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P050 CAH

SALSA MLPA Probemix P050 CAH detects large deletions and large gene conversions in the CYP21A2 gene and its surrounding region on chromosome 6p21.3.

Specifications

Contents: 30 MLPA probes, including 8 probes for CYP21A2 and 4 probes for CYP21A1P.

Tissue: genomic DNA isolated from human peripheral whole blood or specified prenatal samples (see Intended Purpose).

Application: congenital adrenal hyperplasia (CAH).

IVDR certified for in vitro diagnostic (IVD) use.

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This product has recently been CE-marked for in vitro diagnostic (IVD) use under the In Vitro Diagnostic Regulation (IVDR; EU 2017/746), which replaces the former CE-marking under the IVD Directive (IVDD; Directive 98/79/EC). This update was accompanied by a change in the intended purpose and a change in format of the product description. Some information can now be found in a different location (more information).

Intended purpose

The SALSA MLPA Probemix P050 CAH is an in vitro diagnostic (IVD) or research use only (RUO) semi-quantitative manual assay for the detection of large deletions in the CYP21A2 gene. This probemix can also detect copy number variations (CNVs) in the surrounding region to aid in interpretation. P050 CAH is intended to confirm a potential cause for and clinical diagnosis of Congenital Adrenal Hyperplasia (CAH) and for molecular genetic testing of at-risk family members. In addition, this assay can be used for carrier testing. This probemix is for use with genomic DNA isolated from human peripheral whole blood specimens or prenatal DNA isolated from (un)cultured amniotic fluid obtained in week 16 of the pregnancy or later and free from blood contamination, (un)cultured chorionic villi free from maternal contamination, or fetal blood.

For the full intended purpose, see the product description.

Clinical background

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder, which results from a deficiency in one of the enzymes involved in cortisol biosynthesis. In ~95% of cases, CAH is caused by deficiency of the steroid 21-hydroxylating enzyme encoded by the CYP21A2 gene (also known as 21-OHD CAH). 21-OHD CAH affects approximately 1:15,000 births, and among the general population the carrier frequency is estimated at 1:50 to 1:71 (Liu et al. 2022).

The inactive pseudogene CYP21A1P is located closely upstream of CYP21A2. Both the gene and pseudogene have 10 exons and span ~3.2 kilobases (kb). CYP21A2 and CYP21A1P share 98% genetic identity between exons and 96% between introns (Xia et al. 2024). The great majority of the CYP21A2 mutant alleles arise through recombination between CYP21A2 and CYP21A1P due to the high homology. Microconversions between CYP21A2 and CYP21A1P account for approximately 70-80% of 21-OHD CAH cases, while the remaining 20-30% are large rearrangements encompassing large deletions in CYP21A2, large gene conversions between CYP21A2 and CYP21A1P, and CYP21A1P-CYP21A2 chimeric genes (Fernandez et al. 2020, Liu et al. 2022, Xia et al. 2022, Xia et al. 2024, Yuan et al. 2024). Most large rearrangements originate from unequal meiotic cross-overs resulting in intergenic deletions of 30 kb and the formation of a CYP21A1P-CYP21A2 chimeric non-functional gene. Most 21-OHD CAH patients are compound heterozygotes. Each unique combination of alleles results in a different residual enzyme activity, which in turn determines the clinical form of CAH: salt-wasting (SW), simple virilising (SV), nonclassic late onset (NC; attenuated; acquired) and cryptic.

Other genes located on this chromosomal region, also referred to as RCCX module, are the closely related complement genes C4A and C4B, and the TNXB gene and its pseudogene TNXA. Orientation of these genes is as displayed in Figure 1. The TNXB gene spans ~68 kb of genomic DNA and has 44 exons. Many CYP21A2-CYP21A1P gene conversions extend into the TNXB gene and inactivate that copy of the gene. A subset of CAH patients also have genetic deletions of the TNXB gene. This contiguous gene syndrome is referred to as CAH-X. Biallelic mutations in TNXB that do not affect CYP21A2 gene function are associated with the autosomal recessive disorder classic-like Ehlers-Danlos syndrome. Note that for the detection of deletions or duplications in the TNXB gene the SALSA MLPA Probemix P155 EDS is recommended.

