Identification and characterisation of a novel GHR defect disrupting the polypyrimidine tract and resulting in GH insensitivity

Objective: GH insensitivity (GHI) is caused in the majority of cases by impaired function of the GH receptor (GHR). All but one known GHR mutation are in the coding sequence or the exon/intron boundaries. We identified and characterised the first intronic defect occurring in the polypyrimidine tract of the GHR in a patient with severe GHI. Design: We investigated the effect of the novel defect on mRNA splicing using an in vitro splicing assay and a cell transfection system. Methods: GHR was analysed by direct sequencing. To assess the effect of the novel defect, two heterologous minigenes (wild-type and mutant L1-GHR8-L2) were generated by inserting GHR exon 8 and its flanking wild-type or mutant intronic sequences into a well-characterised splicing reporter (Adml-par L1-L2). P-32-labelled pre-mRNA was generated from the two constructs and incubated in HeLa nuclear extracts or HEK293 cells. Results: Sequencing of the GHR revealed a novel homozygous defect in the polypyrimidine tract of intron 7 (IVS7-6T > A). This base change does not involve the highly conserved splice site sequences, and is not predicted in silico to affect GHR mRNA splicing. Nevertheless, skipping of exon 8 from the mutant L1-GHR8-L2 mRNA was clearly demonstrated in the in vitro splicing assay and in transfected HEK293 cells. Conclusion: Disruption of the GHR polypyrimidine tract causes aberrant mRNA splicing leading to a mutant GHR protein. This is predicted to lack its transmembrane and intracellular domains and, thus, be incapable of transducing a GH signal.