MRC-Holland MLPA. Description version 29;

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1 SALSA MLPA KIT P003-B1 MLH1/MSH2 Lot 1209, As compared to the previous lots 0307 and 1006, one MLH1 probe (exon 19) and four MSH2 probes have been replaced. In addition, one extra MSH2 exon 1 probe, two extra EPCAM (=TACSTD1) probes and two extra control fragments at 100 and 105 nt have been included. Germ-line defects in the DNA mismatch repair genes MSH2 and MLH1 are the major cause of hereditary nonpolyposis colon cancer (HNPCC; Lynch syndrome). Detection of the germ-line defect in HNPCC families allows identification of relatives who require appropriate surveillance and prevents useless surveillance in noncarrier relatives. A significant percentage of the HNPCC germ-line mutations are genomic deletions of one or more exons of the MLH1 and MSH2 genes (Wijnen J.T. et al (1998) Nature Genet. 20: ). In a study in which 126 colorectal cancer families were screened, a germ-line mutation was detected in 30% of the families (Gille, J.J.P. et al (2002) British J. of Cancer 87, ). Deletions of one or more MLH1/MSH2 exons were detected with the use of this P003 MLPA kit, and accounted for 46% of all mutations detected. Many studies have shown that genomic rearrangements of MLH1 and MSH2 are also an important cause of HNPCC in other countries. In the study of Gille et al, copy number changes of one or more exons were detected in 17 out of 126 families (13.5%). However, this percentage will depend on the way the cohort is selected. The MLH1 gene contains 19 exons and spans 100 kb on chromosome 3p22.1. The MSH2 gene contains 16 exons and covers 73 kb on chromosome 2p21. The P003 probemix contains probes for each of the 19 exons of the MLH1 gene, for each of the 16 exons of the MSH2 gene. From version B1 onwards, two probes are included for the most 3 exon of EPCAM (formerly known as TACSTD1), a gene located just upstream of MSH2. Deletions of the most 3 exon of EPCAM can result in silencing of the MSH2 gene (Ligtenberg M.J.L. et al. (2009) Nature Genet. 41: ). Finally, 7 probes for other human genes located on different chromosomes are included for reference purposes. This SALSA kit is designed to detect deletions/duplications of one or more sequences in the MLH1 and MSH2 genes and the most 3 exon of EPCAM 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 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 kits are sold by for research purposes and to demonstrate the possibilities of the MLPA technique. This kit is not CE/FDA certified for use in diagnostic procedures. SALSA MLPA kits are supplied with all necessary buffers and enzymes. Purchase of the SALSA MLPA test kits includes a limited license to use these products for research purposes. The use of this SALSA kit 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). Related SALSA MLPA kits P248 MLH1/MSH2: Recommended for confirmation of deletions / duplications detected by the P003 kit. P072 MSH6: Contains probes for MSH6, MUTYH, MLH1 and MSH2 upstream regions. P008 PMS2: Contains probes for the complicated PMS2 gene. ME011 Mismatch Repair genes (MMR): Methylation profiling of MLH1, MSH2, MSH6, PMS2 and MGMT. P043 APC: Hereditary polyposis colon cancer, gene included APC 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 kit P003 MLH1-MSH2 Page 1 of 6

