title: Genome-wide DNA methylation analysis of pseudohypoparathyroidism patients with GNAS imprinting defects.
abstract: BACKGROUND
  Pseudohypoparathyroidism (PHP) is caused by (epi)genetic defects in the imprinted GNAS cluster. Current classification of PHP patients is hampered by clinical and molecular diagnostic overlaps. The European Consortium for the study of PHP designed a genome-wide methylation study to improve molecular diagnosis.
  
  
  METHODS
  The HumanMethylation 450K BeadChip was used to analyze genome-wide methylation in 24 PHP patients with parathyroid hormone resistance and 20 age- and gender-matched controls. Patients were previously diagnosed with GNAS-specific differentially methylated regions (DMRs) and include 6 patients with known STX16 deletion (PHP(Δstx16)) and 18 without deletion (PHP(neg)).
  
  
  RESULTS
  The array demonstrated that PHP patients do not show DNA methylation differences at the whole-genome level. Unsupervised clustering of GNAS-specific DMRs divides PHP(Δstx16) versus PHP(neg) patients. Interestingly, in contrast to the notion that all PHP patients share methylation defects in the A/B DMR while only PHP(Δstx16) patients have normal NESP, GNAS-AS1 and XL methylation, we found a novel DMR (named GNAS-AS2) in the GNAS-AS1 region that is significantly different in both PHP(Δstx16) and PHP(neg), as validated by Sequenom EpiTYPER in a larger PHP cohort. The analysis of 58 DMRs revealed that 8/18 PHP(neg) and 1/6 PHP(Δstx16) patients have multi-locus methylation defects. Validation was performed for FANCC and SVOPL DMRs.
  
  
  CONCLUSIONS
  This is the first genome-wide methylation study for PHP patients that confirmed that GNAS is the most significant DMR, and the presence of STX16 deletion divides PHP patients in two groups. Moreover, a novel GNAS-AS2 DMR affects all PHP patients, and PHP patients seem sensitive to multi-locus methylation defects.
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O&N1, Herestraat 49, Box 911, 3000 Leuven, Belgium ; Department of Pediatrics, University Hospitals Leuven, 3000 Leuven, Belgium.
authors.forename: Anne
authors.initials: A
authors.lastname: Rochtus
authors.affiliation: Laboratory of Genomic Imprinting and Cancer, IDIBELL, 08908 Barcelona, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Martin-Trujillo
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O&N1, Herestraat 49, Box 911, 3000 Leuven, Belgium.
authors.forename: Benedetta
authors.initials: B
authors.lastname: Izzi
authors.affiliation: Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Endocrinology and Diabetology Unit, Department of Clinical Sciences and Community Health, University of Milan, 20122, Milan, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Elli
authors.affiliation: Molecular (Epi)Genetics Laboratory, BioAraba National Health Institute, Hospital Universitario Araba-Txagorritxu, 01009 Vitoria-Gasteiz, Spain.
authors.forename: Intza
authors.initials: I
authors.lastname: Garin
authors.affiliation: Department of Pediatric Endocrinology and Diabetology for Children, APHP, Bicêtre Paris Sud, 94275 Le Kremlin Bicêtre, France ; Reference Center for Rare Disorders of the Mineral Metabolism and Plateforme d'Expertise Paris Sud, APHP, 94275 Le Kremlin Bicêtre, France.
authors.forename: Agnes
authors.initials: A
authors.lastname: Linglart
authors.affiliation: Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Endocrinology and Diabetology Unit, Department of Clinical Sciences and Community Health, University of Milan, 20122, Milan, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Mantovani
authors.affiliation: Molecular (Epi)Genetics Laboratory, BioAraba National Health Institute, Hospital Universitario Araba-Txagorritxu, 01009 Vitoria-Gasteiz, Spain.
authors.forename: Guiomar
authors.initials: G
authors.lastname: Perez de Nanclares
authors.affiliation: Division of Experimental Paediatric Endocrinology and Diabetes, Department of Paediatrics, University of Luebeck, 23560 Luebeck, Germany.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Thiele
authors.affiliation: Department of Clinical and Experimental Endocrinology, University of Leuven, 3000 Leuven, Belgium.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Decallonne
authors.affiliation: Department of Pediatrics, University Hospitals Leuven, 3000 Leuven, Belgium.
authors.forename: Chris
authors.initials: C
authors.lastname: Van Geet
authors.affiliation: Laboratory of Genomic Imprinting and Cancer, IDIBELL, 08908 Barcelona, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Monk
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O&N1, Herestraat 49, Box 911, 3000 Leuven, Belgium.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Freson
mesh_terms: D016022:Case-Control Studies; D002864:Chromogranins; D019175:DNA Methylation; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics*; D055106:Genome-Wide Association Study; D018392:Genomic Imprinting / Q000235:genetics*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D011547:Pseudohypoparathyroidism / Q000235:genetics*
chemical_list: D002864:Chromogranins; C406732:GNAS protein, human; D019205:GTP-Binding Protein alpha Subunits, Gs
keywords: DNA methylation; GNAS; Imprinting disorders; Pseudohypoparathyroidism
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