SALSA MLPA KIT P003-B1 MLH1/MSH2 Lot 1209, 0109. 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: 326-328). 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, 892-897). 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: 112-117). 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 : www.mlpa.com E-mail : 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
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): 119-26. Stella A. et al. (2007). Germline novel MSH2 deletions and a founder MSH2 deletion associated with anticipation effects in HNPCC. Clin Genet. 2007 Feb;71(2):130-139. Pistorius S. et al. (2006). Genomic rearrangements in MSH2, MLH1 or MSH6 are rare in HNPCC patients carrying point mutations. Cancer Lett. 2006 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. 2006 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. 2006 Jan 15;66(2):659-64. 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):138-46. Wehner, M. et al. (2005). Hereditary nonpolyposis colorectal cancer: pitfalls in deletion screening in MSH2 and MLH1 genes. Eur J Hum Genet. 2005 May 4. McVety et al. (2005). Novel genomic insertion-deletion in MLH1: possible mechanistic role for non-homologous endjoining DNA repair. Clin Genet. 2005 Sep;68(3):234-8. 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: 183-188. Bunyan DJ et al. (2004). Dosage analysis of cancer predisposition genes by multiplex ligation-dependent probe amplification. Br J Cancer 13;91(6):1155-9. 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: 428-433. 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: 892-7. 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 64-70-76-82 nt, three DNA denaturation control fragments (D-fragments) at 88-92-96 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 www.mlpa.com. 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 U03911. The MLH1 exon 18 probe is directed to the strand present in U07343. These probes will not detect cdna. All other probes are directed to the complement of NM_000249.3/NM_000251.1 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
Table 1. SALSA MLPA P003-B1 MLH1 / MSH2 probemix Length Chromosomal position SALSA MLPA probe (nt) reference MLH1 MSH2 64-70-76-82 Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA 88-92-96 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 00797-L00463 5q31 136 Reference probe 00981-L00566 10p12 142 MLH1 probe 00886-L00474 Exon 1 148 * MSH2 probe 12036-L02162 Exon 1 154 MLH1 probe 01008-L00577 Exon 2 160 MSH2 probe 00906-L00494 Exon 2 166 MLH1 probe 00888-L00476 Exon 3 172 MSH2 probe 01029-L00601 Exon 3 177 MLH1 probe 00889-L00477 Exon 4 184 MSH2 probe 00908-L00496 Exon 4 190 * MSH2 probe 11287-L12006 Exon 1 196 Reference probe 03861-L03610 3p24 202 MLH1 probe 00890-L06064 Exon 5 211 MSH2 probe 00909-L00497 Exon 5 217 * MSH2 probe 13145-L14624 Exon 1 225 MLH1 probe 00891-L14625 Exon 6 230 ± MSH2 probe 00910-L00498 Exon 6 238 MLH1 probe 00892-L00480 Exon 7 247 * MSH2 probe 11634-L12398 Exon 7 256 MLH1 probe 00893-L00481 Exon 8 265 MSH2 probe 00912-L00582 Exon 8 274 MLH1 probe 00894-L00482 Exon 9 285 Reference probe 00438-L00003 17q21 292 MSH2 probe 00913-L00583 Exon 9 301 MLH1 probe 00895-L00483 Exon 10 310 * MSH2 probe 11288-L12007 Exon 10 319 MLH1 probe 00896-L00484 Exon 11 329 MSH2 probe 00915-L00503 Exon 11 337 MLH1 probe 00897-L00485 Exon 12 346 MSH2 probe 00916-L00504 Exon 12 355 MLH1 probe 00898-L00486 Exon 13 364 MSH2 probe 01013-L00575 exon 13 375 Reference probe 00681-L11147 4q25 382 MLH1 probe 00899-L00586 exon 14 391 MSH2 probe 00918-L00506 Exon 14 401 MLH1 probe 00900-L00488 Exon 15 409 MSH2 probe 00919-L00585 Exon 15 418 MLH1 probe 01009-L00576 Exon 16 427 MSH2 probe 01053-L14623 Exon 16 436 MLH1 probe 01030-L00602 Exon 17 445 MLH1 probe 01031-L00603 Exon 18 454 * MLH1 probe 12094-L12994 Exon 19 463 Reference probe 00979-L00568 10p14 472 * EPCAM probe 13147-L14404 upstream 481 * EPCAM probe 13130-L03603 upstream 490 * Reference probe 04274-L03639 13q12 * 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 rs4987189 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
