Genetic and functional studies of MTTP and PLIN2 in relation to metabolic and cardiac dysfunction

Sammanfattning: Lipids including triglycerides, cholesterol, cholesterol esters and fatty acids are important sources for energy production, act as building blocks for intracellular compartments, are involved in numerous biological pathways and may act as signal molecules. Lipids are transported in blood as lipoproteins between organs, where they are immediately used in cellular processes or stored as cytosolic lipid droplets. The lipoprotein and intracellular lipid metabolism need to be under tight control to avoid adverse cellular events. Dyslipidaemia and ectopic lipid accumulation are associated with metabolic disorders such as obesity, insulin resistance, type 2 diabetes and a spectrum of cardiovascular diseases. Microsomal triglyceride transfer protein (MTTP) and perilipin 2 (PLIN2) are two main players in lipid metabolism. MTTP is crucial for the assembly of apolipoprotein B containing lipoproteins, which are mainly secreted by the liver as very low density lipoprotein, and the intestine as chylomicrons. PLIN2 is the main lipid droplet associated protein in non-adipose tissue and is important for the management of intracellular lipid droplets. This thesis investigates genetic variations in MTTP and PLIN2 and their relation to lipid metabolism and metabolic disorders. A common variant of MTTP, comprising two promoter polymorphisms (rs1800591G>T and rs1800804T>C), and a missense polymorphism (rs3816873/ Ile128Thr), results in decreased expression of MTTP and a less stable protein. The decreased expression, associated with the minor alleles, is mediated by allele-specific binding of nuclear factors to the rs1800804T>C polymorphism. As shown by association studies of cardiovascular diseases, and patients with suspected coronary artery disease undergoing extensively characterisation of their cardiac function, the minor allele of rs1800804T>C confers increased risk for cardiovascular diseases and negatively influences the cardiac function. Decreased cardiac MTTP may impair transport of surplus lipids from heart that may cause lipotoxicity and heart failure. Two patients suffering from Abetalipoproteinaemia were investigated and two novel mutations were identified. Abetalipoproteinaemia is a rare recessive monogenic disease caused by lack-of-function mutations in MTTP. The first proband is homozygous for a missense mutation in exon 13 of MTTP, p.Pro552Leu (NM_000253.2:c.1655C>T). Amino acid 552 is present in an ?-helix domain predicted to bind to protein disulfide isomerase required for functional MTTP. There are three other missense mutations reported in exon 13 of MTTP that cause Abetalipoproteinaemia. The four missense mutations are associated with different severity of disease, and structural analysis of MTTP shows that the position of the mutations may reflect different functional domains of MTTP. The second proband was found to be homozygous for a duplication in the splice junction of intron 17, NM_000253.2:c.2342+2dup. The mother is a heterozygous carrier of this mutation, while no aberrations could be found in MTTP of the father. MTTP is located at 4q22-24, and analysis of microsatellite markers across the complete chromosome 4 showed that the proband has inherited two copies of chromosome 4 from only the mother, a condition called uniparental disomy. As a result of crossing over events, the interstitial region comprising MTTP, is inherited from only one of the mother’s chromosome 4, while the telomeric regions origins from both of the two maternal chromosomes. This explains why the proband is homozygous for the mutation while the mother is heterozygous. Genetic analysis of PLIN2 identified a missense polymorphism in exon 6, rs35568725 (Ser251Pro). The minor Pro251 allele is associated with decreased plasma triglyceride and very low density lipoprotein concentrations. Functional studies showed that the minor Pro251 allele disrupts an ?-helix, is evolutionarily conserved, increases intracellular lipid accumulation and reduces lipolysis. This is the first time a genetic variant of PLIN2 has been shown to influence the lipid metabolism in humans. The Pro251 variant alters the function of PLIN2 and results in more stable lipid droplets, and appears to mediate an increased capacity to store intracellular lipids. The increased understanding of lipid metabolism in the past decade highlights that it is not the amount or concentration of lipid that is the most important issue for maintaining lipid homeostasis. In order to understand the underlying pathophysiology of metabolic disorders we need to address questions related to where, how and why different kinds of lipids are stored and used.