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

David, A; Miraki-Moud, F; Shaw, NJ; Savage, MO; Clark, AJ; Metherell, LA (2010) Identification and characterisation of a novel GHR defect disrupting the polypyrimidine tract and resulting in GH insensitivity. EUROPEAN JOURNAL OF ENDOCRINOLOGY, 162 (1). 37 - 42 (6). ISSN 0804-4643 https://doi.org/10.1530/EJE-09-0583
SGUL Authors: Clark, Adrian John L

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Abstract

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). 32P-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.

Item Type: Article
Additional Information: PubMed ID: 19812236
Keywords: Cell Line, Exons, HeLa Cells, Human Growth Hormone, Humans, Infant, Introns, Laron Syndrome, Male, Membrane Proteins, Mutation, Polypyrimidine Tract-Binding Protein, Protein Structure, Tertiary, RNA Splicing, RNA, Messenger, Receptors, Somatotropin, Signal Transduction, Science & Technology, Life Sciences & Biomedicine, Endocrinology & Metabolism, GROWTH-HORMONE INSENSITIVITY, RNA SPLICE JUNCTIONS, BINDING-PROTEIN, LARON-SYNDROME, RECEPTOR GENE, MUTATIONS, SEQUENCE, EXON, ACTIVATION, EXPRESSION, Cell Line, Exons, HeLa Cells, Human Growth Hormone, Humans, Infant, Introns, Laron Syndrome, Male, Membrane Proteins, Mutation, Polypyrimidine Tract-Binding Protein, Protein Structure, Tertiary, RNA Splicing, RNA, Messenger, Receptors, Somatotropin, Signal Transduction
Journal or Publication Title: EUROPEAN JOURNAL OF ENDOCRINOLOGY
ISSN: 0804-4643
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Dates:
DateEvent
1 January 2010Published
Web of Science ID: WOS:000276611800005
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URI: http://sgultest.da.ulcc.ac.uk/id/eprint/102164
Publisher's version: https://doi.org/10.1530/EJE-09-0583

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