Genetically confirmed thanatophoric dysplasia with fibroblast growth factor receptor 3 mutation.

Thanatophoric dysplasia (TD), the most common lethal skeletal dysplasia, is a de novo genetic disease caused by a mutation in the fibroblast growth factor receptor 3 (FGFR3) gene. "Thanatophoric" means "dead bearing" in Greek. Because FGFR3 is the main modulator of bone maturation, typical features of TD include short extremities, curved femur, clover-leaf skull, small narrow chest, and platyspondyly. TD can be classified into two subgroups according to the morphologic findings, with prominent curved femur suggesting type I TD (TD 1) and with marked clover-leaf skull and relatively straight long bones, favoring type II TD (TD 2). However, considering the genetic profiles, TD 1 and TD 2 could be confidently delineated. Here, we report five genotype-phenotype correlated autopsy cases of TD. Five cases had stigmata of TD on antenatal ultrasonography. Terminations were done at gestational age 16 to 28weeks, after family consultation. In autopsy, all fetuses showed short limbs and clover-leaf skull. The microscopic examination of the bones showed disorganized growth plate, consistent with TD. However, some differences existed in gross and microscopic findings between cases. In genetic analyses, three cases revealed missense mutation of Y373C, while the remaining two cases had missense mutation of S371C and S249C each. They were hot spot mutations of TD 1. A correlation between genotype and phenotype was not apparent due to the limited number of the cases. Therefore, a molecular work up to identify the mutation of FGFR3 is indispensable for TD diagnosis in the era of precision medicine for genetic consultation and future targeted therapy.

[Clinical analysis of 21 cases with short fetal femur in the third trimester].

Objective: To analyze the clinical features and to explore the etiology of short fetal femur during the third trimester. Methods: From January 2010 to June 2016, 21 singleton pregnancies with short fetal femur detected by ultrasonography during the third trimester were referred to the Chinese PLA General Hospital. Clinical data were collected, karyotype or single nucleotide polymorphism microarray was carried out to detect chromosomal abnormalities, and FGFR3 c.1138G>A mutation detection was carried out to detect achondroplasia (ACH) via invasive procedure, respectively. The deviation of femur length from the mean value of the gestational age in ultrasonography was expressed as the Z-score. The difference between ACH and isolated short femur (ISF, in the absence of associated structure abnormality or genetic abnormality) was then explored. Results: In the 21 fetuses, 11 had abnormal genetic test results(52%, 11/21), including 9 cases of ACH, 1 case of Ellis-van Creveld Syndrome and 1 case of Pallister-Killian syndrome. In the 10 ISF fetuses (48%, 10/21), 3 cases were fetal growth restriction, 1 was normal small for gestational age infant and 6 cases were unexplained. The median Z-scores for 9 cases of ACH and 10 cases of ISF in the third trimester were -5.04, -3.20, respectively. The short femur in ACH was more severe than in ISF (P=0.005) in the third trimester. Conclusions: The etiology of short fetal femur is complicated, including skeletal dysplasia, chromosomal abnormality, fetal growth restriction, as well as normal variants during fetal development. Genetic test should be considered during the antenatal consultation.

Novel Biomarkers for Prediction of Response to Preoperative Systemic Therapies in Gastric Cancer.

Preoperative chemo- and radiotherapeutic strategies followed by surgery are currently a standard approach for treating locally advanced gastric and esophagogastric junction cancer in Western countries. However, in a large number of cases, the tumor is extremely resistant to these treatments and the patients are exposed to unnecessary toxicity and delayed surgical therapy. The current clinical trials evaluating the combination of preoperative systemic therapies with modern targeted and immunotherapeutic agents represent a unique opportunity for identifying predictive biomarkers of response to select patients that would benefit the most from these treatments. However, it is of utmost importance that these potential biomarkers are corroborated by extensive preclinical and translational research. The aim of this review article is to present the most promising biomarkers of response to classic chemotherapeutic, anti-HER2, antiangiogenic, and immunotherapeutic agents that can be potentially useful for personalized preoperative systemic therapies in gastric cancer patients.

