OR15-04 Autosomal dominant pseudohypoparathyroidism type Ib (PHP1B) locus, STX16, comprises a biallelically active embryonic enhancer mediating the epigenetic regulation of GNAS

Abstract Y. Iwasaki: None. M. Reyes: None. H. Jüppner: None. M. Bastepe: None. Background and Aim: The GNAS gene encodes Gsα, which mediates a wide range of hormone actions and, thus, plays a critical role in the pathogenesis of various endocrine disorders. The methylation of GNAS is essential for proper expression of Gsα and additional GNAS-derived transcripts including NESP, XL, and A/B. Abnormal GNAS methylation causes pseudohypoparathyroidism type 1B (PHP1B), characterized by end-organ resistance to PTH and other hormones. Developing a human ES cell (hESC)-based PHP1B model, we have recently shown that the STX16 gene, recurrently deleted in PHP1B and located ∼200 kb centromeric of GNAS, comprises an enhancer for maternal NESP transcription, required for normal GNAS methylation. AS2 is a recently defined differentially methylated region (DMR) hypomethylated in most PHP1B patients. It is immediately upstream of XL, altered levels of which are associated with early postnatal neuroendocrine and growth defects. We aimed to determine the broader role of the enhancer at STX16 in regulating GNAS. Methods: We used hESCs with deletions within STX16 on the maternal or paternal allele. The effect of the deletions on allele-specific GNAS expression was analyzed by RT-PCR followed by sequencing of a heterozygous polymorphism and by qPCR. DNA interaction between STX16 and GNAS in an allele-specific manner was examined by chromatin conformation capture (3C) followed by sequencing of heterozygous SNPs. The enhancer activity of STX16 was interrogated using luciferase reporter assays in hESCs. RESULTS: NESP was exclusively maternally expressed, whereas XL was biallelically expressed in hESCs. Maternal STX16 deletion attenuated NESP (9.7% of WT, p<0.0001) and XL expression (63.6% of WT, p = 0.0063), whereas its paternal deletion diminished paternal XL expression only (23.2% of WT, p = 0.0023). Allele-specific XL expression switched to exclusively paternal or maternal upon maternal and paternal STX16 deletion, respectively. Biallelic disruption of an OCT4-SOX2 motif in STX16 intron 4 markedly suppressed transcript levels of both NESP and XL (p = 0.025 and p < 0.0001, respectively). Maternal STX16 deletion also resulted in a complete loss of AS2 methylation. The STX16 region containing the OCT4-SOX2 motif potently enhanced the activity of the XL promoter comprising the AS2 DMR. 3C assays showed that STX16 was in proximity to the NESP DMR on the maternal allele and the XL DMR on both parental alleles. Discussion: We identified the STX16 locus as an embryonic stage GNAS enhancer operating differentially on parental alleles, activating the XL promoter on both parental alleles while enhancing the NESP promoter exclusively on the maternal allele. In addition, regulated by the enhancer at the STX16 locus in hESCs, the AS2 DMR is part of the XL promoter, suggesting that the loss of AS2 methylation in PHP1B may contribute to disease phenotype by affecting XL expression. Saturday, June 1, 2024