Tanaka Chihiro, Kamide Kei, Takiuchi Shin, Miwa Yoshikazu, Yoshii Masayoshi, Kawano Yuhei, Miyata Toshiyuki
Research Institute, National Cardiovascular Center, Suita, Osaka, Japan.
Hypertens Res. 2003 Apr;26(4):301-6. doi: 10.1291/hypres.26.301.
Insertion/deletion (I/D) polymorphisms in intron 16 of the angiotensin converting enzyme gene (ACE) are associated with the plasma angiotensin converting enzyme (ACE) levels, and individuals with the DD allele have been reported to be more susceptible to cardiovascular disease than those without. The conventional genotyping method for the screening of I/D polymorphisms, which involves polymerase chain reaction (PCR)-gel electrophoresis, is laborious and time-consuming. In this study, we assessed the use of TaqMan-PCR genotyping for the screening of I/D polymorphisms as a replacement for the conventional method. We genotyped seven single nucleotide polymorphisms (SNPs) in linkage disequilibrium (LD) with the I/D polymorphisms, and calculated the LD coefficients of the I/D polymorphisms. We found that three polymorphisms, rs4331, rs4334 and rs4341, exhibited the highest LD coefficients (D' = 1.000; r2 = 0.967) and that the genotyping of rs4341 by the TaqMan-PCR method yielded the best discrimination among the different genotypes. Genotyping of 511 samples took only 2 h and the amount of DNA required for each test was only 6 ng by the TaqMan-PCR method using rs4341. In the course of this study, we identified a novel additional polymorphism (a deletion of six amino acids) in exon 13, near rs4316. The deletion allele encoded the testicular ACE, but not the plasma ACE. We concluded that genotyping of the rs4341 ACE polymorphism by the TaqMan-PCR method is a fast and convenient alternative method for direct I/D genotyping. We also concluded that testicular ACE may manifest a deletion of six amino acids that may result in deleterious function of this enzyme.
血管紧张素转换酶基因（ACE）第16内含子中的插入/缺失（I/D）多态性与血浆血管紧张素转换酶（ACE）水平相关，据报道，携带DD等位基因的个体比未携带的个体更容易患心血管疾病。用于筛选I/D多态性的传统基因分型方法涉及聚合酶链反应（PCR）-凝胶电泳，既费力又耗时。在本研究中，我们评估了使用TaqMan-PCR基因分型来筛选I/D多态性以替代传统方法。我们对与I/D多态性处于连锁不平衡（LD）状态的7个单核苷酸多态性（SNP）进行了基因分型，并计算了I/D多态性的LD系数。我们发现，rs4331、rs4334和rs4341这三个多态性表现出最高的LD系数（D' = 1.000；r2 = 0.967），并且通过TaqMan-PCR方法对rs4341进行基因分型在不同基因型之间产生了最佳区分效果。使用rs4341的TaqMan-PCR方法对511个样本进行基因分型仅需2小时，每次检测所需的DNA量仅为6 ng。在本研究过程中，我们在rs4316附近的第13外显子中鉴定出一种新的额外多态性（六个氨基酸的缺失）。该缺失等位基因编码睾丸ACE，但不编码血浆ACE。我们得出结论，通过TaqMan-PCR方法对rs4341 ACE多态性进行基因分型是一种快速便捷的直接I/D基因分型替代方法。我们还得出结论，睾丸ACE可能表现出六个氨基酸的缺失，这可能导致该酶的有害功能。
