Determination of Angiotensin-Converting Enzyme Gene Polymorphisms: Stepdown PCR Increases Detection of Heterozygotes

The angiotensin-converting enzyme (ACE) gene product plays an important role in cardiovascular homeostasis. An insertion/deletion (I/D) polymorphism in intron 16 of the ACE gene, with insertion polymorphism containing three more Alu-repeat sequences, was reported to be a determining factor of the plasma ACE concentration, and the D polymorphism has been found to be associated with certain cardiovascular diseases (1)(2)(3)(4)(5). Controversy exists, however, regarding the strength of the association. The diversity of conclusions has been attributed to methodological and technical variations in detection of the polymorphisms (6)(7). The preferential amplification of the D allele of the ACE gene by the PCR reported by Rigat et al. (8) was thought to be one cause. This PCR method occasionally mistyped ID heterozygotes as DD homozygotes (9). The probability of this mistyping has been estimated to be ∼5–10% (6)(7). A confirmatory PCR method, which requires an additional third PCR primer inside the Alu sequence of the I allele, was proposed to minimize the mistyping of the I allele as a D allele (9). Although this PCR technique was reported to be 100% in the typing of ACE gene polymorphisms, problems with the preferential amplification of multiplexed PCR are not entirely excluded with this method (10)(11). A stepdown PCR method, modified from touchdown PCR, has recently been used in several molecular studies (12). This method involves initial PCR annealing temperatures higher than the melting point of the primers, followed by annealing temperatures reduced stepwise to the melting point. This method should result in higher amplification specificity and greater yield. We compared the stepdown PCR with the conventional method from Rigat et al. (8) and with confirmatory PCR in the amplification efficiency of the ACE gene. We collected blood samples from 60 patients admitted to this hospital for the diagnosis and treatment of various heart diseases from August to December 1996. Leukocyte DNA was extracted by a nonenzymatic method (13). The DNA purity and quantity were assessed by absorbance values and checked by gel electrophoresis. The same materials were used to compare the amplification efficiency of the ACE gene by conventional, confirmatory, and stepdown PCR. Amplification of the ACE gene by conventional PCR was performed as described by Rigat et al. (8). The second method, confirmatory PCR, was done according to the method from Chung et al. (14). The confirmatory PCR condition included an additional, third internal primer to confirm the amplification of the I allele of the ACE gene, which was similar to the method of Shangmugam et al. (9). The third method, stepdown PCR, was developed in this laboratory, and the procedures were modified from Hecker et al. (12). Briefly, the PCR condition was optimized at a magnesium concentration of 2 mmol/L. The PCR primers were the same as those used by Rigat et al. (8). The salt concentration from 0.1 to 50 mmol/L of NaCl did not affect the amplification efficiency. All of the amplifications were carried out in the same thermal cycler (model 480, Perkins-Elmer Cetus). The reaction condition was achieved by denaturing first for 5 min and then repeating the following cycle: denaturing at 95 °C for 1 min, annealing at 70 °C for 1 min, and extension at 72 °C for 1 min. This cycle was repeated five times, and the annealing temperature was reduced to 65 °C and 60 °C each for five cycles and then to 60 °C for 25 cycles, with a final extension for 10 min. Each sample was amplified in duplicate by the same person. The PCR products were resolved by using 2% agarose gel electrophoresis and visualized by ethidium bromide staining. The results were interpreted by two different observers, and consensus opinion was obtained from the third observer, who did not know the results from the two previous observers. Each I or D allele that was detected in any one of the duplicate experiments by the three methods was counted as a positive. Amplification failure was defined as the absence of I and D alleles in both of the duplicate tests. The results of the three PCR methods were analyzed by χ test only after the first run of PCR examining all of the 60 DNA samples. The PCR products generated by the three methods were consistent in duplicate tests for all of the samples. Eight representative heterozygotic samples are shown in Fig. 1 . The I and D alleles amplified by the conventional method are shown in Fig. 1B . The I allele was ∼490 bp in length; the D allele was 190 bp. Those alleles amplified by the confirmatory method are shown in Fig. 1A ; the presence of one shorter DNA fragment represented the D allele (190 bp), whereas only the shorter, confirmatory I allele (277 bp) was amplified from two pairs of primers. The results by stepdown PCR, shown in Fig. 1C , were two DNA fragments (490 and 190 bp), the sizes of which were identical to those generated by the conventional PCR method. The amplification failure rate of the ACE genotyping was the highest in the conventional PCR method (23%, 14 of 60); it was 0% (0 of 60) in the confirmatory method, and 5% (3 of 60) in stepdown PCR. The frequency of ID heterozygote detection was the highest using the stepdown method (47%), whereas it was the lowest using the conventional method (9%; Table 1). Of 27 ID heterozygotes identified by stepdown PCR, 85% (23 of 27) were identified as DD or II homozygotes by conventional PCR, whereas 75% (12 of 16) of ID heterozygotes identified by the confirmatory method were typed as DD or