ESPE Abstracts (2021) 94 P1-173

1Centre for Endocrinology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University London, London, United Kingdom; 2Studies of Campania Luigi Vanvitelli, Department of Woman, Child, General and Specialized Surgery, Naples, Italy; 3Federico II University Hospital: Azienda Ospedaliera Universitaria Federico II, Naples, Italy; 4University of Naples Federico II Department of Translational Medical Sciences: Universita degli Studi di Napoli Federico II Dipartimento di Scienze Mediche Traslazionali, Naples, Italy; 5LMU Klinikum, Medizinische Klinik und Poliklinik IV, München, Munich, Germany; 6Childrens Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, USA

Context: Severe forms of growth hormone insensitivity (GHI) are characterised by extreme short stature, dysmorphism and metabolic anomalies. They are classically caused by homozygous or compound heterozygous mutations of the growth hormone receptor gene (GHR). Genetic analysis traditionally focuses on the exonic regions of genes that encode proteins rather than the intervening non-coding sequences. These seldom explored non-coding regions may harbour numerous disease-causing mutations that are not yet well recognised or understood.

Objective: Identification of the genetic cause of severe postnatal growth failure in 3 ‘classical’ GHI subjects. Characterisation of the novel GHR 6Ω pseudoexon variant identified.

Design: A novel intronic GHR variant was identified by our GHI targeted whole genome custom-made gene panel. In vitro splicing assays were performed to confirm the in silico prediction of aberrant splicing. Patient fibroblast analysis determined the presence of the GHR 6Ω pseudoexon in the cDNA transcripts. A GHR 6Ω pseudoexon vector, created by Gibson assembly, enabled functional assessment of the 6Ω pseudoexon 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 GHR 6Ψ pseudoexon mutation, in the index patient. In the second kindred, two siblings were found to harbour the same novel intronic GHR 6Ω pseudoexon variant in compound heterozygosity with the known GHR c.181C>T (R43X) mutation. RT-PCR of patient fibroblasts demonstrated presence of the 6Ω pseudoexon transcript in the 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. HEK293 cells transfected with 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 loss of GHR function consistent with a severe GHI phenotype. This represents a novel mechanism of growth hormone insensitivity/Laron syndrome and is the first deep intronic variant identified causing severe postnatal growth failure. The two kindreds originate from the same town in Campania, Southern Italy, implying common ancestry. Our findings highlight the importance of studying variation in deep intronic regions as a cause of monogenic disorders.

Volume 94

59th Annual ESPE (ESPE 2021 Online)

22 Sep 2021 - 26 Sep 2021

European Society for Paediatric Endocrinology 

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