Genetic and metabolic studies of APOE, PCSK9, TM6SF2 and PNPLA3

Sammanfattning: 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 ?2/?3/?4 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.