Effects of fibroblast growth factor receptor 3 and human papillomavirus type 2 E2 protein on the differentiation of keratinocytes: a preliminary study / 中华皮肤科杂志

Objective:To evaluate regulatory effects of fibroblast growth factor receptor 3 (FGFR3) and human papillomavirus type 2 (HPV2) E2 protein on the differentiation of an immortalized human keratinocyte line HaCaT and a normal human epidermal keratinocyte line NHEK.Methods:In both HaCaT and NHEK cells, HPV2 E2-stably transfected cell lines (HPV2 E2-transfected groups) were established by using the lentivirus transfection method, wide-type FGFR3-overexpressing cells (FGFR3-WT transfected groups) and FGFR3-K650E mutant-overexpressing cells (FGFR3-K650E transfected groups) were constructed by using the plasmid transfection method, and cells transfected with blank vectors served as control groups (blank vector control groups). Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of HPV2 E2, and Western blot analysis to determine the protein expression of HPV2 E2, FGFR3, and keratinocyte differentiation markers including loricrin, filaggrin, as well as involucrin. Laser scanning confocal microscopy was conducted to observe the spatial localization of HPV2 E2 and FGFR3 in HaCaT cells. Statistical analysis was carried out by using two-independent-sample t test for the comparison between two groups, one-way analysis of variance for the comparison among multiple groups, and Dunnett t-test for multiple comparisons. Results:The HPV2 E2-stably transfected cell lines were successfully constructed, and the expression of HPV2 E2 FLAG protein was significantly higher in the HPV2 E2-transfected groups than in the blank vector control groups in both HaCaT and NHEK cells ( t = 13.71, 25.91, respectively, both P < 0.001) ; both FGFR3-WT and FGFR3-K650E were successfully overexpressed in both HaCaT and NHEK cells, and the FGFR3 protein expression was significantly higher in the FGFR3-WT transfected groups and the FGFR3-K650E transfected groups than in the blank vector control groups ( F = 473.90, 579.90, respectively, both P < 0.001). In both HaCaT and NHEK cells, the expression of keratinocyte differentiation markers including loricrin, filaggrin, and involucrin was significantly upregulated in the HPV2 E2-transfected groups, the FGFR3-WT transfected groups, and the FGFR3-K650E transfected groups than in the blank vector control groups (all P < 0.05). In the HPV2 E2-stably transfected HaCaT and NHEK cells, the expression of loricrin, filaggrin, and involucrin was significantly down-regulated in the HPV2 E2 + FGFR3-WT transfected groups and the HPV2 E2 + FGFR3-K650E transfected groups than in the HPV2 E2 + blank vector groups (all P < 0.05). Laser scanning confocal microscopy showed the spatial co-localization of HPV2 E2 and FGFR3 in the nuclear membrane and cytoplasm of HaCaT cells. Conclusion:HPV2 E2 and FGFR3 could both induce the differentiation of HaCaT and NHEK cells, while FGFR3 could inhibit HPV2 E2-induced differentiation trend of HaCaT and NHEK cells, which may be related to the cellular spatial co-localization of HPV2 E2 and FGFR3.

Diagnosis of acanthosis nigricans in a family by targeted sequencing / 中华皮肤科杂志

A pedigree with familial acanthosis nigricans presenting with atypical clinical symptoms was reported. The 4-year-old female proband began to develop black patches on the neck and abdomen since the age of 1 year, which had gradually spread to the lips and front of the trunk in recent years. Reflectance confocal microscopy of the abdominal skin showed downward extension and twisting of dermal papillary rings with formation of gully-like structures, and moderately to highly refractive particles in the dermal papillary rings. The proband′s father and grandmother had similar medical history, but the pigmentation spontaneously subsided with age, leaving only local thickened skin lines. Peripheral blood samples were collected from the proband, her parents and grandmother, and panel-based targeted sequencing of peripheral blood DNA was performed for the proband. Sequencing showed a missense mutation c.1949A>C (p.Lys650Thr) in exon 14 of the FGFR3 gene in the proband, and Sanger sequencing confirmed the presence of this mutation in the proband and her father and grandmother. A diagnosis of familial acanthosis nigricans was made.

