SAT-039 GNAS-miRNAs Are Likely Involved in the Phenotype of Patients with Pseudohypoparathyroidism 1B/iPPSD3

Pseudohypoparathyroidism 1B (PHP1B) or inactivating PTH/PTHrp Signaling Disorder type 3 (iPPSD3), according to the new classification, presents mainly with hypocalcemia and hyperphosphatemia due to renal resistance towards parathyroid hormone. In addition, patients display early onset obesity and impaired pubertal growth. The disease is due to a methylation defect at one or several Differentially Methylated Regions (DMR) of the GNAS locus, subjected to parental imprinting. 80% of iPPSD3 patients have methylation defect at GNAS A/B:TSS-DMR and at least one other GNAS DMR (most commonly including GNAS-AS1:TSS-DMR). They are known as sporadic cases. GNAS-miRNAs are located on the 3'UTR of the antisense transcript GNAS-AS1. We hypothesize that abnormal expression of the GNAS-miRNAs may contribute to the phenotype of iPPSD3 patients with methylation defect at GNAS-AS1:TSS-DMR. We used fibroblasts obtained from a patient with uniparental maternal disomy of chr. 20 (matUPD20) and leukocytes from a patient with paternal UPD20 (patUPD20) and from controls to measure expression of GNAS-miRNAs. In silico targets of GNAS-miRNAs have been characterized using miRbase. Finally, HEK cells were transfected with GNAS-miRNAs in order to identify targets. We found that GNAS-miRNAs are subjected to parental imprinting. In fact, hsa-miR-296-5p and hsa-miR-296-3p are under-expressed by 400-fold (p=0.0015) and 3.5-fold (p=0.0289) in the fibroblasts of the matUPD20 patient, respectively. We identified in silico, three groups of GNAS-miRNAs targets classified into cAMP signaling, calcium signaling and growth. Overexpression of hsa-miR-296-3p in HEK cells significantly represses PRKAG1 and GNB2 transcripts expression, p<0.001. Overexpression of hsa-miR-298-5p significantly increased transcripts AKAP6 and PRKAB2 expression, p<0.001. Overexpression of hsa-miR-296-5p significantly represses GNB2 transcript expression, p<0.001. In addition, the GNAS-miRNAs target GNAS-AS1, A/B and Gsα the GNAS transcripts. In summary, the GNAS-miRNAs are subjected to parental imprinting. They likely regulate in cis the GNAS transcripts and intrans the expression of transcripts involved in the cAMP/PKA (GNB2 and AKAP6) and in the AMPK (PRKAG1 and PRKAB2) pathways, respectively. PKA and AMPK are involved in lipolysis, fatty acid oxidation and obesity. Therefore, disturbances within AMPK and PKA pathways may explain the early onset obesity phenotype seen in PHP1B patients.