Marker Genotyping. Multiple dinucleotide repeat sequences were identified by visual inspection of the genomic sequence for KLOTHO and flanking regions (GenBank accession nos. Z92540 and Z92541). Marker 1 (sense, 5'-CATACGCAGAC-CCAGAGCC; antisense, 5'-GCTTCTCATTACGAAAGTA-CAGCTGGG, italic sequence is a tail to force reaction to completion) is 11 kb 3 of exon 5, and Marker 2 (sense, wwe5/-ns 5'-CTTACCAGCATGTGAGCTATC; antisense, 5'-TTTTA-an5! CAGGTGATCTGAGACTC) is 66 kb 5 of exon 1. Size fractionation of PCR products derived from multiple unrelated individuals was used to demonstrate polymorphic variation (data not shown). Markers were amplified using AmpliTaq Gold (PerkinElmer). Sense primers were fluorescently labeled, and product size was determined using a PerkinElmer (Abd) 373a DNA sequencer.

Results. The initial cohort was comprised of 435 elderly individuals (≥75 years), and 611 contemporary newborns, both drawn at random from the Bohemian Czech population. This group is well suited for an association study because it represents Central European populations without extensive non-European immigration during the past millennium. Both the World Wars of the last century did not cause disproportionate changes in population sex ratios (7). The elderly are considered to be escapees of strong selective pressure that existed before the introduction of modern health care in the early 1900s (childhood mortality 20%). The majority were stratified into the SENIEUR Apparently Healthy (141 subjects) and SENIEUR Inpatient (291 subjects) categories of the EURAGE SENIEUR protocol (7,17).

Mutation Screening. Exons 15 were analyzed by single-strand conformation polymorphism (SSCP) analysis as described (12), using 1 TME (500 mM Tris 350 mM MES 10 mM EDTA) buffer, pH 6.8, at room temperature. Exon 1 PCRs were performed using the Advantage-GC Genomic PCR kit (CLONTECH). Exons 25 were amplified using AmpliTaq Gold (PerkinElmer). Primer sequences are available on request.

Tissue Culture, Transfections, and Western Analysis. HeLa cells were cultured in DMEM supplemented with 10% FCS and an antibiotic-antimycotic mixture (GIBCO BRL). Transfections were carried out with TransIT-HeLaMonster (Panvera, Madison, WI), using 10 g of plasmid per 10-cm-diameter dish. Twenty-four hours post-transfection, the cell culture media was replaced with fresh DMEM supplemented with antibiotic-antimycotic mixture. Forty-eight hours post-transfection, the cell culture media was collected and cells were removed by pelleting before concentration in a Vivaspin 20 concentrator (Vivascience, Gloucestershire, U.K.). Sample volume was brought up to 450 l with 20 mM Tris (pH 7.5) 50 mM NaCl. After adding 150 l of NuPage LDS sample buffer (Invitrogen) and 100 l of 1 M DTT, samples were heated to 70indicate significant 2 values (P<0.05). Alleles with frequencies less than 1% in both newborn and elderly groups were not included for analysis but were included to derive the total n. They occur within an 800-bp region spanning exon 2 and flanking sequence (Fig. 1A). Allele-specific oligonucleotide hybridization (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. Phenylalanine at this position is likely 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 there is no known klotho substrate, the biochemical consequences of the F352V and C370S amino acid substitutions could not be determined directly. Therefore, we performed enzymatic assays on the closest klotho paralog for which there is a known substrate, human cytosolic β-glucosidase (cBGL1) (20, 21). As in most eukaryotic klotho homologs 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-β-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 elderly Bohemian Czech individuals (P<0.03), with heterozygosity 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 population was stratified by sex or health status (data not shown). The KL-VS allele is found on multiple marker allele haplotypes, and it 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