Endocrine Abstracts (2002) 3 OC8

Identification and characterization of a novel missense mutation in the growth hormone gene in a child with short stature

MD Lewis1, AM Procter2, DS Millar2, M Horan2, TE Easter1, V Newsway2, L Fryklund3, J Gregory4, MF Scanlon1 & DN Cooper2

1Department of Medicine, University of Wales College of Medicine, Cardiff, UK; 2Institute of Medical Genetics, University of Wales College of Medicine, Cardiff, UK; 3Pharmacia AB, Stockholm, Sweden; 4Department of Child Health, University of Wales College of Medicine, Cardiff, UK.

A child with short stature having height greater than 2 standard deviations below the mean for age and bone age delay of two years but with normal growth hormone secretion tests was identified. Since there was no known clinical cause of the short stature, it was decided to sequence both alleles of the patient's GH1 gene that encodes pituitary GH to determine whether there was a genetic defect responsible for the observed phenotype (with local ethical approval). A 3.2kb fragment was PCR amplified from patient lymphocyte genomic DNA using primers specific for the GH1 gene, cloned into pGEM-T and amplified in E. coli. The GH1 gene was sequenced with the Big Dye sequencing kit using 4 primers to span the entire gene and analysed on an ABI Prism 3100 DNA sequencer. We identified a single heterozygous missense mutation that resulted in the substitution of a lysine for an arginine at position 41. This was independently confirmed both by the cloning and sequencing of a second DNA sample and by PCR direct sequencing. A DNA sequence encoding the GH variant was cloned into the insect expression vector pIZ/V5-His and grown in High Five insect cells, medium was harvested and the GH variant quantified by an IRMA. The biological activity of the growth hormone variant was compared to insect expressed wild-type using a Stat5-responsive luciferase reporter gene assay in HK293 cells expressing high levels of the full length human GH receptor. Reduced bioactivity compared to wild-type was noted (41% reduction in assay response at 1nM, p<0.001 vs wild-type). This was due to a two-fold increase in EC50 (1.5nM) compared to wild-type (0.75nM) rather than a reduction of the maximal assay response. Thus the molecular genetic analysis of such patients is likely to shed new light on the aetiology of short stature.

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