In order to determine whether the M TP CC SNP is a buffered - deleterious genotype, we utilized the Buffering Mechanisms in Aging approach, whose statistical consider- ations are described in detail elsewhere ( 18 ). Briefl y, controls and probands were grouped together , and a generalized linear model was used to fi t the pattern of C C genotype prevalence with age. We confi rmed the existence of a signifi cant U - s hape pattern with age using a binomial model incorporating both linear and quadratic terms for age and testing for the sig- nifi cant quadratic component using the equation: P ( Y = 1) = b + b age + b age 2 , where Y = 1 was indicative of an indi- 0 1 2 vidual having the M TP CC genotype. Maximum likelihood estimates of the coeffi cients b , b , and b were then obtained 0 1 2 by the Fisher s coring method , and the signifi cance of the quadratic term (ie , U- s hape trend) was confi rmed using the likelihood ratio test prior to performing interaction analysis on the MTP CC genotype.

R esults

Participant C haracteristics for A ging C ohorts

from http://biomedgerontology.oxfordjournals.org/ at Ondokuz Mayis University on November 9, 2014

offspring , and Ashkenazi controls are presented in Table 1 . There were no signifi cant differences observed between offspring and controls for glucose, insulin, apolipoproteins, LDL-C , or LDL particle size. Offspring did have greater levels of TG ( p < .001) and tended to have elevated chylo- microns ( p = .06), VLDL-TG ( p = .06) , and total cholesterol ( p = .09), as compared with controls. However, offspring also had greater total unadjusted HDL-C ( p < .01) and smaller HDL particle size ( p < .001), though HDL-C was no longer signifi cant after adjusting for several covariates.

Buffering Analysis

Upon confi rmation of a signifi cant U- s hape t rend for the CC genotype in controls and probands, we then combined all offspring and control participants (excluding probands) harboring the CC genotype and subgrouped them by pres- ence or absence of one or more longevity genotypes, includ- ing CETP (homozygosity for the 405 V allele, VV ) [rs5882] ( 13 ), APOC3 (homozygosity for the − 641 C allele, C C ) [rs2542052] ( 14 ) , and ADIPOQ ( del/del APM1+2019) [rs56354395] (1 5) . We next performed logistic regression to test the interaction between the trend of the M TP CC geno- type across age with or without the presence of favorable lon- gevity genotypes. Because we did not detect any additive effect of harboring more than one favorable genotype, any carrier of one or more of the three longevity genotypes were combined as a single group. To test the signifi cance of inter- action effects between age and genotype, the model testing for main effect only and the model testing for the interaction effects were compared using the log-likelihood ratio test.

MTP G enotype and A llele F requency in C entenarians, O ffspring , and C ontrols

The prevalence of the MTP rs2866164 SNP and genotype frequency among centenarians, offspring , and controls are presented in Figure 1A and B , respectively. As compared with Ashkenazi controls, the C C genotype was more prevalent in both centenarians and their offspring ( p < .05 , Figure 1A ), but no signifi cant differences were observed in CG or GG genotype prevalence among groups. When assessing allele frequency in all three groups, the C allele was found to be overrepresented in offspring ( p = .001), as compared w ith controls, but no signifi cant difference was found between centenarians and controls ( p = .26, Figure 1B ).

MTP CC G enotype I s A ssociated W ith an U nfavorable L ipid P rofi le

Statistical A nalysis

Characteristics of offspring and controls by MTP geno- type (C C vs C G/GG ) are presented in Table 2 . There were no signifi cant differences observed for glucose, insulin, choles- terol, apolipoproteins, VLDL particle size, HDL-C , or LDL-C ( Table 2 ). However, individuals with the C C genotype had signifi cantly greater levels of total TG ( p < .05, Figure 2A ), VLDL ( p < .05, Figure 2B ), and chylomicrons ( p < .05, Figure 2C ), even after adjustment for confounders.

We used JMP software, version 9 (SAS Institute Inc., Cary, NC) for data analysis. Plasma VLDL and chylomi- cron were tested for normality of distribution using Shapiro – Wilk test and D ’ Agostino ’s K-squared test and were found to violate the normality assumption. Thus, a comparison between nonparametric variables was performed using Kruskal – Wallis one-way analysis of variance by ranks and Mann – Whitney rank sum test. Data are presented as means ± SE unless cited differently , and a p value less than or equal to .05 was considered statistically signifi cant. The partici- pants’ survival distribution was estimated by the Kaplan – Meier method, and the signifi cance of the difference in survival distribution among the MTP genotypes was tested

The frequency of the MTP CC genotype with normal aging in controls and probands is shown in F igure 3A.

Table 1. Demographic and Metabolic Characteristics of Ashkenazi Probands, Offspring , and Controls