RESULTS

A total of

Italian subjects:

male and

female, aged

–

years (average age:

years), and

unrelated volunteers (average age:

years), who presented themselves at the Department of Senescence, Urological and Neurological Sciences, Catania University Hospital, Catania, Italy. The date of birth of each subject was checked by matching information from the social security document, and by the testimony of a first-degree relative.  A total of

Tunisian subjects:

male and

female, aged

–

years (average age:

years), were recruited from Tunis, Hammamet, Monastir, Sousse, Mahadia, Sidi Bou Said, Djerba, Kairouen, Carthage, Bizerte, Gabes, El Jem, Le Grand Soud, Tozeur, at their home, using the archival records in the City Hall.  A total of

Greek subjects:

male and

female, aged

–

years (average:

years), were recruited from different regions of Greece

A total of 258 90-year-old and 60-year-old subjects were genotyped. Table 1 shows the baseline characteristic of each subject, in which biological characteristics, general health status, disease prevalence and functional status are presented. In terms of biological characteristics, the long-living subjects were older, leaner (lower waist/hip ratio) and had lower triglyceride and glucose levels. These subjects also had better self-rated health and lower prevalence of cardiovascular disease (coronary heart disease (CHD) and stroke) and degenerative disease. Functionally, they seemed better able to walk, but had lower grip strength. There was no difference in the cognitive score between long- living and control subjects.

Table 1 Baseline characteristics on the basis of case–control status

Average control phenotype Average elderly phenotype (age 68.8±2.4 years) (n¼129) (age 97.6±3.6 years) (n¼29) Variables at baseline examination (2003–2008) Mean±s.d. Min–Max Mean±s.d. Min–Max P Biological, fasting values Body mass index (kgm(cid:3)2) 24±2.12 16.89–31.3 21.8±3.07 15.3–30.2 0.9290 Waist/hip ratio 0.93±0.08 0.7–1.12 0.90±0.06 0.71–1.02 0.0012 Total cholesterol (mg100ml(cid:3)1) 187.2±32 99–304 184.6±26.1 95–264 0.2120 HDL (mg100ml(cid:3)1) 51.62±12.6 33–127 50.2±14.5 29–96 0.4921 Triglycerides (mg100ml(cid:3)1) 160.2±88 51–750 147±86.2 42–481 0.0940 Glucose (mg100ml(cid:3)1) 117.2±35 71–312 108.2±22.9 77–281 0.0018 AST (IU/l) 33±29 32–73 30.2±21 19–61 0.1103 ALT (IU/l) 36±21 27–118 28±16 19–74 0.0968 Hemoglobin (g100ml(cid:3)1) 13.5±1.12 11–14 13±1.6 10.5–15 0.8106 General health status Self-rated ‘poor’ health 40.81 — 30.21 — 0.0159 Disease prevalence (%) CHD 28.12 — 6.92 — 0.0001 Stroke 8.21 — 3.3 — 0.0394 Diabetes 65.12 — 54.2 — 0.6587 Degenerative disease 21.6 — 12.42 — 0.0391 Physical/cognitive function Difficulty in walking (%) 24.21 — 18.83 — 0.0622 Grip strength (%) 29.12±7.54 3–30 24.12±3.47 8–45 0.0001 Cognitive score (CASI) 82±14.21 37–100 80.22±16.8 19–96 0.1000

Abbreviations: CASI, cognitive abilities screening instrument; CHD, coronary heart disease; IU, international unit. P value from Student’s t test.

Table 2 Frequencies of wild-type and mutant alleles in elderly (nonagenarians and centenarians) and in control subjects

Elderly Control Populations No Genotype frequency Allele frequency Genotype frequency Allele frequency Wt Wt/H H/H Wt Wt/H Wt Wt/H H/H Wt Wt/H Tunisian 33 36.2 63.8 0 27.3 72.7 71.6 22.9 5.5 74.3 25.7 Greek 46 33.16 66.84 0 19.7 80.03 61.9 33.8 4.3 76.3 23.7 Italian 50 35.2 64.8 0 21.6 78.4 72.2 24.6 3.2 76 24

mutant allele was 5.5, 4.3 and 3.2% in Tunisian, Greek and populations, respectively. We also examined the Chit activity in collected from oldest old subjects and from control subjects. Tunisian, Greek and Italian oldest groups, the plasma Chit activity were higher in subjects homozygous for the wild-type allele than those heterozygous for the mutant allele (Table 3; Figure 1a–c). control subjects, the level of plasma Chit activity was comparable that found in oldest old subjects, in relation to the allele (Table 3; Figure 1a–c).

nonagenarian and centenarian population from the different Medi- terranean areas. The absence of homozygous individuals in the very old group deviates significantly from the expectations of Hardy–Weinberg equilibrium. In the oldest group, the frequency of this mutant allele was 72.7, 80.03 and 78.4% in Tunisian, Greek and Italian populations, respectively, whereas the frequency of the wild- type allele was 27.3, 19.7 and 21.6% in Tunisian, Greek and Italian populations, respectively (Table 2). In contrast, in control subjects, the heterozygote frequency for 24-bp duplication was 25.7, 23.7 and 24% in Tunisian, Greek and Italian populations, respectively (Table 2). In the oldest old group, no subject was found homozygous for CHIT-1 deficiency, whereas in the control group, the incidence of homozygous

DISCUSSION

In this study, we compared the distribution of the CHIT-1 poly- morphism genotype in two groups comprising different populations

deletion of 87 nucleotides and enzyme deficiency, was not significantly different among the oldest old subjects from different Mediterranean populations, whereas it was significantly different between oldest old subjects and control subjects, being highest among oldest old subjects and lowest among control groups. In the oldest old group, no subject was found homozygous for CHIT-1 deficiency, whereas in the control group, the incidence of HH mutant allele was 4.4% in Tunisians, 3.12% in Greeks and 5.31% in Italians. We also found that the mean enzymatic activity for the oldest subjects was lower than that in control groups. This study indicates that the heterozygosity of the 24-bp duplication in the CHIT-1 gene could have a protective effect in human longevity.

Table 3 Chit activity in elderly and control groups

Elderly Controls Chit activity Populations Wt Wt/H Wt Wt/H Tunisian 33 183.4±9 40.4±18.7* 188.5±13.8 50.3±22.8* Greek 46 156.8±42 31.3±8.8* 209.6±17.6 53.7±16.3* Italian 50 177.2±16.3 49±21* 195.3±13.6 58.4±19*