For each nonagenarian and centenarian, a 60–70-year-old control of the same gender was randomly selected from the inhabitants of the same city of the province. This age group control was chosen on the basis of the consideration that at this age both men and women have less than a 1% probability of becoming centenarians.27 Patients with malignant, inflammatory diseases or C- reactive protein levels 420 were excluded from all study groups. Controls living in the same municipality were excluded to avoid excessive consanguinity. Written informed consent was obtained from all study participants or from family representatives if participants were unable to provide consent. Blood specimens were collected in sterile tripotassium-EDTA tubes and immediately stored at (cid:3)701C. Genomic DNA was stored at (cid:3)201C for different genetic analyses.

Chit activity seems to be related to the severity of atherosclerotic lesions, thereby suggesting a possible role for Chit as a marker of atherosclerotic extension.18–20 In addition, it was reported that patients with atherothrombotic stroke (ATS) and ischemic disease (IHD) had significantly higher Chit activities than the control group.19 However, ATS subjects had higher Chit activity than IHD subjects, whose atherosclerosis is localized more specifically in coronary vessels. This was confirmed by the observation that in the ATS group, Chit activity was related to carotid stenosis, in accordance with a clinical picture featured by a more widespread atherosclerosis.19 In addition, the increase in serum Chit activity was found to be age dependent. This phenomenon could be explained by the ongoing accumulation of lipid- laden macrophages during the gradual progression of atherosclerosis in relation to age. Inflammation has a central role in the beginning of the atherosclerosis process and in the mechanism underlying the develop- ment and progression of atherosclerosis complications, plaque rupture and subsequent thrombosis.21–22 Macrophages are present in all phases of atherogenesis and are markers of atherosclerotic plaque formation.23 Macrophage accumulation, localized in supra-aortic and coronary vessels, is associated with increased serum Chit activity, which reflects the state of activation of macrophages within atherosclerotic lesions.18 Interestingly, other investigations have suggested that Chit could have a crucial role even in pathological conditions, such as coronary artery disease,24 acute ischemic stroke,25 cerebrovascular dementia (CVD) and Alzheimer’s disease (AD).26–27 Recently, it has been shown that CHIT-1 may be involved in the progression of nonalcoholic steatohepatitis.28–29 Overall, these data underline the idea that CHIT-1, as an important factor in inflammation,15 could have widespread effects on aging phenotypes, particularly on cardiovascular disease and dementia, and as such may influence the ability to achieve a long and healthy life. In a study conducted on healthy elderly subjects (aged 65–94 years), it has been reported that levels of serum CHITwas higher compared with that in young people.30 However, to date and to the best of our knowledge, there is no information on the association between single-nucleotide polymorphisms (SNPs) in CHIT-1 gene and longevity. The aim of this study was to determine whether common, natural variations in the form of SNPs in the CHIT-1 gene might influence human aging and longevity.

DNA analysis

Genomic DNA was isolated from 5ml of peripheral blood as previously described.14 In total, 30ng of DNA was used as a template in subsequent PCR reactions. The duplication mutation analysis was performed using specific CHIT-1 primers: 5¢-AGCTATCTGAAGCAGAAG-3¢ and 5¢-GGAGAAGCCGG CAAAGTC-3¢. Fragments of 75 and 99bp were amplified from normal and mutant Chit genes, respectively. The DNAwas denatured at 951C for 4min for 1 cycle and amplified at 951C for 1min, 601C for 30s and at 721C for 30s for 35 cycles. The amplified DNA was separated on an 8% acrylamide/0.5(cid:4) Tris borate EDTA (TBE) gel and visualized by ethidium bromide staining. Electro- phoresis in MetaPhor gel (4%) allowed the detection of all fragments. A mixture of both fragments was detected in heterozygous subjects. Amplifica- tion was performed using the conditions described above.

Chit activity

Chit activity determination was performed as previously A volume of 5ml of undiluted plasma was incubated with 100ml of a solution containing 22mmoll(cid:3)l of the artificial substrate 4-methylumbelliferylb- D-N,N0,N00triacetylchitotriose (Sigma, Milan, Italy) in 0.5M citrate-phosphate buffer (pH 5.2) for 15min at 371C. The reaction was stopped using 2ml of 0.5moll(cid:3)1 Na CO –NaHCO buffer (pH 10.7). Fluorescence was read using a 2 3 3 Perkin-Elmer fluorimeter, on 365-nm excitation and 450-nm emission. Chit activity was measured as nanomoles of substrate hydrolyzed per ml per hour (nmolml(cid:3)1h(cid:3)1). The plasma Chit was expressed as nmolml(cid:3)1h(cid:3)1. Samples with Chit levels 411nmolml(cid:3)1h(cid:3)1 were assayed after a 10- or 50-fold dilution with distilled water.

Statistical analysis

were evaluated for deviation from Hardy–Weinberg equilibrium. General model and analysis of covariance were used to compare the proportion of study participants through CHIT-1 genotype. For the analysis of aging in case and controls, Student’s t-test was used for analysis and of plasma Chit activity values. Data are expressed as mean±s.d. was assessed by one-way analysis of variance (ANOVA) and t-test. Po0.05 was considered to be statistically significant.

MATERIALS AND METHODS

This study was carried out between 2003 and 2008. From the time of recruitment, the groups included the following:

RESULTS

A total of

Italian subjects:

male and

female, aged

–

years (average age:

years), and

unrelated volunteers (average age: