Biomedical subjects

Knut Erik Berge

Publications and source records attributed to Knut Erik Berge.

11 recordsLinked to original sources

PubMed2006

Model system for phenotypic characterization of sequence variations in the LDL receptor gene.

BACKGROUND: Sequence variations in the LDL receptor (LDLR) gene cause defects of LDLR protein production and function through different molecular mechanisms. Here we describe a cell model system for the phenotypic characterization of sequence variations in the LDLR gene. Well-known sequence variations belonging to LDLR classes 2 to 5 (p.G565V, p.I161D, p.Y828C, and p.V429M) were studied in CHO and HepG2 cells. METHODS: Expression of LDLR protein on the cell surface was detected by use of fluorescence-conjugated antibodies against the LDLR and the LDLR activity was measured by incubating the cells with fluorescently labeled and radiolabeled LDL. The intracellular locations of the LDLR mutants and wild-type were also investigated. RESULTS: The class 2A p.G565V sequence variant exhibited an intracellular distribution of LDLR with no active receptors on the cell surface. Both the class 3 p.I161D and class 4 p.Y828C sequence variants gave surface staining but had a reduced ability to bind or internalize LDL, respectively. By determining the intracellular locations of the receptors we were able to visualize the accumulation of the class 5 p.V429M sequence variant in endosomes by means of a specific marker, as well as confirming that the class 4 p.Y828C variant was not localized in clathrin-coated pits. Flow cytometry allowed us quantitatively to determine the amount and activity of receptors. To confirm the results of binding and cell association of fluorescently labeled LDL analyzed by flow cytometry, assays using 125I-labeled LDL were performed. In addition to a useful and valid alternative to radiolabeled LDL, the unique properties of fluorescently labeled LDL allowed a variety of detection technologies to be used. CONCLUSIONS: This new approach enables phenotypic characterization of sequence variations in the LDLR gene. The assays developed may be valuable for confirming the pathogenicity of novel missense sequence variations found throughout the LDLR gene.

Animals↗

PubMed2006

Analysis of alternatively spliced isoforms of human LDL receptor mRNA.

BACKGROUND: The low density lipoprotein receptor (LDLR) family is a family of structurally related cell surface receptors with conserved exon/intron organization. Several members of this family have been shown to undergo alternative splicing. However, no alternative splicing of the LDLR pre-mRNA has so far been described. METHODS: In the present study alternative splicing of human LDLR pre-mRNA has been studied in eight different tissues and four different cell lines using reverse transcription (RT) PCR. A quantitative real-time PCR with exon-exon boundary spanning primers was established to measure the relative amount of two novel isoforms. RESULTS: Several novel isoforms were identified by RT-PCR of which the isoforms lacking exon 4 or 12 were two of the most prominent. Although highly detectable by RT-PCR, the quantification by real-time PCR revealed low levels of these isoforms. CONCLUSIONS: Novel isoforms of LDLR mRNA are described. Quantification by real-time PCR of two of the alternatively spliced isoforms revealed low amount of these isoforms in the examined tissues and cell lines. Further investigations are needed to evaluate if these isoforms represent functional transcripts of LDLR mRNA.

Alternative Splicing↗

PubMed2006

Effect of mutations in the PCSK9 gene on the cell surface LDL receptors.

The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene is involved in the post-transcriptional regulation of the low-density lipoprotein (LDL) receptors (LDLR). Mutations in the PCSK9 gene have been associated with both hypocholesterolemia and hypercholesterolemia through 'loss-of-function' and 'gain-of-function' mechanisms, respectively. We have studied the effect of the four loss-of-function mutations R46L, G106R, N157K and R237W and the two gain-of-function mutations S127R and D374Y on the autocatalytic activity of PCSK9, as well as on the amount of the cell surface LDLR and internalization of LDL in transiently transfected HepG2 cells. The two groups of mutations did not differ with respect to autocatalytic activity of PCSK9, but they did differ with respect to the amount of cell surface LDLR and internalization of LDL. The four loss-of-function mutations had a 16% increased level of cell surface LDLR and a 35% increased level of internalization of LDL as compared with WT-PCSK9. The two gain-of-function mutations had a 23% decreased level of cell surface LDLR and a 38% decreased level of internalization of LDL as compared with WT-PCSK9. Our studies have also shown that transfer of media from transiently transfected HepG2 cells to untransfected HepG2 cells, reduces the amount of cell surface LDLR and internalization of LDL in the untransfected cells within 20 min of media transfer. Thus, PCSK9 or a factor acted upon by PCSK9, is secreted from the transfected cells and degrades LDLR both in transfected and untransfected cells.

Amino Acid Substitution↗

PubMed2006

Missense mutations in the PCSK9 gene are associated with hypocholesterolemia and possibly increased response to statin therapy.