2 References for SALSA MLPA kit P003 Aissi-Ben Moussa, S. et al (2009). Identification and characterization of a novel MLH1 genomic rearrangement as the cause of HNPCC in a Tunisian family: evidence for a homologous Alu-mediated recombination. Fam Cancer 8 (2): Stella A. et al. (2007). Germline novel MSH2 deletions and a founder MSH2 deletion associated with anticipation effects in HNPCC. Clin Genet Feb;71(2): Pistorius S. et al. (2006). Genomic rearrangements in MSH2, MLH1 or MSH6 are rare in HNPCC patients carrying point mutations. Cancer Lett Jul 10. Niesen RC et al. (2006). Identification of mismatch repair gene mutations in young colorectal cancer patients and patients with multiple HNPCC-associated tumours. Gut Apr 24 Zhang J et al. (2006). Gene conversion is a frequent mechanism of inactivation of the wild-type allele in cancers from MLH1/MSH2 deletion carriers. Cancer Res Jan 15;66(2): Grabowski, M. et al. (2005). Deletions Account for 17% of Pathogenic Germline Alterations in MLH1 and MSH2 in Hereditary Nonpolyposis Colorectal Cancer (HNPCC) Families. Genet Test. 9(2): Wehner, M. et al. (2005). Hereditary nonpolyposis colorectal cancer: pitfalls in deletion screening in MSH2 and MLH1 genes. Eur J Hum Genet May 4. McVety et al. (2005). Novel genomic insertion-deletion in MLH1: possible mechanistic role for non-homologous endjoining DNA repair. Clin Genet Sep;68(3): Ainsworth, P.J. et al. (2004). Family cancer histories predictive of a high risk of hereditary non-polyposis colorectal cancer associate significantly with a genomic rearrangement in hmsh2 or hmlh1. Clin. Genet. 66: Bunyan DJ et al. (2004). Dosage analysis of cancer predisposition genes by multiplex ligation-dependent probe amplification. Br J Cancer 13;91(6): Taylor, C.F. et al. (2003) Genomic deletions in MSH2 or MLH1 are a frequent cause of hereditary non-polyposis colorectal cancer: identification of novel and recurrent deletions by MLPA. Human Mutation 22: Nakagawa, H. et al. (2003). Identification and characterization of genomic rearrangements of MSH2 and MLH1 in Lynch Syndrome (HNPCC) by novel techniques. Human Mutation 22: 258. Gille, J.J. et al. (2002). Genomic deletions of MSH2 and MLH1 in colorectal cancer families detected by a novel mutation detection approach. Br. J. Cancer 87: Data analysis The P003-B1 MLH1-MSH2 probemix contains 46 different MLPA probes with amplification products between 130 and 490 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 (D-fragments) 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 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 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 or long range PCR. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website mrna analysis We do not recommend the use of this probemix for the analysis of MLH1 and MSH21 mrnas/cdna, although it is possible. The MSH2 exon 11 probe is directed to the strand present in U The MLH1 exon 18 probe is directed to the strand present in U These probes will not detect cdna. All other probes are directed to the complement of NM_ /NM_ and most will bind to cdna. However, some probes extent to within an intron sequence and may not bind to cdna as efficiently as they do to DNA. As the ligation site of probe MLH1 exon 16 is one nucleotide in front of the start of this exon, it will certainly not bind to cdna. Complete probe sequences are available upon request. This probemix was developed by. Info/remarks/suggestions for improvement: info@mlpa.com SALSA kit P003 MLH1-MSH2 Page 2 of 6

