Doria A, Warram J H, Rich S S, Krolewski A S
Joslin Diabetes Center, Department of Medicine, Harvard Medical School, Boston, MA 02215.
Hum Genet. 1994 Aug;94(2):117-23. doi: 10.1007/BF00202855.
The angiotensin I-converting enzyme (ACE) gene (17q23) is a candidate gene for essential hypertension and related diseases, but investigation of its role in human pathology is hampered by a lack of identified polymorphisms. Currently, a 287-bp insertion/deletion (I/D) RFLP in intron 16 represents the only one known. Additional polymorphisms for the ACE gene would make most families informative for linkage studies and would allow haplotypes to be assigned in association studies. To increase the information provided by the ACE gene, we used a sensitive screening technique, denaturing gradient gel electrophoresis (DGGE) blots, to identify polymorphisms and combined this with gene counting to identify haplotypes. Five independent polymorphisms, restriction fragment melting polymorphisms (RFMPs), were identified by four probes (encompassing half of the ACE cDNA) in digests produced by three restriction enzymes (DdeI, RsaI, and AluI). One RFMP has three alleles while the others have two alleles. In a sample of 67 unrelated control subjects, minor allele frequencies ranged from 0.12 to 0.49. A significant level of linkage disequilibrium was found for all pairs of markers. The four most informative RFMPs, taken in combination, define 24 potential haplotypes. Based on gene counting, 11 of the 24 are rare or nonexistent in this population, and the estimated heterozygosity of the remaining 13 haplotypes approaches 80%. Under these conditions for the ACE locus, phase-unknown genotypes could be assigned to haplotype pairs in unrelated subjects with reasonable certainty. Thus, using DGGE blot technique for identifying numerous DNA polymorphisms in a candidate locus, in combination with gene counting, one can often identify DNA haplotypes for both related and unrelated study subjects at a candidate locus. These markers in the ACE gene should be useful for clinical and epidemiologic studies of the role of ACE in human disease.
血管紧张素I转换酶（ACE）基因（17q23）是原发性高血压及相关疾病的一个候选基因，但由于缺乏已识别的多态性，对其在人类病理学中作用的研究受到阻碍。目前，内含子16中一个287bp的插入/缺失（I/D）限制性片段长度多态性（RFLP）是唯一已知的多态性。ACE基因的其他多态性将使大多数家族对连锁研究具有信息价值，并能在关联研究中确定单倍型。为了增加ACE基因提供的信息，我们使用了一种灵敏的筛选技术——变性梯度凝胶电泳（DGGE）印迹来识别多态性，并将其与基因计数相结合以确定单倍型。通过三种限制性内切酶（DdeI、RsaI和AluI）产生的消化产物中的四个探针（涵盖ACE cDNA的一半），鉴定出了五个独立的多态性，即限制性片段熔解多态性（RFMPs）。一个RFMP有三个等位基因，而其他的有两个等位基因。在67名无亲缘关系的对照受试者样本中，次要等位基因频率范围为0.12至0.49。所有标记对之间均发现了显著水平的连锁不平衡。四个信息最丰富的RFMPs组合起来可定义24种潜在单倍型。基于基因计数，这24种单倍型中有11种在该人群中罕见或不存在，其余13种单倍型的估计杂合度接近80%。在ACE基因座的这些条件下，在无亲缘关系的受试者中，可将未知相位的基因型以合理的确定性分配到单倍型对中。因此，使用DGGE印迹技术识别候选基因座中的众多DNA多态性，并结合基因计数，通常可以为候选基因座的相关和无关研究受试者识别DNA单倍型。ACE基因中的这些标记对于ACE在人类疾病中作用的临床和流行病学研究应该是有用的。
