scholarly journals FGFR3 overexpression is a useful detection tool for FGFR3 fusions and sequence variations in glioma

2020 ◽  
Author(s):  
Jens Schittenhelm ◽  
Lukas Ziegler ◽  
Jan Sperveslage ◽  
Michel Mittelbronn ◽  
David Capper ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Growth Factor Receptor ◽  
Gene Mutations ◽  
Diagnostic Tools ◽  
Wild Type ◽  
Rt Pcr ◽  
Fgfr3 Gene ◽  
Detection Tool ◽  
Sequence Variations ◽  
Gene Panel Sequencing

Abstract Background Fibroblast growth factor receptor (FGFR) inhibitors are currently used in clinical development. A subset of glioblastomas carries gene fusion of FGFR3 and transforming acidic coiled-coil protein 3. The prevalence of other FGFR3 alterations in glioma is currently unclear. Methods We performed RT-PCR in 101 glioblastoma samples to detect FGFR3-TACC3 fusions (“RT-PCR cohort”) and correlated results with FGFR3 immunohistochemistry (IHC). Further, we applied FGFR3 IHC in 552 tissue microarray glioma samples (“TMA cohort”) and validated these results in two external cohorts with 319 patients. Gene panel sequencing was carried out in 88 samples (“NGS cohort”) to identify other possible FGFR3 alterations. Molecular modeling was performed on newly detected mutations. Results In the “RT-PCR cohort,” we identified FGFR3-TACC3 fusions in 2/101 glioblastomas. Positive IHC staining was observed in 73/1024 tumor samples of which 10 were strongly positive. In the “NGS cohort,” we identified FGFR3 fusions in 9/88 cases, FGFR3 amplification in 2/88 cases, and FGFR3 gene mutations in 7/88 cases in targeted sequencing. All FGFR3 fusions and amplifications and a novel FGFR3 K649R missense mutation were associated with FGFR3 overexpression (sensitivity and specificity of 93% and 95%, respectively, at cutoff IHC score > 7). Modeling of these data indicated that Tyr647, a residue phosphorylated as a part of FGFR3 activation, is affected by the K649R mutation. Conclusions FGFR3 IHC is a useful screening tool for the detection of FGFR3 alterations and could be included in the workflow for isocitrate dehydrogenase (IDH) wild-type glioma diagnostics. Samples with positive FGFR3 staining could then be selected for NGS-based diagnostic tools.

The Platelet-Derived Growth Factor Receptor beta Fuses to Two Distinct Loci at 3p21 in Imatinib Responsive Chronic Eosinophilic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3253-3253 ◽  
Author(s):  
Claire Curtis ◽  
Jane F. Apperley ◽  
Raymond Dang ◽  
Michael Jeng ◽  
Jason Gotlib ◽  
...  
Keyword(s):  
Amino Acid ◽  
Tyrosine Kinase ◽  
Blast Crisis ◽  
Coiled Coil ◽  
Growth Factor Receptor ◽  
Myeloproliferative Disorders ◽  
Rt Pcr ◽  
Chronic Eosinophilic Leukemia ◽  
Amino Terminal ◽  
Exon 11

Abstract We have identified three patients (2 adults, one infant) who presented with BCR-ABL negative eosinophilic myeloproliferative disorders. Cytogenetic analysis revealed a t(1;3;5)(p36;p21;q33) for case 1 and a t(3;5)(p21–25;q31–35) for cases 2 and 3. Two-color fluorescence in situ hybridization (FISH) using differentially labelled probes flanking PDGFRB indicated that this gene was disrupted in all three cases. 5′ rapid amplification of cDNA ends (5′RACE) for case 1 identified an in-frame mRNA fusion of exon 9 of the WDR48 gene at 3p21 to exon 12 of PDGFRB. The chimeric mRNA is predicted to encode a 872 amino acid fusion protein that retains the amino terminal WD repeat region of WDR48 fused to the transmembrane and intracellular tyrosine kinase domains of PDGFRbeta. Cases 2 and 3 were negative for the WDR48-PDGFRB fusion mRNA by RT-PCR using several combinations of primers. 5′RACE PCR from case 2 RNA identified a fusion involving a second 3p21 gene: GOLGA4 exon 11 was fused in-frame to exon 11 of PDGFRB. Exactly the same fusion was found in case 3. The predicted 991 amino acid protein included the amino terminal coiled-coil domain of GOLGA4 fused to the transmembrane and intracellular tyrosine kinase domains of PDGFRbeta. Interestingly, both WDR48 and GOLGA4 are involved in endocytic shuttling pathways. The presence of all fusions was confirmed by RT-PCR and identification of the genomic breakpoints. Imatinib, a known inhibitor of PDGFRbeta, selectively blocked the growth of patient CFU-GM for case 2. Following the identification of PDGFRB rearrangements, all three patients were treated with imatinib. Case 1 was in transformation, but responded rapidly to minimal doses of imatinib (800mg daily for 4 days) with complete cytogenetic remission but remained pancytopenic. Blast crisis recurred 8 months later, responded similarly to 3 days of imatinib, but the patient died 2 months later of invasive fungal infection. Case 2 responded clinically and remains in sustained cytogenetic and molecular remission (nested RT-PCR negative for GOLGA4-PDGFRB). Case 3 (a 13 month old boy) had a complete hematologic response to 50mg/day imatinib but the t(3;5) was still seen in 40% of metaphases at 3 months. We conclude that PDGFRB fuses to diverse partner genes to give rise to atypical MPDs. Although very rare, identification of these fusions is critical for proper management of affected individuals.

Characterisation and quantification of F8 transcripts of ten putative splice site mutations

2015 ◽  
Vol 113 (03) ◽  
pp. 585-592 ◽  
Author(s):  
Yeling Lu ◽  
Yufeng Ruan ◽  
Qiulan Ding ◽  
Xuefeng Wang ◽  
Xiaodong Xi ◽  
...  
Keyword(s):  
Splice Site ◽  
Mrna Level ◽  
Gene Mutations ◽  
Exon Skipping ◽  
Splice Sites ◽  
Wild Type ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Fluorescent Pcr ◽  
Cryptic Splice Sites

SummaryMutations affecting splice sites comprise approximately 7.5 % of the known F8 gene mutations but only a few were verified at mRNA level. In the present study, 10 putative splice site mutations were characterised by mRNA analysis using reverse transcription PCR (RT-PCR). Quantitative real-time RT-PCR (RT-qPCR) and co-amplification fluorescent PCR were used in combination to quantify the amount of each of multiple F8 transcripts. All of the mutations resulted in aberrant splicing. One of them (c.6187+1del1) generated one form of F8 transcript with exon skipping, and the remaining nine mutations (c.602-6T>C, c.1752+5_1752+6insGTTAG, c.1903+5G>A, c.5219+3A>G, c.5586+3A>T, c.969A>T, c.265+4A>G, c.601+1_601+5del5 and c.1444-8_1444del9) produced multiple F8 transcripts with exon skipping, activation of cryptic splice site and/or normal splicing. Residual wild-type F8 transcripts were produced by the first six of the nine mutations with amounts of 3.9 %>, 14.2 %>, 5.2 %>, 19.2 %>, 1.8 °% and 2.5 %> of normal levels, respectively, which were basically consistent with coagulation phenotypes in the related patients. In comparison with the mRNA findings, software Alamut v2.3 had values in the prediction of pathogenic effects on native splice sites but was not reliable in the prediction of activation of cryptic splice sites. Our quantification of F8 transcripts may provide an alternative way to evaluate the low expression levels of residue wild-type F8 transcripts and help to explain the severity of haemophilia A caused by splicing site mutations.

KIT and Platelet-Derived Growth Factor Receptor Alpha Tyrosine Kinase Gene Mutations and KIT Amplifications in Human Solid Tumors

2005 ◽  
Vol 23 (1) ◽  
pp. 49-57 ◽  
Author(s):  
Harri Sihto ◽  
Maarit Sarlomo-Rikala ◽  
Olli Tynninen ◽  
Minna Tanner ◽  
Leif C. Andersson ◽  
...  
Keyword(s):  
Growth Factor ◽  
Growth Factor Receptor ◽  
Gene Mutations ◽  
Platelet Derived Growth Factor ◽  
Wild Type ◽  
Pdgfra Gene ◽  
Factor Receptor Alpha ◽  
Kit Gene ◽  
Histologic Types ◽  
Tumor Types

