Oligomerization of family I glycosidases is a common char- acteristic (2), and can often be a prerequisite for secretion from the endoplasmic reticulum (ER) (18, 19). We tested whether the F352V mutation alters extracellular levels of klotho. HeLa cells were transiently transfected with an expression construct en- coding either wild-type or mutant klotho (secreted form) fused to a V5 tag. To control for transfection efficiency, the levels of klotho were expressed as a ratio of extracellular to intracellular protein. These ratios were then normalized to wild type. Extra- cellular steady-state levels of klotho harboring V352 are reduced greater than 6-fold (Fig. 3). Surprisingly, extracellular levels of klotho harboring S370 are increased 2.9-fold, and the double vias2/S mutant V352 S370 displays an intermediate phenotype, with a 1.6-fold increase in extracellular klotho levels, demonstrating intragenic complementation in cis (Fig. 3). These results have been replicated in transiently transfected fibroblasts (data not shown). Intracellular levels of klotho harboring V352 alone are wwiix | increased 1.56 0.20-fold, suggesting a defect in klotho secre- tion rather than stability (Fig. 3).

Boldtypeindicatessignificant(cid:3)2values(P(cid:6)0.05).Alleleswithfrequencies less than 1% in both newborn and elderly groups were not included for analysis, but were included to derive the total n.

occur within an 800-bp region spanning exon 2 and flanking sequence (Fig. 1A). Allele-specific oligonucleotide hybridiza- tion (ASO) analysis was used to demonstrate complete linkage disequilibrium for the coding region mutations (data not shown). Of the three mutations in exon 2, two code for amino acid changes and one is silent. The change at amino acid position 352 was a phenylalanine to valine substitution. Phe- nylalanine at this position is likely to be important to klotho function, as it is remarkably conserved in homologous proteins in eukaryotic organisms (Fig. 1B). The change at amino acid position 370 was a cysteine to serine substitution. Cysteine at this position is only seen in the first family I glycosidase domain of human klotho.

Because of the absence of a known klotho substrate, the biochemical consequences of the F352V and C370S amino acid substitutions could not be determined directly. We therefore performed enzymatic assays on the closest klotho paralog for  which there is a known substrate, human cytosolic -glucosidase (cBGL1) (20, 21). As in most eukaryotic klotho homologues identified, phenylalanine is found at the position corresponding to klotho amino acid 352 (amino acid 289); however, neither cysteine nor serine is found at the position corresponding to klotho amino acid 370 (amino acid 307; Fig. 1B). When valine was substituted for phenylalanine at amino acid position 289, cBGL1 demonstrated a complete loss of ability to cleave 6-1 p-nitrophenyl- -D-glucoside (Fig. 4 A and B). The introduction of serine at amino acid position 307 was unable to restore activity (Fig. 4 C and D).

The creation of a Mae III restriction site by the F352V mutation allowed for efficient genotyping of large populations. The genotype distribution was different between newborn and -e<< elderly Bohemian Czech individuals (P 0.03), with heterozy- < gosity for these mutations more prevalent in the elderly (P 0.04) and a trend suggesting that homozygosity is more prevalent  in newborns (P 0.08; Table 2). These data indicate a survival advantage for heterozygosity, with a 1.43-fold (95% CI 1.02– 2.01) increased survival rate to age 75, and suggest a 2.70-fold (95% CI 0.84–8.69) survival disadvantage for homozygosity of this allele. No significant differences were seen when the pop- ulation was stratified by sex or health status (data not shown). The KL-VS allele is found on multiple marker allele haplotypes “pew<< | and is negatively associated with Marker 1 Allele 17 (P 0.04), indicating that there are additional deleterious mutations in KLOTHO that have yet to be identified.

Discussion

Disruption of the mouse klotho gene results in a syndrome resembling human aging, with phenotypes including atheroscle- rosis, 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 d/o for KL-VS carriers was seen in three geographically and or

To determine whether the heterozygote advantage associated with the KL-VS allele was specific to survival or also promoted

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