Materials and methods

Some recent evidence has linked cytokine poly- morphisms with longevity and differential lon- gevity between males and females and reciprocally with age-related diseases, which might cause elderly mortality thus influencing longevity. Differences in the genetic regulation of immune-inflammatory processes might explain why some people but not others develop theses diseases and why some develop a greater inflam- matory response than others (Bonafe et al. 2001; Lio et al. 2002a, b, 2003, 2004). In several cyto- kine genes, polymorphisms (mostly single nucleo- tide polymorphism, SNPs or microsatellites) located within the critical promoter or other reg- ulatory regions, affect gene transcription resulting in inter-individual variation in levels of cytokine production. The polymorphism may either di- rectly influences gene transcription or indirectly via tight linkage with other polymorphisms occurring elsewhere in the cytokine gene. The polymorphic nature of the cytokine genes may confer flexibility on the immune response with certain alleles promoting differential production of cytokines that may influence the outcome of infections or increase susceptibility and resistance to immune mediated disorders (Bidwell et al 1999, 2001).

Subjects and DNA sampling

DNA samples were obtained from 168 centenari- ans (>99 years old, 127 women and 41 men) from Centre (n=68) and South (n=100) Italy. 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, among others). DNA from 214 (112 women and 102 men) <50 years old controls matched for geographical origin were also collected. Written informed consent for enrolling in the study and for personal data management was obtained from all the subjects according to Italian laws. Blood specimens were collected in tripotassium EDTA sterile tubes and immediately stored at ) 70 (cid:2)C. Genomic DNA extraction was carried ) out and subsequently stored at 20 (cid:2)C for the IL-2 gene analysis.

) 330 T/G IL-2 polymorphism was typed by restriction fragment length polymorphism

Statistical analysis

primer with a modified sequence which creates a restriction site for the enzyme MaeI. The sequence modification was introduced to posi- tion -333 T fi G and the primers sequences used for this assay were: Forward 5¢ TAT TCA CAT GTT CAG TGT AGT TGT 3¢; Reverse 5¢TGG ATT CAC ACC CGA TTA CA 3¢. PCR-RFLP was carried out as follows: 10 ng of DNA was amplified in a 25 ll final volume PCR reaction containing 1(cid:1) Buffer (39.6 mM TRIS, 9.8 mM ammonium sulphate) 200 lM dNTPs, 1.5 mM MgCl , 6.25 pmol of each primer; 1 Unit Taq 2 polymerase, and 1 M betaine PCR was carried out on Mastercycler Eppendorf thermal cycler. Cycling was performed at 95 (cid:2)C for 2 min. and 35 cycle at 95 (cid:2)C for 1 min, 48 (cid:2)C for 1 min, and 72 (cid:2)C for 1 min., followed by a final exten- sion of 5 min at 72 (cid:2)C. Following amplification PCR product was visualized on gel a 2% aga- rose gel stained with ethidium bromide This confirmed the presence of a 150 bp product. Mae I enzyme digestion was carried out in 20 ll final volume reaction using 3 Unit MaeI (Boeh- ringer, Mannheim, Germany) and 4 ll of PCR product. The reactions were overlaid with paraf- fin oil and incubated at 45 (cid:2)C overnight. The digestive products were visualized on 4% aga- rose gel stained with ethidium bromide. Materi- als from individuals homozygous for ) 330 T (wild type) don’t cut with MaeI and remains as a 150 bp product. The homozygous mutants () 330G) cuts with MaeI to give 124 and 26 bp fragments.

IL-2 frequencies were evaluated by gene count. The data were tested for the goodness of fit be- tween the observed and expected genotype values and their fit to Hardy-Weinberg equilibrium (HWE) by v2 test. Analysis of statistical differ- ences between genotype frequencies in centenari- ans and controls was performed by v2. Analysis of statistical differences between allele frequencies in centenarians and controls was performed by Fisher exact test. Trend analysis was performed by extended Mantel Haenszel procedure.

Results

Genotypic frequencies of 330 T fi G IL-2 SNP in centenarians and controls from Centre and South Italy are shown in Table 1. The statistical analysis doesn’t show a significant difference of genotypic frequencies at -330 IL-2T/G SNP among centenarians and controls. Absence of statistical difference among centenarians and con- trols was confirmed analysing data stratified according to gender (Table 1). Comparing the two cohorts of subjects by extended Mantel Haenszel procedure a marginally significant trend ) for an increased 330TT genotype frequency was observed. Absence of statistical difference among centenarians and controls was confirmed by studying allele frequencies analysed on the whole (Table 2) or stratified according to the gender (data not shown).

Table 1. Genotypic frequencies of )330 T fi G IL-2 SNP in centenarians and controls from South-Center-Italy (percentages and frequencies per group is reported in parentheses).

TT TG GG n % n % n % Centenarians (168) 68 (40.5) 76 (45.2) 24 Male (41) 16 (39.0) 17 (41.5) 8 Female (127) 52 (41.6) 59 (46.4) 16 Controls (214) 69 (32.2) 110 (51.4) 35 Male (102) 30 (29.4) 52 (51.0) 20 Female (112) 39 (34.8) 58 (51.7) 15

The distribution of genotypes was in HWE in all the groups. No significant different distribution of IL-2 )330T/G genotypes was observed among control and centenarian subjects. No significant different distribution of IL2 )3330T/G genotypes was observed stratifying data according to gender. The analysis of trend by Mantel Haenszel procedure showed a marginally significant increased of - 330TT genotype in centenarians group [P=0.096; OR: 1.43, C.I.: (95%) 0.94–2.18].