This SALSA MLPA probemix P021 SMA is an in vitro diagnostic (IVD)1
or research use only (RUO) assay for the detection of copy number changes in the SMN1
gene for a) patient diagnosis, b) confirmation of a potential cause and clinical diagnosis, and c) carrier testing of spinal muscular atrophy (SMA). Secondly, the assay can be used for SMN2
copy number determination in (presymptomatic) SMA patients as an aid in prognosis and for treatment eligibility.
This assay is for use with human DNA extracted from:
In the majority of SMA patients (>95%), the disease is caused by a homozygous loss of the SMN1
gene, usually detected by the absence of exon 7 specific markers. In a small number of SMA cases, the causative defect concerns a loss of other exon(s) in SMN1
. Both defects can be detected by MLPA probemix P021 SMA. Point mutations, which cause SMA in a small number of cases, will not be detected by MLPA. This assay is not intended to be used as a standalone assay for clinical decisions. The results of this test must be interpreted by a clinical molecular geneticist or equivalent.
Please 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).
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
. The only clinically relevant difference between the two genes is a single nucleotide differences in exon 7. SMN2
is translated much less efficient 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. 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 only 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 0 copies on the other (2+0) (Alias et al. 2004, 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 - 3 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 is important for determining a patient’s eligibility for treatment with Spinraza (nusinersen), a drug that can be used to treat SMA by increasing the amount of full-length SMN protein produced from SMN2
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.
More information is available at: https://www.ncbi.nlm.nih.gov/books/NBK1352/
SALSA MLPA probemix P021-B1 SMA contains 32 probes with amplification products between 175 and 445 nt:
- Four probes specific for sequences in exon 7 or
8 of either SMN1 or SMN2.
- 17 probes detecting sequences that are present in both SMN1 and SMN2
(at least one probe per exon; one additional probe for exon 1, 2b, and 3; seven probes in total for the intron 6-exon 7-intron 7-exon 8 region of both SMN1
- One probe for the NAIP
- Ten reference probes. The identity of the genes detected by the reference probes is available upon request.
• The SMN1
-specific exon 7 probe 21488-L30891 (274 nt) and the SMN2-specific exon 7 probe 21489-L30892
(281 nt) are the most important probes in this mix. These two probes distinguish SMN1
by having their ligation site at the critical single nucleotide difference between these genes in exon 7, which is a site that affects RNA splicing. These probes can be used to quantify respectively SMN1
(important in determining carrier status) and SMN2
(important for disease prognosis).
• The SMN1
-specific exon 8 probe 21490-L29983 (295 nt) and the SMN2-specific exon 8 probe 21491-L29984
(301 nt) distinguish SMN1
at the exon 8 G-to-A transition. In approximately 5-10% of cases, the copy number detected by these exon 8 probes does not correspond to that found by the aforementioned exon 7 probes, due to gene conversion. In such cases, only the exon 7 probes should be used to quantify SMN1
• There are seven SMN exon 7 & 8 probes
(see Table 2) that detect the combined
copy number of SMN1
genes in the exon 7, intron 7 and exon 8 region. In normal individuals carrying two copies of SMN1
and two copies of SMN2
, these probes detect four gene copies in total. In case of a homozygous SMN1
deletion, these probes can be used to more accurately determine the SMN2
• The ten SMN exon 1-6 probes
(see Table 2) are useful to identify patient samples with a gain or loss of these exons as well as rare carriers who have two SMN1
exon 7 sequences but one of the SMN1
genes is non-functional due to a deletion of exon 1-6 (Arkblad et al. 2006; Thauvin-Robinet C et al. 2012). As these probes detect both SMN1
, an exon 1-6 deletion detected by these probes should only be considered pathogenic if this is suggested by the individual’s clinical context. Please note that many healthy individuals have an extra copy of exons 1-6 (SMN1/2Δ7-8
) without known clinical significance.
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.mlpa.com
SALSA Reference selection DNA SD082:
The SD082 Reference selection DNA provided with this probemix can be used to find suitable reference DNA samples from your own sample collection for further use in MLPA experiments. It is strongly advised to use DNA sample and reference DNA samples extracted with the same method and derived from the same source of tissue. For certain applications, the selection of suitable reference DNA samples is complicated. Inclusion of one reaction with 5 μl SD082 Reference selection DNA facilitates the identification of suitable reference DNA samples. We recommend the use of this SD082 Reference selection DNA only for initial experiments on DNA samples from healthy individuals with the intention to identify suitable reference DNA samples. We do not recommend it for use in all experiments. For further instructions, consult the SD082 Reference selection DNA product description provided. This product is for research use only (RUO), except when used in combination with a probemix for in vitro diagnostic (IVD) purpose, as specified at the end of this product description.
Sample DNA developed for this product: