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 like pseudohypoparathyroidism (PHP) originate from a disturbance in this monoallelic expression by disruption or epimutation of imprinted genes (Ishida et al. 2013).

Disorders of GNAS inactivation include the phenotypes pseudohypoparathyroidism Ia, Ib, and Ic (PHP-Ia, -Ib, -Ic), pseudopseudohypoparathyroidism (PPHP), progressive osseous heteroplasia (POH), and osteoma cutis (OC).

PHP-Ia and PHP-Ic are characterised by hormone resistance, mainly to parathyroid hormone (PTH) (leading to obesity and variable degrees of intellectual disability and developmental delay); and the Albright hereditary osteodystrophy (AHO) phenotype characterised by short stature, round face, brachymetacarpia, subcutaneous ossification and various degrees of neurobehavioral defects and developmental delay. PHP-Ib is characterised mainly by PTH resistance, in the absence or presence of only mild features of AHO. AHO in the absence of hormone resistance is called pseudopseudohypoparathyroidism (PPHP). POH and OC are even more restricted variants of PPHP (Haldeman-Englert et al. 2017).

The GNAS locus is a complex imprinted locus that generates multiple transcripts through the use of several alternative first exons that splice into a common set of downstream exons, see Figure 1. Due to differential methylation of their promoters, most gene products originate from one parental allele. Transcripts GNASXL, which encodes XLαs, GNAS1A (also referred to as A/B) and the antisense transcript NESPAS (also referred to as GNAS-AS1) are transcribed from the paternal allele, while NESP55 (also referred to as NESP, or GNAS transcript variant 4) is transcribed from the maternal allele. The most downstream promoter (GNAS exon 1) is not differentially methylated, which results in GNAS expression from both alleles in most tissues.

GNAS encodes the Gsα protein, which is the α-subunit in the heterotrimeric G protein. Gsα is expressed biallelically 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 GNAS1A transcript and the antisense transcript NESPAS 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.

Maternally derived mutations are usually associated with PHPIa, while PPHP is caused by paternally derived mutations. PHPIb is caused by loss-of-methylation (LOM) at GNAS exon A/B located within a differentially methylated region. LOM can also be observed at additional GNAS exons, namely AS and XL, which is usually associated with a gain-of-methylation (GOM) at GNAS exon NESP. 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-B2 GNAS contains 45 (MS-)MLPA probes with amplification products between 126 and 500 nucleotides (nt). Twenty-five probes are specific for the GNAS locus, six for the STX16 gene and one for the TH1L gene (also referred to as NELFCD). Eighteen MS-MLPA probes contain an HhaI recognition site and provide information on the methylation status of the GNAS complex locus. The probe targeting the TH1L gene also contains a HhaI recognition site (unmethylated in normal blood-derived DNA). All probes present will also give information on copy number changes in the analysed sample. In addition, 11 reference probes are included that are not affected by HhaI digestion and detect genes located outside the GNAS region. 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.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 MS-MLPA General Protocol and online at www.mlpa.com.