SAT-272 GNAS AS2 Methylation Status Enables Mechanism-based Categorization of Pseudohypoparathyroidism Type 1B

Abstract Y. Iwasaki: None. M. Reyes: None. H. Jüppner: None. M. Bastepe: None. Background: Pseudohypoparathyroidism type 1B (PHP1B) is caused by aberrant genomic imprinting at the GNAS locus, giving rise to Gsα and additional transcripts derived from differentially methylated regions (DMRs), including NESP, antisense (AS), XL, and A/B. Parent-specific methylation of these DMRs is crucial for normal Gsα expression, while GNAS methylation defects cause PHP1B. Defining the underlying genetic cause in new patients is challenging because various genetic alterations (e.g., deletions, insertions, duplications, or inversions) within the GNAS or the neighboring STX16 regions can cause PHP1B, and a genotype-epigenotype correlation has not been established. Recently, a new DMR located between AS exon 1 and XL, referred to as AS2, was shown to be hypomethylated in some PHP1B variants. In the current study, we categorized GNAS methylation patterns in PHP1B patients based on the AS2 methylation status. Methods: Using MSRE-qPCR, we analyzed AS2 methylation levels in wild-type (WT) human ES cells (hESCs) and after introducing separate deletions of two GNAS imprinting control regions (ICRs) (STX16-ICR or NESP-ICR). We also analyzed, by MS-MLPA and MSRE-qPCR, GNAS methylation status in PHP1B patients (n=31) with various genetic causes or with undetermined defects. Analyzed genetic alterations included those located centromeric of AS2 (e.g., STX16 deletions and a NESP-AS exons 3/4 deletion) and telomeric of AS2 (e.g., a retrotransposon insertion telomeric of XL). We also performed luciferase reporter assays using transfected hESCs to test whether the retrotransposon insertion attenuates transcription. Results: In WT hESCs, maternal deletion of STX16- or NESP-ICRs resulted in a reduction of methylation at AS2 (p<0.0001 for STX16-ICR; p=0.0015 for NESP-ICR). In PHP1B patients, AS2 was hypomethylated only when disease-causing genetic alterations were located centromeric, but not telomeric of this DMR. Methylation levels at a single MS-MLPA probe were significantly correlated with AS2 methylation status (p < 0.0001). Reporter assays using hESCs with insertion of different retrotransposon portions significantly attenuated NESP promoter-driven transcription. Discussion: Based on our findings, normal AS2 methylation is likely to be preserved only when transcription from the upstream NESP promoter is intact during an early embryonic period. AS2 methylation status can help identify the locations of genetic alterations that potentially blunt NESP transcription. By further distinguishing broad GNAS methylation defects, we could classify GNAS methylation defects in PHP1B into three categories: #1, broad methylation defects; #2, loss-of-methylation restricted to A/B and AS2; #3, isolated A/B hypomethylation. Using a single probe in a commercially available MS-MLPA assay, this categorization is widely applicable to guide the molecular diagnosis of PHP1B. Saturday, June 1, 2024