Fibroblast growth factor receptor 3 regulation and mechanism in callus formation / 中国组织工程研究

BACKGROUND: In clinical treatment, patients with traumatic nerve injury and fractures show accelerated fracturehealing and excessive osteophyte growth, and even heterotopic ossification in the muscle, all of which seriously affect the therapeutic efficacy on such fractures. The specific causes and mechanisms for the acceleration of fracture healing after denervation are currently unclear. OBJECTIVE: To investigate the role and expression of fibroblast growth factor receptors 3 (FGFR3) inhibitor in the process of fracture healing. METHODS: The study protocol was approved by the Animal Ethics Committee of Second Hospital of Lanzhou University. Sixty female Sprague-Dawley rats were used to make a transverse humeral fracture model of sciatic nerve injury. They were randomly divided into two groups, experimental group and control group. The experimental group received intraperitoneal injection of FGFR3 blocker; the control group received an equal dose of normal saline. The X-ray films were taken at 4, 7, 10, 14 and 21 days after surgery, and tibia specimens for six animals were subsequently taken at each time point, followed by histological observations using hematoxylin-eosin staining and Masson’s trichrome staining. Osteocyte density and trabecular bone density of the rat tibia were calculated; and the fiber rate of the tibia was determined. RESULTS AND CONCLUSION: There were no significant differences in the X-ray findings of the tibia between the two groups. The experimental group had better bone repair than the control group, shown by hematoxylin-eosin staining and Masson’s trichrome staining. Osteocyte density, trabecular bone density, and fiber rate of the rat tibia were significantly higher in the experimental group than the control group at 7-14 days after treatment. Inhibition of FGFR3 can accelerate fracture healing and promote the shaping of callus in the case of peripheral nerve denervation. FGFR3 is most active at 7-14 days after fracture.

Mutations in FGFR3 gene associated with maxillary retrognathism

Context: Understanding the role of fibroblast growth factor receptor (FGFR) in the regulation of bone development and disease will ultimately lead to better prevention and treatment of related bone deformities and disorders. Aims: To evaluate the role of gene FGFR3 in individuals with retrognathic maxilla by polymerase chain reaction (PCR) technique at molecular level and evaluate the significance of the same. Settings and Design: Hospital based fundamental research involving individuals having maxillary retrognathism. Methodology: A total of 62 individuals (30M and32F) who were willing to take part in the study were selected from cephalometric measurements of N I A and the length PNS to ANS. The institution based basic genetic research study involved collection of fresh blood samples, DNA extraction, PCR analysis, and amplification using the specifically designed forward and reverse primers for targeting the commonly occurring mutations in FGFR3 gene. Further the products were sequenced to evaluate the presence of any novel mutations. Results: The targeted single-nucleotide polymorphisms, at position 1138 in exon 10 of the FGFR3 gene were not identified in the analyzed blood samples. The detailed sequencing of full gene revealed the presence of 2 novel mutations, Exon 3: A213G and Exon 3: A223A/G in one individual. Conclusions: The present study indicated 2 novel mutations in gene FGFR3 in individual with maxillary retrognathism. The genetic–environmental interactions might have played a significant role in the expression of retrognathic maxilla.

Natural history of 39 patients with Achondroplasia

OBJECTIVES: To characterize the natural history of 39 achondroplastic patients diagnosed by clinical, radiological and molecular assessments. METHODS: Observational and retrospective study of 39 patients who were attended at a public tertiary level hospital between 1995 and 2016. RESULTS: Diagnosis was made prenatally in 11 patients, at birth in 9 patients and within the first year of life in 13 patients. The most prevalent clinical findings were short stature, high forehead, trident hands, genu varum and macrocephaly. The most prevalent radiographic findings were rhizomelic shortening of the long bones and narrowing of the interpediculate distance of the caudal spine. There was motor developmental delay in 18 patients and speech delay in 16 patients. The most common clinical intercurrences were middle ear dysfunction, sleep apnea, limb pain and obesity from 2 to 9 years of age. One patient was large for the gestational age but did not develop obesity. One patient developed hydrocephalus at 10 years old. The current age of the patients varies from 15 months to 36 years. The molecular study performed by Sanger sequencing of the common heterozygous mutation 1138G>A in FGFR3 was positive in all patients. Four cases were inherited, and 35 were sporadic (paternal age from 19 to 66 years). CONCLUSIONS: The diagnoses were made early based on clinical and radiographic findings. All cases were confirmed molecularly. Despite presenting a benign course, it is necessary to establish a systematic protocol for the surveillance of these patients due to the common clinical intercurrences.

Association between single nucleotide polymorphisms in FGFR3 gene and risk of breast cancer / 实用肿瘤学杂志

Objective The aim of this study was to investigate the association between single nucleotide polymorphisms(SNPs)in FGFR3 gene and the risk of breast cancer.Methods The frequency of SNP genotypes rs2234909 and rs3135848 of FGFR3 gene in premenopausal breast cancer patients and premenopausal normal fe-males were detected by multiple clonal extension SNP typing technique.The SNP genotypes were compared with different SNP genotypes and the risk of premenopausal breast cancer.Results There was no difference in the genotype frequencies of SNP rs 2234909 and rs3135848 between breast cancer and control groups(P>0.05).Lo-gistic regression analysis showed that there was no correlation between TC and TC +CC genotype and risk of breast cancer(OR=1.035,95% CI:0.680~1.575,P=0.874;OR=0.985,95% CI:0.638~1.521,P=0. 945).For the rs3135848 locus,the genotypes of TC,CC and TC+CC were not associated with the risk of breast cancer(OR=1.177,95%CI:0.846-1.636,P=0.333;OR=0.948,95% CI:0.287-3.137,P=0.931;OR=1.162,95%CI:0.548~1.112,P=0.360).Histological grade was significantly higher in breast cancer with rs2234909 mutation than that of the non-mutation group(dominant model:P=0.032,co-dominant model:P=0.024).The Ki67 index of FGFR3 gene locus rs2234909 mutation was higher than that of the non-mutation (dominant model:P=0.056;co-dominant model:P=0.044).There was no difference between rs3135848 mu-tation and both site mutation with clinicopathological features of breast cancer patients(P>0.05).Conclusion The SNP genotypes of rs2234909 and rs3135848 of FGFR3 gene were not associated with susceptibility to breast cancer in premenopausal women in North of China.Rs2234909 mutation was positively correlated with histological grade and Ki67 index in premenopausal breast cancer patients.

Association between single nucleotide polymorphisms in FGFR3 gene and risk of breast cancer / 实用肿瘤学杂志

Objective The aim of this study was to investigate the association between single nucleotide polymorphisms(SNPs)in FGFR3 gene and the risk of breast cancer.Methods The frequency of SNP genotypes rs2234909 and rs3135848 of FGFR3 gene in premenopausal breast cancer patients and premenopausal normal fe-males were detected by multiple clonal extension SNP typing technique.The SNP genotypes were compared with different SNP genotypes and the risk of premenopausal breast cancer.Results There was no difference in the genotype frequencies of SNP rs 2234909 and rs3135848 between breast cancer and control groups(P>0.05).Lo-gistic regression analysis showed that there was no correlation between TC and TC +CC genotype and risk of breast cancer(OR=1.035,95% CI:0.680~1.575,P=0.874;OR=0.985,95% CI:0.638~1.521,P=0. 945).For the rs3135848 locus,the genotypes of TC,CC and TC+CC were not associated with the risk of breast cancer(OR=1.177,95%CI:0.846-1.636,P=0.333;OR=0.948,95% CI:0.287-3.137,P=0.931;OR=1.162,95%CI:0.548~1.112,P=0.360).Histological grade was significantly higher in breast cancer with rs2234909 mutation than that of the non-mutation group(dominant model:P=0.032,co-dominant model:P=0.024).The Ki67 index of FGFR3 gene locus rs2234909 mutation was higher than that of the non-mutation (dominant model:P=0.056;co-dominant model:P=0.044).There was no difference between rs3135848 mu-tation and both site mutation with clinicopathological features of breast cancer patients(P>0.05).Conclusion The SNP genotypes of rs2234909 and rs3135848 of FGFR3 gene were not associated with susceptibility to breast cancer in premenopausal women in North of China.Rs2234909 mutation was positively correlated with histological grade and Ki67 index in premenopausal breast cancer patients.

A case report of recurrent achondroplasia in fetuses of normal parents.

Achondroplasia, a skeletal dysplasia has an incidence of 1 in 15000 to 1 in 30000 live births. It is inherited in an autosomal dominant manner. The occurrence of recurrent achondroplasia in babies born to normal parents is rare. The present case report is one such type. A female fetus of 27 weeks gestational age was brought to the Department of Anatomy, Karpaga Vinayaga Institute of Medical Sciences, Maduranthagam. There was frontal bossing of forehead, rhizomelic type of limb shortening with limitation of elbow extension in the fetus. The mother of the fetus, who is 26 years old, gave history of recurrence of such condition. Her first pregnancy was a twin pregnancy, conceived by natural methods, where one of the twins was a male baby who also had achondroplasia and died 2 hours after delivery. The other twin is a girl and the child has delayed developmental milestones. Her second pregnancy was uneventful. The present fetus under study is from her third pregnancy. Her marriage is of second degree consanguineous type. The age of her husband is 36 years old. Germinal mosaicism has been attributed for the causation of recurrent achondroplasia in children, whose parents are normal. 80% of a chondroplasia is due to a new mutation. Only 20% of achondroplasia is inherited. Increased paternal age is a risk factor for new mutations to occur. The other investigations of the case and the genetic analysis are described further in the article.

