General information: The SALSA MLPA
Probemix P473 CTNS is a
research use only (RUO) assay for the detection of deletions or duplications in the
CTNS gene, which is associated with cystinosis. This probemix can also be used to detect the presence of the
CTNS c.414G>A (p.Trp138Ter; W138X) point mutation.
Cystinosis is classified as a lysosomal storage disorder and is inherited in an autosomal recessive manner. Cystinosis can be divided in three categories: 1) nephropathic cystinosis, 2) intermediate cystinosis/late-onset juvenile or adolescent nephropathic cystinosis, and 3) ocular non-nephropathic cystinosis. Nephropathic cystinosis is characterised by renal tubular Fanconi syndrome, poor growth, impaired glomerular function and accumulation of cystine in almost all cells, leading to cellular dysfunction with tissue and organ impairment. A typical untreated child has short stature, rickets and photophobia. The first signs of the disease are generally noticed after approximately six months of age. Intermediate cystinosis is characterised by all the typical manifestations of nephropathic cystinosis, but onset is at a later age, usually between the age of 15 and 25 years. Ocular non-nephropathic cystinosis is characterised clinically only by photophobia resulting from corneal cystine crystal accumulation.
Defects in the
CTNS gene on chromosome 17 are the main cause of cystinosis. The
CTNS gene (13 exons) spans ~26.6 kb of genomic DNA and is located on 17p13.3, 3.6 Mb from the p-telomere. The protein encoded by this gene is cystinosin, an integral lysosomal membrane protein. Several articles have described large deletions in the
CTNS gene in individuals affected with cystinosis, such as a 9.5 to 16 kb deletion (Forestier et al. 1999) and a 65 kb deletion (Shotelersuk et al. 1998). The latter deletion was later corrected to a 57 kb deletion that includes the
SHPK (
CARKL) gene (Touchman et al. 2000; Gahl et al. 2002). This 57 kb deletion is found in almost 50% of affected individuals from Northern European descent. The most common point mutation that is found in affected individuals is the c.414G>A (p.Trp138Ter; W138X) mutation. This mutation was found in 14 out of 108 samples (Shotelersuk et al. 1998).
More information is available at
https://www.ncbi.nlm.nih.gov/books/NBK1400/.
Probemix content: The SALSA MLPA Probemix P473-A2 CTNS contains 31 MLPA probes with amplification products between 136 and 364 nucleotides (nt). This includes 16 probes for
CTNS copy number detection, one probe for each exon and two probes for exons 1, 2 and 13, and two flanking probes which detect the adjacent
SHPK gene. Furthermore, this probemix contains one probe specific for the
CTNS c.414G>A (p.Trp138Ter; W138X) mutation which will only generate a signal when the mutation is present. In addition, 12 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.mlpa.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.mlpa.com.
SALSA Binning DNA SD064 The SD064 Binning DNA provided with this probemix can be used for binning of the
CTNS c.414G>A (p.Trp138Ter; W138X) mutation-specific probe (172 nt probe, 21085-L29338). SD064 Binning DNA is a mixture of genomic DNA from healthy individuals and synthetic DNA that contains the target sequence detected by the above mentioned probe. Inclusion of one reaction with 5 μl SD064 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 mutation/SNP 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 SD064 Binning DNA product description, available online:
www.mlpa.com.
Sample DNA
Sample DNA developed for this product: