General information
The SALSA MLPA
Probemix P496 KMT2A is a
research use only (RUO) assay for the detection of deletions or duplications in the
KMT2A gene. This probemix can also be used to detect the presence of the
ASXL1 c.1934dupG mutation.
The histone methyltransferase KMT2A methylates lysine 3 on histone 4 (H3K4), thereby shaping the epigenetic landscape in cells, modulating chromatin accessibility and transcription. A subset of leukemias is characterised by
KMT2A alterations, which typically include amplifications, partial tandem duplications (PTDs) and translocations (Rao et al. 2015).
KMT2A alterations are observed in 5-20% of Acute Myeloid Leukemia (AML) cases, and are associated with poor outcome (Choi et al. 2018; Issa et al. 2021; Yuen et al. 2023).
KMT2A PTDs (also known as
MLL PTDs) are observed in 6-7% of Myelodysplastic Syndrome (MDS) as well, and alongside
TP53 and
FLT3 mutations are considered as top genetic predictors of adverse outcomes according to IPSS-M (Molecular International Prognostic Scoring System for Myelodysplastic Syndromes) (Bernard E et al. 2022; Choi et al. 2018). Accurate detection of
KMT2A PTDs using next-generation sequencing (NGS) or fluorescence
in situ hybridisation (FISH) approaches remains difficult (McKerrell et al. 2016; Afrin et al. 2018; Dai et al. 2021; Tsai et al. 2022), whereas MLPA technique was shown to yield robust and reliable results with a short turnaround time (Balgobind et al. 2010; Kentaro et al. 2013; ) allows more accurate detection of
KMT2A PTDs when combined with DNA structural analysis approaches (Capo-Chichi et al. 2022).
ASXL1 is a gene that is frequently mutated in various haematological malignancies, including AML and MDS. The most common genetic alteration found in the
ASXL1 gene is the c.1934dupG mutation, which leads to a premature stop codon in the
ASXL1 transcript, resulting in a truncated ASXL1 protein which is rapidly degraded. Importantly, loss-of-function
ASXL1 mutations were shown to promote myeloid transformation through loss of PRC2-mediated gene repression (Abdel-Wahab et al. 2012; Gelsi-Boyer et al. 2012). The
ASXL1 c.1934dupG mutation appears to be mostly restricted to the haematopoietic lineage, and is associated with poor prognosis in AML and MDS patients (Gelsi-Boyer et al. 2012).
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 P496-A1 KMT2A contains 60 MLPA probes with amplification products between 64 and 505 nucleotides (nt). This includes 17 probes for the
KMT2A gene and two probes upstream and downstream of
KMT2A gene, namely for the
UBE4A and
TMEM25 genes. This probemix also contains one probe specific for the
ASXL1 c.1934dupG mutation which will only generate a signal when the mutation is present. Furthermore, probes were added for copy number determination of other regions commonly affected by CNAs in AML and MDS. These include two probes targeting
CTNNA1 and
NPM1 (5q deletions), one flanking probe for 5p, two probes for
IKZF1,
CUX1,
KMT2E and
EZH2 (chromosome 7 and 7q deletions); three probes for
ATM (11q deletions), two flanking probes for the 11p arm, one flanking probe for 11q, three probes for
TP53 (17p deletions), two probes for
NF1 and one for
SUZ12 (17q deletions). In addition, 13 reference probes are included that detect autosomal chromosomal locations and target relatively copy number stable regions in various cancer types including hematological cancers. Complete 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 MLPA General Protocol and online at
www.mrcholland.com.
SALSA Binning DNA SD096
The SD096 Binning DNA provided with this probemix can be used for binning of all probes including the mutation-specific probe (ASXL1 probe 18261-SP0848-L2626 for the c.1934dupG mutation). SD096 Binning DNA is a mixture of genomic DNA from healthy individuals and plasmid DNA that contains the target sequence detected by the above mentioned probe. Inclusion of one reaction with 5 μl SD096 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 signal(s). 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 SD096 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: