of ages 75 years, and although a trend for a heterozygous disadvantage for survival was observed for ages 75 to 81 years, it was not statistically significant. The Ashkenazi cohort spanned a greater age range and exhibited a marked decrease in the frequency of heterozygous individuals 63 to 81 years of age. This apparent change in the effect of the KL-VS allele on mortality is consistent in both populations and raises questions as to the mechanism through which klotho exerts its effects on survival. It is possible that the KL-VS allele may increase the risk for causes of mortality earlier in life, such as cancer, but in later life, reduce the risk for CVD. Interestingly, the transition age at which the leading cause of mortality in Caucasian 8  Americans switches between cancer and heart disease is 80 years.19 An alternate hypothesis is that those who have survived to age 80 years with the detrimental KL-VS allele have faced a stronger survival selection and are thus representative of a more robust cohort. However, if this were the case, one would expect that those who survive to age 80 years with two copies of the KL-VS allele would also exhibit this robust phenotype. Instead, these individuals have lower HDL-C, higher SBP, and the highest RR for mortality, as measured prospectively in the proband cohort.

Discussion

Survival is a product of the interaction between environmen- tal and genetic effects, and mortality is determined through numerous mechanisms such as infectious disease, cancer, and CVD. In previous studies of the impact of KLOTHO on human longevity, we had identified a functional variant, KL-VS, for which homozygosity was markedly reduced in the elderly relative to infants, and in the Czech population, showed a heterozygous advantage late in life.4 Analysis of the

Survival (%)8 8 6 8 8 8 81=oo P<0.026|m FF| 

The association of the KL-VS allele with the CVD risk factors SBP and HDL-C is particularly intriguing in light of previous work demonstrating that the effect of the KL-VS allele on early-onset occult CAD is modified by these two factors.5 A recent genome scan for quantitative trait loci influencing HDL-C concentration identified suggestive link- age (LOD, 2.36), with a marker 360 kb 3(cid:7) of KLOTHO,20 providing further evidence for a role for klotho in influencing HDL-C levels. The mechanism through which klotho affects these phenotypes remains to be elucidated, although several studies have already implicated a role for klotho in the

Figure 4. Mortality analysis of Ashkenazi probands stratified by KL-VS genotype. P value indicates overall significance of the KL-VS allele as determined by the (cid:3)2 test.

renin-angiotensin system,10,21,22 as well as in the NO produc- tion pathway.2,3

accurately the magnitude of the risk associated with the KL-VS allele.

Analysis of the history of cardiovascular events revealed an association between KL-VS genotype and stroke. Al- though this result may be anticipated on the basis of the effect of klotho on SBP, a known risk factor for stroke,23 in a multivariate model adjusting for blood pressure, the KL-VS was still a strong predictor of stroke. Wild-type individuals had a 5.88-fold (95% CI, 1.18 to 29.41) and homozygous KL-VS individuals a 30.65-fold (95% CI, 2.55 to 368.00) increased OR for stroke, indicating an effect of the KL-VS allele on stroke independent of its effect on blood pressure. A possible explanation for this result stems from the observa- tion that klotho deficiency in mice increases the levels of PAI-1.11 Increased PAI-1 serum levels have been associated with increased risk for 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 con- firm 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

D.E.A. is supported by National Institute of Mental Health grant MH070172 and by the Donald W. Reynolds Foundation Clinical Cardiovascular Research Center grant to Johns Hopkins Univer- sity. G.A. and N.B. are supported by grants from the Paul Beeson physician faculty scholar in aging award, the Ellison Medical Foundation senior scholar award, RO1 (AG-18728-01A1), Aging Einstein Study (P01 AG-03949-01A1), the General Clinical Research Center (MO1-RR12248) and Diabetes Research and Training Center (DK 20541) at the Albert Einstein College of Medicine, and the Baltimore VA Geriatric Research and Educa- tion Clinical Center. H.C.D. is supported by the Howard Hughes Medical Institute.

allele on longevity have been limited to cross-sectional data, the current study incorporates prospective mortality data generated in individuals   95 years of age. As observed in the cross-sectional analyses of CVD risk factors and events, heterozygous individuals had a marked advantage. Wild-type individuals have an increased RR of 2.15 (95% CI, 1.18 to 3.91) and homozygous KL-VS individuals a 4.49-fold in- creased RR (95% CI, 1.35 to 14.97) for mortality relative to heterozygous individuals, after adjusting for potential con- founders. These data strongly support a role for the KL-VS allele in longevity.

References

Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-Iida T, Nishikawa S, Nagai R, Nabeshima YI. Mutation of the mouse klotho gene leads to a syndrome resembling aging. Nature.

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