Intended purpose
The SALSA MLPA Probemixes P081 NF1 mix 1 and P082 NF1 mix 2 are in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assays² for the detection of deletions or duplications in the NF1 gene in genomic DNA isolated from human peripheral whole blood specimens. P081 NF1 mix 1 and P082 NF1 mix 2 are intended to confirm a potential cause for and clinical diagnosis of Neurofibromatosis type 1 (NF1) and for molecular genetic testing of at-risk family members.

Copy number variations (CNVs) detected with P081 NF1 mix 1 and P082 NF1 mix 2 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 NF1 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 these probemixes are for in vitro diagnostic (IVD) use in the countries specified at the end of this product description. In all other countries, the products are for research use only (RUO).
²To be used in combination with a SALSA MLPA Reagent Kit and Coffalyser.Net analysis software.

Clinical background
Neurofibromatosis is an autosomal dominant disorder characterised particularly by café-au-lait spots and fibromatous tumours of the skin. Neurofibromatosis type 1 is caused by loss-of-function mutations in the NF1 gene on 17q11.2. Neurofibromatosis type 2 is caused by defects in the NF2 gene on chromosome 22q12.2, for which the SALSA MLPA Probemix P044 NF2 can be used.

Estimated birth incidence of Neurofibromatosis type 1 is 1 in 3000, with about half of the NF1 cases caused by de novo sporadic mutations. De novo sporadic mutations may also be the result of germline mosaicism in apparently unaffected parents. Partial deletions and duplications as well as deletions and duplications of the complete NF1 gene have been described. Relatively common (5-10% of NF1 cases) is a deletion of a 1.4 Mb chromosomal region harbouring multiple genes, including the NF1 gene. The phenotype of this 17q11.2 microdeletion is usually more severe than most other NF1 cases and may include developmental delay. Next to the 1.4 Mb deletion described above, a 1.2 Mb microdeletion and nonrecurrent atypical microdeletions of different sizes have been reported. The SALSA MLPA Probemix P122 NF1-area (RUO) can be used to determine the extent of the deletion as it contains many probes for other genes in the frequently deleted 1.4 Mb region. More information is available on https://www.ncbi.nlm.nih.gov/books/NBK1109/.

Probemix content
The P081 and P082 probemixes together contain at least one probe for each exon, three probes for exon 1, one probe for intron 1, and two probes for the exons 15, 21, 23, 51 and 58 of the NF1 gene. Additionally, these probemixes contain one upstream and one downstream probe and two probes for the OMG gene, located within intron 36 of the NF1 gene.

The SALSA MLPA Probemix P081-D1 NF1 mix 1 contains 46 MLPA probes with amplification products between 130 and 463 nucleotides (nt), including 11 reference probes that detect autosomal chromosomal locations. The SALSA MLPA Probemix P082-C2 NF1 mix 2 contains 44 MLPA probes with amplification products between 130 and 483 nt, including nine reference probes 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).

Both probemixes contain 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.