Bidirectional disruption of <i>GNAS</i> transcripts causes broad methylation defects in pseudohypoparathyroidism type 1B

Pseudohypoparathyroidism type 1B (PHP1B) is a multihormone resistance disorder caused by aberrant <i>GNAS</i> methylation. Characteristic epigenetic changes at <i>GNAS</i> differentially methylated regions (DMRs), i.e., NESP, AS1, AS2, XL, and A/B, are associated with specific structural defects in different autosomal dominant PHP1B (AD-PHP1B) subtypes. However, mechanisms underlying abnormal <i>GNAS</i> methylation remain incompletely defined, largely because viable PHP1B mouse models are lacking. Using lymphoblastoid cells and induced pluripotent stem cells, we show that various <i>GNAS</i> methylation patterns in PHP1B reflect differential disruption of sense and antisense <i>GNAS</i> transcripts. In cases with broad <i>GNAS</i> methylation changes, loss of the maternal, sense-transcribed exon H/AS region impairs methylation of the AS1 DMR, which results in biallelic expression of an antisense transcript, <i>GNAS-AS1</i>, and NESP hypermethylation. In contrast, cases with normal AS1 methylation, including <i>STX16</i> deletions, show monoallelic <i>GNAS-AS1</i> expression and normal NESP methylation. The roles of these <i>GNAS</i> transcripts were confirmed by a retrotransposon in <i>GNAS-AS1</i> intron 1, identified in an AD-PHP1B family. This insertion impaired exon H/AS transcription when located on the maternal allele, thus preventing the complete establishment of methylation at all maternal <i>GNAS</i> DMRs, leading to biallelic <i>GNAS-AS1</i> transcription. However, maternal <i>GNAS-AS1</i> transcription was profoundly attenuated, thus allowing only a small gain-of-methylation at NESP. Likewise, on the paternal allele, the retrotransposon attenuated <i>GNAS-AS1</i> transcription, thus preventing complete NESP methylation. Our findings support a model of bidirectional transcription-mediated regulation of methylation at <i>GNAS</i> DMRs and will help to refine systematic approaches for establishing molecular defects underlying different PHP1B subtypes.