0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2010.06.030

2. Methods

Haploview4.2 software and the allele frequency in public database of the National Center for Biotechnology Information (NCBI; http:// www.ncbi.nlm.nih.gov) in SIRT1 gene region (GeneID:23411). We downloaded the China Han population's SNP data of SIRT1 gene from HapMap data base (HapMap Genome Browser ( phases 1, 2 and 3 — merged genotypes and frequencies )). We then analysed these data by Haploview4.2 software [18] and got 3 TagSNPs: rs11596401, rs2236319 and rs4746720. We searched for aging-related articles of 3 SNPs in NCBI database. We did not find any related research article with rs11596401. We found several aging-related articles for rs2236319, but none were found for rs2236319 [12,19]. rs11596401 and rs2236319 both lie in intron regions, but since SNPs in intron region are commonly considered as non-functional SNPs, this study selected the TagSNP rs4746720 which lies in the 3' untranslated region (UTR). After combining the SNP frequency of SIRT1 gene from Hapmap database and NCBI SNP database, we did not find suitable SNPs in SIRT1 gene exon regions, so, we select the rs3758391 near the 5' end of the SIRT1 gene which correlate with aging in other population studies [12,46]. From the selected SNPs, we were able to find rs3758391 near the 5' end of the SIRT1 gene and rs4746720 in the 3' untranslated region. DNA was extracted from white blood cells by a standard phenol/chloroform method [20]. Primers were designed using the Web program Primer3 (http://frodo.wi.mit.edu/primer3/). Primers for SIRT1 rs3758391 and rs4746720, and extension primers are in Table 1. Genotyping of SIRT1 SNPs used Snapshot primer extension assays (ABI). Polymerase chain reactions (PCR) were performed with high-quality hot-start enzyme (HotStarTaq polymer- ase) and landing type (Touch-Down PCR, TD-PCR) technology. PCR products were purified using a Watson DNA Purification Kit (Watson Company, Shanghai, China). The snapshot kit was used for single base extension reaction, with 1 unit SAP for purification of the micro- sequencing product. Resulting fluorescently labeled products were

2.1. Study subjects

Healthy Chinese Han people were recruited for this study. ""Based on “The Developing of China Aging Career”[16], those who are ≥60 years old were classified as aging and those who are b60 years old as young. A representative sample of 669 apparently healthy community-dwelling men and 684 women between 35 and 91 years old were recruited from Shenyang in the LiaoNing Province of China. All participants signed informed consent forms after receiving a clear explanation of the potential risks of the study.

All participants had normal physical examinations. Normal brain function examinations included Pfeffer's Outpatient Disability Ques- tionnaire, Verbal Fluency Test, Mini-Mental State Examination, Activity of Daily Living, Center for Epidemiologic Studies Depression Scale , The Stroop Color–Word Test, Clock Drawing Test, and Digit Span. Laboratory findings for hemogram, peripheral smear, urine, electrolytes, chest X-ray, kidney ureter bladder, abdominal ultra- sound, pulmonary function, echocardiography (including heart and carotid artery), and ECG were all normal (Fig. 1). The final group was 482 healthy unrelated individuals, including 246 aging and 236 control young individuals. The subject selection method was similar with our previous study [17]. This research was approved by the Ethics Committee on Human Experimentation of China Medical University-Shenyang, and was conducted in accordance with the Declaration of Helsinki and subsequent amendments.

The candidate SNPs were selected based on previous study reports. The SNPs associated with aging combined the result analyses by

Neighborhood to provide residents of self-evaluation list of basic health

eT130% of a random sample survey according to sex and age <<LLSurvey done past disease investigation, physical examinationSL Those who meet the inclusion criteriaInvestigation of brain functionsb Those who meet the inclusion criteriaSpecimens from blood, urine samples, testing blood, urine and blood biochemistryJ Those who meet the inclusion criteriaDo electrocardiogram, chest X-ray and cardiovascular ultrasoundJL Those who meet the inclusion criteriaDetermine the selected subject of investigation,do living conditions and food frequency questionnaire Those who meet the inclusion criteria If the selected object is less than the required tasks, then again

130% of a random sample survey according to sex and age

Survey done past disease investigation, physical examination

Specimens from blood, urine samples, testing blood, urine and blood biochemistry

Do electrocardiogram, chest X-ray and cardiovascular ultrasound

Determine the selected subject of investigation,do living conditions and food frequency questionnaire

If the selected object is less than the required tasks, then again

Fig. 1. Healthy subjects selection process.

analyzed by capillary electrophoresis using an ABI3730 (AppliedBio- systems, Foster City, CA) DNA analysis system and Gene Mapper 4.0 software (Applied Biosystems).

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

SNP Young subjects Aging subjects p OR (95%CI); (%) n=236 (%) n=246 rs3758391C/C(%) 14 (5.93) 5 (2.03) 0.027 3.040 (CC/CT+TT) (1.077–8.576) C/T (%) 71 (30.08) 66 (26.83) T/T (%) 151 (63.98) 175 (71.14) C (%) 99 (20.97) 76 (15.45) 0.026 1.453 (1.044–2.021) T (%) 373 (79.03) 416 (84.55) rs4746720C/C(%) 90 (38.14) 73 (29.67) 0.049 1.461 (CC/CT+TT) (1.000–2.135) C/T (%) 103 (43.64) 113 (45.93) T/T (%) 43 (18.22) 60 (24.39) C (%) 283 (59.96) 259 (52.64) 0.022 1.347 (1.043–1.739) T (%) 189 (40.04) 233 (47.35)

2.3. Statistical analysis