Association of human aging with a functional variant of klotho

Dan E. Arking*, Alice Krebsova, Milan Macek Sr., Milan Macek Jr., Albert Arking, I. Saira Mian, Linda Fried, Ada Hamosh*, Srabani Dey*, Iain McIntosh*, and Harry C. Dietz*(cid:1)

*Institute of Genetic Medicine, (cid:1)Howard Hughes Medical Institute, and Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205; Institute of Biology and Medical Genetics, Department of Molecular Genetics, Cystic Fibrosis Centre and Centre for Integrated Genomics, University Motol and 2nd School of Medicine, Charles University, Prague, Czech Republic; Johns Hopkins University, Baltimore, MD 21218; and Department of Cell and Molecular Biology (MS 74-197), Life Science Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720

Edited by William S. Sly, St. Louis University School of Medicine, St. Louis, MO, and approved November 21st, 2001 (received for review September 13th, 2001)

Mice deficient in Klotho gene expression exhibit a syndrome resembling premature human aging. To determine whether variation in the human KLOTHO locus contributes to survival, we applied two newly characterized polymorphic microsatellite markers flanking the gene in a population-based association study. In a cohort chosen for its homogeneity - Bohemian Czechs - we demonstrated significant differences in selected marker allele frequencies between newborn and elderly individuals (P < 0.05). These results precipitated a search for functional variants of klotho. We identified an allele termed KL-VS containing six sequence variants in complete linkage disequilibrium. Two of these variants result in amino acid substitutions F352V and C370S. Homozygous elderly individuals were underrepresented in three distinct populations: Bohemian Czechs, Baltimore Caucasians, and Baltimore African-Americans [combined odds ratio (OR) 2.59, P < 0.0023]. In a transient transfection assay, secreted levels of klotho harboring V352 are reduced six-fold, whereas extracellular levels of the S370 form are increased 2.9-fold. The V352S370 double mutant exhibits an intermediate phenotype (1.6-fold increase), providing a rare example of intragenic complementation in cis by human single nucleotide polymorphisms. The remarkable conservation of F352 among homologous proteins suggests that it is functionally important. The corresponding substitution, F289V, in the closest human klotho paralog with a known substrate, cBGL1, completely eliminates its ability to cleave p-nitrophenyl-D-glucoside. These results suggest that the KL-VS allele influences the trafficking and catalytic activity of klotho and that variation in klotho function contributes to heterogeneity in the onset and severity of human aging.

The mechanism of klotho function remains unclear. Recent experiments have shown that endothelium-dependent vasodilation of the aorta and arterioles was impaired in heterozygous klotho mice but could be restored by parabiosis with wild-type mice. In addition, nitric oxide metabolites (NO and NO2) in urine are significantly lower in heterozygous klotho mice. These results suggest that klotho protein may protect the cardiovascular system through endothelium-derived NO production.

Remarkably, in vivo klotho gene delivery can ameliorate vascular endothelial dysfunction, increase nitric oxide production, reduce elevated blood pressure, and prevent medial hypertrophy and perivascular fibrosis in a rat model with multiple atherogenic risk factors including hypertension, diabetes, obesity, and hyperlipidemia.

Human klotho shows 86% amino acid identity with the mouse protein and is encoded by a gene that spans over 50 kb on chromosome 13q12. To date, no premature aging syndromes have been linked to this region. Like the mouse Klotho gene, human KLOTHO encodes both membrane-bound and secreted forms. However, in humans, the secreted form predominates. Expression of KLOTHO mRNA occurs primarily in the placenta, kidney, prostate, and small intestine.

To determine whether the KLOTHO gene is involved in human aging, we used a population-based association study employing two newly characterized flanking microsatellite markers. Marker 1 is located 11 kb 3' of the last exon, and Marker 2 is located 66 kb 5' of the first exon. We present evidence that the KLOTHO locus is associated with human survival defined as postnatal life expectancy and longevity defined as life expectancy after 75. We also identify and(Clontech) - with primers Glu5 and Glu3, using Pfu turbo polymerase (Stratagene). The PCR product was cloned into the N-terminal GST-fusion expression vector pDEST15 by using the GATEWAY cloning system (GIBCO BRL) and verified by direct sequencing. The substitutions F289V and Q307S were introduced as described above. All primer sequences are available on request.

Statistics. Individual allele distributions were analyzed using the fuhahysek/~a GAS package Version 2.3 (http://users.ox.ac.uk/ayoung), and overall marker comparisons were made using the CLUMP program (15), which employs a Monte Carlo-based method for determining P. Individual genotype distributions were analyzed using Pearson's χ2 test (http://home.clara.net/sisa/binomial.htm) and the CLUMP program was used to determine overall significance. Survival advantage or disadvantage was calculated as an odds ratio (OR) with a 95% confidence interval (CI) Ave://b homa/b (http://home.clara.net/sisa/binomial.htm). MantelHaenszel log rank analysis with one-sided P of KaplanMeier survival curves was performed using PRISM, Version 3.0 for Windows (GraphPad, San Diego). The combined population analysis was calculated using the MantelHaenszel estimate of joint OR and significance (16).