Figure 1. A schematic representation of the 151 kb chromosomal region with the duplicated gene cluster as it is present in the reference genome, containing the TNXA & CYP21P pseudogenes (light) and the TNXB & CYP21A2 genes (dark). Arrows indicate direction of transcription. Note that the 3'-ends of CYP21A2 and TNXB overlap. The figure represents genomic position chr6:31,946-32,097 of the GRCh37/hg19 assembly and is based on Gitelman et al. 1992.

CYP21A2 mutation database[

The CYP21A2 page on the LOVD can be found here. We strongly encourage users to deposit positive results in the CYP21A2 LOVD. Recommendations for the nomenclature to describe deletions/duplications of one or more exons can be found on http://varnomen.hgvs.org/.

Reference sample selection

For correct data analysis using P050 CAH, it is critical to include suitable reference samples in each experiment. Such reference samples should be identified before testing patient samples. Suitable reference samples have one copy of each wildtype allele at the I2G locus, i.e. one I2G-C allele and one I2G-A allele, and a normal copy number with a standard deviation ≤0.10 for all other autosomal sequences detected by P050 CAH. SALSA Reference Selection DNA SD039 should be used for initial selection of suitable reference samples from your own collection. SALSA Reference Selection DNA SD039 should never be used as a reference sample.

In our experience, approximately 1 in 5 (~20%) DNA samples from the general European population are suitable for use as reference sample. By testing 20-25 different DNA samples from unrelated healthy individuals with P050 CAH, and including three reactions of SALSA Reference Selection SD039, there is a good chance of finding at least three different suitable reference samples. Suitable reference samples provide results aligning with SALSA Reference Selection DNA SD039 for each probe, including the I2G-C and I2G-A probes.

For examples on selecting suitable reference samples using SALSA Reference Selection SD039, please consult the Appendix of the P050 CAH Product Description.

Regulatory status

SALSA MLPA Probemix P050 CAH is CE-marked under the IVDR for in vitro diagnostic (IVD) use in Europe.

This assay is for research use only (RUO) in all other territories.

SALSA Sample DNA for this product

SALSA Reference Selection DNA SD039 can be used to aid in the selection of suitable reference samples for the P050 CAH probemix. Reference Selection DNA can only be used in initial experiments on DNA samples from healthy individuals from your sample collection with the intention to identify suitable reference samples. SD039 cannot be used as a reference sample in subsequent experiments.

A vial of SALSA Reference Selection DNA SD039 is included with every order of the P050 CAH probemix, but it is possible to order additional vials separately.

For more information, see the product description.

Product documentation

Version D1

Other versions

Contact us for earlier versions.

Translations and Summary of Safety and Performance

Translations of the product description in selected European languages are available upon request. Please contact us or one of our local sales partners. Translations of the MLPA General Protocol in selected languages are available here.

The Summary of Safety and Performance (SSP) is also available upon request.

List prices

Product

Item no.
Description
Technology
Price
P050-025R
SALSA MLPA Probemix P050 CAH – 25 rxn
MLPA
€ 296.00
P050-050R
SALSA MLPA Probemix P050 CAH – 50 rxn
MLPA
€ 580.00
P050-100R
SALSA MLPA Probemix P050 CAH – 100 rxn
MLPA
€ 1135.00

Required reagents

A general SALSA MLPA Reagent Kit is required for MLPA experiments (to be ordered separately).

Item no.
Description
Technology
Price
EK1-FAM
SALSA MLPA Reagent Kit – 100 rxn – FAM (6 vials)
MLPA MS-MLPA
€ 361.00
EK1-CY5
SALSA MLPA Reagent Kit – 100 rxn – Cy5 (6 vials)
MLPA MS-MLPA
€ 361.00
EK5-FAM
SALSA MLPA Reagent Kit – 500 rxn – FAM (5×6 vials)
MLPA MS-MLPA
€ 1658.00
EK5-CY5
SALSA MLPA Reagent Kit – 500 rxn – Cy5 (5×6 vials)
MLPA MS-MLPA
€ 1658.00
EK20-FAM
SALSA MLPA Reagent Kit – 2000 rxn – FAM (5×6 vials)
MLPA MS-MLPA
€ 6373.00

Sample DNAs (included)

A vial is included with every order of this probemix, but additional vials can also be purchased separately.

