General information
The SALSA MS-MLPA Probemix ME031 GNAS is a research use only (RUO) assay for the detection of aberrant methylation of one or more sequences of the GNAS complex locus. This probemix can also be used to detect deletions/duplications in the GNAS complex locus and the STX16 gene.

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). Loss of methylation in the GNAS locus is associated with the genomic imprinting defect inactivating PTH/PTH-related protein signaling disorders subtype 3 (iPPSD3). Mutations in this locus are associated with iPPSD subtype 2 (iPPSD2) (Turan 2017).

iPPSD3, also referred to as pseudohypoparathyroidism Ib, is caused by loss of methylation (LOM) in the GNAS complex locus. iPPSD3 is characterised mainly by resistance to parathyroid hormone (PTH) and symptoms that include hypocalcemia, numbness, seizures, tetany, cataracts, and dental problems.

The GNAS locus is a complex imprinted locus on chromosome 20 that generates multiple transcripts through the use of several alternative first exons that splice into a common set of downstream exons (see Figure 1). GNAS itself encodes the Gsα protein, which is the α-subunit in the heterotrimeric G protein. Due to differential methylation of their promoters, most gene products originate from one parental allele. Transcripts GNASXL, which encodes XLαs, GNAS A/B (also referred to as 1A) and the antisense transcript GNAS-AS1 (also referred to as NESPAS) are transcribed from the paternal allele, while NESP55 (also referred to as NESP or GNAS transcript variant 4) is transcribed from the maternal allele (Turan and Bastepe 2013). The most downstream promoter (GNAS exon 1) is not differentially methylated, which results in GNAS expression from both alleles in most tissues but its expression is silenced from the paternal allele in a small number of tissues (Turan and Bastepe 2013).

The Gsα and XLαs transcripts are involved in downstream signalling from parathyroid hormone (PTH), parathyroid hormone related protein (PTHrP) receptors and other hormone receptors like TSHR and GHRHR. The GNAS A/B transcript and the antisense transcript GNAS-AS1 are not translated into proteins, but are thought to influence Gsα expression via mechanisms that remain to be determined. The STX16 gene, lastly, is a long range control element of methylation at the GNAS locus, located more than 220 kb centromeric of GNAS (Turan and Bastepe 2013).

PHPIb is caused by loss-of-methylation (LOM) at GNAS A/B located within DMR 1 in the GNAS complex locus (Figure 1). LOM can also be observed at GNAS-AS1 and GNASXL, which can also be associated with a gain-of-methylation (GOM) at NESP55. The autosomal dominant form of PHPIb can be caused by maternal heterozygous deletions in STX16 (Turan and Bastepe 2013).

More information is available at https://www.ncbi.nlm.nih.gov/books/NBK459117/

This SALSA MS-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 MS-MLPA Probemix ME031-C1 GNAS contains 53 (MS-)MLPA probes with amplification products between 126 and 500 nucleotides (nt). This includes 32 probes for the GNAS locus and nine probes for the STX16 gene. 15 MS-MLPA probes contain an HhaI recognition site and provide information on the methylation status of the GNAS complex locus. All probes present will also give information on copy number changes in the analysed sample. In addition, ten reference probes are included that are not affected by HhaI digestion and detect genes located outside the GNAS complex locus. 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.