codes for tyrosine hydroxylase, the rate-limiting enzyme for synthesis of catecholamines, aminoacid- derived molecules which act both as hormone (adrena- lin) and neurotransmitters (dopamine and noradrena- lin). By alternative splicing of the THO transcript, four types of mRNA are produced, which differ in the inclusion or exclusion of exon 2 in the spliced products.7 Specifically in intron 1 an (aagt)n repeat polymorphism (HUMTHO1) is located,8, with 8 alleles varying from 5 to 11 repeats (two alleles have 10 repeats; one of them, 10-1 allele, lacks a single a). Allele frequency distribu- tion is bimodal, with peaks at 6 and 10-1 repeats.9 HUMTHO1 alleles are in linkage disequilibrium with INS-VNTR alleles10,11 which affect the levels of insulin transcription differently in pancreas12,13 and thymus.14,15

Controls The ages of the control group ranged from 10 to 85 years (median ages 41 and 31 years in northern and southern Italian samples, respectively); 119 subjects (70 females and 49 males) were from northern Italy and 239 subjects (126 females and 113 males) were from southern Italy, from the regions listed above. The individuals aged from 10 to 18 years were children from primary and secondary schools, whilst the others were volunteer donors (chiefly undergraduate students and University staff) invited to take part in the study by an appropriate campaign. A total of eight sub-samples were obtained, arranged by age (centenarians/ controls), sex (males/females) and geographic area (northern/ southern Italy).

DNAs were extracted from blood buffy-coats following standard procedures.

HUMREN4 and HUMTHO1 typings were carried out by PCR co-amplification of the DNA target sequences.9 Alleles were detected by silver staining. Home-made allele ladders were used as size markers. The quality of the ladder was tested by comparison with a standard ladder kindly donated by Dr C Previdere`, University of Pavia. Alleles were designed according to the repeat number.9 PARP polymor- (gt)n845nt phism was typed on PCR-amplified DNA products20 stained by silver. Home-made allele ladders were used as size markers.

The PARP gene (23 exons, 1q41–42 chromosome) codes for a zinc finger nuclear enzyme which is strongly activated by DNA breaks and promotes DNA repair.16 PARP activity is positively correlated with species- specific life span in mammalian species.17,18,19 A (gt)n repeat polymorphism lies in exon 1.20 The known six alleles, designated according to size (85 bp–99 bp size range), show bimodal frequency distribution, with peaks at 85 bp and 93 bp alleles.

(t/c) SOD2 polymorphism was investigated by an 401nt original ARMS-PCR assay. PCR reaction was carried out in a mg5 ul  25 l volume containing 80ng genomic DNA, 0.5 M of a sense primer specific for either c (5'gcaggcagctggctccgac3') or t (5'gcaggcagctggctccgat3') at nucleotide + 401 and an anti- bom) um sense primer (5'acgcctcctggtacttctcc3'), 200 of each dNTP, M 1.5U Taq polymerase, 1 3 Taq polymerase buffer under the

SOD2 is a manganese-containing enzyme which is coded by a nuclear gene (6q25 chromosom) and is translationally imported into the

Table 1 REN locus. Genotypic numbers and relative frequencies of the HUMREN4 polymorphism recoded as diallelic

North Italy Males Controls Centenarians Genotypes (n=50) (n=24) P No. Rel. freq St. err No. Rel. freq St. err SS 21 0.420 0.070 15 0.625 0.100 0.137 SL 28 0.560 0.070 7 0.292 0.093 0.046 LL 1 0.020 0.020 2 0.083 0.056 0.213 Females Controls Centenarians Genotypes (n=76) (n=83) P No. Rel. freq St. err No. Rel. freq St. err SS 44 0.579 0.057 54 0.651 0.052 0.328 SL 28 0.368 0.055 25 0.301 0.050 0.406 LL 4 0.053 0.026 4 0.048 0.023 0.711 South Italy Males Controls Centenarians Genotypes (n=38) (n=14) P No. Rel. freq St. err No. Rel. freq St. err SS 20 0.526 0.081 10 0.714 0.121 0.344 SL 16 0.421 0.080 4 0.286 0.121 0.524 LL 2 0.053 0.036 0 0 0 1 Females Controls Centenarians Genotypes (n=54) (n=36) P No. Rel. freq St. err No. Rel. freq St. err SS 27 0.500 0.068 20 0.556 0.083 0.670 SL 24 0.444 0.068 15 0.417 0.082 0.831 LL 3 0.056 0.031 1 0.028 0.027 0.624

S: alleles 7 and 8; L: alleles 10, 11, 12. P: probability of falsely rejecting the hypothesis of equal genotype frequency (SS or SL a LL) in centenarians and controls =0.017.

following conditions. 1min at 94°C, 21 cycles at 94°C for 25s, 60°C for 45s, 72°C for 30s, 9 cycles at 94°C for 25s, 55°C for 4 ~~> 1min, 72°C for 2min. Amplification products (15 l) were detected by ethidium bromide staining after 2% agarose gel electrophoresis.

tenarians; controls) was tested by log-linear analysis of the respective three-way tables.25 The frequency of each genotype was compared between centenarians and controls matched for sex and geographic area by Fisher exact tests. To reject at Æ level ' = 0.05 the null hypothesis that no genotypic fre- quency difference existed between centenarians and controls of specified sex and geographic area, the p values of the three individual tests were compared against Æ

Statistical Analysis

Allele frequencies were computed by counting genes from the observed genotypes and allele frequency distributions were examined. According to allele size and major frequency peaks, each multiallelic system (REN, THO, PARP) was recoded as a diallelic system by grouping the alleles into two classes (small: S and large: L alleles). All the following

Results

Exact tests were applied on each polymorphism to verify Hardy-Weinberg equilibrium24 in each of the eight sub- samples.

Both centenarians and controls showed the known bimodal allele frequency patterns at (REN, THO and PARP loci. According to these patterns, alleles were

The presence of interactions among the three factors genotype/sex/geographic area – within each sample

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