37 centenarians and in 252 controls. However, lon- gevity is strongly affected by environmental factors as well as by the entire genetic background. Different populations are therefore expected to have different gene/longevity associations. On the other hand, sequence analysis of the whole mtDNA is very expen- sive and time-consuming, and it is unfeasible for large size samples from various populations to be analyzed by this approach.

The novelty of our approach is the use of stable, ancestral, and associated polymorphisms, which are currently used in studies of human evolution, as markers of mtDNA inherited variability. Restriction fragment-length polymorphism studies of mtDNA from a wide range of human populations have revealed sets of ancestral mutations that define groups of mtDNA types (haplogroups) that have common ancestry and, because of uniparental inher- itance, evolve independently from each other (13– 15). Each of these haplogroups, which includes evolutionary related types of mtDNAs, is defined by specific sets of associated mutations, thus allowing for a quick and precise classification of the mtDNA molecules within a certain population. Nine mtDNA haplogroups (H, I, J, K, T, U, V, W, and X) have been identified that are characteristic of Europeans and encompass virtually all mtDNAs in Europe (16). We used these haplogroups as mtDNA stable markers to explore the role of mtDNA inherited variability on successful aging and longevity in Europeans.

MtDNA analysis

DNA was extracted from blood buffy coats following standard procedures. Haplogroups typing was carried out by restriction analyses of mtDNA according to reference (16). For each individual, mtDNA fragments encompassing the relevant polymorphic site (Table 1) were amplified by polymerase chain reaction. Primers and amplification conditions are given in Table 2. The amplified fragments were then digested with the appropriate enzyme (Table 1) and separated by a 2% agarose gel electrophoresis. The combination of the results obtained at the different polymorphic sites allowed the mtDNA of the subject to be classified in a certain haplogroup.

Statistical analysis

Permutation tests were used to verify whether the overall distribution of haplogroup frequencies was different between groups (18). For each test, the significance value was com- puted using 10,000 random permutations of the total set of haplogroups.

Fisher exact tests were used to verify whether the frequency of a specific haplogroup was different between sex-matched centenarians and controls. In each comparison, the level of significance was reduced to ad eau!  1–0.951/9 aad  0.0057 (nine independent variables).

Samples

A total of 487 unrelated subjects gathered in both northern and southern Italy were studied. The origin of each individual in the specific geographic area up to the maternal grand- mother was ascertained by interview. Control groups (20–75 years age range, median age 38 years) were made up of 125

RESULTS

Screening for mtDNA haplogroups (Table 1) was carried out in 487 individuals, made up of a group of

Primers Amplification conditions Forward Reverse Denaturation Annealing Polymerization 1561–1581 3717–3701 93°C for 30 51°C for 30 72°C for 60 3951–3970 5917–5890 93°C for 30 61°C for 30 72°C for 60 5720–5738 7608–7588 93°C for 30 57°C for 30 72°C for 60 7392–7410 8628–8608 93°C for 30 57°C for 30 72°C for 60 8232–8305 10107–10088 93°C for 30 57°C for 30 72°C for 60 9911–9932 11873–11851 93°C for 30 69°C for 30 72°C for 60 11711–11727 14208–14190 93°C for 30 49°C for 30 72°C for 60 15553–15569 725–706 93°C for 30 45°C for 30 68°C for 60

Amplification conditions

a The primer coordinates are given according to the Anderson sequence (17). Amplifications were performed in a 9600 Perkin apparatus.

a The primer coordinates are given according to the Anderson sequence (17). Amplifications were performed in a 9600 Perkin Elmer

(P 0.0052), whereas that of the U haplogroup de- _~4 creased from 23,5% in controls to 4% in centenar- Ma ASP=( ians (P 0.050). Since we had a separate interest in each haplogroup, we tested the nine null hypotheses separately: therefore, the correction for multiple tests should not be required in the present case (19). At any rate, the increase of J in centenarians would remain significant even if the level of significance were reduced to AaAee» 2 1–0.951/9  0.0057. In females, the overall frequency distribution was not statistically different between centenarians and controls 0 (P 0.670). However, in this case, too, the J haplo- group showed a tendency to increase (and the U haplogroup to decrease) from controls to centenar- ians (see Table 3).

275 people aged from 20 to 75 years (median age 38 years), and a group of 212 centenarians (100–109 years), all selected for their good clinical health status. As expected, all nine haplogroups character- istic of Europeans were found in both samples. They encompassed 94% and 90% of the mtDNAs in centenarians and younger people, respectively. The remaining mtDNAs were not attributable to any one of the above haplogroups and were grouped as ‘others’, as also found by previous studies (16). These results confirm and extend, over a large group of Italians, the data on mtDNA haplogroups in Europeans.

MtDNA haplogroups observed in northern Italy are shown in Table 3. The overall frequency distri- bution was significantly different between male cen- ~ AN=( tenarians and male controls (P 0.017). The fre- quencies of each of the nine haplogroups observed in the two groups were then compared. We found that the frequency of the J haplogroup increased from 2% in controls to 23% in centenarians