Chen Chia-Hsiang
Department of Psychiatry, Chang Gung Memorial Hospital-Linkou, Taoyuan, Taiwan.
Department and Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan.
PLoS One. 2016 Apr 14;11(4):e0153593. doi: 10.1371/journal.pone.0153593. eCollection 2016.
Genetic polymorphisms of apolipoprotein E (APOE) are associated with various health conditions and diseases, such as Alzheimer's disease, cardiovascular diseases, type 2 diabetes, etc. Hence, genotyping of APOE has broad applications in biomedical research and clinical settings, particularly in the era of precision medicine. The study aimed to develop a convenient and accurate method with flexible throughput to genotype the APOE polymorphisms. A melting curve-based allele-specific PCR method was developed to genotype two single nucleotide polymorphisms (SNPs) of APOE, i.e. rs429358 at codon 112 and rs7412 at codon 158. These two SNPs determine the genotype of APOE2, E3, and E4. PCR-based Sanger sequencing was used as the reference method for APOE genotyping. A 100% concordance rate was obtained in 300 subjects between the melting curve-based allele-specific PCR method and the Sanger sequencing method. This method was applied to a genetic association analysis of APOE and schizophrenia consisting of 711 patients with schizophrenia and 665 control subjects from Taiwan. However, no significant differences in the allele and genotype frequencies were detected between these two groups. Further experiments showed that DNA dissolved from blood collected on Guthrie filter paper and total blood cell lysate without DNA extraction can be used in the melting curve-based allele-specific PCR method. Thus, we suggest that this is a fast, accurate and robust APOE genotyping method with a flexible throughput and suitable for DNA template from different preparations. This convenient method shall meet the different needs of various research and clinical laboratories.
载脂蛋白E（APOE）的基因多态性与多种健康状况和疾病相关，如阿尔茨海默病、心血管疾病、2型糖尿病等。因此，APOE基因分型在生物医学研究和临床环境中具有广泛应用，尤其是在精准医学时代。本研究旨在开发一种便捷、准确且通量灵活的方法来对APOE基因多态性进行基因分型。我们开发了一种基于熔解曲线的等位基因特异性PCR方法，用于对APOE的两个单核苷酸多态性（SNP）进行基因分型，即第112位密码子的rs429358和第158位密码子的rs7412。这两个SNP决定了APOE2、E3和E4的基因型。基于PCR的桑格测序法用作APOE基因分型的参考方法。在300名受试者中，基于熔解曲线的等位基因特异性PCR方法与桑格测序法的一致性率达到100%。该方法应用于一项由711名台湾精神分裂症患者和665名对照受试者组成的APOE与精神分裂症的遗传关联分析。然而，在这两组之间未检测到等位基因和基因型频率的显著差异。进一步的实验表明，从Guthrie滤纸上采集的血液中溶解的DNA以及未经DNA提取的全血细胞裂解物可用于基于熔解曲线的等位基因特异性PCR方法。因此，我们认为这是一种快速、准确且稳健的APOE基因分型方法，通量灵活，适用于不同制备方式的DNA模板。这种便捷的方法应能满足各种研究和临床实验室的不同需求。
