Intended purpose
The SALSA MLPA Probemix P044 NF2 is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions or duplications in the NF2 gene in genomic DNA isolated from human peripheral whole blood specimens. P044 NF2 is intended to confirm a potential cause for and clinical diagnosis of Neurofibromatosis type 2 (NF2) and for molecular genetic testing of at-risk family members. NF2 has a high incidence of mosaicism (~15% of NF2 patients are mosaic) and mosaic mutations may not be detectable in blood.

Copy number variations (CNVs) detected with P044 NF2 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 NF2 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, or for population screening, or for the detection of, or screening for, acquired or somatic genetic aberrations.

Only in a research setting this assay can be used on DNA derived from fresh or formalin-fixed paraffin-embedded (FFPE) tumour tissue.

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
Neurofibromatosis type 2 (NF2) is an autosomal dominant cancer syndrome that is characterized by the development of bilateral vestibular schwannomas (BVSs) in almost all patients. This disease is caused by inactivating mutations of the NF2 tumour-suppressor gene. BVSs result in hearing loss, tinnitus and balance dysfunction. The age of onset is between 18 and 24 years. Patients also suffer from schwannomas of other cranial and peripheral nerves, meningiomas, ependymomas, and rarely, astrocytomas. Although most of these tumours are not malignant, their anatomic location and multiplicity lead to high morbidity and mortality at a low age: the average age of death is 36 years. NF2 occurs in approximately 1 in 25,000-40,000 live births and the estimated prevalence in the general population is 1 in 60,000 without any known ethnic or racial bias. For known pathogenic mutations the penetrance is close to 100% (Asthagiri et al. 2009).

Mutational analysis of the NF2 gene in typical NF2 patients has demonstrated causative mutations in ~70%. The NF2 gene behaves as a typical tumour-suppressor gene, with first hits detectable in both constitutional and tumour specimens and second hits detectable only in tumours. Approximately 50% of NF2 mutation positive patients inherit a germline mutation from an affected parent and the remaining half are sporadic cases due to de novo mutations. Large alterations affecting the NF2 gene account for 15-20% of all known NF2 mutations (Abo-Dalo et al. 2010; Halliday et al. 2017; Kluwe et al. 2005; Smith et al. 2016). Combined with the 70% NF2 mutation detection rate in patients, this means that in 10-15% of NF2 patients large deletions or duplications are detected. A high level of mosaicism is observed in NF2, which can complicate mutation detection. More than 30% of the de novo cases are mosaic for NF2 mutations, which may result in subclinical symptoms and/or difficulties with mutation detection, resulting in a false negative diagnosis (Evans et al. 2007).

Mutations in NF2 are also frequently found in the DNA of sporadic schwannomas and meningiomas (Lassaletta et al. 2013; Mohyuddin et al. 2002; Pathmanaban et al. 2017). Both NF2 syndromic tumours and such sporadic tumours have often lost a large part of chromosome 22 resulting in loss of heterozygosity (LOH) of NF2. These large chromosomal deletions frequently include loss of SMARCB1 and LZTR1, which are also recognized as tumour suppressor genes associated with an NF2-related disorder: schwannomatosis.

More information on NF2 can be found at https://www.ncbi.nlm.nih.gov/books/NBK1201/ and https://omim.org/entry/101000.

Probemix content
The SALSA MLPA Probemix P044-C1 NF2 contains 43 MLPA probes with amplification products between 129 and 472 nucleotides (nt). This includes 21 probes for the NF2 gene; 5 probes on chromosome 22q upstream of NF2, 4 of which target SMARCB1 and LZTR1; and 4 probes on chromosome 22q downstream of NF2. In addition, 13 reference probes are included that detect autosomal chromosomal targets that have stable copy numbers in the general population and have relatively stable copy numbers in various cancer types including schwannomas and meningiomas. 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.