Purpose Mutated KIT and platelet-derived growth factor receptor alpha (PDGFRα) tyrosine kinases are the principal targets for imatinib mesylate in the treatment of gastrointestinal stromal tumors (GISTs). The frequency of activating KIT and PDGFRA gene mutations in most other histologic types of human cancer is not known. Materials and Methods KIT exons 9, 11, 13, and 17 and PDGFRA exons 11 and 17 of 334 human cancers were screened for mutations using sensitive denaturing high-performance liquid chromatography (DHPLC). In addition, all KIT exons from 9 to 21 of 115 tumors were screened. Thirty-two histologic tumor types were examined. Samples with abnormal findings in DHLPC were sequenced. Immunostaining for the KIT protein (CD117) was performed in 322 (96.4%) of the 334 cases. Results Of the 3,039 exons screened, only 17 had mutation. All 17 cases with either mutated KIT (n = 15) or PDGFRA (n = 2) were histologically GIST tumors, whereas none of the other histologic types of cancer (n = 316) harbored KIT or PDGFRA mutation. KIT immunostaining was rarely positive except in GISTs (18 of 18), small-cell lung cancer (10 of 30; 33%), and testicular teratocarcinoma (four of 17; 24%). Wild-type KIT gene amplification or chromosome 4 aneuploidy was common (seven of 12) in non-GIST tumors with strong KIT protein expression when studied with fluorescence in situ hybridization. Conclusion Despite frequent KIT protein expression in some tumor types, KIT and PDGFRA gene mutations are uncommon in most human cancers. Cancer KIT expression is frequently associated with multiple copies of the wild-type KIT gene.

ALAS2 acts as a modifier gene in patients with congenital erythropoietic porphyria

Blood ◽  
2011 ◽  
Vol 118 (6) ◽  
pp. 1443-1451 ◽  
Author(s):  
Jordi To-Figueras ◽  
Sarah Ducamp ◽  
Jerome Clayton ◽  
Celia Badenas ◽  
Constance Delaby ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Gene Mutations ◽  
Wild Type ◽  
Aminolevulinate Synthase ◽  
Sequence Variations ◽  
Congenital Erythropoietic Porphyria ◽  
New Type ◽  
Terminal Amino ◽  
Exon 11 ◽  
Increased Activity

AbstractMutations in the uroporphyrinogen III synthase (UROS) gene cause congenital erythropoietic porphyria (CEP), an autosomal-recessive inborn error of erythroid heme biosynthesis. Clinical features of CEP include dermatologic and hematologic abnormalities of variable severity. The discovery of a new type of erythroid porphyria, X-linked dominant protoporphyria (XLDPP), which results from increased activity of 5-aminolevulinate synthase 2 (ALAS2), the rate-controlling enzyme of erythroid heme synthesis, led us to hypothesize that the CEP phenotype may be modulated by sequence variations in the ALAS2 gene. We genotyped ALAS2 in 4 unrelated CEP patients exhibiting the same C73R/P248Q UROS genotype. The most severe of the CEP patients, a young girl, proved to be heterozygous for a novel ALAS2 mutation: c.1757 A > T in exon 11. This mutation is predicted to affect the highly conserved and penultimate C-terminal amino acid of ALAS2 (Y586). The rate of 5-aminolevulinate release from Y586F was significantly increased over that of wild-type ALAS2. The contribution of the ALAS2 gain-of-function mutation to the CEP phenotype underscores the importance of modifier genes underlying CEP. We propose that ALAS2 gene mutations should be considered not only as causative of X-linked sideroblastic anemia (XLSA) and XLDPP but may also modulate gene function in other erythropoietic disorders.

scholarly journals CD44 Gene Polymorphisms as a Risk Factor for Susceptibility and Their Effect on the Clinicopathological Characteristics of Lung Adenocarcinoma in Male Patients

2020 ◽  
Vol 17 (9) ◽  
pp. 2981 ◽  
Author(s):  
Ju-Pi Li ◽  
Ming-Ju Hsieh ◽  
Ying-Erh Chou ◽  
Yu-Hua Chao ◽  
Thomas Chang-Yao Tsao ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cancer Susceptibility ◽  
Strong Association ◽  
Growth Factor Receptor ◽  
Gene Mutations ◽  
High Morbidity ◽  
Genotype Distribution ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Male Patients

