Methods

Subjects

≥ A total of 493 elderly individuals (females 94 years ≥ old; males 90 years old) from Dujiangyan (Sichuan province, China) agreed to participate in this study. Ages were authenticated by the official certification of the Fifth Nation Census in China as well as by the infer- ence of the number of generations, and the memories of these, their offspring and other local elderly people. The age was confirmed when the certification and the infer- ence were consistent. We also recruited 425 healthy, local individuals between the ages of 32 - 73 years to act as a control group. This control group included 110 spouses of the children of the elderly participants. The ’ subjects information is listed in Table 1.

Statistical analysis

A test of Hardy Weinberg Equilibrium (HWE) was per- formed using Genepop 3.4 [25]. The program PHASE v2.1 [26,27] was used to estimate the haplotype of each individual. Statistical analyses were performed with SPSS (version 13.0, Chicago, IL). Allele distributions and gen- otype frequencies of the elderly (test) and young (con- ’ trol) groups were compared with Pearson s chi square p p test, using a two-tailed value was and considering < 0.05 to be significant. We used a Bonferroni correction to correct for multiple tests of the same data.

All participants indicated informed consent by signing a form after they had been given a clear explanation of the potential risks of the study. This research was approved by the Ethics Committee on human experi- mentation of Kunming Institute of Zoology, Chinese Academy of Sciences and relevant bodies in Dujiangyan. The study was done in accordance with the Declaration of Helsinki and subsequent amendments.

To explore the potential function of this microsatellite locus, we categorized the whole population into three genotype groups depending on owning 2, 1, or no copies of major alleles (19- and 21-repeat): 19/19 (21/ 21), 19/non (21/non), and non/non. We performed chi square test to compare 1) each genotype to the sum of the other two, 2) the frequencies of three genotypes, and 3) the distribution of major allele (19(21)-allele vs. non) in elderly and control groups in order to detect any potential functions of the major alleles.

Genotyping

DNA was extracted from white blood cells in whole blood by the standard phenol/chloroform method. Pri- mers for PCR followed those used in previous study [21]. The forward primer was labeled at the 5’ -end with 6-FAM fluorescent dye. PCR reactions each included 20 μ ng genomic DNA as template, 2.5 L 10× PCR buffer, 1.0 μ L dNTP (2.5 mmol·L-1), 0.5 μ L BSA (bovine serum albumin, 200 μ g·ml-1), 0.5 μ L forward primer (10 pmol·μ L-1), 0.5 μ L reverse primer (10 pmol·μ L-1), 0.125 μ L rTaq polymerase (Takara, Dalian, China) and dis- μ tilled water to a total volume of 25 microliter ( L).

Results

In this study, we examined the microsatellite poly- morphism in IGF1 gene promoter in 493 elderly indivi- duals (241 females and 252 males) and 425 control individuals (223 females and 202 males). The length range of the microsatellite locus was 17 to 23 repeats (only one person has a 13-repeat allele), and the most common alleles are 19-repeat (39.12%) and 21-repeat

Table 1 Demographics of the study participants

Group N (Male/Female) Age (Mean ± SD, years) Nationality Range of age (year) Elderly 493 (252/241) 94.97 ± 3.15 Han Male ≥ 90; Female ≥ 94 Control 425 (202/223) 57.17 ± 9.21 Han 32-73

(26.14%). The genotype distribution followed Hardy- Weinberg Equilibrium (HWE) in both elderly and control groups, as well as when stratified by gender p ( > 0.05).

frequencies in the two groups considered as a whole. However, when we stratified the groups by gender, there were more individuals with genotype 21/non (21-repeat/ non-21-repeat) in the male elderly group than in the p male control group (42.06 vs. 32.18%, = 0.031). After Bonferroni correction, the result was no longer significant.

All alleles and their frequencies are listed in Table 2. Allele frequencies of elderly and control groups were p not significantly different ( = 0.922, df = 9), even when stratified by gender. The results of comparison of the distribution of common alleles (19- and 21-repeat) in both groups are listed in Table 3. There were no signifi- cant differences in genotype distribution and allele

To investigate potential causes of the different fre- quencies of the genotype 21/non in male participants, we compared the distributions of all possible genotypes in the male elderly and male control groups. Altogether,

Table 2 Allele frequencies of the microsatellite in sampled elderly and control groups.

Allele1 All Women Men Elderly(%) Control(%) Elderly(%) Control(%) Elderly(%) Control(%) 13 1(0.10) 0 1(0.21) 0 0 0 16 1(0.10) 1(0.12) 0 1(0.22) 1(0.20) 0 17 79(8.01) 68(8.00) 37(7.68) 34(7.62) 42(8.33) 34(8.42) 18 169(17.14) 138(16.24) 80(16.60) 71(15.92) 89(17.66) 67(16.58) 19 380(38.54) 338(39.76) 198(41.08) 173(38.79) 182(36.11) 165(40.84) 20 69(7.00) 54(6.35) 29(6.02) 28(6.28) 40(7.94) 26(6.44) 21 260(26.37) 220(25.88) 124(25.73) 121(27.13) 136(26.98) 99(24.51) 22 23(2.33) 28(3.29) 12(2.49) 15(3.36) 11(2.18) 13(3.22) 23 4(0.41) 2(0.24) 1(0.21) 2(0.45) 3(0.60) 0 24 0 1(0.12) 0 1(0.22) 0 0 p = 0.922 p = 0.948 p = 0.455

1. The number for an allele is the amount of repeats of this allele.

Table 3 Chi square test results for the genotype distribution and allele frequencies of the 19- and 21-repeat alleles elderly and control groups.