Discussion

Disruption of the mouse klotho gene results in a syndrome resembling human aging, with phenotypes including atherosclerosis, emphysema, osteoporosis, and infertility (1). We therefore decided to explore the role of KLOTHO in human aging by using a population-based association study. We identified an allele of KLOTHO, termed KL-VS, defined by the presence of six SNPs in complete linkage disequilibrium. A homozygous disadvantage for KL-VS carriers was seen in three geographically and/or ethnically distinct populations (P<0.0023). Interestingly, heterozygote advantage was seen in the Bohemian Czech population (P<0.04), but not in either of the Baltimore populations. Multiple examples of heterozygote advantage with homozygote disadvantage exist, including both CFTR allele waif508 (22) and hemoglobin S (23). The heterozygote advantage displayed by these alleles is associated with specific environmental pressures, cholera and malaria, respectively. It is therefore plausible that the apparent lack of heterozygote advantage in the two Baltimore groups may manifest the absence of an environmental pressure that was specific to the Bohemian Czech population. A homozygote disadvantage can generally be observed independent of environmental factors.

One aging theory, antagonistic pleiotropy, proposes that individuals with alleles that are beneficial to survival through reproductive age could be selected for by evolution even if the same alleles had negative effects atCharacteristics of Family 1 glycosidases (2) show that it is essential for export out of the endoplasmic reticulum (ER) (18, 19). Extracellular levels of klotho protein harboring V352 are reduced by 6-fold, but it is yet to be determined whether this mutation influences klotho dimerization or interaction with other proteins required for klotho export, such as ER chaperones. Interestingly, extracellular levels of klotho harboring S370 are increased by 2.9-fold and have the ability to complement the V352 phenotype in cis, as demonstrated by the V352 S370 double mutant which exhibits an intermediate phenotype (1.6-fold increase).

In screening a total of over 300 individuals taken from the geographically and ethnically distinct cohorts used in this study, no individuals were found who harbor only V352 or S370. It is possible that neither genotype is viable if secretion is essential to klotho function. In this scenario, the V352 phenotype would correspond to that of a severe hypomorph similar to the klotho mouse and would be negatively selected. Likewise, if the homozygous disadvantage exhibited by KL-VS allele-containing individuals is due, at least in part, to increased extracellular klotho levels, individuals harboring only S370 would experience an even greater negative selection.

Multiple models of aging invoke accelerated or excessive posttranslational modification of proteins including glycation. Resultant advanced glycation end-products (AGEs) elicit a wide range of responses that have been proposed to contribute to many age-related phenotypes, including atherosclerosis, Alzheimer's disease, diabetic complications, and microvascular changes (29). It is possible that the proposed glycosidase activity of klotho retards the accumulation of AGEs. Alternatively, klotho may contribute to or regulate signaling cascades with downstream targets that influence aging. These hypotheses remain to be tested in humans or mouse models of normal or accelerated aging.

The remarkable evolutionary conservation of the F352 amino acid among homologous proteins suggests that it is important to klotho function. The substitution of valine for phenylalanine at the corresponding residue in the closest human klotho paralog with a known substrate, cBGL1, completely eliminates the ability to cleave p-nitrophenyl- -D-glucoside. The introduction of serine at the position corresponding to klotho aa 370 failed to restore enzymatic activity. These results suggest that the KL-VS allele may exhibit both altered secretion and a loss of catalytic activity.

We thank Charlotte Ferencz for access to epidemiologic data from the Baltimore-Washington Infant Study (supported by NHLBI R37-HL25629) and Christopher Loffredo for access to samples. We thank Aravinda Chakravarti for useful suggestions. This work was supported by National Institutes of Health Grants AR21135 (to H.C.D.) and AR44702 (to I.M.); Grants IGA MZCR (6497-3, 6411-3, 6462-3, 6250-3, 6464-3, 00000064203) and MSMT CR (ME457, LN00AO79, 111300003) from the Czech Republic (to M.M., Jr., A.K., and M.M., Sr.); and U.S. Department of Energy Contract No. DE-AC03-76SF00098 (to I.S.M.). The Women's Health and Aging Study is supported by NOI-AG-1-2112 (to L.F.). H.C.D. is an investigator in the Howard Hughes Medical Institute.

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6. Shiraki-Iida, T., Aizawa, H., Matsumura, Y., Sekine, S., Iida, A., Anazawa, H., Nagai, RI'm sorry, but there is not enough context to determine what corrections or improvements need to be made to the text. Please provide more information or the complete text.