Intended purpose
The SALSA MLPA Probemix ME032 UPD7-UPD14 is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of copy number variations (CNVs) and methylation status of the imprinted 7q32, 7p12 and 14q32 regions, including the GRB10, MEST, DLK1, MEG3 and RTL1 genes in genomic DNA isolated from human peripheral blood, buccal swab and saliva. ME032 UPD7-UPD14 is intended to confirm a potential cause for and clinical diagnosis of Temple syndrome (TS), Kagami-Ogata syndrome (KOS) and Russel-Silver syndrome (RSS) and for molecular genetic testing of at-risk family members.
CNVs and methylation changes detected with ME032 UPD7-UPD14 should be confirmed with a different technique. In particular, CNVs and methylation changes detected by only a single probe always require confirmation by another method.
Assay results are intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, including confirmation by alternative methods, parental evaluation, clinical genetic evaluation, and counselling, as appropriate. The results of this test should be interpreted by a clinical molecular geneticist or equivalent.
This device is not intended to be used for standalone diagnostic purposes, pre-implantation or prenatal testing, population screening, or for the detection of, or screening for, acquired or somatic genetic aberrations.
¹Please note that this probemix is for in vitro diagnostic (IVD) use in the countries specified at the end of this product description. In all other countries, the product is for research use only (RUO).
²To be used in combination with a SALSA MLPA Reagent Kit, SALSA HhaI and Coffalyser.Net analysis software.

Clinical background
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). A frequent cause of imprinting disorders is uniparental disomy (UPD), which 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. Differentially methylated regions (DMRs) act as imprinting control regions to regulate the imprinted expression of the genes. Most frequently, UPD does not result in any phenotypical anomalies but UPD can cause unmasking of an autosomal-recessive disease or can present itself as a syndromic imprinting disorder.

Russell-Silver syndrome (RSS; Silver-Russell syndrome (SRS)) is characterised as a clinically heterogeneous condition with a variable phenotype (Wakeling et al. 2017). Intrauterine and postnatal growth retardation, however, are consistently observed in all RSS patients, who are at significant risk for developmental delay and learning disabilities. Although rare, RSS can be inherited in an autosomal dominant or recessive manner. The prevalence is estimated between 1 and 9 in 1.000.000 (Orphanet, https://www.orpha.net/consor/cgi-bin/OC_Exp.php?Lng=GB&Expert=813).

Temple syndrome (TS; TS14; maternal uniparental disomy 14 (UPD14mat)) is a short stature disorder of imprinting, characterised among other features by low birth weight and length and premature puberty (Hoffmann and Heller, 2011). In TS patients, the proportion of children with learning difficulties or mental retardation is increased compared to the general population. Intrauterine growth retardation occurs in 90% of the TS patients. TS shows an age-dependent overlap with the well-known maternal UPD15 Prader-Willi syndrome. TS is a very rare disease with a prevalence < 1 in 1.000.000 (Orphanet, https://www.orpha.net/consor/cgi-bin/OC_Exp.php?lng=EN&Expert=254516).

Kagami-Ogata syndrome (KOS; KOS14; paternal uniparental disomy 14 (UPD14pat)) is a foetal malformation syndrome, characterised by polyhydramnios and a bell-shaped chest with angulated ribs (“coat-hanger configurations”), which leads to pulmonary hypoplasia and abdominal wall defects (Hoffmann and Heller 2011). The clinical course of KOS is marked by the severe and often lethal respiratory insufficiency, feeding difficulties with postnatal growth failure, and moderate to severe mental retardation. The prognosis in most cases is very poor.

However, there has been no report of death in patients ≥4 years of age (Ogata and Kagami 2016). After intense medical treatment in infancy, these patients become free of mechanical ventilation, tracheostomy and feeding tube. Despite developmental delay, they manage to get on their daily lives from childhood. KOS is a very rare disease with a prevalence < 1 in 1.000.000 (Orphanet, https://www.orpha.net/consor/cgi-bin/OC_Exp.php?lng=EN&Expert=254519).

More information is available at https://www.ncbi.nlm.nih.gov/books/NBK1324/ (RSS); https://www.omim.org/entry/616222 (TS); and https://www.omim.org/entry/608149 (KOS).

Probemix content
The SALSA MLPA Probemix ME032 UPD7-UPD14 contains 48 (MS-)MLPA probes with amplification products between 115 and 511 nt. Nine probes are specific for the 7p12.2 region, eight for the 7q32.2 region, 12 for the 14q32.2 region and three for the 14q32.31 region. Ten of these probes contain an HhaI recognition site and provide information about the methylation status of the target sequence. All probes in the probemix will also give information on copy number changes in the analysed sample. In addition, 14 reference probes are included which are not affected by HhaI digestion and detect genes located outside the 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. 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 MS-MLPA General Protocol and online at www.mrcholland.com.