Fibroblast growth factor receptor 3 gene (FGFR3) mutations in high-grade muscle-invasive urothelial bladder cancer in a Brazilian population: evaluation and prevalence

ABSTRACT Objective To understand the feasibility of FGFR3 tests in the Brazilian public health context, and to sample the mutational burden of this receptor in high-grade muscle invasive bladder cancer. Methods A total of 31 patients with high-grade muscle-invasive bladder cancer were included in the present study. Either transurethral resection of bladder tumor or radical cystectomy specimens were analyzed. Formalin-fixed paraffin-embedded tissue blocks were sectioned, hematoxylin and eosin stained, and histologic sections were reviewed. Total RNA was extracted using the RNeasy DSP formalin-fixed paraffin-embedded kit. Qualitative results were displayed in Rotor-Gene AssayManager software. Results Six patients were excluded. From the samples analyzed, four (16.7%) were considered inadequate and could not have their RNA extracted. Two patients presented FGFR3 mutations, accounting for 9.5% of material available for adequate analysis. The two mutations detected included a Y373C mutation in a male patient and a S249C mutation in a female patient. Conclusion FGFR3 mutations could be analyzed in 84% of our cohort and occurred in 9.5% of patients with high-grade muscle invasive bladder cancer in this Brazilian population. FGFR3 gene mutations are targets for therapeutic drugs in muscle-invasive bladder cancer. For this reason, know the frequency of these mutations can have a significant impact on public health policies and costs provisioning.

Novel Biomarkers for Prediction of Response to Preoperative Systemic Therapies in Gastric Cancer

Preoperative chemo- and radiotherapeutic strategies followed by surgery are currently a standard approach for treating locally advanced gastric and esophagogastric junction cancer in Western countries. However, in a large number of cases, the tumor is extremely resistant to these treatments and the patients are exposed to unnecessary toxicity and delayed surgical therapy. The current clinical trials evaluating the combination of preoperative systemic therapies with modern targeted and immunotherapeutic agents represent a unique opportunity for identifying predictive biomarkers of response to select patients that would benefit the most from these treatments. However, it is of utmost importance that these potential biomarkers are corroborated by extensive preclinical and translational research. The aim of this review article is to present the most promising biomarkers of response to classic chemotherapeutic, anti-HER2, antiangiogenic, and immunotherapeutic agents that can be potentially useful for personalized preoperative systemic therapies in gastric cancer patients.

FGFR3 overexpression is prognostic of adverse outcome for muscle-invasive bladder carcinoma treated with adjuvant chemotherapy.

BACKGROUND: Alterations in fibroblast growth factor receptor 3 (FGFR3) have been implicated in the pathogenesis of urothelial carcinoma. However, its clinicopathological significance has not been clearly established, especially in muscle-invasive bladder cancer (MIBC). OBJECTIVES: The aim of our study was to investigate the mutation and overexpression of FGFR3 in MIBC cases from radical cystectomy and to analyze the prognostic and predictive significance in the groups with or without adjuvant chemotherapy. METHODS AND MATERIALS: Study cohorts included 72 cases of MIBC including 42 patients who were treated with adjuvant chemotherapy. The mutation status of FGFR3 exons 7, 10, and 15 was investigated and protein expression was evaluated. The findings were analyzed for the association with relevant clinicopathological findings. RESULTS: FGFR3 mutations were found in 7 patients (9.7%) and were correlated with a pattern of papillary growth, moderate histologic grade (G2 vs. G3), and moderately advanced TNM stage (II-III vs. IV). FGFR3 protein overexpression was detected in 33 cases (45.8%) but was not associated with the relevant clinicopathological parameters. In patients treated with adjuvant chemotherapy, FGFR3 overexpression was correlated with shorter disease-free survival (P = 0.067, 95% confidence interval: 14.8-29.6, marginal significance) and overall survival (P = 0.035), remaining as a significant independent prognostic factor for disease-free survival and overall survival in multivariate analysis using Cox proportional hazards model. In patients without adjuvant chemotherapy, FGFR3 mutation or overexpression did not have prognostic significance in multivariate analysis. CONCLUSION: We report the FGFR3 alterations in MIBCs, and discuss their biological implication in subsets of patients. FGFR3 overexpression was predictive of adverse outcome in patients with adjuvant cisplatin-based chemotherapy after radical cystectomy. The utility of FGFR3 as a therapeutic target is suggested.

