Intended use: The SALSA MLPA probemix P093 HHT/HPAH is an in vitro diagnostic (IVD)1 or a research use only (RUO) assay for the detection of deletions or duplications in ENG, ACVRL1 and BMPR2 in order to (1) confirm a clinical diagnosis of Hereditary Hemorrhagic Telangiectasia (HHT) or Heritable Pulmonary Arterial Hypertension (HPAH) or to, (2) determine predisposition to HHT or HPAH of at-risk family members/individuals.
This assay is for use with human DNA extracted from peripheral blood, not for use with DNA extracted from formalin-fixed paraffin embedded or fresh tumour materials. Deletions or duplications detected with the P093 HHT/HPAH probemix must be verified by another technique. In particular, deletions or duplications detected by only a single probe always require validation by another method. Most defects in the ENG, ACVRL1 and BMPR2 genes are point mutations, the majority of which will not be detected by MLPA. It is therefore recommended to use this SALSA MLPA probemix in combination with sequence analysis of the ENG, ACVRL1 and BMPR2 genes. This assay is not intended to be used as a standalone assay for clinical decisions. The results of this test must be interpreted by a clinical molecular geneticist or equivalent.
1 Please note that this probemix is for In Vitro Diagnostic use (IVD) in the countries specified at the end of this product description. In all other countries, the product is for Research Use Only (RUO).
Clinical background: Hereditary Hemorrhagic Telangiectasia (HHT) is a disease with an autosomal dominant inheritance pattern and is characterized by the presence of multiple arteriovenous malformations (AVM). In AVMs arteries connect directly to veins instead of through intervening capillaries, resulting in high blood pressure. AVMs occur on the skin, but also in the brain, lungs, liver and intestines. Depending on the location, rupture of these malformations can have catastrophic consequences for the patient. Diagnosis is based on the presence of multiple AVM in the skin, mucus membranes, or visceral organs. Recurrent nosebleeds are also a common finding in HHT patients. Molecular genetic testing is performed to confirm or establish a diagnosis in a proband. HHT is primarily caused by pathogenic variations in the genes endoglin (ENG/HHT1) and activing A receptor like type 1 (ACVLR1/HHT2). Both genes encode endothelial cell surface receptors that are part of a TGF-β/BMP signalling cascade, a pathway involved in angiogenesis, among multiple other developmental processes. Up to 10% of pathogenic variation consists of large deletions/duplications. More information is available at: https://www.ncbi.nlm.nih.gov/books/NBK1351/.
Heritable Pulmonary Arterial Hypertension (HPAH) is inherited in an autosomal dominant manner. This disease is caused by loss or obstruction of the smallest pulmonary arteries, resulting in high blood pressure in the arteries of the lung. Diagnosis is based on the presence of pulmonary hypertension as confirmed through right heart catheterization, and subsequently by identification of a heterozygous pathogenic variant in a known associated gene (simplex cases) and/or confirmation of PAH in one or more of the proband’s family members. Up to 75% of HPAH is caused by variation in the bone morphogenetic protein receptor type 2 (BMPR2) gene. Of this, 12-37% is caused by large duplications/deletions. Similar to ENG and ACVLR1, the BMPR2 gene also encodes a cell surface receptor that is part of the TGF-β/BMP signalling pathway. Sporadically, PAH is observed as a symptom of HHT. The biological similarities between the causative genes suggests a similar aetiology between HPAH and HHT. This is supported by rare observations of mutations in ACVLR1, and even more infrequent in ENG, causing HPAH. In literature, a patient has been described with a combined PAH and HHT phenotype carrying a deletion of exons 6 and 7 in BMPR2 (Handa et al. 2014). In very rare cases, HPAH can be caused by mutations in the KCNK3, SMAD9 or CAV1 gene. To the best of our knowledge, no HPAH causing deletions or duplications have been reported in these genes. More information is available at: https://www.ncbi.nlm.nih.gov/books/NBK1485/.
P093-C2 probemix content: The P093-C2 HHT/HPAH probemix contains 51 probes with amplification products between 130 and 490 nt: 14 probes for the BMPR2 gene (one probe for each exon and one additional probe for exon 1), 11 probes for the ACVRL1 gene (one probe for each exon and one additional probe for exon 1) and 18 probes for the ENG gene (one probe for each exon, and one additional probe for exons 1 and 2, and two additional probes for exon 14b) and 8 reference probes. The identity of the genes detected by the reference probes is available online (www.mlpa.com).
This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity Fragments (Q-fragments), three DNA Denaturation Fragments (D-fragments), and one chromosome X and one chromosome Y-specific fragment (Table 1). The Q-fragments are only visible when less than 100 ng sample DNA is used. Low signal of the 88 or 96 nt fragment indicates incomplete DNA denaturation. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol.