MATERIALS AND METHODS

Centenarians

One hundred twenty-two centenarians from the Danish Centenarian Study, including 28 men, participated in the present study. This cohort has previously been de- scribed.2,3,18 Briefly, The Danish Centenarian Study is a nationwide epidemiological survey of all individuals living in Denmark who celebrated their 100th birthday from April 1, 1995, to May 31, 1996 (N 5 276). All participants were identified in the Civil Registration System in Denmark and admitted to the study as they turned 100. The participation rate was 75% (n 5 207). There was no difference between participants and nonparticipants regarding sex and the proportion living at home or in a nursing home. The same geriatrician (KA-R) and a geriatric nurse performed an in- terview and a physical examination at the place of the sub- ject’s residence within 5 months of the anniversary, and a review of medical records was conducted. Not all subjects agreed to a blood test and a full physical examination; ac- cordingly, the TNF  308 G4A polymorphism was analy-

Plasma Cytokines and CRP in Centenarians

Plasma was stored at   801C. Ethylenediaminetetraacetate was used as an anticoagulant. Levels of TNF-a and IL-6 were measured using high sensitive enzyme-linked immunosorbent assay (ELISA) kits, and IL-8 was meas- ured using Quantikine ELISA kits from R & D Systems (Abingdon, UK), as previously described.3 All samples and standards were run as duplicates, and the mean was used in statistical analyses. The detection limits were 0.1 to 0.2 pg/ mL for TNF-a and IL-6, and 1 ot 2 pg/mL for IL-8. The intra-assay and interassay coefficients of variations were 16% and 25% for TNF-a, 9% and 15% for IL-6, and less than 10% for IL-8. CRP levels were measured using a

Table 1. Prevalence of Tumor Necrosis Factor (TNF)  308 G4A Polymorphism in Different Age Groups Age TNF Polymorphism 18–31 (n547) 85 (n 5178) 100 (n5122) TNF (cid:1)308 GG, n (%) 34 (72) 115 (65) 82 (67) TNF (cid:1)308 GA, n (%) 12 (26) 59 (33) 35 (29) TNF (cid:1)308 AA, n (%) 1 (2) 4 (2) 5 (4)  TNF (cid:1)308 A prevalence 14/94 (15) 67/356 (19) 45/244 (18)

Note: GG/GA/AA versus age group: Fisher exact test P5.7. *) Number of A alleles/number of total alleles (%).

standardized high-sensitivity assay in the central laboratory of Odense University Hospital.

ians) and sex (assuming different baseline hazard functions in men and women) to exclude the possibility of linear and nonlinear interactions between these parameters and the TNF polymorphism. Analyses of variance (ANOVAs) were used to detect differences in circulating levels of inflamma- tory markers (dependent variable) in relation to the TNF-a polymorphism (independent variable). Probit plots were used to check for normality in the distribution of inflam- matory markers. To approximate normality, TNF-a was left untransformed, whereas IL-6, IL-8, and CRP were log transformed. Po.05 was considered significant in all e analyses.

Follow-Up of Centenarians

Vital status and time of death were obtained from the Civil Registration System in Denmark in June 2001. The obser- vation period was 5 to 6 years.

Octogenarians and Young Controls

One hundred seventy-eight 85-year-olds, including 66 men, were from the 1914 cohort, Research Center for Prevention and Health, Glostrup, Denmark.19 Blood samples were collected during a home visit as a part of survey of 85-year- olds in 2000. Collection of blood samples from 47 young volunteers (18–30), including 14 men, took place during the same time period.

RESULTS

The TNF  308 G4A genotypes were in Hardy-Weinberg equilibration in centenarians and in 85-year-olds and young controls, and there was no difference in the distribution of genotypes or the prevalence of A-allele carriers between the three age groups (Table 1). There was no interaction be- tween sex and the  308 genotype in a multinomial logistic regression analysis including the three age groups as the dependent variable and the three genotypes, sex, and an interaction between sex and genotype as independent var- iables (data not shown).

Statistical Analysis

Statistical analyses were performed using SPSS, version 11.0 (SPSS Corp., Chicago, IL) and Stata 8 (Stata Corp., College Station, TX). Fisher exact test and multinomial lo- gistic regression analyses were used to investigate changes in the distribution of the TNF   308 G4A polymorphism across different age groups. A binary logistic regression analysis was used to evaluate whether dementia was asso- ciated with the polymorphism, adjusted for the effect of sex and an interaction between sex and the polymorphism (cross-sectional analysis). Cox regression models were used to explore associations between the polymorphism and survival (longitudinal analysis). Confounders in the survival analysis included dementia and plasma levels of TNF-a representing known predictors of the mortality risk in the population of centenarians.4 Survival analyses were statis- tically stratified by dementia (assuming different baseline hazard functions in demented and nondemented centenar-

The TNF  308 G4A Polymorphism and Dementia in Centenarians

Sixty-one centenarians were classified as being demented, including 11 men. The GA genotype was associated with a lower prevalence of dementia than the GG genotype, whereas such a difference was not detected with regard to the AA genotype (Table 2). There was no interaction be- tween sex and the genotype; furthermore, the effect of the

2. Tumor Necrosis Factor (TNF)  308 G4A Polymorphism and Risk of Dementia in Centenarians at

 Prevalence of Odds Ratio TNF Polymorphism Dementia/total (%) (95% Confidence Interval) TNF (cid:1)308 GG 46/83 (55) 1 TNF (cid:1)308 GA 12/35 (34) 0.43 (0.19–0.98) .02 TNF (cid:1)308 AA 3/5 (60) 1.23 (0.20–7.78) .8

Odds ratio in a logistic regression analysis with GG as reference.

genotype was independent of sex in the logistic regression analysis (data not shown).