BSPED2021 Oral Communications Oral Communications 4 (9 abstracts)
Growth hormone receptor 6Ω pseudoexon activation: a novel cause of severe growth hormone insensitivity
Emily Cottrell 1 , Avinaash Maharaj 1 , Jack Williams 1 , Sumana Chatterjee 1 , Grazia Cirillo 2 , Emanuele Miraglia del Giudice 2 , Adalgisa Festa 2 , Stefania Palumbo 2 , Donatella Capalbo 3 , Mariacarolina Salerno 4 , Claudio Pignata 4 , Martin O. Savage 1 , Katharina Schilbach 5 , Martin Bidlingmaier 5 , Vivian Hwa 6 , Louise A. Metherell 1 , Anna Grandone 2 & Storr Helen L. 1
1Centre for Endocrinology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, United Kingdom; 2Department of Woman, Child, General and Specialized Surgery, The University of Campania Luigi Vanvitelli, Naples, Italy; 3Pediatric Unit, Department of Translational Medical Sciences, University of Naples Federico II, Naples, Italy; 4Pediatric Unit, Department of Mother and Child, University Hospital Federico II of Naples, Naples, Italy; 5Endocrine Research Unit, Medizinische Klinik und Poliklinik IV, LMU Klinikum, Munich, Germany; 6Cincinnati Center for Growth Disorders, Division of Endocrinology, Cincinnati Children’s Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, USA
Context: Severe or ‘classical’ growth hormone insensitivity (GHI) is characterised by extreme short stature, dysmorphism and metabolic anomalies. It is caused by homozygous or compound heterozygous mutations of the Growth Hormone Receptor gene (GHR). Genetic analysis traditionally focuses on the exonic regions of gene(s). The non-coding regions of the genome may harbour numerous disease-causing mutations that are not well recognised or understood.
Objective: Identification of the genetic cause of growth failure in 3 ‘classical’ GHI subjects. Assessment of identified novel 6Ω pseudoexon GHR variant.
Design: Genetic variants of interest were identified using our targeted whole genome gene panel and filtered using our custom bioinformatics pipeline. In vitro splicing assays were performed to confirm aberrant splicing. Patient fibroblast analysis was performed to determine the presence of the GHR 6Ω pseudoexon in cDNA transcripts. A 6Ω pseudoexon GHR vector created by Gibson assembly assessed the functional consequences of the novel inclusion.
Results: We identified a novel homozygous intronic GHR variant (g.5:42700940T>G, c.618+836T> G), 44bp downstream of the previously recognised intronic 6Ψ GHR pseudoexon mutation, in the index patient. In the second kindred, two siblings were also found to harbour this novel intronic 6Ω pseudoexon GHR variant in compound heterozygosity with the known GHR c.181C>T (R43X) mutation. RT-PCR of patient fibroblasts demonstrated the presence of the 6Ω pseudoexon transcript in patient cDNA. In vitro splicing analysis confirmed inclusion of a 151bp mutant 6Ω pseudoexon not identified in wild-type constructs. Inclusion of the 6Ω pseudoexon causes a frameshift resulting in a non-functional truncated GHR lacking the transmembrane and intracellular domains. The 6Ω pseudoexon Gibson construct demonstrated extracellular accumulation of the mutant, truncated GHR protein and diminished activation of STAT5B signalling following growth hormone stimulation.
Conclusion: Novel GHR 6Ω pseudoexon inclusion results in complete loss of GHR function consistent with a severe GHI phenotype. This represents a novel mechanism of GHI and is the first deep intronic variant identified causing severe postnatal growth failure. The two kindreds originate from Campania, Southern Italy, implying common ancestry. Our findings highlight the importance of studying variation in deep intronic regions as a cause of monogenic disorders.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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