Abbreviations: APOA-1, apolipoprotein A-1; APOA4, apolipoprotein A4 gene; APOC3, apolipoprotein C3; APOC3, apolipoprotein C3 gene; CETP, cholesterol ester transfer protein gene; CV, coefficient of variance; CVD, cardiovascular disease; DM, diabetes mellitus; HDL, high density lipoprotein cholesterol; LD, linkage disequalibrium; LDL, low density lipoprotein cholesterol; SNP, single nucleotide polymorphisms; TG, triglycerides; VV, valine–valine

* To whom correspondence should be addressed. E-mail: barzilai@aecom.yu.edu

[ These authors contributed equally to this work.

with longevity gene variants, thereby accounting for period and cohort effects and phenotypic variation that may occur at older age [6].

Percentage in population353025201510APOC3 (-641) CC           APOA4 347 thr/thrAPOC3 (-455) TTCETP 405 val/valAPOA4 360 gin/gin 6080Age100

We recently reported that lipoprotein particle sizes are increased in centenarians and their offspring, and suggested that this trait may increase the likelihood of attaining exceptional longevity [6]. This phenotype was also associated with approximately a 3-fold increased frequency of homo- zygosity (valine–valine [VV]) of a common functional variant (I405V) in the cholesterol ester transfer protein (CETP) gene, and decreased CETP levels. Further, through a genome-wide scan, Geesaman and coworkers found increased frequency of polymorphisms in the microsomal transfer protein in centenarian sibling pairs but not in other populations [7]. Here we implicate a polymorphism in the apolipoprotein C3 gene (APOC3), yet another gene regulating lipoprotein metabolism, in human longevity. We further report associa- tion of this same polymorphism with decreased serum apolipoprotein C3 (APOC3) levels, and favorable lipoprotein profile, outcomes of age-related disease, and lifespan.

Results

APOA4, and CETP in the Study Population The genotype frequencies of the favorable gene polymorphisms of APOC3 C((cid:1)641)A and CETP I405V were analyzed in controls and probands (60 to 100 y old). (Offspring were not included in this analysis due to the genotype dependency of this group on proband genotypes.) The frequency of these two variants was found to be higher among centenarians, with a monotonic increase with age. Other polymorphisms in these same genes (APOC3 ((cid:1)455) TT, APOA4 347 TT, and APOA4 360 EE) showed no differences in genotype frequency with age. DOI: 10.1371/journal.pbio.0040113.g001

genotypes will be present at higher frequency among centenarians than in younger controls. We studied 66 single nucleotide polymorphisms (SNP) in 36 candidate genes in pathways related to lipoprotein metabolism and other risk factors for CVD. After applying the Bonferroni correction for multiple comparisons, two of the associations with excep- tional longevity remained statistically significant. Analysis of the frequency trend with age (in centenarian and control groups combined) for polymorphisms in APOC3 and CETP (Figure 1), revealed a highly significant (p , 0.0006; b ¼ 1.89 [95% CI, 0.2–3.6]) monotonically increasing frequency trend for homozygosity for the APOC3 (cid:1)641 C allele (CC)(rs2542052), and near-significance (b ¼ 3.06 [95% CI, 0.33–5.8, p¼0.069]) for homozygosity for the CETP codon 405 valine allele (VV)(rs5882). The CC genotype at position (cid:1)641 of APOC3 had a prevalence of 25% among the centenarians and 20% in their offspring compared with only 10% in controls (p ¼ 0.0001 and p ¼ 0.001, respectively). Five additional SNPs in APOC3 and six SNPs in CETP (Figure 2) showed no evidence of association with longevity. Conversely, the prevalence of the APOC3 AA genotype was reduced in both centenarians and their offspring compared with con- trols (p ¼ 0.001 both) (Figure 3A).

significantly higher among the control group compared with probands (0.483 versus 0.307, p , 10(cid:1)10). Association analysis between various haplotypes of the APOC3/APOA4 and lipid profile was not significant, neither when the most prevalent haplotypes in the two groups were compared, (CA versus AA) nor the haplotypes that contain the favorable APOC3 allele. The only trend for association (p , 0.07) between haplotype and lipoprotein was for LDL. This suggests that the single genotype APOC3 CC is more prominent in the phenotypes.

lipoprotein metabolism and thus the favorable alleles show- ing monotonic increases in allele frequencies among older age strata would also be expected to be associated with a favorable lipoprotein profile. Indeed, participants of all groups carrying the APOC3 (cid:1)641 CC genotype had lower serum levels of APOC3 compared with those carrying either CA or AA (CA/AA) genotype (mean [SE]: 10.1 [1.1] versus 13.2 [1.1] mg/dl, p , 0.05). Given the higher prevalence of this genotype in centenarians, it is therefore not surprising that serum levels of APOC3 were lower in centenarians and their offspring than in controls (mean [SE]: 9.8 [0.6], 9.3 [0.5] versus 11.7 [0.7] mg/dl; p , 0.05 and 0.01, respectively) (Figure 3B).

Linkage disequilibrium (LD) analysis of the CETP gene revealed that the single nucleotide polymorphism rs1800776 was in LD with rs1800775 just 2 bp apart (D9 ¼ 0.94; r2 ¼ 0.20) (Figure 2). Haplotype analysis with these two SNP revealed that the C-A haplotype was statistically significantly more prevalent among individuals in the control group compared with centenarian probands (0.355 versus 0.278, p ,0.004). LD analysis revealed LD across the neighboring apolipoprotein A4 (APOA4) and apolipoprotein C3 (APOC3) genes as previously reported by others (Figure 2) [8]. In haplotype analysis, the prevalence of the A-C haplotype (corresponding to SNP rs675 in APOA4 and rs2542052 in APOC3) was significantly higher among the centenarian probands com- pared with controls (0.498 versus 0.344, p , 10(cid:1)8). The prevalence of the A-A haplotype of the same SNPs was