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SALSA MLPA KIT P283 TPMT
[http://www.geneclinics.org/profiles/hht/details.html]
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) and 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 nonenzymatically 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 these 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). 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 TPMT gene comprises 9 exons, spanning about 25 kb of genomic DNA. TPMT is located on chromosome 6p22. This P283 TPMT probemix contains one probe for each of the 9 exons of TPMT (two probes for exons 2 and 9). Feedback on this kit is highly appreciated! The DPYD gene is one of the longest human genes. It is located on chromosome 1p22 and comprises 23 exons that span 842 kb of genomic DNA. DPYD defects due to gene rearrangements could be a frequent cause of DPYD deficiency. Probes are present for 3 of the 23 exons in this P283 probemix. 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.
Full mix description (pdf)
Last change in probe mix content: Lot 0907 (September 2007) Current Lot Number.: Lot 0907
IMPORTANT NOTICE: 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)
References
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