(A) Loss of methylation (LOM) can occur at all maternal GNAS DMRs, but LOM at the A/B DMR alone is sufficient to reduce Gsα expression, thus causing PHP1B. Maternal exon H transcripts establish all maternal DMRs during oogenesis, while maternal NESP transcripts facilitate postzygotic remethylation of the A/B and AS2 DMRs. (B) Methylation at GNAS DMRs (%), microsatellite analyses, and the exon H splice donor site (exHss) variant in kindred 285. (C) The exHss variant is located in 285/I-2 on the paternal allele; nucleotide sequence analysis of PCR amplicons of non-digested (top) and HpaII-digested genomic DNA (bottom). (D) Minigene reporter assay revealed in hESCs reduced H exon–derived transcription due to SNVs at the exHss. ****P < 0.0001 by 1-way ANOVA with Tukey’s post hoc test. (E) Disruption of the maternal exHss by introducing 13-bp or 8-bp deletions into hESCs through CRISPR/Cas9. (F) RT-PCR assessing exon H–derived transcripts (left) and schematic presentation of the cryptic splice site (right). Arrows indicate PCR primers (see Supplemental Methods for details). (G) qRT-PCR assessing exon H– and Gsα-derived transcript levels. ***P < 0.001 by 1-sample t test. NS, nonsignificant. (H) Top: The establishment of maternal GNAS methylation imprints occurs during oogenesis and postzygotically. Bottom: Disruption of the exon H splice donor site attenuates transcription from this exon, thus preventing establishment of maternal methylation during oogenesis and leading postzygotically to the Cat1-PHP1B epigenotype. Data are shown as mean ± SEM of 3 independent experiments (D) or independent clones (G).