Intended purpose
The SALSA MLPA Probemix P016 VHL is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions in the VHL gene in genomic DNA isolated from human peripheral whole blood specimens. P016 VHL is intended to confirm a potential cause for and clinical diagnosis of Von Hippel-Lindau (VHL) disease and for molecular genetic testing of at-risk family members.

Copy number variations (CNVs) detected with P016 VHL 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 VHL gene 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, e.g from DNA extracted from formalin-fixed paraffin embedded (FFPE) or fresh tumour materials.

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 and Coffalyser.Net analysis software.

Clinical background
Von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome predisposing to a variety of malignant and benign neoplasms, most frequently retinal, cerebellar and spinal hemangioblastoma, renal cell carcinoma, pheochromocytoma, and pancreatic tumours. The basis of familial inheritance of VHL disease is a germline mutation in the VHL tumour suppressor gene, located in chromosomal region 3p25.3. Approximately 80% of individuals with VHL disease inherit a mutation, whereas 20% of individuals have a de novo mutation (http://www.ncbi.nlm.nih.gov/books/NBK1463/). Of the mutations in the VHL gene, 30-60% are missense mutations, 20-40% are large intragenic deletions (0.5-250 kb), 12-20% are microdeletions or insertions and 7-11% are nonsense mutations (Decker et al. 2014).

Interestingly, loss of the nearby BRK1 gene in combination with loss of (part of) the VHL gene can be associated with a reduced risk of renal cell carcinoma as compared to defects in the VHL gene only (Escobar et al. 2010, McNeill et al. 2009). This probemix contains two probes that target the BRK1 gene.

Probemix content
The SALSA MLPA Probemix P016-C2 VHL contains 29 MLPA probes with amplification products between 166 and 427 nucleotides (nt). This includes nine probes for the VHL gene (two or more probes for each exon), six probes for genes located close to VHL (FANCD2, BRK1, IRAK2 and GHRL), and two probes for genes on 3p located further telomeric or centromeric from VHL (CNTN6 and MLH1). In addition, 12 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.