APOE (e2, e3 and e4 haplotypes corresponding to vari- in positions 3937 T/C and 4075 C/T of the gene) and (I/D in the intron 16 of the gene) polymorphisms were as previously described (13,17). In brief, for PCR amplification was carried out with primers 50 GCT TGG CAC GGC TGT CCA AGG A 30 and GAA TTC GCC CCG GCC TGG TAC AC 30, to am- a 244-bp fragment that was digested with HhaI restric- enzyme (Promega), run on a 12% polyacrylamide gel stained with silver nitrate; APOE genotypes were based on RFLP band combinations, as described (22: 91 and 83 bp; 23: 91, 83 and 48 bp; 24: 83 and 72 bp; 33: 91 and 48 bp; 34: 91 and 72 bp; 72 and 48 bp) (13). For ACE, PCR amplification was out with external primers to the polymorphic Alu 50 CTG GAG ACC ACT CCC ATC CTT TCT and 50 GAT GTG GCC ATC ACA TTC GTC AGA T to amplify 490/190 bp fragments (I/D allele) that were on a 2% agarose gel and stained with ethidium bromide Previous articles have reported a misgenotyping of ID heterozygotes as false ACE DD homozygotes using the external primers. To avoid false ACE DD ho- an additional combination of primers: (50 TGG CAC AGC GCC CGC CAC TAC 30 and 50 TCG CCA CTC CCA TGC CCA TAA T 30) (one primer a sequence in the Alu region) were used for an PCR amplification in DD homozygotes; a spe- 335-bp band was seen only in I allele carriers (on a 2% gel stained with ethidium bromide).

In this study we have analyzed the two common poly- morphisms in APOE and ACE genes in a large sample of Colombian subjects of different ages, to explore the possi- bility that they may be associated with human longevity in this population.

Subjects and Methods

Five hundred thirty eight unrelated subjects (372 women, 69.1%) of different ages (18–106 years) were recruited in Bogota´, the capital city of Colombia (for ACE polymor- phism, a subsample of 406 subjects was analyzed, 18–90 years, 70% women). The differences in the number of sam- ples analyzed for APOE and ACE polymorphisms were due to DNA storage problems of the first recruited samples, which were analyzed for APOE gene but could not be stud- ied for ACE polymorphism (due to DNA degradation). Group of older subjects was recruited in an ongoing popu- lation-based study of genetic factors associated with mental health in three neighborhoods in Bogota´; it was also com- posed of non-demented older subjects participating in ge- netic studies of cognitive impairment in a memory clinic in Bogota´ and by subjects selected in Bogota´ by their high longevity (O85 years). Group of young subjects was composed of university students and subjects participating as volunteers for the genetic analysis of human longevity. Older and younger subjects were functionally independent and living in the community. Each subject signed an in- formed consent, following international recommendations

Statistical Analysis

Genotype and allele frequencies were calculated using direct gene counting methods and the significance of the differ- ences between age groups (the overall sample was divided into two subgroups using the median age as a cut-off point) were evaluated using c2 and Fisher’s tests, with an alpha level of 0.05 (one-sided p value). Hardy-Weinberg equilibrium of genotype frequencies in all subject groups was evaluated. Analysis was also carried out in subgroups stratificated by sex. Statistical analysis of gene frequencies was done with Graphpad Instat program. Structure program was used as described (18) to analyze the presence of stratification in the sample of older subjects in an exploratory fashion.

Results

Table 2. Allele and genotype frequencies for ACE polymorphism in different age groups in Colombia

Genotype frequencies for APOE and ACE polymorphisms in all samples were in Hardy-Weinberg equilibrium ( p O0.05). When the 538 subjects genotyped for APOE were divided into two subgroups of older and younger subjects (mean age: 40 vs. 77 years), there were no significant differences in the distribution of allele or genotype frequencies for APOE polymorphism (Table 1) ( p O0.05). Stratification by sex did not show any significant difference for APOE genotype or allele frequencies between older and younger subjects (data not shown, p O0.05).

45 years (18–64 years) 77 years (65–90 years) Mean age (range) n % n % Genotype II 50 27.6 68 30.2 ID 89 49.2 122 54.2 DD 42 23.2 35 15.6 Total 181 100.0 225 100.0 Alelle n % n % I 189 52.2 258 57.3 D 173 47.8 192 42.7 Total 362 100.0 450 100.0

A significant decrease of the ACE DD genotype (24 vs. 16%, p 5 0.03) was found between subgroups of older and younger subjects (mean age: 45 vs. 77 years) (Table 2; Fig- ure 1). Stratification by sex demonstrated that the ACE DD genotype and D allele significantly decreased with age in women (25 vs. 15% –34 vs. 23 subjects, p 5 0.03 and 50 vs. 42% p 5 0.03, respectively) but not in men (19 vs. 15%–8 vs. 11 subjects, p 5 0.36 and 41 vs. 42%, p 5 0.47, respectively). APOE and ACE genotypes combina- tions did not show significant differences between younger and older subjects (data not shown, p O0.05). An explor- atory analysis of the subsample of older subjects (208 indi- viduals) using five polymorphic autosomal loci and carried out with the Structure program did not show evidence of significant sample stratification (data not shown).

ical interest may be dependent on genetic background and environmental features (4,5). A study in New York analyz- ing subjects of European, African and Latin American descent showed differential values of heritability among these populations (3).