Funktionelle Charakterisierung neu identifizierter Defekte desLDL-Rezeptors und von Apolipoprotein B-100

Atherosclerosis and one of its clinical manifestations Coronary Heart Disease (CHD) are the main cause of death in western industrialized nations. In great epidemiological studies the high impact of hyperlipidemias and especially hypercholesterolemia as a major risk factor for the development of atherosclerosis and CHD have been shown. Familial hypercholesterolemia (FH) is a monogenetic autosomal dominant inherited disorder which results in early onset of atherosclerosis and a premature risk for CHD. Subjects with homozygous FH are at high risk to die from CHD in early childhood. The molecular basis for familial hypercholesterolemia are defects in the genes of the low density lipoprotein receptor (LDLR) and its main ligand Apolipoprotein B-100 (ApoB-100), as a result plasma LDL-levels are dramatically increased in affected patients. In the here presented PhD thesis a DGGE-based mutational screening study of the genes for Apo-B-100 and the LDLR have been performed with patients from the study population of the Marburger Präventions-Allianz. By this, two novel defects of ApoB-100 (ApoB-100 H3543Y) and the LDLR (LDLR W556R) have been identified. The frequency of the new ApoB-100H3543Y mutation within the examined study population was four times higher (1:223) than previously reported ApoB-100 mutations (e.g. ApoB-100R3500Q). As in all other identified ApoB-100 defects with decreased LDLR binding, a positively charged amino acid (H) in the carboxyterminal modulator element of the LDLR binding site of ApoB-100 is substituted by a neutral uncharged amino acid (Y) in ApoB-100H3543Y. Binding and uptake experiments with fluorescence labelled DiI-LDL from ApoB-100 H3543Y subjects, showed decreased binding and uptake of ApoB-100H3453 Y-LDL by the LDLR (-31 to -35%) in HeLa cells. In addition in vivo kinetic turnover experiments showed a delayed catabolism of ApoB-100H3543Y. This data clearly showed that ApoB-100H3543Y is a frequent and functionally relevant mutation within the German population. Despite the novel ApoB-100H3543Y-mutation a homozygous mutation of the LDLR (W556R) was identified for the first time worldwide in 3 years old identical male twins. The same LDLR mutation was then detected a second time in a homozygous affected 4 year old girl from an unrelated FH-family. From the phenotype there was a gender specific expression of the mutation in both FH-families (female subjects from both families had lower plasma LDL and higher HDL values). The LDLR mutation W556R affects the fifth highly conserved YWVD repeat of the six folded ß-propeller motif of the LDLR and leads to a complete cellular LDLR deficiency. Uptake and binding experiments with DiI-LDL in fibroblasts from homozygous subjects showed no detectable DiI-LDL binding and uptake (< 5%). In heterozygous W556R subjects DiI-LDL binding and uptake was decreased to -45/-49% compared to wild type fibroblasts. Experiments with confocal microscopy and western blotting identified a transport defective LDLR (class2A) with complete retention in the endoplasmic reticulum as the molecular basis of the W556R LDLR mutation. Real Time PCR- experiments to study the impact of the W556R LDLR defect on the regulation of cellular cholesterol biosynthesis, revealed increased expression of central genes for cholesterol metabolism. This increased gene expression was predominantly induced by SREBP-2 (sterol regulatory element binding protein 2) transcription factor. In addition mRNA and protein levels of SCARB1 (scavenger receptor type B 1) were increased in fibroblasts from heterozygous and homozygous W556R patients. This points out towards a non endocytotic collateral pathway for direct uptake of cholesterol from HDL and native LDL particles via SCARB1 in heterozygous and homozygous W556R patients. In contrast to SCARB1 the gene expression of cellular cholesterol transporter ABCA1 was strongly decreased in homozygous, but not heterozygous W556R fibroblasts, which seems to be dependant on the availability and amount of intracellular LXR- ligands for activation of ABCA1 gene transcription. Similar to the findings with ApoB-100H3543Y-LDL in vivo kinetic turnover experiments showed a delayed LDL catabolism in subjects with the W556R LDLR mutation. In summary ApoB-100H3543Y and LDLRW556R are frequent mutations with functional relevance. However, in case of the W556R LDLR mutation gender specific factors exists that may contribute to the phenotypic variation observed in male and female subjects with the W556RLDLR mutation. The identification of the underlying mechanisms has a high potential for the development of novel therapeutic options for treatment of homozygous familial hypercholesterolemia (FH), although molecular medicine steadily progressed within the last decades, there is still no adequate medical treatment of homozygous FH-patients until now.