arbitrary age classes, and taking into account cohort effects in mortality changes (28,29). Furthermore, the addition of demographic to genetic data not only is able to reveal sex- and age-specific allelic effects, but also permit a rational definition of the age classes to be screened (30).

The aim of the present study was to investigate if the (A/G)-308 polymorphism affects the probability of reaching extreme old age. To this purpose, we genotyped at the (A/G)-308 locus a wide group of healthy participants ranging from 19 to 110 years, and added demographic information to genetic data to detect possible differential survival of persons carrying different genotypes.

Genotyping

chloroform according to standard procedures (32). Features and relative positions of the four loci were analyzed, and the primers used to amplify each marker are summarized in Table 1. For the analysis of the (A/G)-308 polymorphism, a previously described method (33) was applied. To analyze the TNFa, TNFc, and TNFe microsatellite polymorphisms the original method (34) was modified to allow a simulta- neous analysis of the three markers through multiplex poly- merase chain reaction (PCR) and fluorescence detection of the amplification products. PCR was performed in a 9700 thermal cycler (Perkin-Elmer Applied Biosystems, Foster City, CA) under the following conditions: 2 minutes at 948C; 30 cycles of 30 seconds at 948C, 30 seconds at 56.58C, and 30 seconds at 728C; 6 minutes at 728C for the final extension. For each sample, 100 ng of DNA was amplified in a 25-lL reaction volume composed of [Mg2þ] 1.5 mM, [dNTP] 300 lM, 1 U of Taq polymerase, and the following primers: IR2 (0.12 lM), IR4 (0.12 lM), IR6 (0.4 lM), IR7 (0.4 lM), IR13 (0.08 lM), and IR14 (0.12 lM). Primers IR2, IR7, and IR14 were fluorescence-labeled (see Table 1) to allow for the detection of amplification products per- formed by capillary electrophoresis on an automated DNA sequencer (Abi Prism 310; Applied Biosystems, Foster City, CA). The size of microsatellite DNA fragments was mea- sured by using Genescan software (Applied Biosystems).

Furthermore, due to the high complexity of the chromo- somal region in which the TNF gene is located, we checked also the association with longevity of haplotypes defined by alleles of three microsatellite loci (TNFa, TNFc, TNFe) and the (A/G)-308 polymorphism. In this case, because we were dealing with a multiallelic system, the classic GD algorithm was not appropriate (29), and only the comparison of haplo- type frequencies among age classes was applied.

M ETHODS

Sample

A total of 747 (401 women and 346 men) unrelated individuals, 19–110 years old (median age 73 years) born and residing in northern or central Italy, and whose parents and grandparents were from this same area, participated in the study. Information about health status was obtained for all the participants, and no acute pathological condition was present. The recruitment was carried out as part of a multi- center study on aging conducted in several regions of central and northern Italy during the years1996–2004. Young and middle-aged volunteers were recruited among university students and workers and in blood donor centers, whereas elderly people (excluding centenarians) were mainly re- cruited among guests of elderly residences. Centenarian age was verified by researching archival records in the City Hall and/or church registries, paying attention to the concordance between reported age and personal chronologies (age of marriage and of military service for men, age of first and last pregnancy for women, age of children, etc.); centenarians were contacted via telephone through members of their families, after previous contact of their family doctor. The

GD Analysis

At the basis of the classical GD analysis (28) there is the survival function S(x;t ) of a cohort, defined as the fraction 0 of individuals in a population born at a certain time t and 0 still alive at time t þ x. Because the sample consists of 0 individuals drawn from the Italian population, with ages in the range of 19–110 years, cohorts from the 19th century up to the end of the 20th century are represented in it. For each cohort, the survival function was computed by using the

(Arlequin software) in which the likelihood of the observed data evaluated under the hypothesis of no association between loci is compared to that evaluated under the hypothesis of association. Differences in allele, genotype, and haplotype frequency distribution among age groups were tested by a Monte Carlo likelihood ratio chi-square test implemented in the statistical software package SPSS for Windows (SPSS, Inc., Chicago, IL).