Rapid diagnosis of familial defective apolipoprotein B-100 by Amplification Refractory Mutation System

We report a method for the diagnosis of familial defective apolipoprotein (apo) B-100, using the Amplification Refractory Mutation System (ARMS) and either whole blood or extracted DNA in the polymerase chain reaction. Normal and mutant alleles are identified by using two allele-specific oligonucleotide primers, each with the same common primer, to amplify a 187-bp fragment of the apo B-100 gene. Fragment amplification occurs only when the allele-specific primer matches the nucleotide sequence of the template DNA. The amplification product is detected by agarose gel electrophoresis, followed by staining with ethidium bromide. The technique is simple, reliable, and robust. It avoids the use of radiation or hybridization with allele-specific oligonucleotide probes, and is well suited for use in the routine clinical chemistry department.     

采用ARMS分析口腔脱落细胞ApoE基因型

以口腔脱落细胞ＤＮＡ为模板,采用扩增不应突变系统（ＡＲＭＳ）,设计了等位基因４个特异性寡核苷酸引物和一个公共引物,分析了７４例个体ＡｐｏＥ３个常见共显性等位基因∈２、∈３和∈４。这一系统扩增出１８１ｂｐ和３１９ｂｐ两种ＡｐｏＥ基因顺序,当携某一等位基因的模板ＤＮＡ与相应的等位基因特异性寡核着酸引物及公共引物反应时,扩增出相应的基因顺序。省略了限制性核酸内切酶的消化或等位基因特异性寡核苷酸探针的杂交。方法简单、可靠、非放射性。     

Quantitative assessment of apolipoprotein E genotypes by image analysis of PCR-RFLP fragments

Apolipoprotein E (APOE) genotyping usually involves polymerase chain reaction (PCR) and assessment of restriction fragment length polymorphism (RFLP) by gel electrophoresis. We made determination of HhaI restriction endonuclease digestive patterns more objective and improved diagnostic accuracy with a quantitative approach using sensitive DNA stain (SYBR Green) and image analysis of gel patterns. For distinguishing true and partially-digested restriction fragments, band ratios were calculated for the staining intensity of gel patterns from 116 sample runs of 63 human blood specimens. Each of these specimens was independently genotyped for APOE by at least two (and most of them by three) different PCR-RFLP methods. Based on the distribution of band ratios, decision levels were established and used for developing a program for computer-aided interpretation of APOE genotypes (Microsoft Excel software). Appropriateness of the decision levels for band ratios was validated by APOE genotyping of additional 61 specimens. The approach described here is applicable to a variety of other molecular diagnostic techniques that are based on PCR-RFLP or sequence-specific signal amplifications.     

Detection of human apolipoprotein E genotypes by DNA biosensors coupled with PCR

Apolipoprotein E (apoE) is an important constituent of several plasma lipoproteins and has been associated with the risk of developing cardiovascular diseases and in familiar type III hyperlipoproteinemia. We developed new procedures for the detection of apolipoprotein E polymorphism in human blood based on coupling DNA electrochemical or piezoelectric sensors with polymerase chain reaction (PCR). The electrochemical sensor was obtained by immobilizing single-stranded oligonucleotides onto graphite screen-printed electrodes by adsorption at controlled potential. The hybridization reaction that occurred on the electrode surface was evidenced by chronopotentiometric stripping analysis-using daunomycin as indicator. In the piezoelectric sensor, biotinylated 23-mer probes were immobilized on the streptavidin-coated gold surface of a quartz crystal; streptavidin was covalently bound to the thiol/dextran modified gold surface. The hybridization of the immobilized probes with complementary and mismatched DNA was investigated. With the use of two different probes, it was possible to investigate both positions in which apoE polymorphism takes place and consequently, to distinguish the different genotypes. The procedure was validated with both kinds of biosensor with a reference method based on polyacrilamide gel electrophoresis.     

Detection of human apolipoprotein E genotypes by DNA electrochemical biosensor coupled with PCR

BACKGROUND: Apolipoprotein E (apoE) is an important constituent of several plasma lipoproteins, mainly VLDL, HDL, and chylomicrons. It is involved in the redistribution of lipids in the liver and is implicated in growth and repair of injured neurons in the nervous system. apoE has also been associated with the risk of developing cardiovascular diseases and in familial type III hyperlipoproteinemia. METHODS: We developed a new procedure for detecting genetic polymorphisms of apoE in human blood samples. The procedure is based on coupling of DNA electrochemical sensors with PCR-amplified DNA extracted from human blood. The DNA electrochemical sensor incorporated single-stranded oligonucleotides immobilized on graphite screen-printed electrodes (SPEs) by adsorption at controlled potential. The hybridization reaction on the electrode surface was monitored by chronopotentiometric stripping analysis (PSA), using daunomycin as indicator. RESULTS: With use of two different probes, it was possible to investigate both DNA positions in which the apoE polymorphism takes place and thus to distinguish different genotypes. Real samples containing only complementary sequences gave a good increase in the area of the daunomycin peak ( approximately 600 ms) compared with the peak observed with the buffer. Samples containing 50% complementary sequences gave a much lower increase, and samples containing only mismatch sequences gave a decrease in the daunomycin area. The procedure was validated by comparison with a method based on polyacrylamide gel electrophoresis. CONCLUSION: The coupling of DNA electrochemical sensors with PCR allowed quick discrimination between the different genotypes of apoE.     

建立RT-ARMS-qPCR系统定量测定含A1555G突变的线粒体DNA拷贝数

目的建立RT-ARMS-qPCR（real time-amplification refractory mutation system-quantitative PCR）系统定量测定含线粒体DNA（mitochondrial DNA,mtDNA）A1555G位点突变的片段的拷贝数,为阐明线粒体耳聋临床表型多样性的分子生物学基础奠定基础。方法根据ARMS的基本原理设计引物,在引物3’端插入错配碱基AC建立RT-ARMS-qPCR系统,优化定量PCR体系,并进行方法学评价。经PCR分别扩增相应突变型和野生型的片段,将其克隆到pGEM-T Easy载体上,构建质粒标准品;同时对含突变型和/或野生型mtDNA 1555位点的片段的拷贝数进行定量检测。结果RT-ARMS-qPCR系统用于检测含mtDNA 1555位点的片段,线性检测范围为102-108拷贝数/μl,检测灵敏度为1×102拷贝数/μl,其批内变异系数（CV）为1.34%,批间CV为1.96%,重复性好;突变型和野生型引物分别只扩增相应片段,特异性好。结论RT-ARMS-qPCR系统适合于定量检测含mtDNA A1555G点突变的线粒体DNA片段,结果稳定、准确,有助于阐明线粒体耳聋严重程度的分子基础。     

聚合酶链反应限制性内切酶消化法检测人载脂蛋白E基因多态性

采用聚合酶链反应与限制性核酸内切酶ＨｈａＩ消化相结合的方法建立一种简便。实用的人载脂蛋白Ｅ基因多态性检测方法。ＰＣＲ扩增含有编码１１２和１５８位氨基酸的基因片断序列，产物经ＨｈａⅠ酶消化，经聚丙烯酸 胺凝胶电泳可见２－４条特定长度的ＤＮＡ条带易于判定ａｐｏＥ基因型。     

Factor V Leiden and apolipoprotein E genotypes in severe femoropopliteal atherosclerosis with restenosis

BACKGROUND: The role of factor V (Leiden) mutation, thrombophilia, and apolipoprotein E (apoE) alleles in the pathogenesis of accelerated atherosclerosis and restenosis was studied in patients requiring reoperation within five years after femoropopliteal angioplasty with artificial grafts. METHODS: One hundred ninety-eight consecutive patients with femoropopliteal atherosclerotic disease, reoperated for restenosis were contacted by phone and 100 of them returned for laboratory and clinical work-up. In addition to clinical evaluation and routine laboratory investigations, parameters of lipoprotein metabolism, factor V (Leiden) mutation and apolipoprotein E (apoE) allele were studied by PCR amplification of DNA and endonuclease digestion techniques. RESULTS: A significantly higher incidence of factor V (Leiden) mutation was found in patients with atherosclerosis and restenosis, compared to 445 healthy blood donors (13/200, 6.5% vs. 34/890, 3.8%, p=0.0379). Distribution of the alleles of the apolipoprotein E (apoE) gene was different, when the patients were compared to 372 controls; however, the difference only approached the level of statistical significance (25/200, 12.5% vs. 56/744, 7.5%, p=0.0515). Comparing the two groups, the number of epsilon4 allele carriers was significantly higher among patients with restenosis (25/100, 25% vs. 53/272, 14%, p=0.0147). CONCLUSION: Factor V (Leiden) mutation may influence the progression of atherosclerosis and the development of restenosis after revascularization in patients with accelerated femoropopliteal atherosclerosis. Further investigation is needed whether long-term anticoagulation has an impact or not on the course of disease in such cases. ApoE epsilon4 allele should be screened in patients with femoropopliteal atherosclerosis, because it indicates a faster progression of atherosclerosis and may predict restenosis after revascularization procedure.     

Isolation of human apolipoprotein E by chromatofocusing

Human prolipoprotein E is implicated in the transport of serum cholesterol and the binding of lipoproteins to cell receptors. Further investigations on this apolipoprotein would be facilitated by improved purification methods. We prepared human apo E by the combination of high performance gel filtration and chromatofocusing from serum very low density lipoproteins. Chromatofocusing was performed with a pH gradient from 7 to 4. Apo E contained all isoforms, but was homogeneous in SDS-polyacrylamide gel electrophoresis and in double immunodiffusion against a monospecific antiserum. The reported purification method allows a rapid and simple preparation of large amounts of apo E.     

Association between enzymatic and non-enzymatic antioxidant defense mechanism with apolipoprotein E genotypes in Alzheimer disease

ObjectiveThere are evidence suggesting that APOE-?4 allele play an important role in the pathogenesis of Alzheimer's disease (AD) by reducing peripheral levels and activities of a broad spectrum of nonenzymatic and enzymatic antioxidants systems. However, the link between APOE genotype, oxidative stress, and AD has yet to be established. In this study we examined whether antioxidant defense mechanism exacerbates the risk of AD in individual carrying APOE-?4 allele in a population from Tehran, Iran.MethodWe determined the enzymatic activities of the erythrocyte Cu–Zn superoxide dismutase (Cu–Zn SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and serum level of total antioxidant status(TAS) in various APOE genotypes in 91 patients with AD and 91 healthy subjects as control group (age and sex-matched).ResultThe results showed that the TAS level and the activities of enzymatic antioxidants CAT and GSH-Px were significantly lower and the SOD activity was significantly higher in AD patients compared to controls. The AD patients with APOE-?4 allele genotype had significantly lower serum TAS concentration and lower erythrocytes GSH-Px and CAT activities (p = 0.001) but significantly higher erythrocytes Cu–Zn SOD activity (p = 0.001) than the non-APOE-?4 carrier AD and the control group. In addition, the association observed between the factors involved in an antioxidant defense mechanism and APOE-?4 allele in AD increased with age of the subjects.ConclusionThese data indicate that the reduced serum level of TAS and activity of CAT, GSH-Px and increased SOD exacerbate the risk of AD in individuals carrying APOE-?4 allele. The reduced antioxidants defense in APOE–?4 allele carrier may contribute to beta-amyloidosis. This effect, however, is more pronounced in the AD patients older than 75 years of age. This suggests that a therapeutic modality should be considered for these subjects.     

Detection of human apolipoprotein E3, E2, and E4 genotypes by an allele-specific oligonucleotide-primed polymerase chain reaction assay: development and validation.

A polymerase chain reaction (PCR) assay has been developed and validated by using allele-specific oligonucleotide (ASO) primers to specifically amplify E3, E2, and E4 polymorphic sequences of the human apolipoprotein E (apo E) genes. Degenerate ASOs containing one or two additional 3' mismatches provided greater specificity than did ASOs containing a single mid-sequence or 3' allele-specific mismatch with plasmid pEB4 or genomic DNA as template. Optimal specificity and efficiency of amplification did not correlate with primer annealing conditions, whether determined theoretically or via oligo-melting experiments. Pre-cycling denaturation times and high cycling denaturation temperatures were also required for optimal amplification, presumably because of the high G:C content (75-85%) of apo E gene sequences. Conditions permissive for amplification and discrimination with plasmid DNA did not transpose favorably to amplification from human genomic DNA from peripheral blood leukocytes; the latter required nested primer reactions. These data may be valuable in predicting PCR assay conditions for other G:C-rich sequences containing polymorphic sequence differences. The assay described is both more accurate and rapid (24 h) than previously described methods for phenotyping or genotyping human apo E from blood specimens.     

Effects of apolipoprotein E genotypes and other risk factors on the development of coronary artery disease in Southern Turkey

BACKGROUND: Apolipoprotein E (apoE) plays a major role in lipoprotein metabolism and lipid transport. Associations between apoE genotypes, coronary artery disease (CAD) and other risk factors have been described by many investigators. The aim of this study was to investigate the role of apoE gene polymorphism and other risk factors in the development of CAD in subjects whose coronary arteries were evaluated by means of coronary angiography. METHODS: The study population consisted of 199 subjects (114 male and 55 female). Of the total, 107 had CAD. The apoE gene was amplified by polymerase chain reaction (PCR) and then digested by CfoI restriction enzyme. The plasma lipid levels and other risk factors were also determined in all subjects. RESULTS: The epsilon2 and epsilon4 allele frequencies and genotypes carrying epsilon4 allele were significantly higher in CAD (+) patients. Plasma lipids except triglycerides were increased in CAD (+) cases. We found that apoE genotypes, HT, DM, male gender, age and smoking were the independent predictors of CAD. There was no association between apoE alleles and lipids. CONCLUSION: We conclude that apoE polymorphism (presence of epsilon4 allele) is associated with the development of CAD in Southern Turkey. In our study, we did not observe any effect of apoE alleles on lipid levels.     

Abnormalities in apolipoprotein and lipid levels in an HIV-infected Brazilian population under different treatment profiles: the relevance of apolipoprotein E genotypes and immunological status.

HIV infection is associated with disturbances in lipid metabolism due to a host's response mechanism and the current antiretroviral therapy. The pathological appearance and progression of atherosclerosis is dependent on the presence of injurious agents in the vascular endothelium and variations in different subsets of candidate genes. Therefore, the Hha I polymorphism in the apolipoprotein E gene was evaluated in addition to triglycerides, total cholesterol, very low-density lipoprotein (VLDL), LDL, high-density lipoprotein (HDL), and apolipoprotein (apo) Al, B and E levels in 86 Brazilian HIV-infected patients and 29 healthy controls. The allele frequency for apoE in the HIV-infected group and controls was in agreement with data on the Brazilian population. Dyslipidemia was observed in the HIV group and verified by increased levels of triglycerides, VLDL and apoE, and decreased levels of HDL and apoAl. The greatest abnormalities in these biochemical variables were shown in the HIV-infected individuals whose immune function was more compromised. The effect of the genetic variation at the APOE gene on biochemical variables was more pronounced in the HIV-infected individuals who carried the apoE2/3 genotype. The highly active anti-retroviral therapy (HAART)-receiving group presented increased levels of total cholesterol and apoE. Dyslipidemia was a predictable consequence of HIV infection and the protease inhibitors intensified the increase in apoE values.     

Effect of apolipoprotein E genotypes on incidence and development of coronary stenosis in Iranian patients with coronary artery disease

Background: Apolipoprotein (apo) E polymorphism plays a significant role in the development of coronary disease, but their involvement in coronary artery stenosis (CAS) is controversial. Therefore, the purpose of this study was to investigate the effects of this polymorphism on atherosclerosis, and severity and extent of CAS in unrelated Iranian population. Methods: DNA was isolated from 390 study participants and APOE genotypes were determined utilizing the polymerase chain reaction and restriction fragment length polymorphism. Results: The APOE‐ε4 and ‐ε2 allele frequencies were significantly higher in the CAS patients than in the control group (P<0.05). The association of Apo E polymorphism with the severity of stenosis was evaluated, which is according to the result that apolipoprotein E alleles were not significantly different when compared with the severity of stenosis (χ²=0.84, P>0.05). Conclusion: Our results suggest that APOE‐ε4 is a risk factor for stenosis but does not has any effect on the severity of this disease. J. Clin. Lab. Anal. 25:43–46, 2011. © 2011 Wiley‐Liss, Inc.