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SALSA MLPA Probemix P021 SMA

SALSA® MLPA® Probemix P021 SMA detects copy number variations in the SMN1, SMN2 and in exon 5 of the NAIP genes. P021 SMA has been specifically designed for SMA patient testing and offers the most robust SMN2 copy number detection.

Specifications

Contents: 32 MLPA probes, including 4 probes for exon 7 or 8 of SMN1 or SMN2, 17 probes for sequences present in both SMN1 and SMN2, and 1 probe for NAIP.

Tissue: genomic DNA isolated from human peripheral whole blood, specified prenatal samples (see Intended Purpose) or dry blood spots (DBS) cards.

Application: spinal muscular atrophy (SMA).

CE-marked and registered for in vitro diagnostic (IVD) use in selected territories.

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List Prices Documentation

Key related products

SALSA MLPA Probemix P058 IGHMBP2

Autosomal recessive distal spinal muscular atrophy 1 (DSMA1), gene included IGHMBP2.

SALSA MLPA Probemix P060 SMA Carrier

Spinal Muscular Atrophy (SMA), to determine SMN1 and SMN2 copy number (best suited for carrier testing/screening).

SALSA MLPA Probemix P460 SMA (Silent) Carrier

Spinal Muscular Atrophy (SMA), to determine SMN1 copy number and an increased risk for the 2+0 carrier genotype by detection of two associated polymorphisms (g.27134T>G and g.27706-27707delAT).

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MRC Holland has recently obtained the In Vitro Diagnostic Regulation (IVDR; EU 2017/746) certification for this product. The CE-IVDR version will be sold from early 2025, and will be accompanied by a change in the intended purpose. The probe targeting NAIP, as well as the use of genomic DNA isolated from prenatal samples, will no longer be intended for diagnostic use. Furthermore, SMN2 copy number determination on crude extracts from DBS cards will no longer be included for diagnostic purposes. The composition of this product remains unchanged.

MRC Holland offers four different assays for SMA that fit the complete range of genetic testing needs. Compare our SMA products.

Intended purpose

The SALSA MLPA Probemix P021 SMA is an in vitro diagnostic (IVD) or research use only (RUO) semi-quantitative assay for the detection of deletions or duplication in the SMN1, SMN2 and exon 5 of the NAIP genes in genomic DNA isolated from human peripheral whole blood specimens, prenatal samples, from either (un)cultured amniotic fluid obtained in week 16 of pregnancy or later, free from blood contamination (un)cultured chorionic villi, free from maternal contamination fetal blood or Dried Blood Spot (DBS) cards. P021 SMA is intended to establish or confirm a potential cause for and clinical diagnosis of Spinal Muscular Atrophy (SMA), carrier testing and for molecular genetic testing of at-risk family members. Secondly, the assay can be used for SMN2 (and NAIP) copy number determination in (pre-symptomatic) SMA patients as an aid in prognosis and for treatment eligibility.

For the full intended purpose, see the product description.

Clinical background

Spinal Muscular Atrophy (SMA) is a group of autosomal recessive neuromuscular disorders characterized by degeneration of the anterior horn cells of the spinal cord, leading to symmetrical muscle weakness and atrophy. SMA is a heterogeneous disease, its phenotype ranging from early onset with a life expectancy of less than 2 years to a late onset with very mild symptoms. SMA disease severity is strongly correlated with SMN2 copy number. The estimated incidence of SMA is 1:6,000-1:10,000.

Two highly similar genes play a pivotal role in SMA: SMN1 and SMN2. The only clinically relevant difference between the two genes is a single nucleotide difference in exon 7. SMN2 is translated much less efficiently in a functional SMN protein; therefore, it is the SMN1 gene which is the determinant factor in SMA. Someone lacking functional copies of SMN1 is almost always a SMA patient. In most populations, 95-98% of SMA patients show complete absence of at least exon 7 of the SMN1 gene (this percentage is lower in SMA patients from African descent (Labrum et al. 2007)). Most of the remaining patients have a single copy of the SMN1 gene which is inactive due to a point mutation or a deletion of exons 1-6. Please note that rare cases have been described of healthy individuals with homozygous loss of SMN1 exon 7 and only 2 or 3 SMN2 copies (e.g. Helmken et al. 2003).

SMA carriers are symptom-free. The great majority of SMA carriers can be identified by the presence of a single SMN1 exon 7 copy. The presence of more than two SMN1 copies is a relatively frequent phenomenon in healthy individuals, especially in individuals of African descent. For more details, see Interpretation of Results. About 3-8% of SMA carriers (27% of African Americans) have two SMN1 copies on one chromosome and zero copies on the other (2+0) (Alias et al. 2014, Hendrickson et al. 2009). MLPA cannot distinguish '1+1' from '2+0' (silent carriers) arrangements. Both situations are simply detected as having two SMN1 copies leading to false negative results. A thorough molecular analysis should be performed on samples from parents and blood relatives of SMA patients when initial results indicate two SMN1 copies. Luo et al. (2014) reported that a haplotype block specific for SMN1 duplications is present in a large percentage of Ashkenazi Jews and in other ethnic groups. Identifying this haplotype, e.g. with the use of the SALSA MLPA probemix P460 SMA, will help to identify silent carriers.

