ysis was performed by using the SPSS program (9). No parametric Spearman’s correlation was made to test the as- sociation between ACE genotype, age, and sex. Addition- ally, to determine the starting point of a possible genotype association between age and ACE genotype, a percentile analysis of age (10,25,50,75,90) for each genotype was performed. A one-way analysis of variance followed by Duncan’s test was used to compare the mean ages in the three different genotypes in each ethnic group studied. The chi-square likelihood test was used to compare genotype frequencies between ACE genotype and the following age points: 60, 70, and 80 years old. Also, the odds ratio (OR) and confidence interval (CI) (95%) were calculated. To test the possible association among ACE genotype, age, sex, and ethnic groups, we used the Foward Wald logistic regression. Similar statistics tests were used to analyze allele effect (D [deletion] and I [insertion]) associated with age.

ACE Genotyping

Genomic DNA was isolated from peripheral blood leukocytes by standard methods (8). ACE genotyping was performed using the method described by Schachter and colleagues (3). ACE amplification of DNA was performed by polymerase chain reaction (PCR) with 0.1 mg of the DNA extract and thermostable Taq polymerase (Gibco, Inc., San Diego, CA) according to the manufacturer’s instructions. PCR was performed in a thermal reactor using oligonucle- otide primers 59 CTG CAG ACC ACTCC C ATC CTT TCT 39 and 59GATGTG GCC ATC ACA TTC GTC AGA T 39 (5). In addition, 10% (vol/vol) dimethyl sulfoxide was in- cluded in the reaction mixture to enhance the amplification of I allele. The PCR products were separated by electrophoresis on a 1.5% agarose gel (Figure 1).

R ESULTS

The frequencies of the D and I alleles are presented in Table 1. The genetic frequencies were in Hardy-Weinberg equilibrium for both ethnic groups investigated. Figure 1 shows a PCR product of the ACE polymorphism. The data analysis showed that genotype and allele frequencies were ethnic group related. The JP had a higher II genotype frequency than GP (v2 ¼ 59.6; df ¼ 2, p ¼ .00). We can see that there was a change in allele frequency between the two populations studied. While GP had a higher D allele frequency, JP showed a higher I allele frequency.

Statistical Analysis

The mean age of all participants analyzed was 46.29 6 24.34 years old. The percentile distributions of age con- sidering ACE genotypes were different in the two pop- ulations studied (Figure 2).

Allele frequencies were estimated by the gene-counting method. Chi-square (v2) analysis was used to estimate the Hardy-Weinberg equilibrium and to compare genotypic and allelic frequencies among the ethnic groups. Statistical anal-

In GP, all ACE genotypes had a similar age until the 10th percentile. However, from the 25th percentile, the values changed and the age values for II were less than the DD and ID genotypes. In the JP percentile distribution, the DD genotypes had age values less than II and ID genotypes until the 50th percentile.

Table 1. Angiotensin-Converting Enzyme Genotypes and Allele Frequencies in a Sample of the Japanese Ethnic Population and Gaucha Population (of European Origin) Living in the Southern

JP GP N % N % Genotype II 171 46.9 73 13.9 DD 43 11.8 223 42.5 ID 150 41.2 229 43.6 Allele I 492 0.66 375 0.36 D 236 0.34 675 0.64

When the mean ages were compared among ACE genotypes, the results found were related to ethnic group. The GP showed an association between age and ACE genotype, whereas the JP did not (Table 2). In the GP, the DD and ID genotypes correlated with higher ages, more than the II genotype (F ¼ 5.501, df ¼ 2, p ¼ .004), suggesting a D allele-related dose effect.

Additionally, the following analysis considering different cut-off points was performed ((cid:1)60, (cid:1)70, and (cid:1)80 years old) to determine at which moment the frequency of D allele changed significantly. The D allele frequency increased from (cid:1)60 years old (Table 3) in the GP group. However, no significant differences were found among ACE genotype frequencies in JP at any age cut-points examined.

Note: JP ¼ Japanese population; GP ¼ Gaucha population; I ¼ insertion; D ¼ deletion.

could have influenced the results obtained. For this reason, in the present study we included individuals with a broader range of ages (from 10 to 105 years) to analyze the possible association between D or DD frequencies and longevity.

Also, we found in the GP group a D allele dose effect from (cid:1)60 years old ((cid:1)60 II¼13.9% and IDþDD ¼32.7%, v2 ¼ 10.38, df ¼ 2, p ¼ .001, OR ¼ 2.25, CI 95% ¼ 1.05–4.83).

However, we actually observed a decrease in I and II frequencies in the older population ((cid:1)60 years old) but not necessarily an increase in D and DD frequencies. If we examine the results from this point of view, we can explain the variation among associations obtained in different stud- ies. In this case, we need to determine why the II participants die earlier than ID and DD participants. This question is paradoxical because several studies reported an association between D allele and age-related diseases, such as myocardial

A multivariate analysis using logistic regression that included sex showed that, in the GP sample, the association between ACE polymorphism and age was independent.

D ISCUSSION

We found different results in the two ethnic groups studied. While GP showed genotype and allele frequencies of the ACE gene as a function of age, JP did not show this association. Some questions could be raised to try to understand the results obtained here. During the last years, several articles have been published reporting conflicting results about the association between ACE polymorphism and longevity.