SALSA MLPA probemix P398-A1 CASK Lot A1-1014, Lot A

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1 SALSA MLPA probemix P398-A1 CASK Lot A1-1014, Lot A The CASK gene encodes a calcium/calmodulin-dependent serine protein kinase that is a member of the membrane-associated guanylate kinase (MAGUK) protein family. MAGUKs are scaffolding proteins associated with intercellular junctions. CASK is implicated in X-linked mental retardation and MICPCH (mental retardation and microcephaly with pontine and cerebellar hypoplasia) syndrome. The CASK gene (27 exons) spans ~ 408 kb of genomic DNA and is located on Chr Xp11.4, about 42Mb from the p-telomere. The P398-A1 probemix contains one probe for each exon of the gene, except exon 24, and two probes for exon 1, 2 and 3. Finally, 9 reference probes are included in this probemix, detecting several different locations on the X-chromosome. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned gene(s) in a DNA sample. Deletions of a probe s recognition sequence on the X-chromosome will lead to a complete absence of the corresponding probe amplification product in males, whereas female heterozygotes are recognisable by a 35-50% reduction in relative peak height. Note that a mutation or polymorphism in the sequence detected by a probe can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some probe signals are more sensitive to sample purity and small changes in experimental conditions. Therefore, deletions and duplications detected by MLPA should always be confirmed by other methods. Not all deletions and duplications detected by MLPA will be pathogenic; users should always verify the latest scientific literature when interpreting their findings. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. Finally, note that most defects in this gene are expected to be small (point) mutations which will not be detected by this SALSA test. SALSA probemixes and reagents are sold by for research purposes and to demonstrate the possibilities of the MLPA technique. They are not CE/FDA certified for use in diagnostic procedures. Purchase of the SALSA test probemixes and reagents includes a limited license to use these products for research purposes. The use of a SALSA probemix and reagents requires a thermocycler with heated lid and sequence type electrophoresis equipment. Different fluorescent PCR primers are available. The MLPA technique has been first described in Nucleic Acid Research 30, e57 (2002). More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands Related SALSA probemixes P106 MRX: Contains probes for 16 genes, involved in X-linked mental retardation. P013 ATRX: Contains probes for the ATRX gene on the X chromosome, involved in alphathalassemia/mental retardation. References Moog, U. et al., Phenotypic and molecular insight into CASK-related disorders in male. Orphanet Journal of Rare Diseases. 10(1):44 Data analysis The P398-A1 CASK probemix contains 38 MLPA probes with amplification products between 130 and 445 nt. In addition, it contains 9 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at nt, three DNA Denaturation control fragments (Dfragments) at nt, one X-fragment at 100 nt and one Y-fragment at 105 nt. More information on how to interpret observations on these control fragments can be found in the MLPA protocol. SALSA P398 CASK probemix Page 1 of 6

2 Data generated by this probemix can be intra-sample normalised by dividing the peak height of each amplification product by the total peak height of only the reference probes in the probemix (block normalisation). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalised probe ratio in a sample by the average intra-normalised probe ratio of all reference samples. Please note that this type of normalisation assumes that no changes occurred in the genomic regions recognised by the reference probes. It is recommended to use reference and patient samples of the same sex to minimize variation, but this is not strictly necessary. Sex determination can also be done by visual examination of the electropherogram. Data normalisation should be performed within one experiment. Only samples purified by the same method should be compared. Confirmation of most exons deletions and amplifications can be done by e.g. Southern blotting, long range PCR, qpcr, FISH. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website Many copy number alterations in healthy individuals are described in the database of genomic variants: For example, a duplication of a complete gene might not be pathogenic, while a partial duplication or a deletion may result in disease. For some genes, certain in-frame deletions may result in a very mild, or no disease. Copy number changes of reference probes are unlikely to be the cause of the condition tested for. Users should always verify the latest scientific literature when interpreting their findings. This probemix was developed by R. Boschloo at. In case the results obtained with this probemix lead to a scientific publication, it would be very much appreciated if the probemix designer could be included as co-author. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P398 CASK probemix Page 2 of 6

