Glioblastoma, the most common malignant primary brain tumour, is characterised by aggressive behaviour and a poor survival. 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 with alkylating agents such as temozolomide (Weller M et al. 2009, J Clin Oncol. 27:5743-50; Hegi ME et al. 2005, N Engl J Med. 352:997-1003; Pegg AE 1990, Mutat Res. 233:165-75). Another important diagnostic and prognostic marker in glioma is the IDH1 and IDH2 mutation status (Riemenschneider MJ et al. 2010, Acta Neuropathol. 120:567-84; van den Bent MJ et al. 2010, Clin Cancer Res. 16:1597-604). The presence of IDH1 or IDH2 mutation is suggested to associate with favourable prognosis and a longer survival of glioma patients (Sanson M et al. 2009, J Clin Oncol. 27:4150-54; Zou P et al. 2013, PLoS One. 8(7):e68782). The IDH1/2 mutations described are not activating or inactivating, but probably result in altered enzymatic properties (Hartmann C et al. 2009, Acta Neuropathol. 118:469-74). 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 W et al. 2013, Neurology. 81:1515-22). Combined assessment of IDH1 mutations and MGMT methylation status is suggested to predict survival in glioblastoma better than either IDH1 or MGMT alone (Molenaar RJ et al. 2014, Neuro Oncol. 16:1263-73).
This SALSA® MS-MLPA® probemix ME012-A1 contains six MS-MLPA probes that detect the methylation status of the MGMT gene promoter. 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 that are not affected by HhaI digestion have been included in this probemix, detecting different autosomal chromosomal locations that are relatively stable in glioma samples. Besides detecting aberrant methylation, all MGMT probes present in this probemix will give information on copy number status of the sequences targeted in the analysed sample.
The MS-MLPA probes in this ME012-A1 probemix detect sequences in the promoter region of the MGMT gene that are unmethylated in most blood-derived DNA samples. Upon digestion, the peak signal obtained in unmethylated samples will be very small or absent. In contrast, when tested on in vitro methylated human DNA, these probes do generate a signal. We have no data showing that methylation detected by a particular probe indeed influences the corresponding mRNA levels.
SD054 Sample DNA
Please note that the mutation-specific probes have only been tested on artificial sample DNA and not on positive human DNA samples with 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! This SD054 sample DNA is provided with each probemix vial and can be used in data binning in the fragment analysis and as a positive control for the mutation-specific probes.
This SALSA® MS-MLPA® probemix can be used to detect aberrant methylation of one or more sequences of the MGMT gene. Methylation levels can be different for different tissues. If possible, use identically treated test and reference samples (same tissue type and extraction method). This SALSA® MS-MLPA® probemix can also be used to detect deletions/duplications of one or more sequences in the aforementioned gene and to detect the presence of the aforementioned point mutations in a DNA sample. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak height 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 height, 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. Finally, note that small (point) mutations cannot be detected by this SALSA® MS-MLPA® test. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons.
Sample DNA
Sample DNA developed for this product: