ESPE Abstracts

Background: Patients affected with PHP1B are characterized by resistance to PTH which binds to the PTH receptor and activate the cAMP/Gsa signaling pathway. Gsa is encoded by GNAS, a locus subjected to genomic imprinting. PHP1B patients present with abnormal methylation at the maternal A/B promoter and, in some cases, at the other promoters (XLas, GNAS-AS1 and NESP55) of the GNAS locus, likely leading to a decreased expression of Gsa. Clinical features of PHP1B are not fully explained by the cAMP/Gsa signaling defect. From the 3’ UTR of the GNAS-AS1 transcript, arise 3 miRNAs: hsa-miR-296-5p, hsa-mir-296-3p and hsa-mir-298-5p.

Objectives: Demonstrate that the GNAS-miRs are imprinted. Characterize their expression in controls and PHP1B patients and identify their targets.

Methods: Analysis of the GNAS miRNAS imprinting through pyrosequencing and qPCR. In silico characterization of GNAS miRNAs putative targets. Identification of targets of the GNAS-miRNAs through the overexpression of the 296-3p miRNA in HEK293 cells.

Results: 1- the GNAS-miRNAs display an imprinting expression pattern (in fibroblasts of one matUPD20 patient, down expression by 400 and 3.5 fold of the 296-5p miRNA (P=0.0015) and the 296-3p miRNA (P=0.0289), respectively). 2-Targets of the 3 miRNAs were characterized in silico using mirbase and subsequently classified in three groups: cAMP signaling, calcium signaling and growth. 3- In HEK cells, overexpression of the 296-3p miRNA leads to a significant decrease in the expression of AKAP6 and PRKAG1 by 35% (P=0.000085) and 44% (P=0.000023), respectively. In leukocytes of one patUPD20 patient, we found a significant decrease in AKAP6 (P=0.0015) and a non significant decrease in PRKAG1 expression, respectively. Both factors are involved in cAMP signaling.

Conclusion: The GNAS-296-5p-miRNA and the GNAS-296-3p-miRNA are imprinted and target factors involved in cAMP signaling independently from Gsa. Additional studies are necessary to decipher the precise role of the GNAS-miRNAs on the PHP1B phenotype.