European guidance for the molecular diagnosis of pseudohypoparathyroidism not caused by point genetic variants at GNAS: an EQA study

Abstract

on behalf of the EuroPHP Consortium 12Pseudohypoparathyroidism 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.

Prenatal molecular testing for Beckwith–Wiedemann and Silver–Russell syndromes: a challenge for molecular analysis and genetic counseling

“…However, as shown for the imprinted GNAS locus, the heterogeneity of molecular tests does not affect the correctness of the molecular diagnosis. 43 Thus no specific Prenatal testing in Silver-Russell and Beckwith-Wiedemann syndrome T Eggermann et al Table 3a Data on the prenatal cohort tested for BWS or SRS: overview on the tested tissues and the applied methods in prenatal BWS and SRS testing Prenatal testing in Silver-Russell and Beckwith-Wiedemann syndrome T Eggermann et al assay can be recommended, but in any case a careful validation has to be carried out and advantages and disadvantages in the context of prenatal testing (duration of the protocol, amount of DNA) have to be considered. For the methods analyzed by our centers, between 50 (MS-MLPA) and 100 (HRMA, AMMS-RTqPCR) control samples have been used for validation.…”

Congenital imprinting disorders: Application of multilocus and high throughput methods to decipher new pathomechanisms and improve their management

“…Due to this lack of standardization, it is difficult to compare the molecular results between different studies or laboratories offering molecular diagnosis for the same disorder. However, as data for the GNAS locus reveals, the heterogeneity of molecular tests affects the detection of mosaic (epi)mutations rather than the correctness of the molecular diagnosis [12].…”

Case report: An infantile lethal form of Albright hereditary osteodystrophy due to a GNAS mutation

“…Due to the severity of our case, questions arise as to the possible interaction of modifying genetic or environmental factors, which could worsen the clinical picture . Through MS‐MLPA, we excluded a deletion in the GNAS promotor regions or an abnormal methylation pattern.…”