Item no.
Description
Technology
Price
SD039
SALSA Reference Selection DNA SD039 – 6 rxn
MLPA
€ 25.00

Price details & ordering

The prices above are list prices for direct orders from MRC Holland. Contact us for a quote that takes discounts and additional costs (such as shipping costs) into account. Different prices apply for orders through one of our sales partners; contact your local supplier for a quote.

More information about ordering & prices

Positive samples

Inclusion of a positive sample is usually not required, but can be useful for the analysis of your experiments. MRC Holland has very limited access to positive samples and cannot supply such samples. We recommend using positive samples from your own collection. Alternatively, you can use positive samples from an online biorepository, such as the Coriell Institute.

The commercially available positive samples below can be used with the current (D1) version of this product.

Resources

General MLPA resources

MLPA education

Publications

Selected publications using P050 CAH

  • Baumgartner-Parzer S et al. (2020). EMQN best practice guidelines for molecular genetic testing and reporting of 21-hydroxylase deficiency. Eur J Hum Genet. 28:1341-67.
  • Gangodkar P et al. (2021). Clinical application of a novel next generation sequencing assay for CYP21A2 gene in 310 cases of 21- hydroxylase congenital adrenal hyperplasia from India. Endocrine. 71:189-98.
  • Gao Y et al. (2020). The Prevalence of the Chimeric TNXA/TNXB Gene and Clinical Symptoms of Ehlers-Danlos Syndrome with 21-Hydroxylase Deficiency. J Clin Endocrinol Metab. 105:2288-99.
  • Meinel J et al. (2021). Establishment of Clinical and Lab Algorithms for the Identification of Carriers of Mutations in CYP21A2 - A Study of 365 Children and Adolescents. Exp Clin Endocrinol Diabetes. 129:492-9.
  • Navardauskaité R et al. (2021). Impact of Newborn Screening on Clinical Presentation of Congenital Adrenal Hyperplasia. Medicina (Kaunas). 57:1035.
  • Tolba A et al. (2022). Copy Number Variations in Genetic Diagnosis of Congenital Adrenal Hyperplasia Children. Front Genet. 13:785570.
  • Turan I et al. (2020). 21-Hydroxylase deficiency: Mutational spectrum and Genotype-Phenotype relations analyses by next-generation sequencing and multiplex ligation-dependent probe amplification. Eur J Med Genet. 63:103782.

References

  • Fernández CS et al. (2020). Genetic characterization of a large cohort of Argentine 21-hydroxylase Deficiency. Clin Endocrinol (Oxf). 93:19-27.
  • Gitelman SE et al. (1992). Mechanism and consequences of the duplication of the human C4/P450c21/gene X locus. Mol Cell Biol. 12:2124-34.
  • Liu Y et al. (2022). Comprehensive Analysis of Congenital Adrenal Hyperplasia Using Long-Read Sequencing. Clin Chem. 68:927-39.
  • Xia Y et al. (2022). Genetic analysis and novel variation identification in Chinese patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Steroid Biochem Mol Biol. 222:106156.
  • Xia Y et al. (2024). Novel rapid molecular diagnosis methods for comprehensive genetic analysis of 21-hydroxylase deficiency. Orphanet J Rare Dis. 19:397.
  • Yuan D et al. (2024). Improved Genetic Characterization of Congenital Adrenal Hyperplasia by Long-Read Sequencing Compared with Multiplex Ligation-Dependent Probe Amplification Plus Sanger Sequencing. J Mol Diagn. 26:770-80.
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CE2797

CE-marked products are for In Vitro Diagnostic (IVD) use only in EU (candidate) member states and members of the European Free Trade Association (EFTA), and the UK.