The mechanism by which the DRD4 variants would differentially influence environmental responses is unclear. The variant DRD4 7R protein has a blunted response for cAMP reduction compared with the 4R protein (Asghari et al., 1995; Jovanovic et al., 1999; Swanson et al., 2000; Wang et al., 2004). Also, the DRD4 7R protein, unlike theSignificantly greater (Zuk et al., 2012). Clearly, further studies of DRD4 7R contributions to the variance of activity level in other populations should be pursued. Unfortunately, there are no genetic markers on commercially available genotyping arrays that are in strong LD with the DRD4 7R VNTR (Saccone et al., 2009), making direct genotyping/sequencing essential. In further discussing our findings, it is important to note their limitations. The ideal control for the genotype/haplotype frequencies of DRD4 alleles would have been the non-living birth cohorts of our oldest-old population, which is clearly not possible. However, human gene frequencies cannot change radically in a few generations, except following extreme population bottlenecks, which have not occurred in the last 100 years in European ancestry populations. A possible selective force that could have introduced intergenerational variation is the high mortality due to infectious disease experienced early in life by this oldest-old cohort, which has been largely eliminated through the use of immunization and antibiotics in the younger cohort (Fig. 1). Thus, any hypothetical resistance allele might exhibit higher frequencies in the oldest-old population than currently observed due to this environmental intervention. However, it is unlikely that the DRD4 gene (or any of the three closest genes in modest LD with it: DEAF1, SCT, and MUPCDH) (International Human Genome Sequencing Consortium, 2004) would confer such resistance. Moreover, allele frequencies at 20 randomly chosen polymorphisms in nine unrelated genomic regions (CFTR, ERCC8, ERBB4, FANCC, HTERT, NRF1, RTN1, SLC6A3, and UCP3) did not differ significantly among the oldest-old individuals, younger control individuals or HapMap European ancestry populations (International HapMap Consortium et al., 2007). Nor was the observed DRD4 7R male/female allele frequency bias (Fig. 2) found at any of these randomly chosen sites. It is difficult to conceive of a mechanism that explains the variations in allele frequencies between males and females except for one that involves the functional significance of the underlying variant locus. We cannot, however, rule out alternative behavioral possibilities to explain why 7R/x individuals are disproportionately found in our specific cohort, such as the ability/desire to live in a planned retirement community with good access to leisure activities [see the discussion of the potential impact of socioeconomic status (SES)]. Clearly, further studies of the allele frequencies of DRD4 throughout the lifespan, rather than just at the extremes (Fig. 1), are warranted. For genetic studies of complex phenotypes, most associated variants observed to have relatively small effect sizes (<1%), as expected for populations similar to the oldest-old population studied here (25%; Herskind et al., 1996; Mitchell et al., 2001). Given the estimated effect size for the 7R/longevity association (<2 when s = 0.026; Fig. 2), we can estimate ndt ~ 10.2% of the variance attributable to genes, measured in herita- 0.030-0.04; Fig. 4). These values are quite high for a phenotype expected to be associated with many genes and that variants of DRD4 make a significant impact on longevity.

In conclusion, we observed a striking increase in DRD4 7R allele frequency in the oldest-old population and a strong association of this allele with increased activity levels. These findings are consistent with our animal model results showing that the DRD4 gene moderates the beneficial effects of an enriched environment in increasing rodents' lifespan. We propose that DRD4 gene variants moderate longevity by altering behavioral responses to environmental factors. It is likely that such gene-environmental interactions underlie much human variability and many common disorders afflicting humankind (Wang et al., 2006; Manolio et al., 2009).

A strength and a weakness of the current study are that the oldest-old population studied is of predominantly European ancestry. We specifically chose this population to reduce the genetic diversity and allow for better case-control matching, essential for genetic studies. It should be cautioned, therefore, that it is unclear how broadly such results can be inferred beyond the ancestry group examined. The ubiquitous presence of DRD4 7R (and its derived 2R allele) in most human populations (Ding et al., 2002; Wang et al., 2004), however, makes it likely that this study can be generalized (and potentially replicated) in individuals whose ancestry is from different geographic origins. While a prospective design would allow for much stronger inferences, the logistics and cost of a prospective study to assess the effects of genotype on longevity are enormous, and data would not be available for analysis until the cohorts died.

References