Achondroplasia (ACH) and hypochondroplasia (HCH) are two autosomal-dominant skeletal disorders caused by recurrent missense FGFR3 mutations in the transmembrane (TM) and tyrosine kinase 1 (TK1) domains of the receptor. Although 98% of ACH cases are accounted for by a single G380R substitution in the TM, a common mutation (N540K) in the TK1 region is detected in only 60-65% of HCH cases. The aim of this study was to determine whether the frequency of mutations in patients with HCH was the result of incomplete mutation screening or genetic heterogeneity. Eighteen exons of the FGFR3 gene were entirely sequenced in a cohort of 25 HCH and one ACH patients in whom common mutations had been excluded. Seven novel missense FGFR3 mutations were identified, one causing ACH and six resulting in HCH. Six of these substitutions were located in the extracellular region and four of them creating additional cysteine residues, were associated with severe phenotypes. No mutations were detected in 19 clinically diagnosed HCH patients. Our results demonstrate that the spectrum of FGFR3 mutations causing short-limb dwarfism is wider than originally recognised and emphasise the requirement for complete screening of the FGFR3 gene if appropriate genetic counselling is to be offered to patients with HCH or ACH lacking the most common mutations and their families.

Fibroblast growth factor receptor 3 (FGFR3) belongs to a family of four genes (FGFR1-4) encoding receptors with tyrosine kinase activity (RTK). These structurally related proteins exhibit an extracellular domain (ECD) composed of three immunoglobin-like domains, an acid box, a single transmembrane domain and a Recurrent missense fibroblast growth factor receptor 3 (FGFR3) mutations have been ascribed to skeletal dysplasias of variable severity including the lethal neonatal thanatophoric dysplasia types I (TDI) and II (TDII). To elucidate the role of activating mutations causing TDI on receptor trafficking and endocytosis, a series of four mutants located in different domains of the receptor were generated and transiently expressed. The putatively elongated X807R receptor was identified as three isoforms. The fully glycosylated mature isoform was constitutively but mildly phosphorylated. Similarly, mutations affecting the extracellular domain (R248C and Y373C) induced moderate constitutive receptor phosphorylation. By contrast, the K650M mutation affecting the tyrosine kinase 2 (TK2) domain produced heavy phosphorylation of the nonglycosylated and mannose-rich isoforms that impaired receptor trafficking through the Golgi network. This resulted in defective expression of the mature isoform at the cell surface. Normal processing was rescued by tyrosine kinase inhibitor treatment. Internalization of the R248C and Y373C mutant receptors, which form stable disulfide-bonded dimers at the cell surface was less efficient than the wild-type, whereas ubiquitylation was markedly increased but apparently independent of the E3 ubiquitin-ligase casitas B-lineage lymphoma (c-Cbl). Constitutive phosphorylation of c-Cbl by the K650M mutant appeared to be related to the intracellular retention of the receptor. Therefore, although mutation K650M affecting the TK2 domain induces defective targeting of the overphosphorylated receptor, a different mechanism characterized by receptor retention at the plasma membrane, excessive ubiquitylation and reduced degradation results from mutations that affect the extracellular domain and the stop codon.

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