Intended purpose
The SALSA MLPA Probemix P093 HHT/HPAH is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions or duplications in the ENG, ACVRL1 and BMPR2 genes in genomic DNA isolated from human peripheral whole blood specimens. P093 HHT/HPAH is intended to confirm a potential cause for and clinical diagnosis of Hereditary Hemorrhagic Telangiectasia (HHT) or Heritable Pulmonary Arterial Hypertension (HPAH) and for molecular genetic testing of at-risk family members.

Copy number variations (CNVs) detected with P093 HHT/HPAH 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 ENG, ACVRL1 and BMPR2 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.

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

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 activin A receptor like type 1 (ACVRL1/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 Pulmonary Arterial Hypertension (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 ACVRL1, 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 ACVRL1, and even more infrequent in ENG, causing HPAH. In the 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/.

Exon numbering
The BMPR2, ACVRL1 and ENG exon numbering used in this P093-C2 HHT/HPAH product description is the exon numbering from the RefSeq transcripts NM_001204.7, NM_000020.3 and NM_000118.4, respectively, which is identical to the exon numbering of the LRG_712, LRG_543 and LRG_589 sequences, respectively. As changes to the NCBI database can occur after release of this product description, exon numbering may not be up-to-date.

Probemix content
The SALSA MLPA Probemix P093-C2 HHT/HPAH contains 51 MLPA probes with amplification products between 130 and 490 nucleotides (nt). This includes 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 14(b)) In addition, 8 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 105 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.