II by conventional PCR. The confirmatory and stepdown methods markedly increased the detection rate of the ID heterozygote, but preferential amplification of the I or D allele was occasionally found (Fig. 1 , A and C). The differences in the genotyping of the ACE gene were statistically significant among the three methods (χ = 18.94, P <0.001) and between the conventional and stepdown methods (χ = 18.25, P <0.001). Allele frequencies of the ACE gene between the three methods were not significantly different (χ = 0.39, P = 0.82). Genotype distribution and allele frequencies of the ACE gene in 60 subjects using three PCR strategies. a Typing failures are excluded from percentages. Genotype distribution and allele frequencies of the ACE gene in 60 subjects using three PCR strategies. a Typing failures are excluded from percentages. Comparison of the ACE I/D polymorphism from eight heterozygote samples by the three PCR strategies. (A) Confirmatory PCR shows the D allele (190 bp, lanes 2, 3, and 6–8) and the confirmatory I allele (277 bp, lanes 1 and 3–8). The longer I allele (490 bp) is not amplified. Lanes 1, 4, and 5, overamplification of the I allele; lane 2, overamplification of the D allele. The bottom bands are primer dimers. (B) Conventional PCR shows typical D (190 bp, lanes 2, 3, 6, and 7) and I (490 bp, lanes 1–3 and 5–8) alleles. There is one test failure in Lane 4. Lanes 1, 5, and 8, overamplification of the I alleles. (C) Stepdown PCR clearly shows both D (190 bp, lanes 1–8) and I (490 bp, lanes 1, 3–6, and 8) alleles. However, it occasionally failed to amplify the I allele (lanes 2 and 7). The bottom bands are primer dimers. M, molecular marker. PCR is affected by a variety of factors, including concentrations of DNA templates, primers, Mg2 ion and salts, and annealing or extension temperatures (10)(11). By the conventional PCR method used by Rigat et al. (8) to detect the I/D polymorphism of ACE gene, which is a rather straightforward method, the detection rate of the ID heterozygote of ACE gene has been criticized because of the possibility of underamplifying the I allele. This undertyping is probably because of the stable secondary structure of the genomic DNA during PCR (9). The structural difference between the I and D alleles is the three-Alu repeat sequences. The D allele, which is shorter, is often easily amplified by the conventional method, especially in ID heterozygotes, resulting in a false dominant frequency of the DD genotype. Thus, the PCR results of this protocol are quite variable (6)(7). In our study, the underestimation of heterozygosity was 75–85%. The conventional method had the highest failure rate of amplifying the ACE gene I/D polymorphisms; it underestimated the frequency of the I/D heterozygote because of failure to amplify the I allele and sometimes even the D allele (which has not been reported previously), and thus may have overestimated the frequency of both the DD genotype and the II genotype. Time-consuming, repeated tests are necessary in genotyping ACE by the conventional PCR (9). The I allele confirmatory PCR method was proposed to overcome this shortcoming (9)(14). The advantage of this PCR method is to add a third primer located inside the Alu repeat sequence so that each reaction has an “internal control” and produces two DNA fragments representing the I allele. This method has been reported to have 100% accuracy in genotyping the ID heterozygote (9). However, in our study, the frequency of ID heterozygote detection was still a little lower than with the stepdown PCR method. This was probably due to overamplification of the I or D allele. Preferential amplification is still possible in this kind of multiplexed PCR experiment (10)(11), although a DNA destabilizing agent (i.e., dimethyl sulfoxide) had been added to the reaction mixture. Nevertheless, this method did amplify all of the samples. The stepdown PCR method, derived from touchdown PCR (12), can reduce nonspecific amplification and increase yield. This method starts amplification of the specific product at a temperature (70 °C) higher than the melting point (58 °C) of the designed primers, thus enhancing the specificity, then reducing the temperature at intervals of 5 °C for five cycles down to the melting temperature to increase the yield. Stepdown PCR can thus amplify a specific product more efficiently than conventional PCR. As demonstrated in our study, this method can detect the ID heterozygote more frequently than the other two methods, with an acceptable failure rate. Although the set-up of stepdown PCR is more complex than other methods and preferential amplification still exists, the time required to complete PCR is not increased, and the method avoids the use of an additional internal primer. In conclusion, the conventional PCR method in genotyping ACE gene polymorphism is variable, requires careful control, and often needs repeated testing, especially to verify the ID heterozygote. The confirmatory PCR method may have a higher accuracy but still cannot eliminate the preferential amplification of the multiplexed PCR. Stepdown PCR is a simple and more efficient method in genotyping ACE gene polymorphisms. It can specifically increase the detection rate of the I allele in ID heterozygotes, which is a shortcoming of conventional PCR, and avoid the use of a third primer, which is an advantage over the confirmatory PCR method. It should be the method of choice in screening ACE gene polymorphisms. This study was partly supported by grants from the National Science Council, Taiwan, Republic of China (NSC86-2314-B002-112) and from the National Taiwan University Hospital (NTUH87–1523).