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SALSA MLPA Probemix P155 EDS

Vascular Ehlers-Danlos Syndrome (vEDS); and classic-like EDS (clEDS)

Region: COL3A1 2q32.2; TNXB 6p21.32

MLPA | CE
Intended purpose
The SALSA MLPA probemix P155 EDS is an in vitro diagnostic (IVD)1 or a research use only (RUO) semi-quantitive assay2 for the detection of deletions or duplications in the COL3A1 and TNXB genes in genomic DNA isolated from human peripheral blood specimens in order to confirm a potential cause for and clinical diagnosis of vascular Ehlers-Danlos syndrome (vEDS) and classic-like Ehlers-Danlos syndrome (clEDS), respectively. This product can also be used for molecular genetic testing of at-risk family members. Of note, the majority of copy number variations of TNXB are fusions with the TNXA pseudogene that include loss of the CYP21A2 gene, resulting in the contiguous gene syndrome disorder CAH-X.

Copy number variations (CNVs) detected with P155 EDS should be confirmed with a different technique. In particular, CNVs detected by only a single probe always require confirmation by another method. Most defects in the COL3A1 and TNXB genes are point mutations, none of which will be detected by MLPA. It is therefore recommended to 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, 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 or prenatal testing, population screening, or for the detection of, or screening for, acquired or somatic genetic aberrations.

1 Please 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).
2 To be used in combination with a SALSA MLPA Reagent Kit and Coffalyser.Net analysis software.

Clinical background
Ehlers-Danlos syndrome (EDS) is a group of disorders affecting the connective tissues that support the skin, bones, blood vessels, and many other organs and tissues. The defects that arise in the connective tissue result in the specific symptoms associated with EDS and range from mildly loose joints to life-threatening complications. Initially the different types of EDS were named with roman numerals (EDS I, EDS II, etc.), but in 1997 the Villefrance nosology for the different EDS types was proposed (Beighton et al. 1998), which was revised in 2017 (Malfait et al. 2017). The 2017 International Classification of the Ehlers-Danlos Syndromes has become the generally accepted standard and lists thirteen types, with descriptive names such as classic EDS (cEDS), classic-like EDS (clEDS) and vascular EDS (vEDS), which is also used here.

Vascular EDS is an autosomal dominant disorder solely associated with mutations in the COL3A1 gene. Patients have arterial, intestinal and/or uterine fragility; thin translucent skin that bruises easily; characteristic facial appearance (thin vermilion of the lips, micrognathia, narrow nose, prominent eyes); and an aged appearance to the extremities, particularly the hands. vEDS is a severe disorder: the most prominent signs in patients are vascular dissection or rupture, gastrointestinal perforation, or organ rupture. Approximately 25% of patients experience a severe complication before they reach 20 years of age, and at 40 years ~80% have severe complications. The median age of death is estimated at 50 years. Estimates of the prevalence range from 1:50,000 to 1:200,000. In >95% of patients a COL3A1 mutation can be identified, and >95% of the known COL3A1 mutations are point mutations or small indels that are detected with sequence analysis. 1-2% of the known pathogenic mutations in COL3A1 are large deletions or duplications that can be detected by MLPA (Pepin et al. 2014). More information about vEDS can be found at: https://www.ncbi.nlm.nih.gov/books/NBK1494/ and https://omim.org/entry/130050.

Classic-like EDS is an autosomal recessive disorder associated with mutations in the TNXB gene. clEDS is primarily characterised by hyperextensible skin, generalised joint hypermobility, muscle weakness, intestinal & tracheal tissue weakness, and in some cases mitral valve prolapse, which affects blood flow between the chambers of the heart. clEDS is very rare, but all known patients have mutations in TNXB. Determining the TNXB genotype can be challenging because the gene is located in a complex genomic locus that contains a tandem duplicated region. This cluster of genes (C4, CYP21 and TNX) is also referred to as RCCX module.

TNXB genotyping difficulties are mainly due to the presence of a pseudogene (TNXA), which is almost completely identical to the 3’-end of TNXB (exons 32-44). The only exception is a 120 basepair (bp) TNXB-specific sequence in exon 35. Currently, it is not possible to specifically detect TNXB CNVs in the highly homologous exons 32-34 and 36-44. CNV analysis of exon 35 and of sequences upstream of exon 32 and downstream of exon 44 are used to infer whether deletions or duplications of the highly homologous exons are present. Based on recent research the relative frequency of CNVs among pathogenic TNXB mutations is estimated at ~30%, most of which originate from rearrangements between TNXB and TNXA that include the common 120 bp deletion in exon 35, also described as the c.11435_11524+30 deletion (Demirdas et al. 2017, Green et al. 2020).

