Genotypes obtained for the 90 sample were as follows: 62R/2R, 27 2R/4R, 4 2R/7R, 4 3R/3R, 25 3R/4R, 7 3R/7R, 135 4R/4R, 1 4R/5R, 3 4R/6R, 82 4R/7R, 1 4R/11R, 1 5R/6R, 1 5R/7R, 1 6R/6R, 11 7R/7R, and 1 7R/8R. The genotypes of the control individuals were as follows: 60 2R/2R, 16 2R/3R, 345 2R/4R, 1 2R/5R, 58 2R/7R, 1 2R/8R, 4 3R/3R, 127 3R/4R, 17 3R/7R, 1715 4R/4R, 5 4R/5R, 1 4R/6R, 493 4R/7R, 1 4R/8R, and 1 non-7Rx. There were no significant deviations from the expected Hardy-Weinberg equilibrium. Observed allele frequencies for the oldest-old sample were as follows: 2R (0.07),3 R(0.06),4 R(0.66),5 R(0.005),6 R(0.009),7 R(0.189),and8 R(0.0016). Observed allele frequencies for the population and ancestry-matched controls were as follows:2 R(0.09),3 R(0.03),4 R(0.76),5 R(0.002),6 R(less than or equal to)0.001,and7 R(0.12). The DRD4 7Rx category contains individuals with at least one DRD47Rx allele (or derived allele such as the DRD45Rx, DRD46Rx, DRD48Rx, DRD49Rx, DRD410Rx, and DRD411Rx), and the non-DRD47Rx category contains the remaining individuals. A representative sample of 140 oldest-old individuals was resequenced at the DRD4 exon 3 polymorphism to determine the haplotype (273 alleles). The common 7R (1-2-6-5-2-5-4) haplotype or 7R derivatives were the predominant 7R sequences, with the fraction of common to rare haplotypes similar to that observed in prior population studies.

In the oldest-old, we also observed a significant association between the DRD4 7Rx genotype and higher reported activity levels in both males and females. This association was observed not only for measures obtained in 1981 but also when the measured variable was current self-reported activity. Oldest-old participants with the DRD4 7Rx genotype were twice as likely to report exercising two hours per day in 1981. The correlation of higher activity with DRD4 7Rx genotype was not confounded by age, gender, alcohol intakeIn the larger Leisure World cohort (Paganini-Hill et al., 2011), the proportion of the variance in physical activity explained by DRD4 7R in this oldest-old population was r2 approximately 0.023. 

Results:
To test our hypothesis, we determined DRD4 exon 3 VNTR genotypes/sequences for 310 90+ study individuals (the oldest-old; mean age, 95.2 years; age range, 90-109 years; 91 males) (Fig. 1). These individuals represent 1.4-2.9% of their initial birth cohort and, by definition, should have altered allele frequencies of genes that impact health and longevity. When compared with matched European ancestry controls of individuals aged 50-90 years younger (N=2902; age range, 74-75 years), the oldest-old had a 66% increase in observed 7R/x genotype (Fig. 2) (0.364 vs 0.219, χ2=33.28, p=3.5 x10^-9; χ2=0.026). This extremely small p value would be significant at a genome-wide level following Bonferroni correction. Comparing allele frequency rather than genotype, the frequency of the 7R allele is greater in the oldest-old population by 72% (0.205 vs 0.119, χ2=38.27, p<6.15 x10^-10). This increase was preferentially observed in females (0.393 vs 0.297 males, χ2=9.65, p=0.002), among whom the difference was significant at a genome-wide level (0.393 vs 0.219, χ2=34.66, p<1.7 x10^-9). In contrast, the difference in males, while significant, did not reach genome-wide level significance (0.297 vs 0.219, χ2=3.11, p=0.034) (Fig. 2).

To test the association of the DRD4 gene with longevity and responses to the environment directly, we compared the lifespans of mice that expressed DRD4 (HT and WT) versus those of KO mice (Rubinstein et al., 1997) that lacked the DRD4 gene, when reared in either a DE (standard laboratory cage) or EE (group cages with multiple options for physical activity and exploration) environment (Ullman-Cullere and Foltz, 1999; Quimby et al., 2010). DRD4 genotype significantly affected the lifespan (F=5.74, p=0.004, χ2=0.03; Fig. 4). DRD4 KO mice (N=68) had a 9.7% shorter lifespan (mean and SEs: 94.8 ± 2.08 weeks; 95% CI, 91-99) than HT mice (N=57; 104 ± 1.82 weeks; 95% CI, 100-108; p<0.05), and 7% shorter than WT mice (N=117; 101.4 ± 2.38 weeks; 95% CI, 97-106; p<0.05). The rearing environment also influenced longevity (F=10.58, p=0.001, χ2=0.03; Fig. 4). Mice in EE (N=111; 102.2 ±1 .76 weeks; 95% CI,98-106) lived5.7% longer than mice in DE (N=131; 96.7 ± 1.54 weeks; 95% CI, 94-100; p<0.05), but these effects were moderated by the DRD4 genotype, as shown by a significant environment-by-genotype interaction (F=8.05, p<0.001, χ2=0.04; Fig. 4). Specifically, whereas the lifespan was increased in an EE for HT (9% increase) and WT (16% increase) mice, the longevity of DRD4 KO mice did not differ between DE and EE rearing conditions (p=0.15; Fig. 4, inset).