Inability of the acidic fibroblast growth factor mutant K132E to stimulate DNA synthesis after translocation into cells

Producción CientíficaAcidic fibroblast growth factor (aFGF) is a potent mitogen. It acts through activation of specific cell surface receptors leading to intracellular tyrosine phosphorylation cascades, but several reports also indicate that aFGF enters cells and that it has an intracellular function as well. The aFGF(K132E) mutant binds to and activates fibroblast growth factor receptors equally strongly as the wild-type, but it is a poor mitogen. We demonstrate that aFGF(K132E) enters NIH 3T3 cells and is transported to the nuclear fraction like wild-type aFGF. A fusion protein of aFGF(K132E) and diphtheria toxin A-fragment (aFGF(K132E)-DT-A) and a similar fusion protein containing wild-type aFGF (aFGF-DT-A) were reconstituted with diphtheria toxin B-fragment. Both fusion proteins were translocated to the cytosol by the diphtheria toxin pathway and subsequently recovered from the nuclear fraction. Whereas translocation of aFGF-DT-A stimulated DNA synthesis in U2OSDR1 cells lacking functional fibroblast growth factor receptors, aFGF(K132E)-DT-A did not. The mutation disrupts a protein kinase C phosphorylation site in the growth factor making it unable to be phosphorylated. The data indicate that a defect in the intracellular action of aFGF(K132E) is the reason for its strongly reduced mitogenicity, possibly due to inability to be phosphorylated

VII Scandinavian Copd Research Symposium, Holmenkollen, Oslo 18th-19th November 2016

Peer reviewe

Rapid and reliable detection of α-globin copy number variations by quantitative real-time PCR

Background Alpha-thalassemia is the most common human genetic disease worldwide. Copy number variations in the form of deletions of α-globin genes lead to α-thalassemia while duplications of α-globin genes can cause a severe phenotype in β-thalassemia carriers due to accentuation of globin chain imbalance. It is important to have simple and reliable methods to identify unknown or rare deletions and duplications in cases in which thalassemia is suspected but cannot be confirmed by multiplex gap-PCR. Here we describe a copy number variation assay to detect deletions and duplications in the α-globin gene cluster (HBA-CNV). Results Quantitative real-time PCR was performed using four TaqMan® assays which specifically amplify target sequences representing both the α-globin genes, the –α3.7 deletion and the HS-40 region. The copy number for each target was determined by the 2-ΔΔCq method. To validate our method, we compared the HBA-CNV method with traditional gap-PCR in 108 samples from patients referred to our laboratory for hemoglobinopathy evaluation. To determine the robustness of the four assays, we analyzed samples with and without deletions diluted to obtain different DNA concentrations. The HBA-CNV method identified the correct copy numbers in all 108 samples. All four assays showed the correct copy number within a wide range of DNA concentrations (3.2-100 ng/μL), showing that it is a robust and reliable method. By using the method in routine diagnostics of hemoglobinopathies we have also identified several deletions and duplications that are not detected with conventional gap-PCR. Conclusions HBA-CNV is able to detect all known large deletions and duplications affecting the α-globin genes, providing a flexible and simple workflow with rapid and reliable results

Hemoglobinopathy gone astray—three novel forms of α-thalassemia in Norwegian patients characterized by quantitative real-time PCR and DNA sequencing

α-thalassemia is one of the most common monogenic diseases worldwide and is caused by reduced or absent synthesis of α-globin chains, most commonly due to deletions of one or more of the α-globin genes. α-thalassemia occurs with high frequency in tropical and subtropical regions of the world and are very rarely found in the indigenous Scandinavian population. Here, we describe four rare forms of α-thalassemia out of which three are novel, found in together 20 patients of Norwegian origin. The study patients were diagnosed during routine hemoglobinopathy evaluation carried out at the Department of Medical Biochemistry, Oslo University Hospital, Norway. The patients were selected for their thalassemic phenotype, despite Norway as country of origin. All samples went through standard hemoglobinopathy evaluation. DNA sequencing and copy number variation (CNV) analysis using quantitative real-time polymerase chain reaction (qPCR) was applied to detect sequence variants and uncommon deletions in the α-globin gene cluster, respectively. Deletion breakpoints were characterized using gap-PCR and DNA sequencing. DNA sequencing revealed a single nucleotide deletion in exon 3 of the HBA2 gene (NM_000517.4(HBA2):c.345del) and a novel deletion of 20 nucleotides in exon 2 of the HBA2 gene (NM_000517.4(HBA2):c.142_161del). qPCR CNV analysis detected two novel large deletions in the α-globin gene cluster, –(NOR) deletion covering both α-globin genes and (αα)Aurora Borealis affecting the regulatory region, leaving the downstream α-globin genes intact. Even though inherited globin gene disorders are extremely rare in indigenous Scandinavians, the possibility of a carrier state should not be ignored

