While the concept that alteration of gene expression or function of a particular gene can alter lifespan has been demonstrated convincingly in lower animal models, it is important to determine if those same genes and mechanisms are relevant to the complex biology of mammals and humans. Evidence to support such a connection are the findings that abnormal nematode daf-16 (a transcription factor) or daf-2 (insulin/IGF-1 receptor homolog) causes juvenile animals to enter a state of diapause, called dauer, instead of achieving adulthood, thereby extending lifespan [33]. Both of these genes are part of the insulin–IGF-I signaling pathway, which has been implicated in longevity in mice [34]. Notably the expression of vitellogenin (yolk protein/apolipoprotein-like) genes (vit-2 and vit-5) decreases as lifespan increases [35]. This hypothesis has been independently examined in honeybees where the vitellogenin lipoprotein is associated with both modulation of immune function and longevity [36]. We suggest that modulation of apolipoprotein expression could represent one conserved mechanism of life extension influencing lifespan across species from nematodes to humans. Furthermore, one of the regulators of APOC3 expression is Forkhead Box O1 (FOXO-1), a forkhead tran- scription factor that mediates insulin action [29,30], which is the homolog of the nematode’s daf-16 [33,37]. Deletion of the FOXO-1 binding site in mouse hepatocytes annuls the inhibitory action of insulin on hepatic expression of APOC3, and animals that constitutively express active FOXO-1 have

ipants in the morning to obtain a medical history, perform a physical examination, and draw a fasting venous blood sample. Standardized health histories were obtained using a questionnaire. At that visit, the offspring and the participating spouses underwent similar evalua- tions, as previously described [5,6]. Systolic (first phase) and diastolic (fifth phase) blood pressure were obtained twice, using a standard sphygmomanometer with the patient sitting for at least five minutes, and hypertension was defined according to Joint National Commis- sion VII guidelines [11]. EAS controls underwent a similar evaluation in an outpatient research center at our institution. All blood samples for centenarians, offspring, and controls were processed at the General Clinical Research Center at Albert Einstein College of Medicine, at the clinical laboratories of Montefiore Medical Center Laboratories (Albert Einstein College of Medicine, Bronx, New York, United States), and at LipoScience (Raleigh, North Carolina, Unites States).

using Haploview 3.2 software (http://www.broad.mit.edu/personal/ jcbarret/haploview/index.php) [51].

Lipids and lipoproteins. Total plasma cholesterol, TG, HDL, LDL, APOA1, and apolipoprotein B concentrations for study participants were measured by standard automated methods at the clinical laboratories of Montefiore Medical Center (coefficient of variance [CV]ranged2.5%–3.5%).APOC3concentrationsinhumanserumwere measuredbyELISAusingacommerciallyavailablekit(WakoChemicals USA, Richmond, Virginia, United States) (CV ranged 2%–5%).

The participants’ survival distribution was estimated by the Kaplan-Meier method, and the significance of the difference in survival distribution among the groups was tested by means of a log rank test. Wilcoxon statistics were calculated to test homogeneity between the groups.

LDL and HDL subclass levels and mean particle sizes were determined for all participants by proton nuclear magnetic reso- nance spectroscopy at LipoScience. Each nuclear magnetic resonance measurement produces the concentrations of three LDL subclasses and five HDL subclasses of varying size, as previously described [6] (CV ranged between 3.3 and 5.4). Insulin levels were measured by polyclonal immunoassay at the Hormone Assay Core of the Diabetes Research and Training Center of the Albert Einstein College of Medicine (CV of 8.4%).

Supporting Information

Table S1. Genes and Their Associated SNP Genotyped for This Study The reported values are percent in Control/Proband and its significance of the change (p-Value).

After Bonferroni correction only CETP-VV and APOC-3 CC remain significant (p , 0.0066).

APOC3 genotyping. A multilocus polymerase chain reaction–based assay was utilized to genotype known polymorphisms of APOC3 on Chromosome 11q: C((cid:1)641)A, C((cid:1)482)T, T((cid:1)455)C, C1100T, C3175G, T3206G. Briefly, DNA was amplified using multiplex reaction containing biotinylated primer pairs. Amplified fragments within each polymerase chain reaction product pool were then detected colorimetrically with sequence-specific oligonucleotide probes im- mobilized in a linear array on nylon membranes strips. Probe specificities had previously been confirmed by sequencing and by use of DNA genotyped independently through other methods such as restriction length polymorphism analysis [48].

Found at DOI: 10.1371/journal.pbio.0040113.st001 (135 KB DOC).

Acknowledgments

We thank Mr. William Greiner and Ms. Deborah Davidson for their contributions to this study. We are indebted to all participants and their families for their commitment and enthusiasm. We are also grateful to the following institutions that assisted in recruitment: The Hebrew Home for the Aged, Riverdale, New York; Kittay House, Bronx, New York; the Hebrew Home and Hospital, West Hartford, Connecticut; and the Jewish Home for the Aged, New Haven, Connecticut, all under the aegis of the Association of Jewish Aging Services of North America, Washington, D. C.