ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2019) 66 P73 | DOI: 10.1530/endoabs.66.P73

Three novel Growth Hormone Receptor (GHR) splicing mutations causing a spectrum of Growth Hormone Insensitivity

Emily Cottrell1, Avinaash Maharaj1, Tasneem Ladha1, Sumana Chatterjee1, Anna Grandone2, Grazia Cirillo2, Emanuele Miraglia del Giudice2, Ludmila Kostalova3, Eva Vitariusova3, Vivian Hwa4, Louise A Metherell1 & Helen L Storr1

1Centre for Endocrinology, William Harvey Research Institute, Queen Mary University London, London, UK; 2Department of Woman, Child, General and Specialized Surgery at Universita`egli Studi d’ella Campania ‘L. Vanvitelli’, Naples, Italy; 3Department of Pediatrics, Comenius University Medical School and National Institute of Child Diseases, Bratislava, Slovakia; 4Cincinnati Center for Growth Disorders, Division of Endocrinology, Cincinnati Children’s Hospital Medical Center, Cincinnati, USA

Introduction: Growth Hormone Insensitivity (GHI) is characterised by a triad of short stature (SS), IGF-1 deficiency and normal/high GH levels. ‘Classical’ GHI due to homozygous exonic GHR mutations results in extreme SS with dysmorphic and metabolic abnormalities. Heterozygous exon 9 GHR mutations are rare and exert dominant negative effects due to impairment of GHR dimerization/downstream signalling associated with a milder GHI phenotype. Only seven previous GHR dominant negative defects have been identified.

Objective: To identify the genetic cause of growth failure in undiagnosed patients with GHI phenotypes.

Design: Genetic variants were identified from our SS gene panel which interrogates coding and non-coding regions of known GHI genes using our established bioinformatics pipelines. Aberrant splicing was confirmed by in vitro splicing assays using an exon trap vector (pET01, MoBiTec GmbH, Germany).

Results: A heterozygous GHR variant (42718139T>G, c.810-15T>G) identified in Patient 1 was predicted to decrease splicing efficiency due to disruption of the polypyrimidine tract prior to exon 9. The strongest nearby alternative splice site was 26 bases 3’ from the exon/intron boundary. The variant was inherited from his mother with a similar phenotype. A de novo heterozygous GHR variant (42718180T>G, c.836T>G) identified in Patient 2 was predicted to create a cryptic splice site within exon 9. Splicing assays confirmed the presence of mutant transcripts in both patients. Both novel variants cause frameshift and predicted premature truncation of GHR transcripts. Consistent with published reports, both had less severe growth failure than ‘classical’ GHI (height SDS −3.2 and −2.7, respectively). We also identified a novel homozygous GHR variant (42700940T>G, c.618+836T>G) in Patient 3. In silico analysis predicted donor splice site creation and in vitro splicing analysis confirmed inclusion of a 152bp pseudoexon. This causes frameshift and premature truncation of all GHR mRNA in keeping with his severe GHI phenotype (height SDS −7.5). We predict nonsense mediated mRNA decay and are currently testing this hypothesis.

Discussion: Three novel GHR splicing mutations contribute to our understanding of GHI. Our findings highlight the importance of considering dominant negative mutations in non-classical GHI and studying variation in deep intronic sequence as a cause of monogenic disorders.