Intended purpose
The SALSA MLPA Probemix P213 HSP mix-2 is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions or duplication in REEP1 and SPG7 genes, in order to confirm a potential cause for and clinical diagnosis of spastic paraplegia (SPG) type 31 and SPG type 7, respectively. This assay is for use with genomic DNA isolated from human peripheral whole blood specimens. This product can also be used for molecular genetic testing of at-risk family members.

Copy number variations (CNVs) detected with P213 HSP 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 REEP1 and SPG7 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, 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 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).
²To be used in combination with a SALSA MLPA Reagent Kit and Coffalyser.Net analysis software.

Clinical background
Hereditary spastic paraplegias (HSP) are genetically heterogeneous neurodegenerative disorders characterised by progressive spasticity and weakness of the lower limbs due to axonal degeneration in the pyramidal tract. To date, more than 80 genetic types of HSP have been defined by genetic linkage analysis and identification of HSP-related gene variants.

Spastic paraplegia type 31 (SPG31) is caused by a pathogenic variant in the receptor expression-enhancing protein 1 (REEP1) and accounts for about 5% of all autosomal dominant HSP. The most common type of REEP1 mutations explaining SPG31 are small frameshift mutations, but nonsense, missense, and microRNA target site alterations have been described (Beetz et al. 2008). 9.5% of pathogenic mutations in REEP1 are copy number variations (Goizet et al. 2011).

SPG7 is caused by pathogenic variants in SPG7 and may account for approximately 5% of all autosomal recessive HSP. Of all SPG7 cases, most are caused by SPG7 point mutations (~98%), while less than 2% can be explained by SPG7 copy number variations (Klebe et al. 2012, Pfeffer et al. 2015).

More information on HSP is available on https://www.ncbi.nlm.nih.gov/books/NBK1509/

Probemix content
The SALSA MLPA Probemix P213-B3 HSP mix-2 contains 42 MLPA probes with amplification products between 128 and 436 nucleotides (nt). This includes 10 probes for the REEP1 gene and 21 probes for the SPG7 gene. Additional probes are included for the exons 1 and 3 of REEP1, for exon 1 of SPG7, and for the last exon of alternative splice variant NM_199367.3 of SPG7, which is located within intron 9 of NM_003119.4. In addition, 11 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.