ESPE Abstracts

Autosomal dominant pseudohypoparathyroidism type-Ib is characterized by renal parathyroid hormone resistance, with resultant hypocalcemia and hyperphosphatemia. This disorder is associated with an isolated loss of methylation at GNAS exon A/B and most patients carry maternal microdeletions in the neighboring STX16 gene. The shortest deletion overlap is a 1.2-kb region spanning STX16 exon 4 and thought to harbor a cis-acting element regulating GNAS A/B methylation. However, ablation of the orthologous mouse region does not recapitulate the patient findings and no functional data exists supporting the association between the STX16 gene and GNAS imprinting. The A/B methylation is established in the female germline and maintained throughout the development in somatic cells. We thus investigated the role of the 1.2-kb STX16 region in the maintenance of A/B methylation, employing HCT116 cells, a near-diploid human cell line derived from colorectal carcinoma. To delete this region, we used CRISPR/Cas9 and suitable guide-RNAs targeting upstream and downstream of exon 4. We first generated HCT116 cells stably expressing Cas9 and introduced the guide-RNAs into those cells by lentiviral delivery. After antibiotic enrichment of the transduced cells, the presence of a 2.1-kb deletion spanning STX16 exon 4 and flanking intronic regions was confirmed by PCR, followed by TA-cloning and Sanger sequencing of the products. Multiplex ligation-dependent probe amplification (MLPA) indicated a modest reduction in the STX16 exon 4 copy number, suggesting that the deletion was present only in a subset of cells. Methylation specific-MLPA analysis, which was performed in non-clonal cells, indicated normal methylation status at GNAS exon A/B, and this finding was confirmed by the combined bisulfite restriction analysis. Thus, our initial findings suggest that the putative cis-acting element within STX16 does not regulate the maintenance of exon A/B methylation. To further confirm these results, we are in the process of selecting and analyzing clonal HCT116 cells homozygous or heterozygous for this deletion. In addition, we are introducing the same deletion into human ES cells, so that we can determine its impact on GNAS imprinting during early development.