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Two hundred sixteen probands with exceptional longevity (95 to 109 years of age) were recruited to participate in the study, as described previously. They were required to be living independently at 95 years of age as a reflection of good health, although they could be at any institution or level of dependency at the time of recruitment. Additional samples consisted of spouses of the offspring and age-matched Ashkenazi Jewish individuals recruited from the Einstein Aging Study, 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.
 
The KL-VS allele was genotyped and found to be in Hardy Weinberg equilibrium (P > 0.38) in the entire Ashkenazi cohort, as well as when stratified by proband status (P > 0.15). No genotype frequency differences were observed between males and females in the entire Ashkenazi cohort (P > 0.59) nor in the probands alone (P > 0.90). Figure 1 displays the 5-year average frequency of heterozygous individuals plotted in 1-year increments. For comparison, we plotted 5-year moving averages for Czech elderly individuals reported previously to exhibit a heterozygous advantage for longevity. In Ashkenazi and Czech populations, a significant increase in the frequency of KL-VS heterozygous individuals is observed for ages ≥79 years (P < 0.004 and P < 0.01, respectively). When the Ashkenazi cohort is restricted to the same age range as the Czech (79 to 94 years), the heterozygous advantage is still significant (P < 0.007), and the magnitude of the effect, as indicated by the -estimates, is remarkably similar (0.092 ± 0.034 versus 0.087 ± 0.034). Combining these two data sets, we find a 1.094-fold (95% CI, 1.043 to 1.146) increased odds ratio (OR) for survival per year for heterozygous individuals from age 79 to 94 years in two independent populations. This corresponds to a 1.57-fold (95% CI, 1.23 to 1.98) increased OR for 5-year survival (P < 0.0002).

Clinical evaluation was performed as described previously. Lipid 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 cholesterol (HDL-C) subclass levels and mean particle sizes were determined for all participants by NMR spectroscopy at LipoScience, Inc.

Genotyping for the KL-VS allele was performed as described previously. 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.

Logistic regression analysis of KL-VS frequency and age shows that in the Ashkenazi population, a decrease in the frequency of heterozygous individuals between the ages of 63 to 81 years is observed (OR, 0.908; 95% CI, 0.852 to 0.968; P < 0.003). Although not noted previously, the Czech population shows a similar decrease in the frequency of heterozygous individuals 75 to 81 years of age (OR, 0.845; 95% CI, 0.695 to 1.028; P = 0.092). When the Ashkenazi cohort was restricted to the same age range (75 to 81 years), the negative trend for frequency of heterozygous individuals was also not statistically significant (P > 0.69), nor was the trend significant in the combined data set (P > 0.11).

CVD risk factors stratified by KL-VS genotype indicate that systolic blood pressure (SBP) levels were significantly associated with KL-VS genotype (P < 0.001), and suggestive results (P < 0.10) were seen for LDL-C, HDL-C, and DBPcardiovascular events, including stroke. Therefore, it is plausible that the KL-VS allele may influence stroke risk through its effect on PAI-1 levels. 

Furthermore, the analysis of mortality data showed a significant association between KL-VS genotype and mortality, with wild-type individuals having a 2.15-fold (95% CI, 1.18 to 3.91) increased relative risk for mortality and homozygous KL-VS individuals displaying a 4.49-fold (95% CI, 1.35 to 14.97) increased relative risk compared with heterozygous individuals. These findings support the hypothesis that the KL-VS allele exerts a heterozygous advantage for survival late in life.

In conclusion, our study provides evidence for the association of the KL-VS allele with CVD risk factors, stroke, and mortality in Ashkenazi Jews. These findings suggest that klotho may play a role in the pathogenesis of CVD and aging-related diseases and may have implications for the development of novel therapeutic approaches targeting klotho signaling pathways.Stroke, 24, thus providing an alternate pathway through which Klotho may act.

Conclusions:

In the current study, we presented strong evidence confirming the role of a common variant of KLOTHO, the KL-VS allele, in human longevity. Cross-sectional and prospective studies confirm a heterozygous advantage for survival at later ages in conjunction with a marked homozygous KL-VS disadvantage. This genetic model held true for cardiovascular risk factors HDL-C and SBP, associated previously with KLOTHO, as well as a history of stroke. Collectively, these data implicate an important role for KLOTHO in the etiology of CVD and ultimately mortality.

Acknowledgments: