The SALSA MS-MLPA
Probemix ME034 Multi-locus Imprinting is a research use only (RUO)
assay for the detection of aberrant methylation and/or copy number changes of one or more sequences in 11 different imprinted locations in seven different chromosomal regions. Applications include the study of multi-locus imprinting disorders and finding the parental origin of triploid pregnancies. This probemix can also be used to detect deletions/duplications in these chromosomal regions.
Genomic imprinting is the monoallelic expression of genes, dependent on the parental origin of the chromosome. It plays a role in growth and development. Imprinting disorders originate from a disturbance in this monoallelic expression by disruption or epimutation of imprinted genes (Ishida et al. 2013).
Multi-locus imprinting defects have recently been identified in several patients. For example, a considerable number of patients with 11p15-associated imprinting disorders have been reported to also carry methylation changes at other chromosomal locations (Eggermann T. et al. 2014).
Triploidy is one of the most common chromosomal abnormalities, occurring in 1-2% of all human conceptuses. Triploid pregnancies can have either the chromosomal constitution of two maternal and one paternal set (digyny) or two paternal and one maternal set (diandry). The distinction between digyny and diandry is clinically important because the risk of the conceptus being molar and thereby inducing a risk for maternal complications (such as pre-eclampsia, postpartum haemorrhage and persistent gestational trophoblastic disease) is determined by the parental composition of the genome. As shown by Joergensen et al. (2013, 2014), MS-MLPA analysis of imprinted regions is a reliable method to distinguish triploidies with a double paternal contribution from triploidies with a double maternal contribution.
The SALSA MLPA Probemix ME034-B1 Multi-locus Imprinting contains 36 (MS-)MLPA probes with amplification products between 122 and 466 nt. 21 MS-MLPA probes contain a HhaI recognition site and provide information on the methylation status of different sequences in genes known to be methylated in either the paternal or the maternal allele. This includes three probes for each of the following genes: H19
, two probes for each of the following genes: KCNQ1OT1
, and one probe for each of the following genes: NESP55
, GNAS, GRB10
. All probes present will also give information on copy number changes in the analysed sample. In addition, 11 reference probes are included which are not affected by HhaI digestion and detect genes located outside the chromosomal regions targeted by this probemix. Also, two digestion control probes are included in this probemix indicating whether or not restriction endonuclease digestion in the MS-MLPA reaction was complete. Complete probe sequences and the identity of the genes detected by the reference probes is 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 MLPA General Protocol and online at www.mlpa.com