The SALSA MLPA Probemix P436 ANO5 is a research use only (RUO)
assay for the detection of deletions or duplications in the ANO5
gene, which is associated with limb girdle muscular dystrophy type 2L (LGMD2L). This probemix can also be used to detect the presence of the ANO5
c.191dupA point mutation.
LGMD2L or anoctaminopathy is a condition mainly characterised by adult onset proximal lower limb muscular weakness and raised creatine kinase (CK) values, due to recessive anoctamin 5 (ANO5
) gene mutations. This gene encodes a member of the anoctamin family of transmembrane proteins, and the encoded protein is likely a calcium activated chloride channel. An exon 5 founder mutation (c.191dupA) represents 61% of mutated alleles and appears to be more prevalent in Northern European populations (Sarkozy et al. 2013). The c.191dupA mutation leads to a frameshift and to premature truncation, which strongly suggests that c.191dupA is associated with a loss of ANO5
function (Bolduc et al. 2010).
gene (22 exons) consists of ~90 kb of genomic DNA and is located at 11p14.3, 22 Mb from the p-telomere.
More information is available at https://www.ncbi.nlm.nih.gov/books/NBK114459
The SALSA MLPA Probemix P436-A2 ANO5 contains 37 MLPA probes with amplification products between 136 and 391 nucleotides (nt). This includes 27 probes for ANO5
copy number detection. This probemix contains one probe for every exon, two probes for exon 1, 7 and 19 and three probes for exon 22. Furthermore, this probemix contains one probe specific for the ANO5
c.191dupA mutation which will only generate a signal when the mutation is present (see P436 specific notes). The exon 5 probe detects the wild-type sequence of the c.191dupA mutation, which means that its signal will decrease when the mutation is present. In addition, nine 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 (see table below). 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
SALSA Binning DNA SD033:
The SD033 Binning DNA provided with this probemix can be used for binning of the ANO5
c.191dupA mutation-specific probe (198 nt probe, 18658-SP0690-L24012). SD033 Binning DNA is a mixture of genomic DNA from healthy individuals and plasmid DNA that contains the target sequence detected by the above mentioned probe. Inclusion of one reaction with 5 μl SD033 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 signals, as for this purpose true mutation positive patient samples or cell lines should be used. It is strongly advised that all samples tested are extracted with the same method and derived from the same source of tissue. For further details, please consult the SD033 Binning DNA product description, available online: www.mrcholland.com
Sample DNA developed for this product: