Typing of the 3' hypervariable region of the apolipoprotein B gene: approaches, pitfalls, and applications.
Apolipoprotein B-100 is the principal protein component of lipoproteins with very low, intermediate, and low density. The interaction of apoB-100 with low density lipoprotein (LDL) receptors is responsible for the uptake of LDL into cells. An AT-rich hypervariable region is located adjacent to the 3' end of the apoB gene. It consists of a variable number of tandemly repeated sequences (VNTR). Two approaches were used to analyze this polymorphism. In both, the region harboring the VNTR was amplified with the polymerase chain reaction (PCR). In the first method, fluorescently labeled primers were used in the PCR reactions and products were separated in agarose gels by means of an automated fluorescent fragment analyzer. In the second method, PCR products were analyzed in denaturing polyacrylamide gels and detected with silver staining. Even in the highly sophisticated automated system, agarose gel electrophoresis did not always enable unequivocal assignment of VNTR alleles. In contrast, denaturing polyacrylamide gel electrophoresis made it possible to distinguish the 15 bp differences between the VNTR alleles in a precise and simple manner. The VNTR polymorphism was typed in 234 individuals. Among these were 136 patients with coronary artery disease and 74 healthy controls. Thirteen alleles could be distinguished. The allele containing 49 repeats (VNTR-49) was found in 9.2% of the coronary artery disease patients and in 4.7% of the controls. Thus, the VNTR-49 allele increases relative coronary risk by about twofold. It is concluded that the apoB VNTR polymorphism is a potentially useful genetic marker. Since agarose gel electrophoresis may lead to ambiguous results, we prefer typing by denaturing polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)
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Gustav Embden-Centre of Biological Chemistry, Johann Wolfgang Goethe-University, Frankfurt/Main, Germany.
Ruzicka
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Fisher
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Russ
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AP
Schneider
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Gross
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D000483:Alleles; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D003327:Coronary Disease / Q000235:genetics; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012091:Repetitive Sequences, Nucleic Acid
D001055:Apolipoproteins B; D004247:DNA