| 1. |
- Chernogubova, Ekaterina, et al.
(författare)
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Common and Low-Frequency Genetic Variants in the PCSK9 Locus Influence Circulating PCSK9 Levels
- 2012
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Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology. - 1079-5642 .- 1524-4636. ; 32:6, s. 1526-1534
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Tidskriftsartikel (refereegranskat)abstract
- Objective- Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating protein that influences plasma low-density lipoprotein concentration and susceptibility to coronary heart disease. Circulating PCSK9 levels show considerable interindividual differences, but the factors responsible for this variation are largely unknown. Methods and Results- We analyzed circulating PCSK9 levels in 4 cohorts of healthy, middle-aged Swedes (n=5722) and found that PCSK9 levels varied over approximate to 50-fold range, showed a positive relationship with plasma low-density lipoprotein-cholesterol concentration, and were associated with plasma triglyceride, fibrinogen, insulin, and glucose concentrations. A genome-wide association study conducted in 2 cohorts (n=1215) failed to uncover common genetic variants robustly associated with variation in circulating PCSK9 level. As expected, the minor allele of the PCSK9 R46L variant was in all cohorts associated with reduced PCSK9 levels and decreased plasma low-density lipoprotein-cholesterol concentrations, but no relationship was observed with the plasma triglyceride concentration. Further mapping of the PCSK9 locus revealed a common polymorphism (rs2479415, minor allele frequency 43.9%), located approximate to 6 kb upstream from PCSK9, which is independently associated with increased circulating PCSK9 levels. Conclusion- Common and low-frequency genetic variants in the PCSK9 locus influence the pronounced interindividual variation in circulating PCSK9 levels in healthy, middle-aged white (predominantly Swedish) subjects.
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| 2. |
- Mahdessian, Hovsep
(författare)
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Genetic and metabolic studies of APOE, PCSK9, TM6SF2 and PNPLA3
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lipids – including fatty acids (FAs), triglycerides (TGs), cholesterol and cholesterol esters (CEs) – are hydrophobic molecules involved in several important structural and mechanistic processes. Lipids are found in cellular membranes, give posture and stability to the cell, are actively participating in cellular signaling, and act as mediators in several important biological pathways. Lipids, transported between organs through lipoprotein particles, act as an important energy source. Lipids are either directly utilized in metabolic processes or, if in excess, stored in depots in the cell cytosol. However, imbalances in lipoprotein transport or cellular metabolism of the lipids may give rise to adverse cellular effects leading to metabolic disorders and cardiovascular disease (CVD). The main underlying causes for these imbalances are of dietary, environmental and genetic nature. The focus of this thesis is on the genetic causes of dyslipidemia and the roles of the proprotein convertase subtilisin/kexin type 9 (PCSK9), apolipoprotein (APO) E, transmembrane 6 superfamily member 2 (TM6SF2) and patatin-like phospholipase domain-containing protein 3 (PNPLA3) proteins. PCSK9 is a circulating protein that influences plasma low-density lipoprotein cholesterol (LDL-C) concentration and susceptibility to cardiovascular disease. Circulating PCSK9 levels show considerable inter-individual variation, but the factors responsible for this variability are largely unknown. We analyzed circulating PCSK9 levels in 4 cohorts of healthy, middle-aged Swedes and found that PCSK9 levels varied over ~50-fold range and showed a positive relationship with plasma LDL-C concentration. Mapping of the PCSK9 locus revealed a common polymorphism, (rs2479415, minor allele frequency (MAF) 43.9%), located ~6 kb upstream from PCSK9, which was independently associated with increased circulating PCSK9 levels. It is generally assumed that the APOE concentration, in addition to the APOE ε2/ε3/ε4 genotype, influences plasma lipoprotein levels, but a functional genetic variant influencing the plasma APOE concentration has not been identified. In a genome-wide association (GWA) study, we observed that the APOE locus was the only genetic locus showing robust associations with the plasma APOE concentration. Fine-mapping of the APOE locus showed that rs769446 (-427T/C) in the APOE promoter is independently associated with the plasma APOE concentration. The minor allele of rs769446 is associated with increased APOE mRNA levels (p= 0.015) as analyzed in 199 human liver samples. Transient transfection studies and electrophoretic mobility shift assays in human hepatoma HepG2 cells corroborated the role of rs769446 in transcriptional regulation of APOE. TM6SF2, a gene with unknown function, encodes