An uncommon G375C substitution in a newborn with achondroplasia

We report a G-to-T de novo transversion mutation causing the substitution of a glycine with a cysteine (G375C) in a newborn with achondroplasia. This rare observation confirms allelic heterogeneity.     

Incidence of fibroblast growth factor receptor 3 gene (FGFR3) A248C, S249C, G372C, and T375C mutations in bladder cancer

Bladder cancer is the most frequent cancer of the urinary system. Fibroblast growth factor receptors (FGFR) belong to the tyrosine kinase family and have important roles in cell differentiation and proliferation and embryogenesis. FGFR3 is located on chromosome 4p16.3, and missense mutations of FGFR3 are associated with autosomal dominant human skeletal disorders and have some oncogenic effects. We examined the incidence of FGFR3 thanatophoric dysplasia mutations located in exon 7, A248C and S249C, and in exon 10, G372C and T375C, and their correlation with clinical-pathological parameters in bladder carcinoma patients. Fifty-six paraffin-embedded specimens of transitional cell carcinoma of the urinary bladder were included in this study. Analysis of FGFR3 thanatophoric dysplasia mutations located in exon 7, A248C and S249C, and in exon 10, G372C and T375C, was performed by PCR-restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. FGFR3 thanatophoric dysplasia mutations located in exon 7, A248C and S249C, and in exon 10, G372C and T375C, were detected in 33 of the 56 patients (heterozygous mutant). Among the 56 transitional cell carcinomas, missense point mutations were detected in seven of them at codon A248C, 28 of them at codon S249C, and three of them at codon T375C, similar to data from previous reports. When the results of the FGFR3 thanatophoric dysplasia mutations located in exon 7, A248C and S249C and in exon 10, G372C and T375C, were analyzed one by one or as a group, despite the findings of previous research reports, our data suggest that these mutations are detected homogenously regardless of the tumor classification and tumor grade.     

Dwarfism in Dexter cattle is not caused by the mutations in FGFR3 responsible for achondroplasia in humans

Dexter cattle carry a genetic defect causing a dwarf phenotype in the heterozygotes (Dx +/–), while homozygotes (Dx +/+) are stillborn with extreme shortening of limbs and gross craniofacial defects and are described as 'bulldog' calves. The heterozygous phenotype has been likened to achondroplastic dwarfism in humans (ACH), which has recently been shown to be the result of mutations in the transmembrane region of the fibroblast growth factor receptor 3 (FGFR3) gene. We have sequenced the transmembrane region of bovine FGFR3 from normal Dexter cattle (Dx -/-) and bulldog calves (Dx +/+). The sequence from both is identical and therefore excludes mutations in the transmembrane region of FGFR3 as the cause of Dexter dwarfism.     

Achondroplasia is defined by recurrent G380R mutations of FGFR3.

Genomic DNA from 154 unrelated individuals with achondroplasia was evaluated for mutations in the fibroblast growth factor receptor 3 (FGFR3) transmembrane domain. All but one, an atypical case, were found to have a glycine-to-arginine substitution at codon 380. Of these, 150 had a G-to-A transition at nt 1138, and 3 had a G-to-C transversion at this same position. On the basis of estimates of the prevalence of achondroplasia, the mutation rate at the FGFR3 1138 guanosine nucleotide is two to three orders of magnitude higher than that previously reported for tranversions and transitions in CpG dinucleotides. To date, this represents the most mutable single nucleotide reported in the human genome. The homogeneity of mutations in achondroplasia is unprecedented for an autosomal dominant disorder and may explain the relative lack of heterogeneity in the achondroplasia phenotype.     

FGFR3 heterodimerization in achondroplasia, the most common form of human dwarfism

The G380R mutation in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) causes achondroplasia, the most common form of human dwarfism. Achondroplasia is a heterozygous disorder, and thus the affected individuals express both wild-type and mutant FGFR3. Yet heterodimerization in achondroplasia has not been characterized thus far. To investigate the formation of FGFR3 heterodimers in cellular membranes, we designed an FGFR3 construct that lacks the kinase domain, and we monitored the formation of inactive heterodimers between this construct and wild-type and mutant FGFR3. The formation of the inactive heterodimers depleted the pool of full-length receptors capable of forming active homodimers and ultimately reduced their phosphorylation. By analyzing the effect of the truncated FGFR3 on full-length receptor phosphorylation, we demonstrated that FGFR3 WT/G380R heterodimers form with lower probability than wild-type FGFR3 homodimers at low ligand concentration. These results further our knowledge of FGFR3-associated bone disorders.     

