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Microsoft word - p283 tpmt-v04 0907.doc

Description version 04; 10-10-2008 SALSA MLPA KIT P283-A1 TPMT Lot 0907 Thiopurine S-methyltransferase (TPMT; S-adenosyl-L-methionine: thiopurine S-methyltransferase) catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds, including the antineoplastic agents 6-mercaptopurine (6MP), 6-thioguanine (6TG) and the immunosuppressant azathioprine (Tai et al., 1996). The thiopurines are pro-drugs that require extensive metabolism in order to exert their cytotoxic action. Azathioprine is non-enzymatically reduced to 6MP. 6MP and 6TG are activated by HPRT and subsequent steps to form cytotoxic thioguanine nucleotides (TGNs) which are incorporated into DNA and/or RNA, causing DNA-protein cross-links, single-strand breaks, interstrand cross-links, and sister chromatid exchange. TPMT functions mainly to inactivate aforementioned drugs, thus a deficiency of TPMT results in increased conversion to toxic TGNs (Coulthard and Hogarth, 2005). In addition, 6MP is unique in that it can also be converted via TPMT into a methyl-thioinosine 5-prime monophosphate (MeTIMP), a metabolite that inhibits de novo purine synthesis and likely contributes to the cytotoxic effect of 6MP (Vogt et al., 1993; Krynetski et al., 1995; Coulthard and Hogarth, 2005). The TPMT gene comprises 9 exons, spans about 25 kb of genomic DNA and is located at chromosome 6p22. This P283-A1 TPMT probemix contains one probe for each of the 9 exons of TPMT gene (two probes for exons 2 and 9). Dihydropyrimidine dehydrogenase (DPYD) is the initial and rate-limiting enzyme in the 3-step pathway of uracil and thymidine catabolism and in the pathway leading to the formation of beta-alanine. DPYD is the major enzyme involved in breakdown of 5-Fluorouracil which is one of the most widely used drugs for cancer chemotherapy. The DPYD gene is one of the longest human genes. It comprises 23 exons, spans about 842 kb of genomic DNA and is located at chromosome 1p22. DPYD defects due to gene rearrangements could be a frequent cause of DPYD deficiency. This P283-A1 probemix contains probes for 3 of the 23 exons of the DPYD gene. This SALSA MLPA kit is designed to detect deletions/duplications of one or more exons of the TPMT gene. Heterozygote deletions of probe recognition sequences should give a 35-50% reduced relative peak area of the amplification product of that probe. However, mutations and/or polymorphisms very close to the probe ligation site may also result in a reduced relative peak area. Therefore, apparent deletions detected by a single probe always require confirmation by other methods. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. Please note that most defects in these genes are expected to be small (point) mutations, most of which will not be detected by this MLPA test. SALSA® MLPA® kits are sold by MRC-Holland 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 MLPA 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). P103 DPYD: Probes are included for every DPYD exon. :MRC-Holland bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands Description version 04; 10-10-2008 Data analysis The P283-A1 TPMT probemix contains 22 different MLPA probes with amplification products between 127 and 373 nt. In addition, it contains 7 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at 64-70-76-82 nt and three DNA denaturation control fragments (D-fragments) at 88-92-96 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 be intra-normalized by dividing the peak area of each probe’s amplification product by the total area of only the reference probes in this probemix (block normalization). Secondly, normalisation can be achieved by dividing this intra-normalized probe ratio in a sample by the average intra-normalized probe ratio of all reference samples. Please note that this type of normalization assumes that no changes occurred in the genomic regions targeted by the reference probes. When only small numbers of samples are tested, visual comparison of peak profiles should be sufficient to easily identify copy number changes. Comparison of results should preferably 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 blots or long range PCR Note that Coffalyser, the MLPA analysis tool developed at MRC-Holland, can be downloaded free of charge from our website www.mlpa.com. This probemix was developed by R. Vijzelaar at MRC-Holland. 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 made a coauthor. Info/remarks/suggestions for improvement: info@mlpa.com. Description version 04; 10-10-2008 SALSA MLPA P283-A1 TPMT probemix 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 ‡ Probe designed on the wild type version of Y240C mutation. Apparent deletions could be due to the presence of the Y240C mutation (this probe is not tested on positive samples). Feedback on this kit is highly appreciated! 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. Description version 04; 10-10-2008 TPMT probes arranged according to chromosomal location DPYD probes arranged according to chromosomal location 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 MLPA kit P283-A1 TPMT sample picture Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analyzed with SALSA MLPA kit P283-A1 TPMT (lot 0907).

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