3 Table 1. SALSA MLPA P003-B1 MLH1 / MSH2 probemix Length Chromosomal position SALSA MLPA probe (nt) reference MLH1 MSH 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 MLH1 probe L00474 Exon * MSH2 probe L02162 Exon MLH1 probe L00577 Exon MSH2 probe L00494 Exon MLH1 probe L00476 Exon MSH2 probe L00601 Exon MLH1 probe L00477 Exon MSH2 probe L00496 Exon * MSH2 probe L12006 Exon Reference probe L p MLH1 probe L06064 Exon MSH2 probe L00497 Exon * MSH2 probe L14624 Exon MLH1 probe L14625 Exon ± MSH2 probe L00498 Exon MLH1 probe L00480 Exon * MSH2 probe L12398 Exon MLH1 probe L00481 Exon MSH2 probe L00582 Exon MLH1 probe L00482 Exon Reference probe L q MSH2 probe L00583 Exon MLH1 probe L00483 Exon * MSH2 probe L12007 Exon MLH1 probe L00484 Exon MSH2 probe L00503 Exon MLH1 probe L00485 Exon MSH2 probe L00504 Exon MLH1 probe L00486 Exon MSH2 probe L00575 exon Reference probe L q MLH1 probe L00586 exon MSH2 probe L00506 Exon MLH1 probe L00488 Exon MSH2 probe L00585 Exon MLH1 probe L00576 Exon MSH2 probe L14623 Exon MLH1 probe L00602 Exon MLH1 probe L00603 Exon * MLH1 probe L12994 Exon Reference probe L p * EPCAM probe L14404 upstream 481 * EPCAM probe L03603 upstream 490 * Reference probe L q12 * New in version P003-B1 (from lot 0109 onwards) Changed in version B1 (from lot 0109 onwards). Small change in length. No change in sequence detected. ± SNP rs may influence probe signal. In case of apparent deletions, sequence probe s target region. The 337 nt MLH1 exon 12 has been found to give false deletions, the occurrence of which may be dependent on the SALSA kit P003 MLH1-MSH2 Page 3 of 6

4 extraction method used. The exon 12 probe in the P248 MLPA probemix does not have this problem and can be used for confirmation. Note: The identity of the genes detected by the reference probes is available on request: Table 2. P003 probes arranged according to chromosomal location Table 2a. MLH1 gene Length (nt) SALSA MLPA probe MLH1 exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe startcodon L00474 exon CCAAAATGTCGT-TCGTGGCAGGGG 3.1 Kb L00577 exon GATTCAGATCCA-AGACAATGGCAC 4.3 Kb L00476 exon TGCAGTCCTTTG-AGGATTTAGCCA 3.4 Kb L00477 exon CATAAGCCATGT-GGCTCATGTTAC 2.6 Kb L06064 exon AAACCATGTGCT-GGCAATCAAGGG 1.8 Kb L14625 exon TGGAGGACCTTT-TTTACAACATAG 3.0 Kb L00480 exon ACACAATGCAGG-CATTAGTTTCTC 0.2 Kb L00481 exon TGTTAGGACACT-ACCCAATGCCTC 2.4 Kb L00482 exon CCTAGCCTTCAA-AATGAATGGTTA 3.1 Kb L00483 exon TTCCTTGAGAAA-AGCCATAGAAAC 2.9 Kb L00484 exon GCAGCACATCGA-GAGCAAGCTCCT 5.5 Kb L00485 exon AGGCAGCAAGAT-GAGGAGATGCTT 3.0 Kb L00486 exon TCCCGAAAGGAA-ATGACTGCAGCT 11.4 Kb L00586 exon CGTGGGCTGTGT-GAATCCTCAGTG 2.1 Kb L00488 exon CCAGATACTCAT-TTATGATTTTGC 5.3 Kb L00576 exon CATGCTTGCCTT-AGATAGTCCAGA 1.0 Kb L00602 exon ATCTTCATTCTT-CGACTAGCCACT 0.4 Kb L00603 exon reverse CTCCTCAGATAT-GTACTGCTTCCG 1.8 Kb 454 * L12994 exon reverse TATCAGAAGGCA-AGTATAAGTCTT stopcodon * New in version P003-B1 (from lot 0109 onwards) Changed in version B1 (from lot 0109 onwards). Small change in length. No change in sequence detected. Note: The MLH1 exon numbering in Table 2a is different as compared to the new exon numbering that is used by the NCBI in the NM_ reference sequence! We used the same exon numbering as in all previous versions of this product description. Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA kit P003 MLH1-MSH2 Page 4 of 6