Homology modelling and docking studies of FGFR3 protein to search most effective drug against achondroplasia.

The present investigation was carried out with the aim of modeling the 3D structure of FGFR3 protein and predicting the most effective drug using SU5402 and its analogues. FGFR3 protein, responsible for long bone growth, causes Achondroplasia in H. sapiens when it becomes mutated. When mutated, the dimmer of FGFR3 stabilizes without interacting with its ligand results in constitutive activation of downstream pathway and inhibits the bone growth. No known structure of FGFR3 was available. The 3D modeling of FGFR3 was done using Robetta server and from the various model predicted, model 5 was selected as the best model after evaluating the models using PROCHECK. . The total number of residues in selected model was found as: 589 (86.5%) residues in most favored region, 84 (12.3%) in additional allowed region, 8 (1.2%) in generously allowed region and 0 (0.0%) in disallowed region of the Ramachandran plot. Three analogues were constructed by using the existing FGFR3 specific inhibitor SU5402. Receptor-analogue interaction study was performed in FlexX3 docking software. Ligand 3 (IUPAC Name: 3-{2-[Z)-(4-hydroxy-2-oxo-1,2-dihydro-3Hindol- 3-ylidene) methyl]-4-oxo-4,5-dihydro-1H-pyrrol-3-yl} propanoic acid) was showing the best binding energy (-10.458 KJ/Mol) that can be predicted the most effective inhibitor for FGFR3. It should be noted that these predicted data should be validated using suitable assays for further consideration.

A case of thanatophoric dysplasia type I with an R248C mutation in the FGFR3 gene / 소아과

Thanatophoric dysplasia (TD) is a short-limb neonatal dwarfism syndrome that is usually lethal in the perinatal period. It is characterized by shortening of the limbs, severely small thorax, large head with a prominent forehead, macrocephaly, curved femur, and flattened vertebral bodies. These malformations result from the mutation in fibroblast growth factor receptor 3 (FGFR-3) gene which is located on the short arm of chromosome 4. A definite diagnosis should be established by molecular genetic analysis to find out the abnormal mutations in the FGFR3 gene. We confirmed by detection of a R248C mutation in the FGFR3 gene in DNA analysis.

Immunomagnetic Indirect Positive Sorting of Precartilaginous Stem Cells from Neonatal Rat / 华中科技大学学报（医学）（英德文版）

To investigate the technique of sorting high-purity precartilaginous stem cells from rat's perichondrium, neonatal rat's perichondrium cells suspensions were incubated with monoclone antibody of anti-fibroblast growth factor receptor-3 (anti-FGFR-3), and the labeled cells were separated from the suspension in the magnetic field by immuno-beads coated with the second antibody. Purityof the sorted neural stem cells was found to be 93.0 %-99.0 %, with living cells amounting to 80 %-85 %. The magnetic cell sorting system could effectively separate precartilaginous stem cells fromperichondrium cell suspensions.

Construction and identification of recombinant adenovirus encoding mouse FGFR3 cDNA / 第三军医大学学报

Objective To construct the recombinant adenovirus encoding mouse wild type FGFR3 cDNA. Methods Mouse FGFR3 cDNA obtained from MoFR3/SV was subcloned into plasmid pBluescript KS and further cloned into plasmid pAdTrack-CMV. The plasmid (pAdTR3) was transferred into BJ5183 cells which contained the adenovirus plasmid (pAdeasy-1) to produce recombinant adenovirus vector encoding FGFR3 cDNA (pAdE-FGFR3). The recombinant adenovirus vector was identified and transfected into the adenoviral packaging cell HEK293 by lipofectamine 2000 to get recombinant adenovirus particles. The adenovirus was confirmed by polymerase chain reactin (PCR) and its titer was determined. Then the recombinant vector was transfected into HT29 cells. The expression of FGFR3 mRNA and protein in HT29 cells was assayed by RT-PCR and Western blotting. Results The recombinant adenovirus vector encoding FGFR3 cDNA was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. The transfected HEK 293 cells were lysed by freeze-thawing to obtain the recombinant adenovirus in the lysate. Further, PCR product of the lysate confirmed the presence of recombinant adenovirus. The viral titer was 3?109pfu/ml. RT-PCR and Western blotting showed that pAdE-FGFR3 transfected HT29 group expressed FGFR3 higher than that of GFP controlled group. Conclusion Infective recombinant adenovirus encoding FGFR3 cDNA was successfully obtained by plasmid homogenous recombination in bacteria, and highly expressed in HT29 cells after transfection, which paves a way for studying the effect of FGFR3 in bone fracture healing.

