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HelpOrder Code CYPZ 21-Hydroxylase Gene, CYP21A2, Full Gene Analysis, Varies
Useful For
Carrier screening and diagnosis of 21-hydroxylase deficient congenital adrenal hyperplasia (CAH) in individuals with a personal or family history of 21-hydroxylase deficiency, or as follow-up to positive CAH newborn screens and/or measurement of basal and adrenocorticotropic hormone- 1-24 stimulated 17-hydroxyprogesterone, androstenedione, and other adrenal steroid levels
May identify CYP21A2 variants in individuals with a suspected diagnosis of 21-hydroxylase deficient CAH when a common variant panel is negative or only identifies 1 variant
May identify the CH-1 TNXA::TNXB hybrid associated with CAH-X. Note that the CH-2 and CH-3 TNXA::TNXB hybrids will not be detected by current methodologies.
Evaluation of 21-hydroxlase deficiency in prenatal cases with suspected differences of sex development (such as clitoromegaly) detected by ultrasound, particularly when the fetus is confirmed XX female by chromosome analysis
Due to the complexity of the CYP21A2 locus, site specific testing for known/familial CYP21A2 variants is not offered.
Genetics Test Information
This test includes Sanger sequencing, multiplex ligation-dependent probe amplification, and a droplet digital polymerase chain reaction assay (as needed) to evaluate the CYP21A2 gene for carrier screening and diagnosis of 21-hydroxylase deficient congenital adrenal hyperplasia (CAH).
Reflex Tests
| Test ID | Reporting Name | Available Separately | Always Performed |
|---|---|---|---|
| MATCC | Maternal Cell Contamination, B | Yes | No |
| CULFB | Fibroblast Culture for Genetic Test | Yes | No |
| CULAF | Amniotic Fluid Culture/Genetic Test | Yes | No |
Testing Algorithm
For prenatal specimens only:
If amniotic fluid (nonconfluent cultured cells) is received, amniotic fluid culture will be added and performed at an additional charge.
If chorionic villus specimen (nonconfluent cultured cells) is received, fibroblast culture will be added and performed at an additional charge.
For any prenatal specimen that is received, maternal cell contamination studies will be added and performed at an additional charge.
Special Instructions
Method Name
Polymerase Chain Reaction (PCR) Amplification followed by DNA Sequence Analysis and Gene Dosage Analysis by Multiplex Ligation-Dependent Probe Amplification (MLPA) and Droplet Digital PCR (ddPCR)
Reporting Name
CYP21A2 Gene, Full Gene AnalysisSpecimen Type
VariesOrdering Guidance
This test is a molecular analysis of the CYP21A2 gene and does not include biochemical analysis of steroids. For biochemical analysis for congenital adrenal hyperplasia (CAH), which includes cortisol, androstenedione, and 17-hydroxyprogesterone, see CAH21 / Congenital Adrenal Hyperplasia (CAH) Profile for 21-Hydroxylase Deficiency, Serum.
Additional Testing Requirements
All prenatal specimens must be accompanied by a maternal blood specimen; order MATCC / Maternal Cell Contamination, Molecular Analysis, Varies on the maternal specimen.
Necessary Information
CYP21A2 Gene Testing for Congenital Adrenal Hyperplasia Patient Information (T663) is strongly recommended, but not required, to be filled out and sent with the specimen. This information aids in providing a more thorough interpretation of test results. Ordering healthcare professionals are strongly encouraged to complete the form and send it with the specimen.
Specimen Required
Patient Preparation: A previous bone marrow transplant from an allogenic donor will interfere with testing. For information about testing patients who have received a bone marrow transplant, call 800-533-1710.
Submit only 1 of the following specimens:
Specimen Type: Whole blood
Container/Tube:
Preferred: Lavender top (EDTA) or yellow top (ACD)
Acceptable: None
Specimen Volume: 3 mL
Collection Instructions:
1. Invert several times to mix blood.
2. Send whole blood specimen in original tube. Do not aliquot.
Specimen Stability Information: Ambient 4 days (preferred)/Refrigerated 4 days/Frozen 4 days
Additional Information:
1. Specimens are preferred to be received within 4 days of collection. Extraction will be attempted for specimens received after 4 days, and DNA yield will be evaluated to determine if testing may proceed.
2. To ensure minimum volume and concentration of DNA is met, the requested volume must be submitted. Testing may be canceled if DNA requirements are inadequate.