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: info@mlpa.com. Table 2. P003 probes arranged according to chromosomal location Table 2a. MLH1 gene Length (nt) SALSA MLPA probe MLH1 exon Ligation site NM_000249.3 Partial sequence (24 nt adjacent to ligation site) Distance to next probe startcodon 199-201 142 00886-L00474 exon 1 205-206 CCAAAATGTCGT-TCGTGGCAGGGG 3.1 Kb 154 01008-L00577 exon 2 383-384 GATTCAGATCCA-AGACAATGGCAC 4.3 Kb 166 00888-L00476 exon 3 463-464 TGCAGTCCTTTG-AGGATTTAGCCA 3.4 Kb 177 00889-L00477 exon 4 527-528 CATAAGCCATGT-GGCTCATGTTAC 2.6 Kb 202 00890-L06064 exon 5 627-628 AAACCATGTGCT-GGCAATCAAGGG 1.8 Kb 225 00891-L14625 exon 6 664-665 TGGAGGACCTTT-TTTACAACATAG 3.0 Kb 238 00892-L00480 exon 7 764-765 ACACAATGCAGG-CATTAGTTTCTC 0.2 Kb 256 00893-L00481 exon 8 818-819 TGTTAGGACACT-ACCCAATGCCTC 2.4 Kb 274 00894-L00482 exon 9 920-921 CCTAGCCTTCAA-AATGAATGGTTA 3.1 Kb 301 00895-L00483 exon 10 1019-1020 TTCCTTGAGAAA-AGCCATAGAAAC 2.9 Kb 319 00896-L00484 exon 11 1190-1191 GCAGCACATCGA-GAGCAAGCTCCT 5.5 Kb 337 00897-L00485 exon 12 1482-1483 AGGCAGCAAGAT-GAGGAGATGCTT 3.0 Kb 355 00898-L00486 exon 13 1665-1666 TCCCGAAAGGAA-ATGACTGCAGCT 11.4 Kb 382 00899-L00586 exon 14 1799-1800 CGTGGGCTGTGT-GAATCCTCAGTG 2.1 Kb 401 00900-L00488 exon 15 1892-1893 CCAGATACTCAT-TTATGATTTTGC 5.3 Kb 418 01009-L00576 exon 16 1967-1968 CATGCTTGCCTT-AGATAGTCCAGA 1.0 Kb 436 01030-L00602 exon 17 2172-2173 ATCTTCATTCTT-CGACTAGCCACT 0.4 Kb 445 01031-L00603 exon 18 2269-2268 reverse CTCCTCAGATAT-GTACTGCTTCCG 1.8 Kb 454 * 12094-L12994 exon 19 2595-2594 reverse TATCAGAAGGCA-AGTATAAGTCTT stopcodon 2467-2469 * 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_00249.3 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
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_002354.2 EPCAM stopcodon 1301-1303 481 ** 13130-L03603 EPCAM exon 9 1352-1353 AAATGGACACAA-ATTACAAATGTG 0.1 Kb 472 ** 13147-L14404 EPCAM exon 9 1483-1482 reverse GGTCAAATTTCA-AGATTGGTAAAG 16.0 Kb to MSH2 NM_000251.1 startcodon 69-71 217 * 13145-L14624 exon 1 382 nt before ATG startcodon CCGGGCACATTA-CGAGCTCAGTGC 0.2 Kb 148 * 12036-L02162 exon 1 173 nt before ATG startcodon GCGTGCGCGGGA-AGCTGGGCCGCG 0.7 Kb 190 * 11287-L12006 exon 1 269 nt after exon 1 reverse GAACTAGAACAA-TGCATTAAAATG 4.8 Kb 160 00906-L00494 exon 2 371-372 TATAGAGTTGAA-GTTTATAAGAAT 1.7 Kb 172 01029-L00601 exon 3 519-520 TTGTGGGTGTTA-AAATGTCCGCAG 2.3 Kb 184 00908-L00496 exon 4 806-807 CGGTTGTTGAAA-GGCAAAAAGGGA 1.8 Kb 211 00909-L00497 exon 5 886-887 ACTGTCTGCGGT-AATCAAGTTTTT 2.1 Kb 230 00910-L00498 exon 6 1061-1062 GCCTTGCTGAAT-AAGTGTAAAACC 13.5 Kb 247 * 11634-L12398 exon 7 1265-1266 AGACAAGCAGCA-AACTTACAAGAT 15.7 Kb 265 00912-L00582 exon 8 1392-1393 CTCCTCTTACTG-ATCTTCGTTCTG 17.5 Kb 292 00913-L00583 exon 9 1479-1480 AATTCCTTGTAA-AACCTTCATTTG 3.9 Kb 310 * 11288-L12007 exon 10 190 nt after exon 10 GACTGAAGTGGT-ACTTTGGGTCTA 4.0 Kb 329 00915-L00503 exon 11 1775-1774 reverse GCTTCTTCATAT-TCTGTTTTATTT 4.1 Kb 346 00916-L00504 exon 12 1923-1924 CACCTGTTCCAT-ATGTACGACCAG 1.3 Kb 364 01013-L00575 exon 13 2139-2140 TCATGGCCCAAA-TTGGGTGTTTTG 1.9 Kb 391 00918-L00506 exon 14 2346-2347 CCTACGATGGAT-TTGGGTTAGCAT 2.5 Kb 409 00919-L00585 exon 15 2629-2630 ACTTGAGGAGTT-TCAGTATATTGG 2.0 Kb 427 01053-L14623 exon 16 2705-2706 GTGTTTCAGCAA-GGTGAAAAAATT stopcodon 2871-2873 * 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 RS1803881 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: 112-117). 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
SALSA MLPA kit P003-B1 MLH1-MSH2 sample picture 20000 171,77 128,12 145,50 17500 133,40 182,90 237,99 229,45 328,96 15000 151,48 202,65 223,79 216,23 12500 84,97 141,18 165,28 344,03 105,25 10000 95,21 90,71 195,74 189,00 176,59 210,38 246,49 256,21 263,18 301,96 291,10 283,46 319,18 310,68 352,47 401,53 408,23 391,42 427,69 454,78 482,29 472,71 7500 159,10 272,84 335,03 363,89 374,82 444,91 5000 100,37 382,32 419,15 436,63 462,57 492,25 2500 Dye Signal 0 50 100 150 200 250 300 350 400 450 500 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