genotypic frequency between two groups (young controls and long-lived individuals) as well as monotonic trend anal- ysis in a population containing participants at multiple ages ( 18 ). In order to aid in the discernment between longevity genotypes and deleterious genotypes, we proposed the Buffering Mechanisms in Aging hypothesis. This hypothe- sis states that an important property of many longevity genes (which are enriched in centenarians) is their ability to buffer against the harmful effects of deleterious genotypes ( 18 ). Specifi cally, in a subpopulation lacking longevity genotypes, the prevalence of a deleterious genotype will decline mono- tonically with age, whereas in a subpopulation endowed with a favorable longevity genotype(s), the prevalence of a buffered-deleterious genotype is expected not to vary or even increase with age. Indeed, we have shown that the deleterious variant of two age-related disease genes, Klotho and Lp(a) , demonstrate s a monotonic decline with age, but this is followed by a marked enrichment in prevalence among those surviving beyond 80 y ea rs, forming a U- s hape pattern with aging ( 18 ). Importantly, this pattern in geno- type frequency would have been missed if only a young and old cohort were compared.

Analysis of SNP s

The MTP gene spans 60 kb with 18 exons and is located on the long arm of chromosome 4. We selected the rs2866164 SNP of the M TP gene and genotyped it in probands, off- spring , and controls, using the PSQ HS 96A Pyrosequencer according to the manufacturer ’ s recommendations (Pyrose- quencing, Uppsala, Sweden; www . pyrosequencing . com ). Briefl y, a polymerase chain reaction product was generated from a primer pair that included one primer covalently coupled to biotin, the biotinylated template was bound to streptavidin-coated Sepharose High Performance beads, and this mixture was then annealed to a sequencing primer. Step- wise elongation of a sequencing primer strand upon sequential addition of a specifi ed sequence of deoxynucleotide tri- phosphates and the degradation of nucleotides by apyrase were carried out simultaneously. As the sequencing reaction progressed, the DNA strand was extended , and the sequence was determined from the measured signal output of light upon nucleotide incorporation. The resulting peaks in the pyrogram were analyzed using Pyrosequencing software. Primer sequences are available from the authors upon request. Error rates based on blind replicates were estimated to be 1.5%.

T o determine whether the MTP gene may be a longevity or buffered-deleterious genotype, we characterized the prevalence of SNP rs2866164 in our well-characterized population of Ashkenazi Jew centenarians, their offspring, and age-matched Ashkenazi controls. We then tested its relationship to the metabolic phenotype and performed a buffering analysis for M TP genotype on survivorship. Here , we show that the CC variant of SNP rs2866164, in the MTP gene, is indeed a deleterious genotype that is buffered by longevity genotypes.

M ethods

Lipids and Lipoproteins

Study P opulation

Total plasma cholesterol, high-density lipoprotein- cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), and apolipoprotein A-I (ApoA-I) and B (Apo - B) concentrations for the participants were performed by stan- dard automated methods at the clinical laboratories of Mon- tefi ore Medical Center, Bronx, NY. Plasma VLDL, LDL, triglycerides (TG) and HDL subclass levels, mean particle sizes, and chylomicrons were determined for all partici- pants by nuclear magnetic resonance spectroscopy at Lipo- Science Inc. (Raleigh, NC) as previously described ( 22 , 23 ). The LDL and HDL subclass distributions and particle sizes determined by n uclear magnetic resonance are highly corre- lated with those measured by gradient gel electrophoresis and density gradient ultracentrifugation (2 4, 2 5) . The ana- lytical reproducibility (given by the coeffi cient of variation) of LDL and HDL size is less than 0.5% ( 22 ), and the stability on repeated drawing for LDL size was 0.9% and for HDL size was 1.1%.

141 women, median age = 97 y ea rs and 64 men, median age = 97 y ea rs), their offspring ( n = 145 total, 80 women, median age = 69 y ea rs and 65 men, median age = 68 y ea rs), and a control group of 288 Ashkenazi Jews (167 women, median age = 74 y ea rs and 121 men, median age = 75 y ea rs), not family related to the earlier participants , but from the same geographic area, were similarly assessed. The study population was identifi ed from the Longevity Genes Study at Albert Einstein College of Medicine, as described else- where ( 13 , 14 ), and the Einstein Aging Study, a community- based longitudinal study designed to identify predictive factors for cognitive decline and dementia ( 20 , 21 ). Partici- pants were recruited through publicity, and stated age was verifi ed by checking birth certifi cates or U.S. passports in all participants . Medical history, demographic characteristics, and clinical data were obtained uniformly using a structured

U- S hape T rend Analysis

by means of a log-rank test. Wilcoxon statistics were calculated to test homogeneity between groups , and cox proportional hazard models were used for survival analysis. Average time of follow-up for participants was 8.5 ± 0.3 and 8.7 ± 0.3 y ea rs for the “ b uffered ” and “ u nbuffered ” groups, respectively. Approximately 70% of the b uffered groups and 64% of the u nbuffered groups were still alive at the time of the analysis.