Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency via the CYP21A2 Gene

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  • Clinical Features and Genetics
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Test Code Test Copy GenesPriceCPT Code Copy CPT Codes
1419 CYP21A2$870 81405 Add to Order
Targeted Testing

For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 18 days.

Clinical Sensitivity

Since our test utilizes an integrative strategy via Sanger sequencing to perform a comprehensive evaluation (see Testing Strategy), the detection rate of pathogenic CYP21A2 variants is expected to be equal to what multi-method strategies yielded, which is approximately 98% (Krone et al. 2000; Stikkelbroeck et al. 2003; Finkielstain et al. 2011; Krone et al. 2013).

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Clinical Features

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) (OMIM #201910) is a disease of cortisol synthesis in the adrenal cortex characterized by cortisol deficiency (with or without aldosterone deficiency) and androgen excess (Speiser et al. 2003; Merke et al. 2005; White et al. 2012). The clinical severity corresponds to the degree of 21-hydroxylase enzyme impairment, which is generally correlated with the genotype of the enzyme’s encoding gene CYP21A2. The classic or severe form consists of two types: the salt-wasting type characterized by concurrent cortisol and aldosterone deficiency; and the simple virilizing type characterized by apparently normal aldosterone biosynthesis. In the classic form, newborn girls have prenatal virilization and genital ambiguity; both boys and girls have postnatal virilization. The mild or nonclassic form may be asymptomatic or associated with signs of androgen excess in childhood or early adulthood. Some 21-OHD patients can also be affected by Ehlers-Danlos syndrome if they are homozygous or heterozygous for a contiguous 30kb deletion disrupting both CYP21A2 and TNXB genes (see Test # 843) (Burch et al. 1997; Schalkwijk et al. 2001; Merke et al. 2013).


CAH due to 21-OHD is an autosomal recessive disorder caused by defects in the enzyme’s encoding gene CYP21A2 (Speiser et al. 2003; Merke et al. 2005; White et al. 2012). It is one of the constitutional genes of the RCCX module (RP-C4-CYP21-TNX) in the human leukocyte antigen histocompatibility complex on chromosome 6. The RCCX module represents a complicated genomic region featured by frequent homologous recombination events due to the existence of highly homologous pseudogenes in tandem (Lee 2005; Chen et al. 2012). CYP21A2 and its pseudogene, CYP21A1P, are approximately 98% identical. The pseudogene specific deleterious variants can be obtained by the functional gene through homologous recombination events, accounting for approximately 95% of documented pathogenic CYP21A2 variants: about 65% are the 12 most common pathogenic variants (P30L, In2G, G110Efs, I172N, I236N, V237E, M239K, V281L, Leu307fs, Q318X, R356W and P453S) transferred via miniconversions; nearly 30% are chimeric CYP21A1P/CYP21A2 genes resulting from complex rearrangements. Each chimeric CYP21A1P/CYP21A2 gene contains one to multiple of the 12 most common pathogenic variants depending on the location of junction site (Chen et al. 2012). The classification (determination of junction site) of chimeric CYP21A1P/CYP21A2 genes is clinically relevant. The remaining 5% of pathogenic CYP21A2 variants are rarer aberrations (including missense and nonsense substitutions, splicing variants, and small indels) that occur in the functional CYP21A2 gene (i.e. not originating from the pseudogene CYP21A1P).

Testing Strategy

We utilize a long range PCR strategy to specifically amplify the CYP21A2 gene next to TNXB at the centromeric tail of the RCCX module. Our test involves bidirectional Sanger DNA sequencing of 10 coding exons of CYP21A2 plus ~10 bp of flanking non-coding DNA on either side of each exon. Our test also can target-sequence distinguishing sites outside of coding regions to evaluate the presence of chimeric CYP21A1P/CYP21A2 genes (alternatively called 30kb deletions in the literature). Genetic testing of CYP21A2 at PreventionGenetics utilizes an integrative strategy via Sanger sequencing to provide comprehensive evaluations of CYP21A2 next to TNXB at the centromeric tail of the RCCX module: (1) analysis of 12 most common pathogenic variants (P30L, In2G, G110Efs, I172N, I236N, V237E, M239K, V281L, Leu307fs, Q318X, R356W and P453S); (2) whole gene (coding and flanking intronic sequence) analysis to search for rarer pathogenic variants; (3) identification of chimeric CYP21A1P/CYP21A2 genes (alternatively called 30kb deletions in the literature); (4) further confirmation and classification (determination of junction site) of chimeric CYP21A1P/CYP21A2 genes with assistance of parental testing results (Chen et al. 2012). It should be noted that test results can be complicated by the genomic complexity at the RCCX region and family studies are often required for an informative interpretation of test results. LIMITATIONS OF THIS TEST: A duplicated CYP21A2 gene or a CYP21A2-like gene next to the pseudogene TNXA at the middle of the RCCX module CANNOT be detected via the current strategy (Wedell et al. 1994; Koppens et al. 2002; Parajes et al. 2008; Kleinle et al. 2011; Tsai et al. 2011). Therefore, test results via the current strategy should always be interpreted in context of clinical findings, family history and other laboratory data.

Indications for Test

Candidates for this test are patients with CAH due to 21-OHD. This test is also for a patient who is positive for the 120bp deletion crossing exon 35 and intron 35 of the TNXB gene (Test # 843) and thus needs testing of CYP21A2 to evaluate for the common contiguous 30kb deletion disrupting both CYP21A2 and TNXB genes (Merke et al. 2013). Testing is also indicated for family members of patients who have known pathogenic CYP21A2 variants. PARENTAL TESTING IS PARTICULARLY REQUIRED: (1) to determine the phase of pathogenic variants found in the proband due to the genomic complexity at this region; (2) for confirmation and classification (determination of junction site) of chimeric CYP21A1P/CYP21A2 genes (Chen et al. 2012).