5 Table 2b. MSH2 gene Length (nt) SALSA MLPA probe MSH2 exon Ligation site Partial sequence (24 nt adjacent to ligation site) Distance to next probe NM_ EPCAM stopcodon ** L03603 EPCAM exon AAATGGACACAA-ATTACAAATGTG 0.1 Kb 472 ** L14404 EPCAM exon reverse GGTCAAATTTCA-AGATTGGTAAAG 16.0 Kb to MSH2 NM_ startcodon * L14624 exon nt before ATG startcodon CCGGGCACATTA-CGAGCTCAGTGC 0.2 Kb 148 * L02162 exon nt before ATG startcodon GCGTGCGCGGGA-AGCTGGGCCGCG 0.7 Kb 190 * L12006 exon nt after exon 1 reverse GAACTAGAACAA-TGCATTAAAATG 4.8 Kb L00494 exon TATAGAGTTGAA-GTTTATAAGAAT 1.7 Kb L00601 exon TTGTGGGTGTTA-AAATGTCCGCAG 2.3 Kb L00496 exon CGGTTGTTGAAA-GGCAAAAAGGGA 1.8 Kb L00497 exon ACTGTCTGCGGT-AATCAAGTTTTT 2.1 Kb L00498 exon GCCTTGCTGAAT-AAGTGTAAAACC 13.5 Kb 247 * L12398 exon AGACAAGCAGCA-AACTTACAAGAT 15.7 Kb L00582 exon CTCCTCTTACTG-ATCTTCGTTCTG 17.5 Kb L00583 exon AATTCCTTGTAA-AACCTTCATTTG 3.9 Kb 310 * L12007 exon nt after exon 10 GACTGAAGTGGT-ACTTTGGGTCTA 4.0 Kb L00503 exon reverse GCTTCTTCATAT-TCTGTTTTATTT 4.1 Kb L00504 exon CACCTGTTCCAT-ATGTACGACCAG 1.3 Kb L00575 exon TCATGGCCCAAA-TTGGGTGTTTTG 1.9 Kb L00506 exon CCTACGATGGAT-TTGGGTTAGCAT 2.5 Kb L00585 exon ACTTGAGGAGTT-TCAGTATATTGG 2.0 Kb L14623 exon GTGTTTCAGCAA-GGTGAAAAAATT stopcodon * New in version P003-B1 (from lot 0109 onwards) Changed in version B1 (from lot 0109 onwards). Small change in length. No change in sequence detected. ** The 481 nt EPCAM (=TACSTD1) probe detects the same sequence as the 190 nt probe in P008 PMS2 and the 310 nt probe in P072-B1 MSH6. This probe might be influenced by the RS polymorphism. Simultaneous deletion of the 472 and 481 nt probes is a strong indication that the last EPCAM exon (exon 9) is disrupted, which can lead to methylation and inactivation of MSH2 (Ligtenberg M.J.L. et al.(2009) Nature Genet. 41: ). More probes for EPCAM are available in SALSA MLPA kit P072 MSH6. We recommend usage of SALSA MLPA kit ME011 MMR to test the methylation status of the MSH2 promoter and of other mismatch repair genes. 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 kit P003 MLH1-MSH2 Page 5 of 6

6 SALSA MLPA kit P003-B1 MLH1-MSH2 sample picture ,77 128,12 145, ,40 182,90 237,99 229,45 328, ,48 202,65 223,79 216, ,97 141,18 165,28 344,03 105, ,21 90,71 195,74 189,00 176,59 210,38 246,49 256,21 263,18 301,96 291,10 283,46 319,18 310,68 352,47 401,53 408,23 391,42 427,69 454,78 482,29 472, ,10 272,84 335,03 363,89 374,82 444, ,37 382,32 419,15 436,63 462,57 492, Dye Signal Size (nt) Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analyzed with SALSA MLPA kit P003-B1 MLH1-MSH2 (lot 1209). Implemented Changes the following has been altered compared to the previous product description version(s). Version 29 (45) - Warning added about the possible presence of a SNP in the target sequence of the prober targeting exon 6 of MSH2. Version 28 (45) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Ligation sites updated according to new version of the NM_reference sequence. - Warning added below Table 2a that the MLH1 exon numbering used is different from the NCBI exon numbering in the NM_ mrna reference sequence. Version 27 (45) - Reference added of Aissi-Ben Moussa, S. et al (2009). Minor textual and layout changes. Version 26 (44) - Minor changes in the product description on page 1 and in the data analysis section on page 2. - Minor changes on table titles. SALSA kit P003 MLH1-MSH2 Page 6 of 6