BES2005 Poster Presentations Growth and development (48 abstracts)
In vitro demonstration of the effect on RNA splicing of a novel growth hormone receptor mutation
A David 1 , L Metherell 1 , NJ Shaw 2 , C Camacho-Hubner 1 , SL Chew 1 , MO Savage 1 , B Khoo 1 & AJL Clark 1
1Centre for Molecular Endocrinology, WHRI, Barts and the London School of Medicine and Dentistry, London, UK; 2Department of Endocrinology, Birmingham Children Hospital, Birmingham, UK.
Growth hormone insensitivity, also known as Laron Syndrome (LS), is caused by mutations within the GH receptor (GHR). A 1.5 year-old boy with consanguineous parents was referred with postnatal linear growth failure (length 64 cms, minus 6 SDS). Facial features were typical of LS. Investigation revealed elevated serum GH (1145 mIU per litre) and low IGF-I (4 nmol per litre). Genomic DNA was isolated from peripheral blood leucocytes and all GHR exons, including intron-exon boundaries were amplified by polymerase chain reaction (PCR). Direct sequencing of PCR products revealed a homozygous base change of the acceptor splice site in intron 7 (IVS7as-6, T > A). No other mutations were detected within the GHR sequence. This base change does not involve the highly conserved splice-site sequences and is not recognised as a change that affects RNA splicing. However we investigated the possibility that this mutation interferes with normal GHR splicing, leading to the skipping of exon 8 using an in vitro splicing assay. Exon 8 and the flanking wild type and mutant intronic sequences were inserted into the intron of a well-characterized splicing reporter derived from the adenovirus major late first and second leader exons (AdMLpar), thus generating two heterologous substrates (AdML-8wt and AdML-8mut). 32P-labelled pre-mRNA was generated from the two constructs and incubated in HeLa nuclear extracts for 1 and 2 hours at 30 degC. Under these splicing conditions, exon 8 was included as predicted in AdML-8wt, but skipped in AdML-8mut. In conclusion, we have identified a novel GHR mutation and demonstrated in vitro that it affects pre-mRNA splicing. This is predicted to lead to a GHR translation product with a normal extracellular domain, but no transmembrane domain or intracellular region, which would thus be incapable of transducing a GH signal.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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