2.1. Subjects

In this study, 485 unrelated elderly individuals (age P 94 for fe- male and age P 90 for male) were recruited from Dujiangyan in Sichuan province of China. Ages were authenticated by the official certification of the Fifth Nation Census in China as well as by the inference of the number of offspring generations, and the memo- ries of these, their offspring and other local elderly people. The age was confirmed when the certification and the inference were consistent. At the same time, 392 control individuals, which were healthy local younger people including 110 spouses of the children of the longevity people, were recruited from the same area (age 22–73). The subjects’ information is listed in Table 1. All partici- pants signed informed consent after they had been given a clear explanation of the potential risk of the study. This research was ap- proved by the Ethics Committee on human experimentation of Kunming Institute of Zoology, Chinese Academy of Sciences and relevant bodies in Dujiangyan. The study was done with the Decla- ration of Helsinki and subsequent amendments.

3. Results

In this study, we detected three SNPs ((cid:2)439T/A (rs2288377), (cid:2)541T/C (rs5742612) and (cid:2)1246C/T (rs35767)) in the 2.5 kb DNA fragment, which was consistent with the data in HapMap pro- ject (http://www.hapmap.org/). No novel SNP site was discovered. All of the genotype frequencies of the three SNPs in longevity and control populations followed the Hardy Weinberg equilibrium, even when as stratified by gender. In addition, we explored the linkage disequilibrium between the three SNPs and found that these three SNPs were linked together totally (D0 = 0.997 and r2 = 0.791 for rs35767 and rs5742612, D0 = 1.0 and r2 = 1.0 for rs5742612 and rs2288377).

2.2. Genotyping

The genotype distributions and allele frequencies of the three SNPs are listed in Table 2. Our results indicated that there were no significant differences in the genotype distribution and allele frequencies between the longevity and control groups of the three sites, respectively. Meanwhile, we stratified the genotype distribu- tions and allele frequencies by gender. And there were no signifi- cant differences as well. We also constructed the haplotypes of the three SNPs and performed statistical analyses. The results are the same as allelic results and no significance was found (data not shown).

by the standard phenol/chloroform method (Sambrook et al., 1989). The 2.5 kb DNA fragment located upstream of the transcrip- tion start site of IGF-1 gene exon 1 (chromosome 12: 101, 398, 454–101, 400, 954, NCBI, Build 36.1) was amplified in a long and accurate PCR reaction using an LA-PCR Kit (Takara, Dalian, China). PCR was performed in a 25 lL volume with 20 ng genomic DNA as template, 2.5 lL 10(cid:3) PCR buffer, 1.0 lL dNTP (2.5 mmol L(cid:2)1), 0.5 lL BSA (bovine serum albumin, 200 lg ml(cid:2)1), 0.5 lL forward primer (50-GAGAACATCAAAATGGGTGATAGACT-30, 10 pmol lL(cid:2)1), 0.5 lL reverse primer (50-AAATAATTGGG TTGGAAGACTGCTG-30, 10 pmol lL(cid:2)1), 0.125 lL LA-Taq polymerase (Takara, Dalian, China) and 19.9 lL distilled water. The PCR procedure was as follows: an initial denaturation of 5 min at 95 (cid:3)C, followed by 35 cycles of 95 (cid:3)C for 30 s, 59.5 (cid:3)C for 1 min and 72 (cid:3)C for 2 min, and final extension at 72 (cid:3)C for 10 min. PCR products were purified using a Watson DNA Purification Kit (Watson Company, Shanghai, China) and were directly sequenced with five pairs of inner primers (the primer information can be reached by request to the authors) on an ABI 3700 (AppliedBiosystems, Foster City, CA) according to the manu- facturer’s protocol. In order to collect the information of common SNPs in this fragment, we firstly chose 30 individuals in each of the two groups respectively and performed sequencing of the whole fragment. According to the results, we sequenced the entire two regions containing the SNP sites directly. Any ambiguous re- sult was confirmed by another PCR and sequencing.

4. Discussion

gene at present. In the early 1990s, much research was done on the analyses of the functional mechanism of the promoter region of IGF-1 (Jansen et al., 1992; Kim et al., 1991). However, the results did not provide adequate information to understand the regulation mechanism of IGF-1.There are two promoters located in either exon 1 (P1) or exon 2 (P2) in IGF-1 gene and exon1 50-UTR is con- sidered as a potential region for regulatory elements (Rodriguez et al., 2007), which is also the reason why we chose this segment. Considering the important role of IGF-1 in life processes, it is nec- essary to elucidate the association between the regulation of IGF-1 expression and the ageing process distinctly. By our knowledge, it is the first time to report an association study between the SNPs in the 50 flanking region of IGF-1 and longevity in the Han Chinese population. In our research, the longevity and control populations were recruited from the same area. They shared the same living environment and common diet habits, which exclude the non-ge- netic factors to the greatest extent. Our investigation indicated that individuals in both populations were native Han inhabitants in this research, thus population stratification was avoided to a certain ex- tent in our study (Marchini et al., 2004).

2.3. Statistical analysis

The HWE (Hardy Weinberg equilibrium) test and linkage dis- equilibrium test were performed by Genepop 3.4 software (Ray- mond and Rousset, 1995) and Haploview software (Barrett et al.,

Table 1