a protein of 351 amino acids with 7-10 predicted transmembrane domains. It is located on chromosome 19-12, a locus associated to the plasma TG concentration and hepatic lipid content. Gene expression studies in human liver samples demonstrated that TM6SF2 was the putative causal gene for this association. Subcellular localization studies showed that TM6SF2 is localized in the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment of human liver cells. Functional studies evaluating the secretion of TG-rich lipoproteins (TRLs) and lipid droplet (LD) content in Huh7 and HepG2 cells showed that TM6SF2 inhibition was associated with reduced secretion of TRLs and increased cellular triglyceride concentration and LD content, whereas TM6SF2 overexpression lead to reduced liver cell steatosis. The PNPLA3 gene variant I148M is an important marker of human non-alcoholic fatty liver disease (NAFLD), but the physiological function of PNPLA3 in liver fat metabolism remains unclear. We therefore analyzed PNPLA mRNA levels in human and mouse tissues and evaluated the effect of small interfering RNA (siRNA) silencing of PNPLA3 on TG metabolism in human Huh7 and HepG2 hepatoma cells. Although PNPLA3 had the highest expression level of all PNPLA family members in 91 human liver samples, PNPLA3 silencing in Huh7 and HepG2 cells was not associated with changes in TRL secretion, cellular triglyceride content and the rate of triglyceride synthesis.
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| 3. |
- Nastase Mannila, Maria, et al.
(författare)
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Identification of a Functional Apolipoprotein E Promoter Polymorphism Regulating Plasma Apolipoprotein E Concentration
- 2013
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Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology. - 1079-5642 .- 1524-4636. ; 33:5, s. 1063-1069
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE:There is compelling evidence that the plasma apolipoprotein E (APOE) concentration, in addition to the APOE ε2/ε3/ε4 genotype, influences plasma lipoprotein levels, but the functional genetic variants influencing the plasma APOE concentration have not been identified.APPROACH AND RESULTSGenome-wide association studies in 2 cohorts of healthy, middle-aged subjects identified the APOE locus as the only genetic locus showing robust associations with the plasma APOE concentration. Fine-mapping of the APOE locus confirmed that the rs7412 ε2-allele is the primary genetic variant responsible for the relationship with plasma APOE concentration. Further mapping of the APOE locus uncovered that rs769446 (-427T/C) in the APOE promoter is independently associated with the plasma APOE concentration. Expression studies in 199 human liver samples demonstrated that the rs769446 C-allele is associated with increased APOE mRNA levels (P=0.015). Transient transfection studies and electrophoretic mobility shift assays in human hepatoma HepG2 cells corroborated the role of rs769446 in transcriptional regulation of APOE. However, no relationships were found between rs769446 genotype and plasma lipoprotein levels in 2 cohorts (n=1648 and n=1039) of healthy middle-aged carriers of the APOE ε3/ε3 genotype.CONCLUSIONS:rs769446 is a functional polymorphism involved in the regulation of the plasma APOE concentration.
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| 4. |
- Ståhl, Patrik L., et al.
(författare)
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Translational Database Selection and Multiplexed Sequence Capture for Up Front Filtering of Reliable Breast Cancer Biomarker Candidates
- 2011
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:6, s. e20794-
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Tidskriftsartikel (refereegranskat)abstract
- Biomarker identification is of utmost importance for the development of novel diagnostics and therapeutics. Here we make use of a translational database selection strategy, utilizing data from the Human Protein Atlas (HPA) on differentially expressed protein patterns in healthy and breast cancer tissues as a means to filter out potential biomarkers for underlying genetic causatives of the disease. DNA was isolated from ten breast cancer biopsies, and the protein coding and flanking non-coding genomic regions corresponding to the selected proteins were extracted in a multiplexed format from the samples using a single DNA sequence capture array. Deep sequencing revealed an even enrichment of the multiplexed samples and a great variation of genetic alterations in the tumors of the sampled individuals. Benefiting from the upstream filtering method, the final set of biomarker candidates could be completely verified through bidirectional Sanger sequencing, revealing a 40 percent false positive rate despite high read coverage. Of the variants encountered in translated regions, nine novel non-synonymous variations were identified and verified, two of which were present in more than one of the ten tumor samples.
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