General information
The SALSA MLPA Probemix ME012 MGMT-IDH-TERT 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 IDH1 (p.R132H=c.395G>A and p.R132C=c.394C>T), IDH2 (p.R172K=c.515G>A and p.R172M=c.515G>T) and TERT promoter (C228T and C250T) point mutations in a DNA sample.

Gliomas, glioneuronal and neuronal tumours are the most common central nervous system (CNS) neoplasms, subdivided into six groups according to the fifth edition of the WHO classification of CNS tumours: adult-type diffuse gliomas (encompassing oligodendroglioma, glioblastoma and astrocytoma), paediatric-type diffuse low-grade gliomas, paediatric-type diffuse high-grade gliomas, circumscribed astrocytic gliomas, glioneuronal and neuronal tumours, ependymomas (Torp et al. 2022; Gritsch et al. 2022). Molecular genetic characteristics play an increasingly major role in classification, diagnostics and prognosis of CNS tumours.

IDH1 and IDH2 mutation (IDH-mutation) status represents an important diagnostic and prognostic marker in gliomas (Riemenschneider et al. 2010, van den Bent et al. 2010). The presence of IDH-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 IDH-mutant or IDH-wildtype (Wesseling and Capper 2018).

Point mutations in TERT promoter region generate novel transcription factor binding sites and thus increase the expression of telomerase enzyme encoded by TERT. Common TERT promoter mutations are known as C228T and C250T, referring to C>T transitions at hg19/GCRh37 chr5:1295228 and chr5:1295250 positions, respectively. These mutations are predominantly present in oligodendroglioma and are associated with poor prognosis and reduced survival in the absence of IDH-mutation (Labussière et al. 2014). TERT promoter mutation, in combination with IDH-mutation and 1p/19q codeletion, is characteristic of oligodendroglioma. Absence of TERT promoter mutation, coupled with the presence of IDH-mutation, designates astrocytoma (Cahill et al. 2015; Eckel-Passow et al. 2015).

CpG-islands are located in or near the promoter region or other regulatory regions of approximately 50% of human genes. Aberrant methylation of CpG-islands has been shown to be associated with transcriptional inactivation of genes in a wide spectrum of human cancers. These genes are frequently silenced by methylation in tumours, but are unmethylated in blood-derived DNA. In addition, DNA methylation analysis can indicate in some cases from which type of tissue the tumour was derived.

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). Assessment of both the IDH-mutation 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 IDH-mutation and MGMT methylation status is suggested to predict survival in glioblastoma better than either biomarker alone (Molenaar et al. 2014). The presence of TERT promoter mutation in combination with unmethylated MGMT defines glioblastoma with the poorest prognosis (Arita et al. 2016).

More information is available at https://www.ncbi.nlm.nih.gov/books/NBK558954/ and WHO Classification of Central Nervous System Tumours, 5th Edition, Volume 6.

This SALSA MLPA probemix is not CE/FDA registered for use in diagnostic procedures. Purchase of this product includes a limited license for research purposes.

Probemix content
The SALSA MLPA Probemix ME012-B1 MGMT-IDH-TERT contains 31 (MS-)MLPA probes with amplification products between 123 and 317 nucleotides (nt). Eight MS-MLPA probes contain at least one HhaI recognition site and provide information on the methylation status of the MGMT gene. All probes present will also give information on copy number changes in the analysed sample in the undigested reaction. Moreover, this probemix contains six mutation-specific probes to identify the four most predominant IDH1 (p.R132H and p.R132C) and IDH2 (p.R172K and p.R172M) and two TERT promoter region point mutations (C228T and C250T) in glioma. In addition, 13 reference probes are included that are not affected by HhaI digestion and target relatively copy number stable regions in various cancer types, including gliomas. Also, three digestion control probes are included in this probemix indicating whether or not restriction endonuclease digestion in the MS-MLPA reaction was complete. Furthermore, this probemix contains one DNA depurination-sensitive probe. Complete/partial probe sequences and the identity of the genes detected by the reference probes are available online (www.mrcholland.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.mrcholland.com.

SALSA Binning DNA SD094
The SD094 Binning DNA provided with this probemix can be used for binning of all probes including the six mutation-specific probes: IDH1 probe 19529-L16492 at 203 nt (p.R132H=c.395G>A), IDH1 probe 19926-L32919 at 232 nt (p.R132C=c.394C>T), IDH2 probe 20643-L32911 at 151 nt (p.R172K=c.515G>A), IDH2 probe 20643-L32910 at 154 nt (p.R172M=c.515G>T), TERT probe S1295-L32988 at 127 nt (C250T) and TERT probe S1309-L32882 (C228T) at 146 nt. SD094 Binning DNA is a mixture of genomic DNA from healthy individuals and plasmid DNA that contains the target sequences detected by the above mentioned probes. Inclusion of one reaction with 5 μl SD094 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. 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 SD094 Binning DNA product description, available online: www.mrcholland.com. This product is for research use only (RUO).

Sample DNA
Sample DNA developed for this product:

• SD094 Binning DNA - included with this probemix.