Original received August 17, 2004; resubmission received January 7, 2005; accepted January 14, 2005. From the McKusick-Nathans Institute of Genetic Medicine (D.E.A., H.C.D.), Johns Hopkins University School of Medicine, Baltimore, Md; Institute for Aging Research (G.A., N.B.), Albert Einstein College of Medicine, Bronx, NY; Johns Hopkins University (A.A.), Baltimore, Md; and Howard Hughes

This manuscript was sent to Eugene Braunwald, Consulting Editor, for review by expert referees, editorial decision, and final disposition. Correspondence to Harry C. Dietz, Johns Hopkins University School of Medicine, 733 N Broadway, Room 539, Baltimore, MD 21205. E-mail

Circulation Research is available at http://www.circresaha.org

Statistical

  

association with age, KL-VS allele frequencies were plotted as 5-year averages in 1-year increments. Data points with (cid:1)30 samples were not plotted. Significance for trend was determined using logistic regression with KL-VS allele status (heterozygous versus all others) as the dependent variable and age as the independent variable. For the Ashkenazi cohort, only individuals aged 63 to 104 years were included for this analysis, and for Czech, only individuals aged 75 to 94. These age cutoffs were chosen so that there was no missing data for a given age. The age cutoffs for the uptrend and downtrend in each population were selected to maximize effect and statistical significance. Combined analysis included a term in the logistic regression model to account for allele frequency differences between the populations.

Univariate analyses were assessed by ANOVA for continuous variables and by the (cid:3)2 test for dichotomous variables. Multivariate linear regression was performed for CVD risk factors with sugges- tive univariate associations (P(cid:1)0.10). Because the data did not fit traditional genetic models, genotypes were treated as independent variables. In addition to traditional CVD risk factors, a term for recruitment status (proband versus control) was included to account for any differences attributable to sample collection. The overall significance of the KL-VS allele, P , was determined by compar- robust ing the explained variance, R2, before and after inclusion of the KL-VS allele terms. Multivariate analysis of history of stroke was analyzed using multiple logistic regression with independent vari- ables for each KL-VS genotype. Overall significance, P , was robust calculated by comparing log-likelihood ratios before and after inclusion of the KL-VS allele terms into the model.

Figure 1. Frequency of KL-VS heterozygous individuals plotted as 5-year averages in 1-year increments. Blue line represents Ashkenazi samples. Red lines represents Czech samples.

Materials and Methods

Study Populations

described previously and consists of 435 elderly individuals ((cid:1)75 years) drawn at random from the Bohemian Czech population.14 Ashkenazi Jews were recruited as described previously.15,16 Briefly, 216 probands with exceptional longevity (95 to 109 years of age) were recruited to participate in the study. Probands were required to be living independently at 95 years of age as a reflection of good health, although at the time of recruitment, they could be at any institution or level of dependency. Additional samples consisted of spouses of the offspring and age-matched Ashkenazi Jewish individ- uals recruited from the Einstein Aging Study,17 for a total of 309 subjects (51 to 94 years of age). Informed written consent was obtained in accordance with the policy of the committee on clinical investigations of the Albert Einstein College of Medicine.

Mortality was analyzed using Cox regression survival analysis, with survival measured in 1-year increments. Independent variables and calculations of overall significance were identical to those used for stroke.

Results

KL-VS Genotype Frequencies and Their Association With Age

Clinical Assessment

The KL-VS allele was genotyped and found to be in Hardy– Weinberg equilibrium (P(cid:3) 0.38) in the entire Ashkenazi cohort, as well as when stratified by proband status (P(cid:4) 0.15). No genotype frequency differences were observed between males and females in the entire Ashkenazi cohort (P(cid:4) 0.59) nor in the probands alone (P(cid:4) 0.90). Figure 1 displays the 5-year average frequency of heterozygous individuals plotted in 1-year increments. For a comparison, we plotted 5-year moving averages for Czech elderly individuals reported previously to exhibit a heterozygous advantage for longevi- ty.4 In Ashkenazi and Czech populations, a significant in- crease in the frequency of KL-VS heterozygous individuals is observed for ages   79 years (P(cid:1) 0.004 and P(cid:1) 0.01, respec-

Clinical evaluation was performed as described previously.15,16 Lipids analysis was performed by standard automated methods at the clinical laboratories of Montefiore Medical Center. Low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cho- lesterol (HDL-C) subclass levels and mean particle sizes were determined for all participants by NMR spectroscopy at Lipo- Science, Inc.

Genotyping

Genotyping for the KL-VS allele was performed as described previously.5 Briefly, DNA was amplified using polymerase chain reaction and then digested with Mae III (Roche). The KL-VS allele is characterized by diagnostic Mae III restriction fragments of 265 and 185 bp. Selected genotypes were verified by Applied Biosystems TaqMan assays (primer sequences available on request).

TABLE 1. Logistic Regression Analysis of KL-VS Frequency and Age