body mass index [BMI], 21.8(cid:1)3.5kg=m2), 218 patients younger than 40 who experienced precocious AMI (165 men and 53 women, aged 24–39; BMI, 26.2(cid:1)3.1kg=m2), and a control group of 258 healthy young volunteers (128 men and 120 women, aged 22–39; BMI, 22.7(cid:1)3.8kg=m2). Part of the sample has been described previously.20 Centenarians did not have major age-related disease (i.e., type 2 diabetes mellitus, severe cognitive impairment, clinically evident cancer, ische- mic heart disease, liver or renal insufficiency, severe physical impairment), but some of them showed decreased visual or auditory acuity. Subjects with precocious AMI were 218 nondiabetic patients younger than 40 who were consecutively recruited at the cardiology departments of Alessandro Man- zoni Hospital, Lecco, Italy. Hypertension was present in 32%, obesity in 31%, current smoking in 64%, hypercholesterolemia in 55%, and hypertriglyceridemia in 24%. Healthy young controls were gender- and BMI-matched to healthy cente- narians. Controls were enrolled from within the laboratory staff and the general community. They were all Caucasians residing in northern Italy and had the same geographical or- igin of centenarians. All controls were judged to be in good health on the basis of their clinical history and on complete blood examination. Of these controls, 27% were moderate smokers; no other major cardiac risk factor was present.

formed using online resources (www.kursus.kvl.dk=shares= vetgen=_Popgen=genetik=applets=kitest.htm). Allelic and ge- notypic distributions were estimated by allele counting and compared in centenarians, AMI patients, and controls by the chi-squared test. Univariate and multiple logistic re- gression analyses were performed to test whether the C allele of rs1333049 was associated with longevity after adjustment for gender and BMI. Crude and adjusted odds ratios (ORs) are reported with their 95% confidence intervals (CIs). All statistical analyses were performed using SPSS 16.0 (SPSS Inc., Chicago, IL). The level of significance was set at 0.05 (two-tailed).

Results

Centenarians and healthy young controls were well- matched for gender (w2¼0.10, p¼0.75) and BMI (p¼ 0.89). Table 1 shows genotype distribution and allele frequencies in these two groups. Genotype frequencies of rs1333049 were in Hardy–Weinberg equilibrium and were comparable with those from other studies of individuals of European origin.11,14

Significant differences (p were found between centenarians, AMI patients, and controls in the distribu- tion of the C risk allele of rs1333049. The lowest frequence of the C allele was identified in healthy centenarians (46.3%) and the highest in patients with precocious AMI (65.1%), with an intermediate frequency in healthy young controls

The study protocol was approved by our Institution Re- view Board, and all patients provided their written informed consent.

The multivariable-adjusted OR for longevity for subjects carrying at least one C allele of rs1333049 was 0.64 (95% CI ¼0.39–0.89, p<0.01). As expected, subjects with at least one C allele of rs1333049 had a significantly higher risk of AMI at a young age (OR¼1.68, 95% CI¼ 1.17–2.01, p< 0.01).

Genotyping

Genomic DNA was purified from peripheral blood sam- ples using the QiaAmp DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Geno- typing for rs1333049 was performed using the TaqMan SNP genotyping assay (Applied Biosystems, Foster City, CA) with primer pair rs1333049-F (50-CCTTCATGCTATTTTG AGGAG-30) and rs1333049-R (50-GGAAGATAAGTTGAGA ATGTCA-30) in a final volume of 5 mL. For quality control, genotyping analyses were done blind with respect to the clinical status of the study participants, and a random 20% of the samples were repeated. All genotyping repeated for quality control did not differ from the initial genotyping. Genotyping analyses were done blind with respect to case– control status. Two investigators independently reviewed all results.

Discussion

The interrelationships between successful aging and escape from age-associated illnesses, including atherothrombosis and dementia, have prompted growing research efforts in centenarian subjects aiming at entangling the biological and genetic pathways of human longevity.1,21 In the present work, we provide evidence that Caucasian centenarians have a lower prevalence of the risk allele of rs1333049 at the 9p21.3 locus. This finding, coupled with the high prevalence of this allele observed in patients with precocious AMI, further strengthens the concept that this variant rs1333049 is strongly associated with age-related disorders. The association of the C allele of rs1333049 remained significant after multiple ad- justment for potential confounders. Therefore, this polymor- phism appears to exert an independent effect on human longevity.

Data analysis

The study power was calculated using the StatMate soft- ware, version 2.0 (GraphPad Inc., San Diego, CA). A test for deviation from the Hardy–Weinberg equilibrium was per-

Table Distribution of rs Genotype and Allele Frequency for Centenarians 1. 133304 and Healthy Young Controls

Genotype Allele Study group n GG GC CC G C Centenarians 80 22 (25.0%) 42 (52.5%) 16 (22.5%) 86 (53.7%) 74 (46.3%) Healthy young 258 47 (18.2%) 132 (51.2%) 79 (30.6%) 226 (43.8%) 290 (56.2%) controls

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