Endocrine Abstracts

Introduction: Two heterozygous missense mutations R77C and D112G have been identified in GH-1 gene of patients with short stature by Takahashi et al. These patients had high serum immunoreactive GH concentrations but low IGF-1 concentrations, indicating bioinactivity of their GH. Separation of GH in patients’ serum by isoelectric focusing revealed the coexistence of mutant and wild type (wt) GH. In order to understand the molecular mechanism of the isolated GH deficiency of these patients, we have studied the biological activity of the two mutants in vitro.

Material and methods: The mutations R77C and D112G were produced by site-directed mutagenesis and the cDNA of wt and mutant GH gene were subcloned into the expression vector pcDNA3.1. Human embryonic kidney cells (HEK-293) were transfected with these plasmids, and secreted wt or mutant GH in supernatants was harvested for the experiments. The mutants were studied in comparison to wt GH by fluorescent immunoassays with different monoclonal antibodies to GH, a binding assay with recombinant GH receptor extracellular domain (GHBP), a BaF-B03 cell proliferation assay and a STAT5 transcription assay.

Results: The mutants could be secreted similarly as wt GH from HEK-293 cells. The binding affinity to GHBP was only 25% of wt GH for mutant D112G and 80% for R77C. However, both mutants achieved about 80% of the maximal biological activity induced by wt GH in BaF-B03 cell proliferation assay (R77C and D112G) and in STAT5 transcription assay (D112G).

Conclusion: Receptor binding is much more diminished due to the D112G mutation, which is located close to the receptor binding site 2, than to the R77C mutation, whose position is far away from both binding sites. Both mutants display a modest reduction in their biological activity in comparison to wt GH, which may contribute to the retarded growth of the patients.