Unequal crossovers between the tandem duplicated regions occur frequently and result in chimeric pseudogene-gene products. Bimodular RCCX has an allele frequency of ~69% and is considered the ‘wild type’ situation, but monomodular and trimodular RCCX structures are also frequent and account for ~17% and ~14%, respectively (Blanchong et al. 2000). In addition, due to the high homology between CYP21A2 and CYP21A1P, but also between TNXB and TNXA, gene conversions are likely to occur in these regions. When the functional gene obtains sequence(s) form the non-functional pseudogene or vice versa this is referred to as a gene conversion. Gene conversions can present as apparent CNVs and can be benign, especially when the pseudogene obtains sequences from the functional gene.

When unequal crossover occurs between CYP21A1P and CYP21A2, it will result in a non-transcribed chimeric gene fusion (CYP21A1P-CYP21A2). When both alleles of CYP21A2 are lost or non-functional this leads to the disease Congenital Adrenal Hyperplasia (CAH). The P155 probemix is not suitable to detect all known CYP21A1P-CYP21A2 gene fusions. To determine the copy number of CYP21A2 and detect more CYP21A1P-CYP21A2 fusion genes the SALSA MLPA Probemix P050 CAH is recommended. An unequal crossover between CYP21A1P and CYP21A2 does not affect the TNXB gene. However, ~10% of CAH patients also have TNXB deficiency. This contiguous gene syndrome is referred to as CAH-X. In all known cases this is the result of unequal crossover between TNXA and TNXB, resulting in a non-functional TNXB-TNXA chimeric fusion gene and deletion of the CYP21A2 gene that is located amidst TNXA and TNXB. The known TNXA-TNXB fusions described in literature (Burch et al. 1997; Morissette et al. 2015) can be detected by the SALSA MLPA Probemix P155 EDS. More information about clEDS can be found at: https://omim.org/entry/606408.

Probemix content
The SALSA MLPA Probemix P155-E1 EDS contains 54 MLPA probes with amplification products between 124 and 504 nt. This includes 26 probes for the COL3A1 gene and 20 probes for the TNXB gene region. In addition, eight 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 nine quality control fragments generating amplification products between 64 and 108 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. 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.

Order Items

Probemix

Item no.
Description
Technology
Price
P155-025R
SALSA MLPA Probemix P155 EDS – 25 rxn
€ 281.00
P155-050R
SALSA MLPA Probemix P155 EDS – 50 rxn
€ 550.00
P155-100R
SALSA MLPA Probemix P155 EDS – 100 rxn
€ 1075.00

Required Reagents (Sold Separately)

Item no.
Description
Technology
Price
EK1-FAM
SALSA MLPA Reagent Kit – 100 rxn – FAM
€ 341.00
EK1-Cy5
SALSA MLPA Reagent Kit – 100 rxn – Cy5
€ 341.00
EK5-FAM
SALSA MLPA Reagent Kit – 500 rxn – FAM
€ 1571.00
EK5-Cy5
SALSA MLPA Reagent Kit – 500 rxn – Cy5
€ 1571.00
EK20-FAM
SALSA MLPA Reagent Kit – 2000 rxn – FAM
€ 6037.00

Related Products

SALSA MLPA Probemix P050 CAH

Contains probes for the CYP21A2 gene associated with CAH, probes for the CYP21A1P pseudogene, and several probes for the TNXB gene.

SALSA MLPA Probemix P272 COL1A2

Contains probes for the COL1A2 gene, which is associated to two other types of EDS: arthrochalasia EDS and cardiac-valvular EDS, according to the revised Villefrance nosology. These types were formerly known as EDS VIIA and VIIB.

SALSA MLPA Probemix P331 COL5A1 MIX-1

Contain probes for the COL5A1 gene, which is associated wich classical EDS, according to the revised Villefrance nosology. This type was formerly known as EDS I and II.

SALSA MLPA Probemix P332 COL5A1 MIX-2

Contain probes for the COL5A1 gene, which is associated wich classical EDS, according to the revised Villefrance nosology. This type was formerly known as EDS I and II.

SALSA MLPA Probemix P359 PLOD1

Contains probes for the PLOD1 gene, which is associated with kyphoscoliotic EDS, according to the revised Villefrance nosology. This type was formelery known as EDS VI.

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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.