Official Gene Symbol OMIM ID
CYP21A2 613815
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

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Genetic Counselors
  • Burch GH, Gong Y, Liu W, Dettman RW, Curry CJ, Smith L, Miller WL, Bristow J. 1997. Tenascin-X deficiency is associated with Ehlers-Danlos syndrome. Nat. Genet. 17: 104–108. PubMed ID: 9288108
  • Chen W, Xu Z, Sullivan A, Finkielstain GP, Ryzin C Van, Merke DP, McDonnell NB. 2012. Junction site analysis of chimeric CYP21A1P/CYP21A2 genes in 21-hydroxylase deficiency. Clin. Chem. 58: 421–430. PubMed ID: 22156666
  • Finkielstain GP, Chen W, Mehta SP, Fujimura FK, Hanna RM, Ryzin C Van, McDonnell NB, Merke DP. 2011. Comprehensive genetic analysis of 182 unrelated families with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J. Clin. Endocrinol. Metab. 96: E161–172. PubMed ID: 20926536
  • Kleinle S, Lang R, Fischer GF, Vierhapper H, Waldhauser F, Födinger M, Baumgartner-Parzer SM. 2009. Duplications of the functional CYP21A2 gene are primarily restricted to Q318X alleles: evidence for a founder effect. J. Clin. Endocrinol. Metab. 94: 3954–3958. PubMed ID: 19773403
  • Koppens PFJ, Hoogenboezem T, Degenhart HJ. 2002. Duplication of the CYP21A2 gene complicates mutation analysis of steroid 21-hydroxylase deficiency: characteristics of three unusual haplotypes. Hum. Genet. 111: 405–410. PubMed ID: 12384784
  • Krone N, Braun A, Roscher AA, Knorr D, Schwarz HP. 2000. Predicting phenotype in steroid 21-hydroxylase deficiency? Comprehensive genotyping in 155 unrelated, well defined patients from southern Germany. J. Clin. Endocrinol. Metab. 85: 1059–1065.  PubMed ID: 10720040
  • Krone N, Rose IT, Willis DS, Hodson J, Wild SH, Doherty EJ, Hahner S, Parajes S, Stimson RH, Han TS, Carroll PV, Conway GS, et al. 2013. Genotype-phenotype correlation in 153 adult patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency: analysis of the United Kingdom Congenital adrenal Hyperplasia Adult Study Executive (CaHASE) cohort. J. Clin. Endocrinol. Metab. 98: E346–354. PubMed ID: 23337727
  • Lee H-H. 2005. Chimeric CYP21P/CYP21 and TNXA/TNXB genes in the RCCX module. Mol. Genet. Metab. 84: 4-8. PubMed ID: 15639189
  • Merke DP, Bornstein SR. 2005. Congenital adrenal hyperplasia. Lancet 365: 2125–2136. PubMed ID: 15964450
  • Merke DP, Chen W, Morissette R, Xu Z, Ryzin C Van, Sachdev V, Hannoush H, Shanbhag SM, Acevedo AT, Nishitani M, Arai AE, McDonnell NB. 2013. Tenascin-X haploinsufficiency associated with Ehlers-Danlos syndrome in patients with congenital adrenal hyperplasia. J. Clin. Endocrinol. Metab. 98: E379–387. PubMed ID: 23284009
  • Parajes S, Quinteiro C, Domínguez F, Loidi L. 2008. High frequency of copy number variations and sequence variants at CYP21A2 locus: implication for the genetic diagnosis of 21-hydroxylase deficiency. PLoS ONE 3: e2138. PubMed ID: 18478071
  • Schalkwijk J, Zweers MC, Steijlen PM, Dean WB, Taylor G, Vlijmen IM van, Haren B van, Miller WL, Bristow J. 2001. A recessive form of the Ehlers-Danlos syndrome caused by tenascin-X deficiency. N. Engl. J. Med. 345: 1167–1175. PubMed ID: 11642233
  • Speiser PW, White PC. 2003. Congenital adrenal hyperplasia. N. Engl. J. Med. 349: 776–788. PubMed ID: 12930931
  • Stikkelbroeck NMML, Hoefsloot LH, Wijs IJ de, Otten BJ, Hermus ARMM, Sistermans EA. 2003. CYP21 gene mutation analysis in 198 patients with 21-hydroxylase deficiency in The Netherlands: six novel mutations and a specific cluster of four mutations. J. Clin. Endocrinol. Metab. 88: 3852–3859. PubMed ID: 12915679
  • Tsai L-P, Cheng C-F, Chuang S-H, Lee H-H. 2011. Analysis of the CYP21A1P pseudogene: indication of mutational diversity and CYP21A2-like and duplicated CYP21A2 genes. Anal. Biochem. 413: 133–141. PubMed ID: 21324303
  • Wedell A, Stengler B, Luthman H. 1994. Characterization of mutations on the rare duplicated C4/CYP21 haplotype in steroid 21-hydroxylase deficiency. Hum. Genet. 94: 50–54. PubMed ID: 8034294
  • White PC, Bachega TASS. 2012. Congenital adrenal hyperplasia due to 21 hydroxylase deficiency: from birth to adulthood. Semin. Reprod. Med. 30: 400–409. PubMed ID: 23044877
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Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 10 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of February 2018, we compared 26.8 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 10 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

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  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.


(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
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(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.


(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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