The A391E mutation enhances FGFR3 activation in the absence of ligand

The A391E mutation in the transmembrane domain of fibroblast growth factor receptor 3 leads to aberrant development of the cranium. It has been hypothesized that the mutant glutamic acid stabilizes the dimeric receptor due to hydrogen bonding and enhances its ligand-independent activation. We previously tested this hypothesis in lipid bilayers and showed that the mutation stabilizes the isolated transmembrane domain dimer by -1.3°kcal/mol. Here we further test the hypothesis, by investigating the effect of the A391E mutation on the activation of full-length fibroblast growth factor receptor 3 in human embryonic kidney 293T cells in the absence of ligand. We find that the mutation enhances the ligand-independent activation propensity of the receptor by -1.7°kcal/mol. This value is consistent with the observed strength of hydrogen bonds in membranes, and supports the above hypothesis.     

Chimeras of the native form or achondroplasia mutant (G375C) of human fibroblast growth factor receptor 3 induce ligand-dependent differentiation of PC12 cells.

Mutations in the gene for human fibroblast growth factor receptor 3 (hFGFR3) cause a variety of skeletal dysplasias, including the most common genetic form of dwarfism, achondroplasia (ACH). Evidence indicates that these phenotypes are not due to simple haploinsufficiency of FGFR3 but are more likely related to a role in negatively regulating skeletal growth. The effects of one of these mutations on FGFR3 signaling were examined by constructing chimeric receptors composed of the extracellular domain of human platelet-derived growth factor receptor beta (hPDGFR beta) and the transmembrane and intracellular domains of hFGFR3 or of an ACH (G375C) mutant. Following stable transfection in PC12 cells, which lack platelet-derived growth factor (PDGF) receptors, all clonal cell lines, with either type of chimera, showed strong neurite outgrowth in the presence of PDGF but not in its absence. Antiphosphotyrosine immunoblots showed ligand-dependent autophosphorylation, and both receptor types stimulated strong phosphorylation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, an event associated with the differentiative response of these cells. In addition, ligand-dependent phosphorylation of phospholipase Cgamma and Shc was also observed. All of these responses were comparable to those observed from ligand activation, such as by nerve growth factor, of the native PC12 cells used to prepare the stable transfectants. The cells with the chimera bearing the ACH mutation were more rapidly responsive to ligand with less sustained MAPK activation, indicative of a preactivated or primed condition and consistent with the view that these mutations weaken ligand control of FGFR3 function. However, the full effect of the mutation likely depends in part on structural features of the extracellular domain. Although FGFR3 has been suggested to act as a negative regulator of long-bone growth in chrondrocytes, it produces differentiative signals similar to those of FGFR1, to which only positive effects have been ascribed, in PC12 cells. Therefore, its regulatory effects on bone growth likely result from cellular contexts and not the induction of a unique FGFR3 signaling pathway.     

Effect of pathogenic cysteine mutations on FGFR3 transmembrane domain dimerization in detergents and lipid bilayers

Mutations in fibroblast growth factor receptors are known as the genetic basis of skeletal growth disorders. The mechanism of pathogenesis, as determined by mutation-induced changes in receptor structure, interactions, and function, is elusive. Here we study three pathogenic Cys mutations, associated with either thanatophoric dysplasia or achondroplasia, in the TM domain of fibroblast growth factor receptors 3 (FGFR3). We characterize the dimerization propensities of the mutant TM domains in detergents and in lipid bilayers, in the presence and absence of reducing agents, and compare them to previous measurements of wild-type. We find that the Cys mutations increase the propensity for dimerization in detergent, with the Cys370 mutant exhibiting the highest propensity for disulfide bond formation, the Cys371 mutant having an intermediate propensity, and Cys375 the lowest. Thus, disulfide bonds readily form in detergents, with efficiency that correlates with the severity of the phenotype. In lipid bilayers, however, the Cys370 mutant, which dimerizes strongly in detergent, behaves as the wild-type, suggesting that Cys370-mediated disulfide bonds do not form between the isolated TM domains in bilayers. Thus, the nature of the hydrophobic environment plays an important role in defining the structure and flexibility of transmembrane dimers. These results and previous findings from cellular studies lead us to propose a conformational flexibility mechanism of receptor stabilization as a basis for disregulated FGFR3 signaling in thanatophoric dysplasia and achondroplasia.     