Lung adenocarcinoma is a subtype of lung cancer with high morbidity and mortality. CD44 is instrumental in many physiological and tumor pathological processes. The expression of unique single nucleotide polymorphisms (SNPs) contributes to protein dysfunction and influences cancer susceptibility. In the current study, we investigated the relationship between CD44 polymorphisms and the susceptibility to lung adenocarcinoma with or without epidermal growth factor receptor (EGFR) gene mutations. This study included 279 patients with lung adenocarcinoma. In total, six CD44 SNPs (rs1425802, rs11821102, rs10836347, rs13347, rs187115, and rs713330) were genotyped using a real-time polymerase chain reaction. We found no significant differences in genotype distribution of CD44 polymorphisms between EGFR wild-type and EGFR mutation type in patients with lung adenocarcinoma. We observed a strong association between CD44 rs11821102 G/A polymorphism and EGFR L858R mutation (odds ratio (OR) = 3.846, 95% confidence interval (CI) = 1.018–14.538; p = 0.037) compared with the EGFR wild-type group. In the subgroup of male patients with lung adenocarcinoma harboring the EGFR wild-type, both CD44 rs713330 T/C (OR = 4.317, 95% CI = 1.029–18.115; p = 0.035) and rs10836347 C/T polymorphisms (OR = 9.391, 95% CI = 1.061–83.136; p = 0.019) exhibited significant associations with tumor size and invasion. Data from the present study suggest that CD44 SNPs may help to predict cancer susceptibility and tumor growth in male patients with lung adenocarcinoma.

scholarly journals Psychomotor Delay in a Child with FGFR3 G380R Pathogenic Mutation Causing Achondroplasia

2021 ◽  
Author(s):  
Mahmut C. Ergoren ◽  
Erdal Eren ◽  
Elena Manara ◽  
Stefano Paolacci ◽  
Pinar Tulay ◽  
...  
Keyword(s):  
De Novo ◽  
Growth Factor Receptor ◽  
Gene Mutations ◽  
Pathogenic Mutation ◽  
Phenotypic Characteristics ◽  
Psychomotor Delay ◽  
Fgfr3 Gene ◽  
Proliferation And Differentiation ◽  
Common Causes ◽  
The Common

AbstractAchondroplasia (ACH) is a hereditary disorder of dwarfism that is caused by the aberrant proliferation and differentiation of chondrocyte growth plates. The common findings of macrocephaly and facial anomalies accompany dwarfism in these patients. Fibroblast growth factor receptor 3 (FGFR3) gene mutations are common causes of achondroplasia. The current study presents a case of 2-year-old male presenting with phenotypic characteristics of ACH. The interesting finding of the case is the presence of psychomotor delay that is not very common in these patients. Clinical exome sequencing analyzing 4.813 disease causing genes revealed a de novo c.1138G > A mutation within the FGFR3 gene. In conclusion, the mutation confirms the clinical diagnosis of ACH, and it seems to be causing the psychomotor delay in this patient.

scholarly journals Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis

2009 ◽  
Vol 54 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Hassan Safi ◽  
Robert D. Fleischmann ◽  
Scott N. Peterson ◽  
Marcus B. Jones ◽  
Behnam Jarrahi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
In Vitro Selection ◽  
Gene Mutations ◽  
Wild Type ◽  
Mutant Selection ◽  
Preferential Growth ◽  
Allelic Exchange ◽  
High Level ◽  
Isogenic Mutants

ABSTRACT Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 μg/ml, 5 μg/ml, 12 μg/ml, 3 μg/ml, and 4 μg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 μg/ml, 8 μg/ml, and 2 to 8 μg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.

scholarly journals The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1105-1117 ◽  
Author(s):  
W John Haynes ◽  
Kit-Yin Ling ◽  
Robin R Preston ◽  
Yoshiro Saimi ◽  
Ching Kung
Keyword(s):  
Genomic Library ◽  
Open Reading Frame ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Rt Pcr ◽  
Reading Frame ◽  
Close Proximity ◽  
In The Wild ◽  
Dna Fragment ◽  
Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

scholarly journals Molecular Basis of In Vivo Resistance to Sulfadoxine-Pyrimethamine in African Adult Patients Infected withPlasmodium falciparum Malaria Parasites

