We hypothesized that since only one out of 10,000 people in the general population has survived to 100 y, there should be potent enrichment for favorable genotypes in this unique population. The monotonic, progressive over-representation of the APOC3 (cid:1)641 CC genotype with increasing age, from a relatively infrequent 10% at age ;60 y to 25% in individuals surviving to age ;100 y is statistically robust and provides evidence that this genotype provides a selective advantage for survival to exceptional old age. Underlying population stratification could be responsible for genotypic associations detected in case-control studies, but this is much less likely in this relatively homogeneous Ashkenazi population. Further- more, we demonstrated association of this same genotype with survival in a prospectively followed cohort. Finally, the prevalence of heterozygosity for recessive disease alleles (nearly) unique to the Ashkenazi population, e.g., breast cancer and Tay-Sachs disease, did not differ significantly between longevity cases and controls, suggesting the lack of population stratification (unpublished data).

Discussion

APOC3 is a major component of very low density lip- oproteins and chylomicron remnants; it is also a minor component of HDL [8]. In vitro, it has been shown to inhibit the activity of lipoprotein lipase, resulting in delayed TG clearance from plasma [12]. Several studies have shown that various polymorphisms of the APOC3 gene either within the promoter region, in particular the T-455C variant poly- morphism in the insulin response element, or in the gene, are associated with elevated TG levels but not with significant change in APOC3 levels [13–17]. Previous studies have also associated high levels of APOC3 with macrovascular and microvascular disease and their risks. Increased APOC3 levels were a significant indicator of coronary risk in men and in patients with metabolic syndrome. In particular, those with vascular disease and metabolic syndrome had a markedly increased probability of coronary heart disease in an allele-

In addition to showing statistical associations of the APOC3 genotype with exceptional longevity, we found significant associations between this genotype and several intermediate longevity-associated traits directly related to lipoproteins.

Surviving (%)3wD a3APOC CCAPOC3 CAYAA os  65T80T85 90Age (years)95T100105110

HOMAPercentage of Hypertension5040302010H4poc3 cc Dlapoc3 CA/AA AllMi apoc3 cc [L] APOC3 CA/AA 

Of the 381 participants we genotyped since 1998, 64 had the (cid:1)641 CC genotype. To describe the relationship between genotype and death, we plotted the Kaplan–Meier survival function estimates of probands and controls by APOC3 genotype. Offspring were excluded in this analysis because all participants are currently alive and offspring genotype/ phenotype is not independent of the probands. Log-Rank (p ¼ 0.0008); Wilcoxon (p ¼ 0.0007). CC, homozygous for ((cid:1)641) C; CA/AA, homozygous and heterozygous for ((cid:1)641) A.

suggest that the APOC3 genotype may have favorable pleiotrophic effects on a number of cardiovascular traits and mortality.

Interestingly, while a recent study found that the pheno- typic effects of APOC3 polymorphisms are modified by dietary polyunsaturated fatty acids [22], the interactions of APOC3 levels with exercise, body mass index or other environmental effect have not been well studied. Our population by genotype had similar body mass index, suggesting that body weight is not strongly modulated by genotype.

The APOC3 gene is located on the long arm of Chromo- some 11q23, in tandem with the apolipoprotein A1 and A4 genes [8,26]. The APO A1-C3-A4-A5 gene cluster has been studied extensively in relation to lipoproteins and CVD, and most recently in the oldest old [8,27,28]. Given LD within this gene cluster, we cannot rule out the possibility that the associations we have demonstrated may be due to other functional SNP within this gene cluster. However, our observation that the APOC3 (cid:1)641 CC genotype associates with APOC3 levels suggests that this is a functional SNP or is marking a functional SNP within this gene. Further func- tional studies will be required to determine if this promoter variant directly results in a decrease in transcription.

Figure

Insulin Sensitivity and Hypertension according to APOC

C((cid:

)

)A Genotype All include offspring plus controls. (A) Insulin sensitivity. (B) Hypertension. *p ¼

CC, homozygous for ((cid:

)

) C; CA/AA, homozygous and heterozygous for ((cid:

)

) A. DOI:

/journal.pbio.

g

Our haplotype analysis revealed a 2-SNP haplotype span- ning both APOA4 and APOC3. One haplotype, AA in SNPs (rs675 and rs2542052, respectively), was found to be signifi- cantly more prevalent among controls, whereas the AC haplotype was significantly more prevalent among centenar- ians. The association between these two genes and plasma lipids levels in populations has been reported elsewhere [8,27,28]. Thus, sequence variants in partial LD that constitute one or more haplotypes across more than one of these apolipoprotein genes may influence longevity. However, our findings that Apolipoprotein A1 and Apolipoprotein B levels

First, serum APOC3 levels were significantly (;30%) de- creased in our participants with the favorable APOC3 genotype. Second, lipid profiles were altered in the expected (favorable) direction in participants with this genotype. Finally, this favorable genotype was associated with reduced insulin resistance and less hypertension. These findings

hypertriglyceridemia [38]. Whether this example is relevant to longevity of mammalian models is not known.

are not altered by APOC3 genotype, suggest that the effect of the genotype on the lipoprotein phenotype is mainly mediated by mechanisms that do not involve alterations of these apolipoproteins.