Specimen Type: Cord blood
Container/Tube:
Preferred: Lavender top (EDTA) or yellow top (ACD)
Acceptable: None
Specimen Volume: 3 mL
Collection Instructions:
1. Invert several times to mix blood.
2. Send specimen in original tube. Do not aliquot.
Specimen Stability Information: Ambient (preferred) 4 days/Refrigerated 4 days/Frozen, 4 days
Additional Information:
1. Specimens are preferred to be received within 4 days of collection. Extraction will be attempted for specimens received after 4 days, and DNA yield will be evaluated to determine if testing may proceed.
2. To ensure minimum volume and concentration of DNA is met, the preferred volume of blood must be submitted. Testing may be canceled if DNA requirements are inadequate.
3. While a properly collected cord blood sample may not be at risk for maternal cell contamination, unanticipated complications may occur during collection. Therefore, maternal cell contamination studies are recommended to ensure the test results reflect that of the patient tested and are available at an additional charge.
4. Order MATCC / Maternal Cell Contamination, Molecular Analysis, Varies on the maternal specimen.
Specimen Type: Extracted DNA
Container/Tube:
Preferred: Screw Cap Micro Tube, 2 mL with skirted conical base
Acceptable: Matrix tube, 1 mL
Collection Instructions:
1. The preferred volume is at least 100 mcL at a concentration of 75 ng/mcL.
2. Include concentration and volume on tube.
Specimen Stability Information: Frozen (preferred) 1 year/Ambient/Refrigerated
Additional Information: DNA must be extracted in a CLIA-certified laboratory or equivalent and must be extracted from a specimen type listed as acceptable for this test (including applicable anticoagulants). Our laboratory has experience with Chemagic, Puregene, Autopure, MagnaPure, and EZ1 extraction platforms and cannot guarantee that all extraction methods are compatible with this test. If testing fails, one repeat will be attempted, and if unsuccessful, the test will be reported as failed and a charge will be applied. If applicable, specific gene regions that were unable to be interrogated due to DNA quality will be noted in the report.
Prenatal Specimens
Due to its complexity, consultation with the laboratory is required for all prenatal testing; call 800-533-1710 to speak to a genetic counselor.
Preferred:
Specimen Type: Amniotic fluid
Container/Tube: Amniotic fluid container
Specimen Volume: 20 mL
Specimen Stability Information: Refrigerated (preferred) <24 hours/Ambient <24 hours
Additional information:
1. Specimens are preferred to be received within 24 hours of collection. Culture and extraction will be attempted for specimens received after 24 hours and will be evaluated to determine if testing may proceed.
2. A separate culture charge will be assessed under CULAF / Culture for Genetic Testing, Amniotic Fluid. An additional 2 to 3 weeks is required to culture amniotic fluid before genetic testing can occur.
3. All prenatal specimens must be accompanied by a maternal blood specimen; order MATCC / Maternal Cell Contamination, Molecular Analysis, Varies on the maternal specimen.
Specimen Type: Chorionic villi
Container/Tube: 15-mL tube containing 15 mL of transport media
Specimen Volume: 20 mg
Specimen Stability Information: Refrigerated (preferred) <24 hours/Ambient <24 hours
Additional Information:
1. Specimens are preferred to be received within 24 hours of collection. Culture and extraction will be attempted for specimens received after 24 hours and will be evaluated to determine if testing may proceed.
2. A separate culture charge will be assessed under CULFB / Fibroblast Culture for Biochemical or Molecular Testing. An additional 3 to 4 weeks is required to culture fibroblasts before genetic testing can occur.
3. All prenatal specimens must be accompanied by a maternal blood specimen; order MATCC / Maternal Cell Contamination, Molecular Analysis, Varies on the maternal specimen.
Specimen Type: Prenatal cultured fibroblasts (eg, products of conception), amniocytes, or other confluent cultured cells. This does not include cultured chorionic villi.
Container/Tube: T-25 flask
Specimen Volume: 2 Flasks
Collection Instructions: Submit confluent cultured cells from another laboratory.
Specimen Stability Information: Ambient (preferred) <24 hours/Refrigerated <24 hours)
Additional Information:
1. Specimens are preferred to be received within 24 hours of collection. Culture and extraction will be attempted for specimens received after 24 hours and will be evaluated to determine if testing may proceed.
2. A separate culture charge will be assessed under CULAF / Culture for Genetic Testing, Amniotic Fluid or CULFB / Fibroblast Culture for Biochemical or Molecular Testing.
3. All prenatal specimens must be accompanied by a maternal blood specimen; order MATCC / Maternal Cell Contamination, Molecular Analysis, Varies on the maternal specimen.
