MUTATIONS CAUSING ACHONDROPLASIA AND THANATOPHORIC DYSPLASIA ALTER BFGF-INDUCED CALCIUM SIGNALS IN HUMAN-DIPLOID FIBROBLASTS

Mutations in the fibroblast growth factor receptor (FGFR) gene family recently have been shown to underlie several hereditary disorders of b one development, with specific FGFR3 mutations causing achondroplasia (Ach) and thanatophoric dysplasia (TD), However, for none of these mut ations has the defect in receptor function been demonstrated directly and, therefore, for none has the pathophysiological mechanism of the d isease been defined, Using our established techniques for single-cell ratiometric real-time calcium image analysis, we defined the nature of the basic fibroblast growth factor (bFGF)-induced calcium signal in h uman diploid fibroblasts, and, in blinded studies, have analyzed the b FGF-induced signals from 18 independent fibroblast cell lines, includi ng multiple lines from patients with known mutant alleles of FGFR3 and syndromes of Ach or TD, Control cells responded with transient increa ses in intracellular calcium, with many cells showing oscillatory calc ium waves, Homozygous Ach cell lines failed to signal, whereas heteroz ygous Ach lines responded nearly normally. We observed heterogeneous s ignals in TD heterozygotes: the unresponsive lines all turned out to c arry TD1 alleles, whereas all responsive lines had TD2 alleles. Since FGFR1, 2 and 3 receptors are known to be expressed in fibroblasts, our results suggest that specific mutant FGFR3 alleles can function in a dosage-dependent dominant-negative fashion to inactivate FGFR signalin g.