Material and methods

A sample of 110 unrelated elderly subjects (485 years, mean age 90.2 years) was recruited from different parts of Jordan. Additionally, 120 unrelated control subjects (range from 20 to 50 years, mean age 32.0) were matched to long-lived individuals for geographical origin. Male to female ratio was 1.6:1 in the elderly group and 1.4:1 in the control group (p 4 0.05). Subjects with cardiovascular diseases, diabetes or cognitive impairments were excluded from the study. The experimental design was similar to longevity studies reviewed by Glatt et al. [18]. The age criterion was chosen based on the mortality rate in the Jordanian population which remains approxi- mately constant from childhood until late 40s and then starts inclining exponentially at 50 years of age getting maximum level in the late 70s [19]. Accord- ing to the 2007 census, the total population of Jordan was 5.7 million, percentage of individuals of 65 years of age or older was 4.1%, and life expectancy at birth in the total population was 73 years [20]. Therefore, reaching above 85 year-old is a rarity in Jordan and oldest old people are considered exceptional individ- uals in Jordan. To verify age, an official identification document was required from all participants in the study. Written informed consents were obtained from all participants in accordance with the require- ments of the Institutional Review Board of Jordan University of Science and Technology.

The genotype distributions of the examined polymorphisms were analysed in agreement with Hardy–Weinberg equilibrium. Statistical evaluation of results was done by comparing allele, genotype and predicted phenotype distributions using the w2 test. If n 5 5, exact Fisher statistic was used. The SPSS 15.0 statistical software package (SPSS Inc., Chicago, IL) was used for all statistical evaluations. Because several significant associations were ob- served among the 24 associations examined in the study, which exceeds the number that might be observed by chance (one significant association/ 24 tests), a p-values of 5 0.05 was considered significant without adjustments for multiple com- parisons.

Blood samples (1–3 ml) in ethylenediaminetetra- acetic acid (EDTA) tubes were obtained from all subjects. Deoxyribonucleic acid (DNA) was ex- tracted from all samples using Wizard DNA Extrac- tion Kit (Promega, Madison, USA) according to manufacturer instructions. DNA samples were stored at 7208C until used. The concentration of

Result

Table II shows the frequency of homozygous and heterozygous genotypes for rs2241766, rs1501299 and rs266729 ADIPOQ gene SNPs, and for rs7799039 LEP gene promoter SNP in the elderly

Table I. Primers sequences, PCR conditions and restriction enzymes used for genotyping of SNPs.

Primers Annealing temperature Restriction enzyme Fragment length F: 50-AAGAACCGGCTCAGATCCTGC-30 58 HinP1I (378C) CC Ł Wt 120 R: 50-GCTGCCACCCACTTAGGTGTTC-30 GG Ł Mt 100, 20 F: 50-CTGAGATGGACGGAGTCC TTT-30 58 SmaI (258C) TT Ł Wt 456 R: 50-CCAAATCACTTCAGGTTGCTT-30 GG Ł Mt 313, 143 F: 50-GTCTAGGCCTTAGTTAATAATGAATG-30 58 BsmI (378C) GG Ł Wt 82, 22 R: 50-GAGAAAGGAGATCCAGGTAAGA-30 TT Ł Mt 104 F: 50-TTTCCTGTAATTTTCCCGTGA-30 52 HhaI (378C) AA Ł Wt 242 R: 50-AAAGCAAAGACAGGCATAAAA-30 CC Ł Mt 181, 61

Table II. Frequencies of ADIPOQ and LEP alleles and genotypes in control and elderly groups.

Table III. Frequencies of ADIPQO and LEP alleles and genotypes in control and elderly men subjects.

Genotypes Control Elderly p and alleles group, n (%) group, n (%) value rs2241766 ADIPQO* TT 40 (56.3) 41 (60.3) TG 27 (38.0) 25 (36.8) 0.799{ GG 4 (5.7) 2 (2.9) Allele T 107 (75.4) 107 (78.6) 0.606{ Allele G 35 (24.6 ) 29 (21.4) rs1501299 ADIPQO* GG 21 (29.6) 37 (54.4) GT 42 (59.2) 27 (39.7) 0.011{ TT 8 (11.2) 4 (5.9) Allele G 84 (59.2) 101 (74.3) 0.011{ Allele T 58 (40.8) 35 (25.7) rs266729 ADIPQO* CC 48 (67.6) 43 (63.2) CG 19 (26.8) 23 (33.8) 0.509{ GG 4 (5.6) 2 (3.0) Allele C 115 (81.0) 109 (80.1) 0.980{ Allele G 27 ( 19.0) 27 (19.9) rs7799039 LEP* AA 16 (22.5) 23 (33.8) AC 29 (40.8) 34 (50.0) 0.021{ CC 26 (36.6) 11 (16.2) Allele A 61 (43.0) 80 (58.8) 0.011{ Allele C 81 (57.0 ) 56 ( 41.2)

Genotypes Control Elderly p and Alleles group, n (%) group, n (%) value rs2241766 ADIPOQ* TT 68 (56.6) 70 (63.6) TG 43 (35.8) 37 (33.6) 0.231{ GG 9 (7.6) 3 (2.8) Allele T 179 (74.6) 177 (80.5) 0.164{ Allele G 61 ( 25.4) 43 (19.5) rs1501299 ADIPOQ* GG 49 (40.8) 58 (52.7) GT 56 (46.6) 43 (39.1) 0.172{ TT 15 (12.6) 9 (8.2) Allele G 154 (64.2) 159 (72.3) 0.078{ Allele T 86 (35.8) 61 (27.7) rs266729 ADIPOQ* CC 69 (57.5) 68 (61.8) CG 43 (35.8) 35 (31.8) 0.809{ GG 8 (6.7) 7 (6.4) Allele C 181 (75.4) 171 (77.7) 0.635{ Allele G 59 (24.6 ) 49 (22.3) rs7799039 LEP* AA 29 (24.2) 34 (30.9) AC 53 (44.2) 55 (50) 0.082{ CC 38 (31.6) 21 (19.1) Allele A 111 (46.3) 123 (55.9) 0.051{ Allele C 129 ( 53.7) 97 (44.1 )

*All groups were in Hardy–Weinberg equilibrium (p40.05). {Fisher Exact test. {Chi Square test.

*All groups were in Hardy–Weinberg equilibrium (p40.05). {Fisher Exact test. {Chi Square test.

versus the young group. No differences in the genotype frequencies of the examined ADIPOQ SNPs (p ¼ 0.231 for rs2241766 SNP, p ¼ 0.172 for rs1501299 and p ¼ 0.809 for rs266729 SNP) and the frequency of various alleles (p ¼ 0.164 for rs2241766 T/G alleles, p ¼ 0.078 for rs1501299 G/T alleles and p ¼ 0.635 for rs266729 C/G alleles) were detected between the elderly and the young group. Similarly, rs7799039 LEP gene promoter SNP was not different between the two groups (for genotype frequencies: p ¼ 0.082 and for allele frequencies:

Table IV. Frequencies of ADIPOQ and LEP alleles and genotypes in control and elderly women subjects.