The SALSA MLPA Probemix P460 SMA is a research use only (RUO)
assay for the detection of deletions or duplications in the SMN1
genes, which are associated with Spinal Muscular Atrophy (SMA). This Probemix can also be used to detect the presence of two polymorphisms, g.27134T>G and g.27706-27707delAT, for haplotype identification.
Spinal Muscular Atrophy (SMA) is a neuromuscular disorder characterised by degeneration of the anterior horn cells of the spinal cord, leading to symmetrical muscle weakness and atrophy. SMA is the second most common lethal autosomal recessive disorder in Caucasians, after cystic fibrosis. SMA is usually divided into three clinical groups. Patients with type I SMA (MIM# 253300) show onset at birth or before six months, and usually die of respiratory insufficiency within two years. Type I SMA patients are never able to sit or walk. Patients with type II SMA (MIM# 253550) show onset after 6 months. They can sit but are never able to walk unaided, and their life expectancy is significantly reduced. Type III SMA (MIM# 253400) patients show the first symptoms after 18 months and are able to stand and walk, but often become wheelchair-bound during youth or adulthood.
There are two (highly-similar) genes playing a pivotal role in SMA: SMN1
. The telomeric SMN1
and the centromeric SMN2
genes are located in a complicated inverted repeat area spanning ~500 kb on chromosome 5q13.2. This area displays high instability, leading to frequent deletions and gene conversions. Most individuals have two copies each of SMN1
. The SMN1
genes, each having nine exons (exons 1, 2a, 2b, and 3-8), can only be distinguished by two single nucleotide differences: one in exon 7 and one in exon 8. The exon 8 difference has no effect on the transcript, however, the exon 7 difference disrupts a putative exonic splicing enhancer in SMN2
. Only 10-15% of the SMN2
transcripts are functional. This SALSA MLPA probemix P460 SMA detects the copy number of exon 7 and exon 8 of the SMN1
gene and exon 7 of the SMN2
Absence of any functional SMN1
copy results in insufficient amounts of full length transcripts. More than 95% of SMA patients show homozygous deletion of at least exon 7 of the telomeric SMN1
gene. The great majority of SMA carriers can be identified by the presence of only a single SMN1
exon 7 copy. The one copy frequency in the US is estimated to be 1:37 for Caucasians, 1:46 for Ashkenazi Jews, 1:56 for Asians, 1:91 for African-Americans and 1:125 for Hispanics (Hendrickson et al. 2009). The SMN2
copy number is very variable with only 60-70% of individuals having two copies.
The presence of SMN2
results in a small amount of full length transcripts. Establishing the number of the SMN2
copy number is important for SMA patients: the more SMN2
copies a patient has, the more functional SMN protein is produced and the milder the SMA phenotype is. The majority of type I SMA patients carry a homozygous deletion of SMN1
and a normal or reduced number of SMN2
copies. The majority of the type II and III SMA patients show homozygous absence of SMN1
and an increased number of SMN2
copies (3-4 copies). A homozygous deletion of SMN2
is found in about 5% of healthy individuals. This has no clinical phenotype when at least one functional SMN1
copy is present. Healthy individuals lacking SMN1
but having five or more SMN2
copies have been described.
More information on Spinal Muscular Atrophy is available at http://www.ncbi.nlm.nih.gov/books/NBK1352/
This SALSA MLPA Probemix is not CE/FDA registered for use in diagnostic procedures. Purchase of this product includes a limited MLPA license for research purposes.
The SALSA MLPA Probemix P460-A1 SMA contains 23 MLPA probes with amplification products between 131 and 331 nt. This includes 3 probes for the SMN1
genes. Furthermore, it also contains 2 probe(s) specific for the polymorphisms g.27134T>G and g.27706-27707delAT, which will only generate a signal when a polymorphism is present. In addition, 18 reference probes are included and detect 18 different autosomal chromosomal locations. Complete probe sequences and the identity of the genes detected by the reference probes is available online (www.mlpa.com).
This Probemix contains nine quality control fragments generating amplification products between 64 and 121 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 Binning DNA SD042:
The SD042 Binning DNA provided with this Probemix can be used as Binning DNA sample for binning of the g.27134T>G and g.27706-27707delAT polymorphism-specific probes (143 nt SMN1 probe S0938-L26163 g.27134T>G and 148 nt SMN1 probe S0961-L25586 g.27706-27707delAT). SD042 Binning DNA is a mixture of female genomic DNA from healthy individuals and plasmid DNA that contains the target sequence detected by the above mentioned probes. Inclusion of one reaction with 5 μl SD042 Binning DNA in initial MLPA experiments is essential as it can be used to aid in data binning of the peak pattern using Coffalyser.Net software. Furthermore, Binning DNA should be included in the experiment whenever changes have been applied to the set-up of the capillary electrophoresis device (e.g. when capillaries have been renewed). Binning DNA should never be used as a reference sample in the MLPA data analysis, neither should it be used in quantification of mutation signal(s), as for this purpose true polymorphism positive patient samples or cell lines should be used. 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 further details, please consult the SD042 Binning DNA product description provided. This product is for research use only (RUO).
Sample DNA developed for this product: