Abstract
BackgroundAn apolipoprotein C1 gene promoter polymorphism (CGTT insertion at position -317) is associated with familial dysbetalipoprotemia, cardiovascular diseases, and Alzheimer's disease. Restriction site polymorphism (RSP) assays were previously established to detect this polymorphism. In this study, we introduce an improved RSP assay to detect this polymorphism.MethodsThis method included newly designed primers and only one round of PCR amplification which yields one short and specific APOC1 fragment followed by HpaI digestion. Briefly, It consists of three steps: 1) one round of PCR amplification of DNA sample, 2) HpaI enzyme digestion of PCR products, and 3) electrophoresis on an agarose gel to visualize the genotypes. This improved RSP method was applied to genotype 92 human samples collected from The Johns Hopkins Hospital.ResultsThe observed allele frequencies for H1 and H2 from 92 genotyped human subjects were 0.707 and 0.293 respectively. The H2 allele frequency in the black subjects (0.350) was significantly (p = 0.024) higher than that in the white subjects (0.177). This method was more economical and convenient than the methods previously reported to detect this mutation in the APOC1 gene.ConclusionsThis assay will be readily applied to screen large sample sizes for population studies in a simple and cost effective way.
Highlights
An apolipoprotein C1 gene promoter polymorphism (CGTT insertion at position 317) is associated with familial dysbetalipoprotemia, cardiovascular diseases, and Alzheimer's disease
Our new protocol consists of only three steps: one PCR amplification, HpaI digestion and agarose gel electrophoresis
That the H1-allele frequency is 0.707 and the H2-allele frequency is 0.293, are in good agreement with the findings of Xu et al (1999)[4] and Hubacek et al [2]. This new Restriction site polymorphism (RSP) assay should facilitate the screening of this mutation in large populations and contribute to the understanding of the molecular mechanism underlying the association of this mutation with a number of diseased conditions
Summary
An apolipoprotein C1 gene promoter polymorphism (CGTT insertion at position 317) is associated with familial dysbetalipoprotemia, cardiovascular diseases, and Alzheimer's disease. Restriction site polymorphism (RSP) assays were previously established to detect this polymorphism. We introduce an improved RSP assay to detect this polymorphism. APOC1 is a constituent of triglyceride-rich lipoproteins and HDL. The presence of the Hpa I restriction site (CGTT insertion at position -317) in the APOC1 promoter has a significant effect on APOC1 transcription [3,4]. In one population-based genetic association study, this polymorphism was associated with lipoprotein metabolism disorder [5]. The association of APOC1 gene polymorphism with both late-onset Alzheimer's disease [6] and age-associated memory impairment [7] has been reported. Protection from obesity and insulin resistance has been found in mice over expressing human APOC1 [8]
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