2. Methods

The local Ethical Committee approved the study. A detailed medical history was obtained from the patients and centenarians and they or their caregivers gave a written informed consent after receiving clear explanation of the study. We analyzed a sample of 691 subjects: 235 consecutive sporadic AD patients: 148 females and 87 males, age at onset 66.7    8.12 (mean    S.D.) years defined as the onset of the first cognitive changes, Mini Mental State Examination (MMSE) score 21.2    4.4 points, with a diagnosis of probable AD. Moreover, we studied 153 patients with FAD (88 females and 65 males, mean age 71.3    5.4 years, MMSE score 23.2  3.4 points). Patients were considered familial patients if at least two first-degree relatives with dementia in two generations were documented. If reliable family history was not available, patients were termed sporadic, early-onset FAD (EOFAD). They were defined by the occurrence of AD with a onset at age <65 years and late-onset FAD (LOFAD) by the occurrence of AD with a onset age >65 years. All patients were enrolled at the Department of Neurology of the University of Florence and Villa Serena Hospital, Pescara. Clinical assessments of the patients were done according to published guidelines and the AD diagnosis fulfilled the DSM-IV criteria

Moreover, we also examined a sample of 192, apparently normal, Caucasian controls (110 females and 82 males, mean age 84.2    18.2 years). All controls were carefully assessed using a rigorous clinical history and general/neurological examination in order to exclude the presence of any neurological disorder.

In addition, a sample of 111 centenarians (23 men and 88 women, mean age 102.4    2.6 years) were included in this study. DNA from the affected and control individuals were extracted from peripheral blood samples using the phenol–chloroform procedure. The polymorphisms of ACE and APOE gene were determined in 691 subjects using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methods as previously described (Rigat et al., 1992; Sorbi et al., 1994). ACE genotypes were followed by a quality control amplification step necessary in detecting under-amplified ACE I alleles (Kehoe et al., 1999). Briefly, the reaction volume of 25 ml contained 200 mM each of deoxynucleoside triphosphate, 1.5 mM MgCl , enzyme buffer recommended by 2 manufacturer, 1.25 units of Taq DNA polymerase (Roche) and 10 pmol of each of the following primers: 50-CTG-GAG-ACC-ACT-CCC-ATC-CTT-TCT-30 and 50-GAT-GTG- GCC-ATC-ACA-TTC-GTC-AGA-T-30. Follow-ing denaturation at 96 8C for 20 s, amplification was achieved by 30 cycles of polymerization (10 s at 96 8C, 20 s at 55 8C, 30 s at 74 8C) with a final incubation for 1.30 min at 74 8C to ensure complete elongation. The amplification product was resolved through 2% standard agarose and stained with ethidium bromide. A band of 190 basepairs (bp) was present in the absence of the insertion (allele D) and a 490 bp fragment in the presence of the insertion (allele I).

2.1. Statistical analysis

The frequencies of ACE and APOE alleles and genotypes were estimated by gene counting. Statistical analysis was done using the chi-square test with the Yates correction. Odds ratios corresponding to 95% confidence intervals were calculated with standard

Distribution of ACE I/D polymorphismgenotypesand alleles, stratified by APOE e4 allele carrier status invarious types of AD and in centenarians

Group ACE genotypes ACE alleles DD (%) ID (%) II (%) D (%) I (%) AD APOE e4(cid:3) (126) 54 (42.8) 57 (45.2) 15 (11.9) 165 (65.4) 87 (34.5) AD APOE e4+ (109) 48 (44) 51 (46.7) 10 (9.2) 147 (67.4) 71 (32.5) FAD APOE e4(cid:3) (109) 55 (50.4) 39 (35.8) 15 (13.7) 149 (68.3) 69 (31.6) FAD APOE e4+ (44) 24 (54.5) 14 (31.8) 6 (13.6) 62 (70.4) 26 (29.5) Cent APOE e4(cid:3) (102) 54 (52.9) 35 (34.3) 13 (12.7) 143 (70.1) 61 (39.8) Cent APOE e4+ (9) 3 (33.3) 5 (55.5) 1 (11.1) 11 (61.1) 7 (38.8) Contr APOE e4(cid:3) (168) 71 (42.2) 74 (44) 23 (13.7) 216 (64.3) 120 (35.6) Contr APOE e4+ (24) 11 (45.8) 10 (41.6) 3 (12.5) 32 (66.6) 16 (33.3)

Notes: APOE e4(cid:3): non-carrier; APOE e4+: carrier.

methods. Significance limit was defined as p < 0.05. Statistical analysis was performed using SSPS software (SPSS, Inc., Evanston, IL, USA). To test the effects of the APOE e4 allele status and ACE genotypes (Table 1) alone and in combination, a logistic regression analysis was used. In detail, diagnosis served as a dependent variable and the presence/ absence of the APOE e4 allele and/or the ACE I/D and ACE I/I genotypes was pooled into a single referent group versus the ACE D/D genotype as the covariates. We also did the same calculation in the presence/absence of the APOE e4 allele and/or the ACE I/D and ACE D/ D genotypes pooled into a single referent group versus the ACE II genotype as the covariates. Comparison of the ages at onset between the genotype groups was made using the ANOVA test. The power analysis was performed using Power Calculator software (Department of Statistics at the University of Los Angeles, CA, USA;

3. Results

Both polymorphisms followed a distribution in the Hardy–Weinberg equilibrium and did not significantly differ from the controls ( p > 0.1). As shown in Table 2, out of the 235 AD patients, 10.6% were homozygous (II) and 46% were heterozygous (ID). We did not find any significant differences (13.5% II; 43.7% ID) with respect to the controls. There

ACE I/D polymorphism: allele and genotype distributions in sporadic and familial AD and in centenarians

Group No. Genotype Allele frequency DD (%) ID II D I AD 235 102 (43.4) 108 (46) 25 (10.6) 0.663 0.336 FAD 153 79 (51.6) 53 (34.6) 21 (13.7) 0.689 0.310 Centenarians 111 57 (51.4) 40(36.0) 14 (12.6) 0.694 0.306 Controls 192 82 (42.7) 84 (43.7) 26 (13.5) 0.645 0.354