Most healthy individuals have 0 - 4 copies of SMN2. Provided that at least one functional SMN1 copy is present, complete absence of the centromeric SMN2 gene seems to have no clinical consequences.

Most patients have 1 - 4 copies of SMN2. Establishing the SMN2 copy number is of importance for SMA patients: the more SMN2 copies present, the less severe the disease usually is (Feldkötter et al. 2002). Accurate SMN2 copy number quantification can be important for determining a patient’s eligibility for treatment.

Another factor that influences disease severity is the presence of the c.859G>C polymorphism in SMN2 (Prior et al. 2009). Please note that the SMN2 copy number and the presence of the c.859G>C variant do not completely explain the differences in disease severity between SMA patients. The c.859G>C polymorphism cannot be detected by P021-B1 SMA.

More information is available at: https://www.ncbi.nlm.nih.gov/books/NBK1352/.

Regulatory status

SALSA MLPA Probemix P021 SMA is CE-marked for in vitro diagnostic (IVD) use. This assay has also been registered for IVD use in Colombia and Israel.

This assay is for research use only (RUO) in all other territories.

SALSA Sample DNA for this product

SALSA Reference Selection DNA SD082 can be used to aid in the selection of suitable reference samples for the P021 SMA probemix. Reference Selection DNA can only be used in initial experiments on DNA samples from healthy individuals from your sample collection with the intention to identify suitable reference samples. SD082 cannot be used as a reference sample in subsequent experiments.

A vial of SALSA Reference Selection DNA SD082 is included with every order of the P021 SMA probemix, but it is possible to order additional vials separately.

For more information, see the product description.

Product documentation

Version B1

Other versions

Contact us for earlier versions.

List prices

Product

Item no.
Description
Technology
Price
P021-025R
SALSA MLPA Probemix P021 SMA – 25 rxn
MLPA
€ 281.00
P021-050R
SALSA MLPA Probemix P021 SMA – 50 rxn
MLPA
€ 550.00
P021-100R
SALSA MLPA Probemix P021 SMA – 100 rxn
MLPA
€ 1075.00

Required reagents

A general SALSA MLPA Reagent Kit is required for MLPA experiments (to be ordered separately).

Item no.
Description
Technology
Price
EK1-FAM
SALSA MLPA Reagent Kit – 100 rxn – FAM (6 vials)
MLPA MS-MLPA
€ 341.00
EK1-Cy5
SALSA MLPA Reagent Kit – 100 rxn – Cy5 (6 vials)
MLPA MS-MLPA
€ 341.00
EK5-FAM
SALSA MLPA Reagent Kit – 500 rxn – FAM (5×6 vials)
MLPA MS-MLPA
€ 1571.00
EK5-Cy5
SALSA MLPA Reagent Kit – 500 rxn – Cy5 (5×6 vials)
MLPA MS-MLPA
€ 1571.00
EK20-FAM
SALSA MLPA Reagent Kit – 2000 rxn – FAM (5×6 vials)
MLPA MS-MLPA
€ 6037.00

Sample DNAs (included)

A vial is included with every order of this probemix, but additional vials can also be purchased separately.

Item no.
Description
Technology
Price
SD082
SALSA Reference Selection DNA SD082 – 6 rxn
MLPA
€ 23.70

Price details & ordering

The prices above are list prices for direct orders from MRC Holland. Contact us for a quote that takes discounts and additional costs (such as shipping costs) into account. Different prices apply for orders through one of our sales partners; contact your local supplier for a quote.

More information about ordering & prices

Positive samples

Inclusion of a positive sample is usually not required, but can be useful for the analysis of your experiments. MRC Holland has very limited access to positive samples and cannot supply such samples. We recommend using positive samples from your own collection. Alternatively, you can use positive samples from an online biorepository, such as the Coriell Institute.

The commercially available positive samples below have been tested with current (B1) and have been shown to produce useful results.

