Pseudohypoparathyroidism type 1B (PHP1B), also known as inactivating PTH/PTHrP signaling disease (iPPSD), is characterised by proximal renal tubular resistance to PTH, resulting in hypocalcemia, hyperphosphatemia, and high PTH levels. Maternal deletions involving STX16 can result in autosomal dominant PHP1B (AD-PHP1B) with a lack of methylation at the maternal GNAS A/B: TSS-DMR. Characterize and evaluate GNAS-AS2:TSS-DMR methylation in a previously unreported AD-PHP1B family with the loss of methylation at GNAS A/B: TSS-DMR but no indication of an STX16 deletion on the maternal allele. A total of 24 patients and 10 controls had their DNA analyzed for the study. A single-family of AD-PHP1B patients without STX16 deletion, AD-PHP1B patients with STX16 deletion, sporPHP1B, unaffected controls, patUPD20, and matUPD20 were all tested. Pyrosequencing, methylation-sensitive multiplex ligation-dependent probe amplification, and multiplex ligation-dependent probe amplification were used to analyze methylation and copy number. GNAS-AS2:TSS-DMR subdomains called AS2-1 and AS2-2 were found in the bisulfite-treated genomic DNA of healthy controls after polymerase chain reaction amplification. The methylation levels of AS2-1 and AS2-2 were respectively 16.0% and 31.00%. Without any known genetic abnormalities, DNA from afflicted members of a previously unreported AD-PHP1B family showed partial loss of methylation at GNAS A/B: TSS-DMR and normal DMR levels in both parents. 

GNAS-AS2:TSS-DMR methylation alterations may assist characterize PHP1B/iPPSD3 variants and drive the search for underlying genetic abnormalities, which may give unique insights into the processes of GNAS methylation.