OBJECTIVE: The proprotein convertase subtilisin/kexin type 9 (PCSK9) gene encodes a proprotein convertase that causes degradation of cell surface low-density lipoprotein receptors (LDLRs). Mutations in the PCSK9 gene that disrupt the normal function of PCSK9 could therefore result in increased number of LDLRs and hypocholesterolemia. Also, the cholesterol-lowering effect of statins could be increased in subjects carrying mutations in the PCSK9 gene. METHODS AND RESULTS: We have screened 38 unrelated hypocholesterolemic subjects as well as 25 unrelated familial hypercholesterolemia (FH) heterozygotes who responded particularly well to statin therapy for mutations in the 12 exons of the PCSK9 gene by DNA sequencing. Six of the 38 (15.8%) hypocholesterolemic subjects were heterozygous for 1 of the 3 mutations R46L, G106R, or R237W in the PCSK9 gene. In the group of 25 FH heterozygotes who responded particularly well to statin therapy, 3 (8.8%) were heterozygous for mutations R46L or N157K in the PCSK9 gene. None of 441 hypercholesterolemic subjects without mutations in the LDLR gene or in the apolipoprotein B-100 gene possessed any of the 4 mutations. CONCLUSIONS: The 4 missense mutations R46L, G106R, N157K, and R237W are associated with hypocholesterolemia and possibly increased response to statin therapy.

Adult↗

PubMed2005

Differential regulation of vacuolar H+ -ATPase and Na+/H+ exchanger 3 in rat cholangiocytes after bile duct ligation.

The cholangiocytes lining the intrahepatic bile ducts modify the primary secretion from the hepatocytes. The cholangiocytes secrete HCO (3)(-) into bile when stimulated with secretin in many species, including man. However, in rats, secretin stimulation neither affects biliary HCO (3)(-) concentration nor bile flow, whereas following bile duct ligation (BDL) it induces hypercholeresis with significant increase of NaHCO(3) concentration. We hypothesized that BDL might affect the expression of cholangiocyte H(+) transporters and thereby choleresis, and determined the expression and localization of the 31 kDa vacuolar type H(+)-ATPase (V-ATPase) subunit and of Na(+)/H(+) exchanger NHE3 in the livers of control and BDL rats by real-time PCR, in situ hybridization, immunoblotting, and immunohistochemistry. In controls, secretin had no effect on bile flow, whereas following BDL, secretin increased bile flow approximately threefold. V-ATPase and NHE3 were expressed in control cholangiocytes showing intracellular and apical distribution, respectively. BDL significantly up-regulated V-ATPase mRNA and protein expression and was associated with redistribution to the apical pole in approximately 60% of the cholangiocytes lining the small bile ductules. In contrast, NHE3 expression was significantly down-regulated by BDL at the mRNA and protein level. The data demonstrate expression of V-ATPase in rat cholangiocytes. BDL-induced down-regulation of NHE3 may contribute to a reduction of Na(+) and HCO (3)(-) reabsorption and thus to their net secretion into bile. Apical localization of V-ATPase in cholangiocytes may indicate its involvement in pH regulation and/or HCO (3)(-) salvage to compensate for NHE3 down-regulation in BDL.

Animals↗

PubMed2005

[Cardiac ion channel disorders--diagnosis and treatment].

BACKGROUND: Inherited arrhythmogenic disorders are a group of genetically determined diseases characterised by ventricular tachyarrhythmias sometimes leading to sudden death. The molecular bases of these disorders are mutations in genes coding for various cardiac ion channels. The most common cardiac ion channel disease is the long QT syndrome. This syndrome is rare, but probably more common in Norway than previously expected. We have recently started genetic testing for cardiac ion channel disorders at Rikshospitalet University Hospital in Oslo. This review describes the current understanding of the etiology, prognosis and management of cardiac ion channel disorders, based on literature and our own clinical experience. INTERPRETATION: Cardiac ion channel disorders may lead to sudden cardiac death. Prophylactic and life-saving therapies are available for many of these disorders. Therapy and risk stratification depend on the clinical presentation, the ECG pattern, and which gene is mutated. Genetic testing offers the opportunity to exclude individual family members as mutation carriers.

Adult↗

PubMed2005

[DNA-based diagnostics of long QT syndrome].

BACKGROUND: Long QT syndrome is characterised by inherited long QT interval on the ECG and increased risk for syncope and sudden death caused by arrhythmias. For Romano-Ward syndrome and Jervell and Lange-Nielsen syndrome DNA based diagnostics are available. MATERIALS AND METHODS: This paper is a summary of our experience with DNA-based diagnostics of LQTS since the autumn of 2003. The diagnostic analyses are performed by sequencing the exons of five genes, KCNQ1, HERG, SCN5A, minK and MiRP1. RESULTS AND INTERPRETATIONS: As of mid-January 2005, 56 probands with long QT syndrome have been referred for genetic testing. We have identified an underlying mutation in 64% of the patients. Mutations in the KCNQ1 gene are most frequent in Norwegian long QT syndrome patients, as 61% of the patients have their mutation in this gene. The detection of a mutation in the probands has led to genetic testing of 215 relatives; 99 out of these are heterozygous for the mutation present in the family. Heterozygous patients have been referred to a cardiologist. Of the 43 that have been referred to follow up at the department of cardiology at Rikshospitalet, 35 have started treatment with beta blockers to reduce the risk of arrhythmias. Thus, DNA-based diagnostics has clinical significance leading to prophylactic treatment of long QT syndrome patients. Compared to evaluation of ECG, which is negative in 30% of mutation carriers, the sensitivity of DNA-based diagnostics of relatives of probands with a known mutation, is close to 1.