1998 ◽  
Vol 42 (7) ◽  
pp. 1811-1814 ◽  
Author(s):  
Leonardo K. Basco ◽  
Rachida Tahar ◽  
Pascal Ringwald
Keyword(s):  
Dihydrofolate Reductase ◽  
Point Mutations ◽  
Gene Mutations ◽  
Adult Patients ◽  
Wild Type ◽  
Dihydropteroate Synthase ◽  
Reductase Gene ◽  
Parasite Populations

ABSTRACT In vitro sulfadoxine and pyrimethamine resistance has been associated with point mutations in the dihydropteroate synthase and dihydrofolate reductase domains, respectively, but the in vivo relevance of these point mutations has not been well established. To analyze the correlation between genotype and phenotype, 10 Cameroonian adult patients were treated with sulfadoxine-pyrimethamine and followed up for 28 days. After losses to follow-up (n = 1) or elimination of DNA samples due to mixed parasite populations with pyrimethamine-sensitive and pyrimethamine-resistant profiles (n = 3), parasite genomic DNA from day 0 blood samples of six patients were analyzed by DNA sequencing. Three patients who were cured had isolates characterized by a wild-type or mutant dihydrofolate reductase gene (with one or two mutations) and a wild-type dihydropteroate synthase gene. Three other patients who failed to respond to sulfadoxine-pyrimethamine treatment carried isolates with triple dihydrofolate reductase gene mutations and either a wild-type or a mutant dihydropteroate synthase gene. Three dihydrofolate reductase gene codons (51, 59, and 108) may be reliable genetic markers that can accurately predict the clinical outcome of sulfadoxine-pyrimethamine treatment in Africa.

scholarly journals Primary and Secondary Kinase Genotypes Correlate With the Biological and Clinical Activity of Sunitinib in Imatinib-Resistant Gastrointestinal Stromal Tumor

2008 ◽  
Vol 26 (33) ◽  
pp. 5352-5359 ◽  
Author(s):  
Michael C. Heinrich ◽  
Robert G. Maki ◽  
Christopher L. Corless ◽  
Cristina R. Antonescu ◽  
Amy Harlow ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Progression Free Survival ◽  
Clinical Results ◽  
Clinical Activity ◽  
Wild Type ◽  
Mutational Status ◽  
Imatinib Resistant ◽  
Exon 9 ◽  
Exon 11 ◽  
The Impact

PurposeMost gastrointestinal stromal tumors (GISTs) harbor mutant KIT or platelet-derived growth factor receptor α (PDGFRA) kinases, which are imatinib targets. Sunitinib, which targets KIT, PDGFRs, and several other kinases, has demonstrated efficacy in patients with GIST after they experience imatinib failure. We evaluated the impact of primary and secondary kinase genotype on sunitinib activity.Patients and MethodsTumor responses were assessed radiologically in a phase I/II trial of sunitinib in 97 patients with metastatic, imatinib-resistant/intolerant GIST. KIT/PDGFRA mutational status was determined for 78 patients by using tumor specimens obtained before and after prior imatinib therapy. Kinase mutants were biochemically profiled for sunitinib and imatinib sensitivity.ResultsClinical benefit (partial response or stable disease for ≥ 6 months) with sunitinib was observed for the three most common primary GIST genotypes: KIT exon 9 (58%), KIT exon 11 (34%), and wild-type KIT/PDGFRA (56%). Progression-free survival (PFS) was significantly longer for patients with primary KIT exon 9 mutations (P = .0005) or with a wild-type genotype (P = .0356) than for those with KIT exon 11 mutations. The same pattern was observed for overall survival (OS). PFS and OS were longer for patients with secondary KIT exon 13 or 14 mutations (which involve the KIT-adenosine triphosphate binding pocket) than for those with exon 17 or 18 mutations (which involve the KIT activation loop). Biochemical profiling studies confirmed the clinical results.ConclusionThe clinical activity of sunitinib after imatinib failure is significantly influenced by both primary and secondary mutations in the predominant pathogenic kinases, which has implications for optimization of the treatment of patients with GIST.

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