Intended purpose
The SALSA MLPA Probemix P165 HSP mix-1 is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions or duplications in ATL1 and SPAST genes, in order to confirm a potential cause for and clinical diagnosis of spastic paraplegia (SPG) type 3A and SPG type 4, 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 P165 HSP mix-1 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 ATL1 and SPAST 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 3A (SPG3A) is caused by pathogenic mutations in the ATL1 gene and accounts for approximately 10% of all autosomal dominant HSP. Currently, there are more than 60 different ATL1 mutations described for SPG3A patients, which are divided into five broad groups: 91.5% missense mutations, 4% small insertions, 2.8% small deletions, 0.7% splice site mutation, and one (0.7%) whole exon deletion (exon 4) (Sulek et al. 2013).

Mutations in the SPAST gene are responsible for both autosomal dominant HSP (40-50%) and sporadic cases (10-15%), causing spastic paraplegia type 4 (SPG4). The most common type of SPAST mutations are point mutations (75-80%), and large genomic abnormalities, such as exon deletions, account for up to 20-29% of disease-associated SPAST mutations (Beetz et al. 2006, d’Amore et al. 2018, Depienne et al. 2006, Kadnikova et al. 2019).

More information on HSP is available on https://www.ncbi.nlm.nih.gov/books/NBK1509/, https://www.ncbi.nlm.nih.gov/books/NBK1160/, https://www.ncbi.nlm.nih.gov/books/NBK45978/.

Probemix content
The SALSA MLPA Probemix P165-C3 HSP mix-1 contains 47 MLPA probes with amplification products between 130 and 481 nucleotides (nt). This includes 16 probes for the ATL1 gene and 20 probes for the SPAST gene. 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.