Table 2 THO locus. Genotypic numbers and relative frequencies of the HUMTHO1 polymorphism recoded as diallelic

North Italy Males Controls Centenarians Genotypes (n=49) (n=26) P No. Rel. freq St. err No. Rel. freq St. err SS 17 0.347 0.068 10 0.385 0.095 0.803 SL 20 0.408 0.070 15 0.577 0.097 0.225 LL 12 0.245 0.061 1 0.038 0.038 0.027 Females Controls Centenarians Genotypes (n=70) (n=83) P No. Rel. freq St. err No. Rel. freq St. err SS 15 0.214 0.049 14 0.169 0.041 0.537 SL 38 0.543 0.059 48 0.578 0.054 0.744 LL 17 0.243 0.051 21 0.253 0.048 0.994 South Italy Males Controls Centenarians Genotypes (n=113) (n=27) P No. Rel. freq St. err No. Rel. freq St. err SS 33 0.292 0.043 10 0.370 0.093 0.488 SL 56 0.496 0.047 17 0.630 0.093 0.284 LL 24 0.212 0.038 0 0 0 0.004 Females Controls Centenarians Genotypes (n=126) (n=60) P No. Rel. freq St. err No. Rel. freq St. err SS 48 0.381 0.043 20 0.333 0.061 0.626 SL 64 0.508 0.044 26 0.433 0.064 0.352

S: alleles 6, 7 and 8; L: alleles 9, 10–1, and 10. P: probability of falsely rejecting the hypothesis of equal genotype frequency (SS or a SL or LL) in centenarians and controls =0.017.

grouped as follows. Locus REN: 7, 8 small (S) alleles; 10,11,12 large (L) alleles. Locus THO: 6, 7, 8 small (S) alleles; 9, 10-1, 10 large (L) alleles. Locus PARP: 85, 87, 89 small (S) alleles; 93, 95, 97, 99 large (L) alleles.

LL genotypes was observed in male centenarians (Table 2). The loss was statistically significant Æ (P = 0.004 with = 0.017) in southern Italy and had borderline significance (P = 0.027) in northern Italy. On the contrary, no significant case/control difference was found at loci REN (Table 1), PARP (Table 3) and SOD2 (Table 4). An analysis of interactions between genotypes/sex/geographic area within both the sample selected for longevity and the controls confirmed the peculiarity observed at the THO locus. The geographic factor interacted with genotypic frequencies in controls (P = 0.040) but not in centenarians (P = 0.142), while sex strongly interacted with the genotypic frequencies in centenarians (P = 0.0002) but not in controls (P = 0.083). These findings suggested that:

The observed genotypes of the recoded systems, as well as those of the SOD2 diallelic system, are reported in Table 1, Table 2, Table 3, Table 4. They were in agreement (P > 0.05) with those expected at Hardy- Weinberg equilibrium in every sub-sample at each locus, except for male centenarians at the THO locus (P = 0.028) in southern Italy. Direct comparisons between genotypes in centenarians and controls matched for sex and geographic area (last column of Table 1, Table 2, Table 3, Table 4) showed sex-specific THO locus/longevity association. A loss of homozygous

Table 3 PARP locus. Genotypic numbers and relative frequencies of the PARP (gt)n polymorphism recoded as diallelic

North Italy Males Controls Centenarians Genotypes (n=38) (n=24) P No. Rel. freq St. err No. Rel. freq St. err SS 17 0.447 0.081 10 0.417 0.101 1 SL 16 0.421 0.080 10 0.417 0.101 1 LL 5 0.132 0.055 4 0.166 0.076 0.725 Females Controls Centenarians Genotypes (n=39) (n=63) P No. Rel. freq St. err No. Rel. freq St. err SS 20 0.513 0.080 39 0.619 0.061 0.310 SL 12 0.308 0.074 19 0.302 0.058 1 LL 7 0.179 0.061 5 0.079 0.034 0.204 South Italy Males Controls Centenarians Genotypes (n=46) (n=19) P No. Rel. freq St. err No. Rel. freq St. err SS 27 0.587 0.073 9 0.474 0.114 0.426 SL 13 0.283 0.066 9 0.474 0.114 0.159 LL 6 0.130 0.050 1 0.052 0.051 0.663 Females Controls Centenarians Genotypes (n=41) (n=41) P No. Rel. freq St. err No. Rel. freq St. err SS 23 0.561 0.077 19 0.463 0.078 0.508 SL 15 0.366 0.075 18 0.439 0.077 0.653

S: alleles 85, 87 and 89; L: alleles 93, 95, 97 and 99. P: probability of falsely rejecting the hypothesis of equal genotype a (SS or SL or LL) in centenarians and controls =0.017.

approach was the genotypic analysis of individuals selected for longevity and younger people matched for sex and geographic area. Genotypes rather than alleles were considered because of a non-additive variance of the longevity trait26 and because Hardy-Weinberg equilibrium was not fulfilled in the sample of southern Italian male centenarians (P = 0.028).

i) starting from different frequency patterns in northern and southern Italian controls, the THO genotypic pools attained the same pattern in northern and southern Italian centenarians by means of a hypothetical selection affecting survival;

approach to identify the genes that affect inter- individual variability in life-expectancy, but three points should be taken into account. Firstly, the risk of false positive associations caused by confounding heteroge- neity factors. To reduce such a risk, both cases and controls examined in the present study were born and resident in the specific geographic area (northern or

Discussion

The aim of the present study was to test whether the variability of four genes controlling fundamental meta- bolic pathways is associated with human longevity. The

southern Italy) for three generations at least. Fur- thermore, sex-matched cases and controls were com- pared, on the assumption that the effect of a certain gene could vary between males and females (as expected for multifactorial traits) according to sex- specific physiological scenarios. Secondly, the age of the control group. Usually people aged 20–60 years are tested as controls with respect to centenarians, since this age range should minimise the risk of case/control overlap.3 However, this choice introduces selective criteria for the control group which reduce its random- ness. In the present study, whilst a stringent selection was made as to cases (people older than 100 years), the only selection as to controls was the availability of each person to take part in the study. Thirdly, the choice of candidate loci. The loci tested in our study are good