Materials and methods

Study population

Healthy individuals >85 and between 20 and 50 years old were recruited to serve in the experimental and control groups, respectively. A total of 119 elderly and 118 young unrelated individuals from the same geo- graphical areas volunteered for the study. All subjects were asked to obtain medical clearance before partici- pation in the study, for verifications of chronic dis- eases and medications. Subjects with cardiovascular diseases, diabetes, or cognitive impairment were excluded from the study. The experimental design and the sample size are similar to most longevity studies reviewed by Glatt et al. (2007). Written informed con- sent was obtained from all participants in accordance with the requirements of the Institutional Review Boards of Jordan University of Science and Technol- ogy. An official identification document was required to participate in the study. Acceptable documents include civil ID card, birth certificate, family book, passport and military card, otherwise enrolment in the study was denied.

IL-10 )1082 G⁄A genotyping

) The IL-10 1082 G⁄A polymorphism was analysed by an original ARMS-PCR assay as described by Howell et al. (2001). PCR reaction was performed in a 10 lL volume containing 100 ng genomic DNA, 1 l of a M forward primer specific for either A (5¢-act act aag gcttct ttg gga a-3¢) or G (5¢-cta cta agg cttctttgggag-3¢) and a reverse primer (5¢-ctt gga tta aat tgg cct tag a- 3¢), 200 l of each dNTP, 4.8 l MgCl , and M M 2 0.25 units ThermoprimePLUS (TP) DNA polymerase (Abgene, Epsom, UK) and 1X TP polymerase reaction buffer. Also the reaction mixture contains 1 l of a M forward (5¢-tgc caa gtg gag cac cca a-3¢) and a reverse (5¢-gca tct tgc tct gtg cag at-3¢) primer specific for amplification of the internal control fragment (796 bp). PCR reaction was performed under the fol- lowing conditions: 1 min at 96 (cid:3)C, ten cycles at 96 (cid:3)C for 15 s, 65 (cid:3)C for 50 s, 72 (cid:3)C for 40 s, 20 cycles at 96 (cid:3)C for 10 s, 60 (cid:3)C for 50 s, 72 (cid:3)C for 40 s. Ampli- fication products (194 and 796 bp) were detected by ethidium bromide staining after 2% agarose gel elec- trophoresis.

The experimental and control groups mean ages were 90.2 (range: 85–113 years) and 31.9 years (range: 16–50 years), respectively. The ranges were selected based on the mortality rate in the Jordanian population which stays approximately constant from childhood to late forties, then it starts gradually inclin- ing to reach maximum in late seventies (Khoury et al., 1999). Therefore, individuals who reach more than 85 years constitute a tiny fraction of the total popula- tion cohort (<1%). Men to women ratio was 1.9:1 in the elderly group and 1.6:1 in the control group (P = 0.41).

DNA extraction

EDTA venous blood samples (1–3 mL) were obtained from both groups. DNA was extracted from all sam- ples using Wizard DNA Extraction Kit (Promega, Madison, WI, USA) according to the manufacturer instructions. The concentration of the extracted DNA was measured using SmartSpect(cid:2)3000 (Bio-Rad, Hertfordshire, UK). DNA samples were stored at ) 20 (cid:3)C until used.

Statistical analysis

The genotype distributions of the studied polymor- phisms were tested in accordance with Hardy–Wein- berg equilibrium. Statistical analysis of results was performed by comparing allele, genotype, and pre- dicted phenotype distributions using the chi-squared test. If n < 5, exact Fisher test statistic was used. The

TNF-a )308 G⁄A genotyping

TNF-a ) 308 G⁄A polymorphism was typed by restric- tion fragment length polymorphism (RFLP) according to Garnacho-Montero et al. (2006), using a primer

Table 2. Functional IL-10⁄TNF-a genotypes and longevity in the elderly and control subjects

test power was calculated using Power and Sample size calculation program (PS version 3.0.1; Vanderbilt University Medical Center, Nashville, TN, USA). For all analysis, the power was more than 80%. The SPSS 15.0 statistical software package (SPSS Inc., Chicago, IL, USA) was used for all statistical evaluations. P-val- ues of <0.05 were considered significant.

Controls Elderly Combined Functional group group TNF-a⁄IL-10 genotypes N (%) N (%) TNF-a GG⁄IL-10 Low⁄low 91 (77.1) 93 (78.2) 0.814 (AA⁄AG) TNF-a GG⁄ Low⁄high 6 (5.1) 3 (2.5) IL-10 GG TNF-a (AA⁄AG)⁄ High⁄high 1 (0.8) 1 (0.8) IL-10 GG TNF-a (AA⁄AG)⁄ High⁄low 20 (16.9) 22 (18.5) IL-10 (AA⁄AG)

Results

Table 1 shows the frequency of homozygous and het- ) erozygous genotypes for 1082 G⁄A IL-10 promoter SNP and for ) 308 G⁄A TNF-a promoter SNP in the experimental and control groups. No differences in the genotype frequencies of the IL-10 promoter SNP (Fish- er’s exact, P = 0.384) and the frequency of IL-10 G allele to A allele were found between the groups (Fish- er’s exact, P = 0.225). Similarly, ) 308 G⁄A TNF-a promoter SNP were not different between the groups (Fisher’s exacts; for genotype frequencies: P = 0.828 and for allele frequencies: P = 1.000).

effect of these polymorphisms on longevity was sepa- rately investigated in men and women between the groups. Genotype frequency and allele frequency for ) 1082 G⁄A IL-10 promoter SNP were different between the groups in men (Fisher’s exact, P < 0.05, Table 3) but not in women (Fisher’s exact, P > 0.05, Table 4). On the other hand, no significant difference was found between the experimental and control groups when gender was considered in the TNF-a ) 308 G⁄A polymorphisms (Fisher’s exact, men: P > 0.05, Table 3, and women: P > 0.05, Table 4).