MRC-Holland MLPA. Description version 12;

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1 SALSA MLPA probemix P083-C1 CDH1 Lot C As compared to previous B1 version, new in version C1: two CDH1 probes and several reference probes have been replaced/added. In addition, the 88 and 96nt control fragments have been replaced (QDX2). Germline mutations in the CDH1 gene have been reported in families with a hereditary predisposition to gastric cancer. CDH1 or E-cadherin is a classical cadherin from the cadherin superfamily. The encoded protein is a calcium dependent cell-cell adhesion glycoprotein. Reduced expression of E-cadherin is regarded as one of the main molecular events involved in dysfunction of the cell-cell adhesion system, triggering cancer invasion and metastasis. Loss of function is thought to contribute to progression in cancer by increasing proliferation, invasion, and/or metastasis. The ectodomain of this protein mediates bacterial adhesion to mammalian cells and the cytoplasmic domain is required for internalization. The CDH1 gene (16 exons) spans ~98.2 kb of genomic DNA and is located on chromosome 16q22.1, ~67.4 Mb from the p-telomere. The P083-C1 probemix contains probes for each CDH1 exon. In addition, 17 reference probes are included in this probemix, detecting several different autosomal chromosomal locations. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned gene in a DNA sample. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak area of the amplification product of that probe. Note that a mutation or polymorphism in the sequence detected by a probe can also cause a reduction in relative peak area, 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 a single MLPA probe 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 this gene is caused by deletions/duplications of complete exons. Finally, note that most defects in this gene are expected to be small (point) mutations, most of 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). References for SALSA probemix P083 CDH1 Oliveira C et al. (2009) Germline CDH1 deletions in hereditary diffuse gastric cancer families. Human Molecular Cancer 18: More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands SALSA probemix P083 CDH1 Page 1 of 6

2 Data analysis The P083-C1 CDH1 probemix contains 34 MLPA probes with amplification products between 130 and 409 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. Data generated by this probemix can first be normalised intra-sample by dividing the peak area of each probe s amplification product by the total area of only the reference probes in this probemix (block normalisation). Secondly, inter-sample normalisation can be achieved by dividing this 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 no changes occurred in the genomic regions recognised by the reference probes. 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 at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix P083 CDH1 Page 2 of 6

3 Table 1. SALSA MLPA P083-C1 CDH1 probemix Length (nt) SALSA MLPA probe Chromosomal position reference CDH 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 L q * Reference probe L p * Reference probe L p CDH1 probe L19725 Exon CDH1 probe L19938 Exon CDH1 probe L19374 Exon * Reference probe L p * CDH1 probe L19717 Exon * Reference probe L q CDH1 probe L19369 Exon CDH1 probe L01857 Exon * Reference probe L p Reference probe L q CDH1 probe L01858 Exon Reference probe L q * Reference probe L q CDH1 probe L19937 Exon CDH1 probe L19371 Exon * Reference probe L p CDH1 probe L01860 Exon * Reference probe L q CDH1 probe L01853 Exon * Reference probe L p CDH1 probe L19372 Exon CDH1 probe L13240 Exon CDH1 probe L01862 Exon * Reference probe L p * Reference probe L q CDH1 probe L19936 Exon Reference probe L q * CDH1 probe L19718 Exon CDH1 probe L14803 Exon * Reference probe L q * Reference probe L q26 * New in version C1 (from lot 0211 onwards). Changed in version C1 (from lot 0211 onwards): Small change in length, no change in sequence detected. Note: Exon numbering might be different as compared to 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 probemix P083 CDH1 Page 3 of 6

4 Table 2. CDH1 probes arranged according to chromosomal location Length (nt) SALSA MLPA probe CDH1 exon Ligation site in NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 1) L19938 Exon nt after ex 1 AGCGGCCTGGAA-GCCTCGCGCGCT 0.3 kb L14803 Exon nt before ex 2 CCGGGGATAAGA-AAGTGAGGTCGG 0.4 kb L19369 Exon CGCCGAGAGCTA-CACGTTCACGGT 63.5 kb L19937 Exon ACTCCACCTACA-GAAAGTTTTCCA 6.7 kb L19936 Exon TCAGAAGACAGA-AGAGAGACTGGG 0.2 kb L01853 Exon AGGTTTTCTACA-GCATCACTGGCC 1.5 kb L13240 Exon TCCAACGGGAAT-GCAGTTGAGGAT 1.5 kb L19374 Exon GACGCGGACGAT-GATGTGAACACC 0.4 kb L19725 Exon CCTGGTGGTTCA-AGCTGCTGACCT 1.3 kb 174 * L19717 Exon AGTGAACAACGA-TGGCATTTTGAA 2.2 kb L01857 Exon CAGGAAATCACA-TCCTACACTGCC 3.7 kb L01858 Exon GGGAGGATTTTG-AGCACGTGAAGA 2.8 kb 382 * L19718 Exon CATTCAGTACAA-CGACCCAAGTGG 1.3 kb L19371 Exon AGTGACCACCTT-AGAGGTCAGCGT 4.7 kb L01860 Exon TGCTGTTTCTTC-GGAGGAGAGCGG 1.5 kb L19372 Exon GTGACTCGTAAC-GACGTTGCACCA 3.7 kb L01862 Exon CCGAAGCTGCTA-GTCTGAGCTCCC stop codon (ex 16) * New in version C1 (from lot 0211 onwards). Changed in version C1 (from lot 0211 onwards): Small change in length, no change in sequence detected. The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Note: Exon numbering might be different as compared to literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA probemix P083 CDH1 Page 4 of 6

5 SALSA MLPA probemix P083-C1 CDH1 sample picture D ye S ign al Size (nt) Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P083-C1 CDH1 (lot 0211). The old MLPA buffer (replaced in December 2012) was used. Vials with the old MLPA buffer have a white label D ye S ign al Size (nt) Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P083-C1 CDH1 (lot 0211). The new MLPA buffer (introduced in December 2012) was used. Vials with the new MLPA buffer have a yellow label. SALSA probemix P083 CDH1 Page 5 of 6

6 Implemented Changes compared to the previous product description versions Version 12 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 10 (48) - Remark on RefSeqGene standard added below Table 2. - Various minor textual changes. Version 10 (46) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Reference article has been added on page 1 - Various minor layout changes. Version 09 (46) - Warning added: SNP at ligation site of probe L Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Data analysis method has been modified. - Various minor textual changes on page 1. - Various minor layout changes. SALSA probemix P083 CDH1 Page 6 of 6