Hemoglobinopathy gone astray—three novel forms of α-thalassemia in Norwegian patients characterized by quantitative real-time PCR and DNA sequencing

α-thalassemia is one of the most common monogenic diseases worldwide and is caused by reduced or absent synthesis of α-globin chains, most commonly due to deletions of one or more of the α-globin genes. α-thalassemia occurs with high frequency in tropical and subtropical regions of the world and are very rarely found in the indigenous Scandinavian population. Here, we describe four rare forms of α-thalassemia out of which three are novel, found in together 20 patients of Norwegian origin. The study patients were diagnosed during routine hemoglobinopathy evaluation carried out at the Department of Medical Biochemistry, Oslo University Hospital, Norway. The patients were selected for their thalassemic phenotype, despite Norway as country of origin. All samples went through standard hemoglobinopathy evaluation. DNA sequencing and copy number variation (CNV) analysis using quantitative real-time polymerase chain reaction (qPCR) was applied to detect sequence variants and uncommon deletions in the α-globin gene cluster, respectively. Deletion breakpoints were characterized using gap-PCR and DNA sequencing. DNA sequencing revealed a single nucleotide deletion in exon 3 of the HBA2 gene (NM_000517.4(HBA2):c.345del) and a novel deletion of 20 nucleotides in exon 2 of the HBA2 gene (NM_000517.4(HBA2):c.142_161del). qPCR CNV analysis detected two novel large deletions in the α-globin gene cluster, –(NOR) deletion covering both α-globin genes and (αα)Aurora Borealis affecting the regulatory region, leaving the downstream α-globin genes intact. Even though inherited globin gene disorders are extremely rare in indigenous Scandinavians, the possibility of a carrier state should not be ignored

Hb Aalesund (HBA2: c.400_406delAGCACCG), an Unstable α-Globin Variant Found in a Norwegian Patient Causing Moderate Hemolytic Anemia and Falsely High Hb A1c Using Ion Exchange High Performance Liquid Chromatography

A new unstable hemoglobin (Hb) variant, named Hb Aalesund, was detected during Hb A1c measurement in a patient with a nearly compensated hemolytic anemia. Sequencing of the α-globin genes revealed a 7 bp deletion in exon 3 of the HBA2 gene (HBA2: c.400_406delAGCACCG) (NM_000517.4) causing a frameshift and a premature termination codon (PTC) two positions downstream. Apparently, the transcript bypassed nonsense-mediated decay (NMD), and a truncated protein was translated. The unstable Hb variant presumably underwent rapid denaturation, as heterozygosity of Hb Aalesund was associated with mild hemolytic anemia. In addition, the Hb variant interfered with Hb A1c measurement by cation exchange high performance liquid chromatography (HPLC), causing a falsely high Hb A1c result when using the Bio-Rad D10™ Hemoglobin Analyzer fast Hb A1c Program

Hb Aalesund (HBA2: c.400_406delAGCACCG), an Unstable α-Globin Variant Found in a Norwegian Patient Causing Moderate Hemolytic Anemia and Falsely High Hb A1c Using Ion Exchange High Performance Liquid Chromatography.

A new unstable hemoglobin (Hb) variant, named Hb Aalesund, was detected during Hb A1c measurement in a patient with a nearly compensated hemolytic anemia. Sequencing of the α-globin genes revealed a 7 bp deletion in exon 3 of the HBA2 gene (HBA2: c.400_406delAGCACCG) (NM_000517.4) causing a frameshift and a premature termination codon (PTC) two positions downstream. Apparently, the transcript bypassed nonsense-mediated decay (NMD), and a truncated protein was translated. The unstable Hb variant presumably underwent rapid denaturation, as heterozygosity of Hb Aalesund was associated with mild hemolytic anemia. In addition, the Hb variant interfered with Hb A1c measurement by cation exchange high performance liquid chromatography (HPLC), causing a falsely high Hb A1c result when using the Bio-Rad D10™ Hemoglobin Analyzer fast Hb A1c Program