Specimen Type: Cultured chorionic villi
Container/Tube: T-25 flasks
Specimen Volume: 2 Full flasks
Collection Instructions: Submit confluent cultured cells from another laboratory.
Specimen Stability Information: Ambient (preferred) <24 hours/Refrigerated <24 hours
Additional Information:
1. Specimens are preferred to be received within 24 hours of collection. Culture and/or extraction will be attempted for specimens received after 24 hours and will be evaluated to determine if testing may proceed.
2. A separate culture charge will be assessed under CULFB / Fibroblast Culture for Biochemical or Molecular Testing
3. All prenatal specimens must be accompanied by a maternal blood specimen; order MATCC / Maternal Cell Contamination, Molecular Analysis, Varies on the maternal specimen.
Specimen Minimum Volume
See Specimen Required.
Specimen Stability Information
| Specimen Type | Temperature | Time |
|---|---|---|
| Varies | Varies | |
Reject Due To
All specimens will be evaluated at Mayo Clinic Laboratories for test suitability.Reference Values
An interpretive report will be provided.
Method Description
A combined testing approach involving polymerase chain reaction (PCR) amplification, bi-directional sequence analysis, multiplex ligation-dependent probe amplification (MLPA), and droplet digital (ddPCR), when necessary, is used to identify sequence variants and copy number variation within the CYP21A2 gene (GenBank accession number NM_000500; build GRCh37 [hg19]).
Four sets of PCR primer pairs amplify the CYP21A2 gene, the inactive CYP21A1P pseudogene, and the CYP21A2::CYP21A1P and CYP21A1P::CYP21A2 hybrids to determine the presence or absence of amplification product.
Bi-directional full gene sequence analysis, including a portion of the promoter, 5'-untranslated region, and 3'-untranslated region, is then performed on the CYP21A2 gene and the CYP21A2::CYP21A1P hybrid (if present) to test for the presence of sequence variants. Because the CYP21A1P::CYP21A2 hybrid and the CYP21A1P pseudogene are expected to be inactive, sequencing is not performed unless required for interpretation.
Multiplex ligation-dependent probe amplification is performed to determine the copy number of the 5-prime and 3-prime regions of the CYP21A2 gene and the CYP21A1P pseudogene. In certain cases, a ddPCR assay is used in conjunction with MLPA to determine copy number. Quantification and comparison of results is used to determine the copy number of the CYP21A2 gene, the CYP21A1P pseudogene, and the CYP21A2::CYP21A1P and CYP21A1P::CYP21A2 hybrids.
Correlation of all performed test components is used to determine the CYP21A2 genotype.
This technology cannot always determine the cis/trans status (cis:=same chromosome, trans:=opposite chromosomes) of the identified genes, rearrangements, or variants. Family studies of blood relatives may assist in determination of the cis/trans status.(Cradic KW, Grebe SK. A diagnostic sequencing assay for CYP21 based on promoter activity provides better understanding of gene rearrangements. Abstract. Endocrine Society Annual Meeting, ENDO 2005)
Day(s) Performed
Varies
Performing Laboratory
Mayo Clinic Laboratories in Rochester
CPT Code Information
81405-CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide2) (eg, steroid 21-hydroxylase isoform, congenital adrenal hyperplasia), full gene sequence
81402-CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide2) (eg, congenital adrenal hyperplasia, 21-hydroxylase deficiency), common variants (eg, IVS2-13G, P30L, I172N, exon 6 mutation cluster [I235N, V236E, M238K], V281L, L307FfsX6, Q318X, R356W, P453S, G110VfsX21, 30-kb deletion variant)
88233-Tissue culture, skin or solid tissue biopsy (if appropriate)
88235-Tissue culture for amniotic fluid (if appropriate)
88240-Cryopreservation (if appropriate)
81265-Comparative analysis using Short Tandem Repeat (STR) markers; patient and comparative specimen (eg, pre-transplant recipient and donor germline testing, post-transplant non-hematopoietic recipient germline [eg, buccal swab or other germline tissue sample] and donor testing, twin zygosity testing or maternal cell contamination of fetal cells (if appropriate)
Forms
1. New York Clients-Informed consent is required. Document on the request form or electronic order that a copy is on file. The following documents are available:
-Informed Consent for Genetic Testing (T576)
-Informed Consent for Genetic Testing-Spanish (T826)
2. CYP21A2 Gene Testing for Congenital Adrenal Hyperplasia Patient Information (T663) is recommended.
3. If not ordering electronically, complete, print, and send a Biochemical Genetics Test Request (T798) with the specimen.