milder, non-classic form of the disease, such as the Val281Leu variant (aka V281L/c.844G>T).  Affected individuals who have at least 1 copy of the V281L variant are expected to be affected with the milder, non-classic form of CAH.  The correlation between genotype and phenotype may be less certain for other known genetic variants.

Why is 21-OHD testing a laboratory challenge?
Twenty one-OHD poses a significant challenge for genetic laboratories because of the complexity of the gene itself along with presence of a pseudogene, CYP21A1, that lies next to and is almost identical with the CYP21A2 gene, both of which can complicate result interpretation. It is important for molecular laboratories to be able to differentiate between the CYP21A1 gene and the pseudogene, such as offered by Quest Diagnostics, to ensure accurate interpretation.

How can this information help providers?
Expanded carrier screening is now making it possible to identify carriers for both classic and non-classic forms of 21-OHD allowing at-risk couples to make decisions earlier in pregnancy or in some cases, prior to pregnancy.  Healthcare providers should discuss with at-risk couples the most up-to-date information regarding genotype and phenotype associations, and the range of symptoms for an affected child.  It is important for couples to understand the differences between the classic (severe) and non-classic (mild) forms so the couple can make the most informed decision.  

Quest Diagnostics offers a CYP21A2 common variant panel (90 to 95% detection rate) as a standalone carrier screen or for diagnostic testing. In addition, CYP21A2 gene sequencing (>95% detections rate) is available; both tests are performed using next generation sequencing (NGS). As our QHerit ™Expanded Carrier Screen was developed based on guidelines and recommendations by the American Congress of Obstetrics and Gynecology (ACOG) [4], 21-OHD carrier screening is not currently included in this test. Quest Diagnostics offers fetal testing for CYP21A2 when parental variants are known; please consult with a Quest Diagnostic Genetic Counselor at 866-436-3463 prior to submission of any fetal sample.  


  1. White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-hydroylase deficiency. Endocrine Reviews. 2000:21(3); 245-291.
  2. Nimkarn S, Gangishetti PK, Yau M, et al. 21-Hydroxylase-Deficient Congenital Adrenal Hyperplasia. Updated Feb 4, 2016 . In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mirzaa G, Amemiya A, editors. GeneReviews® [Internet].: University of Washington, Seattle; 1993–2021.
  3. Krone N, Braun A, Roscher AA, Knorr D, et al. Predicting phenotype in steroid 21-hydroxylase deficiency? Comprehensive genotyping in 155 unrelated, well defined patients from southern Germany. J Clin Endocrinol Metab. 2000;85:1059–1065. doi:10.1210\jcem.85.3.6441
  4. Britton Rink, Stephanie Romero, Joseph R. Briggo Jr, Devereux N. Saller Jr, and Rose Giardine. Carrier Screening for Genetic Conditions. The American College of Obstetricians and Gynecologists. Committee Opinion Number 691. March 2017