Fig. 1. Sequence variation in the KLOTHO locus. (A) Schematic of mutations detected in KLOTHO. (B) Amino acid sequence of klotho amino acid positions 346–374 and homologous proteins. Klotho has two homologous domains, here labeled ‘‘Klotho1’’ and ‘‘Klotho2.’’ The common allele of klotho is labeled ‘‘Klotho1-wt’’ and the rare allele is labeled ‘‘Klotho1-mt.’’ Alignment was performed using the CLUSTALW program (MACVECTOR 6.5.1). Amino acids found in at least 9of16positionsareshadedinblack.Conservationofaminoacidcharacterinatleast9of16positionsisindicatedbygrayshading.Theblackarrowheadsindicate amino acid positions at which mutations in klotho have been detected.

with homozygote disadvantage exist, including both CFTR allele wai F508 (22) and hemoglobin S (23). The heterozygote advantage displayed by these alleles is associated with specific environmen- tal pressures, cholera and malaria, respectively. It is therefore

 ethnically distinct populations (P 0.0023). Interestingly, heterozygote advantage was seen in the Bohemian Czech oe>< ( ulation (P 0.04), but not in either of the Baltimore Multiple examples of heterozygote advantage in

Table 2. KLOTHO genotypes in newborn and elderly individuals

haloSoa3xoQDoEFFFVSurvival (%)ao403020107576 77 78 79 80 81 82 83 84 85 86 87 88 89 9091 92 93 94 9F

Genotype at codon 352 (frequency, %) (frequency, %) (cid:3)2 P Bohemian Czech FF 307 (78.7) 308 (74.2) 2.26 0.13 FV 73 (18.7) 103 (24.8) 4.38 0.036 VV 10 (2.6) 4 (1.0) 3.01 0.08 Total 390 415 Baltimore Caucasian FF 309 (73.6) 530 (73.3) 0.01 0.92 FV 100 (23.8) 185 (25.6) 0.45 0.50 VV 11 (2.6) 8 (1.1) 3.72 0.05 Total 420 723 Baltimore African- American FF 156 (69.0) 169 (69.8) 0.04 0.85 FV 58 (25.7) 68 (28.1) 0.35 0.55 VV 12 (5.3) 5 (2.1) 3.60 0.06 Total 226 242 Combined analysis* VV 9.31 0.0023

The common allele is denoted as F and the rare allele, defined by six mutations in complete linkage disequilibrium, is denoted as V. *P is the probability that the joint odds ratio for the 3 populations is not greater than 1.

Fig.2. Kaplan–MeiersurvivalcurveforCzechelderlystratifiedbygenotype. Error bars depict standard error. Individuals homozygous for V352 were too few to generate a survival curve.

plausible that the apparent lack of heterozygote advantage in the two Baltimore groups may manifest the absence of an environ- mental pressure that was specific to the Bohemian Czech population. A homozygote disadvantage can generally be ob- served independent of environmental factors.

AYSYO <pSE PLSMedia te = PP" G5 kDklotho [ACells 9 8 = =" 60 kDneo Cells = a ee = 32 kD4.0 ————_—_                 3.5 +3.02.5 +2.0 51.5 41.0 +0.5 50.0 +10 Exirecellilet intracellularra

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 at later ages (24). To test whether the KL-VS allele of KLOTHO followed this model, we generated Kaplan–Meier survival curves for the Czech elderly and demonstrated that heterozygosity for KL-VS contributed to improved longevity, defined as life expectancy after age 75, and 7<< |   not just survival (P 0.05 for individuals 80 years).

Although cryptic population stratification has the potential to the interpretation of association studies, it is unlikely be a significant factor in the study described here. First, the survival disadvantage for homozygosity of the KL-VS was observed in three distinct populations [odds ratio  2.70, 2.40, 2.66]. Second, large differences in allele akd/o were not observed between ethnically and or geo- diverse populations for a given age group. For the KL-VS genotype frequencies were not significantly am> ( for Baltimore Caucasian and Czech infants (P 0.20) aR RAE> > ( for Baltimore Caucasian and Czech elderly (P 0.93). Yet the in allele frequencies between the combined Cauca- (Baltimore and Czech) infants and combined Caucasian << | was significant (P 0.006). Third, if one considers the similarities within an age group, the differences between groups that we observe would require truly massive migra- to have occurred within one generation.

Fig. 3. Extracellular and intracellular levels of klotho from transiently Extracellular klotho is glycosylated, resulting in a 5-kDa increase in mass (data not shown). (B) Bar graph depicting the extracellular to lular ratio of klotho protein normalized to wild-type from at least independent assays. Error bars represent 1 SD. All ratios are different from each other and wild-type (P >< ( 0.05).

Dimerization is a common characteristic of family 1 glycosi- dases (2) and, for many integral membrane proteins, is essential for export out of the endoplasmic reticulum (ER) (18, 19). Indeed, extracellular levels of klotho protein harboring V352 are reduced 6-fold. It remains to be determined whether this mu- tation influences klotho dimerization or interaction with other

    %Ro |%,“~% |%,YayP8288 8o o 10oO QAnhx N“efin |“ep|a |sos cs so a9< m

Fig. 4. Enzymatic activity of wild-type and F289V substituted human cytosolic (cid:1)-glucosidase (cBGL1). (A) Coomassie-stained gel demonstrating protein purificationofcBGL1-GSTfusionsinelutionfractionsE2(cid:10)E3(cBGL1-F)andE1(cid:10)E2(cBGL1-V).Nootherproteinswerevisualizedintheselanes(datanotshown). (BandD)Bargraphsdepictingenzymaticactivityagainstp-nitrophenyl-(cid:1)-D-glucoside.(C)Coomassie-stainedgeldemonstratingproteinexpressionofcBGL1-FS and cBGL1-VS. *, No measurable enzymatic activity.