title: European guidance for the molecular diagnosis of pseudohypoparathyroidism not caused by point genetic variants at GNAS: an EQA study.
abstract: Pseudohypoparathyroidism is a rare endocrine disorder that can be caused by genetic (mainly maternally inherited inactivating point mutations, although intragenic and gross deletions have rarely been reported) or epigenetic alterations at GNAS locus. Clinical and molecular characterization of this disease is not that easy because of phenotypic, biochemical and molecular overlapping features between both subtypes of the disease. The European Consortium for the study of PHP (EuroPHP) designed the present work with the intention of generating the standards of diagnostic clinical molecular (epi)genetic testing in PHP patients. With this aim, DNA samples of eight independent PHP patients carrying GNAS genetic and/or epigenetic defects (three patients with GNAS deletions, two with 20q uniparental disomy and three with a methylation defect of unknown origin) without GNAS point mutations were anonymized and sent to the five participant laboratories for their routine genetic analysis (methylation-specific (MS)-MLPA, pyrosequencing and EpiTYPER) and interpretations. All laboratories were able to detect methylation defects and, after the data analysis, the Consortium compared the results to define technical advantages and disadvantages of different techniques. To conclude, we propose as first-level investigation in PHP patients copy number and methylation analysis by MS-MLPA. Then, in patients with partial methylation defect, the result should be confirmed by single CpG bisulphite-based methods (ie pyrosequencing), whereas in case of a complete methylation defect without detectable deletion, microsatellites or SNP genotyping should be performed to exclude uniparental disomy 20.
authors.affiliation: Molecular (Epi)Genetics Laboratory, BioAraba National Health Institute, Hospital Universitario Araba-Txagorritxu, Vitoria-Gasteiz, Spain.
authors.forename: Intza
authors.initials: I
authors.lastname: Garin
authors.affiliation: Endocrinology and Diabetology Unit, Department of Clinical Sciences and Community Health, University of Milan, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Mantovani
authors.affiliation: Research Unit, REDISSEC: Health Services Research on Chronic Patients Network, Hospital Galdakao-Usansolo, Galdakao, Bizkaia, Spain.
authors.forename: Urko
authors.initials: U
authors.lastname: Aguirre
authors.affiliation: APHM, Hôpital la Conception, Laboratory of Molecular Biology, Aix Marseille Université, CRNS, CRN2M-UMR 7286, Marseille, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Barlier
authors.affiliation: Division of Experimental Paediatric Endocrinology and Diabetes, Department of Paediatrics, University of Luebeck, Luebeck, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Brix
authors.affiliation: Endocrinology and Diabetology Unit, Department of Clinical Sciences and Community Health, University of Milan, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.
authors.forename: Francesca M
authors.initials: FM
authors.lastname: Elli
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Freson
authors.affiliation: INSERM U986, Hôpital Bicêtre, Le Kremlin-Bicêtre, France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Grybek
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.
authors.forename: Benedetta
authors.initials: B
authors.lastname: Izzi
authors.affiliation: 1] INSERM U986, Hôpital Bicêtre, Le Kremlin-Bicêtre, France [2] Service d'Endocrinologie Pédiatrique, Hôpital Bicêtre-Paris Sud-Assistance-Publique-Hôpitaux-de Paris, Le Kremlin-Bicêtre, France [3] Centre de Référence des Maladies Rares du Métabolisme du calcium et du phosphore, Hôpital Bicêtre Paris Sud, Le Kremlin-Bicêtre, France.
authors.forename: Agnès
authors.initials: A
authors.lastname: Linglart
authors.affiliation: Molecular (Epi)Genetics Laboratory, BioAraba National Health Institute, Hospital Universitario Araba-Txagorritxu, Vitoria-Gasteiz, Spain.
authors.forename: Guiomar
authors.initials: G
authors.lastname: Perez de Nanclares
authors.affiliation: 1] INSERM U986, Hôpital Bicêtre, Le Kremlin-Bicêtre, France [2] Centre de Référence des Maladies Rares du Métabolisme du calcium et du phosphore, Hôpital Bicêtre Paris Sud, Le Kremlin-Bicêtre, France [3] Laboratoire de Biochimie Hormonale et Génétique, Hôpital Bichat Claude Bernard-Assistance Publique Hôpitaux de Paris, Paris, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Silve
authors.affiliation: Division of Experimental Paediatric Endocrinology and Diabetes, Department of Paediatrics, University of Luebeck, Luebeck, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Thiele
authors.affiliation: Division of Experimental Paediatric Endocrinology and Diabetes, Department of Paediatrics, University of Luebeck, Luebeck, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Werner
mesh_terms: D002864:Chromogranins; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005060:Europe; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics*; D017353:Gene Deletion; D005820:Genetic Testing / Q000592:standards*; D014644:Genetic Variation; D060005:Genotyping Techniques; D006801:Humans; D017410:Practice Guidelines as Topic; D011547:Pseudohypoparathyroidism / Q000175:diagnosis* / Q000235:genetics*; D017422:Sequence Analysis, DNA; D051438:Syntaxin 16 / Q000235:genetics; D024182:Uniparental Disomy / Q000235:genetics
chemical_list: D002864:Chromogranins; C496118:STX16 protein, human; D051438:Syntaxin 16; C406732:GNAS protein, human; D019205:GTP-Binding Protein alpha Subunits, Gs