ESPE Abstracts (2022) 95 P1-148

1University of Sao Paulo Medical School, Sao Paulo, Brazil; 2Developmental Endocrine Unit - Endocrinology and Metabolism Division, Sao Paulo, Brazil; 3Neuroendocrinology Unit - Endocrinology and Metabolism Division, Sao Paulo, Brazil; 4Metabolic Bone Disease Unit - Endocrinology and Metabolism Division, Sao Paulo, Brazil; 5University of Sao Paulo Chemistry Department, Sao Paulo, Brazil

Introduction: McCune-Albright Syndrome (MAS) is a rare congenital disorder caused by post-zygotic activating mutations in GNAS gene. Due to the mosaic pattern of this disease, mutation abundance is frequently low in several tissues, including blood cells. The emergence of next-generation sequencing (NGS) methodologies has allowed the analysis of millions of DNA fragments simultaneously and independently, enabling detection of low mutation abundance. Aim: To establish the molecular diagnosis of patients with MAS using NGS analysis. Methods: We selected 18 patients (13 girls and 5 boys) with at least 2 out of 3 classic clinical features of MAS. Of them, 14 and 17 patients had café au lait skin spots and fibrous dysplasia, respectively. All girls presented peripheral precocious puberty. In addition, hyperthyroidism and acromegaly were identified in 5 (2 girls e 3 boys) and 3 (all boys) patients, respectively. DNA was extracted from peripheral blood and analyzed through a targeted gene panel for endocrinopathies by NGS (Agilent SureSelect XT). DNA samples were prepared and pair-end sequenced on the Illumina NextSeq 500 platform (High Output - 300Cycles). The sequences were aligned using GTKA program. Using the Integrative Genomics Viewer (IGV) program, BAM files were visually analyzed for genomic regions 20:57484420-57484421 and 20:57484595-57484597 (GRCh37/hg19), where GNAS mutations associated with MAS are located. Allelic variants represented for 6 or more independent reads with no strand bias were considered as a positive result after 35 negative controls analyzes. Results: The mean read depth per sample for two codons (201 and 227) was 269 (range 180-317) and 499 (range 363-686), respectively. Conventional base calling programs (Freebayes) using protocols for analysis of germline variants did not detect variants in GNAS associated with MAS. The visual analysis in IGV revealed GNAS mutations in 7 of 18 (38.8%) patients. The p.R201C and p.R201H mutations were identified in 4 and 3 patients, respectively. The allele mutation abundance ranged from 2.8% to 19%. Inconclusive molecular results were obtained in 5 patients due to low reads (≤ 5) and/or strand bias: p.R201H (≤5 reads), p.G227H (strand bias), and p.G227R (3 reads and strand bias). Conclusion: NGS represents a more sensitive tool for identifying GNAS mutations in peripheral blood, even with low abundance, in patients with MAS, with the advantage of analyzing multiple positions.

Volume 95

60th Annual ESPE (ESPE 2022)

Rome, Italy
15 Sep 2022 - 17 Sep 2022

European Society for Paediatric Endocrinology 

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