The SALSA MS-MLPA
Probemix ME032 UPD7-UPD14 is a research use only (RUO)
assay for the detection of aberrant methylation of one or more sequences of the GRB10
genes. This probemix can also be used to detect deletions/duplications in the aforementioned genes as well as of DLK1, MIR380
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 like uniparental disomy (UPD) originate from a disturbance in this monoallelic expression by disruption or epimutation of imprinted genes (Ishida et al., 2013).
UPD occurs when both alleles are inherited from a single parent instead of one copy from each parent. This aberrant genetic inheritance causes disturbed genomic imprinting and results in either the overexpression or complete silencing of genes that are expressed parent-specifically. Most frequently, UPD does not results in any phenotypical anomalies but UPD can cause unmasking of an autosomal-recessive disease or can present itself as a syndromic imprinting disorder. The UPD phenotypes depend on the specific chromosome/segment involved and whether both copies are of maternal or paternal origin. For example, paternal UPD7 is clinically unapparent while maternal UPD7 is one of the several causes of Russell-Silver syndrome. Moreover, paternal UPD14, or Kagami-Ogata syndrome, is a thoracic dysplasia syndrome with mental retardation and limited survival (Kagami et al., 2005). Maternal UPD14, also called Temple syndrome, shows an age-dependent overlap with the well-known maternal UPD15 Prader-Willi syndrome and is dominated by initial failure to thrive followed by obesity, learning difficulties and precocious puberty (Hoffmann and Heller, 2011).
The ME032-A1 probemix contains probes for the target imprinted gene PLAGL1
on chromosome 6, the genes GRB10
on chromosome 7 and the DLK1
genes in the imprinted region on chromosome 14.
gene (11 exons) spans ~124 kb of genomic DNA and is located on 6q24.2. PLAGL1
is involved in transient neonatal diabetes mellitus (TNDM), a form of diabetes that occurs in infants and is characterised by severe intra-uterine growth retardation, hyperglycemia, dehydration and absence of ketoacidosis. Approximately 70% of transient diabetes in newborns is related to the 6q24 chromosomal region (Flanagan et al., 2008).
gene (21 exons) spans ~203 kb of genomic DNA and is located on 7p12.2. This gene encodes a growth factor receptor-binding protein that interacts with insulin receptors and insulin-like growth-factor receptors. The gene is mono-allelic or bi-allelic expressed, depending on the tissue-type.
gene (12 exons) spans ~14 kb of genomic DNA and is located on 7q32.2. This gene encodes a member of the a/b hydrolase superfamily. It is imprinted, exhibiting preferential expression from the paternal allele in fetal tissues.
gene (5 exons) spans ~11 kb of genomic DNA and is located on 14q32.2. DLK1
is a paternally expressed imprinted gene and encodes a trans-membrane protein involved in the differentiation of several cell types.
gene (13 exons) spans ~35 kb of genomic DNA and is located on 14q32.2. MEG3
is a maternally expressed imprinted gene which appears to function as an RNA molecule; multiple splice variants are observed in the available sequence data and a pituitary transcript variant has been associated with inhibited cell proliferation.
gene (1 exon) spans ~4 kb of genomic DNA and is located on 14q32.31. This gene is a retrotransposon-derived, paternally expressed imprinted gene that is highly expressed at the late fetal stage in both the fetus and placenta.
The microRNA MIR380
(1 exon) spans 61 bp of genomic DNA and is located on 14q32.31. This microRNA is involved in regulation of p53.
More information is available at https://www.ncbi.nlm.nih.gov/books/NBK1534/
(TNDM) and https://www.ncbi.nlm.nih.gov/books/NBK1324/
The SALSA MS-MLPA Probemix ME032 UPD7-UPD14 contains 46 (MS-)MLPA probes with amplification products between 115 and 454 nt. Six probes are specific for the 6q24.2 region, six for the 7p12.2 region, five for the 7q32.2 region, nine for the 14q32.2 region and three for the 14q32.31 region. 13 of these probes contain an HhaI recognition site and provide information about the methylation status of the target sequence. Except for one probe specific for the MIR380 region which contains an HhaI recognition site, but will only give information on copy number changes in the analysed sample, since this HhaI recognition site is fully methylated in normal tissue. All probes present will also give information on copy number changes in the analysed sample. In addition, 15 reference probes are included which are not affected by HhaI digestion and detect genes located outside the 6q24, 7p12, 7q32 and 14q32 regions. Also, two digestion control probes are included in this probemix indicating whether or not restriction endonuclease digestion in the MS-MLPA reaction was complete.
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