Mistyping ACE heterozygotes.

Abstract

Depar tment of Pathology, Emory University School of Medicine, Atlanta, Georgia 30322 Human angiotensin I-converting enzyme (ACE) insertion/deletion (I/D) polymorphism has been proposed to be a significant predictor of risk for myocardial infarction (MI) among individuals otherwise at low risk for heart attack. (1~ The insertion sequence (288 bp) located within intron 16 of the h u m a n dipeptidyl carboxypeptidase 1 (DCP1) gene is a member of the Alu family. (2) Recently, Rigat et al. (3~ developed a rapid PCRbased assay for identifying the ACE genotypes. The procedure involves PCR amplification of genomic DNA utilizing a set of primers flanking the insertion sequence, which allows discrimination among the three ACE genotypes: II, DD, and ID. In genotyping a large pedigree in which one of the parents was an II homozygote, we were disturbed to encounter several DD genotypes among the offspring (Fig. 1, left). This was not a case of mistaken paternity as judged by several other criteria (not presented here). Furthermore, on repeating the PCR amplification under a slightly different condition, all of the DD genotypes amplified as ID (Fig. 1, right). However, on several other occasions, we observed that ID genotypes amplify as DDs. This led us to conclude that amplification of the I allele is sometimes suppressed in an ID heterozygote so that the latter can be mistyped as DD. This p h e n o m e n o n was also observed by Perna et al. (4) during amplification of an Alu I/D polymorphism within the h u m a n TPA gene. Although the mechan ism for this suppression is not currently understood, the mistyping is of u tmost concern because it is the ACE/DD genotype that has been shown to be significantly at a higher risk for MI when compared with the ID or the II genotype. (1) To resolve the mistyping of ID to DD, we examined several experimental parameters and found that inclusion of 5% dimethylsulfoxide (DMSO) in the reaction mixture greatly improved amplification of the I allele in an ID heterozygote (Fig. 2, left). In several trials with DMSO the ID heterozygotes amplified faithfully. However, to safeguard against any ID to DD mistyping we devised an additional PCR amplification protocol. This included utilization of a new sense primer (see legend to Fig. 2) from the 5' end of the insertion sequence, along with the standard antisense primer, (3~ re-