Intended purpose
The SALSA MLPA Probemix P050 CAH is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² 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.

Copy number variations (CNVs) detected with P050 CAH should always be confirmed with a different technique. In particular, CNVs detected by only a single probe always require confirmation by another method. Reciprocal exchanges between CYP21A2 and its pseudogene CYP21A1P will not be detected. It is therefore recommended to always use this assay in combination with sequence analysis.

Assay results are intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, including confirmation by alternative methods, parental evaluation, clinical genetic evaluation, and counselling, as appropriate. The results of this test should be interpreted by a clinical molecular geneticist or equivalent.

This device is not intended to be used for standalone diagnostic purposes, pre-implantation testing, population screening, or for the detection of, or screening for, acquired or somatic genetic aberrations.

¹Please note that this probemix is for in vitro diagnostic (IVD) use in the countries specified at the end of this product description. In all other countries, the product is for research use only (RUO).
²To be used in combination with a SALSA MLPA Reagent Kit, Reference Selection DNA SD039 and Coffalyser.Net analysis software.

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.

Probemix content
The SALSA MLPA Probemix P050-D1 CAH contains 30 MLPA probes with amplification products between 130 and 382 nt. This includes eight probes for the CYP21A2 gene and four probes for the CYP21A1P pseudogene. Furthermore the probemix contains six probes for the TNXB gene and one for the ATF6B gene in order to further delineate CYP21A2 gene deletions. In addition, eleven reference probes are included that detect autosomal chromosomal locations. Complete probe sequences and the identity of the genes detected by the reference probes are available online (www.mrcholland.com).

This probemix contains ten quality control fragments generating amplification products between 64 and 118 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and two chromosome Y-specific fragments. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at www.mrcholland.com.

SALSA Reference Selection DNA SD039
As described above the selection of suitable reference DNA samples for P050 CAH is complicated. To facilitate this selection from your own sample collection, a reference selection DNA sample (catalogue number SD039) is provided with this probemix from MRC Holland. Reference Selection DNA SD039 should only be used for initial experiments on DNA samples from healthy individuals with the intention to identify suitable reference samples and it should never be used as a reference sample in subsequent experiments. For further details, consult the Reference Selection DNA SD039 product description, available online: www.mrcholland.com.

Sample DNA
Sample DNA developed for this product:

• SD039 Reference Selection DNA - included with this probemix.