Expression of fibroblast growth factor receptor-3 during fracture healing / 第三军医大学学报

Objective To observe the spatial and temporal expression patterns of FGFR3 gene and analyze the potential roles of FGFR3 during fracture healing and explore the technique of in situ hybridization of paraffin sections of bone tissue. Methods The fracture model of mouse tibia was established by the standard methods. The expression of FGFR3 mRNA was studied by in situ hybridization during the various stages of bone repair. Results There was faint FGFR3 positive-signal in the cytoplasm of prehypertrophic chondrocytes in soft callus on day 5. FGFR3 mRNA was strongly expressed in prehypertrophic chondrocytes and hypertrophic chondrocytes in soft callus on day 7, but disappeared on day 14, 28 because chondrocytes was replaced by osteoblasts. Conclusion FGFR3 mRNA was strongly expressed in prehypertrophic chondrocytes and hypertrophic chondrocytes in the fracture callus, which shows that FGFR3, a gene that regulates the proliferation of chondrocytes during bone development, may participate in the regulation of differentiation of chondrocytes during fracture healing.

Mutation Analysis in Fibroblast Growth Factor Receptor 3 Gene in Korean Children with Simple Craniosynostosis

The C749G(Pro250Arg) mutation in the gene for fibroblast growth factor receptor 3 (FGFR3) has been found in patients with various types of craniosynostosis. In this study, the blood of 9 Korean children with non-syndromic craniosynostosis were collected and mutation analyses were performed to screen whether this Pro250Arg mutation is also prevalent in Korean population. The genomic DNA samples were analysed by PCR amplification to amplify exon 7 and flanking intron sequence of FGFR3 (341 bp). Restriction digests were analysed by gel electrophoresis. There were no heterozygous for Pro250Arg mutation. No mutations in restriction enzyme digestion were confirmed by direct DNA sequencing. In this study, only 9 patients with simple craniosynostosis were subjected to mutation detection. Therefore, it is necessary to study a large number of patients in order to understand the proportion of non-syndromic craniosynostosis attributalbe to FGFR3 mutation. The epidemiologic study of this disease should be also combined in addition.

Mutation Analysis in Fibroblast Growth Factor Receptor 3 Gene in Korean Children with Simple Craniosynostosis

The C749G(Pro250Arg) mutation in the gene for fibroblast growth factor receptor 3 (FGFR3) has been found in patients with various types of craniosynostosis. In this study, the blood of 9 Korean children with non-syndromic craniosynostosis were collected and mutation analyses were performed to screen whether this Pro250Arg mutation is also prevalent in Korean population. The genomic DNA samples were analysed by PCR amplification to amplify exon 7 and flanking intron sequence of FGFR3 (341 bp). Restriction digests were analysed by gel electrophoresis. There were no heterozygous for Pro250Arg mutation. No mutations in restriction enzyme digestion were confirmed by direct DNA sequencing. In this study, only 9 patients with simple craniosynostosis were subjected to mutation detection. Therefore, it is necessary to study a large number of patients in order to understand the proportion of non-syndromic craniosynostosis attributalbe to FGFR3 mutation. The epidemiologic study of this disease should be also combined in addition.

Mutation analysis of Fibroblast Growth Factor Receptor 3 (FGFR3) Gene in Korean Patients with Achondroplasia and Hypochondroplasia / 대한임상병리학회지

BACKGROUND: Achondroplasia is the most common form of dwarfism. The achondroplasia class consists of achondroplasia, thanatophoric dysplasia and hypochondroplasia. Clinical symptoms are variable, but the common gene, fibroblast growth factor receptor 3 (FGFR3), could account for these variable conditions. We tried to isolate the molecular defects in Korean patients with achondroplasia and hypochondroplasia. METHODS: The sites frequently mutated (G380R and N540K) of the FGFR3 gene of seventeen Korean patients with skeletal dysplasia (16 cases of achondroplasia and one of hypochondroplasia) were analyzed by PCR-RFLP and confirmed by direct sequencing. RESULTS: Missense mutations, which cause G380R of the FGFR3, were present in 15/16 (93.7%) achondroplasia patients. Among these, G to A transition was found in 14 of the 15 (93.3%) patients, and a G to C transversion in a single (6.6%) patient. One case did not show any mutation of the FGFR3 gene reported in achondroplasia, including G375C. A patient with suspected hypochondroplasia exhibited the common C to G transversion mutation, resulting in N540K. CONCLUSIONS: The mutations at codons 380 and 540 of the FGFR3 gene were also found to be common causative mutations of achondroplasia and hypochondroplasia, respectively, in Koreans. These mutations could be used as the target of molecular diagnosis. Based on this simple molecular study, genetic counseling for skeletal dysplasia and prenatal diagnosis will be possible.