In light of these findings, we analyzed the genotype and allele distribution of the rs3851179 polymorphism in PICALM in Italian LOAD patients, extending our study to centenarians.

The studied group consisted of 813 Italian subjects: 349 sporadic LOAD patients, 359 sex-and age matched

Controls n (%) Centenarians n (%) LOAD patients n (%) Total 359 105 349 Age (mean±SD) 74.5±6.2◦ 99.6±3.3◦ 74.04±6.1 54.3% aged >100 years MMSE (mean±SD) 28.4±1.2 27.5±1.3 21.2±4.4 points Gender Females 201 (55.9) 81 (77.2) 229 (65.6) 58% aged >100 years Males 158 (44.1) 24 (22.8) 120 (34.4) 42% aged >100 years ApoE(cid:2)4 Carriers No 305 (84.9) 95 (90.5) 210 (60.2) ∗ Yes 54 (15.1) 10 (9.5) 139 (39.8)

LOAD patients: Late Onset Alzheimer’s Disease patients are defined by the occurrence of AD with an onset age ≥65 years. MMSE: Mini Mental State Examination score. * p-value<0.00001; OR 3.74, 95% CI: 2.57–5.45. ◦

I. Piaceri et al. / Implication of a Genetic Variant at PICALM in Alzheimer’s Disease Patients and Centenarians

the different PICALM genotypes was found (F[2, 346] = 0.81, p = 0.45 and F[2, 102] = 0.91, p = 0.405,

evident cancer or renal insufficiency), although many had decreased auditory and visual acuity as observed in other studies on healthy centenarians [15].

Total DNA was isolated from peripheral blood using standard methods. The analysis of the rs3851179 was performed using High Resolution Melting Analysis (HRMA) and the three genotypes were identified through sequencing (310 ABI PRISM Genetic Ana- lyzer, Applied Biosystem).

e4 The ApoE 4 allele was significantly associated with AD ( p < 0.00001; OR 3.74; 95% CI: 2.57–5.45) (Table 1).

The distribution of the rs3851179 genotypes and allele frequencies in the centenarian group shows a sta- tistically significant difference compared to the group of healthy subjects: OR 1.92 for the AA genotype (95% CI:1.00–3.67,p = 0.033)and1.44fortheAallele(95% CI: 1.04–1.98, p = 0.021) (Table 2). Comparing the LOAD group to the controls our data was not demon- strated to be statistically significant, the OR being 0.93 (95% CI: 0.59–1.47) for the AA genotype versus GG, and 0.98, 95% CI: 0.79–1.22, for the A allele (Table 2). Our data is in accordance with previous data ranging from OR 0.82 (95% CI: 0.76–0.88) [2], to 0.98 (95% CI: 0.69–1.40) [8].

Moreover,wedeterminedtheAPOEgenotypesinall the studied subjects using polymerase chain reaction (PCR) and the restriction fragment length polymor- phism (RFLP) method [16].

Moreover, the frequency (38.3%) of the minor allele in our control group is in agreement with the Caucasian control studied, ranging from 34% to 40% [2, 4, 6, 7].

After stratification for gender, the statistically sig- nificant value of the distribution of the A allele was confirmed among centenarian females: OR was 1.46 (95% CI: 1.00-.14, p = 0.042), while no differences

All genotype distributions of rs3851179 were in Hardy-Weinberg equilibrium in all the studied groups. No significant association between mean age-at-onset of LOAD or mean age of the centenarians and

Distribution, odds ratios (ORs) with corresponding 95% confidence intervals (CIs), p-value of PICALM rs3851179 polymorphism in the studied groups

Total Genotypes Alleles GG GA AA G A Control, n (%) 359 145 (40.4) 153 (42.6) 61 (16.9) 443 (61.7) 275 (38.3) Females 201 78 (38.8) 85 (42.3) 38 (18.9) 241 (59.9) 161 (40) Males 158 67 (42.4) 68 (43) 23 (14.5) 202 (64) 114 (36) Centenarians n (%) 105 31 (29.5) 49 (46.7) 25 (23.8) 111 (52.8) 99 (47.2) OR (95% CI), p-value 1* 1.50 (0.88–2.56), 0.11 1.92 (1.00–3.67 ), 0.033 1* 1.44 (1.04–1.98), 0.021 Females n (%) 81 21 (25.9) 40 (49.4) 20 (24.7) 82 (50.6) 80 (49.4) OR (95% CI), p-value 1* 1.75 (0.91–3.37), 0.07 1.95 (0.89–4.3), 0.067 1* 1.46 (1.00–2.14), 0.042 Males 38 (18.9) n (%) 24 10 (41.6) 9 (37.6) 5 (23.8) 29 (60.4) 19 (39.6) OR (95% CI), p-value 1* 0.89 (031–2.55), 0.80 1.3 (0.59–2.9), 0.52 1* 1.19 (0.60–2.26), 0.63 LOAD n (%) 349 140 (40.1) 154 (44.1) 55 (15.7) 434 (62.1) 264 (37.8) OR (95% CI), p-value 1* 1.04 (0.75–1.46), 0.80 0.93 (0.59–1.47), 0.75 1* 0.98 (0.79–1.22), 0.85 Females n (%) 229 92 (40.2) 101 (44.1) 36 (15.7) 285 (62.2) 173 (37.8) OR (95% CI), p-value 1* 1.01 (0.65–1.56), 0.97 0.80 (0.45–1.44), 0.43 1* 0.91 (0.68–1.21), 0.49 Males n (%) 120 49 (40.8) 54 (45) 17 (14.2) 152 (63.3) 88 (36.7)

I. Piaceri et al. / Implication of a Genetic Variant at PICALM in Alzheimer’s Disease Patients and Centenarians

were shown in LOAD (Table 2). After stratification, no differences were observed among all studied subjects Le4 with the presence/absence of APOE 4 allele (data not

negative finding for the limited number of men (24 out of 105) in the centenarians.

We have analyzed for the first time PICALM on cen- tenarians and here provide evidence of its implication in the genetic architecture of longevity. It could there- fore be hypothesized that PICALM, possibly involved  in amyloid- clearance from the brain, could have a functional role in healthy aging.

The sample size analysis demonstrated a power of 95.5% in the LOAD group versus controls, with an OR = 0.5 being the allele associated with a reduced risk in developing LOAD.

Additionally, our sample of 105 centenarians was evaluated as having an 87.1% power to detect an OR of 2.0 at a 5% significance rate.

ACKNOWLEDGMENTS

We analyzed the distribution of genotypes and alle- les of the PICALM SNP (rs3851179) in Italian LOAD patients and, as expected, we observed a decreased fre- quency of the A allele and AA genotype in AD cases compared to controls, although it was not statistically significant.

REFERENCES