Coriell Sample ID Copy Number
SMN1 exon 7 SMN2 exon 7 SMN1 exon 8 SMN2 exon 8
NA00232; NA106840202
NA22592; NA09677; NA038130303
NA03815; NA20760; NA207871111
NA23687; NA23688; NA207641212
HG00346; HG002811313
HG01773; HG01774; HG021321414
NA038141515
NA19122; HG01941; NA197942020
HG02514; HG03663; HG036362121
HG01701; HG01942; HG019352222
HG01748; HG01971; HG003292323
HG036252424
NA19123; HG03258; HG02891; HG00255; NA19437; HG013773030
HG01755; HG03650; NA20775; HG011373131
NA12548; NA207553232
NA12552; NA205153333
NA19235; HG03027; HG027694040
NA19429; HG028364141

Samples with a different copy number for exon 7 and 8 due to gene conversion

Coriell Sample ID Copy Number
SMN1 exon 7 SMN2 exon 7 SMN1 exon 8 SMN2 exon 8
NA191772130
NA215272213
NA192492231
NA215262314
NA197903113
NA193273122
NA215133140
NA193604031
NA190264150
HG026974132
NA190194352

Resources

General MLPA resources

MLPA education

Publications

Selected publications using P021 SMA

  • Bai JL et al. (2014). Subtle mutation detection of SMN1 gene in Chinese spinal muscular atrophy patients: implication of molecular diagnostic procedure for SMN1 gene mutations. Genet Test Mol Biomarkers. 18:546-51.
  • Boemer F et al. (2021). Three years pilot of spinal muscular atrophy newborn screening turned into official program in Southern Belgium. Sci Rep. 11:19922.
  • Brkušanin M et al. (2015). Joint effect of the SMN2 and SERF1A genes on childhood-onset types of spinal muscular atrophy in Serbian patients. J Hum Genet. 60:723-8.
  • Kaneko K et al. (2017). Relationships between long-term observations of motor milestones and genotype analysis results in childhood-onset Japanese spinal muscular atrophy patients. Brain Dev. 39:763-773.
  • Kekou K et al. (2020). Evaluation of Genotypes and Epidemiology of Spinal Muscular Atrophy in Greece: A Nationwide Study Spanning 24 Years. J Neuromuscul Dis. 7:247-56.
  • Strunk A et al. (2019). Validation of a Fast, Robust, Inexpensive, Two-Tiered Neonatal Screening Test algorithm on Dried Blood Spots for Spinal Muscular Atrophy. Int J Neonatal Screen. 5:21.
  • Theodorou L et al. (2015). Genetic findings of Cypriot spinal muscular atrophy patients. Neurol Sci. 36:1829-34.
  • Veldhoen ES et al. (2022). Natural history of respiratory muscle strength in spinal muscular atrophy: a prospective national cohort study. Orphanet J Rare Dis. 17:70.
  • Vijzelaar R et al. (2019). The frequency of SMN gene variants lacking exon 7 and 8 is highly population dependent. PLoS One. 14:e0220211.
  • Wadman RI et al. (2020). Intragenic and structural variation in the SMN locus and clinical variability in spinal muscular atrophy. Brain Commun. 2:fcaa075.

Selected publications using SALSA MLPA Probemix P021 SMA and P060 SMA Carrier

  • Cheung VCK et al. (2024). Motor patterns of patients with spinal muscular atrophy suggestive of sensory and corticospinal contributions to the development of locomotor muscle synergies. J Neurophysiol. 131:338-59.
  • Jiang Y et al. (2024). Development of a low-cost and accurate carrier screening method for spinal muscular atrophy in developing countries. Eur J Med Genet. 68:104921.
  • Laurito S et al. (2018). The impact of paralog genes: detection of copy number variation in spinal muscle atrophy patients. Biocell. 42:87-92.
  • Qu Y et al. (2024). Variants located in intron 6 of SMN1 lead to misdiagnosis in genetic detection and screening for SMA. Heliyon. 10:e28015.
  • Ricci M et al. (2023). Clinical Phenotype of Pediatric and Adult Patients With Spinal Muscular Atrophy With Four SMN2 Copies: Are They Really All Stable? Ann Neurol. 94:1126-35.

References

  • Alías L et al. (2014). Improving detection and genetic counseling in carriers of spinal muscular atrophy with two copies of the SMN1 gene. Clin Genet. 85:470-5.
  • Feldkötter M et al. (2002). Quantitative analyses of SMN1 and SMN2 based on real-time lightCycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy. Am J Hum Genet. 70:358-68.
  • Helmken C et al. (2003). Evidence for a modifying pathway in SMA discordant families: reduced SMN level decreases the amount of its interacting partners and Htra2-beta1. Hum Genet. 114:11-21.
  • Hendrickson BC et al. (2009). Differences in SMN1 allele frequencies among ethnic groups within North America. J Med Genet. 49:641-4.
  • Labrum R et al. (2007). The molecular basis of spinal muscular atrophy (SMA) in South African black patients. Neuromuscul Disord. 17:684-92.
  • Luo M et al. (2014). An Ashkenazi Jewish SMN1 haplotype specific to duplication alleles improves pan-ethnic carrier screening for spinal muscular atrophy. Genet Med. 16:149-56.
  • Prior TW et al. (2009). A positive modifier of spinal muscular atrophy in the SMN2 gene. Am J Hum Genet. 85:408-13.
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CE

CE-marked products are for In Vitro Diagnostic (IVD) use only in EU (candidate) member states and members of the European Free Trade Association (EFTA), and the UK.

CO

IVD-registered in Colombia.

IL

IVD-registered in Israel.