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SALSA MLPA Probemix 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, 4 probes for CYP21A1P, 6 probes for TNXB and 1 probe for ATF6B.

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

Application: congenital adrenal hyperplasia (CAH).

CE-marked for in vitro diagnostic (IVD) use.

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Intended purpose

The SALSA MLPA Probemix P050 CAH is an in vitro diagnostic (IVD) or research use only (RUO) semi-quantitative assay for the detection of large deletions and large gene conversions in the CYP21A2 gene and its surrounding region located on chromosome 6p21.3. 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. 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. CAH affects approximately 1:5,000 births, and among the general population the carrier frequency is estimated at 1:35. In ~95% of cases, CAH is caused by deficiency of the steroid 21-hydroxylating enzyme encoded by the CYP21A2 gene. The inactive pseudogene CYP21A1P is located closely upstream of CYP21A2. Both the gene and pseudogene have 10 exons and span ~3.2 kilobases (kb). The great majority of the CYP21A2 mutant alleles arise through recombination between CYP21A2 and CYP21A1P. In most populations ~65-75% of the pathogenic CYP21A2 mutations are point mutations or small indels, the far majority (>90%) of which are microconversions where the CYP21A2 gene has obtained a single small inactivating mutation from the CYP21A1P pseudogene. The frequency of large deletions and large conversions ranges from ~10 to 35%, two-thirds of which originate from unequal meiotic cross-overs resulting in intergenic deletions of 30 kb and the formation of a CYP21A1P-CYP21A2 chimeric non-functional gene.

Other genes located in 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. Some large rearrangements in this region affect both CYP21A2 and TNXB. Orientation of these genes is as displayed in Figure 1. When both functional alleles of CYP21A2 and both alleles of TNXB are lost this leads to the contiguous gene syndrome CAH-X, where the patient develops symptoms of classic-like Ehlers-Danlos syndrome (a disorder affecting the connective tissues) in combination with CAH. 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.

Regulatory status

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

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.

List prices

Product

Item no.
Description
Technology
Price
P050-025R
SALSA MLPA Probemix P050 CAH – 25 rxn
MLPA
€ 281.00
P050-050R
SALSA MLPA Probemix P050 CAH – 50 rxn
MLPA
€ 550.00
P050-100R
SALSA MLPA Probemix P050 CAH – 100 rxn
MLPA
€ 1075.00

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
€ 341.00
EK1-Cy5
SALSA MLPA Reagent Kit – 100 rxn – Cy5 (6 vials)
MLPA MS-MLPA
€ 341.00
EK5-FAM
SALSA MLPA Reagent Kit – 500 rxn – FAM (5×6 vials)
MLPA MS-MLPA
€ 1571.00
EK5-Cy5
SALSA MLPA Reagent Kit – 500 rxn – Cy5 (5×6 vials)
MLPA MS-MLPA
€ 1571.00
EK20-FAM
SALSA MLPA Reagent Kit – 2000 rxn – FAM (5×6 vials)
MLPA MS-MLPA
€ 6037.00

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
€ 23.70

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 have been tested with the current (D1) version of this product and have been shown to produce useful results.

  • Coriell NA12217: Heterozygous deletion affecting the probes for CYP21A2 exon 1-4.
    This sample has a typical, more complex, genotype: a large deletion/conversion spanning exon 1-3 on one allele, and a micro-conversion of the I173N locus in exon 4 on the second allele. Note that this and other complex genotypes cannot be determined with MLPA alone and require allele/locus-specific long range PCR amplification, sequencing data and often parental evaluation for full delineation of the genotype.
  • Coriell NA14734: Homozygous deletion affecting the probes for CYP21A2.

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

  • 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.
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CE

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.