3 Table 1. SALSA MLPA P398-A1 CASK probemix Length Chromosomal position SALSA MLPA probe (nt) reference CASK Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA D-fragments: Low signal of 88 or 96 nt fragment indicates incomplete denaturation 100 X-fragment: Specific for the X chromosome 105 Y-fragment: Specific for the Y chromosome 130 Reference probe L18702 Xq CASK probe L19245 Exon «CASK probe L19246 Exon CASK probe L19247 Exon Reference probe L15127 Xp CASK probe L20191 Exon CASK probe L19249 Exon CASK probe L20192 Exon CASK probe L19251 Exon CASK probe L19252 Exon CASK probe L20193 Exon CASK probe L20194 Exon CASK probe L19255 Exon Reference probe L17182 Xq CASK probe L19256 Exon CASK probe L19257 Exon CASK probe L19258 Exon CASK probe L19259 Exon Reference probe L14320 Xq CASK probe L19260 Exon CASK probe L19261 Exon CASK probe L19262 Exon CASK probe L19263 Exon Reference probe L09794 Xp CASK probe L19264 Exon CASK probe L19265 Exon CASK probe L19267 Exon Reference probe L13690 Xq CASK probe L19268 Exon CASK probe L19269 Exon CASK probe L19270 Exon CASK probe L19271 Exon Reference probe L06668 Xp CASK probe L19272 Exon CASK probe L19273 Exon «CASK probe L19274 Exon Reference probe L13929 Xq Reference probe L15488 Xq22 «This probe is located within, or close to, a very strong CpG island. A low signal of this probe can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. Note: Exon numbering used here may differ from literature! Please notify us of any mistakes. The identity of the genes detected by the reference probes is available on request: info@mlpa.com. SALSA P398 CASK probemix Page 3 of 6

4 Table 2. CASK probes arranged according to chromosomal location Length (nt) SALSA MLPA probe CASK exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (exon 1) 427 «16701-L19274 Exon CCGACGACGACG-TGCTGTTCGAGG kb 142 «16673-L19246 Exon 1 26nt after exon 1 GAGGGAGCGGGA-GTGTGGAAAAAT 0.1 kb L19263 Exon GGGTCCCTTCAG-TGTTGTACGACG 69.7 kb L19251 Exon 2 1nt after exon 2 AAGTACAGAAGG-TAAGAGATGGAT 0.1 kb L20194 Exon TCTAAAGCGGGA-AGCCAGTATCTG 65.8 kb L19268 Exon TTTACATGGTTT-TCGAATTGTGAG 0.1 kb L19249 Exon GTTTTGAAATCG-TAAAGCGAGCTG 41.6 kb L19267 Exon CATGATAATAAC-ATAATTCACAGG 6.2 kb L19272 Exon CTTTGGGGTAGC-TATTCAATTAGG 67.9 kb L19261 Exon TTATCCTGCTCA-GTGGTTGTTTGC 6.1 kb L19265 Exon TGAAAGTGCCAA-AGACCTAGTACG 4.8 kb L19257 Exon CGCCTACAAGAT-TCATCTTCCAGA 23.9 kb L19259 Exon AGTCACAAATTC-AACTCATTCTAT 10.0 kb L19270 Exon 11 56nt after exon 11 AAGTGTTTGGCT-TTGCATGGTTCG 4.1 kb L19252 Exon GACAGCCTGGAA-GAGATTCATGCG 12.6 kb L19264 Exon 13 87nt after exon 13 TTAAGAGGCAGT-TTCCCTGGAGTC 20.6 kb L19258 Exon TTTAACACAACC-TCATTTCATGGT 2.5 kb L19273 Exon GACGTAGTGGCA-CATGAAGTTTAC 8.4 kb L19269 Exon AATCATTGTATT-GTTGCAAGAATT 8.8 kb L19271 Exon ATCAGTGTGGCT-AACCAAACAGTG 8.1 kb L19255 Exon 18 27nt after exon 18 AACCACCCCCAT-CTGCTCTGAGTT 1.8 kb L19262 Exon ACTTCCAGACAG-TCCCCAGCTAAT 2.7 kb L20191 Exon CTACAGGACTTG-CCATCAACTACC 1.4 kb L19260 Exon 21 9nt before exon 21 TTACGAACTCCT-GTTTTATAGATC 1.7 kb L19256 Exon CATGGAGAAGAC-CAAACAGGAGCA 11.1 kb L19247 Exon ACACTAGTCTTA-TTAGGTAAGTTT 7.9 kb No probe Exon L20193 Exon AGACATCTCTAA-TAACGAGTACTT 3.8 kb L20192 Exon 26 93nt after exon 26 ACATTTGCAGCA-AGGCTGCTCTGA 7.3 kb L19245 Exon ACTCTTAATGCA-TGTGGACAGTCG 4.1 kb stop codon (exon 27) «This probe is located within, or close to, a very strong CpG island. A low signal of this probe can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Note: Exon numbering used here may differ from literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA P398 CASK probemix Page 4 of 6

5 SALSA MLPA probemix P398-A1 CASK sample pictures Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P398-A1 CASK (lot A1-1014). Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng human female control DNA analysed with SALSA MLPA probemix P398-A1 CASK (lot A1-1014). SALSA P398 CASK probemix Page 5 of 6

6 Implemented Changes compared to the previous product description versions. Version November 2015 (55) - Product description adapted to a new lot (lot number added, new picture included). Version June 2015 (54) - Electropherogram pictures of the old buffer (introduced Dec. 2012) removed. Version 02 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 01 (47) - Not applicable, new document. SALSA P398 CASK probemix Page 6 of 6