The ACE genotypes were identified as previously described (21). Briefly, the forward CTG GAG ACC ACT CCC ATC CTT TCT and the reverse GAT GTG GCC ATC ACA TTC GTC AGA T primers were used for PCR in a total of 30 cycles at 958C for 30 seconds, 608C for 30 seconds, and 728C for 45 seconds. Primers were synthesized by Invitrogen (Invitrogen Corporation). Reaction buffer included 1 U of Taq DNA polymerase (Platinum Taq, Invitrogen Corporation), 10 pmol of each primer, 100 lM each deoxynucleotide triphosphate, and 1.5 mM MgCl . 2 Given the 287 bp size difference, a direct analysis of PCR products on a 1.8%–2.0% agarose gel identified the three ACE genotypes.

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Statistical Analysis

Agreement of the observed genotype frequencies with the expected Hardy–Weinberg frequencies was verified with Arlequin Software, version 2.000 (22,23). Relative allele frequencies were estimated by the gene counting method (24), and the 95% confidence intervals (CIs) expressing variances of the estimated allele frequencies were also calculated (23).

Additionally, a sample of 64 centenarians (18 men, 46 women; mean age ¼ 100.28 6 1.86, range 98–108 years) was recruited through the Central Population Register of Apulia and included in the study. They were all Caucasians residing in southern Italy. The inclusion criteria were: (a) an age of at least 98 years on the day of blood collection, (b) no known diagnosis of dementia or other chronic neurological diseases or psychiatric syndromes with cognitive impairment (16), and (c) no cerebrovascular dis- ease, nephropathy, or end-stage renal disease, and no severe functional limitations.

We considered genotypes according to the presence of at least one allele. For the apoE polymorphism, we reported e2 (e2/) and e4 (e4/) carriers. e2 carriers were defined as those participants showing at least one e2 allele, that is, having a e2/e2, a e2/e3, or a e2/e4 genotype. Similarly, e4 carriers were defined as those participants showing at least one e4 allele, that is, having a e4/e2, a e4/e3, or a e4/e4 genotype. For ACE polymorphism we reported D (D/) and I (I/) carriers. D carriers were defined as those participants showing at least one D allele, that is, having a D/D or a D/I genotype. Similarly, I carriers were those participants showing at least one I allele, that is, having an I/I or an I/D genotype.

ApoE and ACE Genotyping

Genomic DNA was manually purified from 4 mL of frozen blood samples by organic protein extraction and ethanol precipitation according to standard methods (17). The apoE and ACE genotypes were determined by polymerase chain reaction (PCR) and agarose gel electro- phoresis as follows.

Pearson’s chi-square test and Fisher’s Exact test were used to compare genotype and estimated allele frequencies. Binary logistic regression analysis was used to verify the associations between apoE and ACE alleles with age and sex, as well as to verify interactions between apoE and ACE alleles. All statistical analyses were made with SPSS statistical software (version 10.1.3; SPSS Inc., Chicago, IL). A value of p , .05 was considered to be statistically significant.

Briefly, the two transition C3937 !T (Arg112 !Cys) and C4075 !T (Arg158 !Cys) resulting in the apoE polymor- phism, that is, the two single nucleotide polymorphisms rs429358 and rs7412, abolished two HhaI restriction sites (GCGC!GTGC), corresponding to codons 112 and 158 of the protein. We used the forward primer described by Houlston and colleagues (18) (TCC AAG GAG CTG CAG GCG GCG CA) and a reverse primer modified from Emi and colleagues (19) (CCC TCG CGG GCC CCG GCC TGG TAC AC). Primers were synthesized by Invitrogen (Invitrogen Corporation, Carlsbad, CA). A total of 100 ng of genomic DNA was amplified on an Applied Biosystems GeneAmp PCR System 9700 (Applied Biosystems Inc., Foster City, CA). Amplification conditions were 948C for 2 minutes, followed by 35 cycles at 968C for 30 seconds, 658C for 45 seconds, and 728C for 45 seconds. Reaction buffer included 1.5 U of Taq DNA polymerase (Platinum Taq; Invitrogen Corporation), 10 pmol of each primer, 100 lM of each deoxynucleotide triphosphate, and 1 mM MgCl . 2

R ESULTS

The overall study population of 1344 participants was divided into four groups according to age, as follows: Group 1 (n ¼ 292, 21.73%), participants aged   30 years (range from 22 to 30 years of age, mean age 27.29 6 2.10 years); Group 2 (n ¼ 629, 46.80%), participants aged from 31 to 44 years (mean age 36.76 6 3.94 years); Group 3 (n ¼ 270, 20.09%), participants aged from 45 to 59 years (mean age 50.44 6 4.17 years); and Group 4 (n ¼ 153, 11.38%), participants aged from 60 to 90 years (mean age 70.71 6

Table 1. Apolipoprotein E (ApoE) Genotypes in Relation to Sex and Age in 1344 Healthy Elderly Persons and 64 Centenarians