Chinese achondroplasia is also defined by recurrent G380R mutations of the fibroblast growth factor receptor-3 gene

Achondroplasia is the most common form of dwarfism in humans. A recurrent glycine-to-arginine mutation at codon 380 (G380R) of the transmembrane domain of fibroblast growth factor receptor-3 (FGFR-3) was identified in the majority of Western and Japanese patients, which is uncommon in other autosomal dominant genetic diseases. To determine whether this mutation is also common in Chinese patients, we examined the G380R mutation in Chinese patients with achondroplasia. Of ten patients studied, including eight sporadic cases and one family with two affected members, all have the same G380R mutation with a G-to-A transition. Our results support the argument that the G380R mutation of FGFR-3 is the most frequent mutation causing achondroplasia across different populations. Received: 8 January 1996     

Paternal origin of FGFR3 mutations in Muenke-type craniosynostosis

Muenke syndrome, also known as FGFR3-associated coronal synostosis, is defined molecularly by the presence of a heterozygous nucleotide transversion, c.749C>G, encoding the amino acid substitution Pro250Arg, in the fibroblast growth factor receptor type 3 gene (FGFR3). This frequently occurs as a new mutation, manifesting one of the highest documented rates for any transversion in the human genome. To understand the biology of this mutation, we have investigated its parental origin, and the ages of the parents, in 19 families with de novo c.749C>G mutations. All ten informative cases originated from the paternal allele (95% confidence interval 74–100% paternal); the average paternal age at birth overall was 34.7 years. An exclusive paternal origin of mutations, and increased paternal age, were previously described for a different mutation (c.1138G>A) of the FGFR3 gene causing achondroplasia, as well as for mutations of the related FGFR2 gene causing Apert, Crouzon and Pfeiffer syndromes. We conclude that similar biological processes are likely to shape the occurrence of this c.749C>G mutation as for other mutations of FGFR3 as well as FGFR2.S.V. Rannan-Eliya and I.B. Taylor contributed equally to this work.     

Distinct missense mutations of the FGFR3 lys650 codon modulate receptor kinase activation and the severity of the skeletal dysplasia phenotype

The fibroblast growth factor-receptor 3 (FGFR3) Lys650 codon is located within a critical region of the tyrosine kinase-domain activation loop. Two missense mutations in this codon are known to result in strong constitutive activation of the FGFR3 tyrosine kinase and cause three different skeletal dysplasia syndromes-thanatophoric dysplasia type II (TD2) (A1948G [Lys650Glu]) and SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans) syndrome and thanatophoric dysplasia type I (TD1) (both due to A1949T [Lys650Met]). Other mutations within the FGFR3 tyrosine kinase domain (e.g., C1620A or C1620G [both resulting in Asn540Lys]) are known to cause hypochondroplasia, a relatively common but milder skeletal dysplasia. In 90 individuals with suspected clinical diagnoses of hypochondroplasia who do not have Asn540Lys mutations, we screened for mutations, in FGFR3 exon 15, that would disrupt a unique BbsI restriction site that includes the Lys650 codon. We report here the discovery of three novel mutations (G1950T and G1950C [both resulting in Lys650Asn] and A1948C [Lys650Gln]) occurring in six individuals from five families. Several physical and radiological features of these individuals were significantly milder than those in individuals with the Asn540Lys mutations. The Lys650Asn/Gln mutations result in constitutive activation of the FGFR3 tyrosine kinase but to a lesser degree than that observed with the Lys540Glu and Lys650Met mutations. These results demonstrate that different amino acid substitutions at the FGFR3 Lys650 codon can result in several different skeletal dysplasia phenotypes.     

