Mutationssuche in regulatorischen Sequenzen der Gene der Apolipoproteine AI, AIV und CIII mithilfe der denaturierenden Gradientengelelektrophorese
Loading...
Files
Date
relationships.isAuthorOf
Publisher
Philipps-Universität Marburg
item.page.supervisor-of-thesis
Abstract
Adipositas and high concentrations of triglycerides in human plasma apart from high LDL, cholesterol, lipoprotein(a) and low HDL concentrations and diabetes
mellitus, smoking, arterial hypertension, age and familial predisposition are known risk factors for the development of coronary artery disease (CAD). In general, adipositas leads to the release of free fatty acids leading to hypertriglyceridemia. However, some patients develop hypertriglyceridemia although suffering from only mild adipositas. The apolipoproteins AI, AIV and CIII are key players in the metabolism of triglycerides. Their regulatory elements, the promoters and enhancers, are crucial for sufficient expression. The goal of this work was to search for mutations in regulatory regions of the genes of apolipoproteins AI, AIV and CIII that were able to explain a low body mass index (BMI) and high triglyceride concentration in plasma at the same time. We designed a case control study to be able to proof a correlation of found mutations and the BMI-triglyceride-constellation. The DNA target segments have then been screened for mutations. 264 patients of the “Marburger Praeventionsallianz” were assigned to a case group (BMI under 25kg/m2 and triglycerides above 150mg/dl, 134 patients) and a control group (BMI under 25kg/m2 and triglycerides under 150mg/dl, 130 patients). The DNA of all patients was isolated from EDTA blood and the target sequences amplificated by polymerase chain reaction.
A denaturing gradient gel electrophoresis (DGGE) followed to show differences in electrophoretic mobility. At present, the DGGE is described as the most effective method for screening a population for specific mutations. Its sensitivity reaches about 95 per cent and it is possible to examine a rather big number of samples at a time. Samples showing a mobility shift were sequenced. The segments examined in this study are organized in a joint gene cluster on the long arm of chromosome 11. The ApoAI promoter sequence in this work consisted of 229bp reaching from nucleotide 116708439 to 116708667 (position -329 to -101 with regard to the transcription start point). The ApoAIV promoter sequence covered nucleotides 116693995 to 11669444231 (-439 bis -3, 437bp). The ApoCIII promoter sequence spanned 226bp (nucleotides 116700604 to116700351, -251 bis -26). Different mutations are known in all regions. However, their clinical significance has not been clarified yet. Only the ApoCIII enhancer gene (340bp, nucleotides 116700210 to 116699819, -800 to -471) has certain polymorphisms with known effects that were examined in different studies. In this work we found one mutation in the gene of apolipoprotein AIV (-63C-->G), that was validated by the 1000genome project and published as rs5090 by now. The high sensitivity of the DGGE that has been described in different studies could not be supported by our study. Although a frequency of 4 to 6% for the C-allel of the rs5090 SNP has been described only one out of 264 samples showed a variation. No more polymorphisms could be found in the study population. More studies have to be carried out to show the mutation’s possible effect on triglyceride metabolism, for example, in an expression model.
Review
Metadata
Contributors
Supervisor:
Dates
Created: 2013Issued: 2013-11-06Updated: 2013-11-06
Faculty
Medizin
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
ApolipoproteinLipoproteinstoffwechselapolipoproteindenaturing gradient gel electrophoresislipoprotein metabolismpolymerase chain reactiontriglyceridesDenaturierende Gradientengelelektrophorese
DFG-subjects
TriglyceridePolymerase-KettenreaktionKoronare Herzkrankheit
DDC-Numbers
610
show more
Stünckel, Malte Kristof (1044455896): Mutationssuche in regulatorischen Sequenzen der Gene der Apolipoproteine AI, AIV und CIII mithilfe der denaturierenden Gradientengelelektrophorese. : Philipps-Universität Marburg 2013-11-06. DOI: https://doi.org/10.17192/z2013.0637.
License
This item has been published with the following license: In Copyright