Successful birth with preimplantation genetic diagnosis using single-cell allele-specific PCR and sequencing in a woman with hypochondroplasia due to FGFR3 mutation (c.1620C>A, p.N540K).

Hypochondroplasia (HCH) is an autosomal dominant inherited skeletal dysplasia, usually caused by a heterozygous mutation in the fibroblast growth factor receptor 3 gene (FGFR3). A 27-year-old HCH woman with a history of two consecutive abortions of HCH-affected fetuses visited our clinic for preimplantation genetic diagnosis (PGD). We confirmed the mutation in the proband (FGFR3:c.1620C>A, p.N540K), and established a nested allele-specific PCR and sequence analysis for PGD using single lymphocyte cells. We performed this molecular genetic analysis to detect the presence of mutation among 20 blastomeres from 18 different embryos, and selected 9 embryos with the wild-type sequence (FGFR3:c.1620C). A successful pregnancy was achieved through a frozen-thawed cycle and resulted in the full-term birth of a normal neonate. To the best of our knowledge, this is the first report of a successful pregnancy and birth using single-cell allele-specific PCR and sequencing for PGD in an HCH patient.

Prenatal diagnosis and mutation analysis of fibroblast growth factor receptor 3 gene in achondroplasia / 中华围产医学杂志

Objective To explore the value of prenatal genetical diagnosis by mutation analysis of achondroplasia (ACH) fibroblast growth factor receptor 3 (FGFR3) gene.Methods Genomic DNA from nine ACH patients and their parents in Gansu Maternal and Child Health Hospital from July,2010 to December,2014 was prepared for polymerase chain reaction.Direct sequencing revealed the samples were performed after amplification of exon 10 of FGFR3 containing the potential mutation.Fetal DNA was extracted from cells in both amniotic fluid and umbilical cord,and then exon 10 of FGFR3 was also tested.Three fetuses with short-limb dysplasia were also included and prenatal diagnosis was offered to them through amniocentesis or cordocentesis.Results Prenatal ultrasonography test showed shorter femoral length,which was less than 2-3 standard deviation of normal reference dysplasia fetal performance for femoral short.Femur length is lower than 2-3 standard deviation minus normal value,and discrepancy in biparietal diameter compared with fetuses at the same gestational age.In the four families with one ACH parent,c.1138G ＞ A heterozygous mutation was detected in all of the four mothers,while two fetuses among them showed c.1138G ＞ A heterozygous mutation mutation and the other two were normal.There were other two fetuses with c.1138G ＞ A heterozygous mutation from other two families,one's father had c.1138G ＞ A heterozygous mutations,but not the mother,the other had c.1138G ＞ A heterozygous mutations in both the mother and father.Among the three families with unaffected parents but each had a de novo c.1138G ＞ A mutation child,no mutation of c.1138G ＞ A genotype was detected in their fetuses,neither in the three fetus with short limb dysplasia.Four fetuses with a c.1138G ＞ A mutation and three with short-limb dysplasia were terminated.The other five fetuses whose genotype was normal were born and healthy with normal phenotype at one-year-old follow-up.Conclusion FGFR3 genetic analysis could provide information for genetic counseling and prenatal diagnosis for ACH parents or parents who had an ACH baby to prevent birth defect.

The progress of fibroblast growth factor receptor 3 in breast cancer / 国际肿瘤学杂志

Fibroblast growth factor receptor 3(FGFR3)plays important roles in cell proliferation,diffe rentiation,and angiogenesis.Recent studies have demonstrated that FGFR3 is associated with progression of breast cancer and has effects in endocrine therapy resistance breast cancer.It has also been showed that FGFR3 is correlated with breast cancer prognosis.

