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 dur- ing the past millennium. Both the World Wars of 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 am care in the early 1900s (childhood mortality 20%). The majority were stratified into the ‘‘SENIEUR –Apparently Healthy’’ (141 subjects) and ‘‘SENIEUR –Inpatient’’ (291 sub- jects) categories of the EURAGE ‘‘SENIEUR protocol’’ (7, 17).

Mutation Screening. Exons 1–5 were analyzed by single-strand conformation polymorphism (SSCP) analysis as described (12),  Wwws/3 a5/1 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 (CLON- TECH). Exons 2–5 were amplified using AmpliTaq Gold (Per- kin–Elmer). Primer sequences are available on request.

Tissue Culture, Transfections, and Western Analysis. HeLa cells were cultured in DMEM supplemented with 10% FCS and an anti- WitO/E biotic–antimycotic mixture (GIBCO BRL). Transfections were carried out with TransIT-HeLaMonster (Panvera, Madison, ) eg 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, Is) pul Gloucestershire, U.K.). Sample volume was brought up to 450 l conan)/5 ) pal with 20 mM Tris (pH 7.5) 50 mM NaCl. After adding 150 l of AREA) pal NuPage LDS sample buffer (Invitrogen) and 100 l of 1 M DTT, samples were heated to 70°C for 10 min and centrifuged briefly. Cell extracts were also prepared 48 h after transfection. Follow- 2x ing a brief wash with PBS, cells were lysed in 2 SDS sample buffer (13), collected by scraping, and samples were boiled for S/ 5 min and centrifuged briefly. Samples were subjected to SDS PAGE [NuPage 7% Tris-Acetate (Invitrogen)] and transferred to nitrocellulose.

  Marker 1 allele frequencies were significantly different (P 0.04) between newborn and elderly individuals (Table 1). This difference was largely attributable to allele 17, which was more ee prevalent in newborns (P 0.0002). These data indicate a detrimental role for this allele in survival, with a 2.19-fold (95% CI 1.45–3.31) increase in the risk of death before age 75. This Muve>< ( difference is still highly significant (P 0.0031) after applying a Bonferroni correction for the number of alleles tested, and did not depend on the sex or health of the individuals (data not shown). The distribution of Marker 2 alleles did not differ   significantly between the groups (P 0.25). These marker data strongly suggest a role for KLOTHO in human survival and precipitated a search for functional variants.

We used SSCP analysis and DNA sequencing to screen for mutations in KLOTHO that could influence human aging and identified an allele, termed KL-VS, that was defined by the presence of six single nucleotide polymorphisms (SNPs) that

Immunoblotting with anti-V5 (Invitrogen) and anti-neomycin

longevity, we generated Kaplan–Meier survival curves for the Czech elderly population stratified by genotype (Fig. 2). A positive effect on lifespan was seen in individuals over 80 years wi ©<< | old who were heterozygous for the KL-VS allele (P 0.05).

Table 1. KLOTHO allele distribution in newborn and elderly individuals

Allele Newborn, n Elderly, n (cid:3)2 P Marker 1 5 274 196 0.14 0.71 7 396 295 0.03 0.87 8 13 12 0.32 0.57 12 20 17 0.19 0.66 13 29 16 0.89 0.34 14 36 31 0.42 0.52 15 162 137 1.60 0.20 16 78 62 0.21 0.65 17 91 32 14.0 0.00014 18 21 21 1.00 0.32 19 8 10 1.30 0.26 Totals 1,153 850 Overall comparison 19.3 P < 0.04 Marker 2 1 54 29 1.50 0.23 2 12 12 0.76 0.38 5 15 8 0.41 0.52 6 71 34 3.60 0.06 7 542 369 0.45 0.50 8 285 217 1.00 0.32 9 56 55 3.30 0.07 10 42 37 1.00 0.31 11 44 33 0.08 0.78 12 65 41 0.32 0.57 Totals 1,196 842 Overall comparison 11.5 P > > ( 0.25

We analyzed two additional populations for the presence of the KL-VS allele. The cohorts used consisted of 965 elderly   individuals ( 65 years) collected for the Baltimore Women’s Health and Aging Study (WHAS) (8, 9) and 646 control infants collected for the Baltimore Washington Infant Study (BWIS) (10) stratified by race (Caucasian and African-American). ASO analysis was performed on at least 120 individuals from each race (60 infant, 60 elderly), and again demonstrated complete linkage disequilibrium for all of the coding region mutations in the allelic variant marked by the presence of V352 (data not shown). Whereas the heterozygous advantage was not observed, the frequency of elderly individuals homozygous for the KL-VS allele was decreased relative to that observed in infants in both ne St the Caucasian (P 0.05), and African-American populations   (P 0.06; Table 2). Thus, a homozygous disadvantage for survival was observed in three distinct populations. Use of the Mantel–Haenszel (16) estimate of joint odds ratio and signifi- cance demonstrates a 2.59-fold survival disadvantage for indi- viduals homozygous for the KL-VS allele, independent of race   (P 0.0023).