Adult↗

PubMed2005

Identification of deletions and duplications in the low density lipoprotein receptor gene by MLPA.

BACKGROUND: Familial hypercholesterolemia (FH) is caused by mutations in the low density lipoprotein (LDL) receptor gene. In this study we have compared multiplex ligation-dependent probe amplification (MLPA) and long-range PCR to detect large deletions/duplications in the LDL receptor gene. METHOD: DNA from 431 unrelated FH patients without mutations in the LDL receptor gene detectable by DNA sequencing and who had total serum cholesterol levels above 10.0 mmol/l, was subjected to analyses by MLPA and by five long-range PCRs. RESULT: Eleven deletions and two duplications were detected by MLPA. Six of the deletions and one of the duplications were also detected by long-range PCR. A total of 44 of the 431 (10.2%) FH patients possessed a deletion or a duplication. CONCLUSION: MLPA has a higher sensitivity than five long-range PCRs to detect large deletions/duplications in the LDL receptor gene. Even though the direct cost of MLPA is twice that of five long-range PCRs, it has replaced long-range PCR for routine diagnostics in our laboratory because of the higher sensitivity and the 30-50% reduction in hands-on time.

Adult↗

PubMed2005

A daily glass of red wine: does it affect markers of inflammation?

AIMS: Epidemiological studies have shown that moderate consumption of alcohol is associated with a decreased risk of developing cardiovascular disease, but the causal mechanisms are only partly understood. As inflammation is an important process in the progression of atherosclerosis, we hypothesized that the protective effect of red wine is partly mediated through a reduction in inflammation. METHODS: We conducted a randomized controlled crossover trial to study the effect of red wine on the levels of the inflammatory markers serum C-reactive protein (CRP) and plasma fibrinogen in healthy, non-smoking individuals. The subjects were randomized to drink one glass of red wine (150 ml, 15 g alcohol) every day ('wine period') or to undergo a period of total abstention from alcohol ('abstention period'). After 3 weeks they switched intervention group. Eighty-seven volunteers completed the study (mean age 50 years). RESULTS: Red wine did not reduce CRP levels and only marginally reduced fibrinogen levels compared with a similar period without alcohol. CONCLUSIONS: Consumption of 150 ml of red wine slightly reduced fibrinogen levels but did not reduce CRP levels.

Alcohol Drinking↗

PubMed2003

Sitosterolemia: a gateway to new knowledge about cholesterol metabolism.

Sitosterolemia (OMIM 210250) is a rare, autosomal recessive lipid disorder initially described almost 30 years ago. The disease is characterized by elevated plasma levels of plant sterols due to increased intestinal absorption and reduced biliary secretion of neutral sterols. Patients with sitosterolemia are frequently hypercholesterolemic, and develop xanthomas and premature coronary heart disease (CHD). Hemolysis, arthralgias and arthritis are also frequently associated with the disorder. Recently, sitosterolemia has been revealed to be due to mutations in either of the two ATP-binding cassette (ABC) half-transporters. ABCG5 or ABCG8. These two genes are expressed almost exclusively in the liver and intestine, and are co-regulated by the nuclear hormone receptor, liver X receptor (LXR). Genetically modified mice, which express either high levels or no ABCG5 and ABCG8 have been developed. Analyses of these mice confirm that these two transporters play key roles in regulating the absorption of dietary and biliary sterols, and in mediating the excretion of neutral sterols from the liver to the bile. The elucidation of the gene defects responsible for sitosterolemia provides potential therapeutic targets for the treatment of hyperlipidemias in the general population.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2002

Human umbilical vein endothelial cells express complement receptor 1 (CD35) and complement receptor 4 (CD11c/CD18) in vitro.

We have examined complement receptors on human umbilical vein endothelial cells (HUVEC) and found that they express complement receptor 1 (CR1, CD35) and complement receptor 4 (CR4, CD11c/CD18), but not complement receptor 3 (CR3, CD11b/CD18). Binding of monoclonal antibodies against CR1 (CD35) and CR4 (CD11c/CD18) to HUVEC was demonstrated by flow cytometry. The presence of the corresponding mRNAs was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing of the amplified cDNA fragments. When HUVEC were treated with inflammatory mediators, chemotactic agents or the secretagogue phorbol-12-myristate-13-acetate (PMA), no change in reactivity to CR1 (CD35) or CR4 (CD11c/CD18) monoclonal antibodies was detected on the surface of the cells compared with untreated cells. The presence of CR1 (CD35) and CR4 (CD1c/CD18) on HUVEC indicates that endothelial cells (EC) have the potential to bind C3b and iC3b, respectively, which both mediate biological effects in the course of complement activation.

Antibodies, Monoclonal↗