SALSA MLPA probemix P083-C2 CDH1 Lot C & lot C As compared to previous C version, five reference probes have been replaced.

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1 SALSA MLPA probemix P083-C2 CDH1 Lot C & lot C As compared to previous C version, five reference probes have been replaced. 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, ~68.8 Mb from the p-telomere. The P083-C2 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 height 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 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 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 Molinaro V et al. (2014) Complementary Molecular Approaches Reveal Heterogeneous CDH1 Germline Defects in Italian Patients with Hereditary Diffuse Gastric Cancer (HDGC) Syndrome. Genes Chromosomes Cancer. 53: Sugimoto S et al. (2014) Early-onset diffuse gastric cancer associated with a de novo large genomic deletion of CDH1 gene. Gastric Cancer. 17: Kim S et al. (2012) Searching for E-cadherin gene mutations in early onset diffuse gastric cancer and hereditary diffuse gastric cancer in Korean patients. Fam Cancer. 12: Yamada H et al. (2011) Germline alterations in the CDH1 gene in familial gastric cancer in the Japanese population. Cancer Sci. 102: Oliveira C et al. (2009) Germline CDH1 deletions in hereditary diffuse gastric cancer families. Hum Mol Genet. 18: More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands SALSA P083 CDH1 probemix Page 1 of 5

2 Data analysis The P083-C2 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 height of each probe s amplification product by the total peak height 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 and derived from the same source of tissue. 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. Warning, if this probemix is used on tumour DNA: MLPA analysis on tumour samples provides information on the average situation in the cells from which the DNA sample was purified. Gains or losses of genomic regions or genes may not be detected if the percentage of tumour cells is low. Furthermore, although reference probes are located in stable regions that are not frequently altered in copy number in cancer sample, there is always a possibility that one or more reference probes do show a copy number alteration in a sample. This probemix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P083 CDH1 probemix Page 2 of 5

3 Table 1. SALSA MLPA P083-C2 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 q 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 L26561 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 q11 * New in version C2 (from lot C onwards). ± SNP rs could influence the probe signal at 154 nt, SNPs rs & rs & rs probe signal at 174 nt, rs probe signal at 202 nt, and SNP rs could influence the probe signal at 258 nt. In case of apparent deletions, it is recommended to sequence the region targeted by these probes. Note: Exon numbering might be different as compared to literature! Please notify us of any mistakes: info@mlpa.com. The identity of the genes detected by the reference probes is available in table 2b. SALSA P083 CDH1 probemix Page 3 of 5

4 Table 2. P083 probes arranged according to chromosomal location Table 2a. CDH1 gene. 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) 154 ± 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 258 ± 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 202 ± L01857 Exon CAGGAAATCACA-TCCTACACTGCC 3.7 kb L01858 Exon GGGAGGATTTTG-AGCACGTGAAGA 2.8 kb 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) ± SNP rs could influence the probe signal at 154 nt, SNPs rs & rs & rs probe signal at 174 nt, rs probe signal at 202 nt, and SNP rs could influence the probe signal at 258 nt. In case of apparent deletions, it is recommended to sequence the region targeted by these probes. The NM_ (NG_ ) sequence is a reference standard in the NCBI RefSeqGene project. Table 2b. Reference probes. Length SALSA MLPA Partial sequence Gene Location (nt) probe (24 nt adjacent to ligation site) MV location L05368 LMNA 1q22 CTCCTCTGTTTT-CTCTCTTAGAGC L22132 PPM1B 2p21 AGCAGAAAATCA-TTAGCATTTCCC L06145 SCN2A 2q24.3 TGGTCAACAACT-ACAGTGAGTGCA L06284 WFS1 4p16.1 GGAGACATGGAA-ATCCCCTTTGAA L00463 IL4 5q31.1 ATCGACACCTAT-TAATGGGTCTCA L11281 PKHD1 6p12.2 AGGGTCTGTACT-TCCTGGAAGCAT L16707 LAMA2 6q22.33 GCACCACCTAGG-AGAAAACGAAGG L15525 CLCN1 7q34 TCAAAGGGCATA-TACTGGTACTGG L08733 PCSK5 9q21.13 GTGCAGAGCTGT-AGTATCAGCTAT L06296 ZNF25 10p11.1 CTTGACTGAATT-AATCCTTCAAAA L01250 YAP1 11q22.1 ACAGTGTCCCTC-GAACCCCAGATG L12371 KCNA1 12p13.32 GAGAGTGCTGTT-TATCGTCATTTG L26820 MYH7 14q11.2 GCAGGGAAGACA-GTCAACACCAAG L01904 FBN1 15q21.1 ATCAGTGTGCCT-GCAACCCTGGCT L09234 CACNA1A 19p13.2 CTCAGGCCTTCT-ACTGGACTGTAC L06584 JAM2 21q21.3 TCGTTGTGAAGT-TAGTGCCCCATC L13451 LARGE 22q12.3 AGTACCTCAGGT-AAGGACCTGCAG 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 P083 CDH1 probemix Page 4 of 5

5 SALSA MLPA probemix P083-C2 CDH1 sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P083-C2 CDH1 (lot C2-0215). Implemented Changes compared to the previous product description versions Version 14 (54) 21 April Product description adapted to a new lot (lot number added, new picture included). - Table 2 divided into tables 2a and 2b to include information about the reference probes included in the probemix. - Warnings added to table 1 & 2a: SNP near the ligation site of probe L19938, L19717, L01857, and L Various minor textual changes. Version 13 (53) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - New references added on page 1. - Warning added on page 2, in case this probemix is being used for tumour DNA analysis. Version 12 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 11 (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. SALSA P083 CDH1 probemix Page 5 of 5