Construction of eukaryotic expression vectors of FGFR3 gene and their expressions in human leukemia K562 cell line / 吉林大学学报(医学版)

Objective To construct the eukaryotic expression vectors of fibroblast growth factor receptor 3(FGFR3) MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN, and to detect their expressions in human chronic myeloid leukemia(CML)K562 cell line.Methods The full-length FGFR3 (fgfr3-WT)and dominant negative FGFR3 (fgfr3-DN)were amplified by polymerase chain reaction (PCR). The two genes were respectively digested with EcoRⅠand BamHⅠ,and then ligated into MSCV/puro to construct the recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN which were tranduced into K562 cells by LipofectaminTM 2000 after PCR,double digestion and DNA sequencing.The expressions of FGFR3 protein in K562 cells were detected by Western blotting and flow cytometry. Results The recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN were amplified by PCR method, and the results showed fgfr3-WT of 2 400 bp and fgfr3-DN of 1 200 bp had been successfully cloned into MSCV-puro vector. The 2 400 bp fragment was oblained after double digestion of recombinant plasmid.The sequencing results showed that the size of fgfr3-WT was 2 400 bp which was the same as the sequence from GeneBank.Fgfr3-DN of 1 200 bp was also in conformity with the expected sequence.Compared with control (K562 MSCV)group,the expression level of FGFR3-WT in MSCV/puro-fgfr3-WT transfection (K562-WT)group was increased to above 10 times.There was high expression of FGFR3-DN in MSCV/puro-FGFR3-DN transfection (K562-DN)group,but there was no expressions in control(K562 MSCV)group and K562-WT group.The flow cytometry results showed that the high expressions of FGFR3-WT were in 57.5% cells in K562-DN group and the high expressions of FGFR3-DN were in 41.5% cells in K562-DN group. Conclusion The K562 cell lines highly expressing FGFR3-WT and FGFR3-DN are constructed successfully.

Successful birth with preimplantation genetic diagnosis using single-cell allele-specific PCR and sequencing in a woman with hypochondroplasia due to FGFR3 mutation (c.1620C>A, p.N540K) / 대한생식의학회지

Hypochondroplasia (HCH) is an autosomal dominant inherited skeletal dysplasia, usually caused by a heterozygous mutation in the fibroblast growth factor receptor 3 gene (FGFR3). A 27-year-old HCH woman with a history of two consecutive abortions of HCH-affected fetuses visited our clinic for preimplantation genetic diagnosis (PGD). We confirmed the mutation in the proband (FGFR3:c.1620C>A, p.N540K), and established a nested allele-specific PCR and sequence analysis for PGD using single lymphocyte cells. We performed this molecular genetic analysis to detect the presence of mutation among 20 blastomeres from 18 different embryos, and selected 9 embryos with the wild-type sequence (FGFR3:c.1620C). A successful pregnancy was achieved through a frozen-thawed cycle and resulted in the full-term birth of a normal neonate. To the best of our knowledge, this is the first report of a successful pregnancy and birth using single-cell allele-specific PCR and sequencing for PGD in an HCH patient.

Gene mutation in fetuses with short limbs deformity / 中华围产医学杂志

Objective To identify the genetic mechanism of fetuses with short limbs deformity.Methods From Aug.2008 to Aug.2011,ten fetuses with obvious short limbs were found in ultrasound screening performed at 18-24 and (or) 30-32 gestational weeks and underwent artificial induced labor with the patient' consent.Amniotic fluid or cord blood of the fetuses was collected for karyotyping analysis and detection of mutation point of fibroblast growth factor receptor 3 (FGFR3)gene by polymerase chain reaction and gene sequencing.One fetus (case 3) who presented with achondrogenesis underwent sequencing of SLC26A2 and Trip11 gene meanwhile.Results Among the 10 fetuses with short limbs deformity,five cases were found during second trimester and five during third trimester.Nine cases were identified as normal karyotype and one was chimera (46,XY/45,XY,- 18).One fetus carried a rare FGFR3 mutation of c.1108G＞T (G370C) and was diagnosed as thanatophoric dysplasia at 21+3 weeks.Three fetus carried c.1138G＞A (G380R) mutation and were diagnosed as achondroplasia.These four families had low recurrent risk because no gene mutations were found in the parents.Three mothers of these four fetuses were pregnant again and had normal neonates now.No mutations were found in all gene sequencing in case 3.Conclusions Karyotyping analysis and sequencing of FGFR3 gene could find causative gene mutations and provide genetic counselling and prenatal diagnosis for some fetuses with short limbs deformity.In the third trimester,achondroplasia is the most possible diagnosis when short limbs fetus is found by ultrasound.