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SALSA MLPA Probemix ME012 MGMT-IDH1-IDH2

Gliomas

Region: MGMT, IDH1, IDH2

General information: The SALSA MS-MLPA Probemix ME012 MGMT-IDH1-IDH2 is a research use only (RUO) assay for the detection of aberrant methylation of one or more sequences of the MGMT gene. This probemix can also be used to detect the presence of the IDH1 (p.R132H=c.395G>A and p.R132C=c.394C>T) or IDH2 (p.R172K=c.515G>A and p.R172M=c.515G>T) point mutations in a DNA sample.

Glioma is the most common neoplasm of central nervous system, comprising three phenotypic groups based on the type of glial cell forming tumour: astrocytomas (including the most aggressive type of brain cancer -glioblastomas), ependymomas, and oligodendrogliomas. Hypermethylation in the promoter region of the MGMT gene, encoding for the DNA repair enzyme O6-methylguanine DNA methyltransferase, is an important prognostic marker and predictor for response to treatment in glioma with alkylating agents such as temozolomide (Weller et al. 2009, Hegi et al. 2005, Pegg 1990). Another important diagnostic and prognostic marker in glioma is the IDH1 and IDH2 mutation status (Riemenschneider et al. 2010, van den Bent et al. 2010). The presence of IDH1 or IDH2 mutation is suggested to associate with favourable prognosis and a longer survival of glioma patients (Sanson et al. 2009, Zou et al. 2013). The IDH1/2 mutations described are not activating or inactivating, but probably result in altered enzymatic properties (Hartmann et al. 2009). IDH mutation is a marker for glioma classification since 2016, defining glioblastomas as IDH1-mutant or IDH1-wildtype (Wesseling and Capper 2018). Assessment of both the IDH1 mutation status and the MGMT methylation status is proposed to be used as a combined predictor for glioblastoma patient survival (Wick et al. 2013). Combined assessment of IDH1 mutations and MGMT methylation status is suggested to predict survival in glioblastoma better than either IDH1 or MGMT alone (Molenaar et al. 2014).

Probemix content:
The SALSA MS-MLPA Probemix ME012 MGMT-IDH1-IDH2 contains 31 (MS-)MLPA probes with amplification products between 121 and 317 nucleotides (nt). Six MS-MLPA probes contain an HhaI recognition site and provide information on the methylation status of MGMT promoter region. All probes present will also give information on copy number changes in the analysed sample. Moreover, this probemix contains four mutation-specific probes to identify the four most predominant IDH1 (p.R132H and p.R132C) and IDH2 (p.R172K and p.R172M) point mutations in glioma. In addition, 18 reference probes are included that are not affected by HhaI digestion and target relatively copy number stable regions in glioma. Also, two digestion control probes and one depurination control probe are included in this probemix, indicating whether or not restriction endonuclease digestion in the MS-MLPA reaction was complete, and whether DNA has been affected by depurination, respectively. Identity of the genes detected by the reference probes are available and complete probe sequences are available online (www.mlpa.com).

This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MS-MLPA General Protocol and online at www.mlpa.com.

SALSA Binning DNA SD054: The SD054 Binning DNA provided with this probemix can be used for binning of four mutation-specific probes IDH1 probe 19529-L16492 at 203 nt (p.R132H=c.395G>A), IDH1 probe 19926-L29107 at 225 nt (p.R132C=c.394C>T), IDH2 probe 20643-L29002 at 145 nt (p.R172K=c.515G>A) and IDH2 probe 20643-L29001 at 151 nt (p.R172M=c.515G>T). SD054 Binning DNA is a mixture of genomic DNA from healthy individuals and synthetic DNA that contains the target sequence detected by the above mentioned probes. Inclusion of one reaction with 5 μl of SD054 Binning DNA in initial MLPA experiments is essential as it can be used to aid in data binning of the peak pattern using Coffalyser.Net software. Furthermore, Binning DNA should be included in the experiment whenever changes have been applied to the set-up of the capillary electrophoresis device (e.g. when capillaries have been renewed). Binning DNA should never be used as a reference sample in the MLPA data analysis, neither should it be used in quantification of mutation signals, as for this purpose true mutation/SNP positive patient samples or cell lines should be used. It is strongly advised that all samples tested are extracted with the same method and derived from the same source of tissue. For further details, please consult the SD054 Binning DNA product description, available online: www.mlpa.com.

Sample DNA
Sample DNA developed for this product:

Order Items

Probemix

Item no.
Description
Technology
Price
ME012-025R
SALSA MLPA Probemix ME012 MGMT-IDH1-IDH2 – 25 rxn
€ 243.00
ME012-050R
SALSA MLPA Probemix ME012 MGMT-IDH1-IDH2 – 50 rxn
€ 486.00
ME012-100R
SALSA MLPA Probemix ME012 MGMT-IDH1-IDH2 – 100 rxn
€ 972.00

Required Reagents

Item no.
Description
Technology
Price
EK1-FAM
SALSA MLPA EK1 reagent kit – 100 rxn – FAM
€ 300.00
EK1-Cy5
SALSA MLPA EK1 reagent kit – 100 rxn – Cy5
€ 300.00
EK5-FAM
SALSA MLPA EK5 reagent kit – 500 rxn – FAM
€ 1380.00
EK5-Cy5
SALSA MLPA EK5 reagent kit – 500 rxn – Cy5
€ 1380.00
EK20-FAM
SALSA MLPA EK20 reagent kit – 2000 rxn – FAM
€ 5295.00
SMR50
SALSA HhaI – 200 rxn
€ 40.00

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