ESPE Abstracts

Background: Growth Hormone Insensitivity (GHI) is usually caused by mutations in the Growth Hormone receptor (GHR). Patients present with short stature associated with high GH and low IGF-I levels and often have midfacial hypoplasia (typical Laron syndrome facial features). Our centre previously described the first GHR pseudoexon mutation (42700896A>G, c. 618+792A>G). The inclusion of this 108bp pseudoexon is predicted to lead to in-frame insertion of 36 amino acid residues, between exons 6 and 7. This 36-residue insertion in the dimerization domain of the GHR results in defective trafficking rather than impaired signalling, and thus causes a partial loss-of function. As such, the observed phenotype is usually moderate postnatal growth failure (Height SDS −3.3 to −6.0).

Objective and Hypothesis: Pseudoexons outnumber exons by 10 to 1 and variants in them may be a major contributor to disease burden in short stature.

Methods: We designed a custom short stature gene panel that interrogates both coding and non-coding regions to uncover such mutations. In vitro splicing assays, using an exon trap vector (pET01, MoBiTec GmbH, Göttingen, Germany) were utilised to mimic the splicing process.

Results: We identified a homozygous GHR variant (g.5:42700940 T>G, c.618+836T>G) in an Italian patient with severe postnatal growth failure, classical Laron phenotype and height SDS −7.5. Both unaffected, non-consanguineous parents were heterozygous for the mutation. This mutation was 44bp downstream of the previous pseudoexon mutation and was predicted in silico to create a donor splice site. Splicing analysis of this variant confirmed inclusion of the 152bp mutant pseudoexon in all transcripts with no evidence of normal splicing in contrast to the wild-type pseudoexon which showed no such inclusion. Inclusion of the pseudoexon will lead to a frameshift and premature truncation of the mRNA.

Discussion: This novel pseudoexon inclusion event will result in a truncated message which may be destroyed by nonsense mediated mRNA decay or, if not, will lead to a truncated protein lacking the transmembrane and intracellular domains responsible for anchoring the protein in the membrane and signalling respectively. Given the undetectable GHBP levels in this patient, the former effect is considered most likely. Either scenario will lead to complete loss-of-function, consistent with the more severe growth phenotype exhibited by this patient in comparison to the previously described, in-frame, pseudoexon mutation with milder phenotype. Our work highlights the potential for such splicing events to be more commonly causal for this and other rare diseases.