GNAS Methylation-Specific MLPA

Clinical Information

Pseudohypoparathyroidism (PHP) is caused by inactivation of GNAS. There are multiple PHP phenotypes resulting from GNAS inactivation, including PHP -Ia, -Ib, and –Ic. Additional phenotypes include pseudopseudohypoparathyroidism (PPHP), progressive osseous heteroplasia (POH), and osteoma cutis (OC). These disorders are associated with varying degrees of hormone resistance and skeletal abnormalities including short stature, subcutaneous ossifications, and brachydactyly.

GNAS is an imprinted locus on chromosome 20q13.32 with a primary mechanism for GNAS inactivation resulting from methylation defects. Epigenetic inactivation of GNAS can occur through paternal uniparental disomy, isolated epivariants, and alterations in regulatory elements of GNAS or STX16.

Disorders of GNAS inactivation are examples of disorders involving imprinted genes. Imprinted genes are only expressed from either the maternally or paternally derived member of a homologous chromosome pair. Disorders of GNAS inactivation are caused by the lack of expression of GNAS. PHP -Ia, -Ib, and –Ic are caused by inactivation of the maternal GNAS complex locus while PPHP, POH, and OC are caused by inactivation of the paternal GNAS complex locus.

Technical Information

Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) analysis is performed to determine if deletions, duplications or methylation defects involving the GNAS complex locus on 20q13.32 are present. MS-MLPA analysis allows for the determination of dosage for specific sequences of interest as well as for methylation status at several differentially methylated HhaI restriction sites (Schouten et al. (2002) Nucleic Acids Research 30, e57).

Genomic DNA is hybridized with probes that correspond to CpG islands in the promoters of GNAS-NESP, GNAS-AS1, GNAS-XL, and GNAS-A/B to determine dosage and methylation status of the genes in those regions. This assay also includes probes to detect deletions or duplications in the STX16 gene, which serves as a long-range control element of methylation at the GNAS locus.

Specimen Requirements

• The preferred sample type is 3-4 ml of peripheral blood collected in an EDTA (purple top) tube. Extracted DNA is also accepted for this test. Blood kits are available by request.
• Send approximately 5µg of extracted DNA at a requested concentration of 90-130 ng/µl.

Transport Instructions

• The blood specimen should be kept at room temperature and delivered via overnight shipping. If shipment is delayed by one or two days, the specimen should be refrigerated and shipped at room temperature. Do not freeze the specimen. Samples collected on Friday can be safely designated for Monday delivery.
• Extracted DNA should be sent at room temperature via overnight delivery.