FGFR3 intracellular mutations induce tyrosine phosphorylation in the Golgi and defective glycosylation

Mutations of the Fibroblast Growth Factor Receptor 3 (FGFR3) gene have been implicated in a series of skeletal dysplasias including hypochondroplasia, achondroplasia and thanatophoric dysplasia. The severity of these diseases ranges from mild dwarfism to severe dwarfism and to perinatal lethality, respectively. Although it is considered that the mutations give rise to constitutively active receptors, it remains unclear how the different mutations are functionally linked to the severity of the different pathologies. By examining various FGFR3 mutations in a HEK cell culture model, including the uncharacterized X807R mutation, it was found that only the mutations affecting the intracellular domain, induced premature receptor phosphorylation and inhibited receptor glycosylation, suggesting that premature receptor tyrosine phosphorylation of the native receptor inhibits its glycosylation. Moreover, these mutations appeared to be associated with elevated receptor signaling in the Golgi apparatus. In conclusion, although pathological severity could not be correlated with a single factor arising from FGFR3 mutations, these results suggest that intracellular domain mutations define a distinct means by which mutated FGFR3 could disrupt bone development.     

Sprouty 2 disturbs FGFR3 degradation in thanatophoric dysplasia type II: a severe form of human achondroplasia

Thanatophoric dysplasia is a member of the achondroplasia family of human skeletal dysplasias, which result from FGFR3 mutations that exaggerate this receptor's inhibitory influence on chondrocyte proliferation and differentiation in the skeletal growth plate. We have previously reported that defective lysosomal degradation of activated receptor contributes to the gain-of-function of the mutant FGFR3. We now provide evidence that this disturbance is mediated by the receptor's kinase activity and involves constitutive induction and activation of Spry2. Our findings suggest that activated Spry2 may interfere with c-Cbl-mediated ubiquitination of FGFR3 by sequestering c-Cbl. They provide novel insight into the pathogenesis of this group of human skeletal dysplasias and identify a mechanism that potentially could be targeted therapeutically.     

Unraveling the toxic effects mediated by the neurodegenerative disease–associated S375G mutation of TDP-43 and its S375E phosphomimetic variant

TAR DNA-binding protein 43 (TDP-43) is a nucleic acid–binding protein found in the nucleus that accumulates in the cytoplasm under pathological conditions, leading to proteinopathies, such as frontotemporal dementia and ALS. An emerging area of TDP-43 research is represented by the study of its post-translational modifications, the way they are connected to disease-associated mutations, and what this means for pathological processes. Recently, we described a novel mutation in TDP-43 in an early onset ALS case that was affecting a potential phosphorylation site in position 375 (S375G). A preliminary characterization showed that both the S375G mutation and its phosphomimetic variant, S375E, displayed altered nuclear–cytoplasmic distribution and cellular toxicity. To better investigate these effects, here we established cell lines expressing inducible WT, S375G, and S375E TDP-43 variants. Interestingly, we found that these mutants do not seem to affect well-studied aspects of TDP-43, such as RNA splicing or autoregulation, or protein conformation, dynamics, or aggregation, although they do display dysmorphic nuclear shape and cell cycle alterations. In addition, RNA-Seq analysis of these cell lines showed that although the disease-associated S375G mutation and its phosphomimetic S375E variant regulate distinct sets of genes, they have a common target in mitochondrial apoptotic genes. Taken together, our data strongly support the growing evidence that alterations in TDP-43 post-translational modifications can play a potentially important role in disease pathogenesis and provide a further link between TDP-43 pathology and mitochondrial health.     

Effect of Thanatophoric Dysplasia Type I Mutations on FGFR3 Dimerization

Thanatophoric dysplasia type I (TDI) is a lethal human skeletal growth disorder with a prevalence of 1 in 20,000 to 1 in 50,000 births. TDI is known to arise because of five different mutations, all involving the substitution of an amino acid with a cysteine in fibroblast growth factor receptor 3 (FGFR3). Cysteine mutations in receptor tyrosine kinases (RTKs) have been previously proposed to induce constitutive dimerization in the absence of ligand, leading to receptor overactivation. However, their effect on RTK dimer stability has never been measured experimentally. In this study, we characterize the effect of three TDI mutations, Arg248Cys, Ser249Cys, and Tyr373Cys, on FGFR3 dimerization in mammalian membranes, in the absence of ligand. We demonstrate that the mutations lead to surprisingly modest dimer stabilization and to structural perturbations of the dimers, challenging the current understanding of the molecular interactions that underlie TDI.