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Bone Abstracts (2017) 6 OC2 | DOI: 10.1530/boneabs.6.OC2

ICCBH2017 Oral Communications (1) (26 abstracts)

Methylation patterns at the novel DMR of GNAS (GNAS-AS2) in pseudohypoparathyroidism 1B (PHP1B or iPPSD3) subtypes

Patrick Hanna 1 , Anne Rochtus 2 , Harald Jueppner 3 , Deborah Mackay 4 , Bruno Francou 5 , Jerôme Bouligand 5 , Anne Mantel 5 , Elli Anagnostou 6 , Elpis Vlachopapadopoulou 6 , Dominique Gaillard 7 , Brigitte Delemer 9 & Agnès Linglart 1,

1INSERM U1169, Bicêtre Paris Sud, France; 2Department of Pediatrics, University Hospitals, Leuven, Belgium; 3MGH and Harvard Medical School, Boston, MA, USA; 4Human Genetics and Genomic Medicine, Faculty of Medicine, University of Southampton, Southampton, UK; 5Laboratory of Molecular Genetics, APHP Bicêtre, Bicêtre Paris Sud, France; 6Division of Human Reproduction, Athens University Medical School, Athens, Greece; 7Génétique et Biologie de la Reproduction, Centre Hospitalier Universitaire, Reims, France; 8APHP, CMR Calcium-Phosphore, Bicêtre Paris Sud, France; 9Endocrinologie, CHU, Reims, France.

PHP1B -iPPSD3 per the new proposed classification- is a rare disorder characterized in most patients by proximal tubular resistance to PTH resulting in hypocalcemia, hyperphosphatemia and elevated PTH. Loss-of-methylation (LOM) at the Differentially Methylated Region (DMR) at GNAS exon A/B occurs in all PHP1B patients, but methylation changes at other DMRs within GNAS occur in some familial and most sporadic PHP1B cases. All patients with autosomal dominant PHP1B (AD-PHP1B) due to a maternal deletion that comprises the STX16 region (delSTX16+) present with LOM restricted to the GNAS-A/B DMR, while sporadic cases (sporPHP1B) present with broad GNAS methylation defects, including LOM at a novel, recently identified DMR within the GNAS locus referred to as antisense DMR2 (GNAS-AS2).

Objectives and patients: Characterize the methylation pattern at the GNAS-AS2 DMR in AD-PHP1B delSTX16+ (n=9) and delSTX16− (n=5) patients; furthermore, sporPHP1B (n=10) and healthy controls (n=10) were investigated. STX16 and GNAS deletions were excluded in the delSTX16− patients by MLPA, genomic multiplex and quantitative PCR of the GNAS and STX16 regions.

Results: 1- The mean methylation index at the GNAS-AS2-DMR was significantly higher in delSTX16− patients (32±14%) than in controls (24±6%), delSTX16+(5±2%) and sporPHP1B patients (3+1%) (P<0.0001).

2- Bisulfite-treated DNA of PHP1B patients with delSTX16− was PCR amplified across the GNAS-AS2-DMR and products were cloned into pcDNA3.1. First, we identified 2 CG-rich subdomains (SD1 and SD2) within the GNAS-AS2 DMR that are separated by 184 bp. Second, in delSTX16− patients we observed a unique pattern of methylation including an gain of methylation at SD1 and a methylation pattern at SD2 similar to that of controls, whereas both delSTX16+ and sporPHP1B patients displayed full LOM at SD1 and SD2.

Conclusion: We have further refined the GNAS-AS2-DMR and identified a subgroup of PHP1B patients with a specific pattern of methylation at the GNAS-AS2-DMR. Our findings reinforce the hypothesis that delSTX16− patients carry a defect in an element that controls the methylation both at the GNAS-A/B DMR and at the GNAS-AS2 DMR.

Disclosure: The authors declared no competing interests.

Volume 6

8th International Conference on Children's Bone Health


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