2.3. Statistical analysis

The HWE (Hardy Weinberg equilibrium) test and linkage disequilibrium test were performed by Genepop 3.4 software [21] and Haploview software [18], respectively. The haplotype estimation was conducted by Phase 2.1 software [22,23]. Statistical analyses were performed by SPSS software package (SPSS ver. 13.0, Chicago, IL). The genotype, alleles and haplotype frequencies in both groups were compared by Pearson's chi square test. The 2-tailed pb0.05 was considered significant.

3. Results

We screened 482 unrelated healthy Chinese subjects for 2 well- known functional polymorphisms near the 5' end of SIRT1, and in the 3' UTR. For a comparative analysis of genotypes, the cohort of subjects was divided into 2 groups: 236 young subjects, from 35 to 59 years old (mean±SD: 47.3±7.2 years; 45.3% male); and 246 aging subjects, from 60 to 91 years old (71.2±7.1 years; 49.2% male). All SIRT1 polymorphisms were in HWE for the entire cohort of subjects (pN0.05). We explored linkage disequilibrium between the 2 SNPs, and found linkage (D' value: 0.64, r2=0.07 for rs3758391 and rs4746720).

of SIRT1 with the NF-kappaB, FoxO and p53 signaling systems might resist stress, modulate aging-associated disease, delay the aging process and extend lifespan [29,36,37]. As a key regulator of energy and metabolic homeostasis [35,38], SIRT1 has also been linked to increases in maximum lifespan mediated by calorie restriction. SIRT1 activates a critical component of calorie restriction in mammals [39,40], and calorie restriction activates SIRT1 [41] . SIRT1 interacts with the insulin/IGF-1 signaling pathway [42–44], promotes fat mobilization [40], improves insulin sensitivity [45], reduces the incidence of age-related disease, slows the pace of aging, and increases maximum lifespan [10,11].

Differences in allele and genetype frequency were seen between the groups (Table 2): rs3758391/C is more common than rs3758391/T in aging subjects (odds ratio=1.453, p=0.026), rs3758391/CC is more common than rs3758391/CT and rs3758391/TT in aging subjects (odds ratio=3.040, p=0.027). For the 3' SNP, rs4746720/C was more common than rs4746720/T in aging subjects (odds ratio=1.347, p=0.022), rs4746720/CC was more common than rs4746720/CT and rs4746720/CT in aging subjects (odds ratio=1.461, p=0.049). Genotype distributions and allele frequen- cies showed no differences in different genders (Table 3).

We have little knowledge about the polymorphism of SIRT1 gene with healthy aging at present, although there are several studies on the association between the SNPs and aging. In 2006, the relationship between 5 SNPs (rs3758391, rs1885472, rs2273773, rs10997870, and rs2234975) in SIRT1 gene and 1026 unrelated German long-lived individuals (mean age: 98.3 years) were studied but found no association between SIRT1 genes polymorphisms and exceptional human longevity [14]. In 2007, the relationship between five SNPs (rs3758391, rs3740051, rs2236319, rs2273773, and rs3818291) in SIRT1 gene and 1245 (mean age: 85+ years) unrelated Netherlands people were studied. They were able to observe associations between better cognitive functioning and rs3758391 T-allele [12]. In 2008, the relationship between four SNPs (rs12778366, rs3740051, rs2236319, and rs2273773) inSIRT1geneand326FinnishAlzheimer's disease (mean age: 72.1±6.8 years) were studied but found no association between SIRT1 genes polymorphisms and AD risk in the Finnish population [19]. In 2010, the relationship between a SNPs rs3758391 in SIRT1 gene and 519 Mexico City subjects (mean age:

Statistical analysis on the SNP haplotypes showed that the CC haplotype was significantly increased in the aging group compared to the younger individuals (odds ratio=1.63, p=0.01; Table 4). A frequency of less than 0.03 was considered negligible. As shown in Table 4, the other estimated haplotypes of CC, TC and TT were distributed similarly among young and aging subjects, and showed no significant differences.

4. Discussion

SIRT1 induces cell cycle arrest and DNA repair, attenuates apoptosis [5,24], and regulates premature cellular senescence [25]. Our previous work suggested that apoptosis might be an important factor in endothelial senescence and vascular aging [26], and SIRT1 might influence aging through attenuation of p53-dependent apo- ptosis [27,28]. SIRT1 evolved to bind or deacetylate a wide variety of factors that affect aging [29,30], including NF-kappaB [31], FOXO [32,33], p53 [27,34], and heat shock factor 1 (HSF1) [35]. Interaction

Table 3

Genotype and allele frequencies of rs3758391 and rs4746720 in different gender groups.

SNP Male subjects Female subjects p OR (95%CI); (%) n=228 (%) n=254 rs3758391C/C(%) 9 (3.95) 10 (3.94) 0.995 1.003 (CC/CT+TT) (0.400−2.513) C/T (%) 64 (28.07) 73 (28.74) T/T (%) 155 (67.98) 171 (67.32) C (%) 82 (17.98) 93 (18.31) 0.896 0.978 (0.705−1.358) T (%) 374 (82.02) 415 (81.69) rs4746720C/C(%) 76 (33.33) 87 (34.25) 0.831 0.960 (CC/CT+TT) (0.658−1.401) C/T (%) 101 (44.30) 115 (45.28) T/T (%) 51 (22.37) 52 (20.47) C (%) 253 (55.48) 289 (56.89) 0.660 0.944 (0.732−1.219) T (%) 203 (44.52) 219 (43.11)

Table 1

Primers for rs3758391 and rs4746720 in SIRT1 gene.

SNP Oligo Amplicon rs3758391 C/T F 151 TGTACCATAAACTCGTAGAGGTCC R CCTGAAATTCTGGCACACTG A 28 ACTAAGGACCCATATAACCC rs4746720 C/T F 239 AGGGAACAGCTAATCTAGACCA R AAAGTAAAGACAACCGAGTGCT A 36 CAAAATCTGTTACGCTAAACTT

F: forward, R: reverse, A: extension primer.

work was supported by the National Basic Research Program of China (No.2007CB507405).

Table 4

Haplotypes for rs3758391 and rs4746720 in SIRT1 gene.

Haplotype Frequency of Frequency of chi2 P OR(95%CI) haplotype aging group young group 1—C C 0.18 0.12 7.44 0.01 1.63 (1.14–2.33) 2—C T 0.02 0.03 0.53 0.92 0.75 (0.35–1.61) 3—T C 0.41 0.40 0.09 1.00 1.04 (0.80–1.34) 4—T T 0.37 0.44 4.24 0.15 0.76 (0.58–0.98)

References

[

] Yashi

I,Iachine IA,Harri